US20090098594A1 - Methods for diagnosis prognosis and methods of treatment - Google Patents

Methods for diagnosis prognosis and methods of treatment Download PDF

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US20090098594A1
US20090098594A1 US12/229,476 US22947608A US2009098594A1 US 20090098594 A1 US20090098594 A1 US 20090098594A1 US 22947608 A US22947608 A US 22947608A US 2009098594 A1 US2009098594 A1 US 2009098594A1
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cell
level
activation
activatable
cells
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Wendy J. Fantl
Santosh K. Putta
Omar D. Perez
Helen L. Francis-Lang
Aileen C. Cohen
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Nodality Inc
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Nodality Inc
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Priority to US12/229,476 priority Critical patent/US20090098594A1/en
Assigned to NODALITY, INC. reassignment NODALITY, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: PEREZ, OMAR D., COHEN, AILEEN C., FRANCIS-LANG, HELEN L., PUTTA, SANTOSH K., FANTL, WENDY J.
Publication of US20090098594A1 publication Critical patent/US20090098594A1/en
Priority to US13/493,857 priority patent/US9182385B2/en
Priority to US14/825,486 priority patent/US20160097770A1/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
    • G01N33/57426—Specifically defined cancers leukemia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5041—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving analysis of members of signalling pathways
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • 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
    • G01N2333/4703—Regulators; Modulating activity
    • 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
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/7051—T-cell receptor (TcR)-CD3 complex
    • 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
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70514—CD4
    • 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
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70517—CD8
    • 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
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)

Definitions

  • a method for classifying a cell comprising contacting the cell with an inhibitor, determining the presence or absence of a change in activation level of an activatable element in the cell, and classifying the cell based on the presence or absence of the change in the activation level of the activatable element.
  • the change in activation level of an activatable element is an increase in the activation level of an activatable element.
  • the activatable element is a protein subject to phosphorylation or dephosphorylation.
  • the invention provides a method for classifying a cell by contacting the cell with an inhibitor; determining the activation levels of a plurality of activatable elements in the cell; and classifying the cell based on the activation level.
  • the inhibitor is a kinase or phosphatase inhibitor, such as adaphostin, AG 490, AG 825, AG 957, AG 1024, aloisine, aloisine A, alsterpaullone, aminogenistein, API-2, apigenin, arctigenin, AY-22989, BAY 61-3606, bisindolylmaleimide IX, chelerythrine, 10-[4′-(N,N-Diethylamino)butyl]-2-chlorophenoxazine hydrochloride, dasatinib, 2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole, 5,7-Dimeth
  • the cell is a hematopoietic-derived cell.
  • the hematopoietically derived cell is selected from the group consisting of pluripotent hematopoietic stem cells, B-lymphocyte lineage progenitor or derived cells, T-lymphocyte lineage progenitor or derived cells, NK cell lineage progenitor or derived cells, granulocyte lineage progenitor or derived cells, monocyte lineage progenitor or derived cells, megakaryocyte lineage progenitor or derived cells and erythroid lineage progenitor or derived cells.
  • the hematopoietic derived cell is a B-lymphocyte lineage progenitor and derived cell, e.g., an early pro-B cell, late pro-B cell, large pre-B cell, small pre-B cell, immature B cell, mature B cell, plasma cell and memory B cell, a CD5+ B cell, a CD38+ B cell, a B cell bearing a mutatated or non mutated heavy chain of the B cell receptor, or a B cell expressing Zap70.
  • a B-lymphocyte lineage progenitor and derived cell e.g., an early pro-B cell, late pro-B cell, large pre-B cell, small pre-B cell, immature B cell, mature B cell, plasma cell and memory B cell, a CD5+ B cell, a CD38+ B cell, a B cell bearing a mutatated or non mutated heavy chain of the B cell receptor, or a B cell expressing Zap70.
  • the classification includes classifying the cell as a cell that is correlated with a clinical outcome.
  • the clinical outcome is the prognosis and/or diagnosis of a condition.
  • the clinical outcome is the presence or absence of a neoplastic or a hematopoietic condition, such as Non-Hodgkin Lymphoma, Hodgkin or other lymphomas, acute or chronic leukemias, polycythemias, thrombocythemias, multiple myeloma or plasma cell disorders, e.g., amyloidosis and Waldenstrom's macroglobulinemia, myelodysplastic disorders, myeloproliferative disorders, myelofibrosis, or atypical immune lymphoproliferations.
  • a neoplastic or a hematopoietic condition such as Non-Hodgkin Lymphoma, Hodgkin or other lymphomas, acute or chronic leukemias, polycythemias, thrombocy
  • the neoplastic or hematopoietic condition is non-B lineage derived, such as acute myeloid leukemia (AML), Chronic Myeloid Leukemia (CML), non-B cell acute lymphocytic leukemia (ALL), non-B cell lymphomas, myelodysplastic disorders, myeloproliferative disorders, myelofibrosis, polycythemias, thrombocythemias, or non-B atypical immune lymphoproliferations.
  • AML acute myeloid leukemia
  • CML Chronic Myeloid Leukemia
  • ALL non-B cell acute lymphocytic leukemia
  • non-B cell lymphomas myelodysplastic disorders
  • myeloproliferative disorders myelofibrosis
  • polycythemias thrombocythemias
  • non-B atypical immune lymphoproliferations non-B lineage derived
  • the neoplastic or hematopoietic condition is a B-Cell or B cell lineage derived disorder, such as Chronic Lymphocytic Leukemia (CLL), B lymphocyte lineage leukemia, B lymphocyte lineage lymphoma, Multiple Myeloma, acute lymphoblastic leukemia (ALL), B-cell pro-lymphocytic leukemia, precursor B lymphoblastic leukemia, hairy cell leukemia or plasma cell disorders, e.g., amyloidosis or Waldenstrom's macroglobulinemia, B cell lymphomas including but not limited to diffuse large B cell lymphoma, follicular lymphoma, mucosa associated lymphatic tissue lymphoma, small cell lymphocytic lymphoma and mantle cell lymphoma.
  • CLL Chronic Lymphocytic Leukemia
  • ALL acute lymphoblastic leukemia
  • B-cell pro-lymphocytic leukemia precursor B lymphoblastic leukemia,
  • the condition is CLL.
  • the CLL is defined by a monoclonal B cell population that co-expresses CD5 with CD19 and CD23 or CD5 with CD20 and CD23 and by surface immunoglobulin expression.
  • the clinical outcome is the staging or grading of a neoplastic or hematopoietic condition.
  • staging in methods provided by the invention include aggressive, indolent, benign, refractory, Roman Numeral staging, TNM Staging, Rai staging, Binet staging, WHO classification, FAB classification, IPSS score, WPSS score, limited stage, extensive stage, staging according to cellular markers such as ZAP70 and CD38, occult, including information that may inform on time to progression, progression free survival, overall survival, or event-free survival.
  • classifying the cell based on activation levels of activatable element includes classifying the cell as a cell that is correlated to a patient response to a treatment, such as complete response, partial response, nodular partial response, no response, progressive disease, stable disease, relapse or adverse reaction.
  • the method may further comprise determining a method of treatment, e.g., chemotherapy, biological therapy, radiation therapy, bone marrow transplantation, Peripheral stem cell transplantation, umbilical cord blood transplantation, autologous stem cell transplantation, allogeneic stem cell transplantation, syngeneic stem cell transplantation, surgery, induction therapy, maintenance therapy, watchful waiting, or holistic/alternative therapy.
  • the classifying of the cell based on activation level includes classifying the cell as a cell that is correlated with minimal residual disease or emerging resistance.
  • the activation level of the plurality of activatable elements in the cell is selected from the group consisting of cleavage by extracellular or intracellular protease exposure, novel hetero-oligomer formation, glycosylation level, phosphorylation level, acetylation level, methylation level, biotinylation level, glutamylation level, glycylation level, hydroxylation level, isomerization level, prenylation level, myristoylation level, lipoylation level, phosphopantetheinylation level, sulfation level, ISGylation level, nitrosylation level, palmitoylation level, SUMOylation level, ubiquitination level, neddylation level, citrullination level, deamidation level, disulfide bond formation level, proteolytic cleavage level, translocation level, changes in protein turnover, multi-protein complex level, oxidation level, multi-lipid complex, and biochemical changes in cell membrane.
  • the activation level is a phosphorylation level.
  • the activatable element is selected from the group consisting of proteins, carbohydrates, lipids, nucleic acids and metabolites.
  • the activatable element is a protein.
  • the activatable element is a change in metabolic state, temperature, or local ion concentration.
  • the protein is a protein subject to phosphorylation or dephosphorylation, such as kinases, phosphatases, adaptor/scaffold proteins, ubiquitination enzymes, adhesion molecules, contractile proteins, cytoskeletal proteins, heterotrimeric G proteins, small molecular weight GTPases, guanine nucleotide exchange factors, GTPase activating proteins, caspases and proteins involved in apoptosis (e.g.
  • PARP ion channels
  • molecular transporters molecular chaperones
  • metabolic enzymes vesicular transport proteins
  • hydroxylases isomerases
  • transferases deacetylases
  • methylases demethylases
  • proteases esterases
  • hydrolases DNA binding proteins or transcription factors.
  • the protein is selected from the group consisting of PI3-Kinase (p85, p110a, p110b, p110d), Jak1, Jak2, SOCs, Rac, Rho, Cdc42, Ras-GAP, Vav, Tiam, Sos, Dbl, Nck, Gab, PRK, SHP1, and SHP2, SHIP1, SHIP2, sSHIP, PTEN, Shc, Grb2, PDK1, SGK, Akt1, Akt2, Akt3, TSC1,2, Rheb, mTor, 4EBP-1, p70S6Kinase, S6, LKB-1, AMPK, PFK, Acetyl-CoAa Carboxylase, DokS, Rafs, Mos, Tp12, MEK1/2, MLK3, TAK, DLK, MKK3/6, MEKK1,4, MLK3, ASK1, MKK4/7, SAPK/JNK1,2,3, p38s, Erk1/2, Syk,
  • the protein selected from the group consisting of Erk, Syk, Zap70, Lck, Btk, BLNK, Cbl, PLC ⁇ 2, Akt, RelA, p38, S6. In some embodiments the protein is S6.
  • the protein is selected from the group consisting of HER receptors, PDGF receptors, Kit receptor, FGF receptors, Eph receptors, Trk receptors, IGF receptors, Insulin receptor, Met receptor, Ret, VEGF receptors, TIE1, TIE2, FAK, Jak1, Jak2, Jak3, Tyk2, Src, Lyn, Fyn, Lck, Fgr, Yes, Csk, Abl, Btk, ZAP70, Syk, IRAKs, cRaf, ARaf, BRAF, Mos, Lim kinase, ILK, Tpl, ALK, TGF ⁇ receptors, BMP receptors, MEKKs, ASK, MLKs, DLK, PAKs, Mek 1, Mek 2, MKK3/6, MKK4/7, ASK1, Cot, NIK, Bub, Myt 1, Wee1, Casein kinases, PDK1, SGK1, SGK2, SGK3, Akt1, Akt2, Akt
  • the invention provides methods for determining the presence or absence of a condition in an individual by subjecting a cell from the individual to a modulator or an inhibitor, determining the activation level of an activatable element in the cell and determining the presence or absence of the condition based on the activation level.
  • the cell is a hematopoietic derived cell.
  • the hematopoietically derived cell is selected from the group consisting of pluripotent hematopoietic stem cells, B-lymphocyte lineage progenitor or derived cells, T-lymphocyte lineage progenitor or derived cells, NK cell lineage progenitor or derived cells, granulocyte lineage progenitor or derived cells, monocyte lineage progenitor or derived cells, megakaryocyte lineage progenitor or derived cells and erythroid lineage progenitor or derived cells.
  • the hematopoietic derived cell is a B-lymphocyte lineage progenitor and derived cell, e.g., an early pro-B cell, late pro-B cell, large pre-B cell, small pre-B cell, immature B cell, mature B cell, plasma cell and memory B cell, a CD5+ B cell, a CD38+ B cell, a B cell bearing a mutatated or non mutated heavy chain of the B cell receptor, or a B cell expressing Zap70.
  • the condition is a neoplastic or hematopoietic condition.
  • the modulator to which the cell is subjected is an activator or an inhibitor.
  • the modulator is, e.g., a growth factor, cytokine, adhesion molecule modulator, hormone, small molecule, polynucleotide, antibodies, natural compounds, lactones, chemotherapeutic agents, immune modulator, carbohydrate, proteases, ions, reactive oxygen species, or radiation.
  • the modulator is a B cell receptor modulator, e.g., a B cell receptor activator such as a cross-linker of the B cell receptor complex or the B-cell co-receptor complex.
  • the cross-linker is an antibody, or molecular binding entity.
  • the cross-linker is an antibody, such as a multivalent antibody.
  • the antibody is a monovalent, bivalent, or multivalent antibody made more multivalent by attachment to a solid surface or tethered on a nanoparticle surface to increase the local valency of the epitope binding domain.
  • the cross-linker is a molecular binding entity, such as an entity that acts upon or binds the B cell receptor complex via carbohydrates or an epitope in the complex.
  • the molecular binding entity is a monovalent, bivalent, or multivalent binding entity that is made more multivalent by attachment to a solid surface or tethered on a nanoparticle surface to increase the local valency of the epitope binding domain.
  • the modulator is a B cell receptor modulator, e.g., a B cell receptor activator such as a cross-linker of the B cell receptor complex or the B-cell co-receptor complex
  • cross-linking includes binding of an antibody or molecular binding entity to the cell and then causing its crosslinking via interaction of the cell with a solid surface that causes crosslinking of the BCR complex via antibody or molecular binding entity.
  • the crosslinker is selected from the group consisting of F(ab)2 IgM, IgG, IgD, polyclonal BCR antibodies, monoclonal BCR antibodies, Fc receptor derived binding elements.
  • the Ig may be derived from a species selected from the group consisting of mouse, goat, rabbit, pig, rat, horse, cow, shark, chicken, or llama.
  • the crosslinker is F(ab)2 IgM, Polyclonal IgM antibodies, Monoclonal IgM antibodies, Biotinylated F(ab)2 IgCM, Biotinylated Polyclonal IgM antibodies, Biotinylated Monoclonal IgM antibodies and/or a combination thereof.
  • the inhibitor is a kinase or phosphatase inhibitor, such as adaphostin, AG 490, AG 825, AG 957, AG 1024, aloisine, aloisine A, alsterpaullone, aminogenistein, API-2, apigenin, arctigenin, AY-22989, BAY 61-3606, bisindolylmaleimide IX, chelerythrine, 10-[4′-(N,N-Diethylamino)butyl]-2-chlorophenoxazine hydrochloride, dasatinib, 2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole, 5,7-Dimethoxy-3-(4-pyridinyl)quinoline dihydrochloride, edelfosine, ellagic acid, enzastaurin, ER 27319 maleate, erlotinib, ET18OCH3, f
  • the cell is subjected to a B cell receptor activator and a phosphatase inhibitor or kinase inhibitor, such as F(ab) 2 IgM or biotinylated F(ab) 2 IgM and a phosphatase inhibitor (e.g. H 2 O 2 ).
  • a phosphatase inhibitor or kinase inhibitor such as F(ab) 2 IgM or biotinylated F(ab) 2 IgM and a phosphatase inhibitor (e.g. H 2 O 2 ).
  • the invention provides a method of determining a tonic signaling status of a cell by subjecting the cell to a modulator, determining the activation level of an activatable element that participates in a tonic signaling pathway in the cell, and determining the status of a tonic signaling pathway in the cell from the activation level.
  • a condition of an individual is determined based on tonic signaling status of a cell.
  • the condition is a neoplastic and/or hematopoietic condition.
  • the neoplastic or hematopoietic condition is selected from the group consisting of Non-Hodgkin Lymphoma, Hodgkin or other lymphomas, acute or chronic leukemias, polycythemias, thrombocythemias, multiple myeloma and plasma cell disorders, e.g., amyloidosis and Waldenstrom's macroglobulinemia, myelodysplastic disorders, myeloproliferative disorders, myelofibrosis, and atypical immune lymphoproliferations.
  • the neoplastic or hematopoietic condition is non-B lineage derived, such as acute myeloid leukemia (AML), Chronic Myeloid Leukemia (CML), non-B cell acute lymphocytic leukemia (ALL), non-B cell lymphomas, myelodysplastic disorders, myeloproliferative disorders, myelofibrosis, polycythemias, thrombocythemias, and non-B atypical immune lymphoproliferations.
  • AML acute myeloid leukemia
  • CML Chronic Myeloid Leukemia
  • ALL non-B cell acute lymphocytic leukemia
  • non-B cell lymphomas myelodysplastic disorders
  • myeloproliferative disorders myelofibrosis
  • polycythemias thrombocythemias
  • non-B atypical immune lymphoproliferations non-B lineage derived
  • the neoplastic or hematopoietic condition is a B-Cell or B cell lineage derived disorder such as B-Cell or B cell lineage derived disorder is selected from the group consisting of Chronic Lymphocytic Leukemia (CLL), B lymphocyte lineage leukemia, B lymphocyte lineage lymphoma, Multiple Myeloma, and plasma cell disorders, e.g., amyloidosis and Waldenstrom's macroglobulinemia.
  • CLL Chronic Lymphocytic Leukemia
  • B lymphocyte lineage leukemia B lymphocyte lineage lymphoma
  • Multiple Myeloma Multiple Myeloma
  • plasma cell disorders e.g., amyloidosis and Waldenstrom's macroglobulinemia.
  • the condition is CLL.
  • the CLL is defined by a monoclonal B cell population that co-expresses CD5 with CD19 and CD23 or CD5 with CD20 and CD23 and by surface immunoglobulin
  • the tonic signaling status of a cell is correlated with a clinical outcome such as prognosis or diagnosis of the condition.
  • the clinical outcome is the staging or grading of a neoplastic or hematopoietic condition, such as aggressive, indolent, benign, refractory, Roman Numeral staging, TNM Staging, Rai staging, Binet staging, WHO classification, FAB classification, IPSS score, WPSS score, limited stage, extensive stage, staging according to molecular markers such as ZAP70, the mutational status of the heavy chain of the B-cell receptor (IgVH) and CD38, occult, including information that may inform on time to progression, progression free survival, overall survival, or event-free survival.
  • a neoplastic or hematopoietic condition such as aggressive, indolent, benign, refractory, Roman Numeral staging, TNM Staging, Rai staging, Binet staging, WHO classification, FAB classification, IPSS score, WPSS score, limited stage, extensive stage
  • the correlation is determining the individual's response to a treatment, e.g., normal responder, hyper responder, poor responder, having emerging resistance, non-compliant, and adverse reaction.
  • a treatment e.g., normal responder, hyper responder, poor responder, having emerging resistance, non-compliant, and adverse reaction.
  • the correlation includes classifying the cell as minimal residual disease or emerging resistance.
  • the correlation may further include determining a method of treatment, such as chemotherapy, biological therapy, radiation therapy, bone marrow transplantation, Peripheral stem cell transplantation, umbilical cord blood transplantation, autologous stem cell transplantation, allogeneic stem cell transplantation, syngeneic stem cell transplantation, surgery, induction therapy, maintenance therapy, or watchful waiting.
  • the invention provides a method of correlating an activation level of a B-lymphocyte lineage derived cell with a neoplastic or hematopoietic condition in an individual by subjecting the B-lymphocyte lineage derived cell from the individual to a modulator; determining the activation levels of a plurality of activatable elements that participate in a tonic signaling pathway in the B-lymphocyte lineage derived cell; and identifying a pattern of the activation levels of the plurality of activatable elements in the tonic signaling pathway in the cell that correlates with a clinical outcome, such as the prediction of outcome for a particular treatment, a prognosis or diagnosis of a certain condition (e.g.
  • the B-lymphocyte lineage progenitor or derived cell is selected from the group consisting of early pro-B cell, late pro-B cell, large pre-B cell, small pre-B cell, immature B cell, mature B cell, plasma cell and memory B cell, a CD5+ B cell, a CD38+ B cell, a B cell bearing a mutatated or non mutated heavy chain of the B cell receptor and a B cell expressing Zap70.
  • the correlation is determining a clinical outcome, such as prognosis or diagnosis of the condition.
  • the modulator to which the cell is subjected is an activator or an inhibitor.
  • the modulator is, e.g., a growth factor, cytokine, adhesion molecule modulator, hormone, small molecule, polynucleotide, antibodies, natural compounds, lactones, chemotherapeutic agents, immune modulator, carbohydrate, proteases, ions, reactive oxygen species, or radiation.
  • the modulator is a B cell receptor modulator, e.g., a B cell receptor activator such as a cross-linker of the B cell receptor complex or the B-cell co-receptor complex.
  • the cross-linker is an antibody, or molecular binding entity.
  • the cross-linker is an antibody, such as a multivalent antibody.
  • the antibody is a monovalent, bivalent, or multivalent antibody made more multivalent by attachment to a solid surface or tethered on a nanoparticle surface to increase the local valency of the epitope binding domain.
  • the cross-linker is a molecular binding entity, such as an entity that acts upon or binds the B cell receptor complex via carbohydrates or an epitope in the complex.
  • the molecular binding entity is a monovalent, bivalent, or multivalent binding entity that is made more multivalent by attachment to a solid surface or tethered on a nanoparticle surface to increase the local valency of the epitope binding domain.
  • the modulator is a B cell receptor modulator, e.g., a B cell receptor activator such as a cross-linker of the B cell receptor complex or the B-cell co-receptor complex
  • cross-linking includes binding of an antibody or molecular binding entity to the cell and then causing its crosslinking via interaction of the cell with a solid surface that causes crosslinking of the BCR complex via antibody or molecular binding entity.
  • the crosslinker is selected from the group consisting of F(ab)2 IgM, IgG, IgD, polyclonal BCR antibodies, monoclonal BCR antibodies, Fc receptor derived binding elements and/or a combination thereof.
  • the Ig may be derived from a species selected from the group consisting of mouse, goat, rabbit, pig, rat, horse, cow, shark, chicken, or llama.
  • the crosslinker is F(ab)2 IgM, Polyclonal IgM antibodies, Monoclonal IgM antibodies, Biotinylated F(ab)2 IgCM, Biotinylated Polyclonal IgM antibodies, Biotinylated Monoclonal IgM antibodies and/or a combination thereof.
  • the modulator to which the cell is subjected is an inhibitor of a cellular factor or a plurality of factors that participates in a signaling cascade in the cell.
  • the inhibitor is a kinase or phosphatase inhibitor, such as adaphostin, AG 490, AG 825, AG 957, AG 1024, aloisine, aloisine A, alsterpaullone, aminogenistein, API-2, apigenin, arctigenin, AY-22989, BAY 61-3606, bisindolylmaleimide IX, chelerythrine, 10-[4′-(N,N-Diethylamino)butyl]-2-chlorophenoxazine hydrochloride, dasatinib, 2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole, 5,7-Dimethoxy-3-(4-pyridiny
  • the cell is further subjected to a second modulator, e.g., the cell may be subjected to a B cell receptor activator and a phosphatase inhibitor, such as F(ab) 2 IgM or biotinylated F(ab) 2 IgM and a phosphatase inhibitor (e.g. H 2 0 2 ).
  • a phosphatase inhibitor such as F(ab) 2 IgM or biotinylated F(ab) 2 IgM and a phosphatase inhibitor (e.g. H 2 0 2 ).
  • the activation level of the plurality of activatable elements in the cell is selected from the group consisting of cleavage by extracellular or intracellular protease exposure, novel hetero-oligomer formation, glycosylation level, phosphorylation level, acetylation level, methylation level, biotinylation level, glutamylation level, glycylation level, hydroxylation level, isomerization level, prenylation level, myristoylation level, lipoylation level, phosphopantetheinylation level, sulfation level, ISGylation level, nitrosylation level, palmitoylation level, SUMOylation level, ubiquitination level, neddylation level, citrullination level, deamidation level, disulfide bond formation level, proteolytic cleavage level, translocation level, changes in protein turnover, multi-protein complex level, oxidation level, multi-lipid complex, and biochemical changes in cell membrane.
  • the activation level is a phosphorylation level.
  • the activatable element is selected from the group consisting of proteins, carbohydrates, lipids, nucleic acids and metabolites.
  • the activatable element is a protein.
  • the activatable element is a change in metabolic state, temperature, or local ion concentration.
  • the protein is a protein subject to phosphorylation or dephosphorylation, such as kinases, phosphatases, adaptor/scaffold proteins, ubiquitination enzymes, adhesion molecules, contractile proteins, cytoskeletal proteins, heterotrimeric G proteins, small molecular weight GTPases, guanine nucleotide exchange factors, GTPase activating proteins, caspases and proteins involved in apoptosis (e.g.
  • PARP ion channels
  • molecular transporters molecular chaperones
  • metabolic enzymes vesicular transport proteins
  • hydroxylases isomerases
  • transferases deacetylases
  • methylases demethylases
  • proteases esterases
  • hydrolases DNA binding proteins or transcription factors.
  • the protein is selected from the group consisting of PI3-Kinase (p85, p110a, p110b, p110d), Jak1, Jak2, SOCs, Rac, Rho, Cdc42, Ras-GAP, Vav, Tiam, Sos, Dbl, Nck, Gab, PRK, SHP1, and SHP2, SHIP1, SHIP2, sSHIP, PTEN, Shc, Grb2, PDK1, SGK, Akt1, Akt2, Akt3, TSC1,2, Rheb, mTor, 4EBP-1, p70S6Kinase, S6, LKB-1, AMPK, PFK, Acetyl-CoAa Carboxylase, DokS, Rafs, Mos, Tp12, MEK1/2, MLK3, TAK, DLK, MKK3/6, MEKK1,4, MLK3, ASK1, MKK4/7, SAPK/JNK1,2,3, p38s, Erk1/2, Syk,
  • the protein selected from the group consisting of Erk, Syk, Zap70, Lck, Btk, BLNK, Cbl, PLC ⁇ 2, Akt, RelA, p38, S6. In some embodiments the protein is S6.
