WO2020163591A1 - Compositions and methods for characterizing pancreatic ductal adenocarcinoma - Google Patents

Compositions and methods for characterizing pancreatic ductal adenocarcinoma Download PDF

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Publication number
WO2020163591A1
WO2020163591A1 PCT/US2020/017005 US2020017005W WO2020163591A1 WO 2020163591 A1 WO2020163591 A1 WO 2020163591A1 US 2020017005 W US2020017005 W US 2020017005W WO 2020163591 A1 WO2020163591 A1 WO 2020163591A1
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marker
panel
cd44v6
polypeptide
markers
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PCT/US2020/017005
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French (fr)
Inventor
Senthil K. MUTHUSWAMY
Manuel Hidalgo
Bruno BOCKORNY
Ling Huang
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Beth Israel Deaconess Medical Center, Inc.
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Publication of WO2020163591A1 publication Critical patent/WO2020163591A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4727—Calcium binding proteins, e.g. calmodulin
    • 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
    • G01N2333/70585—CD44
    • 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
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates

Definitions

  • pancreatic ductal adenocarcinoma has an overall five-year survival rate of just 9%, but if it is detected at an early stage, the five-year survival rate increases to 32%.
  • pancreatic ductal adenocarcinoma is very often hard to diagnosis.
  • CA 19-9 which is an antigen released by the pancreas, is commonly used to detect pancreatic ductal adenocarcinoma, but diagnostics used to detect this marker have only a 75.4% pooled sensitivity and a specificity of 77.6%.
  • Improved methods for early detection of pancreatic ductal adenocarcinoma are urgently required.
  • the present invention features compositions and methods for characterizing pancreatic ductal adenocarcinoma, as well as other pancreatic neoplasias.
  • Some aspects of the present invention provide a panel for characterizing pancreatic ductal adenocarcinoma, wherein the panel includes two or more capture molecules each bound to a substrate, where each capture molecule specifically binds a marker polypeptide that is any one or more of ANXA11, CD14, CD44v6, and GPC4, or a polynucleotide encoding said polypeptide.
  • Some aspects of the present disclosure provide a panel for characterizing pancreatic ductal adenocarcinoma, and the panel includes two or more capture molecules each bound to a substrate, where each capture molecule specifically binds a marker polypeptide selected from the group consisting of CD44v6, MUC1, CLDN4, TSPAN8, CD 147, and CD 104, or the marker is a polynucleotide encoding said polypeptide.
  • the capture molecule is a polypeptide, polynucleotide probe, or fragment thereof.
  • the polypeptide is an antibody that specifically binds the marker polypeptide.
  • the polynucleotide is an aptamer that specifically binds the marker polypeptide, and in some embodiments, polynucleotide probe specifically hybridizes to the polynucleotide encoding the marker polypeptide.
  • the substrate is a bead or planar surface.
  • the planar surface is a membrane, filter, chip, glass slide, or other solid support.
  • the panel in some embodiments, includes capture molecules each of which bind ANXA11, CD 14, CD44v6, and GPC4; ANXA11, CD14, and CD44v6; ANXA11, CD14, and GPC4; ANXA11, CD44v6, and GPC4; ANXA11 and CD14; ANXA11 and CD44v6;
  • ANXA11 and GPC4 CD14, CD44v6, and GPC4; CD14 and CD44v6;CD14 and GPC4; or CD44v6, and GPC4.
  • the panel includes capture molecules that bind CD44v6 and one or more of MUC1, CLDN4, TSPAN8, CD147, and CD104. In some embodiments, the panel includes capture molecules that bind MUC1 and one or more of CD44v6, CLDN4, TSPAN8, CD147, and CD104. In some embodiments, the panel includes capture molecules that bind TSPAN8 and one or more of CD44v6, MUC1, CLDN4, CD 147, and CD 104. In some embodiments, the panel includes capture molecules that bind CD147 and one or more of CD44v6, MUC1, CLDN4, TSPAN8, and CD104.
  • Another aspect of the present disclosure provides a panel for characterizing pancreatic ductal adenocarcinoma, and the panel includes two or more capture molecules each bound to a substrate, where each capture molecule specifically binds a marker polypeptide selected from those listed in Table 1.
  • each capture molecule specifically binds a marker polypeptide selected from those listed in Table 1.
  • one or more of the capture molecules are antibodies.
  • a method for detecting a marker in a sample derived from a subject, the method involving detecting two or more markers selected from the group consisting of CD44v6, MUC1, CLDN4, TSPAN8, CD147, and CDKMor a marker of Table 1 in the sample.
  • Another aspect provides a method for detecting a marker in a sample derived from a subject, the method involving detecting two or more markers selected from the group consisting of ANXA11, CD14, CD44v6, and GPC4, or a marker of Table 1 in the sample.
  • a method for characterizing pancreatic ductal adenocarcinoma the method involving a) culturing a cell from a subject having or suspected of having pancreatic ductal adenocarcinoma, thereby generating an organoid culture; b) isolating extracellular vesicles present in culture media of the organoid culture; and c) detecting a marker present in or on the extracellular vesicles, where the marker is selected from the group consisting of CD44v6, MUC1, CLDN4, TSPAN8, CD147, and CD 104 or the group consisting of ANXA11, CD 14, CD44v6, and GPC or a marker of Table 1 using a capture molecule that specifically binds the marker.
  • the capture molecule is bound to a substrate surface.
  • the substrate surface has a spectral reflectance signature.
  • the method further involves (d) exposing the substrate surface to a light source; and (e) detecting the presence or absence of extracellular vesicles on the substrate surface, where the presence of the extracellular vesicles indicates the presence of one of the markers.
  • the sample is a biological fluid selected from the group consisting of culture media, blood, blood serum, plasma, urine, abdominal fluids, or pancreatic secretions.
  • the sample is a biopsy.
  • the sample includes extracellular vesicles, and in some embodiments, these extracellular vesicles are exosomes or microvesicles.
  • the detecting involves RT-PCR, Northern blotting, Western blotting, flow cytometry, immunocytochemistry, binding to magnetic and/or antibody-coated beads, in situ
  • the detecting involves liquid chromatography-mass spectrometry (LC-MS/MS).
  • the detecting involves using a capture molecule that specifically binds the marker.
  • the capture molecule is an antibody, while in other embodiments, the capture molecule is an aptamer.
  • the capture molecule is bound to a substrate surface, which has, in some embodiments, a spectral reflectance signature.
  • kits for characterizing markers in a sample includes two or more capture molecules fixed to a substrate surface, where each capture molecule specifically binds a marker polypeptide selected from the group consisting of CD44v6, MUC1, CLDN4, TSPAN8, CD147, and CD104 or the group consisting of
  • the substrate surface has a spectral reflectance signature.
  • the present disclosure provides methods for characterizing markers associated with pancreatic ductal adenocarcinoma, as well as other pancreatic neoplasias.
  • the method involves analyzing markers present in extracellular vesicles, such as exosomes, isolated from the supernatant of an organoid model of pancreatic disease (e.g., pancreatic ductal adenocarcinoma or another pancreatic neoplasia).
  • an organoid model of pancreatic disease e.g., pancreatic ductal adenocarcinoma or another pancreatic neoplasia.
  • pancreatic ductal adenocarcinoma The methods disclosed herein were used to identify subjects having pancreatic ductal adenocarcinoma and distinguish these subjects from others having a benign pancreatic disease (e.g., pancreatitis), as is explained the examples provided below.
  • a benign pancreatic disease e.g., pancreatitis
  • alteration is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein.
  • an alteration includes a 10% change, a 25% change, a 40% change, a 50% or a greater change.
  • ANXA11 a polypeptide or fragment thereof having at least about 85% amino acid identity to GenBank Accession No. AAV38737.1 and having anti- ANXA11 antibody binding activity.
  • An exemplary ANXA11 amino acid sequence is provided below:
  • CLDN4 refers to a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_001296 and having anti-CLDN4 antibody binding activity.
  • An exemplary CLDN4 amino acid sequence is provided below:
  • CD9 Cluster of Differentiation 9
  • NCBI Accession No. NP_001760.1 An exemplary CD9 amino acid sequence is provided below:
  • CD14 Cluster of Differentiation 14
  • NP_001167576.1 amino acid identity to NCBI Accession No. NP_001167576.1 and having anti-CD14 antibody binding activity.
  • An exemplary CD14 amino acid sequence is provided below:
  • CD44v6 Cluster of Differentiation 44, transcript variant 6
  • a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_001189484.1 and having anti-CD44v6 antibody binding activity.
  • An exemplary CD44v6 amino acid sequence is provided below: 1 mdkfwwhaaw glclvplsla qidlnitcrf agvfhvekng rysisrteaa dlckafnstl
  • CD81 Cluster of Differentiation 81
  • Cluster of Differentiation 104 is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_000204.3 and having anti-CD 104 antibody binding activity.
  • Detect refers to identifying the presence, absence or amount of the analyte to be detected.
  • decreases is meant a reduction by at least about 5% relative to a reference level.
  • a decrease may be by 5%, 10%, 15%, 20%, 25% or 50%, or even by as much as 75%, 85%, 95% or more.
  • detectable label is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
  • useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.
  • disease is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.
  • Pancreatic ductal adenocarcinoma is a disease, for example.
  • an effective amount is meant the amount of an agent required to ameliorate the symptoms of a disease relative to an untreated subject.
  • the effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.
  • the invention provides targets that are useful for the development of highly specific drugs to treat or a disorder characterized by the methods delineated herein.
  • the methods of the invention provide a facile means to identify therapies that are safe for use in subjects.
  • the methods of the invention facilitate analysis of virtually any compound for effects on a disease described herein.
  • EPCAM Epidermal Cell Adhesion Molecule
  • EPHA2 EPH Receptor A2
  • extracellular vesicle a membrane surrounded structure that is released by a cell.
  • exemplary extracellular vesicles include exosomes and microvesicles.
  • Exosomes include small membrane bound extracellular vesicles of -30-300 nm diameter that are secreted by cells into the extracellular environment.
  • the surface of an exosome comprises a lipid bilayer from the membrane of the donor cell, and the lumen of the exosome is topologically the same as the cytosol from the cell that produces the exosome.
  • An exosome contains, for example, proteins, RNAs, lipids, and/or carbohydrates of the producing cell, though the contents of the exosome may be modified after the exosome’ s release from the producing cell, either through natural processes or by experimental manipulation.
  • fragment is meant a portion of a polypeptide or nucleic acid molecule. This portion comprises, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide.
  • a fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.
  • GPC1 GPC1
  • GPC4 Glypican 4
  • GenBank Accession No. EAX11772.1 GenBank Accession No. EAX11772.1
  • An exemplary GPC4 amino acid sequence is provided below:
  • a healthy cell culture is a culture derived from cells that exhibit no signs of a disease.
  • healthy refers to cells derived from an organoid model, wherein the organoid model does not manifest any disease traits.
  • Diseased cells may be obtained from a diseased subject, a disease model (e g., an organoid model of disease), or are otherwise derived from diseased cells.
  • a disease model e g., an organoid model of disease
  • HER2 Human Epidermal Growth Factor Receptor 2
  • HER2 Human Epidermal Growth Factor Receptor 2
  • HER2 amino acid sequence is provided below:
  • Hybridization means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases.
  • By“increases” is meant a reduction by at least about 5% relative to a reference level.
  • a increase may be by 5%, 10%, 15%, 20%, 25% or 50%, or even by as much as 75%, 85%, 95% or more.
  • isolated refers to material that is free to varying degrees from components which normally accompany it as found in its native state.
  • Isolate denotes a degree of separation from original source or surroundings.
  • Purify denotes a degree of separation that is higher than isolation.
  • a “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high-performance liquid chromatography.
  • the term "purified" can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel.
  • modifications for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.
  • isolated polynucleotide is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene.
  • the term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences.
  • the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.
  • an "isolated polypeptide” is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated.
  • the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention.
  • An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
  • marker is meant any analyte having an alteration in structure, expression level or activity that is associated with a disease or disorder.
  • MUC1 MUC1
  • NP_001018017.1 amino acid identity to NCBI Accession No. NP_001018017.1 and having anti- MUC1 antibody binding activity.
  • An exemplary MUC1 amino acid sequence is provided below:
  • “obtaining” as in“obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent.
  • organoid refers to a three-dimensional tissue culture having a microanatomy similar to an intact organ.
  • the organoid generated from a tumor tissue recreate the histomorphology present in the patient tumor, retain the differentiation station, retain major genetic mutations and H3K27 acetylation profile.
  • the organoid can be a subject derived organoid, wherein the cells cultured to generate the organoid are obtained from a subject having or suspected of having a disease.
  • Subject derived organoids can be tumor organoids, wherein the cells used to generate the organoid are obtained from a subject’s tumor.
  • a“normal reference” is a reference obtained from an otherwise healthy subject or subjects.
  • a normal reference for the quantity of a marker in an exosome is the quantity of the marker in an exosome obtained from a healthy subject or subjects, a culture of healthy cells, or an organoid model of a healthy organ, such as a pancreas.
  • the quantity may be the average of quantities detected in each subject.
  • the normal reference may also be a published reference.
  • a“disease reference” is a known quantity of the marker in an exosome obtained from a subject or subjects having the disease, a culture of diseased cells, or an organoid model of the disease. When the quantity is determined using more than one subject having the disease, the quantity may be the average of quantities detected in each subject.
  • the disease reference may also be a published reference.
  • a “reference sequence” is a defined sequence used as a basis for sequence comparison.
  • a reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
  • the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids.
  • the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween.
  • telomere binding By “specifically binds” is meant a compound or antibody that recognizes and binds a polypeptide of the invention, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the invention.
  • Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having“substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having“substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule.
  • hybridize pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency.
  • complementary polynucleotide sequences e.g., a gene described herein
  • stringency See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).
  • stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate.
  • Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide.
  • Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C.
  • hybridization time the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA
  • concentration of detergent e.g., sodium dodecyl sulfate (SDS)
  • SDS sodium dodecyl sulfate
  • Various levels of stringency are accomplished by combining these various conditions as needed.
  • hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS.
  • hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 pg/ml denatured salmon sperm DNA (ssDNA).
  • ssDNA denatured salmon sperm DNA
  • hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate,
  • wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature.
  • stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.
  • Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, more preferably of at least about 42° C, and even more preferably of at least about 68° C.
  • wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS.
  • Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.
  • substantially identical is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). In embodiments, such a sequence is at least 60%, at least 80% or 85%, or at least 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.
  • Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705,
  • BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications.
  • Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine;
  • a BLAST program may be used, with a probability score between e 3 and e 100 indicating a closely related sequence.
  • TSPAN8 Tetraspanin 8
  • the terms“treat,” treating,”“treatment,” and the like refer to reducing or ameliorating a disorder and/or symptom associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
  • To“vary” as used herein means to differ from a reference in a quantitative manner, and to“vary significantly” is to differ from the reference in an amount greater than a predetermined threshold.
  • concentration of a marker in a sample isolated from a model of a disease can be said to vary significantly from a reference concentration if the marker’s concentration is at least 2-fold greater or at least 50% below the reference concentration.
  • the reference concentration can be known in the art or experimentally determined.
  • Ranges provided herein are understood to be shorthand for all of the values within the range.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
  • the term“about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.
  • the recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups.
  • the recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
  • compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
  • FIG. l is a graph illustrating the results of a principle component analysis performed on patient derived organoid models.
  • “PCI” and“PC2” refer to principle components 1 and 2, which are sets of variables defined by the Principle Component Analysis.
  • FIG. 2. is an illustration depicting the functional clustering of potential markers identified in patient derived organoid models of pancreatic ductal adenocarcinoma.
  • FIG. 3 is a graph depicting normalized counts of extracellular vesicles comprising particular proteins.
  • FIGs. 4A-4C present clinical validation of a selected group of markers including ANNXA11, CD14, GPC4, and CD44v6.
  • FIG. 4A is an image of a western blot of proteins in extracellular vesicles from patients. Extracellular vesicles from 10 m ⁇ of plasma were loaded in each lane. “GI Benign” denotes benign gastrointestinal disorders, and“PDACnf’ denotes pancreatic ductal adenocarcinoma no treatment.” Throughout the figures,“GP” and“PA” identify individual subjects diagnosed with benign gastrointestinal disorders and pancreatic ductal adenocarcinoma, respectively.
  • FIG. 4B is a heatmap of protein quantification from FIG. 4A.
  • FIG. 4C is a graph depicting the geometric means of protein marker scores for each patient sample. Protein marker score is calculated from western blot: intensity per band divided by the median band intensity of each marker in whole cohort and multiplied by 100.
  • compositions and methods that are useful for the characterization of pancreatic ductal adenocarcinoma (PDAC).
  • PDAC pancreatic ductal adenocarcinoma
  • the invention is based, at least in part, on the discovery of markers including CD44v6, MUC1, CLDN4, TSPAN8, CD147, and CD104, (the“Group I markers”) that are increased in extracellular vesicles isolated from organoids derived from a subject that has PD AC.
  • markers including CD44v6, MUC1, CLDN4, TSPAN8, CD147, and CD104, (the“Group I markers”) that are increased in extracellular vesicles isolated from organoids derived from a subject that has PD AC.
  • a second set of markers was identified that is increased in extracellular vesicles derived from reference subject-derived organoid samples. These markers are CD9, CD81, GPC1, EPHA2, EPCAM, EGFR, HER2, and CD63 (the“Group II markers”).
  • a further group of markers that is increased in extracellular vesicles of patients with pancreatic cancer includes ANXA11, GPC4, CD14, and CD44v6.
  • the identification of these sets of markers provides a blood-based assay for the detection of pancreatic ductal adenocarcinoma.
  • the Group I markers were shown to be present in pancreatic cancer patient blood samples at levels about 100 to 1000-fold greater than observed in the blood obtained from patients having pancreatitis.
  • pancreatic adenocarcinoma PDAC
  • One promising approach involves identification of secreted extracellular vesicles in the blood of PDAC patients that comprise markers that are specific for the disease. These markers would allow differentiation of PDAC patients from both others who are disease-free or afflicted with chronic pancreatitis, a benign condition.
  • Several studies have attempted to identify disease associated protein markers using extracellular vesicles isolated from cultured cells. To date, it is not yet established if organoid cultures can be used to discover new, clinically significant, secreted markers.
  • a typical extracellular vesicle identification effort using cells in culture requires large amount of media supernatant (0.1 -1.0 liters), coupled with elaborate analytical centrifugation and purification steps, which would be difficult to implement when working with patient derived tumor models that are not amenable to large scale cultures.
  • the present disclosure provides methods for identifying markers associated with a disease.
  • the methods comprise isolating extracellular vesicles derived from an organoid model of the disease and from a reference organoid and then quantifying the concentration of markers in or on the extracellular vesicles.
  • extracellular vesicles are released by cells (for example, tumor cells) into the extracellular environment, and the extracellular vesicles nucleic acids polypeptides that identify from what type of cell the exosome is derived.
  • Extracellular vesicles as defined herein are isolated from a sample as described herein below.
  • Extracellular vesicles are isolated from a variety of biological fluids, including but not limited to, blood, plasma, serum, urine, abdominal fluids, and pancreatic secretions n some embodiments, the extracellular vesicles are enriched prior to being isolated.
  • extracellular vesicles within a sample can be enriched using a 100 kDa concentration step that is subsequently followed by purification of the extracellular vesicles.
  • the 100 kDa concentration step comprises passing the sample comprising the extracellular vesicles through a high molecular weight filter.
  • extracellular vesicles can be purified using any means known in the art.
  • Concentrating and purifying the extracellular vesicles allows removal of free proteins, cell membranes, and other cellular debris that may confound marker detection and quantification.
  • Markers derived from the extracellular vesicles isolated from the organoid model of the disease with at least a 2-fold difference in marker concentration relative to the extracellular vesicles isolated from the reference organoid are identified as markers associated with the disease.
  • the marker associated with disease will have about a 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or even a 1000-fold.
  • the difference in expression of a marker associated with disease between the expression of the marker in the organoid model of disease and the marker’s expression in a reference sample is between about 2-fold and about 1000-fold, between about 5-fold and about 1000-fold, between about 10-fold and about 1000-fold, between about 20-fold and about 1000-fold, between about 50-fold and about 1000-fold, between about 100-fold and about 1000-fold, between about 200-fold and about 1000-fold, between about 300-fold and about 1000-fold, between about 400-fold and about 1000-fold, between about 500-fold and about 1000-fold, between about 600-fold and about 1000-fold, between about 700-fold and about 1000-fold, between about 800-fold and 1000-fold, and between about 900-fold and
  • the difference in expression of a marker associated with disease between the expression of the marker in the organoid model of disease and the marker’s expression in a reference sample is about 2-fold.
  • Table l is a listing of markers identified as having at least a two-fold increase in extracellular vesicles isolated from an organoid model of pancreatic ductal adenocarcinoma (PDAC).
  • PDAC pancreatic ductal adenocarcinoma
  • a marker can be any marker of Table 1
  • a panel of markers can be any combination of markers of Table 1.
  • a panel of markers comprises ANXA11, CD14, CD44v6, and GPC4. In some embodiments, a panel of markers comprises ANXA11, CD14, and CD44v6. In some embodiments, a panel of markers comprises ANXA11, CD14, and GPC4. In some embodiments, a panel of markers comprises ANXA11, CD44v6, and GPC4. In some embodiments, a panel of markers comprises ANXA11 and CD14. In some embodiments, a panel of markers comprises ANXA11 and CD44v6. In some embodiments, a panel of markers comprises ANXA11 and GPC4. In some embodiments, a panel of markers comprises CD14, CD44v6, and GPC4. In some embodiments, a panel of markers comprises CD 14 and CD44v6. In some embodiments, a panel of markers comprises CD 14 and GPC4. In some embodiments, a panel of markers comprises CD44v6, and GPC4.
  • the markers associated with disease can be clustered into functional groups.
  • a marker associated with a disease such as pancreatic ductal adenocarcinoma can be clustered into a group of proteins involved in RNA splicing, histone or chromatin maintenance, proteasomes, translation, cytoskeleton regulation, cell adhesion, and membrane trafficking.
  • the neoplasia is pancreatic ductal adenocarcinoma.
  • Any suitable method can be used to detect markers (e.g., ANXA11, GPC4, CD14, CD44v6, MUC1, CLDN4, TSPAN8, CD147, and CD104) associated with pancreatic ductal adenocarcinoma.
  • Successful practice of the invention can be achieved with one or a combination of methods that can detect and, in some embodiments, quantify the markers.
  • These methods include, without limitation, hybridization-based methods, including those employed in biochip arrays, mass spectrometry (e.g., laser desorption/ionization mass spectrometry), fluorescence (e.g. sandwich immunoassay), surface plasmon resonance, ellipsometry and atomic force microscopy.
  • Expression levels of markers e.g.,
  • polynucleotides or polypeptides are compared by procedures well known in the art, such as RT-PCR, Northern blotting, Western blotting, flow cytometry, immunocytochemistry, binding to magnetic and/or antibody-coated beads, in situ hybridization, fluorescence in situ hybridization (FISH), flow chamber adhesion assay, ELISA, microarray analysis, or colorimetric assays.
  • procedures well known in the art such as RT-PCR, Northern blotting, Western blotting, flow cytometry, immunocytochemistry, binding to magnetic and/or antibody-coated beads, in situ hybridization, fluorescence in situ hybridization (FISH), flow chamber adhesion assay, ELISA, microarray analysis, or colorimetric assays.
  • Methods may further include, one or more of electrospray ionization mass spectrometry (ESI-MS), ESI-MS/MS, ESI-MS/(MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS), matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), surface-enhanced laser desorption/ionization time-of- flight mass spectrometry (SELDI-TOF-MS), desorption/ionization on silicon (DIOS), secondary ion mass spectrometry (SIMS), quadrupole time-of-flight (Q-TOF), atmospheric pressure chemical ionization mass spectrometry (APCI-MS), APCI-MS/MS, APCI-(MS), atmospheric pressure photoionization mass spectrometry (APPI-MS), APPI-MS/MS, and APPI-(MS), quadrupole mass spectrometry, fourier transform
  • Markers may be captured with capture reagents immobilized to a solid support, such as a biochip, a multi-well microtiter plate, a resin, or a nitrocellulose membrane that is subsequently probed for the presence or level of a marker.
  • a solid support such as a biochip, a multi-well microtiter plate, a resin, or a nitrocellulose membrane that is subsequently probed for the presence or level of a marker.
  • Capture can be on a
  • a sample containing the markers such as serum
  • a sample containing the markers may be used to contact the active surface of a biochip for a sufficient time to allow binding. Unbound molecules are washed from the surface using a suitable eluent, such as phosphate buffered saline.
  • a suitable eluent such as phosphate buffered saline.
  • the more stringent the eluent the more tightly the proteins must be bound to be retained after the wash.
  • analytes Upon capture on a biochip, analytes can be detected by a variety of detection methods selected from, for example, a gas phase ion spectrometry method, an optical method, an electrochemical method, atomic force microscopy and a radio frequency method.
  • mass spectrometry is used.
  • the mass spectrometry used is LC -MS/MS.
  • the mass spectrometry is SELDI.
  • Optical methods include, for example, detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, birefringence or refractive index (e.g., surface plasmon resonance, ellipsometry, a resonant mirror method, a grating coupler waveguide method or interferometry).
  • Optical methods include microscopy (both confocal and non- confocal), imaging methods and non-imaging methods.
  • Immunoassays in various formats e.g., ELISA are popular methods for detection of analytes captured on a solid phase.
  • Electrochemical methods include voltammetry and amperometry methods.
  • Radio frequency methods include multipolar resonance spectroscopy.
  • Mass spectrometry is a well-known tool for analyzing chemical compounds.
  • the methods of the present invention comprise performing quantitative MS to measure the serum peptide marker.
  • the method may be performed in an automated (Villanueva, e/ a/., Nature Protocols (2006) 1(2):880-891) or semi-automated format. This can be accomplished, for example with MS operably linked to a liquid chromatography device (LC-MS/MS or LC-MS) or gas chromatography device (GC-MS or GC -MS/MS).
  • Methods for performing MS are known in the field and have been disclosed, for example, in US Patent Application Publication Nos: 20050023454; 20050035286; US Patent No: 5,800,979; and references disclosed therein.
  • the protein fragments are collected on the collection layer. They may then be analyzed by a spectroscopic method based on matrix-assisted laser desorption/ionization (MALDI) or electrospray ionization (ESI).
  • MALDI matrix-assisted laser desorption/ionization
  • ESI electrospray ionization
  • the preferred procedure is MALDI with time of flight (TOF) analysis, known as MALDI-TOF MS. This involves forming a matrix on the membrane, e.g. as described in the literature, with an agent which absorbs the incident light strongly at the particular wavelength employed.
  • the sample is excited by UV, or IR laser light into the vapour phase in the MALDI mass spectrometer.
  • Ions are generated by the vaporization and form an ion plume.
  • the ions are accelerated in an electric field and separated according to their time of travel along a given distance, giving a mass/charge (m/z) reading which is very accurate and sensitive.
  • MALDI spectrometers are commercially available from PerSeptive Biosystems, Inc. (Frazingham, Mass., USA) and are described in the literature, e.g. M. Kussmann and P. Roepstorff, cited above.
  • Magnetic-based serum processing can be combined with traditional MALDI-TOF. Through this approach, improved peptide capture is achieved prior to matrix mixture and deposition of the sample on MALDI target plates. Accordingly, methods of peptide capture are enhanced through the use of derivatized magnetic bead based sample processing.
  • MALDI-TOF MS allows scanning of the fragments of many proteins at once.
  • many proteins can be run simultaneously on a polyacrylamide gel, subjected to a method of the invention to produce an array of spots on the collecting membrane, and the array may be analyzed.
  • automated output of the results is provided by using the ExPASy server, as at present used for MIDI-TOF MS and to generate the data in a form suitable for computers.
  • MALDI-TOF MS can be used to analyze the fragments of protein obtained on the collection membrane. These include the use of delayed ion extraction, energy reflectors and ion-trap modules. In addition, post source decay and MS-MS analysis are useful to provide further structural analysis. With ESI, the sample is in the liquid phase and the analysis can be by ion-trap,
  • Capillary infusion may be employed to introduce the marker to a desired MS implementation, for instance, because it can efficiently introduce small quantities of a sample into a mass spectrometer without destroying the vacuum.
  • Capillary columns are routinely used to interface the ionization source of a MS with other separation techniques including gas chromatography (GC) and liquid chromatography (LC).
  • GC and LC can serve to separate a solution into its different components prior to mass analysis.
  • Such techniques are readily combined with MS, for instance.
  • One variation of the technique is that high performance liquid chromatography (HPLC) can now be directly coupled to mass spectrometer for integrated sample separation/and mass spectrometer analysis.
  • HPLC high performance liquid chromatography
  • Quadrupole mass analyzers may also be employed as needed to practice the invention.
  • Fourier-transform ion cyclotron resonance (FTMS) can also be used for some invention embodiments. It offers high resolution and the ability of tandem MS experiments.
  • FTMS is based on the principle of a charged particle orbiting in the presence of a magnetic field.
  • FTMS couples high accuracy with errors as low as 0.001%.
  • the marker qualification methods of the invention may further comprise identifying significant peaks from combined spectra.
  • the methods may also further comprise searching for outlier spectra.
  • the method of the invention further comprises determining distant dependent K-nearest neighbors.
  • an ion mobility spectrometer can be used to detect and characterize serum peptide markers.
  • the principle of ion mobility spectrometry is based on different mobility of ions. Specifically, ions of a sample produced by ionization move at different rates, due to their difference in, e.g., mass, charge, or shape, through a tube under the influence of an electric field. The ions (typically in the form of a current) are registered at the detector which can then be used to identify a marker or other substances in a sample.
  • One advantage of ion mobility spectrometry is that it can operate at atmospheric pressure.
  • multiple markers are measured.
  • the use of multiple markers increases the predictive value of the test and provides greater utility in diagnosis, toxicology, patient stratification and patient monitoring.