  • the protein is selected from the group consisting of HER receptors, PDGF receptors, Kit receptor, FGF receptors, Eph receptors, Trk receptors, IGF receptors, Insulin receptor, Met receptor, Ret, VEGF receptors, TIE1, TIE2, FAK, Jak1, Jak2, Jak3, Tyk2, Src, Lyn, Fyn, Lck, Fgr, Yes, Csk, Abl, Btk, ZAP70, Syk, IRAKs, cRaf, ARaf, BRAF, Mos, Lim kinase, ILK, Tpl, ALK, TGF ⁇ receptors, BMP receptors, MEKKs, ASK, MLKs, DLK, PAKs, Mek 1, Mek 2, MKK3/6, MKK4/7, ASK1, Cot, NIK, Bub, Myt 1, Wee1, Casein kinases, PDK1, SGK1, SGK2, SGK3, Akt1, Akt2, Akt
  • the methods for classifying a cell further comprise determining the level of an additional intracellular marker and/or a cell surface marker. In some embodiments the methods for classifying a cell comprise determining the level of an additional intracellular marker. In some embodiments the intracelluar marker is a captured intracellular cytokine. In some embodiments the methods for classifying a cell comprise determining the level of an additional cell surface marker. In some embodiments the cell surface marker is a cell surface ligand or receptor. In some embodiments the cell surface marker is a component of a B-cell receptor.
  • the cell surface marker is CD45, CD5, CD19, CD20, CD22, CD23, CD27, CD37, CD40, CD52, CD79, CD38, CD96, major histocompatability antigen (MHC) Class1 or MHC Class 2.
  • MHC major histocompatability antigen
  • the methods of the invention for prognosis, diagnosis, or determination of treatment further comprise determining the level of an additionalserum marker.
  • the serum marker comprises a protein.
  • the serum marker is a cytokine, growth factor, chemokine, soluble receptor, small compound, or pharmaceutical drug.
  • the serum marker comprises a component or product of a pathogen or parasite.
  • the serum marker is selected from a group consisting of beta-2-microglobulin, calcitonin, thymidine kinase and ferritin.
  • the invention provides a method of correlating an activation level of B-lymphocyte lineage derived cells with a neoplastic or hematopoietic condition in an individual by subjecting the B-lymphocyte lineage derived cell from the individual to a modulator; determining the activation levels of a plurality of activatable elements in the B-lymphocyte lineage derived cell; and identifying a pattern of the activation levels of the plurality of activatable elements in the cell that correlates with the neoplastic condition.
  • the activatable element is selected from the group consisting of elements selected from the group consisting of Erk, Syk, Zap70, Lck, Btk, BLNK, Cbl, PLC ⁇ 2, Akt, RelA, p38, S6. In some embodiments, the activatable element is selected from the group consisting of Cbl, PLC ⁇ 2, and S6. In some embodiments, the activatable element is S6.
  • the B-lymphocyte lineage progenitor or derived cell is selected from the group consisting of early pro-B cell, late pro-B cell, large pre-B cell, small pre-B cell, immature B cell, mature B cell, plasma cell and memory B cell, a CD5+ B cell, a CD38+ B cell, a B cell bearing a mutatated or non mutated heavy chain of the B cell receptor, or a B cell expressing Zap70.
  • the invention provides methods for correlating and/or classifying an activation state of a CLL cell with a clinical outcome in an individual by subjecting the CLL cell from the individual to a modulator, where the CLL cell expresses a B-Cell receptor (BCR), determining the activation levels of a plurality of activatable elements, and identifying a pattern of the activation levels of the plurality of activatable elements to determine the presence or absence of an alteration in signaling proximal to the BCR, wherein the presence of the alteration is indicative of a clinical outcome.
  • BCR B-Cell receptor
  • the method comprises identifying a pattern of said activation levels of said plurality of activatable elements in said cell, wherein said pattern is correlated to a disease or condition.
  • the correlation is determining a clinical outcome, such as prognosis or determination of treatment of the condition.
  • the neoplastic or hematopoietic condition is selected from the group consisting of Non-Hodgkin Lymphoma, Hodgkin or other lymphomas, acute or chronic leukemias, polycythemias, thrombocythemias, multiple myeloma and plasma cell disorders, such as amyloidosis and Waldenstrom's macroglobulinemia, myelodysplastic disorders, myeloproliferative disorders, myelofibrosis, or atypical immune lymphoproliferations.
  • the neoplastic or hematopoietic condition is non-B lineage derived, such as acute myeloid leukemia (AML), Chronic Myeloid Leukemia (CML), non-B cell acute lymphocytic leukemia (ALL), non-B cell lymphomas, myelodysplastic disorders, myeloproliferative disorders, myelofibrosis, polycythemias, thrombocythemias, and non-B atypical immune lymphoproliferations.
  • AML acute myeloid leukemia
  • CML Chronic Myeloid Leukemia
  • ALL non-B cell acute lymphocytic leukemia
  • non-B cell lymphomas myelodysplastic disorders
  • myeloproliferative disorders myelofibrosis
  • polycythemias thrombocythemias
  • non-B atypical immune lymphoproliferations non-B lineage derived
  • the neoplastic or hematopoietic condition is a B-Cell or B cell lineage derived disorder, such as B-Cell or B cell lineage derived disorder is selected from the group consisting of Chronic Lymphocytic Leukemia (CLL), B lymphocyte lineage leukemia, B lymphocyte lineage lymphoma, Multiple Myeloma, or plasma cell disorders, e.g., amyloidosis and Waldenstrom's macroglobulinemias.
  • the condition is CLL.
  • the CLL is defined by a monoclonal B cell population that co-expresses CD5 with CD19 and CD23 or CD5 with CD20 and CD23 and dim surface immunoglobulin expression.
  • the clinical outcome is the staging or grading of a neoplastic or hematopoietic condition, such as aggressive, indolent, benign, refractory, Roman Numeral staging, TNM Staging, Rai staging, Binet staging, WHO classification, FAB classification, IPSS score, WPSS score, limited stage, extensive stage, staging according to cellular markers such as ZAP70, IgV H mutational status and CD38, occult, including information that may inform on time to progression, progression free survival, overall survival, or event-free survival.
  • a neoplastic or hematopoietic condition such as aggressive, indolent, benign, refractory, Roman Numeral staging, TNM Staging, Rai staging, Binet staging, WHO classification, FAB classification, IPSS score, WPSS score, limited stage, extensive stage, staging according to cellular markers such as ZAP70, IgV H mutational status and CD38, occult, including information that may inform on time to progression, progression free survival, overall survival,
  • the correlation is determining the individual's response to a specific treatment, e.g., normal responder, hyper responder, poor responder, having emerging resistance, non-compliant, and adverse reaction.
  • a specific treatment e.g., normal responder, hyper responder, poor responder, having emerging resistance, non-compliant, and adverse reaction.
  • the correlation includes classifying the cell as minimal residual disease or emerging resistance.
  • the correlation may further include determining a method of treatment, such as chemotherapy, biological therapy, targeted therapy, radiation therapy, bone marrow transplantation, Peripheral stem cell transplantation, umbilical cord blood transplantation, autologous stem cell transplantation, allogeneic stem cell transplantation, syngeneic stem cell transplantation, surgery, induction therapy, maintenance therapy, or watchful waiting.
  • the correlation may further include determination of the appropriate dosage or timing of a given treatment.
  • the modulator to which the cell is subjected is an activator or an inhibitor.
  • the modulator is, e.g., a growth factor, cytokine, adhesion molecule modulator, hormone, small molecule, polynucleotide, antibodies, natural compounds, lactones, chemotherapeutic agents, immune modulator, carbohydrate, proteases, ions, reactive oxygen species, or radiation.
  • the modulator is an antibody e.g. anti-CD20 (Rituxan), anti-CD22 (epratuzumab), anti-CD23 (lumiliximab) or anti-CD52 (Alemtuzumab), that recognize antigens on the cell surface.
  • the modulator is a B cell receptor complex modulator, e.g., anti-CD20, which recognizes a component of the B cell receptor co-complex, or a B cell receptor activator such as a cross-linker of the B cell receptor complex or the B-cell co-receptor complex.
  • the cross-linker is an antibody, or molecular binding entity.
  • the cross-linker is an antibody, such as a multivalent antibody.
  • the antibody is a monovalent, bivalent, or multivalent antibody made more multivalent by attachment to a solid surface or tethered on a nanoparticle surface to increase the local valency of the epitope binding domain.
  • the cross-linker is a molecular binding entity, such as an entity that acts upon or binds the B cell receptor complex via carbohydrates or an epitope in the complex.
  • the molecular binding entity is a monovalent, bivalent, or multivalent binding entity that is made more multivalent by attachment to a solid surface or tethered on a nanoparticle surface to increase the local valency of the epitope binding domain.
  • cross-linking includes binding of an antibody or molecular binding entity to the cell and then causing its crosslinking via interaction of the cell with a solid surface that causes crosslinking of the BCR complex via antibody or molecular binding entity.
  • the crosslinker is selected from the group consisting of F(ab)2 IgM, IgG, IgD, polyclonal BCR antibodies, monoclonal BCR antibodies, Fc receptor derived binding elements and/or a combination thereof.
  • the Ig may be derived from a species selected from the group consisting of mouse, goat, rabbit, pig, rat, horse, cow, shark, chicken, or llama.
  • the crosslinker is F(ab)2 IgM, Polyclonal IgM antibodies, Monoclonal IgM antibodies, Biotinylated F(ab)2 IgCM, Biotinylated Polyclonal IgM antibodies, Biotinylated Monoclonal IgM antibodies and/or a combination thereof.
  • the modulator to which the cell is subjected is an inhibitor of a cellular factor or a plurality of factors that participates in a signaling cascade in the cell.
  • the inhibitor is a kinase or phosphatase inhibitor, such as adaphostin, AG 490, AG 825, AG 957, AG 1024, aloisine, aloisine A, alsterpaullone, aminogenistein, API-2, apigenin, arctigenin, AY-22989, BAY 61-3606, bisindolylmaleimide IX, chelerythrine, 10-[4′-(N,N-Diethylamino)butyl]-2-chlorophenoxazine hydrochloride, dasatinib, 2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole, 5,7-Dimethoxy-3-(4-pyridiny
  • the cell is further subjected to a second modulator, e.g., the cell may be subjected to a B cell receptor activator and a kinase inhibitor or a phosphatase inhibitor, such as F(ab) 2 IgM or biotinylated F(ab) 2 IgM and H 2 0 2 .
  • a second modulator e.g., the cell may be subjected to a B cell receptor activator and a kinase inhibitor or a phosphatase inhibitor, such as F(ab) 2 IgM or biotinylated F(ab) 2 IgM and H 2 0 2 .
  • the activation level of the activatable element in the cell is selected from the group consisting of cleavage by extracellular or intracellular protease exposure, novel hetero-oligomer formation, glycosylation level, phosphorylation level, acetylation level, methylation level, biotinylation level, glutamylation level, glycylation level, hydroxylation level, isomerization level, prenylation level, myristoylation level, lipoylation level, phosphopantetheinylation level, sulfation level, ISGylation level, nitrosylation level, palmitoylation level, SUMOylation level, ubiquitination level, neddylation level, citrullination level, deamidation level, disulfide bond formation level, proteolytic cleavage level, translocation level, changes in protein turnover, multi-protein complex level, oxidation level, multi-lipid complex, and biochemical changes in cell membrane.
  • the activation level is a phosphorylation level.
  • the activatable element is selected from the group consisting of proteins, carbohydrates, lipids, nucleic acids and metabolites.
  • the activatable element is a protein.
  • the activatable element is a change in metabolic state, temperature, or local ion concentration.
  • the protein is a protein subject to phosphorylation or dephosphorylation, such as kinases, phosphatases, adaptor/scaffold proteins, ubiquitination enzymes, adhesion molecules, contractile proteins, cytoskeletal proteins, heterotrimeric G proteins, small molecular weight GTPases, guanine nucleotide exchange factors, GTPase activating proteins, caspases and proteins involved in apoptosis (e.g.
  • PARP ion channels
  • molecular transporters molecular chaperones
  • metabolic enzymes vesicular transport proteins
  • hydroxylases isomerases
  • transferases deacetylases
  • methylases demethylases
  • proteases esterases
  • hydrolases DNA binding proteins or transcription factors.
  • the invention provides for methods for determining a phenotypic profile of a population of cells by exposing the population of cells to a plurality of modulators in separate cultures, where at least one of the modulators is an inhibitor, determining the presence or absence of an increase in activation level of an activatable element in the cell population from each of the separate culture and classifying the cell population based on the presence or absence of the increase in the activation of the activatable element from each of the separate culture.
  • the inhibitor is an inhibitor of a cellular factor or a plurality of factors that participates in a signaling cascade in the cell.
  • the inhibitor is a phosphatase or kinase inhibitor.
  • kinase inhibitors include adaphostin, AG 490, AG 825, AG 957, AG 1024, aloisine, aloisine A, alsterpaullone, aminogenistein, API-2, apigenin, arctigenin, AY-22989, BAY 61-3606, bisindolylmaleimide IX, chelerythrine, 10-[4′-(N,N-Diethylamino)butyl]-2-chlorophenoxazine hydrochloride, dasatinib, 2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole, 5,7-Dimethoxy-3-(4-pyridinyl)quinoline dihydrochloride, edelfosine, ellagic acid, enzastaurin, ER 27319 maleate, erlotinib, ET18OCH3, fasudil, flavopiridol, gefitin
  • the modulator is an activator or an inhibitor.
  • the modulators are independently selected from the group consisting of growth factor, cytokine, adhesion molecule modulator, hormone, small molecule, polynucleotide, antibodies, natural compounds, lactones, chemotherapeutic agents, immune modulator, carbohydrate, proteases, ions, reactive oxygen species, and radiation.
  • at least one modulator is a B cell receptor modulator.
  • the B cell receptor modulator is a B cell receptor activator, such as Rituxan or a cross-linker of the B cell receptor complex or the B-cell co-receptor complex.
  • the modulator is PMA, BAFF, April, SDF1a, SCF, CD40L, IGF-1, Imiquimod, polyCpG, fludarabine, cyclophosphamide, chlorambucil IL-7, IL-6, IL-10, IL-27, IL-4, IL-2, IL-3, thapsigargin and/or a combination thereof.
  • the activatable element is a protein.
  • the protein is selected from the group consisting of Akt1, Akt2, Akt3, SAPK/JNK1,2,3, p38s, Erk1/2, Syk, ZAP70, Btk, BLNK, Lck, PLC ⁇ , PLC 1 ⁇ 2, STAT1, STAT 3, STAT 4, STAT 5, STAT 6, CREB, Lyn, p-S6, Cbl, NF- ⁇ B, GSK3 ⁇ , CARMA/Bcl10 and Tcl-1.
  • the invention provides methods of classifying a cell population by contacting the cell population with at least one modulator, where the modulator is F(ab)2 IgM, Rituxan, Alemtuzumab, anti CD22 (epratuzumab), anti-CD23 (lumiliximab), Campath, H 2 O 2 , PMA, BAFF, April, SDF1a, CD40L, IGF-1, Imiquimod, polyCpG, fludarabine, cyclophosphamide, chlorambucil, IL-7, IL-6, IL-10, IL-27, IL-4, IL-2, IL-3, thapsigargin and/or a combination thereof, determining the presence or absence of an increase in activation level of an activatable element in the cell population, and classifying the cell population based on the presence or absence of the increase in the activation of the activatable element.
  • the modulator is F(ab)2 IgM, Rituxan, Alemtuzum
  • the cell population is a hematopoietic-derived cell population.
  • the hematopoietically derived cell population is selected from the group consisting of pluripotent hematopoietic stem cells, B-lymphocyte lineage progenitor or derived cells, T-lymphocyte lineage progenitor or derived cells, NK cell lineage progenitor or derived cells, granulocyte lineage progenitor or derived cells, monocyte lineage progenitor or derived cells, megakaryocyte lineage progenitor or derived cells and erythroid lineage progenitor or derived cells.
  • the hematopoietic derived cell population is a B-lymphocyte lineage progenitor and derived cell population, e.g., an early pro-B cell population, late pro-B cell population, large pre-B cell population, small pre-B cell population, immature B cell population, mature B cell population, plasma cell and memory B cell population, a CD5+ B cell population, a CD38+ B cell, a B cell bearing a mutatated or non mutated heavy chain of the B cell receptor, or a B cell population expressing Zap70.
  • a B-lymphocyte lineage progenitor and derived cell population e.g., an early pro-B cell population, late pro-B cell population, large pre-B cell population, small pre-B cell population, immature B cell population, mature B cell population, plasma cell and memory B cell population, a CD5+ B cell population, a CD38+ B cell, a B cell bearing a mutatated or non mutated heavy chain of the B cell
  • the classification includes classifying the cell population as a cell population that is correlated with a clinical outcome.
  • the clinical outcome is the predicted respose to a specific therapy, or the prognosis and/or diagnosis of a condition.
  • the clinical outcome is the presence or absence of a neoplastic or a hematopoietic condition, such as Non-Hodgkin Lymphoma, Hodgkin or other lymphomas, acute or chronic leukemias, polycythemias, thrombocythemias, multiple myeloma or plasma cell disorders, e.g., amyloidosis and Waldenstrom's macroglobulinemia, myelodysplastic disorders, myeloproliferative disorders, myelofibrosis, or atypical immune lymphoproliferations.
  • a neoplastic or a hematopoietic condition such as Non-Hodgkin Lymphoma, Hodgkin or other lymphomas, acute or chronic leukemias, polycythemias, thrombocythemias, multiple myeloma or plasma cell disorders, e.g., amyloidosis and Waldenstrom's macroglobulinemia, myel
  • the neoplastic or hematopoietic condition is non-B lineage derived, such as acute myeloid leukemia (AML), Chronic Myeloid Leukemia (CML), non-B cell acute lymphocytic leukemia (ALL), non-B cell lymphomas, myelodysplastic disorders, myeloproliferative disorders, myelofibrosis, polycythemias, thrombocythemias, or non-B atypical immune lymphoproliferations.
  • AML acute myeloid leukemia
  • CML Chronic Myeloid Leukemia
  • ALL non-B cell acute lymphocytic leukemia
  • non-B cell lymphomas myelodysplastic disorders
  • myeloproliferative disorders myelofibrosis
  • polycythemias thrombocythemias
  • non-B atypical immune lymphoproliferations non-B lineage derived
  • the neoplastic or hematopoietic condition is a B-Cell or B cell lineage derived disorder, such as Chronic Lymphocytic Leukemia (CLL), B lymphocyte lineage leukemia, B lymphocyte lineage lymphoma, Multiple Myeloma, or plasma cell disorders, e.g., amyloidosis or Waldenstrom's macroglobulinemia.
  • CLL Chronic Lymphocytic Leukemia
  • B lymphocyte lineage leukemia B lymphocyte lineage lymphoma
  • Multiple Myeloma or plasma cell disorders, e.g., amyloidosis or Waldenstrom's macroglobulinemia.
  • the condition is CLL.
  • the CLL is defined by a monoclonal B cell population that co-expresses CD5 with CD19 and CD23 or CD5 with CD20 and CD23 and dim surface immunoglobulin expression.
  • the clinical outcome is the staging or grading of a neoplastic or hematopoietic condition.
  • staging in methods provided by the invention include aggressive, indolent, benign, refractory, Roman Numeral staging, TNM Staging, Rai staging, Binet staging, WHO classification, FAB classification, IPSS score, WPSS score, limited stage, extensive stage, staging according to cellular markers such as ZAP70, IgV H mutation status and CD38, occult, including information that may inform on time to progression, progression free survival, overall survival, or event-free survival.
  • the classifying of the cell population based on activation level includes classifying the cell population as a cell population that is correlated to a patient response to a treatment, such as complete response, partial response, nodular partial response, no response, progressive disease, stable disease, relapse or adverse reaction.
  • the method may further comprise determining a method of treatment, e.g., chemotherapy, biological therapy, targeted therapy, radiation therapy, bone marrow transplantation, Peripheral stem cell transplantation, umbilical cord blood transplantation, autologous stem cell transplantation, allogeneic stem cell transplantation, syngeneic stem cell transplantation, surgery, induction therapy, maintenance therapy, watchful waiting, or holistic/alternative therapy.
  • the classifying of the cell population based on activation level includes classifying the cell population as a cell population that is correlated with minimal residual disease or emerging resistance.
  • FIG. 1 depicts histograms showing the activation of p-Erk and p-Syk/pZap70 in Ramos cells following F(ab) 2 IgM stimulation.
  • FIG. 2 depicts histograms showing the phosphorylation of BLNK, Cbl, PLC ⁇ 2, Lck, and p38 in Ramos Cell lines following treatment with F(ab) 2 IgM.
  • FIG. 3 depicts histograms showing increased phosphorylation of Erk in Pheresed CLL Samples and in the Ramos cell lines following PMA activation.
  • FIG. 4 depicts a histogram showing the activation of Erk in Pheresed CLL Samples and Ramos cells following activation with increasing amounts of F(ab) 2 IgM for 15 Min.
  • FIG. 5 depicts contour plots showing increased phosphorylation of Erk and Syk/Zap 70 in CLL Samples following treatment with PMA or F(ab) 2 IgM.
  • FIG. 6 depicts contour plots showing phosphorylation of Erk and Syk/Zap70 in CLL samples following treatment with H 2 O 2 alone or in combination with F(ab) 2 IgM.
  • FIG. 7 depicts histograms showing the phosphorylation of Erk and Syk/Zap70 in healthy B cells following treatment with H 2 O 2 .
  • FIG. 8 depicts contour plots showing the phosphorylation of Erk and Syk/Zap70 in CLL cells upon treatment with H 2 O 2 and F(ab) 2 IgM.
  • FIG. 9 depicts contour plots showing Erk and Syk in CLL samples following activation with PMA or F(ab) 2 IgM.
  • FIG. 10 depicts contour plots showing the phosphorylation of Erk and Syk/Zap70 following treatment with H 2 O 2 alone or in combination with F(ab) 2 IgM.
  • FIG. 11 depicts contour plots comparing CLL samples with low and high Frequency ZAP70 the phosphorylation or Erk and Syk/Zap70 following treatment with H 2 O 2 alone or in combination with F(ab) 2 IgM.
  • FIG. 12 depicts contour plots showing Syk/Zap70 and Erk phosphorylation after treatment F(ab) 2 IgM and H 2 O 2 at different times in CLL samples.
  • FIG. 13 depicts contour plots showing Syk/Zap70 and Erk phosphorylation after treatment with F(ab) 2 IgM and H 2 O 2 in CLL samples.
  • FIG. 14 depicts histograms showing the kinetics of signaling mediated by F(ab) 2 IgM and H 2 O 2 in CLL.
  • FIG. 15 depicts contour plots showing the influence of % ZAP70 on BLNK phosphorylation following treatment with H 2 O 2 alone or in combination with F(ab) 2 IgM.
  • FIG. 16 depicts histograms showing the influence of ZAP70 status on BLNK phosphorylation.
  • FIG. 17 depicts histograms showing the kinetics of phosphorylation of pPLC ⁇ 2, prpS6, and pCb1 after B-Cell Receptor crosslinking by F(ab) 2 IgM Alone in CD20+/CD5+ population of CLL samples.
  • FIG. 18 depicts histograms showing the kinetics of phosphorylation of pPLC ⁇ 2, prpS6, and pCb1 after B-Cell Receptor crosslinking by F(ab) 2 IgM and H 2 O 2 in CD20+/CD5+ population in CLL samples.
  • FIG. 19 depicts histograms showing the kinetics of phosphorylation of pPLC ⁇ 2, prpS6, and pCb1 after B-Cell Receptor crosslinking by F(ab) 2 IgM Alone in CD20+/CD5+ population in CLL samples.
  • FIG. 20 depicts histograms showing the kinetics of phosphorylation of pPLC ⁇ 2, prpS6, and pCb1 after B-Cell Receptor crosslinking by F(ab) 2 IgM and H 2 O 2 in CD20+/CD5+ population in CLL samples.
  • FIG. 21 depicts contour plots showing the phosphorylation of Syk/Zap 70 and Erk by F(ab) 2 IgM alone over time.
  • FIG. 22 depicts contour plots showing the phosphorylation of Syk/Zap 70 and Erk in response to F(ab) 2 IgM alone over time.
  • FIG. 23 depicts histograms showing phosphorylation of Syk, Erk and BLNK in response to F(ab) 2 IgM over time in the CD20+/CD5+ population of CLL samples.
  • FIG. 24 depicts histograms showing the kinetics of phosphorylation of pPLC ⁇ 2, prpS6, and pCb1 after H 2 O 2 treatment in CD20+/CD5+ population of CLL samples.
  • FIG. 25 depicts histograms showing the kinetics of phosphorylation of pPLC ⁇ 2, prpS6, and pCb1 after H 2 O 2 treatment in CD20+/CD5+ population of CLL Samples.
  • FIG. 26 depicts a heat-map showing intracellular responses of CLL patient peripheral blood samples to BCR and/or H 2 O 2 stimulation.
  • CLL indicates that the sample was taken from a patient diagnosed with CLL.
  • CON indicates that the sample was taken from a healthy subject.
  • the patient sample numbers are indicated at the top of the heat map and each column represents a single patient.
  • the shaded squares within the column represent a phospho-protein node. The different shades represent the degree of phosphorylation of each node (see scale at top of figure).
  • Bright white located to the far right of the scale) represents the greatest increase (i.e. +3.0 Log Change).
  • Black located in the center of the scale indicates little or no change and dark grey (located to the far left of the scale) represents the greatest decrease in phosphorylation status (i.e. ⁇ 3.0 Log Change).
  • MFI median fluorescence intensity
  • the label “p-Blnk/H 2 O 2 /F(ab)2 IgM” indicates that the phosphorylation status of phosphorylated BLNK was measured in response to hydrogen peroxide (H 2 O 2 ) and the Fab fragment that recognizes IgM (F(ab)2 IgM).
  • US indicates that the sample was unstimulated.
  • FIG. 27 depicts the lower portion of the heat map shown in FIG. 26 .
  • White framed boxes show 2 distinct clusters of CLL patient samples in which signaling increases in response to H 2 O 2 treatment (left hand side) or signaling decreases in response to H 2 O 2 treatment (righthand side).
  • FIG. 28 depicts the lower portion of the heat map shown in FIG. 26 further illustrating the two patient clusters, 10/22 patients (top left white framed box) and 11/22 patients (bottom right white framed box), that are distinguished by H 2 O 2 treatment.
  • the cartoon on the right of the figure depicts the B cell receptor signaling pathway.
  • this invention is directed to methods and compositions for diagnosis, prognosis and to methods of treatment.
  • the physiological status of cells present in a sample e.g. clinical sample
  • a sample e.g. clinical sample
  • monitor treatment e.g., to monitor treatment
  • modify therapeutic regimens e.g., to further optimize the selection of therapeutic agents; which may be administered as one or a combination of agents.
  • therapeutic regimens can be individualized and tailored according to the data obtained prior to, and at different times over the course of treatment, thereby providing a regimen that is individually appropriate.
  • the present invention is directed to methods for classifying a sample derived from an individual having or suspected of having a condition, e.g., a neoplastic or a hematopoietic condition.
  • a condition e.g., a neoplastic or a hematopoietic condition.
  • the invention allows for identification of prognostically and therapeutically relevant subgroups of conditions and prediction of the clinical course of an individual.
  • the methods of the invention provide tools useful in the treatment of an individual afflicted with a condition, including but not limited to methods of choosing a therapy for an individual, methods of predicting response to a therapy for an individual, methods of determining the efficacy of a therapy in an individual, methods for assigning a risk group, methods of predicting an increased risk of relapse, methods of predicting an increased risk of developing secondary complications, and methods of determining the prognosis for an individual.
  • the present invention provides methods that can serve as a prognostic indicator to predict the course of a condition, e.g. whether the course of a neoplastic or a hematopoietic condition in an individual will be aggressive or indolent, thereby aiding the clinician in managing the patient and evaluating the modality of treatment to be used.
  • the invention is directed to methods for determining the activation level of one or more activatable elements in a cell upon treatment with one or more modulators.
  • the activation of an activatable element in the cell upon treatment with one or more modulators can reveal operative pathways in a condition that can then be used, e.g., choose a therapy for an individual, predict response to a therapy for an individual, determine the efficacy of a therapy in an individual.
  • the modulators may themselves be used directly within individuals as therapeutic agents.
  • the activation of an activatable agent may be used as an indicator to predict course of the condition, identify risk group, predict an increased risk of developing secondary complications, and determine the prognosis for an individual.
  • the invention is directed to methods for classifying a cell by contacting the cell with an inhibitor, determining the presence or absence of an increase in activation level of an activatable element in the cell, and classifying the cell based on the presence or absence of the increase in the activation of the activatable element. In some embodiments, the invention is directed to methods of determining the presence or absence of a condition in an individual by subjecting a cell from the individual to a modulator and an inhibitor, determining the activation level of an activatable element in the cell, and determining the presence or absence of the condition based on the activation level upon treatment with a modulator and an inhibitor.
  • the invention is directed to methods for classifying a cell by contacting the cell with an inhibitor, determining the presence or absence of a change in activation level of an activatable element in the cell, and classifying the cell based on the presence or absence of the change in the activation of the activatable element.
  • the change is an increase. In some embodiments the change is a decrease.
  • the invention is directed to methods of determining tonic signaling status of a cell by subjecting the cell to a modulator, determining the activation level of an activatable element that participates in a tonic signaling pathway in the cell, and determining the status of a tonic signaling pathway in the cell from the activation level.
  • Tonic signaling in a cell may have functional consequences, for instance, to maintain certain differentiated cellular properties or functions.
  • the status of a tonic signaling pathway is used to correlate the status to differences in populations.
  • the invention is directed to methods of determining a phenotypic profile of a population of cells by exposing the population of cells in separate cultures to a plurality of modulators, wherein at least one of the modulators is an inhibitor, determining the presence or absence of an increase in activation level of an activatable element in the cell population from each of the separate culture and classifying the cell population based on the presence or absence of the increase in the activation of the activatable element from populations of cells in each separate culture.
  • the invention is directed to methods of classifying a cell population by contacting the cell population with at least one modulator, where the modulator is F(ab)2 IgM, H 2 O 2 , PMA, BAFF, April, SDF1a, CD40L, IGF-1, Imiquimod, polyCpG, IL-7, IL-6, IL-10, IL-27, IL-4, IL-2, IL-3, thapsigargin and/or a combination thereof, determining the presence or absence of an increase in activation level of an activatable element in the cell population, and classifying the cell population based on the presence or absence of the increase in the activation of the activatable element.
  • the modulator is F(ab)2 IgM, H 2 O 2 , PMA, BAFF, April, SDF1a, CD40L, IGF-1, Imiquimod, polyCpG, IL-7, IL-6, IL-10, IL-27, IL-4, IL-2, IL-3,
  • the invention is directed to methods of correlating and/or classifying an activation state of a CLL cell with a clinical outcome in an individual by subjecting the CLL cell from the individual to a modulator, where the CLL cell expresses a B-Cell receptor (BCR), determining the activation levels of a plurality of activatable elements, and identifying a pattern of the activation levels of the plurality of activatable elements to determine the presence or absence of an alteration in signaling proximal to the BCR, where the presence of the alteration is indicative of a clinical outcome.
  • BCR B-Cell receptor
  • a method for classifying a cell comprises contacting the cell with an inhibitor, determining the presence or absence of a change in an activation level of at least one activatable element in said cell, and classifying said cell based on said presence or absence of said change in the activation level of said at least one activatable element.
  • the change is an increase. In some embodiments the change is a decrease.
  • the method of classifying a cell further comprises determining the level of an intracellular marker, cell surface marker or any combination thereof.
  • a cell may be classified by a change in activation level of an activatable element and also by the level of one or more cell surface markers.
  • a cell may be classified by a change in activation level of an activatable element and by the level of an intracellular marker. Combinations may also be used. Serum markers are also useful in methods of diagnosis, prognosis, determining treatments effects and/or choosing a treatment.
  • One or more cell surface markers may also be used in the method of the invention in addition to intracellular markers (e.g. phospho-proteins).
  • the method comprises determining the level of a plurality of cell surface markers.
  • Cell surface markers may include any cell surface molecule that is detected by flow cytometry.
  • the cell surface marker is a human leukocyte differentiation antigen.
  • the human leukocyte differentiation antigen is selected from the list: CD1, CD2, CD3, CD4, CD5, CD8, CD10, CD14, CD19, CD20, CD22, CD23, CD40, CD52, CD100, CD280, CD281, CD282, CD283, CD284, and CD289.
  • the human leukocyte differentiation antigen is selected from the list comprising CD1 though CD300.
  • the cell surface marker is any cell surface receptor or ligand. Examples of cell surface ligands and receptors include, but are not limited to, members of the TNF superfamily, interleukins, hormones, neurotransmitters, interferons, growth factor,s chemokines, integrins, toll receptor ligands, prostaglandins, or leukotriene families. Other examples of cell surface markers include, but are not limited to metalloproteases. In some embodiments the cell surface marker is membrane bound IgM. In some embodiments the cell surface marker is a B-cell receptor (BCR) or a component of a BCR.
  • BCR B-cell receptor
  • the marker is CD45, CD5, CD14, CD19, CD20, CD22, CD23, CD27, CD37, CD40, CD52, CD79, CD38, CD96, major histocompatability antigen (MHC) Class1 or MHC Class 2.
  • MHC major histocompatability antigen
  • the cell surface marker is membrane bound IgD.
  • the cell surface marker is membrane bound IgG.
  • the method of classifying a cell comprises determining a level of at least one cell surface marker on said cell and an activation level of at least one activatable element on said cell.
  • the method of classifying a cell comprises determining the level of cell surface IgM on said cell.
  • the method comprises determining the level of cell surface IgD on said cell. In some embodiments, the method comprises determining the level of a BCR on said cell.
  • the cell surface marker is associated with a diesease or conditions. In some embodiments the maker is CD38 or CD96. In some embodiments the marker is CD38 and the condition is leukemia. In some embodiments the marker is CD96 and the condition is leukemia.
  • intracellular markers may be used in the method of the invention.
  • the levels of these markers can be determined before they are secreted and are referred to as “captured”.
  • captured intracellular markers include, but are not limited to, TNF superfamily members, interleukins, hormones, neurotransmitters, interferons, growth factors, chemokines, integrins, prostaglandins, leukotrines and receptors for all of the above.
  • intracellular markers also include, but are not limited to, metalloproteases.
  • intracellular markers also include, but are not limited to, proteins involved in programmed cell death and proliferation.
  • intracellular markers also include, but are not limited to viruses, pathogens, parasites and components or products thereof.
  • the method of classifying a cell further comprises determining the level of an intracellular pathogen or component of a pathogen.
  • the intracellular pathogen is HIV.
  • the intracellular pathogen is EBV.
  • the intracellular component of a pathogen is a nucleic acid sequence derived from said pathogen.
  • the intracellular component of a pathogen is a pathogen derived polypeptide.
  • the method of the invention may comprise determining the level of one or more serum markers.
  • the serum marker is a marker of a condition.
  • the serum marker is a marker of inflammation.
  • the serum marker is a soluble cytokine, TNF superfamily member, interleukin, hormone, neurotransmitter, interferon, growth factor, chemokine, integrin, prostaglandin, leukotriene or any soluble receptor thereof.
  • the serum marker is a marker of a specific disease or condition.
  • the serum marker is a cancer marker.
  • the serum marker is a leukemia marker.
  • the serum marker is beta-2-microglobulin, calcitonin, CD20, CD23, CD52, IL6, IL2R, ICAM-1, CD14, IgG, thymidine kinase or ferritin.
  • the serum marker is a pharmaceutical drug, pathogen, virus, parasite, small compound or toxin. Therefore, in some embodiments, the methods described herein are for diagnosis, prognosis or determining a method of treatment for a subject or patient. In some embodiments the methods comprise classifying a cell or population of cells. In certain embodiments, the methods of diagnosis, prognosis or determining a method of treatment comprise determining the level of at least one serum marker derived from the subject or patient. In some embodiments the serum marker is a cytokine, chemokine, soluble receptor, growth factor, antibody or binding protein. In some embodiments the serum marker is a pathogen. In some embodiments the serum marker is a pharmaceutical compound or drug.
  • kits for use in determining the physiological status of cells in a sample comprising one or more specific binding elements for signaling molecules, and may additionally comprise one or more therapeutic agents.
  • the kit may further comprise a software package for data analysis of the physiological status, which may include reference profiles for comparison with the test profile.
  • the invention provides methods, including methods to determine the physiological status of a cell, e.g., by determining the activation level of an activatable element upon contact with one or more modulators. In some embodiments, the invention provides methods, including methods to classify a cell according to the status of an activatable element in a cellular pathway.
  • the information can be used in prognosis and diagnosis, including susceptibility to disease(s), status of a diseased state and response to changes, in the environment, such as the passage of time, treatment with drugs or other modalities.
  • the physiological status of the cells provided in a sample may be classified according to the-activation of cellular pathways of interest. The cells can also be classified as to their ability to respond to therapeutic agents and treatments.
  • One or more cells, or samples containing one or more cells can be isolated from body samples, such as, but not limited to, smears, sputum, biopsies, secretions, cerebrospinal fluid, bile, blood, lymph fluid, urine and feces, a lavage of a tissue or organ (e.g. lung) or tissue which has been removed from organs, such as breast, lung, intestine, skin, cervix, prostate, and stomach.
  • a tissue sample can comprise a region of functionally related cells or adjacent cells.
  • Such samples can comprise complex populations of cells, which can be assayed as a population, or separated into sub-populations.
  • Such cellular and acellular samples can be separated by centrifugation, elutriation, density gradient separation, apheresis, affinity selection, panning, FACS, centrifugation with Hypaque, etc.
  • a relatively homogeneous population of cells may be obtained.
  • a heterogeneous cell population can be used.
  • Cells can also be separated by using filters.
  • whole blood can also be applied to filters that are engineered to contain pore sizes that select for the desired cell type or class.
  • Rare pathogenic cells can be filtered out of diluted, whole blood following the lysis of red blood cells by using filters with pore sizes between 5 to 10 ⁇ m, as disclosed in U.S.
  • Suitable cells include those cell types associated in a wide variety of disease conditions, even while in a non-diseased state. Accordingly, suitable eukaryotic cell types include, but are not limited to, tumor cells of all types (e.g. melanoma, myeloid leukemia, carcinomas of the lung, breast, ovaries, colon, kidney, prostate, pancreas and testes), cardiomyocytes, dendritic cells, endothelial cells, epithelial cells, lymphocytes (T-cell and B cell), mast cells, eosinophils, vascular intimal cells, macrophages, natural killer cells, erythrocytes, hepatocytes, leukocytes including mononuclear leukocytes, stem cells such as haemopoetic, neural, skin, lung, kidney, liver and myocyte stem cells (for use in screening for differentiation and de-differentiation factors), osteoclasts, chondrocytes and other connective tissue cells, keratinocytes
  • Suitable cells also include primary disease state cells, such as primary tumor cells.
  • Suitable cells also include known research cells, including, but not limited to, Jurkat T cells, NIH3T3 cells, CHO, COS, etc. See the ATCC cell line catalog, hereby expressly incorporated by reference.
  • the cells are cultured post collection in a media suitable for revealing the activation level of an activatable element (e.g. RPMI, DMEM) in the presence, or absence, of serum such as fetal bovine serum, bovine serum, human serum, porcine serum, horse serum, or goat serum.
  • an activatable element e.g. RPMI, DMEM
  • serum such as fetal bovine serum, bovine serum, human serum, porcine serum, horse serum, or goat serum.
  • serum is present in the media it could be present at a level ranging from 0.0001% to 100%.
  • serum is present in the media at a level ranging from 0.0001% to 90%.
  • serum is present in the media at a level ranging from 0.01% to 30%.
  • serum is present in the media at 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10%.
  • serum is present in the media at any suitable level.
  • the cell is a hematopoietic cell.
  • hematopoietic cells include but are not limited to pluripotent hematopoietic stem cells, B-lymphocyte lineage progenitor or derived cells, T-lymphocyte lineage progenitor or derived cells, NK cell lineage progenitor or derived cells, granulocyte lineage progenitor or derived cells, monocyte lineage progenitor or derived cells, megakaryocyte lineage progenitor or derived cells and erythroid lineage progenitor or derived cells.
  • the cells used in the present invention are taken from a patient.
  • Cells used in the present invention can be purified from whole blood by any suitable method.
  • the term “patient” or “individual” as used herein includes humans as well as other mammals.
  • the methods generally involve determining the status of an activatable element.
  • the methods also involve determining the status of a plurality of activatable elements.
  • the invention provides a method of classifying a cell by determining the presence or absence of a change in activation level of an activatable element in the cell upon treatment with one or more modulators, and classifying the cell based on the presence or absence of the change in the activation of the activatable element.
  • the change is a decrease.
  • the change is an increase.
  • the activation level of the activatable element is determined by contacting the cell with a binding element that is specific for an activation state of the activatable element.
  • a cell is classified according to the activation level of a plurality of activatable elements after the cell have been subjected to a modulator.
  • the activation levels of a plurality of activatable elements are determined by contacting a cell with a plurality of binding element, where each binding element is specific for an activation state of an activatable element.
  • the classification of a cell according to the status of an activatable element can comprise classifying the cell as a cell that is correlated with a clinical outcome.
  • the clinical outcome is the prognosis and/or diagnosis of a condition.
  • the clinical outcome is the presence or absence of a neoplastic or a hematopoietic condition such as Non-Hodgkin Lymphoma, Hodgkin or other lymphomas, acute or chronic leukemias, polycythemias, thrombocythemias, multiple myeloma or plasma cell disorders, e.g., amyloidosis and Waldenstrom's macroglobulinemia, myelodysplastic disorders, myeloproliferative disorders, myelofibrosis, or atypical immune lymphoproliferations.
  • the neoplastic or hematopoietic condition is non-B lineage derived, such as Acute myeloid leukemia (AML), Chronic Myeloid Leukemia (CML), non-B cell Acute lymphocytic leukemia (ALL), non-B cell lymphomas, myelodysplastic disorders, myeloproliferative disorders, myelofibrosis, polycythemias, thrombocythemias, or non-B atypical immune lymphoproliferations, Chronic Lymphocytic Leukemia (CLL), B lymphocyte lineage leukemia, B lymphocyte lineage lymphoma, Multiple Myeloma, or plasma cell disorders, e.g., amyloidosis or Waldenstrom's macroglobulinemia.
  • AML Acute myeloid leukemia
  • CML Chronic Myeloid Leukemia
  • ALL non-B cell Acute lymphocytic leukemia
  • non-B cell lymphomas myelodysplastic
  • the clinical outcome is the presence or absence of a neoplastic or a hematopoietic condition, such as Chronic Lymphocytic Leukemia (CLL), B lymphocyte lineage leukemia, B lymphocyte lineage lymphoma, Multiple Myeloma, acute lymphoblastic leukemia (ALL), B-cell pro-lymphocytic leukemia, precursor B lymphoblastic leukemia, hairy cell leukemia or plasma cell disorders, e.g., amyloidosis or Waldenstrom's macroglobulinemia, B cell lymphomas including but not limited to diffuse large B cell lymphoma, follicular lymphoma, mucosa associated lymphatic tissue lymphoma, small cell lymphocytic lymphoma, mantle cell lymphoma and marginal zone lymphoma.
  • CLL Chronic Lymphocytic Leukemia
  • ALL acute lymphoblastic leukemia
  • B-cell pro-lymphocytic leukemia precursor B lymphoblastic
  • the condition is CLL.
  • the clinical outcome is the staging or grading of a neoplastic or hematopoietic condition.
  • staging include, but are not limited to, aggressive, indolent, benign, refractory, Roman Numeral staging, TNM Staging, Rai staging, Binet staging, WHO classification, FAB classification, IPSS score, WPSS score, limited stage, extensive stage, staging according to cellular markers such as ZAP70 and CD38, occult, including information that may inform on time to progression, progression free survival, overall survival, or event-free survival.
  • the classification of a cell according to the status of an activatable element can comprise classifying a cell as a cell that is correlated to a patient response to a treatment.
  • the patient response is selected from the group consisting of complete response, partial response, nodular partial response, no response, progressive disease, stable disease and adverse reaction.
  • the classification of a cell according to the status of an activatable element can comprise classifying the cell as a cell that is correlated with minimal residual disease or emerging resistance.
  • the classification of a cell according to the status of an activatable element can comprise selecting a method of treatment.
  • Method of treatments include, but are not limited to, chemotherapy, biological therapy, radiation therapy, bone marrow transplantation, Peripheral stem cell transplantation, umbilical cord blood transplantation, autologous stem cell transplantation, allogeneic stem cell transplantation, syngeneic stem cell transplantation, surgery, induction therapy, maintenance therapy, watchful waiting, and holistic/alternative therapy.
  • Modulators include compounds or conditions capable of impacting cellular signaling networks.
  • a modulator can be an activator or an inhibitor.
  • Modulators can take the form of a wide variety of environmental inputs. Examples of modulators include but are not limited to growth factors, cytokines, chemokines, soluble receptors, Toll-like receptor ligands, pathogens, parasites, components of pathogens or parasites, adhesion molecule modulators, pharmaceutical compounds, drugs, hormones, small molecules, polynucleotides, antibodies, natural compounds, lactones, chemotherapeutic agents, immune modulators, carbohydrates, proteases, ions, reactive oxygen species, radiation, physical parameters such as heat, cold, UV radiation, peptides, and protein fragments, either alone or in the context of cells, cells themselves, viruses, and biological and non-biological complexes (e.g.
  • modulators include, but are not limited to, F(ab)2 IgM, Rituxan, alemtuzumab, fludarabine, cyclophosphamide, chlorambucil, anti CD22 (epratuzumab), anti CD23 (lumiliximab), H 2 O 2 , PMA, BAFF, April, SDF1a, CD40L, IGF-1, Imiquimod, polyCpG, IL-7, IL-6, IL-10, IL-27, IL-4, IL-2, and IL-3. Additional modulators, inhibitors and activators are disclosed in U.S. 61/085,789 which is hereby incorporated by reference in its entirety.
  • the modulator is an activator. In some embodiments the modulator is an inhibitor. In some embodiments, the invention provides methods for classifying a cell by contacting the cell with an inhibitor, determining the presence or absence of a change in activation level of an activatable element in the cell, and classifying the cell based on the presence or absence of the change in the activation of the activatable element. In some embodiments the change is a decrease. In some embodiments the change is an increase. In some embodiments, a cell is classified according to the activation level of a plurality of activatable elements after the cell have been subjected to an inhibitor.
  • the inhibitor is an inhibitor of a cellular factor or a plurality of factors that participates in a signaling cascade in the cell.
  • the inhibitor is a kinase or phosphatase inhibitor.
  • kinase inhibitors include adaphostin, AG 490, AG 825, AG 957, AG 1024, aloisine, aloisine A, alsterpaullone, aminogenistein, API-2, apigenin, arctigenin, AY-22989, BAY 61-3606, bisindolylmaleimide IX, chelerythrine, 10-[4′-(N,N-Diethylamino)butyl]-2-chlorophenoxazine hydrochloride, dasatinib, 2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole, 5,7-Dimethoxy-3-(4-pyridinyl)quinoline dihydroch
  • the methods of the invention provide methods for determining the presence or absence of a condition in an individual by subjecting a cell from the individual to a modulator and an inhibitor, determining the activation level of an activatable element in the cell, and determining the presence or absence of a condition based on the activation level.
  • the activation level of a plurality of activatable elements in the cell is determined.
  • the inhibitor can be an inhibitor as described herein.
  • the inhibitor is a phosphatase inhibitor.
  • the inhibitor is H 2 O 2 .
  • the modulator can be any modulator described herein.
  • the modulator is a B cell receptor modulator.
  • the B cell receptor modulator is a B cell receptor activator.
  • An example of B cell receptor activator is a cross-linker of the B cell receptor complex or the B-cell co-receptor complex.
  • cross-linker is an antibody or molecular binding entity.
  • the cross-linker is an antibody.
  • the antibody is a multivalent antibody.
  • the antibody is a monovalent, bivalent, or multivalent antibody made more multivalent by attachment to a solid surface or tethered on a nanoparticle surface to increase the local valency of the epitope binding domain.
  • the cross-linker can be a molecular binding entity.
  • the molecular binding entity acts upon or binds the B cell receptor complex via carbohydrates or an epitope in the complex.
  • the molecular is a monovalent, bivalent, or multivalent is made more multivalent by attachment to a solid surface or tethered on a nanoparticle surface to increase the local valency of the epitope binding domain.
  • the cross-linking of the B cell receptor complex or the B-cell co-receptor complex can comprise binding of an antibody or molecular binding entity to the cell and then causing its crosslinking via interaction of the cell with a solid surface that causes crosslinking of the BCR complex via antibody or molecular binding entity.
  • the crosslinker can be F(ab)2 IgM, IgG, IgD, polyclonal BCR antibodies, monoclonal BCR antibodies, Fc receptor derived binding elements and/or a combination thereof.
  • the Ig can be derived from a species selected from the group consisting of mouse, goat, rabbit, pig, rat, horse, cow, shark, chicken, llama or human.
  • the Ig or binding element can be fully human or partially human and can be generated by any suitable method known in the art.
  • the crosslinker is F(ab)2 IgM, Polyclonal IgM antibodies, Monoclonal IgM antibodies, Biotinylated F(ab)2 IgCM, Biotinylated Polyclonal IgM antibodies, Biotinylated Monoclonal IgM antibodies and/or a combination thereof.
  • the methods of the invention provides for the use of more than one modulator.
  • the methods of the invention utilize a B cell receptor activator and a phosphatase inhibitor.
  • the methods of the invention utilize F(ab) 2 IgM or biotinylated F(ab) 2 IgM and H 2 0 2 .
  • the methods of the invention provides for methods of classifying a cell population by exposing the cell population in separate cultures to a plurality of modulators and determining the status of activatable elements in the cell populations. In some embodiments, the status of a plurality of activatable elements in the cell population is determined. In some embodiments, at least one of the modulators of the plurality of modulators is an inhibitor.
  • the modulator can be any modulators described herein.
  • the modulator is selected from the group consisting of F(ab)2 IgM, H 2 O 2 , PMA, BAFF, April, SDF1a, CD40L, IGF-1, Imiquimod, polyCpG, IL-7, IL-6, IL-10, IL-27, IL-4, IL-2, IL-3, thapsigargin and a combination thereof.
  • the status of an activatable element is determined by contacting the cell population with a binding element that is specific for an activation state of the activatable element.