  • the process called“Pattern recognition” detects the patterns formed by multiple markers greatly improves the sensitivity and specificity of clinical proteomics for predictive medicine. Subtle variations in data from clinical samples indicate that certain patterns of protein expression can predict phenotypes such as the presence or absence of a certain disease, a particular stage of neoplasia progression, or a positive or adverse response to drug treatments.
  • detection and quantitation of markers is accomplished by using an affinity microarray-based technology (Nanoview Biosciences) coupled with a Reflectance Imaging Sensor detection methodology as described in International Application No.
  • extracellular vesicles may be bound to a substrate surface that has a spectral reflectance signature.
  • This surface comprises binding probes that specifically bind to exosomes.
  • the binding probe may be an antibody that specifically binds to a surface marker on an exosome (e.g., a glypican protein).
  • the substrate comprising the bound exosomes is exposed to a light source, and the reflectance of the substrate surface is altered compared to a substrate without bound exosomes.
  • the exosomes may be observed as discrete dots in an image.
  • the light source may be a light emitting diode (LED) light source.
  • the binding probe specifically binds to a marker.
  • Expression levels of the markers described herein are correlated with pancreatic ductal adenocarcinoma, and can be used to identify subjects with high probability of having the disease.
  • Antibodies that specifically bind a marker described herein or any other method known in the art may be used to monitor expression of a marker of interest. Detection of an alteration relative to a normal, reference sample can be used as a diagnostic indicator of prostate carcinoma. In some embodiments, at least a 2-fold change in the level of a
  • ANXA11, GPC4, CD 14, CD44v6, MUC1, CLDN4, TSPAN8, CD 147, and CD 104 marker of the present disclosure is indicative of pancreatic ductal adenocarcinoma (PD AC) or the propensity to develop PD AC.
  • an expression profile that characterizes alterations in the expression of two or more markers is correlated with a particular disease state (e.g., PDAC). Such correlations are indicative of PD AC or the propensity to develop PDAC.
  • a pancreatic ductal adenocarcinoma can be monitored using the methods and compositions of the invention.
  • the present invention features diagnostic assays for the detection of PDAC or the propensity to develop such a condition.
  • levels of any one or more of a group of markers consisting of ANXA11, GPC4, CD14, CD44v6, MUC1, CLDN4, TSPAN8, CD 147, and CD 104 are measured in a subject sample and used to characterize PDAC or the propensity to develop such a condition.
  • levels of any two or more of a group of markers consisting of ANXA11, GPC4, CD14, CD44v6, MUC1, CLDN4, TSPAN8, CD 147, and CD 104 are measured in a subject sample and used to characterize PDAC or the propensity to develop such a condition.
  • the levels of any three or more of a group of markers consisting of ANXA11, GPC4, CD14, CD44v6, MUC1, CLDN4, TSPAN8, CD147, and CD104 are measured in a subject sample and used to characterize PDAC or the propensity to develop such a condition.
  • the levels of any four or more of a group of markers consisting of ANXA11, GPC4, CD14, CD44v6, MUC1, CLDN4, TSPAN8, CD 147, and CD 104 are measured in a subject sample and used to characterize PD AC or the propensity to develop such a condition.
  • the level of any five or more of a group of markers consisting of ANXA11, GPC4, CD14, CD44v6, MUC1, CLDN4, TSPAN8, CD147, and CD104 are measured in a subject sample and used to characterize PD AC or the propensity to develop such a condition.
  • the levels of any six or more of a group of markers consisting of ANXAl 1, GPC4, CD 14, CD44v6, MUC1, CLDN4, TSPAN8, CD 147, and CD 104 are measured in a subject sample and used to characterize PD AC or the propensity to develop such a condition.
  • the levels of any seven or more of a group of markers consisting of ANXAl 1, GPC4, CD14, CD44v6, MUC1, CLDN4, TSPAN8, CD147, and CD104 are measured in a subject sample and used to characterize PD AC or the propensity to develop such a condition.
  • the level of one or more markers is measured on at least two separate occasions and an increase in the level is an indication of PD AC progression.
  • the level of the marker decreases over time, which may indicate a slowing of the progression or a regression of the disease.
  • the level of marker in the bodily fluid e g., blood, blood serum, plasma, saliva, urine, abdominal fluid, or pancreatic secretions
  • the level of marker in the bodily fluid may be altered by as little as 10%, 20%, 30%, or 40%, or by as much as 50%, 60%, 70%, 80%, or 90% or more relative to the level of the marker in the normal control.
  • a healthy subject generally expresses
  • a subject sample of a bodily fluid e.g., blood, blood serum, plasma, urine, abdominal fluid, or pancreatic secretions
  • a sample can be a tissue or cell collected prior to the onset of PD AC symptoms.
  • the diagnostic methods described herein can be used to provide a diagnosis individually or to confirm the results of another diagnostic method. Additionally, the methods described herein can be use used with any other diagnostic method described herein for a more accurate diagnosis of the presence or severity of PD AC.
  • the markers identified using the methods described herein can be used individually, in combination with other identified markers, or with other markers known in the art that are associated with cancer or cancer treatment.
  • the markers are differentially present in samples from a subject having cancer and from a normal subject in whom cancer is undetectable. Detecting one or more of these markers in a subject would provide useful information regarding the probability that the subject may have, or be susceptible to, PD AC, the aggressiveness of the neoplasia, and the susceptibility of the neoplasia to treatment.
  • the detection of the peptide marker is then correlated with a probable diagnosis of cancer.
  • the mere detection of a marker e.g., MUC1
  • the analysis of markers present in a sample may also involve quantifying the markers to correlate the detection of markers with a probable diagnosis of cancer. For example, if a detected marker (or set of markers) in a subject is present in a sample in a different amount compared to a control, or reference, amount (i.e., higher than the control), then the subject being tested has a higher probability of having cancer.
  • the correlation of a marker or markers in a sample to a probable diagnosis of cancer may consider the amount of the marker or markers in the sample compared to a control, or reference, amount of the marker or markers (e.g., in subjects having no detectable cancer).
  • the control amount of the marker is measured under the same or substantially similar experimental conditions as used when measuring the amount of the marker in a subject having, suspected of having, or at risk of developing PD AC.
  • the control, or reference, amount of the marker is known, and result obtained from test samples can be compared to that standard, rather than re-running a control.
  • a marker profde may be obtained from a subject sample and compared to a reference marker profde obtained from a reference population, enabling classifying the subject as belonging to or not belonging to the reference population.
  • the correlation of the marker profde to a neoplasia diagnosis may consider the presence or absence of the markers in test and control samples. The correlation may consider both factors when making a cancer status determination.
  • the methods further comprise managing subject treatment based on the status.
  • the invention also provides for such methods where the markers (or specific combination of markers) are measured again after subject management. In these cases, the methods are used to monitor the status of the cancer, e.g., response to cancer treatment, remission of the disease or progression of the disease.
  • markers disclosed herein have uses other than just diagnostic. In some embodiments, they can be used in monitoring responses to neoplasia therapy. In another embodiment, the markers can be used to study the heredity of the disease. For example, markers may be genetically linked, which be detected by analyzing samples from a population of subjects whose families have a history of neoplasia. The results can then be compared with data obtained from subjects whose families do not have a history of neoplasia. The markers that are genetically linked may facilitate determination if a subject is predisposed to neoplasia based on a family.
  • the individual markers disclosed herein is useful in determining the status or stage of a subject’s neoplasia.
  • a marker detected in a subject sample using the methods described herein is compared with the marker in a control sample, wherein differences in the expression or amounts of the marker distinguishes neoplasia status from non-neoplasia status.
  • the techniques can be adjusted, as is well understood in the art, to increase the sensitivity or specificity of the diagnostic assay.
  • markers While individual markers are useful diagnostic markers, in some instances, a combination of markers provides greater predictive value than a single marker. Detection the presence or absence of a plurality of markers in a sample can decrease false positives and false negative diagnoses, while increasing the occurrence of true positives and true negatives.
  • the methods described herein can also be used to monitor the progression of a subject’s PD AC or to assist in the management of the disease.
  • therapeutic options may vary at different stages of the disease, which may be reflected in changes in marker expression over time as detected by the methods described herein. These methods may also be used to distinguish PDAC from pancreatitis.
  • markers e.g., ANXA11, GPC4, CD14, CD44v6, MUC1, CLDN4, TSPAN8, CD147, and CD104
  • Methods for assaying expression of these polypeptides may be used to characterize PDAC.
  • the present disclosure provides diagnostic methods and compositions useful for identifying a polypeptide expression profile that identifies a subject as having or having a propensity to develop prostate carcinoma. Such assays can be used to measure an alteration in the level of a polypeptide.
  • the polypeptides of the invention are useful as hybridizable array elements in a microarray.
  • the array elements are organized in an ordered fashion such that each element is present at a specified location on the substrate.
  • Useful substrate materials include membranes, composed of paper, nylon or other materials, filters, chips, glass slides, and other solid supports. The ordered arrangement of the array elements allows hybridization patterns and intensities to be interpreted as expression levels of particular genes or proteins.
  • Methods for making nucleic acid microarrays are known to the skilled artisan and are described, for example, in U.S. Pat. No. 5,837,832, Lockhart, et al. (Nat. Biotech. 14: 1675-1680, 1996), and Schena, et al. (Proc. Natl. Acad.
  • Proteins may be analyzed using protein microarrays. Such arrays are useful in high- throughput low-cost screens to identify alterations in the expression or post-translation modification of a polypeptide of the invention, or a fragment thereof. In particular, such microarrays are useful to identify a protein whose expression is altered in prostate carcinoma.
  • a protein microarray of the invention binds a marker present in a subject sample and detects an alteration in the level of the marker.
  • a protein microarray features a protein, or fragment thereof, bound to a solid support.
  • Suitable solid supports include membranes (e.g., membranes composed of nitrocellulose, paper, or other material), polymer-based films (e.g., polystyrene), beads, or glass slides.
  • proteins e.g., antibodies that bind a marker of the invention
  • a substrate using any convenient method known to the skilled artisan (e.g., by hand or by inkjet printer).
  • the protein microarray is hybridized with a detectable probe.
  • probes can be polypeptide, nucleic acid molecules, antibodies, or small molecules.
  • polypeptide and nucleic acid molecule probes are derived from a biological sample taken from a subject, such as a bodily fluid (such as blood, blood serum, plasma, saliva, urine, abdominal fluid, or pancreatic secretions); a homogenized tissue sample (e.g. a tissue sample obtained by biopsy); or a cell isolated from a subject sample.
  • Probes can also include antibodies, candidate peptides, nucleic acids, or small molecule compounds derived from a peptide, nucleic acid, or chemical library.
  • Hybridization conditions e.g., temperature, pH, protein concentration, and ionic strength
  • the microarray is an affinity microarray- based technology (Nanoview Biosciences) coupled with a Reflectance Imaging Sensor detection methodology as described in International Application No. PCT/US2017/016434.
  • the present disclosure provides methods of treating a disease, which comprise detecting the level of at least one marker as disclosed above and administering a
  • a pretreatment level of at least one marker is determined in a subject prior to treatment.
  • the marker’s pretreatment level may be compared to the marker’s level in the subject during and/or after treatment, to monitor the efficacy of the treatment.
  • the level of the marker is measured by any suitable method as described herein. In some embodiments, the level of the marker is measured
  • the terms“treat,” treating,”“treatment,” and the like refer to reducing or ameliorating a disorder and/or symptom associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
  • the terms“prevent,”“preventing,”“prevention,”“prophylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.
  • the pharmaceutical composition may be administered by any appropriate route for the treatment or prevention of a neoplasia.
  • administration may be accomplished by parenteral, intravenous, intra-arterial, subcutaneous, intramuscular, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, by aerosol, by suppositories, or by oral administration.
  • Pharmaceutical compositions for the treatment of a neoplasia may be administered to humans, domestic pets, livestock, or any other animals with a pharmaceutically acceptable diluent, carrier, or excipient, in unit dosage form.
  • the invention provides a method of monitoring treatment progress.
  • the method includes the step of determining a level of a marker (e.g., ANXA11, GPC4, CD 14, CD44v6, MUC1, CLDN4, TSPAN8, CD 147, and CD 104) in a subject suffering from or susceptible to a PD AC or symptoms thereof, in which the subject has been administered a therapeutic amount of a compound sufficient to treat the disease or symptoms thereof.
  • a marker e.g., ANXA11, GPC4, CD14,CD44v6, MUC1, CLDN4, TSPAN8, CD147, and CD104
  • the level of a marker e.g., ANXA11, GPC4, CD14,CD44v6, MUC1, CLDN4, TSPAN8, CD147, and CD104
  • a second level of the marker in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy.
  • a pre-treatment level of a marker e.g., ANXA11, GPC4, CD14, CD44v6, MUC1, CLDN4, TSPAN8, CD147, and CD104
  • this pre-treatment level of the marker can then be compared to the level of the marker in the subject after the treatment commences, to determine the efficacy of the treatment.
  • kits for characterizing markers associated with pancreatic neoplasia in a sample The invention provides kits for the characterization of pancreatic ductal adenocarcinoma that expresses one, two, three, or four or all of ANXA11, CD14, CD44v6, or GPC4.
  • the kit includes a capture molecule (e.g., antibody or polynucleotide probe) that binds a ANXA11, CD14, CD44v6, or GPC4 polynucleotide or polypeptide.
  • kits for the characterization of pancreatic ductal adenocarcinoma that expresses one, two, three, four, five, six, seven or all of CD44v6, MUC1, CLDN4, TSPAN8, CD147, and CD104.
  • the kit includes a capture molecule (e.g., antibody or polynucleotide probe) that binds a CD44v6, MUC1, CLDN4, TSPAN8, CD147, and CD104 polynucleotide or polypeptide.
  • the kit comprises a sterile container.
  • Such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art.
  • kits can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding reagents.
  • the kit is provided together with instructions for using the kit for the detection of the markers.
  • the instructions will generally include information about the use of the kit for the characterization of PD AC.
  • the instructions include at least one of the following: precautions; warnings; clinical studies; and/or references.
  • the instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
  • a kit can comprise instructions in the form of a label or separate insert (package insert) for suitable operational parameters.
  • the kit can comprise one or more containers with appropriate positive and negative controls or control samples, to be used as standard(s) for detection, calibration, or normalization.
  • a subject can be diagnosed with PD AC by adding a biological sample (e.g., blood or serum) from the subject to the kit, or components thereof, and detecting the relevant biomarkers that are specifically bound by capture molecules.
  • a biological sample e.g., blood or serum
  • the method comprises: (i) collecting a sample from the subject; (ii) adding subject’s sample to the components in the kit, e.g., a holding tube or a substrate; and (iii) detecting the capture molecules to which the markers in the sample have bound.
  • PDAC Adenocarcinoma
  • Markers present in an organoid model of PDAC were assessed to determine if the markers were increased in PDAC or were simply present at levels observed in healthy cells.
  • 4.0 ml of supernatant from an organoid culture were collected and subjected to a 100 kDa vesicle enrichment method followed by extracellular vesicle purification. The purified extracellular vesicles were then subjected to liquid chromatography-mass spectrometry (LC- MS/MS).
  • LC- MS/MS liquid chromatography-mass spectrometry
  • W020160] 5158A 1, the contents of which are incorporated herein by reference in their entirety, and processed in the same manner as performed for the sample collected from the organoid model of disease.