  • the status of a plurality of activatable elements is determined by contacting the cell population with a plurality of binding elements, where each binding element is specific for an activation state of an activatable element.
  • the methods of the invention provide for methods for classifying a cell population by contacting the cell population with at least one modulator, where the modulator is to F(ab)2 IgM, Rituxan, Alemtuzumab, anti CD22 (epratuzumab), anti-CD23 (lumiliximab), Campath, H 2 O 2 , PMA, BAFF, April, SDF1a, CD40L, IGF-1, Imiquimod, polyCpG, fludarabine, cyclophosphamide, chlorambucil, IL-7, IL-6, IL-10, IL-27, IL-4, IL-2, IL-3, thapsigargin and/or a combination thereof, and determining the status of an activatable element in the cell population.
  • the modulator is to F(ab)2 IgM, Rituxan, Alemtuzumab, anti CD22 (epratuzumab), anti-CD23 (lumiliximab), Campath
  • the status of a plurality of activatable elements in the cell population is determined. In some embodiments of the invention, the status of an activatable element is determined by contacting the cell population with a binding element that is specific for an activation state of the activatable element. In some embodiments, the status of a plurality of activatable elements is determined by contacting the cell population with a plurality of binding elements, where each binding element is specific for an activation state of an activatable element.
  • the methods of the invention provide for determining a phenotypic profile of a population of cells by exposing the population of cells in separate cultures to a plurality of modulators, wherein at least one of the modulators is an inhibitor, determining the presence or absence of a change in activation level of an activatable element in the cell population from each of the separate cultures and classifying the cell population based on the presence or absence of the change in the activation of the activatable element from each of the separate cultures.
  • the change is a decrease. In some embodiments the change is an increase.
  • the modulator is selected from the group consisting of F(ab)2 IgM, Rituxan, Alemtuzumab, anti CD22 (epratuzumab), anti-CD23 (lumiliximab), Campath, H 2 O 2 , PMA, BAFF, April, SDF1a, CD40L, IGF-1, Imiquimod, polyCpG, fludarabine, cyclophosphamide, chlorambucil, IL-7, IL-6, IL-10, IL-27, IL-4, IL-2, IL-3, thapsigargin and combination thereof.
  • the status of a plurality of activatable elements in the cell population is determined.
  • the phenotypic profile of a population of cells is used to classify the population as described herein.
  • the presence or absence of an increase in the activation level of an activatable element is determined by contacting the cell population with a binding element that is specific for an activation state of the activatable element.
  • the status of a plurality of activatable elements is determined by contacting the cell population with a plurality of binding elements, where each binding element is specific for an activation state of an activatable element.
  • the invention provides a method for classifying a B-lymphocyte progenitor or derived cell as described herein by contacting the cell with a modulator, determining the presence or absence of a change in activation level of an activatable element in the cell, and classifying the cell based on the presence or absence of the change in the activation of the activatable element.
  • the change is a decrease.
  • the change is an increase.
  • the presence or absence of a change in the activation level of an activatable element is determined by contacting the cell with a binding element that is specific for an activation state of the activatable element.
  • a B-lymphocyte progenitor or derived cell is classified according to the activation level of a plurality of activatable elements after the cells have been subjected to a modulator.
  • the presence or absence of a change in the activation levels of a plurality of activatable elements is determined by contacting the cell population with a plurality of binding elements, where each binding elements is specific for an activation state of an activatable element.
  • the method for classifying a B-lymphocyte progenitor or derived cell further comprises determining the level of at least one cell-surface marker.
  • the method for classifying a B-lymphocyte progenitor or derived cell further comprises determining the level of at least one intracellular marker, for example a captured intracellular cytokine.
  • the B-lymphocyte progenitor or derived cell is associated with a condition such a neoplastic or hematopoetic condition.
  • the invention provides methods for classifying a B-lymphocyte progenitor or derived cell associated with a condition (e.g.
  • the change is a decrease. In some embodiments the change is an increase.
  • the invention provides methods for correlating and/or classifying an activation state of a CLL cell with a clinical outcome in an individual by subjecting the CLL cell from the individual to a modulator, wherein the CLL cell expresses B-Cell receptor (BCR), determining the activation levels of a plurality of activatable elements, and identifying a pattern of the activation levels of the plurality of activatable elements to determine the presence or absence of an alteration in signaling proximal to the BCR, wherein the presence of the alteration is indicative of a clinical outcome.
  • BCR B-Cell receptor
  • the activation levels of a plurality of activatable elements are determined by contacting the cell with a plurality of binding elements, where each binding element is specific for an activation state of an activatable element.
  • the clinical outcome can be any clinical outcome described herein.
  • the methods of the invention provide methods for determining tonic signaling status of a cell by subjecting the cell to a modulator, determining the activation level of an activatable element that participates in a tonic signaling pathway in the cell, and determining the status of a tonic signaling pathway in the cell from the activation level.
  • the status of a plurality of activatable elements in the cell population is determined.
  • the activation level of an activatable element is determined by contacting the cell with a binding element that is specific for an activation state of the activatable element.
  • the activation level of a plurality of activatable elements is determined by contacting the cell with a plurality of binding elements, where each binding element is specific for an activation state of an activatable element.
  • the tonic signaling is a cellular receptor tonic signaling.
  • the tonic signaling is a T-cell receptor (TCR) tonic signaling.
  • the tonic signaling is a BCR tonic signaling.
  • the tonic signaling status in the cell is used to classify the cell as described herein.
  • Patterns and profiles of one or more activatable elements are detected using the methods known in the art including those described herein.
  • patterns and profiles of activatable elements that are cellular components of a cellular pathway are detected using the methods described herein.
  • patterns and profiles of activatable elements that are cellular components of a signaling pathway are detected using the methods described herein.
  • patterns and profiles of activatable elements that are cellular components of a tonic signaling pathway are detected using the methods described herein.
  • patterns and profiles of one or more phosphorylated polypeptide are detected using methods known in art including those described herein.
  • cells e.g. normal non-transformed cells
  • cells other than the cells associated with a condition (e.g. cancer cells) can be used to make clinical decisions. That is that cells, other than cells associated with a condition (e.g. cancer cells), are in fact reflective of the condition process.
  • Normal cells e.g. healthy cells or non-transformed cells
  • cells other than cells associated with a condition are in fact reflective of the condition process.
  • infiltrating immune cells can determine the outcome of the disease.
  • a combination of information from a cancer cell plus responding immune cells in the blood of a cancer patient can be used for diagnosis or prognosis of the cancer.
  • the methods of the invention are applicable to any condition in an individual involving, indicated by, and/or arising from, in whole or in part, altered physiological status in a cell.
  • physiological status includes mechanical, physical, and biochemical functions in a cell.
  • the physiological status of a cell is determined by measuring characteristics of cellular components of a cellular pathway.
  • Cellular pathways are well known in the art.
  • the cellular pathway is a signaling pathway.
  • Signaling pathways are also well known in the art (see, e.g., Hunter T., Cell (2000) 100(1): 113-27; Cell Signaling Technology, Inc., 2002 Catalogue, Pathway Diagrams pgs. 232-253).
  • a condition involving or characterized by altered physiological status may be readily identified, for example, by determining the state in a cell of one or more activatable elements, as taught herein.
  • the condition is a neoplastic or hematopoietic condition.
  • the neoplastic or hematopoietic condition is selected from the group consisting of Non-Hodgkin Lymphoma, Hodgkin or other lymphomas, acute or chronic leukemias, polycythemias, thrombocythemias, multiple myeloma and plasma cell disorders, including amyloidosis and Waldenstrom's macroglobulinemia, myelodysplastic disorders, myeloproliferative disorders, myelofibrosis, and atypical immune lymphoproliferations.
  • the neoplastic or hematopoietic condition is non-B lineage derived.
  • non-B lineage derived neoplastic or hematopoietic condition include, but are not limited to, Acute myeloid leukemia (AML), Chronic Myeloid Leukemia (CML), non-B cell Acute lymphocytic leukemia (ALL), non-B cell lymphomas, myelodysplastic disorders, myeloproliferative disorders, myelofibrosis, polycythemias, thrombocythemias, and non-B atypical immune lymphoproliferations.
  • AML Acute myeloid leukemia
  • CML Chronic Myeloid Leukemia
  • ALL non-B cell Acute lymphocytic leukemia
  • non-B cell lymphomas myelodysplastic disorders, myeloproliferative disorders, myelofibrosis, polycythemias, thrombocythemias, and non-B
  • the neoplastic or hematopoietic condition is a B-Cell or B cell lineage derived disorder.
  • B-Cell or B cell lineage derived neoplastic or hematopoietic condition include but are not limited to Chronic Lymphocytic Leukemia (CLL), B lymphocyte lineage leukemia, B lymphocyte lineage lymphoma, Multiple Myeloma, and plasma cell disorders, including amyloidosis and Waldenstrom's macroglobulinemia.
  • CLL Chronic Lymphocytic Leukemia
  • B lymphocyte lineage leukemia B lymphocyte lineage lymphoma
  • Multiple Myeloma Multiple Myeloma
  • plasma cell disorders including amyloidosis and Waldenstrom's macroglobulinemia.
  • the condition is CLL.
  • CLL is defined by a monoclonal B cell population that co-expresses CD5 with CD19 and CD23 or CD5 with CD20 and CD23 and by surface immunoglobulin expression.
  • CLL is defined by a monoclonal B cell population that co-expresses CD5 with CD19 and CD23 or CD5 with CD20 and CD23 and dim surface immunoglobulin expression.
  • cancers such as gliomas, lung cancer, colon cancer and prostate cancer.
  • Specific signaling pathway alterations have been described for many cancers, including loss of PTEN and resulting activation of Akt signaling in prostate cancer (Whang Y E. Proc Natl Acad Sci USA Apr. 28, 1998; 95(9):5246-50), increased IGF-1 expression in prostate cancer (Schaefer et al., Science Oct. 9, 1998, 282: 199a), EGFR over expression and resulting ERK activation in glioma cancer (Thomas C Y. Int J Cancer Mar. 10, 2003; 104(1):19-27), expression of HER2 in breast cancers (Menard et al. Oncogene. Sep. 29, 2003, 22(42):6570-8), and APC mutation and activated Wnt signaling in colon cancer (Bienz M. Curr Opin Genet Dev 1999 October, 9(5):595-603).
  • Diabetes involves underlying signaling changes, namely resistance to insulin and failure to activate downstream signaling through IRS (Burks D J, White M F. Diabetes 2001 February; 50 Suppl 1:S140-5).
  • cardiovascular disease has been shown to involve hypertrophy of the cardiac cells involving multiple pathways such as the PKC family (Malhotra A. Mol Cell Biochem 2001 September; 225 (1-):97-107).
  • Inflammatory diseases such as rheumatoid arthritis, are known to involve the chemokine receptors and disrupted downstream signaling (D'Ambrosio D. J Immunol Methods 2003 February; 273 (1-2):3-13).
  • the invention is not limited to diseases presently known to involve altered cellular function, but includes diseases subsequently shown to involve physiological alterations or anomalies.
  • the present invention is directed to methods for classifying one or more cells in a sample derived from an individual having or suspected of having condition.
  • the invention allows for identification of prognostically and therapeutically relevant subgroups of the conditions and prediction of the clinical course of an individual.
  • the invention provides method of classifying a cell according to the activation level of one or more activatable element in a cell from an individual having or suspected of having condition.
  • the classification includes classifying the cell as a cell that is correlated with a clinical outcome.
  • the clinical outcome can be the prognosis and/or diagnosis of a condition, and/or staging or grading of a condition.
  • the classifying of the cell includes classifying the cell as a cell that is correlated to a patient response to a treatment. In some embodiments, the classifying of the cell includes classifying the cell as a cell that is correlated with minimal residual disease or emerging resistance.
  • the methods and compositions of the invention may be employed to examine and profile the status of any activatable element in a cellular pathway, or collections of such activatable elements.
  • Single or multiple distinct pathways may be profiled (sequentially or simultaneously), or subsets of activatable elements within a single pathway or across multiple pathways may be examined (again, sequentially or simultaneously).
  • activation events can find use in the present invention.
  • the basic requirement is that the activation results in a change in the activatable protein that is detectable by some indication (termed an “activation state indicator”), preferably by altered binding of a labeled binding element or by changes in detectable biological activities (e.g., the activated state has an enzymatic activity which can be measured and compared to a lack of activity in the non-activated state).
  • an activation state indicator preferably by altered binding of a labeled binding element or by changes in detectable biological activities (e.g., the activated state has an enzymatic activity which can be measured and compared to a lack of activity in the non-activated state).
  • What is important is to differentiate, using detectable events or moieties, between two or more activation states (e.g. “off” and “on”).
  • the activation state of an individual activatable element is either in the on or off state.
  • an individual phosphorylatable site on a protein can activate or deactivate the protein.
  • the terms “on” and “off,” when applied to an activatable element that is a part of a cellular constituent, are used here to describe the state of the activatable element, and not the overall state of the cellular constituent of which it is a part.
  • a cell possesses a plurality of a particular protein or other constituent with a particular activatable element and this plurality of proteins or constituents usually has some proteins or constituents whose individual activatable element is in the on state and other proteins or constituents whose individual activatable element is in the off state. Since the activation state of each activatable element is measured through the use of a binding element that recognizes a specific activation state, only those activatable elements in the specific activation state recognized by the binding element, representing some fraction of the total number of activatable elements, will be bound by the binding element to generate a measurable signal. The measurable signal corresponding to the summation of individual activatable elements of a particular type that are activated in a single cell is the “activation level” for that activatable element in that cell.
  • Activation levels for a particular activatable element may vary among individual cells so that when a plurality of cells is analyzed, the activation levels follow a distribution.
  • the distribution may be a normal distribution, also known as a Gaussian distribution, or it may be of another type. Different populations of cells may have different distributions of activation levels that can then serve to distinguish between the populations.
  • the basis for classifying cells is that the distribution of activation levels for one or more specific activatable elements will differ among different phenotypes.
  • a certain activation level, or more typically a range of activation levels for one or more activatable elements seen in a cell or a population of cells, is indicative that that cell or population of cells belongs to a distinctive phenotype.
  • cellular levels e.g., expression levels
  • biomolecules that may not contain activatable elements may also be used to classify cells in addition to activation levels of activatable elements; it will be appreciated that these levels also will follow a distribution, similar to activatable elements.
  • the activation level or levels of one or more activatable elements optionally in conjunction with levels of one or more levels of biomolecules that may not contain activatable elements, of cell or a population of cells may be used to classify a cell or a population of cells into a class.
  • the activation level of intracellular activatable elements of individual single cells can be placed into one or more classes, e.g., a class that corresponds to a phenotype.
  • a class encompasses a class of cells wherein every cell has the same or substantially the same known activation level, or range of activation levels, of one or more intracellular activatable elements. For example, if the activation levels of five intracellular activatable elements are analyzed, predefined classes that encompass one or more of the intracellular activatable elements can be constructed based on the activation level, or ranges of the activation levels, of each of these five elements. It is understood that activation levels can exist as a distribution and that an activation level of a particular element used to classify a cell may be a particular point on the distribution but more typically may be a portion of the distribution.
  • intracellular activatable elements In addition to activation levels of intracellular activatable elements, expression levels of intracellular or extracellular biomolecues, e.g., proteins can be used alone or in combination with activation states of activatable elements to classify cells. Further, additional cellular elements, e.g., biomolecules or molecular complexes such as RNA, DNA, carbohydrates, metabolites, and the like, may be used in conjunction with activatable states or expression levels in the classification of cells encompassed here.
  • additional cellular elements e.g., biomolecules or molecular complexes such as RNA, DNA, carbohydrates, metabolites, and the like, may be used in conjunction with activatable states or expression levels in the classification of cells encompassed here.
  • other characteristics that affect the status of a cellular constituent may also be used to classify a cell. Examples include the translocation of biomolecules or changes in their turnover rates and the formation and disassociation of complexes of biomolecule. Such complexes can include multi-protein complexes, multi-lipid complexes, homo- or hetero-dimers or oligomers, and combinations thereof. Other characteristics include proteolytic cleavage, e.g. from exposure of a cell to an extracellular protease or from the intracellular proteolytic cleavage of a biomolecule.
  • Additional elements may also be used to classify a cell, such as the expression level of extracellular or intracellular markers, nuclear antigens, enzymatic activity, protein expression and localization, cell cycle analysis, chromosomal analysis, cell volume, and morphological characteristics like granularity and size of nucleus or other distinguishing characteristics.
  • B cells can be further subdivided based on the expression of cell surface markers such as CD45, CD5, CD19, CD20, CD22, CD23, CD27, CD37, CD40, CD52, CD79, CD38, CD96, major histocompatability antigen (MHC) Class or MHC Class 2.
  • MHC major histocompatability antigen
  • predefined classes of cells can be classified based upon shared characteristics that may include inclusion in one or more additional predefined class or the presence of extracellular and/or intracellular markers, a similar gene expression profile, mutational status, epigenetic silencing, nuclear antigens, enzymatic activity, protein expression and localization, cell cycle analysis, chromosomal analysis, cell volume, and morphological characteristics like granularity and size of nucleus or other distinguishing characteristics.
  • the physiological status of one or more cells is determined by examining and profiling the activation level of one or more activatable elements in a cellular pathway.
  • a cell is classified according to the activation level of a plurality of activatable elements.
  • a hematopoietic cell is classified according to the activation levels of a plurality of activatable elements.
  • the activation level of one or more activatable elements of a hematopoietic cell is correlated with a condition.
  • the activation level of one or more activatable elements of a hematopoietic cell is correlated with a neoplastic or hematopoietic condition as described herein.
  • hematopoietic cells include but are not limited to pluripotent hematopoietic stem cells, myeloid progenitors, B-lymphocyte lineage progenitor or derived cells, T-lymphocyte lineage progenitor or derived cells, NK cell lineage progenitor or derived cells, granulocyte lineage progenitor or derived cells, monocyte lineage progenitor or derived cells, megakaryocyte lineage progenitor or derived cells and erythroid lineage progenitor or derived cells.
  • the hematopoietic cell is a B-lymphocyte lineage progenitor or derived cell as described herein.
  • the activation level of one or more activatable elements in single cells within the sample is determined.
  • Cellular constituents that may include activatable elements include without limitation, proteins, carbohydrates, lipids, nucleic acids and metabolites.
  • the activatable element may be a portion of the cellular constituent, for example, an amino acid residue in a protein that may undergo phosphorylation, or it may be the cellular constituent itself, for example, a protein that is activated by translocation from one part of the cell to another, change in conformation (due to, e.g., change in pH or ion concentration), by proteolytic cleavage, and the like.
  • a change occurs to the activatable element, such as covalent modification of the activatable element (e.g., binding of a molecule or group to the activatable element, including but not limited to, phosphorylation, acetylation, methylation, ubiquitination) or a conformational change.
  • covalent modification of the activatable element e.g., binding of a molecule or group to the activatable element, including but not limited to, phosphorylation, acetylation, methylation, ubiquitination
  • a conformational change e.g., phosphorylation, acetylation, methylation, ubiquitination
  • Such changes generally contribute to changes in particular biological, biochemical, or physical properties of the cellular constituent that contains the activatable element.
  • the state of the cellular constituent that contains the activatable element is determined to some degree, though not necessarily completely, by the state of activation of a particular activatable element of the cellular constituent.
  • a protein may have multiple activatable elements, and the particular activation states of these elements may overall determine the activation state of the protein; the state of a single activatable element is not necessarily determinative. Additional factors, such as the binding of other proteins, pH, ion concentration, interaction with other cellular constituents, and the like, can also affect the state of the cellular constituent.
  • the activation levels of a plurality of intracellular activatable elements in single cells are determined. In some embodiments, at least about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 intracellular activatable elements are determined.
  • Activation states of activatable elements may result from chemical additions or modifications of biomolecules and include biochemical processes such as glycosylation, phosphorylation, acetylation, methylation, biotinylation, glutamylation, glycylation, hydroxylation, isomerization, prenylation, myristoylation, lipoylation, phosphopantetheinylation, sulfation, ISGylation, nitrosylation, palmitoylation, SUMOylation, ubiquitination, neddylation, citrullination, amidation, and disulfide bond formation, disulfide bond reduction.
  • biochemical processes such as glycosylation, phosphorylation, acetylation, methylation, biotinylation, glutamylation, glycylation, hydroxylation, isomerization, prenylation, myristoylation, lipoylation, phosphopantetheinylation, sulfation, ISGylation,
  • biomolecules include the formation of protein carbonyls, direct modifications of protein side chains, such as o-tyrosine, chloro-, nitrotyrosine, and dityrosine, and protein adducts derived from reactions with carbohydrate and lipid derivatives.
  • modifications may be non-covalent, such as binding of a ligand or binding of an allosteric modulator.
  • proteins that may include activatable elements include, but are not limited to kinases, phosphatases, lipid signaling molecules, adaptor/scaffold proteins, cytokines, cytokine regulators, ubiquitination enzymes, adhesion molecules, cytoskeletal/contractile proteins, heterotrimeric G proteins, small molecular weight GTPases, guanine nucleotide exchange factors, GTPase activating proteins, caspases, proteins involved in apoptosis (e.g.
  • PARP cell cycle regulators
  • molecular chaperones metabolic enzymes
  • vesicular transport proteins hydroxylases
  • isomerases deacetylases
  • methylases demethylases
  • tumor suppressor genes proteases
  • ion channels molecular transporters
  • transcription factors/DNA binding factors regulators of transcription, and regulators of translation.
  • activatable elements, activation states and methods of determining the activation level of activatable elements are described in US Publication Number 20060073474 entitled “Methods and compositions for detecting the activation state of multiple proteins in single cells” and U.S. Pat. No. 7,393,656 entitled “Methods and compositions for risk stratification” the content of which are incorporate here by reference.
  • the protein is selected from the group consisting of HER receptors, PDGF receptors, Kit receptor, FGF receptors, Eph receptors, Trk receptors, IGF receptors, Insulin receptor, Met receptor, Ret, VEGF receptors, TIE1, TIE2, FAK, Jak1, Jak2, Jak3, Tyk2, Src, Lyn, Fyn, Lck, Fgr, Yes, Csk, Abl, Btk, ZAP70, Syk, IRAKs, cRaf, ARaf, BRAF, Mos, Lim kinase, ILK, Tpl, ALK, TGF ⁇ receptors, BMP receptors, MEKKs, ASK, MLKs, DLK, PAKs, Mek 1, Mek 2, MKK3/6, MKK4/7, ASK1, Cot, NIK, Bub, Myt 1, Wee1, Casein kinases, PDK1, SGK1, SGK2, SGK3, Akt1, Akt2, Akt
  • the classification of a cell according to activation level of an activatable element, e.g., in a cellular pathway comprises classifying the cell as a cell that is correlated with a clinical outcome.
  • the clinical outcome is the prognosis and/or diagnosis of a condition.
  • the clinical outcome is the presence or absence of a neoplastic or a hematopoietic condition.
  • the clinical outcome is the staging or grading of a neoplastic or hematopoietic condition.
  • staging examples include, but are not limited to, aggressive, indolent, benign, refractory, Roman Numeral staging, TNM Staging, Rai staging, Binet staging, WHO classification, FAB classification, IPSS score, WPSS score, limited stage, extensive stage, staging according to cellular markers such as ZAP70, IgV H mutational status and CD38, occult, including information that may inform on time to progression, progression free survival, overall survival, or event-free survival.
  • methods and compositions are provided for the classification of a cell according to the activation level of an activatable element, e.g., in a cellular pathway wherein the classification comprises classifying a cell as a cell that is correlated to a patient response to a treatment.
  • the patient response is selected from the group consisting of complete response, partial response, nodular partial response, no response, progressive disease, stable disease and adverse reaction.
  • methods and compositions are provided for the classification of a cell according to the activation level of an activatable element, e.g., in a cellular pathway wherein the classification comprises classifying the cell as a cell that is correlated with minimal residual disease or emerging resistance.
  • methods and compositions are provided for the classification of a cell according to the activation level of an activatable element, e.g., in a cellular pathway wherein the classification comprises selecting a method of treatment.
  • Method of treatments include, but are not limited to, chemotherapy, biological therapy, radiation therapy, bone marrow transplantation, Peripheral stem cell transplantation, umbilical cord blood transplantation, autologous stem cell transplantation, allogeneic stem cell transplantation, syngeneic stem cell transplantation, surgery, induction therapy, maintenance therapy, and watchful waiting.
  • the methods of the invention involve determining the activation levels of an activatable element in a plurality of single cells in a sample.
  • the methods of the invention are employed to determine the status of an activatable element in a signaling pathway.
  • a cell is classified, as described herein, according to the activation level of one or more activatable elements in one or more signaling pathways. Signaling pathways and their members have been extensively described. See (Hunter T. Cell (2000) 100(1): 13-27).
  • Exemplary signaling pathways include the following pathways and their members: The MAP kinase pathway including Ras, Raf, MEK, ERK and elk; the PI3K/Akt pathway including PI-3-kinase, PDK1, Akt and Bad; the NF- ⁇ B pathway including IKKs, IkB and NF- ⁇ B and the Wnt pathway including frizzled receptors, beta-catenin, APC and other co-factors and TCF (see Cell Signaling Technology, Inc. 2002 Catalog pages 231-279 and Hunter T., supra.).
  • the correlated activatable elements being assayed are members of the MAP kinase, Akt, NFkB, WNT, STAT and/or PKC signaling pathways.
  • the methods of the invention also comprise the methods, signaling pathways and signaling molecules disclosed in U.S. 61/085,789 which is hereby incorporated by reference in its entirety.
  • the methods of the invention are employed to determine the status of a signaling protein in a signaling pathway known in the art including those described herein.
  • exemplary types of signaling proteins within the scope of the present invention include, but are not limited to, kinases, kinase substrates (i.e. phosphorylated substrates), phosphatases, phosphatase substrates, binding proteins (such as 14-3-3), receptor ligands and receptors (cell surface receptor tyrosine kinases and nuclear receptors)).
  • Exemplary signaling proteins include, but are not limited to, kinases, HER receptors, PDGF receptors, Kit receptor, FGF receptors, Eph receptors, Trk receptors, IGF receptors, Insulin receptor, Met receptor, Ret, VEGF receptors, TIE1, TIE2, FAK, Jak1, Jak2, Jak3, Tyk2, Src, Lyn, Fyn, Lck, Fgr, Yes, Csk, Abl, Btk, ZAP70, Syk, IRAKs, cRaf, ARaf, BRAF, Mos, Lim kinase, ILK, Tpl, ALK, TGF ⁇ receptors, BMP receptors, MEKKs, ASK, MLKs, DLK, PAKs, Mek 1, Mek 2, MKK3/6, MKK4/7, ASK1, Cot, NIK, Bub, Myt 1, Wee1, Casein kinases, PDK1, SGK1, SGK2, SGK3, Akt1,
  • the protein is selected from the group consisting of PI3-Kinase (p85, p110a, p110b, p110d), Jak1, Jak2, SOCs, Rac, Rho, Cdc42, Ras-GAP, Vav, Tiam, Sos, Dbl, Nck, Gab, PRK, SHP1, and SHP2, SHIP1, SHIP2, sSHIP, PTEN, Shc, Grb2, PDK1, SGK, Akt1, Akt2, Akt3, TSC1,2, Rheb, mTor, 4EBP-1, p70S6Kinase, S6, LKB-1, AMPK, PFK, Acetyl-CoAa Carboxylase, DokS, Rafs, Mos, Tp12, MEK1/2, MLK3, TAK, DLK, MKK3/6, MEKK1,4, MLK3, ASK1, MKK4/7, SAPK/JNK1,2,3, p38s, Erk1/2, Syk, B
  • the methods of the invention are employed to determine the status of an activatable element in the MAP kinase pathway.
  • the MAP Kinase pathway is a signal transduction pathway that couples intracellular responses to the binding of growth factors to cell surface receptors. This pathway is very complex and includes many protein components. In many cell types, activation of this pathway promotes cell division.
  • Receptor-linked tyrosine kinases such as the epidermal growth factor receptor (EGFR) are activated by extracellular ligands. Binding of epidermal growth factor (EGF) to the EGFR activates the tyrosine kinase activity of the cytoplasmic domain of the receptor. The EGFR becomes phosphorylated on tyrosines. Docking proteins such as GRB2 contain SH2 domains that bind to the phosphotyrosines of the activated receptor. GRB2 binds to the guanine nucleotide exchange factor SOS by way of an SH3 domain of GRB2. When the GRB2-SOS complex docks to phosphorylated EGFR, SOS becomes activated.
  • Activated SOS promotes the removal of GDP from Ras. Ras can then bind GTP and become active. Other small G proteins can be activated in a similar way, but are not discussed further here.
  • Activated Ras activates the protein kinase activity of RAF kinase, a serine/threonine-selective protein kinase.
  • RAF kinase phosphorylates and activates MEK, another serine/threonine kinase.
  • MEK phosphorylates and activates mitogen-activated protein kinase (MAPK).
  • RAF, MEK and MAPK are all mitogen-activated kinases, as is MNK.
  • MAPK was originally called “extracellular signal-regulated kinases” (ERKs) and microtubule-associated protein kinase (MAPK).
  • ERKs extracellular signal-regulated kinases
  • MAPK microtubule-associated protein kinase
  • One of the first proteins known to be phosphorylated by ERK was a microtubule-associated protein.
  • MAPK mitogen-activated protein kinase
  • the series of kinases from RAF to MEK to MAPK is an example of a protein kinase cascade. Such series of kinases provide opportunities for feedback regulation and signal amplification.
  • RAS is activated in a wide range of cancers (see Cell Signaling Technology, Inc. Catalog, supra. at pages 231-279 and Hunter T, supra. and references therein).
  • PI3K/Akt pathway In some embodiments, the methods of the invention are employed to determine the status of an activatable element in a PI3K/Akt pathway.
  • the PI3K/Akt pathway plays a role in effecting alterations in abroad range of cellular functions in response to extracellular signals.
  • a downstream effector of PI3K is the serine-threonine kinase Akt which in response to PI3K activation, phosphorylates and regulates the activity of a number of targets including kinases, transcription factors and other regulatory molecules.
  • the serine/threonine kinase Akt functions intracellularly as a nodal point for a constellation of converging upstream signaling pathways, which involve stimulation of receptor tyrosine kinases such as IGF-1R, HER2/Neu, VEGF-R, PDGF-R), and an assembly of membrane-localized complexes of receptor-PI3K and activation of Akt through the second messenger PIP3.
  • receptor tyrosine kinases such as IGF-1R, HER2/Neu, VEGF-R, PDGF-R
  • the integration of these intracellular signals at the level of Akt and its kinase activity regulates the phosphorylation of its several downstream effectors, such as NF-B, mTOR, Forkhead, Bad, GSK-3 and MDM-2.
  • Akt and its upstream regulators are deregulated in a wide range of solid tumors and hematologic malignancies.
  • the Akt pathway is the central cell survival pathway that is activated by such oncogenic events as over expression of an upstream receptor tyrosine kinase such as EGFR (ibid) or loss of an upstream regulatory protein such as PTEN (ibid).
  • the methods of the invention are employed to determine the status of an activatable element in a NF- ⁇ B pathway.
  • the NF- ⁇ B pathway is involved in regulating many aspects of cellular activity, in stress, injury and especially in pathways of the immune response.
  • Some examples are the response to and induction of IL-2, the induction of TAP1 and MHC molecules by NF- ⁇ B, and many aspects of the inflammatory response, e.g. induction of IL-1 (alpha and beta), TNF-alpha and leukocyte adhesion molecules (E-selectin, VCAM-1 and ICAM-1).
  • NF- ⁇ B is involved in many aspects of cell growth, differentiation and proliferation via the induction of certain growth and transcription factors (e.g. c-myc, ras and p53).
  • the NF- ⁇ B signal transduction pathway is misregulated in a variety of human cancers, especially those of lymphoid cell origin.
  • Several human lymphoid cancer cells are reported to have mutations or amplifications of genes encoding NF- ⁇ B transcription factors.
  • NF- ⁇ B is constitutively active and resides in the nucleus. In some cases, this may be due to chronic stimulation of the IKK pathway, while in others the gene encoding IkBa may be defective.
  • Such continuous nuclear NF- ⁇ B activity not only protects cancer cells from apoptotic cell death, but may even enhance their growth activity. Designing anti-tumor agents to block NF- ⁇ B activity or to increase their sensitivity to conventional chemotherapy may have great therapeutic value.
  • the methods of the invention are employed to determine the status of an activatable element in a WNT pathway.
  • the Wnt signaling pathway describes a complex network of proteins most well known for their roles in embryogenesis and cancer, but also involved in normal physiological processes in adult animals.
  • the canonical Wnt pathway describes a series of events that occur when Wnt proteins bind to cell-surface receptors of the Frizzled family, causing the receptors to activate Dishevelled family proteins and ultimately resulting in a change in the amount of ⁇ -catenin that reaches the nucleus.
  • Dishevelled is a key component of a membrane-associated Wnt receptor complex which, when activated by Wnt binding, inhibits a second complex of proteins that includes axin, GSK-3, and the protein APC.
  • the axin/GSK-3/APC complex normally promotes the proteolytic degradation of the ⁇ -catenin intracellular signaling molecule.
  • ⁇ -catenin destruction complex After this “ ⁇ -catenin destruction complex” is inhibited, a pool of cytoplasmic ⁇ -catenin stabilizes, and some ⁇ -catenin is able to enter the nucleus and interact with TCF/LEF family transcription factors to promote specific gene expression.
  • PKC pathway In some embodiments, the methods of the invention are employed to determine the status of an activatable element in a PKC pathway. Without intending to be limited to any theory, PKC pathway is associated with cell proliferation, differentiation, and apoptosis. At least eleven closely related PKC isozymes have been reported that differ in their structure, biochemical properties, tissue distribution, subcellular localization, and substrate specificity. They are classified as conventional ( ⁇ , ⁇ 1, ⁇ 2, ⁇ ), novel ( ⁇ , ⁇ , ⁇ , ⁇ , ⁇ ), and atypical ( ⁇ , ⁇ ) isozymes. Conventional PKC isozymes are Ca2+-dependent, while novel and atypical isozymes do not require Ca2+ for their activation.
  • PKC isozymes are activated by diacylglycerol (DAG).
  • DAG diacylglycerol
  • PKC isozymes negatively or positively regulate critical cell cycle transitions, including cell cycle entry and exit and the G1 and G2 checkpoints. Altered PKC activity has been linked with various types of malignancies. Higher levels of PKC and differential activation of various PKC isozymes have been reported in breast tumors, adenomatous pituitaries, thyroid cancer tissue, leukemic cells, and lung cancer cells. Down regulation of PKC ⁇ is reported in the majority of colon adenocarcinomas and in the early stages of intestinal carcinogenesis. Thus, PKC inhibitors have become important tools in the treatment of cancers. The involvement of PKC in the regulation of apoptosis adds another dimension to the effort to develop drugs that will specifically target PKC. PKC pathway activation is thought to also play a role in diseases such as cardiovascular disease and diabetes.
  • the methods described herein are employed to determine the status of an activatable element in a signaling pathway.
  • Methods and compositions are provided for the classification of a cell according to the status of an activatable element in a signaling pathway.
  • the cell can be a hematopoietic cell.
  • hematopoietic cells include but are not limited to pluripotent hematopoietic stem cells, B-lymphocyte lineage progenitor or derived cells, T-lymphocyte lineage progenitor or derived cells, NK cell lineage progenitor or derived cells, granulocyte lineage progenitor or derived cells, monocyte lineage progenitor or derived cells, megakaryocyte lineage progenitor or derived cells and erythroid lineage progenitor or derived cells.
  • the classification of a cell according to the status of an activatable element in a signaling pathway comprises classifying the cell as a cell that is correlated with a clinical outcome.
  • the clinical outcome is the prognosis and/or diagnosis of a condition.
  • the clinical outcome is the presence or absence of a neoplastic or a hematopoietic condition.
  • the clinical outcome is the staging or grading of a neoplastic or hematopoietic condition.
  • staging examples include, but are not limited to, aggressive, indolent, benign, refractory, Roman Numeral staging, TNM Staging, Rai staging, Binet staging, WHO classification, FAB classification, IPSS score, WPSS score, limited stage, extensive stage, staging according to cellular markers such as ZAP70, IgV H mutational status and CD38, occult, including information that may inform on time to progression, progression free survival, overall survival, or event-free survival.
  • methods and compositions are provided for the classification of a cell according to the status of an activatable element in a signaling pathway wherein the classification comprises classifying a cell as a cell that is correlated to a patient response to a treatment.
  • the patient response is selected from the group consisting of complete response, partial response, nodular partial response, no response, progressive disease, stable disease and adverse reaction.
  • methods and compositions are provided for the classification of a cell according to the status of an activatable element in a signaling pathway wherein the classification comprises classifying the cell as a cell that is correlated with minimal residual disease or emerging resistance.
  • methods and compositions are provided for the classification of a cell according to the status of an activatable element in a signaling pathway wherein the classification comprises selecting a method of treatment.
  • Method of treatments include, but are not limited to, chemotherapy, biological therapy, radiation therapy, bone marrow transplantation, Peripheral stem cell transplantation, umbilical cord blood transplantation, autologous stem cell transplantation, allogeneic stem cell transplantation, syngeneic stem cell transplantation, surgery, induction therapy, maintenance therapy, watchful waiting, and holistic/alternative therapy.
  • the invention is not limited to presently elucidated signaling pathways and signal transduction proteins, and encompasses signaling pathways and proteins subsequently identified.
  • the methods and compositions of the invention may be employed to examine and profile the status of any activatable element in B-Cell Receptor (BCR) signaling, or collections of such activatable elements in a B-lymphocyte lineage progenitor or derived cell.
  • BCR B-Cell Receptor
  • the physiological status of one or more B-lymphocyte lineage progenitor or derived cell is determined by examining and profiling the status of one or more activatable element in BCR signaling.
  • a B-lymphocyte lineage progenitor or derived cell is classified, as described herein, according to the activation level of one or more activatable elements in BCR signaling.
  • B-lymphocyte lineage derived cell examples include, but are not limited to, B-lymphocyte lineage early pro-B cell, late pro-B cell, large pre-B cell, small pre-B cell, immature B cell, mature B cell, plasma cell, memory B cell, a CD5+ B cell, a CD38+ B cell, a B cell bearing a mutatated or non mutated heavy chain of the B cell receptor and a B cell expressing Zap70.
  • the B-lymphocyte lineage progenitor or derived cell is a cell associated with a condition as described herein.
  • BCR cross-linking triggers phosphorylation of tyrosines within the ITAM motif domains of Ig ⁇ and Ig ⁇ by Src family member tyrosine kinases (e.g., Lyn, Lck, Blk, Fyn).
  • Src family member tyrosine kinases e.g., Lyn, Lck, Blk, Fyn.
  • the phosphorylated ITAMs of Ig ⁇ recruit and enhance phosphorylation of Syk (directly) and Btk (via Syk).
  • BCR cross-linking also brings together numerous regulator and adapter molecules (e.g., SLP-65/BLNK, Grb2, CD22, SHP-1) and compartmentalizes the BCR in lipid rafts with coreceptors CD19 and CD21.
  • PLC ⁇ 2 activation generates calcium flux, inositol-1,4,5-triphosphate, and diacylglycerol, and results in activation of protein kinase C and NF- ⁇ B.
  • Syk interacts with PLC ⁇ 2 via adapters, whereas Btk can interact directly, and each is required for PLC ⁇ 2 activity following BCR cross-linking. Both Syk and Btk can activate PI3K following BCR cross-linking.
  • PI3K Activation of PI3K enables Akt-mediated survival signaling, and PI3K is required for BCR-mediated survival during B cell development.
  • PLC ⁇ 2 and PI3K also initiate kinase cascades that result in phosphorylation of the MAPK family proteins ERK1/2 and p38.
  • Activation of the Ras-Raf-ERK1/2 signaling cascade is considered a central event in BCR signaling, and decreased Ras activation due to RasGRP1 and RasGRP3 loss in mouse impairs B cell proliferation.
  • p38 is a stress response protein that interacts with p53 and regulates cell cycle checkpoints. Differential activation of ERK1/2 and p38 might enable the BCR to drive diverse cellular outcomes, but the question arises whether a given B cell activates these two pathways simultaneously or favors one pathway depending on additional signaling context.
  • BCR signaling depends on generation of H 2 O 2 and inactivation of negative regulatory protein tyrosine phosphatases (PTPs). Following BCR cross-linking, recruitment and activation of calcium-dependent NADPH oxidases (NOX) proteins, such as NOX5, enables production of H 2 O 2 and lowers the signaling threshold for the BCR.
  • NOX calcium-dependent NADPH oxidases
  • BCR-induced H 2 O 2 transiently inactivates membrane proximal PTPs, including SHP-1, via reversible oxidation of the catalytic cysteine to sulfenic acid.
  • the invention provides a method for classifying a B-lymphocyte lineage progenitor or derived cell upon treatment with a modulator and/or inhibitor.
  • B-lymphocyte lineage progenitor or derived cells include, but are not limited to an early pro-B cell, late pro-B cell, large pre-B cell, small pre-B cell, immature B cell, mature B cell, plasma cell and memory B cell, a CD5+ B cell, a CD38+ B cell, a B cell bearing a mutatated or non mutated heavy chain of the B cell receptor, or a B cell expressing Zap70.
  • the classification includes classifying the cell according to the status of an activatable element in a BCR pathway as a cell that is correlated with a clinical outcome.
  • the invention provides methods for classifying a B-lymphocyte lineage progenitor or derived cell based on an alteration in signaling proximal to the BCR.
  • the clinical outcome is the prognosis and/or diagnosis of a condition.
  • the clinical outcome is the presence or absence of a neoplastic or a hematopoietic condition, such as Chronic Lymphocytic Leukemia (CLL), B lymphocyte lineage leukemia, B lymphocyte lineage lymphoma, Multiple Myeloma, or plasma cell disorders, e.g., amyloidosis or Waldenstrom's macroglobulinemia.
  • CLL Chronic Lymphocytic Leukemia
  • B lymphocyte lineage leukemia B lymphocyte lineage lymphoma
  • Multiple Myeloma or plasma cell disorders, e.g., amyloidosis or Waldenstrom's macroglobulinemia.
  • the condition is CLL.
  • the invention provides methods for classifying a CLL cell based on an alteration in signaling proximal to the BCR. The presence of the alteration is indicative of a clinical outcome.
  • CLL is defined by a monoclonal B cell population that co-expresses CD5 with CD19 and CD23 or CD5 with CD20 and CD23 and by surface immunoglobulin expression. In some embodiments, CLL is defined by a monoclonal B cell population that co-expresses CD5 with CD19 and CD23 or CD5 with CD20 and CD23 and dim surface immunoglobulin expression. Additional B-cell markers can be used to identify or classify a B-lymphocyte lineage progenitor or derived cell.
  • markers include CD45, CD5, CD14, CD19, CD20, CD22, CD23, CD27, CD37, CD40, CD52, CD79, CD38, CD96, major histocompatability antigen (MHC) Class1 and MHC Class 2.
  • MHC major histocompatability antigen
  • the clinical outcome is the staging or grading of a neoplastic or hematopoietic condition.
  • staging in methods provided by the invention include aggressive, indolent, benign, refractory, Roman Numeral staging, TNM Staging, Rai staging, Binet staging, WHO classification, FAB classification, IPSS score, WPSS score, limited stage, extensive stage, staging according to cellular markers such as ZAP70, IgV H mutational status and CD38, occult, including information that may inform on time to progression, progression free survival, overall survival, or event-free survival.
  • the classifying of the B-lymphocyte lineage progenitor or derived cell based on activation level of an activatable element in BCR pathway includes classifying the cell as a cell that is correlated to a patient response to a treatment, such as complete response, partial response, nodular partial response, no response, progressive disease, stable disease, relapse or adverse reaction.
  • the method may further comprise determining a method of treatment, e.g., chemotherapy, biological therapy, radiation therapy, bone marrow transplantation, Peripheral stem cell transplantation, umbilical cord blood transplantation, autologous stem cell transplantation, allogeneic stem cell transplantation, syngeneic stem cell transplantation, surgery, induction therapy, maintenance therapy, watchful waiting, or holistic/alternative therapy.
  • a method of treatment e.g., chemotherapy, biological therapy, radiation therapy, bone marrow transplantation, Peripheral stem cell transplantation, umbilical cord blood transplantation, autologous stem cell transplantation, allogeneic stem cell transplantation, syngeneic stem cell transplantation, surgery, induction therapy, maintenance therapy, watchful waiting, or holistic/alternative therapy.
  • the classifying of the B-lymphocyte lineage progenitor or derived cells based on activation of an activatable element in BCR pathway includes classifying the cell as a cell that is correlated with minimal residual disease or emerging resistance.
  • the methods and compositions of the invention may be employed to determine the status of a tonic signaling pathway in a cell. In some embodiments, the methods and compositions of the invention may be employed to examine and profile the status of any activatable element in a tonic signaling pathway, or collections of such activatable elements in a cell. In some embodiments, the physiological status of a cell is determined by examining and profiling the status of one or more activatable elements in a tonic signaling pathway. In some embodiments, a cell is classified, as described herein, according to the status of one or more activatable elements in a tonic signaling pathway.
  • the term “tonic signaling” includes antigen-independent signaling, independent basal signaling and non-induced or ligand-independent signaling.
  • the invention provides for methods of determining tonic signaling status of a cell.
  • the tonic signaling is a cellular receptor tonic signaling.
  • the tonic signaling is a BCR tonic signaling. Methods and compositions are provided for the classification of a cell according to the status of an activatable element in a tonic signaling pathway.
  • the cell can be a hematopoietic cell.
  • hematopoietic cells include but are not limited to pluripotent hematopoietic stem cells, B-lymphocyte lineage progenitor or derived cells, T-lymphocyte lineage progenitor or derived cells, NK cell lineage progenitor or derived cells, granulocyte lineage progenitor or derived cells, monocyte lineage progenitor or derived cells, megakaryocyte lineage progenitor or derived cells and erythroid lineage progenitor or derived cells.
  • the classification of a cell according to the status of an activatable element in a tonic signaling pathway comprises classifying the cell as a cell that is correlated with a clinical outcome.
  • the clinical outcome is the prognosis and/or diagnosis of a condition.
  • the clinical outcome is the presence or absence of a neoplastic or a hematopoietic condition.
  • neoplastic or hematopoietic conditions include, but are not limited to, such as Chronic Lymphocytic Leukemia (CLL), B lymphocyte lineage leukemia, B lymphocyte lineage lymphoma, Multiple Myeloma, or plasma cell disorders, e.g., amyloidosis or Waldenstrom's macroglobulinemia.
  • CLL Chronic Lymphocytic Leukemia
  • B lymphocyte lineage leukemia B lymphocyte lineage lymphoma
  • Multiple Myeloma Multiple Myeloma
  • plasma cell disorders e.g., amyloidosis or Waldenstrom's macroglobulinemia.
  • the condition is CLL.
  • CLL is defined by a monoclonal B cell population that co-expresses CD5 with CD19 and CD23 or CD5 with CD20 and CD23 and by surface immunoglobulin expression.
  • the clinical outcome is the staging or grading of a neoplastic or hematopoietic condition.
  • staging include, but are not limited to, aggressive, indolent, benign, refractory, Roman Numeral staging, TNM Staging, Rai staging, Binet staging, WHO classification, FAB classification, IPSS score, WPSS score, limited stage, extensive stage, staging according to cellular markers such as ZAP70, IgV H mutational status and CD38, including information that may inform on time to progression, progression free survival, overall survival, or event-free survival.
  • the invention provides methods for classifying a CLL cell based on an alteration in signaling proximal to the BCR that is indicative of the presence of tonic signaling.
  • the presence of the alteration is indicative of a clinical outcome, where the clinical outcome is as described herein.
  • methods and compositions are provided for the classification of a cell according to the status of an activatable element in a tonic signaling pathway wherein the classification comprises classifying a cell as a cell that is correlated to a patient response to a treatment.
  • the patient response is selected from the group consisting of complete response, partial response, nodular partial response, no response, progressive disease, stable disease and adverse reaction.
  • methods and compositions are provided for the classification of a cell according to the status of an activatable element in a tonic signaling pathway wherein the classification comprises classifying the cell as a cell that is correlated with minimal residual disease or emerging resistance.
  • methods and compositions are provided for the classification of a cell according to the status of an activatable element in a tonic signaling pathway wherein the classification comprises selecting a method of treatment.
  • Method of treatments include, but are not limited to, chemotherapy, biological therapy, radiation therapy, bone marrow transplantation, Peripheral stem cell transplantation, umbilical cord blood transplantation, autologous stem cell transplantation, allogeneic stem cell transplantation, syngeneic stem cell transplantation, surgery, induction therapy, maintenance therapy, watchful waiting, and holistic/alternative therapy.
  • the activation level of an activatable element is determined by contacting a cell with a binding element that is specific for an activation state of the activatable element.
  • Binding element includes any molecule, e.g., peptide, nucleic acid, small organic molecule which is capable of detecting an activation state of an activatable element over another activation state of the activatable element.
  • the binding element is a peptide, polypeptide, oligopeptide or a protein.
  • the peptide, polypeptide, oligopeptide or protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures.
  • amino acid or “peptide residue”, as used herein include both naturally occurring and synthetic amino acids.
  • homo-phenylalanine, citrulline and norleucine are considered amino acids for the purposes of the invention.
  • the side chains may be in either the (R) or the (S) configuration.
  • the amino acids are in the (S) or L-configuration.
  • non-amino acid substituents may be used, for example to prevent or retard in vivo degradation.
  • Proteins including non-naturally occurring amino acids may be synthesized or in some cases, made recombinantly; see van Hest et al., FEBS Lett 428:(1-2) 68-70 May 22, 1998 and Tang et al., Abstr. Pap Am. Chem. S218: U138 Part 2 Aug. 22, 1999, both of which are expressly incorporated by reference herein.
  • Methods of the present invention may be used to detect any particular activatable element in a sample that is antigenically detectable and antigenically distinguishable from other activatable element which is present in the sample.
  • the activation state-specific antibodies of the present invention can be used in the present methods to identify distinct signaling cascades of a subset or subpopulation of complex cell populations; and the ordering of protein activation (e.g., kinase activation) in potential signaling hierarchies.
  • protein activation e.g., kinase activation
  • the expression and phosphorylation of one or more polypeptides are detected and quantified using methods of the present invention.
  • the expression and phosphorylation of one or more polypeptides that are cellular components of a cellular pathway are detected and quantified using methods of the present invention.
  • the term “activation state-specific antibody” or “activation state antibody” or grammatical equivalents thereof refer to an antibody that specifically binds to a corresponding and specific antigen.
  • the corresponding and specific antigen is a specific form of an activatable element.
  • the binding of the activation state-specific antibody is indicative of a specific activation state of a specific activatable element.
  • the binding element is an antibody. In some embodiment, the binding element is an activation state-specific antibody. In some embodiment, the binding element is an phospho-specific antibody.