  • the purified extracellular vesicles were then subjected to liquid chromatography-mass spectrometry (LC-MS/MS).
  • LC-MS/MS liquid chromatography-mass spectrometry
  • Group I proteins are listed in Table 1.
  • Group I proteins include CD44v6, MUC1, CLDN4, TSPAN8, CD147, and CD104.
  • Blood was obtained from five PD AC patients having metastatic or premalignant disease and from four chronic pancreatitis patients with no evidence of PDAC.
  • an affinity microarray-based technology (NanoView Biosciences) coupled with a Reflectance Imaging Sensor detection methodology was used to identify and count extracellular vesicles.
  • Validated antibodies against the selected proteins were arrayed on the chip and supernatants from pancreatic derived organoid (PDO) cultures were analyzed for presence of extracellular vesicles (FIG. 3).
  • the extracellular vesicles containing antigens in Group I were very low or undetectable in samples derived from pancreatitis patients, but were present at levels 100 to 1000-fold greater in blood derived from PDAC patients.
  • antigens identified in Group II were not differentially present in blood derived from patients with PDAC, demonstrating the ability of patient derived organoid cultures to identify extracellular vesicle-associated proteins that can be used as diagnostic markers of PDAC.
  • Example 3 Clinical Validation of a Selected Group of Putative Secreted Markers
  • Clinical validation was performed for a selected group of putative secreted markers including ANNXA11, CD14, GPC4, and CD44v6.
  • the expression of the marker proteins was analyzed in the plasma of patients with an established diagnosis of pancreatic cancer, as well as in individuals with a variety of benign gastrointestinal disorders.
  • a summary of the PDAC and benign gastrointestinal disorder patients is provided in Table 2.
  • Plasma samples were processed following standard operating procedures to isolate secreted proteins, including those from extracellular vesicles. Subsequently, western blot analyses were conducted to confirm that the expression of four proteins was increased in the plasma of patients with pancreatic cancer compared to the levels present in the plasma from patients with benign disease (FIGs. 4A-4C).
  • Table 2 Subjects Diagnosed with PDAC or a Benign Gastrointestinal Disorder

Abstract

The invention features compositions and methods for characterizing pancreatic ductal adenocarcinoma, as well as other pancreatic neoplasias. Some aspects of the present invention provide a panel for characterizing pancreatic ductal adenocarcinoma, wherein the panel includes two or more capture molecules each bound to a substrate, where each capture molecule specifically binds a marker polypeptide that is any one or more of ANXA11, CD14, CD44v6, and GPC4, or a polynucleotide encoding said polypeptide.

Description

COMPOSITIONS AND METHODS FOR CHARACTERIZING PANCREATIC
DUCTAL ADENOCARCINOMA
CROSS-REFERENCE TO RELATED APPLICATION
This application claims the benefit of the following U.S. Provisional Application No. : 62/802,000, filed February 6, 2019, the entire contents of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
Early diagnosis of cancer can greatly increase a patient’s chance of survival. For example, pancreatic ductal adenocarcinoma has an overall five-year survival rate of just 9%, but if it is detected at an early stage, the five-year survival rate increases to 32%.
Unfortunately, pancreatic ductal adenocarcinoma is very often hard to diagnosis. CA 19-9, which is an antigen released by the pancreas, is commonly used to detect pancreatic ductal adenocarcinoma, but diagnostics used to detect this marker have only a 75.4% pooled sensitivity and a specificity of 77.6%. Improved methods for early detection of pancreatic ductal adenocarcinoma are urgently required.
SUMMARY OF THE INVENTION
As described below, the present invention features compositions and methods for characterizing pancreatic ductal adenocarcinoma, as well as other pancreatic neoplasias.
Some aspects of the present invention provide a panel for characterizing pancreatic ductal adenocarcinoma, wherein the panel includes two or more capture molecules each bound to a substrate, where each capture molecule specifically binds a marker polypeptide that is any one or more of ANXA11, CD14, CD44v6, and GPC4, or a polynucleotide encoding said polypeptide.
Some aspects of the present disclosure provide a panel for characterizing pancreatic ductal adenocarcinoma, and the panel includes two or more capture molecules each bound to a substrate, where each capture molecule specifically binds a marker polypeptide selected from the group consisting of CD44v6, MUC1, CLDN4, TSPAN8, CD 147, and CD 104, or the marker is a polynucleotide encoding said polypeptide.
In some embodiments of the above aspects, the capture molecule is a polypeptide, polynucleotide probe, or fragment thereof. In some embodiments, the polypeptide is an antibody that specifically binds the marker polypeptide. In some embodiments of the panel, the polynucleotide is an aptamer that specifically binds the marker polypeptide, and in some embodiments, polynucleotide probe specifically hybridizes to the polynucleotide encoding the marker polypeptide.
In some embodiments of the panel, the substrate is a bead or planar surface. In some embodiments, the planar surface is a membrane, filter, chip, glass slide, or other solid support.
The panel, in some embodiments, includes capture molecules each of which bind ANXA11, CD 14, CD44v6, and GPC4; ANXA11, CD14, and CD44v6; ANXA11, CD14, and GPC4; ANXA11, CD44v6, and GPC4; ANXA11 and CD14; ANXA11 and CD44v6;
ANXA11 and GPC4; CD14, CD44v6, and GPC4; CD14 and CD44v6;CD14 and GPC4; or CD44v6, and GPC4.
The panel, in some embodiments, includes capture molecules that bind CD44v6 and one or more of MUC1, CLDN4, TSPAN8, CD147, and CD104. In some embodiments, the panel includes capture molecules that bind MUC1 and one or more of CD44v6, CLDN4, TSPAN8, CD147, and CD104. In some embodiments, the panel includes capture molecules that bind TSPAN8 and one or more of CD44v6, MUC1, CLDN4, CD 147, and CD 104. In some embodiments, the panel includes capture molecules that bind CD147 and one or more of CD44v6, MUC1, CLDN4, TSPAN8, and CD104.
Another aspect of the present disclosure provides a panel for characterizing pancreatic ductal adenocarcinoma, and the panel includes two or more capture molecules each bound to a substrate, where each capture molecule specifically binds a marker polypeptide selected from those listed in Table 1. In some embodiments, one or more of the capture molecules are antibodies.
In one aspect, a method is disclosed for detecting a marker in a sample derived from a subject, the method involving detecting two or more markers selected from the group consisting of CD44v6, MUC1, CLDN4, TSPAN8, CD147, and CDKMor a marker of Table 1 in the sample.
Another aspect provides a method for detecting a marker in a sample derived from a subject, the method involving detecting two or more markers selected from the group consisting of ANXA11, CD14, CD44v6, and GPC4, or a marker of Table 1 in the sample.
In yet another aspect of the present disclosure, a method is provided for characterizing pancreatic ductal adenocarcinoma, the method involving a) culturing a cell from a subject having or suspected of having pancreatic ductal adenocarcinoma, thereby generating an organoid culture; b) isolating extracellular vesicles present in culture media of the organoid culture; and c) detecting a marker present in or on the extracellular vesicles, where the marker is selected from the group consisting of CD44v6, MUC1, CLDN4, TSPAN8, CD147, and CD 104 or the group consisting of ANXA11, CD 14, CD44v6, and GPC or a marker of Table 1 using a capture molecule that specifically binds the marker. In some embodiments, the capture molecule is bound to a substrate surface. In some embodiments, the substrate surface has a spectral reflectance signature. In some embodiments, the method further involves (d) exposing the substrate surface to a light source; and (e) detecting the presence or absence of extracellular vesicles on the substrate surface, where the presence of the extracellular vesicles indicates the presence of one of the markers.
In some embodiments, the sample is a biological fluid selected from the group consisting of culture media, blood, blood serum, plasma, urine, abdominal fluids, or pancreatic secretions. In some embodiments, the sample is a biopsy. In some embodiments, the sample includes extracellular vesicles, and in some embodiments, these extracellular vesicles are exosomes or microvesicles.
In some embodiments of the method of treating a selected subject having PD AC, the detecting involves RT-PCR, Northern blotting, Western blotting, flow cytometry, immunocytochemistry, binding to magnetic and/or antibody-coated beads, in situ
hybridization, fluorescence in situ hybridization (FISH), flow chamber adhesion assay, an immunoassay such as ELISA or a radioimmunoassay, microarray analysis, colorimetric assays, mass spectrometry, liquid chromatography-mass spectrometry (LC-MS/MS), or high- performance liquid chromatography. In some embodiments, the detecting involves liquid chromatography-mass spectrometry (LC-MS/MS). In some embodiments, the detecting involves using a capture molecule that specifically binds the marker. In some embodiments, the capture molecule is an antibody, while in other embodiments, the capture molecule is an aptamer. In some embodiments, the capture molecule is bound to a substrate surface, which has, in some embodiments, a spectral reflectance signature.
Also disclosed herein are kits for characterizing markers in a sample, andthe kit includes two or more capture molecules fixed to a substrate surface, where each capture molecule specifically binds a marker polypeptide selected from the group consisting of CD44v6, MUC1, CLDN4, TSPAN8, CD147, and CD104 or the group consisting of
ANXA11, CD14, CD44v6, and GPC or a marker polypeptide of Table 1, or a polynucleotide encoding said marker polypeptide. In some embodiments of the kits, the substrate surface has a spectral reflectance signature.
The present disclosure provides methods for characterizing markers associated with pancreatic ductal adenocarcinoma, as well as other pancreatic neoplasias. In some embodiments, the method involves analyzing markers present in extracellular vesicles, such as exosomes, isolated from the supernatant of an organoid model of pancreatic disease (e.g., pancreatic ductal adenocarcinoma or another pancreatic neoplasia). Methods and compositions are also provided for detecting pancreatic ductal adenocarcinoma in a subject. The methods disclosed herein were used to identify subjects having pancreatic ductal adenocarcinoma and distinguish these subjects from others having a benign pancreatic disease (e.g., pancreatitis), as is explained the examples provided below. Other features and advantages of the disclosures contained herein will be apparent from the detailed description, and from the claims.
Definitions
Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et ah, Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.
By "alteration" is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes a 10% change, a 25% change, a 40% change, a 50% or a greater change.
By“Annexin Al 1 (ANXA11)” is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to GenBank Accession No. AAV38737.1 and having anti- ANXA11 antibody binding activity. An exemplary ANXA11 amino acid sequence is provided below:
1 msypgypppp ggyppaapgg gpwggaaypp ppsmppigld nvatyagqfn qdylsgmaan 61 msgtfgganm pnlypgapga gyppvppggf gqppsaqqpv ppygmypppg gnppsrmpsy
121 ppypgapvpg qpmpppgqqp pgaypgqppv typgqppvpl pgqqqpvpsy pgypgsgtvt
181 pavpptqfgs rgtitdapgf dplrdaevlr kamkgfgtde qaiidclgsc snkqrqqill
241 sfktaygkdl ikdlkselsg nfektilalm ktpvlfdiye ikeaikgvgt deaclieila
301 srsnehirel nraykaefkk tleeairsdt sghfqrllis lsqgnrdest nvdmslaqrd
361 aqelyaagen rlgtdeskfn avlcsrsrah lvavfneyqr mtgrdieksi cremsgdlee
421 gmlavvkclk ntpaffaerl nkamrgagtk drtlirimvs rsetdlldir seykrmygks
481 lyhdisgdts gdyrkillki cggnd
By“Claudin 4 (CLDN4)” is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_001296 and having anti-CLDN4 antibody binding activity. An exemplary CLDN4 amino acid sequence is provided below:
1 masmglqvmg ialavlgwla vmlccalpmw rvtafigsni vtsqtiwegl wmncvvqstg 61 qmqckvydsl lalpqdlqaa ralviisiiv aalgvllsvv ggkctncled esakaktmiv
181 lccncpprtd kpysakysaa rsaaasnyv
By“Cluster of Differentiation 9 (CD9)” is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_001760.1 and having anti-CD9 antibody binding activity. An exemplary CD9 amino acid sequence is provided below:
1 mpvkggtkci kyllfgfnfi fwlagiavla iglwlrfdsq tksifeqetn nnnssfytgv 61 yiligagalm mlvgflgccg avqesqcmlg lffgfllvif aieiaaaiwg yshkdevike 121 vqefykdtyn klktkdepqr etlkaihyal nccglaggve qfisdicpkk dvletftvks 181 cpdaikevfd nkfhiigavg igiavvmifg mifsmilcca irrnremv
By“Cluster of Differentiation 14 (CD14)” is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_001167576.1 and having anti-CD14 antibody binding activity. An exemplary CD14 amino acid sequence is provided below:
1 merascllll llplvhvsat tpepceldde dfrcvcnfse pqpdwseafq cvsaveveih
61 agglnlepfl krvdadadpr qyadtvkalr vrrltvgaaq vpaqllvgal rvlaysrlke
121 ltledlkitg tmpplpleat glalsslrlr nvswatgrsw laelqqwlkp glkvlsiaqa
181 hspafsceqv rafpaltsld lsdnpglger glmaalcphk fpaiqnlalr ntgmetptgv
241 caalaaagvq phsldlshns lratvnpsap rcmwssalns lnlsfagleq vpkglpaklr
301 vldlscnrln rapqpdelpe vdnltldgnp flvpgtalph egsmnsgvvp acarstlsvg
361 vsgtlvllqg argfa
By“Cluster of Differentiation 44, transcript variant 6 (CD44v6)” is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_001189484.1 and having anti-CD44v6 antibody binding activity. An exemplary CD44v6 amino acid sequence is provided below: 1 mdkfwwhaaw glclvplsla qidlnitcrf agvfhvekng rysisrteaa dlckafnstl
61 ptmaqmekal sigfetcryg fieghvvipr ihpnsicaan ntgvyiltsn tsqydtycfn
121 asappeedct svtdlpnafd gpititivnr dgtryvqkge yrtnpediyp snptdddvss
181 gssserssts ggyifytfst vhpipdedsp witdstdrip atnrndvtgg rrdpnhsegs
241 ttllegytsh yphtkesrtf ipvtsaktgs fgvtavtvgd snsnvnrsls gdqdtfhpsg
301 gshtthgses dghshgsqeg ganttsgpir tpqipewlii lasllalali lavciavnsr
361 rrcgqkkklv insgngaved rkpsglngea sksqemvhlv nkessetpdq fmtadetrnl
421 qnvdmkigv
By“Cluster of Differentiation 81 (CD81)” is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_004347.1 and having anti-CD81 antibody binding activity.
1 mgvegctkci kyllfvfnfv fwlaggvilg valwlrhdpq ttnllylelg dkpapntfyv
61 giyiliavga vmmfvgflgc ygaiqesqcl lgtfftclvi lfacevaagi wgfvnkdqia
121 kdvkqfydqa lqqavvddda nnakavvktf hetldccgss tltalttsvl knnlcpsgsn
181 iisnlfkedc hqkiddlfsg klyligiaai vvavimifem ilsmvlccgi rnssvy
By“Cluster of Differentiation 104 (CD 104)” is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_000204.3 and having anti-CD 104 antibody binding activity.