  • antibody includes full length antibodies and antibody fragments, and may refer to a natural antibody from any organism, an engineered antibody, or an antibody generated recombinantly for experimental, therapeutic, or other purposes as further defined below.
  • antibody fragments as are known in the art, such as Fab, Fab′, F(ab′)2, Fv, scFv, or other antigen-binding subsequences of antibodies, either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies.
  • antibody comprises monoclonal and polyclonal antibodies. Antibodies can be antagonists, agonists, neutralizing, inhibitory, or stimulatory.
  • the antibodies of the present invention may be nonhuman, chimeric, humanized, or fully human.
  • Chimeric antibodies comprise the variable region of a nonhuman antibody, for example VH and VL domains of mouse or rat origin, operably linked to the constant region of a human antibody (see for example U.S. Pat. No. 4,816,567).
  • the antibodies of the present invention are humanized.
  • humanized antibody as used herein is meant an antibody comprising a human framework region (FR) and one or more complementarity determining regions (CDR's) from a non-human (usually mouse or rat) antibody.
  • the non-human antibody providing the CDR's is called the “donor” and the human immunoglobulin providing the framework is called the “acceptor”.
  • Humanization relies principally on the grafting of donor CDRs onto acceptor (human) VL and VH frameworks (Winter U.S. Pat. No. 5,225,539). This strategy is referred to as “CDR grafting”. “Backmutation” of selected acceptor framework residues to the corresponding donor residues is often required to regain affinity that is lost in the initial grafted construct (U.S.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin, and thus will typically comprise a human Fc region.
  • humanized murine monoclonal antibodies are also known in the art, for example antibodies binding human protein C(O'Connor et al., 1998, Protein Eng 11:321-8), interleukin 2 receptor (Queen et al., 1989, Proc Natl Acad Sci, USA 86:10029-33), and human epidermal growth factor receptor 2 (Carter et al., 1992, Proc Natl. Acad Sci USA 89:4285-9).
  • the antibodies of the present invention may be fully human, that is the sequences of the antibodies are completely or substantially human.
  • aglycosylated antibodies Specifically included within the definition of “antibody” are aglycosylated antibodies.
  • aglycosylated antibody as used herein is meant an antibody that lacks carbohydrate attached at position 297 of the Fc region, wherein numbering is according to the EU system as in Kabat.
  • the aglycosylated antibody may be a deglycosylated antibody, which is an antibody for which the Fc carbohydrate has been removed, for example chemically or enzymatically.
  • the aglycosylated antibody may be a nonglycosylated or unglycosylated antibody, that is an antibody that was expressed without Fc carbohydrate, for example by mutation of one or residues that encode the glycosylation pattern or by expression in an organism that does not attach carbohydrates to proteins, for example bacteria.
  • activation state specific antibodies can be used to detect kinase activity, however additional means for determining kinase activation are provided by the present invention.
  • substrates that are specifically recognized by protein kinases and phosphorylated thereby are known.
  • Antibodies that specifically bind to such phosphorylated substrates but do not bind to such non-phosphorylated substrates may be used to determine the presence of activated kinase in a sample.
  • an element activation profile is determined using a multiplicity of activation state antibodies that have been immobilized.
  • Antibodies may be non-diffusibly bound to an insoluble support having isolated sample-receiving areas (e.g. a microtiter plate, an array, etc.).
  • the insoluble supports may be made of any composition to which the compositions can be bound, is readily separated from soluble material, and is otherwise compatible with the overall method of screening.
  • the surface of such supports may be solid or porous and of any convenient shape. Examples of suitable insoluble supports include microtiter plates, arrays, membranes, and beads.
  • Microtiter plates and arrays are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples. In some cases magnetic beads and the like are included.
  • the particular manner of binding of the composition is not crucial so long as it is compatible with the reagents and overall methods of the invention, maintains the activity of the composition and is non-diffusible.
  • Methods of binding include the use of antibodies (which do not sterically block either the ligand binding site or activation sequence when the protein is bound to the support), direct binding to “sticky” or ionic supports, chemical cross-linking, the synthesis of the antibody on the surface, etc. Following binding of the antibody, excess unbound material is removed by washing. The sample receiving areas may then be blocked through incubation with bovine serum albumin (BSA), casein or other innocuous protein or other moiety.
  • BSA bovine serum albumin
  • an activated isoform of an activatable element is distinguishable from the antigenicity of non-activated isoform of an activatable element or from the antigenicity of an isoform of a different activation state.
  • an activated isoform of an element possesses an epitope that is absent in a non-activated isoform of an element, or vice versa.
  • this difference is due to covalent addition of moieties to an element, such as phosphate moieties, or due to a structural change in an element, as through protein cleavage, or due to an otherwise induced conformational change in an element which causes the element to present the same sequence in an antigenically distinguishable way.
  • such a conformational change causes an activated isoform of an element to present at least one epitope that is not present in a non-activated isoform, or to not present at least one epitope that is presented by a non-activated isoform of the element.
  • the epitopes for the distinguishing antibodies are centered around the active site of the element, although as is known in the art, conformational changes in one area of an element may cause alterations in different areas of the element as well.
  • proteins that can be analyzed with the methods described herein include, but are not limited to, kinases, HER receptors, PDGF receptors, Kit receptor, FGF receptors, Eph receptors, Trk receptors, IGF receptors, Insulin receptor, Met receptor, Ret, VEGF receptors, TIE1, TIE2, FAK, Jak1, Jak2, Jak3, Tyk2, Src, Lyn, Fyn, Lck, Fgr, Yes, Csk, Abl, Btk, ZAP70, Syk, IRAKs, cRaf, ARaf, BRAF, Mos, Lim kinase, ILK, Tpl, ALK, TGF ⁇ receptors, BMP receptors, MEKKs, ASK, MLKs, DLK, PAKs, Mek 1, Mek 2, MKK3/6, MKK4/7, ASK1, Cot, NIK, Bub, Myt 1, Wee1, Casein kinases, PDK1, SGK1, SGK2,
  • an epitope-recognizing fragment of an activation state antibody rather than the whole antibody is used.
  • the epitope-recognizing fragment is immobilized.
  • the antibody light chain that recognizes an epitope is used.
  • a recombinant nucleic acid encoding a light chain gene product that recognizes an epitope may be used to produce such an antibody fragment by recombinant means well known in the art.
  • Non-activation state antibodies may also be used in the present invention.
  • non-activation state antibodies bind to epitopes in both activated and non-activated forms of an element. Such antibodies may be used to determine the amount of non-activated plus activated element in a sample.
  • non-activation state antibodies bind to epitopes present in non-activated forms of an element but absent in activated forms of an element. Such antibodies may be used to determine the amount of non-activated element in a sample. Both types of non-activation state antibodies may be used to determine if a change in the amount of activation state element, for example from samples before and after treatment with a candidate bioactive agent as described herein, coincide with changes in the amount of non-activation state element. For example, such antibodies can be used to determine whether an increase in activated element is due to activation of non-activation state element, or due to increased expression of the element, or both.
  • antibodies are immobilized using beads analogous to those known and used for standardization in flow cytometry. Attachment of a multiplicity of activation state specific antibodies to beads may be done by methods known in the art and/or described herein. Such conjugated beads may be contacted with sample, preferably cell extract, under conditions that allow for a multiplicity of activated elements, if present, to bind to the multiplicity of immobilized antibodies. A second multiplicity of antibodies comprising non-activation state antibodies which are uniquely labeled may be added to the immobilized activation state specific antibody-activated element complex and the beads may be sorted by FACS on the basis of the presence of each label, wherein the presence of label indicates binding of corresponding second antibody and the presence of corresponding activated element.
  • aromatic amino acids of protein binding elements may be replaced with D- or L-naphylalanine, D- or L-phenylglycine, D- or L-2-thieneylalanine, D- or L-1-, 2-, 3- or 4-pyreneylalanine, D- or L-3-thieneylalanine, D- or L-(2-pyridinyl)-alanine, D- or L-(3-pyridinyl)-alanine, D- or L-(2-pyrazinyl)-alanine, D- or L-(4-isopropyl)-phenylglycine, D-(trifluoromethyl)-phenylglycine, D-(trifluoromethyl)-phenylalanine, D-p-fluorophenylalanine, D- or L-p-biphenylphenylalanine, D- or L-p-methoxybiphenylphenylalanine, D- or L
  • Acidic amino acids can be substituted with non-carboxylate amino acids while maintaining a negative charge, and derivatives or analogs thereof, such as the non-limiting examples of (phosphono)alanine, glycine, leucine, isoleucine, threonine, or serine; or sulfated (e.g., —SO3H) threonine, serine, or tyrosine.
  • (phosphono)alanine glycine, leucine, isoleucine, threonine, or serine
  • sulfated e.g., —SO3H
  • alkyl refers to a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isoptopyl, n-butyl, isobutyl, t-butyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracisyl and the like.
  • Alkyl includes heteroalkyl, with atoms of nitrogen, oxygen and sulfur.
  • alkyl groups herein contain 1 to 12 carbon atoms.
  • Basic amino acids may be substituted with alkyl groups at any position of the naturally occurring amino acids lysine, arginine, ornithine, citrulline, or (guanidino)-acetic acid, or other (guanidino)alkyl-acetic acids, where “alkyl” is define as above.
  • Nitrile derivatives e.g., containing the CN-moiety in place of COOH
  • methionine sulfoxide may be substituted for methionine.
  • any amide linkage in any of the polypeptides may be replaced by a ketomethylene moiety.
  • Such derivatives are expected to have the property of increased stability to degradation by enzymes, and therefore possess advantages for the formulation of compounds which may have increased in vivo half lives, as administered by oral, intravenous, intramuscular, intraperitoneal, topical, rectal, intraocular, or other routes.
  • Additional amino acid modifications of amino acids of variant polypeptides of to the present invention may include the following: Cysteinyl residues may be reacted with alpha-haloacetates (and corresponding amines), such as 2-chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives.
  • Cysteinyl residues may also be derivatized by reaction with compounds such as bromotrifluoroacetone, alpha-bromo-beta-(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.
  • compounds such as bromotrifluoroacetone, alpha-bromo-beta-(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol,
  • Histidyl residues may be derivatized by reaction with compounds such as diethylprocarbonate e.g., at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain, and para-bromophenacyl bromide may also be used; e.g., where the reaction is preferably performed in 0.1M sodium cacodylate at pH 6.0.
  • compounds such as diethylprocarbonate e.g., at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain, and para-bromophenacyl bromide may also be used; e.g., where the reaction is preferably performed in 0.1M sodium cacodylate at pH 6.0.
  • Lysinyl and amino terminal residues may be reacted with compounds such as succinic or other carboxylic acid anhydrides. Derivatization with these agents is expected to have the effect of reversing the charge of the lysinyl residues.
  • Suitable reagents for derivatizing alpha-amino-containing residues include compounds such as imidoesters, e.g., as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.
  • Arginyl residues may be modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin according to known method steps.
  • arginine residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.
  • the specific modification of tyrosyl residues per se is well known, such as for introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane.
  • N-acetylimidizol and tetranitromethane may be used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.
  • Carboxyl side groups (aspartyl or glutamyl) may be selectively modified by reaction with carbodiimides (R′—N—C—N—R′) such as 1-cyclohexyl-3-(2-morpholiny-1-(4-ethyl) carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.
  • aspartyl and glutamyl residues may be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
  • Glutaminyl and asparaginyl residues may be frequently deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues may be deamidated under mildly acidic conditions. Either form of these residues falls within the scope of the present invention.
  • the activation state-specific binding element is a peptide comprising a recognition structure that binds to a target structure on an activatable protein.
  • recognition structures are well known in the art and can be made using methods known in the art, including by phage display libraries (see e.g., Gururaja et al. (2000) Chem. Biol. 7:515-27; Houimel et al., (2001) Eur. J. Immunol. 31:3535-45; Cochran et al. (2001) J. Am. Chem. Soc. 123:625-32; Houimel et al. (2001) Int. J. Cancer 92:748-55, each incorporated herein by reference).
  • fluorophores can be attached to such antibodies for use in the methods of the present invention.
  • the activation state-specific antibody is a protein that only binds to an isoform of a specific activatable protein that is phosphorylated and does not bind to the isoform of this activatable protein when it is not phosphorylated or non-phosphorylated.
  • the activation state-specific antibody is a protein that only binds to an isoform of an activatable protein that is intracellular and not extracellular, or vice versa.
  • the recognition structure is an anti-laminin single-chain antibody fragment (scFv) (see e.g., Sanz et al., (2002) Gene Therapy 9:1049-53; Tse et al., (2002) J. Mol. Biol. 317:85-94, each expressly incorporated herein by reference).
  • the binding element is a nucleic acid.
  • nucleic acid include nucleic acid analogs, for example, phosphoramide (Beaucage et al., (1993) Tetrahedron 49(10):1925 and references therein; Letsinger, J. (1970) Org. Chem. 35:3800; SRocl et al., (1977) Eur. J. Biochem. 81:579; Letsinger et al., (1986) Nucl. Acids Res. 14:3487; Sawai et al, (1984) Chem. Lett. 805, Letsinger et al., (1988) J. Am. Chem. Soc.
  • nucleic acid analogs may find use in the present invention.
  • mixtures of naturally occurring nucleic acids and analogs can be made.
  • mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made.
  • peptide nucleic acids PNA which includes peptide nucleic acid analogs are used.
  • PNA peptide nucleic acids
  • These backbones are substantially non-ionic under neutral conditions, in contrast to the highly charged phosphodiester backbone of naturally occurring nucleic acids.
  • the nucleic acids may be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence.
  • the nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid contains any combination of deoxyribo- and ribo-nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xathanine hypoxathanine, isocytosine, isoguanine, etc.
  • the binding element is a synthetic compound. Any numbers of techniques are available for the random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. See for example WO 94/24314, hereby expressly incorporated by reference, which discusses methods for generating new compounds, including random chemistry methods as well as enzymatic methods.
  • some embodiments utilize natural compounds, as binding elements, in the form of bacterial, fungal, plant and animal extracts that are available or readily produced.
  • binding element is a small organic compound.
  • Binding elements can be synthesized from a series of substrates that can be chemically modified. “Chemically modified” herein includes traditional chemical reactions as well as enzymatic reactions. These substrates generally include, but are not limited to, alkyl groups (including alkanes, alkenes, alkynes and heteroalkyl), aryl groups (including arenes and heteroaryl), alcohols, ethers, amines, aldehydes, ketones, acids, esters, amides, cyclic compounds, heterocyclic compounds (including purines, pyrimidines, benzodiazepins, beta-lactams, tetracylines, cephalosporins, and carbohydrates), steroids (including estrogens, androgens, cortisone, ecodysone, etc.), alkaloids (including ergots, vinca, curare, pyrollizdine, and mitomycines), organometallic compounds, hetero-atom bearing compounds, amino acids
  • the binding element is a carbohydrate.
  • carbohydrate is meant to include any compound with the general formula (CH 2 O) n .
  • Examples of carbohydrates are di-, tri- and oligosaccharides, as well polysaccharides such as glycogen, cellulose, and starches.
  • the binding element is a lipid.
  • lipid herein is meant to include any water insoluble organic molecule that is soluble in nonpolar organic solvents. Examples of lipids are steroids, such as cholesterol, and phospholipids such as sphingomeylin.
  • activatable elements Examples of activatable elements, activation states and methods of determining the activation level of activatable elements are described in US publication number 20060073474 entitled “Methods and compositions for detecting the activation state of multiple proteins in single cells” and US publication number 20050112700 entitled “Methods and compositions for risk stratification” the content of which are incorporate here by reference.
  • label a molecule that can be directly (i.e., a primary label) or indirectly (i.e., a secondary label) detected; for example a label can be visualized and/or measured or otherwise identified so that its presence or absence can be known.
  • a compound can be directly or indirectly conjugated to a label which provides a detectable signal, e.g. radioisotopes, fluorescers, enzymes, antibodies, particles such as magnetic particles, chemiluminescers, or specific binding molecules, etc.
  • Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin etc.
  • labels include, but are not limited to, optical fluorescent and chromogenic dyes including labels, label enzymes and radioisotopes.
  • one or more binding elements are uniquely label.
  • uniquely labeled is meant that a first activation state antibody recognizing a first activated element comprises a first label, and second activation state antibody recognizing a second activated element comprises a second label, wherein the first and second labels are detectable and distinguishable, making the first antibody and the second antibody uniquely labeled.
  • labels fall into four classes: a) isotopic labels, which may be radioactive or heavy isotopes; b) magnetic, electrical, thermal labels; c) colored, optical labels including luminescent, phosphorous and fluorescent dyes or moieties; and d) binding partners. Labels can also include enzymes (horseradish peroxidase, etc.) and magnetic particles.
  • the detection label is a primary label.
  • a primary label is one that can be directly detected, such as a fluorophore.
  • Labels include optical labels such as fluorescent dyes or moieties.
  • Fluorophores can be either “small molecule” fluors, or proteinaceous fluors (e.g. green fluorescent proteins and all variants thereof).
  • Suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade BlueTM, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705 and Oregon green.
  • Suitable optical dyes are described in the 1996 Molecular Probes Handbook by Richard P. Haugland, hereby expressly incorporated by reference.
  • Suitable fluorescent labels also include, but are not limited to, green fluorescent protein (GFP; Chalfie, et al., Science 263(5148):802-805 (Feb. 11, 1994); and EGFP; Clontech—Genbank Accession Number U55762), blue fluorescent protein (BFP; 1. Quantum Biotechnologies, Inc. 1801 de Maisonneuve Blvd. West, 8th Floor, Montreal (Quebec) Canada H3H 1J9; 2. Stauber, R. H. Biotechniques 24(3):462-471 (1998); 3. Heim, R. and Tsien, R. Y. Curr. Biol. 6:178-182 (1996)), enhanced yellow fluorescent protein (EYFP; 1.
  • GFP green fluorescent protein
  • EGFP blue fluorescent protein
  • EYFP enhanced yellow fluorescent protein
  • labels for use in the present invention include: Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, Cascade Yellow and R-phycoerythrin (PE) (Molecular Probes) (Eugene, Oreg.), FITC, Rhodamine, and Texas Red (Pierce, Rockford, Ill.), Cy5, Cy5.5, Cy7 (Amersham Life Science, Pittsburgh, Pa.).
  • Alexa-Fluor dyes Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680
  • Cascade Blue Cascade Yellow and R-phycoerythrin (PE
  • Tandem conjugate protocols for Cy5PE, Cy5.5PE, Cy7PE, Cy5.5APC, Cy7APC are known in the art. Quantitation of fluorescent probe conjugation may be assessed to determine degree of labeling and protocols including dye spectral properties are also well known in the art.
  • the fluorescent label is conjugated to an aminodextran linker which is conjugated to a binding element or antibody. Additional labels listed in and are available through the on-line and hard copy catalogues of BD Biosciences, Beckman Coulter, AnaSpec, Invitrogen, Cell Signaling Technology, Millipore, eBioscience, Caltag, Santa Cruz Biotech, Abcam and Sigma, the contents of which are incorporated herein by reference.
  • the fluorescent label is a GFP and, more preferably, a Renilla, Ptilosarcus , or Aequorea species of GFP.
  • a secondary detectable label is used.
  • a secondary label is one that is indirectly detected; for example, a secondary label can bind or react with a primary label for detection, can act on an additional product to generate a primary label (e.g. enzymes), etc.
  • Secondary labels include, but are not limited to, one of a binding partner pair; chemically modifiable moieties; nuclease inhibitors, enzymes such as horseradish peroxidase, alkaline phosphatases, luciferases, etc.
  • the secondary label is a binding partner pair.
  • the label may be a hapten or antigen, which will bind its binding partner.
  • suitable binding partner pairs include, but are not limited to: antigens (such as proteins (including peptides) and small molecules) and antibodies (including fragments thereof (FAbs, etc.)); proteins and small molecules, including biotin/streptavidin; enzymes and substrates or inhibitors; other protein-protein interacting pairs; receptor-ligands; and carbohydrates and their binding partners. Nucleic acid-nucleic acid binding proteins pairs are also useful. Binding partner pairs include, but are not limited to, biotin (or imino-biotin) and streptavidin, digeoxinin and Abs, and ProlinxTMreagents.
  • the binding partner pair comprises an antigen and an antibody that will specifically bind to the antigen.
  • specifically bind herein is meant that the partners bind with specificity sufficient to differentiate between the pair and other components or contaminants of the system. The binding should be sufficient to remain bound under the conditions of the assay, including wash steps to remove non-specific binding.
  • the dissociation constants of the pair will be less than about 10 ⁇ 4 to 10 ⁇ 9 M ⁇ 1 , with less than about 10 ⁇ 5 to 10 ⁇ 9 M ⁇ 1 being preferred and less than about 10 ⁇ 7 to 10 ⁇ 9 M ⁇ 1 being particularly preferred.
  • the secondary label is a chemically modifiable moiety.
  • labels comprising reactive functional groups are incorporated into the molecule to be labeled.
  • the functional group can then be subsequently labeled (e.g. either before or after the assay) with a primary label.
  • Suitable functional groups include, but are not limited to, amino groups, carboxy groups, maleimide groups, oxo groups and thiol groups, with amino groups and thiol groups being particularly preferred.
  • primary labels containing amino groups can be attached to secondary labels comprising amino groups, for example using linkers as are known in the art; for example, homo- or hetero-bifunctional linkers as are well known (see 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages 155-200, incorporated herein by reference).
  • each lable is distinct and distinguishable from other labels.
  • antibody-label conjugation may be performed using standard procedures or by using protein-protein/protein-dye cross-linking kits from Molecular Probes (Eugene, Oreg.).
  • labeled antibodies are used for functional analysis of activatable proteins in cells.
  • several areas of the experiment are considered: (1) identification of the proper combination of antibody cocktails for the stains (2), identification of the sequential procedure for the staining using the antigens (i.e., the activatable protein) and antibody clones of interest, and (3) thorough evaluation of cell culture conditions' effect on cell stimulation.
  • Antigen clone selection is of particular importance for surface antigens of human cells, as different antibody clones yield different result and do not stain similarly in different protocols. Selection of cell types and optimization of culture conditions is also a critical component in detecting differences. For example, some cell lines have the ability to adapt to culture conditions and can yield heterogeneous responses.
  • activation state-specific antibodies are labeled with quantum dots as disclosed by Chattopadhyay, P. K. et al. Quantum dot semiconductor nanocrystals for immunophenotyping by polychromatic flow cytometry. Nat. Med. 12, 972-977 (2006). Quantum dot labels are commercially available through Invitrogen, http://probes.invitrogen.com products/qdot/.
  • Quantum dot labeled antibodies can be used alone or they can be employed in conjunction with organic fluorochrome-conjugated antibodies to increase the total number of labels available. As the number of labeled antibodies increase so does the ability for subtyping known cell populations. Additionally, activation state-specific antibodies can be labeled using chelated or caged lanthanides as disclosed by Erkki, J. et al. Lanthanide chelates as new fluorochrome labels for cytochemistry. J. Histochemistry Cytochemistry, 36:1449-1451, 1988, and U.S. Pat. No. 7,018,850, entitled Salicylamide-Lanthanide Complexes for Use as Luminescent Markers.
  • Quantum dot methods see, e.g., Goldman et al., J. Am. Chem. Soc. (2002) 124:6378-82; Pathak et al. J. Am. Chem. Soc. (2001) 123:4103-4; and Remade et al., Proc. Natl. Sci. USA (2000) 18:553-8, each expressly incorporated herein by reference) as well as confocal microscopy.
  • the activatable elements are labeled with tags suitable for Inductively Coupled Plasma Mass Spectrometer (ICP-MS) as disclosed in Tanner et al. Spectrochimica Acta Part B: Atomic Spectroscopy, 2007 March; 62(3):188-195; Ornatsky et al, mRNA Detection in Leukemia Cell lines by Novel Metal-Tagged in situ Hybridization using Inductively Coupled Plasma Mass Spectometry, Translational Oncogenomics (2006): 1, 1-9; Ornatsky et al, Multiple Cellular Antigen Detection by ICP-MS, J. Imm. Methods 308 (2006) 68-76; and Lou et al., Polymer-Based Elemental Tags for Sensitive Bioassays, Angew. Chem. Int. Ed., (2007) 46, 6111-6114.
  • ICP-MS Inductively Coupled Plasma Mass Spectrometer
  • FRET fluorescence resonance energy transfer
  • FRET Fluorescence Reduction Activated FRET is phenomenon known in the art wherein excitation of one fluorescent dye is transferred to another without emission of a photon.
  • a FRET pair consists of a donor fluorophore and an acceptor fluorophore.
  • the fluorescence emission spectrum of the donor and the fluorescence absorption spectrum of the acceptor must overlap, and the two molecules must be in close proximity.
  • the distance between donor and acceptor at which 50% of donors are deactivated (transfer energy to the acceptor) is defined by the Forster radius (Ro), which is typically 10-100 ⁇ . Changes in the fluorescence emission spectrum comprising FRET pairs can be detected, indicating changes in the number of that are in close proximity (i.e., within 100 521 of each other).
  • FRET pairs (donor/acceptor) useful in the invention include, but are not limited to, EDANS/fluorescein, IAEDANS/fluorescein, fluorescein/tetramethylrhodamine, fluorescein/LC Red 640, fluorescein/Cy 5, fluorescein/Cy 5.5 and fluorescein/LC Red 705.
  • a fluorescent donor molecule and a non-fluorescent acceptor molecule may be employed.
  • fluorescent emission of the donor will increase when quencher is displaced from close proximity to the donor and fluorescent emission will decrease when the quencher is brought into close proximity to the donor.
  • Useful quenchers include, but are not limited to, TAMRA, DABCYL, QSY 7 and QSY 33.
  • Useful fluorescent donor/quencher pairs include, but are not limited to EDANS/DABCYL, Texas Red/DABCYL, BODIPY/DABCYL, Lucifer yellow/DABCYL, coumarin/DABCYL and fluorescein/QSY 7 dye.
  • FRET and fluorescence quenching allow for monitoring of binding of labeled molecules over time, providing continuous information regarding the time course of binding reactions.
  • changes in the degree of FRET are determined as a function of the change in the ratio of the amount of fluorescence from the donor and acceptor moieties, a process referred to as “ratioing.”
  • ratioing Changes in the absolute amount of substrate, excitation intensity, and turbidity or other background absorbances in the sample at the excitation wavelength affect the intensities of fluorescence from both the donor and acceptor approximately in parallel. Therefore the ratio of the two emission intensities is a more robust and preferred measure of cleavage than either intensity alone.
  • the ratio-metric fluorescent reporter system described herein has significant advantages over existing reporters for protein integration analysis, as it allows sensitive detection and isolation of both expressing and non-expressing single living cells.
  • the assay system uses a non-toxic, non-polar fluorescent substrate that is easily loaded and then trapped intracellularly. Modification of the fluorescent substrate by a cognate protein yields a fluorescent emission shift as substrate is converted to product.
  • the reporter readout is ratiometric it is unique among reporter protein assays in that it controls for variables such as the amount of substrate loaded into individual cells.
  • the stable, easily detected, intracellular readout eliminates the need for establishing clonal cell lines prior to expression analysis.
  • This system and other analogous flow sorting systems can be used to isolate cells having a particular receptor element clustering and/or activation profile from pools of millions of viable cells.
  • label enzyme an enzyme that may be reacted in the presence of a label enzyme substrate that produces a detectable product.
  • Suitable label enzymes for use in the present invention include but are not limited to, horseradish peroxidase, alkaline phosphatase and glucose oxidase. Methods for the use of such substrates are well known in the art.
  • the presence of the label enzyme is generally revealed through the enzyme's catalysis of a reaction with a label enzyme substrate, producing an identifiable product.
  • Such products may be opaque, such as the reaction of horseradish peroxidase with tetramethyl benzedine, and may have a variety of colors.
  • label enzyme substrates such as Luminol (available from Pierce Chemical Co.) have been developed that produce fluorescent reaction products.
  • Methods for identifying label enzymes with label enzyme substrates are well known in the art and many commercial kits are available. Examples and methods for the use of various label enzymes are described in Savage et al., Previews 247:6-9 (1998), Young, J. Virol. Methods 24:227-236 (1989), which are each hereby incorporated by reference in their entirety.
  • radioisotope any radioactive molecule. Suitable radioisotopes for use in the invention include, but are not limited to 14 C, 3 H, 32 P, 33 P, 35 S, 125 I, and 131 I. The use of radioisotopes as labels is well known in the art.
  • labels may be indirectly detected, that is, the tag is a partner of a binding pair.
  • partner of a binding pair is meant one of a first and a second moiety, wherein the first and the second moiety have a specific binding affinity for each other.
  • Suitable binding pairs for use in the invention include, but are not limited to, antigens/antibodies (for example, digoxigenin/anti-digoxigenin, dinitrophenyl (DNP)/anti-DNP, dansyl-X-anti-dansyl, Fluorescein/anti-fluorescein, lucifer yellow/anti-lucifer yellow, and rhodamine anti-rhodamine), biotin/avidin (or biotin/streptavidin) and calmodulin binding protein (CBP)/calmodulin.
  • antigens/antibodies for example, digoxigenin/anti-digoxigenin, dinitrophenyl (DNP)/anti-DNP, dansyl-X-anti-dansyl, Fluorescein/anti-fluorescein, lucifer yellow/anti-lucifer yellow, and rhodamine anti-rhodamine
  • biotin/avidin or biotin/streptavidin
  • binding pair pairs include polypeptides such as the FLAG-peptide [Hopp et al., BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin et al., Science, 255: 192-194 (1992)]; tubulin epitope peptide [Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990)] and the antibodies each thereto.
  • binding pair partners may be used in applications other than for labeling, as is described herein.
  • a partner of one binding pair may also be a partner of another binding pair.
  • an antigen first moiety
  • first moiety may bind to a first antibody (second moiety) that may, in turn, be an antigen for a second antibody (third moiety).
  • second moiety an antigen for a second antibody (third moiety).
  • a partner of a binding pair may comprise a label, as described above. It will further be appreciated that this allows for a tag to be indirectly labeled upon the binding of a binding partner comprising a label. Attaching a label to a tag that is a partner of a binding pair, as just described, is referred to herein as “indirect labeling”.
  • surface substrate binding molecule or “attachment tag” and grammatical equivalents thereof is meant a molecule have binding affinity for a specific surface substrate, which substrate is generally a member of a binding pair applied, incorporated or otherwise attached to a surface.
  • Suitable surface substrate binding molecules and their surface substrates include, but are not limited to poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags and Nickel substrate; the Glutathione-S Transferase tag and its antibody substrate (available from Pierce Chemical); the flu HA tag polypeptide and its antibody 12CA5 substrate [Field et al., Mol. Cell.
  • surface binding substrate molecules useful in the present invention include, but are not limited to, polyhistidine structures (His-tags) that bind nickel substrates, antigens that bind to surface substrates comprising antibody, haptens that bind to avidin substrate (e.g., biotin) and CBP that binds to surface substrate comprising calmodulin.
  • His-tags polyhistidine structures
  • antigens that bind to surface substrates comprising antibody
  • haptens that bind to avidin substrate (e.g., biotin)
  • CBP that binds to surface substrate comprising calmodulin.
  • tag-components of the invention can be made in various ways, depending largely upon the form of the tag.
  • Components of the invention and tags are preferably attached by a covalent bond.
  • tag-polypeptides by recombinant means when the tag is also a polypeptide is described below.
  • Production of tag-labeled proteins is well known in the art and kits for such production are commercially available (for example, from Kodak and Sigma).
  • Examples of tag labeled proteins include, but are not limited to, a Flag-polypeptide and His-polypeptide. Methods for the production and use of tag-labeled proteins are found, for example, in Winston et al., Genes and Devel. 13:270-283 (1999), incorporated herein in its entirety, as well as product handbooks provided with the above-mentioned kits.
  • Biotinylation of target molecules and substrates is well known, for example, a large number of biotinylation agents are known, including amine-reactive and thiol-reactive agents, for the biotinylation of proteins, nucleic acids, carbohydrates, carboxylic acids; see chapter 4, Molecular Probes Catalog, Haugland, 6th Ed. 1996, hereby incorporated by reference.
  • a biotinylated substrate can be attached to a biotinylated component via avidin or streptavidin.
  • haptenylation reagents are also known (Id.).
  • kits for producing such proteins are commercially available.
  • such a kit and its use are described in the QIAexpress Handbook from Qiagen by Joanne Crowe et al., hereby expressly incorporated by reference.
  • the tag is functionalized to facilitate covalent attachment.
  • the covalent attachment of the tag may be either direct or via a linker.
  • the linker is a relatively short coupling moiety, which is used to attach the molecules.
  • a coupling moiety may be synthesized directly onto a component of the invention and contains at least one functional group to facilitate attachment of the tag.
  • the coupling moiety may have at least two functional groups, which are used to attach a functionalized component to a functionalized tag, for example.
  • the linker is a polymer.
  • covalent attachment is accomplished either directly, or through the use of coupling moieties from the component or tag to the polymer.
  • the covalent attachment is direct, that is, no linker is used.
  • the component preferably contains a functional group such as a carboxylic acid that is used for direct attachment to the functionalized tag. It should be understood that the component and tag may be attached in a variety of ways, including those listed above.
  • the tag is attached to the amino or carboxy terminus of the polypeptide. As will be appreciated by those in the art, the above description of the covalent attachment of a label applies to the attachment of virtually any two molecules of the present disclosure.
  • the tag is functionalized to facilitate covalent attachment, as is generally outlined above.
  • tags are commercially available which contain functional groups, including, but not limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which may be used to covalently attach the tag to a second molecule, as is described herein.
  • the choice of the functional group of the tag will depend on the site of attachment to either a linker, as outlined above or a component of the invention.
  • amino modified or hydrazine modified tags will be used for coupling via carbodiimide chemistry, for example using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimi-de (EDAC) as is known in the art (see Set 9 and Set 11 of the Molecular Probes Catalog, supra; see also the Pierce 1994 Catalog and Handbook, pages T-155 to T-200, both of which are hereby incorporated by reference).
  • the carbodiimide is first attached to the tag, such as is commercially available for many of the tags described herein.
  • An alternative activation state indicator useful with the instant invention is one that allows for the detection of activation by indicating the result of such activation.
  • phosphorylation of a substrate can be used to detect the activation of the kinase responsible for phosphorylating that substrate.
  • cleavage of a substrate can be used as an indicator of the activation of a protease responsible for such cleavage. Methods are well known in the art that allow coupling of such indications to detectable signals, such as the labels and tags described above in connection with binding elements. For example, cleavage of a substrate can result in the removal of a quenching moiety and thus allowing for a detectable signal being produced from a previously quenched label.
  • the methods and composition utilize a modulator.
  • a modulator can be an activator, an inhibitor or a compound capable of impacting a cellular pathway.
  • Modulators can take the form of environmental cues and inputs.
  • Modulation can be performed in a variety of environments.
  • cells are exposed to a modulator immediately after collection.
  • purification of cells is performed after modulation.
  • whole blood is collected to which a modulator is added.
  • cells are modulated after processing for single cells or purified fractions of single cells.
  • whole blood can be collected and processed for an enriched fraction of lymphocytes that is then exposed to a modulator.
  • Modulation can include exposing cells to more than one modulator. For instance, in some embodiments, cells are exposed to at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 modulators.
  • cells are cultured post collection in a suitable media before exposure to a modulator.
  • the media is a growth media.
  • the growth media is a complex media that may include serum.
  • the growth media comprises serum.
  • the serum is selected from the group consisting of fetal bovine serum, bovine serum, human serum, porcine serum, horse serum, and goat serum.
  • the serum level ranges from 0.0001% to 30%. In some embodiments any suitable amount of serum is used.
  • the growth media is a chemically defined minimal media and is without serum.
  • cells are cultured in a differentiating media.
  • Modulators include chemical and biological entities, and physical or environmental stimuli. Modulators can act extracellularly or intracellularly. Chemical and biological modulators include growth factors, cytokines, neurotransmitters, adhesion molecules, hormones, small molecules, inorganic compounds, polynucleotides, antibodies, natural compounds, lectins, lactones, chemotherapeutic agents, biological response modifiers, carbohydrate, proteases and free radicals. Modulators include complex and undefined biologic compositions that may comprise cellular or botanical extracts, cellular or glandular secretions, physiologic fluids such as serum, amniotic fluid, or venom.
  • Physical and environmental stimuli include electromagnetic, ultraviolet, infrared or particulate radiation, redox potential and pH, the presence or absences of nutrients, changes in temperature, changes in oxygen partial pressure, changes in ion concentrations and the application of oxidative stress.
  • Modulators can be endogenous or exogenous and may produce different effects depending on the concentration and duration of exposure to the single cells or whether they are used in combination or sequentially with other modulators. Modulators can act directly on the activatable elements or indirectly through the interaction with one or more intermediary biomolecule. Indirect modulation includes alterations of gene expression wherein the expressed gene product is the activatable element or is a modulator of the activatable element.
  • modulators produce different activation states depending on the concentration of the modulator, duration of exposure or whether they are used in combination or sequentially with other modulators.
  • the modulator is selected from the group consisting of growth factor, cytokine, adhesion molecule modulator, drugs, hormone, small molecule, polynucleotide, antibodies, natural compounds, lactones, chemotherapeutic agents, immune modulator, carbohydrate, proteases, ions, reactive oxygen species, peptides, and protein fragments, either alone or in the context of cells, cells themselves, viruses, and biological and non-biological complexes (e.g. beads, plates, viral envelopes, antigen presentation molecules such as major histocompatibility complex).
  • the modulator is a physical stimuli such as heat, cold, UV radiation, and radiation.
  • modulators include but are not limited to, F(ab)2 IgM, Rituxan, Alemtuzumab, anti CD22 (epratuzumab), anti-CD23 (lumiliximab), Campath, H 2 O 2 , PMA, BAFF, April, SDF1a, CD40L, IGF-1, Imiquimod, polyCpG, fludarabine, cyclophosphamide, chlorambucil, IL-7, IL-6, IL-10, IL-27, IL-4, IL-2, IL-3, and thapsigargin.
  • the modulator is an activator. In some embodiments the modulator is an inhibitor. In some embodiments, cells are exposed to one or more modulator. In some embodiments, cells are exposed to at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 modulators. In some embodiments, cells are exposed to at least two modulators, wherein one modulator is an activator and one modulator is an inhibitor. In some embodiments, cells are exposed to at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 modulators, where at least one of the modulators is an inhibitor.
  • the modulator is a B cell receptor modulator.
  • the B cell receptor modulator is a B cell receptor activator.
  • An example of B cell receptor activator is a cross-linker of the B cell receptor complex or the B-cell co-receptor complex.
  • cross-linker is an antibody or molecular binding entity. In some embodiments, the cross-linker is an antibody. In some embodiments, the antibody is a multivalent antibody. In some embodiments, the antibody is a monovalent, bivalent, or multivalent antibody made more multivalent by attachment to a solid surface or tethered on a nanoparticle surface to increase the local valency of the epitope binding domain.
  • the cross-linker is a molecular binding entity.
  • the molecular binding entity acts upon or binds the B cell receptor complex via carbohydrates or an epitope in the complex.
  • the molecular is a monovalent, bivalent, or multivalent is made more multivalent by attachment to a solid surface or tethered on a nanoparticle surface to increase the local valency of the epitope binding domain.
  • the cross-linking of the B cell receptor complex or the B-cell co-receptor complex comprises binding of an antibody or molecular binding entity to the cell and then causing its crosslinking via interaction of the cell with a solid surface that causes crosslinking of the BCR complex via antibody or molecular binding entity.
  • the crosslinker is F(ab)2 IgM, IgG, IgD, polyclonal BCR antibodies, monoclonal BCR antibodies, Fc receptor derived binding elements and/or a combination thereof.
  • the Ig can be derived from a species selected from the group consisting of mouse, goat, rabbit, pig, rat, horse, cow, shark, chicken, or llama.
  • the crosslinker is F(ab)2 IgM, Polyclonal IgM antibodies, Monoclonal IgM antibodies, Biotinylated F(ab)2 IgCM, Biotinylated Polyclonal IgM antibodies, Biotinylated Monoclonal IgM antibodies and/or combination thereof.
  • the inhibitor is an inhibitor of a cellular factor or a plurality of factors that participates in a cellular pathway (e.g. signaling cascade) in the cell.
  • the inhibitor is a kinase or phosphatase inhibitor.
  • kinase inhibitors include adaphostin, AG 490, AG 825, AG 957, AG 1024, aloisine, aloisine A, alsterpaullone, aminogenistein, API-2, apigenin, arctigenin, AY-22989, BAY 61-3606, bisindolylmaleimide IX, chelerythrine, 10-[4′-(N,N-Diethylamino)butyl]-2-chlorophenoxazine hydrochloride, dasatinib, 2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole, 5,7-Dimethoxy-3-(4-pyridinyl)quinoline dihydrochloride, edelfosine, ellagic acid, enzastaurin, ER 27319 maleate, erlotinib, ET18OCH3, fasudil, flavopiridol, gefitin
  • H 2 O 2 is administered as an inhibitor. In some embodiments H 2 O 2 is administered at between 0.01 and 50 mM. In some embodiments H 2 O 2 is administered at between 0.1 and 10 mM. In some embodiments H 2 O 2 is administered at between 1 and 10 mM. In some embodiments H 2 O 2 is administered at between 1 and 5 mM. In some embodiments H 2 O 2 is administered at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 mM. In certain embodiments, H 2 O 2 is administered at 3.0 mM. In certain embodiments, H 2 O 2 is administered at 3.3 mM.
  • the duration of exposure of H 2 O 2 is between 0.01 and 360 minutes. In some embodiments the duration of exposure of H 2 O 2 is between 0.1 and 240 minutes. In some embodiments the duration of exposure of H 2 O 2 is between 0.5 and 180 minutes. In some embodiments the duration of exposure of H 2 O 2 is between 0 and 120 minutes. In some embodiments the duration of exposure to H 2 O 2 is between 5 and 15 minutes. In some embodiments the duration of exposure of H 2 O 2 is 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 120, 140, 160 or 180 minutes. In some embodiments the duration of exposure of H 2 O 2 is 10 minutes.
  • H 2 O 2 is administered as an inhibitor with at least one other modulator. In some embodiments H 2 O 2 is administered as an inhibitor with F(ab)2 IgM or any suitable BCR agonist. In some embodiments H 2 O 2 is administered before administration of F(ab)2 IgM. In some embodiments H 2 O 2 is administered simultaneously with F(ab)2 IgM. In some embodiments H 2 O 2 is administered after F(ab)2 IgM.
  • the activation level of an activatable element in a cell is determined after contacting the cell with at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 modulators. In some embodiments, the activation level of an activatable element in a cell is determined after contacting the cell with at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 modulators where at least one of the modulators is an inhibitor. In some embodiments, the activation level of an activatable element in a cell is determined after contacting the cell with an inhibitor and a modulator, where the modulator can be an inhibitor or an activator. In some embodiments, the activation level of an activatable element in a cell is determined after contacting the cell with an inhibitor and an activator. In some embodiments, the activation level of an activatable element in a cell is determined after contacting the cell with two or more modulators.
  • a phenotypic profile of a population of cells is determined by measuring the activation level of an activatable element when the population of cells is exposed to a plurality of modulators in separate cultures.
  • the modulators include F(ab)2 IgM, Rituxan, Alemtuzumab, anti CD22 (epratuzumab), anti-CD23 (lumiliximab), Campath, H 2 O 2 , PMA, BAFF, April, SDF1a, CD40L, IGF-1, Imiquimod, polyCpG, fludarabine, cyclophosphamide, chlorambucil, IL-7, IL-6, IL-10, IL-27, IL-4, IL-2, IL-3, thapsigardin and/or a combination thereof.
  • a population of cells can be exposed to one or more, all or a combination of the following combination of modulators: (i) F(ab)2 IgM; (ii) Rituxan; (iii) Campath; (iv) H 2 O 2 ; (v) PMA; (vi) BAFF; (vii) April; (viii) SDF1a; (ix) CD40L; (x) IGF-1; (xi) Imiquimod; (xii) polyCpG; (xiii) fludarabine; (xiv) cyclophosphamide; (xv) chlorambucil; IL-7; (xvi) IL-6; (xvii) IL-10; (xviii) IL-27; (xx) IL-4; (xx) IL-2; (xxi) IL-3; (xxii) Alemtuzumab, (xxiii) anti CD22 (epratuzumab), (xxiv) anti-CD23 (lum
  • the detection of the status of the one or more activatable elements can be carried out by a person, such as a technician in the laboratory. Alternatively, the detection of the status of the one or more activatable elements can be carried out using automated systems. In either case, the detection of the status of the one or more activatable elements for use according to the methods of this invention is performed according to standard techniques and protocols well-established in the art.
  • One or more activatable elements can be detected and/or quantified by any method that detect and/or quantitates the presence of the activatable element of interest.
  • Such methods may include radioimmunoassay (RIA) or enzyme linked immunoabsorbance assay (ELISA), immunohistochemistry, immunofluorescent histochemistry with or without confocal microscopy, reversed phase assays, homogeneous enzyme immunoassays, and related non-enzymatic techniques, Western blots, whole cell staining, immunoelectronmicroscopy, nucleic acid amplification, gene array, protein array, mass spectrometry, patch clamp, 2-dimensional gel electrophoresis, differential display gel electrophoresis, microsphere-based multiplex protein assays, label-free cellular assays and flow cytometry, etc.
  • RIA radioimmunoassay
  • ELISA enzyme linked immunoabsorbance assay
  • immunohistochemistry immunofluorescent histochemistry with or without confocal micros
  • U.S. Pat. No. 4,568,649 describes ligand detection systems, which employ scintillation counting. These techniques are particularly useful for modified protein parameters. Cell readouts for proteins and other cell determinants can be obtained using fluorescent or otherwise tagged reporter molecules. Flow cytometry methods are useful for measuring intracellular parameters.
  • the present invention provides methods for determining an activatable element's activation profile for a single cell.
  • the methods may comprise analyzing cells by flow cytometry on the basis of the activation level of at least two activatable elements.
  • Binding elements e.g. activation state-specific antibodies
  • non-binding elements systems as described above can be used in any system described herein.
  • fluorescent monitoring systems e.g., Cytometric measurement device systems
  • flow cytometric systems are used or systems dedicated to high throughput screening, e.g. 96 well or greater microtiter plates.
  • Methods of performing assays on fluorescent materials are well known in the art and are described in, e.g., Lakowicz, J. R., Principles of Fluorescence Spectroscopy, New York: Plenum Press (1983); Herman, B., Resonance energy transfer microscopy, in: Fluorescence Microscopy of Living Cells in Culture, Part B, Methods in Cell Biology, vol. 30, ed.
  • Fluorescence in a sample can be measured using a fluorimeter.
  • excitation radiation from an excitation source having a first wavelength, passes through excitation optics.
  • the excitation optics cause the excitation radiation to excite the sample.
  • fluorescent proteins in the sample emit radiation that has a wavelength that is different from the excitation wavelength.
  • Collection optics then collect the emission from the sample.
  • the device can include a temperature controller to maintain the sample at a specific temperature while it is being scanned.
  • a multi-axis translation stage moves a microtiter plate holding a plurality of samples in order to position different wells to be exposed.
  • the multi-axis translation stage, temperature controller, auto-focusing feature, and electronics associated with imaging and data collection can be managed by an appropriately programmed digital computer.
  • the computer also can transform the data collected during the assay into another format for presentation.
  • known robotic systems and components can be used.
  • Quantum dot methods See, e.g., Goldman et al., J. Am. Chem. Soc. (2002) 124:6378-82; Pathak et al. J. Am. Chem. Soc. (2001) 123:4103-4; and Remade et al., Proc. Natl. Sci. USA (2000) 18:553-8, each expressly incorporated herein by reference) as well as confocal microscopy.
  • flow cytometry involves the passage of individual cells through the path of a laser beam. The scattering the beam and excitation of any fluorescent molecules attached to, or found within, the cell is detected by photomultiplier tubes to create a readable output, e.g. size, granularity, or fluorescent intensity.
  • the detecting, sorting, or isolating step of the methods of the present invention can entail fluorescence-activated cell sorting (FACS) techniques, where FACS is used to select cells from the population containing a particular surface marker, or the selection step can entail the use of magnetically responsive particles as retrievable supports for target cell capture and/or background removal.
  • FACS fluorescence-activated cell sorting
  • a variety of FACS systems are known in the art and can be used in the methods of the invention (see e.g., WO99/54494, filed Apr. 16, 1999; U.S. Ser. No. 20010006787, filed Jul. 5, 2001, each expressly incorporated herein by reference).
  • a FACS cell sorter e.g. a FACSVantageTM Cell Sorter, Becton Dickinson Immunocytometry Systems, San Jose, Calif.
  • a FACS cell sorter e.g. a FACSVantageTM Cell Sorter, Becton Dickinson Immunocytometry Systems, San Jose, Calif.
  • the change is a decrease. In some embodiments the change is an increase.
  • the cells are first contacted with fluorescent-labeled activation state-specific binding elements (e.g. antibodies) directed against specific activation state of specific activatable elements.
  • the amount of bound binding element on each cell can be measured by passing droplets containing the cells through the cell sorter. By imparting an electromagnetic charge to droplets containing the positive cells, the cells can be separated from other cells. The positively selected cells can then be harvested in sterile collection vessels.
  • positive cells can be sorted using magnetic separation of cells based on the presence of an isoform of an activatable element.
  • cells to be positively selected are first contacted with specific binding element (e.g., an antibody or reagent that binds an isoform of an activatable element).
  • the cells are then contacted with retrievable particles (e.g., magnetically responsive particles) that are coupled with a reagent that binds the specific element.
  • retrievable particles e.g., magnetically responsive particles
  • the cell-binding element-particle complex can then be physically separated from non-positive or non-labeled cells, for example, using a magnetic field.
  • magnetically responsive particles the positive or labeled cells can be retained in a container using a magnetic filed while the negative cells are removed.
  • methods for the determination of a receptor element activation state profile for a single cell comprise providing a population of cells and analyze the population of cells by flow cytometry. Preferably, cells are analyzed on the basis of the activation level of at least two activatable elements. In some embodiments, a multiplicity of activatable element activation-state antibodies is used to simultaneously determine the activation level of a multiplicity of elements.
  • cell analysis by flow cytometry on the basis of the activation level of at least two elements is combined with a determination of other flow cytometry readable outputs, such as the presence of surface markers, granularity and cell size to provide a correlation between the activation level of a multiplicity of elements and other cell qualities measurable by flow cytometry for single cells.
  • the present invention also provides for the ordering of element clustering events in signal transduction.
  • the present invention allows the artisan to construct an element clustering and activation hierarchy based on the correlation of levels of clustering and activation of a multiplicity of elements within single cells. Ordering can be accomplished by comparing the activation level of a cell or cell population with a control at a single time point, or by comparing cells at multiple time points to observe subpopulations arising out of the others.