1 magprpspwa rlllaalisv slsgtlanrc kkapvkscte cvrvdkdcay ctdemfrdrr
61 cntqaellaa gcqresivvm essfqiteet qidttlrrsq mspqglrvrl rpgeerhfel
121 evfeplespv dlyilmdfsn smsddldnlk kmgqnlarvl sqltsdytig fgkfvdkvsv
181 pqtdmrpekl kepwpnsdpp fsfknvislt edvdefrnkl qgerisgnld apeggfdail
241 qtavctrdig wrpdsthllv fstesafhye adganvlagi msrnderchl dttgtytqyr
301 tqdypsvptl vrllakhnii pifavtnysy syyeklhtyf pvsslgvlqe dssnivelle
361 eafnrirsnl diraldsprg lrtevtskmf qktrtgsfhi rrgevgiyqv qlralehvdg
421 thvcqlpedq kgnihlkpsf sdglkmdagi icdvctcelq kevrsarcsf ngdfvcgqcv
481 csegwsgqtc ncstgslsdi qpclregedk pcsgrgecqc ghcvcygegr yegqfceydn
541 fqcprtsgfl cndrgrcsmg qcvcepgwtg pscdcplsna tcidsnggic ngrghcecgr
601 chchqqslyt dticeinysa ihpglcedlr scvqcqawgt gekkgrtcee cnfkvkmvde
661 lkraeevvvr csfrdedddc tysytmegdg apgpnstvlv hkkkdcppgs fwwlipllll
721 llpllallll lcwkycacck aclallpccn rghmvgfked hymlrenlma sdhldtpmlr
781 sgnlkgrdw rwkvtnnmqr pgfathaasi nptelvpygl slrlarlcte nllkpdtrec
841 aqlrqeveen lnevyrqisg vhklqqtkfr qqpnagkkqd htivdtvlma prsakpallk
901 ltekqveqra fhdlkvapgy ytltadqdar gmvefqegve lvdvrvplfi rpedddekql
961 lveaidvpag tatlgrrlvn itiikeqard vvsfeqpefs vsrgdqvari pvirrvldgg
1021 ksqvsyrtqd gtaqgnrdyi pvegellfqp geawkelqvk llelqevdsl lrgrqvrrfh
1081 vqlsnpkfga hlgqphstti iirdpdeldr sftsqmlssq ppphgdlgap qnpnakaags
1141 rkihfnwlpp sgkpmgyrvk ywiqgdsese ahlldskvps veltnlypyc dyemkvcayg
1201 aqgegpyssl vscrthqevp sepgrlafnv vsstvtqlsw aepaetngei tayevcyglv 1261 nddnrpigpm kkvlvdnpkn rmllienlre sqpyrytvka rngagwgper eaiinlatqp
1321 krpmsipiip dipivdaqsg edydsflmys ddvlrspsgs qrpsvsddtg cgwkfepllg
1381 eeldlrrvtw rlppeliprl sassgrssda eaphgppddg gaggkggslp rsatpgppge
1441 hlvngrmdfa fpgstnslhr mtttsaaayg thlsphvphr vlstsstltr dynsltrseh
1501 shsttlprdy stltsvsshd srltagvpdt ptrlvfsalg ptslrvswqe prcerplqgy
1561 sveyqllngg elhrlnipnp aqtsvvvedl lpnhsyvfrv raqsqegwgr eregvities
1621 qvhpqsplcp lpgsaftlst psapgplvft alspdslqls werprrpngd ivgylvtcem
1681 aqgggpataf rvdgdspesr ltvpglsenv pykfkvqart tegfgpereg iitiesqdgg
1741 pfpqlgsrag lfqhplqsey ssittthtsa tepflvdglt lgaqhleagg sltrhvtqef
1801 vsrtlttsgt lsthmdqqff qt
In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes," "including," and the like; "consisting essentially of' or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
“Detect” refers to identifying the presence, absence or amount of the analyte to be detected.
By“decreases” is meant a reduction by at least about 5% relative to a reference level. A decrease may be by 5%, 10%, 15%, 20%, 25% or 50%, or even by as much as 75%, 85%, 95% or more.
By "detectable label" is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.
By“disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Pancreatic ductal adenocarcinoma is a disease, for example.
By "effective amount" is meant the amount of an agent required to ameliorate the symptoms of a disease relative to an untreated subject. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.
The invention provides targets that are useful for the development of highly specific drugs to treat or a disorder characterized by the methods delineated herein. In addition, the methods of the invention provide a facile means to identify therapies that are safe for use in subjects. In addition, the methods of the invention facilitate analysis of virtually any compound for effects on a disease described herein.
By“Epithelial Cell Adhesion Molecule (EPCAM)” is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_002345.2 and having anti-EPCAM antibody binding activity. An exemplary EPCAM amino acid sequence is provided below:
1 mappqvlafg lllaaatatf aaaqeecvce nyklavncfv nnnrqcqcts vgaqntvics
61 klaakclvmk aemngsklgr rakpegalqn ndglydpdcd esglfkakqc ngtstcwcvn
121 tagvrrtdkd teitcservr tywiiielkh karekpydsk slrtalqkei ttryqldpkf
181 itsilyennv itidlvqnss qktqndvdia dvayyfekdv kgeslfhskk mdltvngeql
241 dldpgqtliy yvdekapefs mqglkagvia vivvvviavv agivvlvisr kkrmakyeka
301 eikemgemhr elna
By“EPH Receptor A2 (EPHA2)” is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_004422.2 and having anti- EPHA2 binding activity. An exemplary EPHA2 amino acid sequence is provided below:
1 melqaaracf allwgcalaa aaaaqgkevv lldfaaagge lgwlthpygk gwdlmqnimn
61 dmpiymysvc nvmsgdqdnw lrtn vyrge aerifielkf tvrdcnsfpg gasscketfn
121 lyyaesdldy gtnfqkrlft kidtiapdei tvssdfearh vklnveersv gpltrkgfyl
181 afqdigacva llsvrvyykk cpellqglah fpetiagsda pslatvagtc vdhavvppgg
241 eeprmhcavd gewlvpigqc lcqagyekve dacqacspgf fkfeasespc lecpehtlps
301 pegatscece egffrapqdp asmpctrpps aphyltavgm gakvelrwtp pqdsggredi
361 vysvtceqcw pesgecgpce asvrysepph gltrtsvtvs dlephmnytf tvearngvsg
421 lvtsrsfrta svsinqtepp kvrlegrstt slsvswsipp pqqsrv kye vtyrkkgdsn
481 synvrrtegf svtlddlapd ttylvqvqal tqegqgagsk vhefqtlspe gsgnlavigg
541 vavgvvlllv lagvgffihr rrknqrarqs pedvyfskse qlkplktyvd phtyedpnqa
601 vlkftteihp scvtrqkvig agefgevykg mlktssgkke vpvaiktlka gytekqrvdf
661 lgeagimgqf shhniirleg viskykpmmi iteymengal dkflrekdge fsvlqlvgml
721 rgiaagmkyl anmnyvhrdl aarnilvnsn lvckvsdfgl srvleddpea tyttsggkip
781 irwtapeais yrkftsasdv wsfgivmwev mtygerpywe lsnhevmkai ndgfrlptpm
841 dcpsaiyqlm mqcwqqerar rpkfadivsi ldklirapds lktladfdpr vsirlpstsg
901 segvpfrtvs ewlesikmqq ytehfmaagy taiekvvqmt nddikrigvr lpghqkriay
961 sllglkdqvn tvgipi By“extracellular vesicle” is meant a membrane surrounded structure that is released by a cell. Exemplary extracellular vesicles include exosomes and microvesicles. Exosomes include small membrane bound extracellular vesicles of -30-300 nm diameter that are secreted by cells into the extracellular environment. The surface of an exosome comprises a lipid bilayer from the membrane of the donor cell, and the lumen of the exosome is topologically the same as the cytosol from the cell that produces the exosome. An exosome contains, for example, proteins, RNAs, lipids, and/or carbohydrates of the producing cell, though the contents of the exosome may be modified after the exosome’ s release from the producing cell, either through natural processes or by experimental manipulation.
By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion comprises, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.
“Glypican 1 (GPC1)” as used herein refers to a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_002072.2 and having anti- GPC1 antibody binding activity. An exemplary GPC1 amino acid sequence is provided below:
1 melrargwwl lcaaaalvac argdpasksr scgevrqiyg akgfslsdvp qaeisgehlr
61 icpqgytcct semeenlanr shaeletalr dssrvlqaml atqlrsfddh fqhllndser
121 tlqatfpgaf gelytqnara frdlyselrl yyrganlhle etlaefwarl lerlfkqlhp
181 qlllpddyld clgkqaealr pfgeaprelr lratrafvaa rsfvqglgva sdvvrkvaqv
241 plgpecs rav mklvycahcl gvpgarpcpd ycrnvlkgcl anqadldaew rnlldsmvli
301 tdkfwgtsgv esvigsvhtw laeainalqd nrdtltakvi qgcgnpkvnp qgpgpeekrr
361 rgklaprerp psgtleklvs eakaqlrdvq dfwislpgtl csekmalsta sddrcwngma
421 rgrylpevmg dglanqinnp evevditkpd mtirqqimql kimtnrlrsa yngndvdfqd
481 asddgsgsgs gdgclddlcg rkvsrkssss rtplthalpg lseqegqkts aascpqpptf
541 llplllflal tvarprwr
By“Glypican 4 (GPC4)” is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to GenBank Accession No. EAX11772.1 and having anti- GPC4 antibody binding activity. An exemplary GPC4 amino acid sequence is provided below:
1 marfglpall ctlavlsaal laaelksksc sevrrlyvsk gfnkndaplh eingdhlkic 61 pqgstccsqe meekyslqsk ddfksvvseq cnhlqavfas rykkfdeffk ellenaeksl 121 ndmfvktygh lymqnselfk dlfvelkryy vvgnvnleem lndfwarlle rmfrlvnsqy 181 hftdeylecv skyteqlkpf gdvprklklq vtrafvaart faqglavagd vvskvsvvnp 241 taqcthallk miycshcrgl vtvkpcynyc snimrgclan qgdldfewnn fidamlmvae 301 rlegpfnies vmdpidvkis daimnmqdns vqvsqkvfqg cgppkplpag risrsisesa 361 fsarfrphhp eerpttaagt sldrlvtdvk eklkqakkfw sslpsnvcnd ermaagngne 421 ddcwngkgks rylfavtgng lanqgnnpev qvdtskpdil ilrqimalrv mtskmknayn 481 gndvdffdis dessgegsgs gceyqqcpse fdynatdhag ksanekadsa gvrpgaqayl 541 ltvfcilflv mqrewr
By“healthy” is meant not having or suspected of having a disease and/or not being derived from a diseased source. For example, a healthy cell culture is a culture derived from cells that exhibit no signs of a disease. In some instances, healthy refers to cells derived from an organoid model, wherein the organoid model does not manifest any disease traits.
Diseased cells, conversely, may be obtained from a diseased subject, a disease model (e g., an organoid model of disease), or are otherwise derived from diseased cells.
By“Human Epidermal Growth Factor Receptor 2 (HER2)” is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No.
NP 004439.2 and having anti- HER2 antibody binding activity. An exemplary HER2 amino acid sequence is provided below:
1 melaalcrwg lllallppga astqvctgtd mklrlpaspe thldmlrhly qgcqvvqgnl
61 eltylptnas lsflqdiqev qgyvliahnq vrqvplqrlr ivrgtqlfed nyalavldng
121 dplnnttpvt gaspgglrel qlrslteilk ggvliqrnpq lcyqdtilwk difhknnqla
181 ltlidtnrsr achpcspmck gsrcwgesse dcqsltrtvc aggcarckgp lptdccheqc
241 aagctgpkhs dclaclhfnh sgicelhcpa lvtyntdtfe smpnpegryt fgascvtacp
301 ynylstdvgs ctlvcplhnq evtaedgtqr cekcskpcar vcyglgmehl revravtsan
361 iqefagckki fgslaflpes fdgdpasnta plqpeqlqvf etleeitgyl yisawpdslp
421 dlsvfqnlqv irgrilhnga ysltlqglgi swlglrslre lgsglalihh nthlcfvhtv
481 pwdqlfrnph qallhtanrp edecvgegla chqlcarghc wgpgptqcvn csqflrgqec
541 veecrvlqgl preyvnarhc lpchpecqpq ngsvtcfgpe adqcvacahy kdppfcvarc
601 psgvkpdlsy mpiwkfpdee gacqpcpinc thscvdlddk gcpaeqrasp ltsiisavvg
661 illvvvlgvv fgilikrrqq kirkytmrrl lqetelvepl tpsgampnqa qmrilketel
721 rkvkvlgsga fgtvykgiwi pdgenvkipv aikvlrents pkankeilde ayvmagvgsp
781 yvsrllgicl tstvqlvtql mpygclldhv renrgrlgsq dlln cmqia kgmsyledvr
841 lvhrdlaarn vlvkspnhvk itdfglarll dideteyhad ggkvpikwma lesilrrrft
901 hqsdvwsygv tvwelmtfga kpydgipare ipdllekger lpqppictid vymimvkcwm
961 idsecrprfr elvsefsrma rdpqrfvviq nedlgpaspl dstfyrslle dddmgdlvda
1021 eeylvpqqgf fcpdpapgag gmvhhrhrss strsgggdlt lglepseeea prsplapseg 1081 agsdvfdgdl gmgaakglqs lpthdpsplq rysedptvpl psetdgyvap ltcspqpeyv 1141 nqpdvrpqpp spregplpaa rpagatlerp ktlspgkngv vkdvfafgga venpeyltpq 1201 ggaapqphpp pafspafdnl yywdqdpper gappstfkgt ptaenpeylg ldvpv "Hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.
By“increases” is meant a reduction by at least about 5% relative to a reference level. A increase may be by 5%, 10%, 15%, 20%, 25% or 50%, or even by as much as 75%, 85%, 95% or more.
The terms "isolated," "purified," or "biologically pure" refer to material that is free to varying degrees from components which normally accompany it as found in its native state. "Isolate" denotes a degree of separation from original source or surroundings. "Purify" denotes a degree of separation that is higher than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term "purified" can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.
By "isolated polynucleotide" is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence. By an "isolated polypeptide" is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
By“marker” is meant any analyte having an alteration in structure, expression level or activity that is associated with a disease or disorder.
By“Mucin 1 (MUC1)” is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_001018017.1 and having anti- MUC1 antibody binding activity. An exemplary MUC1 amino acid sequence is provided below:
1 mtpgtqspff llllltvltv vtgsghasst pggeketsat qrssvpsste knafnssled
61 pstdyyqelq rdisemflqi ykqggflgls nikfrpgsvv vqltlafreg tinvhdvetq
121 fnqykteaas rynltisdvs vsdvpfpfsa qsgagvpgwg iallvlvcvl valaivylia
181 lavcqcrrkn ygqldifpar dtyhpmseyp tyhthgryvp psstdrspye kvsagnggss
241 lsytnpavaa tsanl
As used herein,“obtaining” as in“obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent.
As used herein,“organoid” refers to a three-dimensional tissue culture having a microanatomy similar to an intact organ. In this case, the organoid generated from a tumor tissue recreate the histomorphology present in the patient tumor, retain the differentiation station, retain major genetic mutations and H3K27 acetylation profile. In some instances, the organoid can be a subject derived organoid, wherein the cells cultured to generate the organoid are obtained from a subject having or suspected of having a disease. Subject derived organoids can be tumor organoids, wherein the cells used to generate the organoid are obtained from a subject’s tumor.
By“reduces” is meant a negative alteration of at least 10%, 25%, 50%, 75%, or
100%. By“reference” is meant a standard or control condition. As used herein, a“normal reference” is a reference obtained from an otherwise healthy subject or subjects. For example, a normal reference for the quantity of a marker in an exosome is the quantity of the marker in an exosome obtained from a healthy subject or subjects, a culture of healthy cells, or an organoid model of a healthy organ, such as a pancreas. When the quantity is determined using more than one healthy subject, the quantity may be the average of quantities detected in each subject. The normal reference may also be a published reference. As used herein, a“disease reference” is a known quantity of the marker in an exosome obtained from a subject or subjects having the disease, a culture of diseased cells, or an organoid model of the disease. When the quantity is determined using more than one subject having the disease, the quantity may be the average of quantities detected in each subject.
The disease reference may also be a published reference.
A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween.
By "specifically binds" is meant a compound or antibody that recognizes and binds a polypeptide of the invention, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the invention.
Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having“substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having“substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule.
By "hybridize" is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).
For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred: embodiment, hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 pg/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate,
1% SDS, 50% formamide, and 200 pg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, more preferably of at least about 42° C, and even more preferably of at least about 68° C. In a preferred embodiment, wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS.
Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.
By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). In embodiments, such a sequence is at least 60%, at least 80% or 85%, or at least 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.
Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705,
BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine;
aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e 3 and e 100 indicating a closely related sequence.
By "subject" is meant a mammal, including, but not limited to, a human or non human mammal, such as a bovine, equine, canine, ovine, or feline. By“Tetraspanin 8 (TSPAN8)” is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_004607.1 and having anti- TSPAN8 antibody binding activity. An exemplary TSPAN8 amino acid sequence is provided below:
1 magvsaciky smftfnflfw lcgililala iwvrvsndsq aifgsedvgs ssyvavdili
61 avgaiimilg flgccgaike srcmlllffi glllilllqv atgilgavfk sksdrivnet 121 lyentkllsa tgesekqfqe aiivfqeefk ccglvngaad wgnnfqhype lcacldkqrp 181 cqsyngkqvy ketcisfikd flaknliivi gisfglavie ilglvfsmvl ycqignk
As used herein, the terms“treat,” treating,”“treatment,” and the like refer to reducing or ameliorating a disorder and/or symptom associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
To“vary” as used herein means to differ from a reference in a quantitative manner, and to“vary significantly” is to differ from the reference in an amount greater than a predetermined threshold. For example, the concentration of a marker in a sample isolated from a model of a disease can be said to vary significantly from a reference concentration if the marker’s concentration is at least 2-fold greater or at least 50% below the reference concentration. The reference concentration can be known in the art or experimentally determined.
Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.
Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40
41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
Unless specifically stated or obvious from context, as used herein, the term“about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about. The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. l is a graph illustrating the results of a principle component analysis performed on patient derived organoid models. In this figure,“PCI” and“PC2” refer to principle components 1 and 2, which are sets of variables defined by the Principle Component Analysis.
FIG. 2. is an illustration depicting the functional clustering of potential markers identified in patient derived organoid models of pancreatic ductal adenocarcinoma.
FIG. 3 is a graph depicting normalized counts of extracellular vesicles comprising particular proteins.
FIGs. 4A-4C present clinical validation of a selected group of markers including ANNXA11, CD14, GPC4, and CD44v6. FIG. 4A is an image of a western blot of proteins in extracellular vesicles from patients. Extracellular vesicles from 10 mΐ of plasma were loaded in each lane. “GI Benign” denotes benign gastrointestinal disorders, and“PDACnf’ denotes pancreatic ductal adenocarcinoma no treatment.” Throughout the figures,“GP” and“PA” identify individual subjects diagnosed with benign gastrointestinal disorders and pancreatic ductal adenocarcinoma, respectively. FIG. 4B is a heatmap of protein quantification from FIG. 4A. FIG. 4C is a graph depicting the geometric means of protein marker scores for each patient sample. Protein marker score is calculated from western blot: intensity per band divided by the median band intensity of each marker in whole cohort and multiplied by 100.
DETAILED DESCRIPTION OF THE INVENTION
The present disclosure features compositions and methods that are useful for the characterization of pancreatic ductal adenocarcinoma (PDAC).
The invention is based, at least in part, on the discovery of markers including CD44v6, MUC1, CLDN4, TSPAN8, CD147, and CD104, (the“Group I markers”) that are increased in extracellular vesicles isolated from organoids derived from a subject that has PD AC. A second set of markers was identified that is increased in extracellular vesicles derived from reference subject-derived organoid samples. These markers are CD9, CD81, GPC1, EPHA2, EPCAM, EGFR, HER2, and CD63 (the“Group II markers”). A further group of markers that is increased in extracellular vesicles of patients with pancreatic cancer includes ANXA11, GPC4, CD14, and CD44v6. The identification of these sets of markers provides a blood-based assay for the detection of pancreatic ductal adenocarcinoma. As reported in greater detail below, the Group I markers were shown to be present in pancreatic cancer patient blood samples at levels about 100 to 1000-fold greater than observed in the blood obtained from patients having pancreatitis.
There is a demonstrated need for diagnostic markers specific for pancreatic adenocarcinoma (PDAC). One promising approach involves identification of secreted extracellular vesicles in the blood of PDAC patients that comprise markers that are specific for the disease. These markers would allow differentiation of PDAC patients from both others who are disease-free or afflicted with chronic pancreatitis, a benign condition. Several studies have attempted to identify disease associated protein markers using extracellular vesicles isolated from cultured cells. To date, it is not yet established if organoid cultures can be used to discover new, clinically significant, secreted markers. A typical extracellular vesicle identification effort using cells in culture requires large amount of media supernatant (0.1 -1.0 liters), coupled with elaborate analytical centrifugation and purification steps, which would be difficult to implement when working with patient derived tumor models that are not amenable to large scale cultures.
The present disclosure provides methods for identifying markers associated with a disease. In some embodiments, the methods comprise isolating extracellular vesicles derived from an organoid model of the disease and from a reference organoid and then quantifying the concentration of markers in or on the extracellular vesicles. In general, extracellular vesicles are released by cells (for example, tumor cells) into the extracellular environment, and the extracellular vesicles nucleic acids polypeptides that identify from what type of cell the exosome is derived. Extracellular vesicles as defined herein are isolated from a sample as described herein below. Extracellular vesicles are isolated from a variety of biological fluids, including but not limited to, blood, plasma, serum, urine, abdominal fluids, and pancreatic secretions n some embodiments, the extracellular vesicles are enriched prior to being isolated. For example, extracellular vesicles within a sample can be enriched using a 100 kDa concentration step that is subsequently followed by purification of the extracellular vesicles. In some embodiments, the 100 kDa concentration step comprises passing the sample comprising the extracellular vesicles through a high molecular weight filter. In other embodiments, extracellular vesicles can be purified using any means known in the art.
Concentrating and purifying the extracellular vesicles allows removal of free proteins, cell membranes, and other cellular debris that may confound marker detection and quantification.
Markers (e.g., ANXA11, GPC4, CD14, CD44v6, MUC1, CLDN4, TSPAN8, CD147, and CD104) derived from the extracellular vesicles isolated from the organoid model of the disease with at least a 2-fold difference in marker concentration relative to the extracellular vesicles isolated from the reference organoid are identified as markers associated with the disease. In some embodiments, the marker associated with disease will have about a 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or even a 1000-fold. In some embodiments, the difference in expression of a marker associated with disease between the expression of the marker in the organoid model of disease and the marker’s expression in a reference sample is between about 2-fold and about 1000-fold, between about 5-fold and about 1000-fold, between about 10-fold and about 1000-fold, between about 20-fold and about 1000-fold, between about 50-fold and about 1000-fold, between about 100-fold and about 1000-fold, between about 200-fold and about 1000-fold, between about 300-fold and about 1000-fold, between about 400-fold and about 1000-fold, between about 500-fold and about 1000-fold, between about 600-fold and about 1000-fold, between about 700-fold and about 1000-fold, between about 800-fold and 1000-fold, and between about 900-fold and
1000-fold. In some embodiments, the difference in expression of a marker associated with disease between the expression of the marker in the organoid model of disease and the marker’s expression in a reference sample is about 2-fold.
Table l is a listing of markers identified as having at least a two-fold increase in extracellular vesicles isolated from an organoid model of pancreatic ductal adenocarcinoma (PDAC). In some embodiments of the present disclosure, a marker can be any marker of Table 1, and a panel of markers can be any combination of markers of Table 1.
In some embodiments, a panel of markers comprises ANXA11, CD14, CD44v6, and GPC4. In some embodiments, a panel of markers comprises ANXA11, CD14, and CD44v6. In some embodiments, a panel of markers comprises ANXA11, CD14, and GPC4. In some embodiments, a panel of markers comprises ANXA11, CD44v6, and GPC4. In some embodiments, a panel of markers comprises ANXA11 and CD14. In some embodiments, a panel of markers comprises ANXA11 and CD44v6. In some embodiments, a panel of markers comprises ANXA11 and GPC4. In some embodiments, a panel of markers comprises CD14, CD44v6, and GPC4. In some embodiments, a panel of markers comprises CD 14 and CD44v6. In some embodiments, a panel of markers comprises CD 14 and GPC4. In some embodiments, a panel of markers comprises CD44v6, and GPC4.
Table 1: PDAC markers
Figure imgf000022_0001
In some embodiments, the markers associated with disease can be clustered into functional groups. For example, in some embodiments, a marker associated with a disease, such as pancreatic ductal adenocarcinoma can be clustered into a group of proteins involved in RNA splicing, histone or chromatin maintenance, proteasomes, translation, cytoskeleton regulation, cell adhesion, and membrane trafficking. In some embodiments, the neoplasia is pancreatic ductal adenocarcinoma.
Methods for Detecting Markers
Any suitable method can be used to detect markers (e.g., ANXA11, GPC4, CD14, CD44v6, MUC1, CLDN4, TSPAN8, CD147, and CD104) associated with pancreatic ductal adenocarcinoma. Successful practice of the invention can be achieved with one or a combination of methods that can detect and, in some embodiments, quantify the markers. These methods include, without limitation, hybridization-based methods, including those employed in biochip arrays, mass spectrometry (e.g., laser desorption/ionization mass spectrometry), fluorescence (e.g. sandwich immunoassay), surface plasmon resonance, ellipsometry and atomic force microscopy. Expression levels of markers (e.g.,
polynucleotides or polypeptides) are compared by procedures well known in the art, such as RT-PCR, Northern blotting, Western blotting, flow cytometry, immunocytochemistry, binding to magnetic and/or antibody-coated beads, in situ hybridization, fluorescence in situ hybridization (FISH), flow chamber adhesion assay, ELISA, microarray analysis, or colorimetric assays. Methods may further include, one or more of electrospray ionization mass spectrometry (ESI-MS), ESI-MS/MS, ESI-MS/(MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS), matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), surface-enhanced laser desorption/ionization time-of- flight mass spectrometry (SELDI-TOF-MS), desorption/ionization on silicon (DIOS), secondary ion mass spectrometry (SIMS), quadrupole time-of-flight (Q-TOF), atmospheric pressure chemical ionization mass spectrometry (APCI-MS), APCI-MS/MS, APCI-(MS), atmospheric pressure photoionization mass spectrometry (APPI-MS), APPI-MS/MS, and APPI-(MS), quadrupole mass spectrometry, fourier transform mass spectrometry (FTMS), and ion trap mass spectrometry, where n is an integer greater than zero. Detection methods may include use of a biochip array. Biochip arrays contemplated in the present disclosure include protein arrays. One or more markers are captured on the biochip array and subjected to analysis to detect the level of the markers in a sample.
Markers may be captured with capture reagents immobilized to a solid support, such as a biochip, a multi-well microtiter plate, a resin, or a nitrocellulose membrane that is subsequently probed for the presence or level of a marker. Capture can be on a
chromatographic surface or a biospecific surface. For example, a sample containing the markers, such as serum, may be used to contact the active surface of a biochip for a sufficient time to allow binding. Unbound molecules are washed from the surface using a suitable eluent, such as phosphate buffered saline. In general, the more stringent the eluent, the more tightly the proteins must be bound to be retained after the wash.
Upon capture on a biochip, analytes can be detected by a variety of detection methods selected from, for example, a gas phase ion spectrometry method, an optical method, an electrochemical method, atomic force microscopy and a radio frequency method. In one embodiment, mass spectrometry is used. In some embodiments, the mass spectrometry used is LC -MS/MS. In some embodiments, the mass spectrometry is SELDI. Optical methods include, for example, detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, birefringence or refractive index (e.g., surface plasmon resonance, ellipsometry, a resonant mirror method, a grating coupler waveguide method or interferometry). Optical methods include microscopy (both confocal and non- confocal), imaging methods and non-imaging methods. Immunoassays in various formats (e.g., ELISA) are popular methods for detection of analytes captured on a solid phase.
Electrochemical methods include voltammetry and amperometry methods. Radio frequency methods include multipolar resonance spectroscopy.
Mass spectrometry (MS) is a well-known tool for analyzing chemical compounds. Thus, in one embodiment, the methods of the present invention comprise performing quantitative MS to measure the serum peptide marker. The method may be performed in an automated (Villanueva, e/ a/., Nature Protocols (2006) 1(2):880-891) or semi-automated format. This can be accomplished, for example with MS operably linked to a liquid chromatography device (LC-MS/MS or LC-MS) or gas chromatography device (GC-MS or GC -MS/MS). Methods for performing MS are known in the field and have been disclosed, for example, in US Patent Application Publication Nos: 20050023454; 20050035286; US Patent No: 5,800,979; and references disclosed therein.
The protein fragments, whether they are peptides derived from the main chain of the protein or are residues of a side-chain, are collected on the collection layer. They may then be analyzed by a spectroscopic method based on matrix-assisted laser desorption/ionization (MALDI) or electrospray ionization (ESI). The preferred procedure is MALDI with time of flight (TOF) analysis, known as MALDI-TOF MS. This involves forming a matrix on the membrane, e.g. as described in the literature, with an agent which absorbs the incident light strongly at the particular wavelength employed. The sample is excited by UV, or IR laser light into the vapour phase in the MALDI mass spectrometer. Ions are generated by the vaporization and form an ion plume. The ions are accelerated in an electric field and separated according to their time of travel along a given distance, giving a mass/charge (m/z) reading which is very accurate and sensitive. MALDI spectrometers are commercially available from PerSeptive Biosystems, Inc. (Frazingham, Mass., USA) and are described in the literature, e.g. M. Kussmann and P. Roepstorff, cited above.
Magnetic-based serum processing can be combined with traditional MALDI-TOF. Through this approach, improved peptide capture is achieved prior to matrix mixture and deposition of the sample on MALDI target plates. Accordingly, methods of peptide capture are enhanced through the use of derivatized magnetic bead based sample processing.
MALDI-TOF MS allows scanning of the fragments of many proteins at once. Thus, many proteins can be run simultaneously on a polyacrylamide gel, subjected to a method of the invention to produce an array of spots on the collecting membrane, and the array may be analyzed. Subsequently, automated output of the results is provided by using the ExPASy server, as at present used for MIDI-TOF MS and to generate the data in a form suitable for computers.
Other techniques for improving the mass accuracy and sensitivity of the MALDI-TOF MS can be used to analyze the fragments of protein obtained on the collection membrane. These include the use of delayed ion extraction, energy reflectors and ion-trap modules. In addition, post source decay and MS-MS analysis are useful to provide further structural analysis. With ESI, the sample is in the liquid phase and the analysis can be by ion-trap,
TOF, single quadrupole or multi-quadrupole mass spectrometers. The use of such devices (other than a single quadrupole) allows MS— MS or MS analysis to be performed. Tandem mass spectrometry allows multiple reactions to be monitored at the same time.
Capillary infusion may be employed to introduce the marker to a desired MS implementation, for instance, because it can efficiently introduce small quantities of a sample into a mass spectrometer without destroying the vacuum. Capillary columns are routinely used to interface the ionization source of a MS with other separation techniques including gas chromatography (GC) and liquid chromatography (LC). GC and LC can serve to separate a solution into its different components prior to mass analysis. Such techniques are readily combined with MS, for instance. One variation of the technique is that high performance liquid chromatography (HPLC) can now be directly coupled to mass spectrometer for integrated sample separation/and mass spectrometer analysis.
Quadrupole mass analyzers may also be employed as needed to practice the invention. Fourier-transform ion cyclotron resonance (FTMS) can also be used for some invention embodiments. It offers high resolution and the ability of tandem MS experiments. FTMS is based on the principle of a charged particle orbiting in the presence of a magnetic field.
Coupled to ESI and MALDI, FTMS offers high accuracy with errors as low as 0.001%.
In one embodiment, the marker qualification methods of the invention may further comprise identifying significant peaks from combined spectra. The methods may also further comprise searching for outlier spectra. In another embodiment, the method of the invention further comprises determining distant dependent K-nearest neighbors.
In another embodiment of the method of the invention, an ion mobility spectrometer can be used to detect and characterize serum peptide markers. The principle of ion mobility spectrometry is based on different mobility of ions. Specifically, ions of a sample produced by ionization move at different rates, due to their difference in, e.g., mass, charge, or shape, through a tube under the influence of an electric field. The ions (typically in the form of a current) are registered at the detector which can then be used to identify a marker or other substances in a sample. One advantage of ion mobility spectrometry is that it can operate at atmospheric pressure.
In an additional embodiment of the methods of the present invention, multiple markers are measured. The use of multiple markers increases the predictive value of the test and provides greater utility in diagnosis, toxicology, patient stratification and patient monitoring. The process called“Pattern recognition” detects the patterns formed by multiple markers greatly improves the sensitivity and specificity of clinical proteomics for predictive medicine. Subtle variations in data from clinical samples indicate that certain patterns of protein expression can predict phenotypes such as the presence or absence of a certain disease, a particular stage of neoplasia progression, or a positive or adverse response to drug treatments.
In some embodiments, detection and quantitation of markers is accomplished by using an affinity microarray-based technology (Nanoview Biosciences) coupled with a Reflectance Imaging Sensor detection methodology as described in International Application No.
PCT/US2017/016434, which is incorporated herein in its entirety. For example, extracellular vesicles may be bound to a substrate surface that has a spectral reflectance signature. This surface comprises binding probes that specifically bind to exosomes. For example, the binding probe may be an antibody that specifically binds to a surface marker on an exosome (e.g., a glypican protein). Upon binding, the substrate comprising the bound exosomes is exposed to a light source, and the reflectance of the substrate surface is altered compared to a substrate without bound exosomes. The exosomes may be observed as discrete dots in an image. The light source may be a light emitting diode (LED) light source. In some embodiments, the binding probe specifically binds to a marker.
Diagnostic Assays
Expression levels of the markers described herein are correlated with pancreatic ductal adenocarcinoma, and can be used to identify subjects with high probability of having the disease. Antibodies that specifically bind a marker described herein or any other method known in the art may be used to monitor expression of a marker of interest. Detection of an alteration relative to a normal, reference sample can be used as a diagnostic indicator of prostate carcinoma. In some embodiments, at least a 2-fold change in the level of a
ANXA11, GPC4, CD 14, CD44v6, MUC1, CLDN4, TSPAN8, CD 147, and CD 104 marker of the present disclosure is indicative of pancreatic ductal adenocarcinoma (PD AC) or the propensity to develop PD AC. In yet another embodiment, an expression profile that characterizes alterations in the expression of two or more markers is correlated with a particular disease state (e.g., PDAC). Such correlations are indicative of PD AC or the propensity to develop PDAC. In one embodiment, a pancreatic ductal adenocarcinoma can be monitored using the methods and compositions of the invention.
The present invention features diagnostic assays for the detection of PDAC or the propensity to develop such a condition. In one embodiment, levels of any one or more of a group of markers consisting of ANXA11, GPC4, CD14, CD44v6, MUC1, CLDN4, TSPAN8, CD 147, and CD 104 are measured in a subject sample and used to characterize PDAC or the propensity to develop such a condition. In some embodiments, levels of any two or more of a group of markers consisting of ANXA11, GPC4, CD14, CD44v6, MUC1, CLDN4, TSPAN8, CD 147, and CD 104 are measured in a subject sample and used to characterize PDAC or the propensity to develop such a condition. In some embodiments, the levels of any three or more of a group of markers consisting of ANXA11, GPC4, CD14, CD44v6, MUC1, CLDN4, TSPAN8, CD147, and CD104 are measured in a subject sample and used to characterize PDAC or the propensity to develop such a condition. In some embodiments, the levels of any four or more of a group of markers consisting of ANXA11, GPC4, CD14, CD44v6, MUC1, CLDN4, TSPAN8, CD 147, and CD 104 are measured in a subject sample and used to characterize PD AC or the propensity to develop such a condition. In some embodiments, the level of any five or more of a group of markers consisting of ANXA11, GPC4, CD14, CD44v6, MUC1, CLDN4, TSPAN8, CD147, and CD104 are measured in a subject sample and used to characterize PD AC or the propensity to develop such a condition. In some embodiments, the levels of any six or more of a group of markers consisting of ANXAl 1, GPC4, CD 14, CD44v6, MUC1, CLDN4, TSPAN8, CD 147, and CD 104 are measured in a subject sample and used to characterize PD AC or the propensity to develop such a condition. In some embodiments, the levels of any seven or more of a group of markers consisting of ANXAl 1, GPC4, CD14, CD44v6, MUC1, CLDN4, TSPAN8, CD147, and CD104 are measured in a subject sample and used to characterize PD AC or the propensity to develop such a condition.
In one embodiment, the level of one or more markers is measured on at least two separate occasions and an increase in the level is an indication of PD AC progression. In some instances, the level of the marker decreases over time, which may indicate a slowing of the progression or a regression of the disease. The level of marker in the bodily fluid (e g., blood, blood serum, plasma, saliva, urine, abdominal fluid, or pancreatic secretions) of a subject having PD AC or the susceptible to develop the disease may be altered by as little as 10%, 20%, 30%, or 40%, or by as much as 50%, 60%, 70%, 80%, or 90% or more relative to the level of the marker in the normal control. A healthy subject generally expresses
ANXAl 1, GPC4, CD14, CD44v6, MUC1, CLDN4, TSPAN8, CD147, and CD104 at lower levels relative to subjects having or at risk of developing PD AC). In one embodiment, a subject sample of a bodily fluid (e.g., blood, blood serum, plasma, urine, abdominal fluid, or pancreatic secretions) is collected prior to the onset of symptoms of PD AC. In another example, the sample can be a tissue or cell collected prior to the onset of PD AC symptoms.
The diagnostic methods described herein can be used to provide a diagnosis individually or to confirm the results of another diagnostic method. Additionally, the methods described herein can be use used with any other diagnostic method described herein for a more accurate diagnosis of the presence or severity of PD AC.
The markers identified using the methods described herein can be used individually, in combination with other identified markers, or with other markers known in the art that are associated with cancer or cancer treatment. The markers are differentially present in samples from a subject having cancer and from a normal subject in whom cancer is undetectable. Detecting one or more of these markers in a subject would provide useful information regarding the probability that the subject may have, or be susceptible to, PD AC, the aggressiveness of the neoplasia, and the susceptibility of the neoplasia to treatment.
The detection of the peptide marker is then correlated with a probable diagnosis of cancer. In some embodiments, the mere detection of a marker (e.g., MUC1) is useful and can be correlated with a probable diagnosis of cancer. The analysis of markers present in a sample may also involve quantifying the markers to correlate the detection of markers with a probable diagnosis of cancer. For example, if a detected marker (or set of markers) in a subject is present in a sample in a different amount compared to a control, or reference, amount (i.e., higher than the control), then the subject being tested has a higher probability of having cancer. The correlation of a marker or markers in a sample to a probable diagnosis of cancer may consider the amount of the marker or markers in the sample compared to a control, or reference, amount of the marker or markers (e.g., in subjects having no detectable cancer). In some embodiments, the control amount of the marker is measured under the same or substantially similar experimental conditions as used when measuring the amount of the marker in a subject having, suspected of having, or at risk of developing PD AC. In some embodiments, the control, or reference, amount of the marker is known, and result obtained from test samples can be compared to that standard, rather than re-running a control.
A marker profde may be obtained from a subject sample and compared to a reference marker profde obtained from a reference population, enabling classifying the subject as belonging to or not belonging to the reference population. The correlation of the marker profde to a neoplasia diagnosis may consider the presence or absence of the markers in test and control samples. The correlation may consider both factors when making a cancer status determination.
In certain embodiments of the methods of qualifying cancer status, the methods further comprise managing subject treatment based on the status. The invention also provides for such methods where the markers (or specific combination of markers) are measured again after subject management. In these cases, the methods are used to monitor the status of the cancer, e.g., response to cancer treatment, remission of the disease or progression of the disease.
The markers disclosed herein have uses other than just diagnostic. In some embodiments, they can be used in monitoring responses to neoplasia therapy. In another embodiment, the markers can be used to study the heredity of the disease. For example, markers may be genetically linked, which be detected by analyzing samples from a population of subjects whose families have a history of neoplasia. The results can then be compared with data obtained from subjects whose families do not have a history of neoplasia. The markers that are genetically linked may facilitate determination if a subject is predisposed to neoplasia based on a family.
The individual markers disclosed herein is useful in determining the status or stage of a subject’s neoplasia. A marker detected in a subject sample using the methods described herein is compared with the marker in a control sample, wherein differences in the expression or amounts of the marker distinguishes neoplasia status from non-neoplasia status. The techniques can be adjusted, as is well understood in the art, to increase the sensitivity or specificity of the diagnostic assay.
While individual markers are useful diagnostic markers, in some instances, a combination of markers provides greater predictive value than a single marker. Detection the presence or absence of a plurality of markers in a sample can decrease false positives and false negative diagnoses, while increasing the occurrence of true positives and true negatives.
The methods described herein can also be used to monitor the progression of a subject’s PD AC or to assist in the management of the disease. For example, therapeutic options may vary at different stages of the disease, which may be reflected in changes in marker expression over time as detected by the methods described herein. These methods may also be used to distinguish PDAC from pancreatitis.
Microarrays
As disclosed herein, markers (e.g., ANXA11, GPC4, CD14, CD44v6, MUC1, CLDN4, TSPAN8, CD147, and CD104) have been associated with PDAC. Methods for assaying expression of these polypeptides may be used to characterize PDAC. For example, the present disclosure provides diagnostic methods and compositions useful for identifying a polypeptide expression profile that identifies a subject as having or having a propensity to develop prostate carcinoma. Such assays can be used to measure an alteration in the level of a polypeptide.
The polypeptides of the invention are useful as hybridizable array elements in a microarray. The array elements are organized in an ordered fashion such that each element is present at a specified location on the substrate. Useful substrate materials include membranes, composed of paper, nylon or other materials, filters, chips, glass slides, and other solid supports. The ordered arrangement of the array elements allows hybridization patterns and intensities to be interpreted as expression levels of particular genes or proteins. Methods for making nucleic acid microarrays are known to the skilled artisan and are described, for example, in U.S. Pat. No. 5,837,832, Lockhart, et al. (Nat. Biotech. 14: 1675-1680, 1996), and Schena, et al. (Proc. Natl. Acad. Sci. 93 : 10614-10619, 1996), herein incorporated by reference. Methods for making polypeptide microarrays are described, for example, by Ge (Nucleic Acids Res. 28: e3. i-e3. vii, 2000), MacBeath et al., (Science 289: 1760-1763, 2000), Zhu et al, (Nature Genet. 26:283-289), and in U.S. Pat. No. 6,436,665, hereby incorporated by reference.
Proteins (e.g., ANXA11, GPC4, CD14, CD44v6, MUC1, CLDN4, TSPAN8, CD147, and CD 104) may be analyzed using protein microarrays. Such arrays are useful in high- throughput low-cost screens to identify alterations in the expression or post-translation modification of a polypeptide of the invention, or a fragment thereof. In particular, such microarrays are useful to identify a protein whose expression is altered in prostate carcinoma. In one embodiment, a protein microarray of the invention binds a marker present in a subject sample and detects an alteration in the level of the marker. Typically, a protein microarray features a protein, or fragment thereof, bound to a solid support. Suitable solid supports include membranes (e.g., membranes composed of nitrocellulose, paper, or other material), polymer-based films (e.g., polystyrene), beads, or glass slides. For some applications, proteins (e.g., antibodies that bind a marker of the invention) are spotted on a substrate using any convenient method known to the skilled artisan (e.g., by hand or by inkjet printer).
The protein microarray is hybridized with a detectable probe. Such probes can be polypeptide, nucleic acid molecules, antibodies, or small molecules. For some applications, polypeptide and nucleic acid molecule probes are derived from a biological sample taken from a subject, such as a bodily fluid (such as blood, blood serum, plasma, saliva, urine, abdominal fluid, or pancreatic secretions); a homogenized tissue sample (e.g. a tissue sample obtained by biopsy); or a cell isolated from a subject sample. Probes can also include antibodies, candidate peptides, nucleic acids, or small molecule compounds derived from a peptide, nucleic acid, or chemical library. Hybridization conditions (e.g., temperature, pH, protein concentration, and ionic strength) are optimized to promote specific interactions.
Such conditions are known to the skilled artisan and are described, for example, in Harlow,
E. and Lane, D., Using Antibodies: A Laboratory Manual. 1998, New York: Cold Spring Harbor Laboratories. After removal of non-specific probes, specifically bound probes are detected, for example, by fluorescence, enzyme activity (e.g., an enzyme-linked calorimetric assay), direct immunoassay, radiometric assay, or any other suitable detectable method known to the skilled artisan. In some embodiments, the microarray is an affinity microarray- based technology (Nanoview Biosciences) coupled with a Reflectance Imaging Sensor detection methodology as described in International Application No. PCT/US2017/016434.
Methods of Treatment
The present disclosure provides methods of treating a disease, which comprise detecting the level of at least one marker as disclosed above and administering a
therapeutically effective amount of a pharmaceutical composition. In certain embodiments, a pretreatment level of at least one marker is determined in a subject prior to treatment. The marker’s pretreatment level may be compared to the marker’s level in the subject during and/or after treatment, to monitor the efficacy of the treatment. In some embodiments, the level of the marker is measured by any suitable method as described herein. In some embodiments, the level of the marker is measured
As used herein, the terms“treat,” treating,”“treatment,” and the like refer to reducing or ameliorating a disorder and/or symptom associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
As used herein, the terms“prevent,”“preventing,”“prevention,”“prophylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.
In some embodiments, the pharmaceutical composition may be administered by any appropriate route for the treatment or prevention of a neoplasia. For example, administration may be accomplished by parenteral, intravenous, intra-arterial, subcutaneous, intramuscular, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, by aerosol, by suppositories, or by oral administration. Pharmaceutical compositions for the treatment of a neoplasia may be administered to humans, domestic pets, livestock, or any other animals with a pharmaceutically acceptable diluent, carrier, or excipient, in unit dosage form. In one embodiment, the invention provides a method of monitoring treatment progress. The method includes the step of determining a level of a marker (e.g., ANXA11, GPC4, CD 14, CD44v6, MUC1, CLDN4, TSPAN8, CD 147, and CD 104) in a subject suffering from or susceptible to a PD AC or symptoms thereof, in which the subject has been administered a therapeutic amount of a compound sufficient to treat the disease or symptoms thereof The level of a marker (e.g., ANXA11, GPC4, CD14,CD44v6, MUC1, CLDN4, TSPAN8, CD147, and CD104) determined in the method can be compared to known levels of the marker in either healthy normal controls or in other afflicted subjects to establish the subject’s disease status. In some embodiments, a second level of the marker in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy. In some embodiments, a pre-treatment level of a marker (e.g., ANXA11, GPC4, CD14, CD44v6, MUC1, CLDN4, TSPAN8, CD147, and CD104) in the subject is determined prior to beginning treatment according to this invention; this pre-treatment level of the marker can then be compared to the level of the marker in the subject after the treatment commences, to determine the efficacy of the treatment.
Kits for characterizing markers associated with pancreatic neoplasia
Some aspects of the present disclosure provide kits for characterizing markers associated with pancreatic neoplasia in a sample. The invention provides kits for the characterization of pancreatic ductal adenocarcinoma that expresses one, two, three, or four or all of ANXA11, CD14, CD44v6, or GPC4. In one embodiment, the kit includes a capture molecule (e.g., antibody or polynucleotide probe) that binds a ANXA11, CD14, CD44v6, or GPC4 polynucleotide or polypeptide. The invention also provides kits for the characterization of pancreatic ductal adenocarcinoma that expresses one, two, three, four, five, six, seven or all of CD44v6, MUC1, CLDN4, TSPAN8, CD147, and CD104. In one embodiment, the kit includes a capture molecule (e.g., antibody or polynucleotide probe) that binds a CD44v6, MUC1, CLDN4, TSPAN8, CD147, and CD104 polynucleotide or polypeptide. In some embodiments, the kit comprises a sterile container. Such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding reagents. If desired the kit is provided together with instructions for using the kit for the detection of the markers. The instructions will generally include information about the use of the kit for the characterization of PD AC. In other embodiments, the instructions include at least one of the following: precautions; warnings; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container. In a further embodiment, a kit can comprise instructions in the form of a label or separate insert (package insert) for suitable operational parameters. In yet another embodiment, the kit can comprise one or more containers with appropriate positive and negative controls or control samples, to be used as standard(s) for detection, calibration, or normalization.
In certain embodiments, a subject can be diagnosed with PD AC by adding a biological sample (e.g., blood or serum) from the subject to the kit, or components thereof, and detecting the relevant biomarkers that are specifically bound by capture molecules. By way of example, the method comprises: (i) collecting a sample from the subject; (ii) adding subject’s sample to the components in the kit, e.g., a holding tube or a substrate; and (iii) detecting the capture molecules to which the markers in the sample have bound.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.
EXAMPLES
Example 1: Identification of Markers Associated with Pancreatic Ductal
Adenocarcinoma (PDAC)
Markers present in an organoid model of PDAC were assessed to determine if the markers were increased in PDAC or were simply present at levels observed in healthy cells. 4.0 ml of supernatant from an organoid culture were collected and subjected to a 100 kDa vesicle enrichment method followed by extracellular vesicle purification. The purified extracellular vesicles were then subjected to liquid chromatography-mass spectrometry (LC- MS/MS). To differentiate cancer-associated from extracellular vesicle secreted by normal human pancreatic epithelial, media was collected from human embryonic stem cell derived exocrine pancreas organoids generated using a proprietary method described in
W020160] 5158A 1, the contents of which are incorporated herein by reference in their entirety, and processed in the same manner as performed for the sample collected from the organoid model of disease. The purified extracellular vesicles were then subjected to liquid chromatography-mass spectrometry (LC-MS/MS). Among the 1465 proteins identified, 263 proteins that were at least two-fold higher in tumor organoid extracellular vesicles compared to exocrine organoids and expressed in at least four out of the six tumor organoid lines.
Referring to FIG. 1, principle component analysis showed that the models differ from each other in the extracellular vesicles that they secrete (i.e., the extracellular vesicles comprised different sets of markers or different levels of markers). This demonstrated the ability of the method to detect distinct secreted protein profiles from patient derived models. Furthermore, it also highlights the fact that the culture/media conditions used to generate and maintain these organoid models is effective in retaining interpatient heterogeneity for secretion of vesicles. The 263 proteins identified (Table 1), clustered into functional groups including RNA splicing, histone/chromatin, proteasome and translation, in addition to vesicles containing proteins involved in cytoskeleton regulation, cell adhesion and membrane trafficking (Figure 2).
Example 2: Validation of Markers as Associated with Pancreatic Ductal
Adenocarcinoma
Experiments were carried out to determine a) if vesicles can be detected in the circulating blood of pancreatic cancer patients, and b) if the markers present in such vesicles are present in blood samples derived from patients with pancreatic cancer, but are not present in blood samples derived from patients with a non-malignant pancreatic disease. Two groups of proteins were selected for analysis. Group I proteins are listed in Table 1. Group I proteins include CD44v6, MUC1, CLDN4, TSPAN8, CD147, and CD104. Group P proteins include= GPC1, EPHA2, EPCAM, EGFR, HER2, CD63, CD9, and CD81. Blood was obtained from five PD AC patients having metastatic or premalignant disease and from four chronic pancreatitis patients with no evidence of PDAC. To enable detection of secreted vesicles in patient blood, an affinity microarray-based technology (NanoView Biosciences) coupled with a Reflectance Imaging Sensor detection methodology was used to identify and count extracellular vesicles. Validated antibodies against the selected proteins were arrayed on the chip and supernatants from pancreatic derived organoid (PDO) cultures were analyzed for presence of extracellular vesicles (FIG. 3). Interestingly, the extracellular vesicles containing antigens in Group I, were very low or undetectable in samples derived from pancreatitis patients, but were present at levels 100 to 1000-fold greater in blood derived from PDAC patients. In contrast, antigens identified in Group II were not differentially present in blood derived from patients with PDAC, demonstrating the ability of patient derived organoid cultures to identify extracellular vesicle-associated proteins that can be used as diagnostic markers of PDAC.
Interestingly, both the total number of extracellular vesicle particles and the serum concentration of CA 19-9, the clinically approved diagnostic marker for PDAC, varied greatly between the patients analyzed suggesting that neither the absolute levels of extracellular vesicle nor the levels of CA 19-9 are as reliable as the extracellular vesicle- associated proteins of Group I that were identified herein as markers for PDAC. These results demonstrate that patient derived organoid supernatants are an effective platform to identify clinically relevant diagnostic markers for PDAC.
Example 3: Clinical Validation of a Selected Group of Putative Secreted Markers
Clinical validation was performed for a selected group of putative secreted markers including ANNXA11, CD14, GPC4, and CD44v6. The expression of the marker proteins was analyzed in the plasma of patients with an established diagnosis of pancreatic cancer, as well as in individuals with a variety of benign gastrointestinal disorders. A summary of the PDAC and benign gastrointestinal disorder patients is provided in Table 2.
Plasma samples were processed following standard operating procedures to isolate secreted proteins, including those from extracellular vesicles. Subsequently, western blot analyses were conducted to confirm that the expression of four proteins was increased in the plasma of patients with pancreatic cancer compared to the levels present in the plasma from patients with benign disease (FIGs. 4A-4C).
Table 2: Subjects Diagnosed with PDAC or a Benign Gastrointestinal Disorder
Figure imgf000036_0001
Figure imgf000037_0001
Other Embodiments
From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.
The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

What is claimed:
1. A panel for characterizing pancreatic ductal adenocarcinoma, the panel comprising two or more capture molecules each bound to a substrate, wherein each capture molecule specifically binds a marker polypeptide selected from the group consisting of ANXA11, CD14, CD44v6, and GPC4, or a polynucleotide encoding said polypeptide.
2. The panel of claim 1, wherein the panel comprises capture molecules that bind ANXA11 and one or more of CD 14, CD44v6, and GPC4.
3. The panel of claim 1, wherein the panel comprises capture molecules that bind CD14 and one or more of ANXA1, CD44v6, and GPC4.
4. The panel of any one of claims 1, wherein the panel comprises capture molecules that bind CD44v6 and one or more of ANXA11, CD14, and GPC4.
5. The panel of claim 1, wherein the panel comprises capture molecules that bind GPC4 and one or more of ANXA11, CD 14, and CD44v6.
6. A panel for characterizing pancreatic ductal adenocarcinoma, the panel comprising two or more capture molecules each bound to a substrate, wherein each capture molecule specifically binds a marker polypeptide selected from the group consisting of CD44v6, MUC1, CLDN4, TSPAN8, CD147, and CD104, or the marker is a polynucleotide encoding said polypeptide.
7. The panel of any one of claims 1-6, wherein the capture molecule is a polypeptide, polynucleotide probe, or fragment thereof.
8. The panel of any one of claims 1-6, wherein the polypeptide is an antibody that specifically binds the marker polypeptide.
9. The panel of any one of claims 1-6, wherein the polynucleotide is an aptamer that specifically binds the marker polypeptide.
10. The panel of any one of claims 1-6, wherein the polynucleotide probe specifically hybridizes to the polynucleotide encoding the marker polypeptide.
11. The panel of any one of claims 1-6, wherein the substrate is a bead or planar surface.
12. The panel of claim 10, wherein the planar surface is a membrane, filter, chip, glass slide, or other solid support.
13. The panel of claim 6, wherein the panel comprises capture molecules that bind CD44v6 and one or more of MUC1, CLDN4, TSPAN8, CD147, and CD104.
14. The panel of claim 6, wherein the panel comprises capture molecules that bind MUC1 and one or more of CD44v6, CLDN4, TSPAN8, CD147, and CD104.
15. The panel of claim 6, wherein the panel comprises capture molecules that bind TSPAN8 and one or more of CD44v6, MUC1, CLDN4, CD 147, and CD 104.
16. The panel of claim 6, wherein the panel comprises capture molecules that bind CD147 and one or more of CD44v6, MUC1, CLDN4, TSPAN8, and CD104.
17. A panel for characterizing pancreatic ductal adenocarcinoma, the panel comprising two or more capture molecules each bound to a substrate, wherein each capture molecule specifically binds a marker polypeptide selected from those listed in Table 1.
18. The panel of claim 17, wherein one or more of the capture molecules are antibodies.
19. A method for detecting a marker in a sample derived from a subject, the method comprising detecting two or more markers selected from the group consisting of CD44v6, MUC1, CLDN4, TSPAN8, CD147, and CD104, or a marker of Table 1 in the sample.
20. A method for detecting a marker in a sample derived from a subject, the method comprising detecting a marker selected from the group consisting of ANXA11, CD14, CD44v6, and GPC4, and a marker of Table 1 in the sample.
21. A method for characterizing pancreatic ductal adenocarcinoma in a subject, the method comprising
a) culturing a cell from a subject having or suspected of having pancreatic ductal adenocarcinoma, thereby generating an organoid culture;
b) isolating extracellular vesicles present in culture media of the organoid culture; and c) detecting a marker present in or on the extracellular vesicles, wherein the marker is selected from the group consisting of CD44v6, MUC1, CLDN4, TSPAN8, CD147, CD104, ANXA11, CD14, CD44v6, and GPC or a marker of Table 1 using a capture molecule that specifically binds the marker.
22. The method of claim 21, wherein the capture molecule is bound to a substrate surface.
23. The method of claim 21, wherein the substrate surface has a spectral reflectance signature.
24. The method of claim 21, further comprising:
(d) exposing the substrate surface to a light source; and
(e) detecting the presence or absence of extracellular vesicles on the substrate surface, wherein the presence of the extracellular vesicles indicates the presence of one of the markers.
25. A method of treating a selected subject having pancreatic ductal adenocarcinoma (PDAC), the method comprising administering an agent to the subject, wherein the subject is selected by detecting a marker selected from the group consisting of CD44v6, MUC1, CLDN4, TSPAN8, CD 147, and CD 104 in a sample derived from the subject.
26. The method of any one of claims 19-24, wherein the sample is a biological fluid selected from the group consisting of culture media, blood, blood serum, plasma, urine, abdominal fluids, or pancreatic secretions.
27. The method of any one of claims 19-24, wherein the sample is a biopsy.
28. The method of any one of claims 19-24, wherein the sample comprises extracellular vesicles.
29. The method of claim 28, wherein the extracellular vesicles are exosomes or
microvesicles.
30. The method of any one of claims 19-24, wherein the detecting comprises RT-PCR, Northern blotting, Western blotting, flow cytometry, immunocytochemistry, binding to magnetic and/or antibody-coated beads, in situ hybridization, fluorescence in situ hybridization (FISH), flow chamber adhesion assay, an immunoassay such as ELISA or a radioimmunoassay, microarray analysis, colorimetric assays, mass spectrometry, liquid chromatography mass spectrometry (LC-MS/MS), or high-performance liquid
chromatography.
31. The method of claim 30, wherein the detecting comprises liquid chromatography-mass spectrometry (LC-MS/MS).
32. The method of any one of claims 19-24, wherein the detecting comprises using a capture molecule that specifically binds the marker.
33. The method of claim 32, wherein the capture molecule is an antibody
34. The method of claim 32, wherein the capture molecule is an aptamer.
35. The method of any one of claims 19-34, wherein the capture molecule is bound to a substrate surface.
36. The method of claim 35, wherein the substrate surface has a spectral reflectance signature.
37. A kit for characterizing markers in a sample, the kit comprising two or more capture molecules fixed to a substrate surface, wherein each capture molecule specifically binds a marker polypeptide selected from the group consisting of CD44v6, MUC1, CLDN4, TSPAN8, CD147, CD104 ANXA11, CD14, and GPC4 or a marker polypeptide of Table 1, or a polynucleotide encoding said marker polypeptide.
38. The kit of claim 37, wherein the substrate surface has a spectral reflectance signature.
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