  • the present invention provides a valuable method of determining the presence of cellular subsets within cellular populations. Ideally, signal transduction pathways are evaluated in homogeneous cell populations to ensure that variances in signaling between cells do not qualitatively nor quantitatively mask signal transduction events and alterations therein. As the ultimate homogeneous system is the single cell, the present invention allows the individual evaluation of cells to allow true differences to be identified in a significant way.
  • the invention provides methods of distinguishing cellular subsets within a larger cellular population.
  • these cellular subsets often exhibit altered biological characteristics (e.g. activation levels, altered response to modulators) as compared to other subsets within the population.
  • the methods of the invention allow the identification of subsets of cells from a population such as primary cell populations, e.g. peripheral blood mononuclear cells that exhibit altered responses (e.g. response associated with presence of a condition) as compared to other subsets.
  • this type of evaluation distinguishes between different activation states, altered responses to modulators, cell lineages, cell differentiation states, etc.
  • these methods provide for the identification of distinct signaling cascades for both artificial and stimulatory conditions in complex cell populations, such a peripheral blood mononuclear cells, or naive and memory lymphocytes.
  • cells are dispersed into a single cell suspension (e.g. by enzymatic digestion with a suitable protease, collagenase, dispase, etc; and the like).
  • An appropriate solution is used for dispersion or suspension.
  • Such solution will generally be a balanced salt solution, e.g. normal saline, PBS, Hanks balanced salt solution, etc., conveniently supplemented with fetal calf serum or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration, generally from 5-25 mM.
  • Convenient buffers include HEPES1 phosphate buffers, lactate buffers, etc.
  • the cells may be fixed, e.g. with 3% paraformaldehyde, and are usually permeabilized, e.g. with ice cold methanol; HEPES-buffered PBS containing 0.1% saponin, 3% BSA; covering for 2 min in acetone at ⁇ 200C; and the like as known in the art and according to the methods described herein
  • one or more cells are contained in a well of a 96 well plate or other commercially available multi-well plate.
  • the reaction mixture or cells are in a cytometric measurement device.
  • Other multi-well plates useful in the present invention include, but are not limited to 384 well plates and 1536 well plates. Still other vessels for containing the reaction mixture or cells and useful in the present invention will be apparent to the skilled artisan.
  • the addition of the components of the assay for detecting the activation level or activity of an activatable element, or modulation of such activation level or activity may be sequential or in a predetermined order or grouping under conditions appropriate for the activity that is assayed for. Such conditions are described here and known in the art. Moreover, further guidance is provided below (see, e.g., in the Examples).
  • the activation level of an activatable element is measured using Inductively Coupled Plasma Mass Spectrometer (ICP-MS).
  • ICP-MS Inductively Coupled Plasma Mass Spectrometer
  • a binding element that has been labeled with a specific element binds to the activatable element.
  • the elemental composition of the cell, including the labeled binding element that is bound to the activatable element is measured.
  • the presence and intensity of the signals corresponding to the labels on the binding element indicates the level of the activatable element on that cell (Tanner et al. Spectrochimica Acta Part B: Atomic Spectroscopy, (2007), 62(3): 188-195.).
  • a chip analogous to a DNA chip can be used in the methods of the present invention.
  • Arrayers and methods for spotting nucleic acid to a chip in a prefigured array are known.
  • protein chips and methods for synthesis are known. These methods and materials may be adapted for the purpose of affixing activation state binding elements to a chip in a prefigured array.
  • such a chip comprises a multiplicity of element activation state binding elements, and is used to determine an element activation state profile for elements present on the surface of a cell.
  • a chip comprises a multiplicity of the “second set binding elements,” in this case generally unlabeled.
  • sample preferably cell extract
  • a second multiplicity of binding elements comprising element activation state specific binding elements is used in the sandwich assay to simultaneously determine the presence of a multiplicity of activated elements in sample.
  • each of the multiplicity of activation state-specific binding elements is uniquely labeled to facilitate detection.
  • confocal microscopy can be used to detect activation profiles for individual cells.
  • Confocal microscopy relies on the serial collection of light from spatially filtered individual specimen points, which is then electronically processed to render a magnified image of the specimen.
  • the signal processing involved confocal microscopy has the additional capability of detecting labeled binding elements within single cells, accordingly in this embodiment the cells can be labeled with one or more binding elements.
  • the binding elements used in connection with confocal microscopy are antibodies conjugated to fluorescent labels, however other binding elements, such as other proteins or nucleic acids are also possible.
  • the methods and compositions of the instant invention can be used in conjunction with an “In-Cell Western Assay.”
  • an assay cells are initially grown in standard tissue culture flasks using standard tissue culture techniques. Once grown to optimum confluency, the growth media is removed and cells are washed and trypsinized. The cells can then be counted and volumes sufficient to transfer the appropriate number of cells are aliquoted into microwell plates (e.g., NuncTM 96 MicrowellTM plates). The individual wells are then grown to optimum confluency in complete media whereupon the media is replaced with serum-free media. At this point controls are untouched, but experimental wells are incubated with a modulator, e.g. EGF.
  • a modulator e.g. EGF.
  • the plates can be scanned using an imager such as the Odyssey Imager (LiCor, Lincoln Nebr.) using techniques described in the Odyssey Operator's Manual v1.2., which is hereby incorporated in its entirety. Data obtained by scanning of the multi-well plate can be analyzed and activation profiles determined as described below.
  • an imager such as the Odyssey Imager (LiCor, Lincoln Nebr.) using techniques described in the Odyssey Operator's Manual v1.2., which is hereby incorporated in its entirety. Data obtained by scanning of the multi-well plate can be analyzed and activation profiles determined as described below.
  • the detecting is by high pressure liquid chromatography (HPLC), for example, reverse phase HPLC, and in a further aspect, the detecting is by mass spectrometry.
  • HPLC high pressure liquid chromatography
  • the living cells may be grown under controlled growth conditions, with controls for temperature, humidity, and gas for time series of the live cell assays. Automated transformation of cells and automated colony pickers may facilitate rapid screening of desired cells.
  • Flow cytometry or capillary electrophoresis formats can be used for individual capture of magnetic and other beads, particles, cells, and organisms.
  • the software program modules allow creation, modification, and running of methods.
  • the system diagnostic modules allow instrument alignment, correct connections, and motor operations.
  • Customized tools, labware, and liquid, particle, cell and organism transfer patterns allow different applications to be performed.
  • Databases allow method and parameter storage. Robotic and computer interfaces allow communication between instruments.
  • the methods of the invention include the use of liquid handling components.
  • the liquid handling systems can include robotic systems comprising any number of components.
  • any or all of the steps outlined herein may be automated; thus, for example, the systems may be completely or partially automated.
  • Fully robotic or microfluidic systems include automated liquid-, particle-, cell- and organism-handling including high throughput pipetting to perform all steps of screening applications.
  • This includes liquid, particle, cell, and organism manipulations such as aspiration, dispensing, mixing, diluting, washing, accurate volumetric transfers; retrieving, and discarding of pipet tips; and repetitive pipetting of identical volumes for multiple deliveries from a single sample aspiration.
  • These manipulations are cross-contamination-free liquid, particle, cell, and organism transfers.
  • This instrument performs automated replication of microplate samples to filters, membranes, and/or daughter plates, high-density transfers, full-plate serial dilutions, and high capacity operation. Additional examples of automation, automated sample collection and analysis are disclosed in U.S. 61/048,657 which is hereby incorporated by reference in its entirety.
  • chemically derivatized particles, plates, cartridges, tubes, magnetic particles, or other solid phase matrix with specificity to the assay components are used.
  • the binding surfaces of microplates, tubes or any solid phase matrices include non-polar surfaces, highly polar surfaces, modified dextran coating to promote covalent binding, antibody coating, affinity media to bind fusion proteins or peptides, surface-fixed proteins such as recombinant protein A or G, nucleotide resins or coatings, and other affinity matrix are useful in this invention.
  • platforms for multi-well plates, multi-tubes, holders, cartridges, minitubes, deep-well plates, microfuge tubes, cryovials, square well plates, filters, chips, optic fibers, beads, and other solid-phase matrices or platform with various volumes are accommodated on an upgradeable modular platform for additional capacity.
  • This modular platform includes a variable speed orbital shaker, and multi-position work decks for source samples, sample and reagent dilution, assay plates, sample and reagent reservoirs, pipette tips, and an active wash station.
  • the methods of the invention include the use of a plate reader.
  • thermocycler and thermoregulating systems are used for stabilizing the temperature of heat exchangers such as controlled blocks or platforms to provide accurate temperature control of incubating samples from 0° C. to 100° C.
  • interchangeable pipet heads with single or multiple magnetic probes, affinity probes, or pipetters robotically manipulate the liquid, particles, cells, and organisms.
  • Multi-well or multi-tube magnetic separators or platforms manipulate liquid, particles, cells, and organisms in single or multiple sample formats.
  • the instrumentation will include a detector, which can be a wide variety of different detectors, depending on the labels and assay.
  • useful detectors include a microscope(s) with multiple channels of fluorescence; plate readers to provide fluorescent, ultraviolet and visible spectrophotometric detection with single and dual wavelength endpoint and kinetics capability, fluorescence resonance energy transfer (FRET), luminescence, quenching, two-photon excitation, and intensity redistribution; CCD cameras to capture and transform data and images into quantifiable formats; and a computer workstation.
  • the robotic apparatus includes a central processing unit which communicates with a memory and a set of input/output devices (e.g., keyboard, mouse, monitor, printer, etc.) through a bus. Again, as outlined below, this may be in addition to or in place of the CPU for the multiplexing devices of the invention.
  • a central processing unit which communicates with a memory and a set of input/output devices (e.g., keyboard, mouse, monitor, printer, etc.) through a bus.
  • input/output devices e.g., keyboard, mouse, monitor, printer, etc.
  • this may be in addition to or in place of the CPU for the multiplexing devices of the invention.
  • the general interaction between a central processing unit, a memory, input/output devices, and a bus is known in the art. Thus, a variety of different procedures, depending on the experiments to be run, are stored in the CPU memory.
  • robotic fluid handling systems can utilize any number of different reagents, including buffers, reagents, samples, washes, assay components such as label probes, etc.
  • flow cytometry applications require software for different phases of operation and analysis, see Ser. No. 61/079,579; 61/079,551; 61/079,537; 61/087,555; 61/085,789 which are hereby incorporated by reference in their entireties.
  • flow cytometry experiments are arrayed and the results are approximated as fold changes using a heat map to facilitate evaluation.
  • arrayed flow cytometry experiments simplify multidimensional flow cytometry data based on experimental design and observed differences between flow cytometry samples.
  • One common way of comparing changes in a set of flow cytometry samples is to overlay histograms of one parameter on the same plot.
  • Arrayed flow cytometry experiments ideally contain a reference sample against which experimental samples are compared. This reference sample is placed in the first position of the array, and subsequent experimental samples follow the control in the sequence. Reference samples can include normal and/or cells associated with a condition (e.g. tumor cells).
  • the populations of interest and the method for characterizing these populations are determined prior to analyzing of data. For instance, there are at least two general ways of identifying populations for data analysis: (i) “Outside-in” comparison of Parameter sets for individual samples or subset (e.g., patients in a trial). In this more common case, cell populations are homogenous or lineage gated in such a way as to create distinct sets considered to be homogenous for targets of interest.
  • An example of sample-level comparison would be the identification of signaling profiles in tumor cells of a patient and correlation of these profiles with non-random distribution of clinical responses. This is considered an outside-in approach because the population of interest is pre-defined prior to the mapping and comparison of its profile to other populations.
  • Each of these techniques capitalizes on the ability of flow cytometry to deliver large amounts of multiparameter data at the single cell level.
  • a condition e.g. neoplastic or hematopoetic condition
  • a third “meta-level” of data exists because cells associated with a condition (e.g. cancer cells) are generally treated as a single entity and classified according to historical techniques.
  • These techniques have included organ or tissue of origin, degree of differentiation, proliferation index, metastatic spread, and genetic or metabolic data regarding the patient.
  • the present invention uses variance mapping techniques for mapping condition signaling space. These methods represent a significant advance in the study of condition biology because it enables comparison of conditions independent of a putative normal control.
  • Traditional differential state analysis methods e.g., DNA microarrays, subtractive Northern blotting
  • They rely on the comparison of cells associated with a condition from each patient sample with a normal control, generally adjacent and theoretically untransformed tissue.
  • they rely on multiple clusterings and re-clusterings to group and then further stratify patient samples according to phenotype.
  • variance mapping of condition states compares condition samples first with themselves and then against the parent condition population.
  • activation states with the most diversity among conditions provide the core parameters in the differential state analysis. Given a pool of diverse conditions, this technique allows a researcher to identify the molecular events that underlie differential condition pathology (e.g., cancer responses to chemotherapy), as opposed to differences between conditions and a proposed normal control.
  • conditions whose signaling response to modulators is similar are grouped together, regardless of tissue or cell type of origin.
  • two conditions e.g. two tumors
  • two tumors that are thought to be relatively alike based on lineage markers or tissue of origin could have vastly different abilities to interpret environmental stimuli and would be profiled in two different groups.
  • CLL serves as an example of the methods of the invention.
  • the data shown in FIGS. 26 , 27 and 28 is a heat map comparing the activation states of multiple activatable elements in 22 CLL patients and 4 control patients. This data demonstrates that B-cells from various CLL patients display distinguishable patterns of activatable elements as visualized by a heat map. An inhibitor or inhibitor plus another modulator further define additional patterns of activatable elements that allow identification, classification and grouping of cryptic or aberrant hematopoetic populations (i.e. patient clustering).
  • patient samples are indicated at the top of the heat map. Each column represents a single patient.
  • CLL indicates that the sample was obtained from a patient diagnosed with CLL.
  • CON indicates that the sample was obtained from a control patient.
  • the heat map legend is indicated at the top of the figure and uses a shaded scale based on the log 10-fold increase, or decrease, in mean fluorescence intensity (MFI), relative to the unstimulated control (0 min).
  • MFI mean fluorescence intensity
  • the heat map defines the activation state of various activatable elements by denoting a change, or lack thereof, in the level of an activatable element revealed by the presence of an inhibitor and/or additional modulator.
  • the heat map can define the presence or absence of an increase in the activation level of a plurality of activatable elements in a cell upon contacting said cell with an inhibitor or a modulator.
  • Labels to the right of the heat map indicate the activatable element detected, e.g. a phospho-protein. Labels to the right also indicate the modulator or inhibitor treatment for that row. “US” indicates unstimulated or untreated.
  • FIG. 28 illustrates a pattern of activation levels of a plurality of activatable elements in a cell.
  • a patient clustering group is comprised of samples from patients that display similar or distinct patterns of activation levels in one or more activatable elements in response to one or more modulators (e.g., an inhibitor, or an inhibitor and another modulator).
  • FIG. 28 illustrates a clustering group comprised of samples from patients in which the activation levels of p-PLC ⁇ 2, p-SyK/Zap-70, p-BLNK and p-Lck are similar in response to the same stimulus.
  • Some patient clustering groups are revealed upon modulation or treatment with an inhibitor as illustrated by the boxed regions.
  • Treatment with H 2 O 2 reveals a patient clustering group defined by the levels of p-PLC ⁇ 2, p-SyK/Zap-70, p-BLNK and p-Lck ( FIG. 28 , bottom right boxed area) that are similar to those of the four control patients ( FIG. 28 , bottom center box). Treatment with H 2 O 2 further reveals a patient clustering group that is distinct from the controls ( FIG. 28 , 9 patients to the left of bottom boxed area). Modulation with H 2 O 2 and BCR crosslinking defines another patient clustering group comprised of samples from patients that display the activation levels of p-BLNK, p-Syk and p-PLC ⁇ 2 ( FIG. 28 , top left boxed area) that are similar to the control patients (top center box). In addition, modulation with H 2 O 2 and BCR crosslinking further reveals another clustering group distinct from the controls (10 patients to the right of top boxed area).
  • a method of deriving a classification involves defining a clustering group.
  • a clustering group is defined by determining the activation state of a plurality of activatable elements from a plurality of cells wherein each cell is derived from an individual with a known conditions and/or known clinical outcome.
  • a clustering group may define a pattern that associated with a known condition or known clinical outcome. Any suitable activatable element can be used wherein the activation level of said activatable element provides useful information regarding a known condition or clinical outcome of a patient.
  • a cell derived from a patient with an unknown condition and/or unknown clinical outcome may be classified depending upon which clustering group it is identified with. This can further lead to diagnosis, prognosis, and/or evaluation or choice of treatment for the patient.
  • kits provided by the invention may comprise one or more of the state-specific binding element described herein, such as phospho-specific antibodies.
  • the kit comprises one or more of the phospho-specific antibodies specific for the proteins selected from the group consisting of PI3-Kinase (p85, p110a, p110b, p110d), Jak1, Jak2, SOCs, Rac, Rho, Cdc42, Ras-GAP, Vav, Tiam, Sos, Dbl, Nck, Gab, PRK, SHP1, and SHP2, SHIP1, SHIP2, sSHIP, PTEN, Shc, Grb2, PDK1, SGK, Akt1, Akt2, Akt3, TSC1,2, Rheb, mTor, 4EBP-1, p70S6Kinase, S6, LKB-1, AMPK, PFK, Acetyl-CoAa Carboxylase, DokS, Rafs, Mos, Tpl
  • the kit comprises one or more of the phospho-specific antibodies specific for the proteins selected from the group consisting of Erk, Syk, Zap70, Lck, Btk, BLNK, Cbl, PLC ⁇ 2, Akt, RelA, p38, S6.
  • the kit comprises one or more of the phospho-specific antibodies specific for the proteins selected from the group consisting of Akt1, Akt2, Akt3, SAPK/JNK1,2,3, p38s, Erk1/2, Syk, ZAP70, Btk, BLNK, Lck, PLC ⁇ , PLC 1 ⁇ 2, STAT1, STAT 3, STAT 4, STAT 5, STAT 6, CREB, Lyn, p-S6, Cbl, NF- ⁇ B, GSK3 ⁇ , CARMA/Bcl10 and Tcl-1.
  • the proteins selected from the group consisting of Akt1, Akt2, Akt3, SAPK/JNK1,2,3, p38s, Erk1/2, Syk, ZAP70, Btk, BLNK, Lck, PLC ⁇ , PLC 1 ⁇ 2, STAT1, STAT 3, STAT 4, STAT 5, STAT 6, CREB, Lyn, p-S6, Cbl, NF- ⁇ B, GSK3 ⁇ , CARMA/Bcl10 and Tcl-1
  • Kits provided by the invention may comprise one or more of the modulators described herein.
  • the kit comprises one or more modulators selected from the group consisting of F(ab)2 IgM, H 2 O 2 , PMA, BAFF, April, SDF1a, CD40L, IGF-1, Imiquimod, polyCpG, IL-7, IL-6, IL-10, IL-27, IL-4, IL-2, IL-3, thapsigargin and a combination thereof.
  • the state-specific binding element of the invention can be conjugated to a solid support and to detectable groups directly or indirectly.
  • the reagents may also include ancillary agents such as buffering agents and stabilizing agents, e.g., polysaccharides and the like.
  • the kit may further include, where necessary, other members of the signal-producing system of which system the detectable group is a member (e.g., enzyme substrates), agents for reducing background interference in a test, control reagents, apparatus for conducting a test, and the like.
  • the kit may be packaged in any suitable manner, typically with all elements in a single container along with a sheet of printed instructions for carrying out the test.
  • kits enable the detection of activatable elements by sensitive cellular assay methods, such as IHC and flow cytometry, which are suitable for the clinical detection, prognosis, and screening of cells and tissue from patients, such as leukemia patients, having a disease involving altered pathway signaling.
  • sensitive cellular assay methods such as IHC and flow cytometry
  • kits may additionally comprise one or more therapeutic agents.
  • the kit may further comprise a software package for data analysis of the physiological status, which may include reference profiles for comparison with the test profile.
  • kits may also include information, such as scientific literature references, package insert materials, clinical trial results, and/or summaries of these and the like, which indicate or establish the activities and/or advantages of the composition, and/or which describe dosing, administration, side effects, drug interactions, or other information useful to the health care provider.
  • kits may also include instructions to access a database such as described in U.S. Ser. No. 61/087,555 for selecting an antibody specific for the pathway of interest.
  • Such information may be based on the results of various studies, for example, studies using experimental animals involving in vivo models and studies based on human clinical trials.
  • Kits described herein can be provided, marketed and/or promoted to health providers, including physicians, nurses, pharmacists, formulary officials, and the like. Kits may also, in some embodiments, be marketed directly to the consumer.
  • BCR B cell receptor
  • CLL chronic lymphocytic leukemia
  • CLL samples from patients showed weak or minimal signaling activity at p72SYK/p70ZAP, Erk1/2, B-Cell linker protein (BLNK) and phospholipase-C ⁇ -2, (PLC ⁇ 2) when stimulated only at the BCR with anti- ⁇ crosslinking, whereas a robust signal was observed in a control Ramos B cell line.
  • the low-level signaling in CLL cells could be accounted for by either a defect in activation of a key protein required for signaling, or by enhanced inhibition mediated by phosphatases such as SHIP-1, SHIP-2, SHP-1, or SHP-2.
  • H 2 O 2 hydrogen peroxide
  • Ramos cells were maintained and cultured using methods known in the art. Cells from CLL patients were obtained using methods known in the art.
  • BCR cross-linking and preparation for staining Cells were thawed at 37° C. in a water bath until partially thawed ( ⁇ 50%). Cells were thawed into 5% FBS/RPMI at room temperature. The cell suspension was added drop-wise into media. Cells were centrifuged at 900 RPM for 10 minutes with no brake. The supernatants were decanted and pellets resuspended in 25 milliliters (ml) of media. Cells were re-centrifuged at 900 RPM for 10 minutes, decanted again and resuspended in 5 ml of 1% FBS/RPMI. Cells were counted with a hemocytometer using trypan blue staining.
  • the concentration of the cells was adjusted as necessary to reduce crowding.
  • Cells were incubated at 37° C. for 2 hours.
  • the final cell concentration, including additives and reagents e.g. stimulant, Fab fragment, H 2 0 2 , aqua, etc.
  • additives and reagents e.g. stimulant, Fab fragment, H 2 0 2 , aqua, etc.
  • Cells were incubated with the different treatments for 15 minutes, unless a time course was performed.
  • the cells were incubated with the different treatments for 5, 10, 30, 60, or 120 minutes. After incubation, the cells were fixed with 1.6% paraformaldehyde for 5 minutes at room temperature in the dark. Cells were then washed with 0.1% BSA/PBS (2 ml) and centrifuged at 2000 RPM for 5 minutes. The supernatant were decanted and pellet resuspended in 1 ml of 100% MeOH (methanol).
  • Tubes were washed with 2 ml of 0.4% BSA PBS (ice cold) and centrifuged at 2000 RPM for 10 minutes at 4° C. Cells were washed twice. Then cells were stained with fluorescent conjugated antibodies specific for CD20, CD3, CD4, CD8, CD5, p-Erk, p-BLNK, p-syk/Zap70, for 25 min. at RT. Cells were placed into 80 wells making a total of 2 plates. The cocktail mix used for the staining is described below:
  • a probability density estimate of log 10 of the fluorescence intensity value of each of the intra-cellular markers was computed for the cells in the CD20+ and CD5+ using the kernel density estimation function on the R-Statistics package (http://www.r-project.org/). The probability density estimates provided were then plotted to visualize the change in the phopho-levels of the various markers from the base-line.
  • FIG. 1 shows that both F(ab) 2 IgM and PMA activates p-Erk and p-Syk/pZap70 in Ramos cells.
  • the large dashed line on the histograms represent the level of fluorescence of unstimulated and unstained cells referred herein after as autofluorescence.
  • the thick solid line represents the level of fluorescence of unstimulated and stained cells referred hereinafter as background.
  • the p-Erk and p-Syk fluorescence is above the autofluorescence and background signal indicating activation of those proteins upon stimulation.
  • Ramos cells (Ramos cell lines) are used as positive control throughout the experiments performed herein.
  • FIG. 3 shows that PMA (thin solid line) activates Erk in pheresed CLL samples.
  • PMA thin solid line
  • F(ab) 2 IgM for 15 min minimally activated signaling in CLL samples.
  • CLL samples were divided into populations with low and high frequency of ZAP70.
  • Zap70 can be a prognostic indicator used for CLL.
  • CLL samples show modest p-Erk (y-axis) and p-Syk (x-axis) phosphorylation when activated with PMA ( FIG. 5 ). These cells show weak activation when activated with F(ab) 2 IgM as shown in FIG. 5 .
  • H 2 O 2 is a known inhibitor of phosphatases. Inhibition of phosphatases activates Erk and Syk as shown in FIG. 6 . Without intending to be limited to any theory, inhibition of phosphatases reveals strong tonic BCR signaling. This tonic signaling is not apparent by F(ab) 2 IgM alone ( FIG. 4-5 ). F(ab) 2 IgM and H 2 O 2 reveals different kinetics subpopulation (heterogeneity) differences between CLL patients ( FIG. 6 ). In contrast, in normal B cells H 2 O 2 blocks phosphatases, but is incapable of activating post BCR by itself ( FIG. 7 ).
  • CLL H 2 O 2 reveals an underlying signaling event, possibly a tonic signaling, that can drive signaling events downstream of BCR (such as Syk and Erk) as shown in FIG. 8 .
  • CLL samples show modest and weak Erk and Syk phosphorylation when activated with PMA and F(ab) 2 IgM, respectively ( FIG. 9 ).
  • PMA PMA
  • F(ab) 2 IgM F(ab) 2 IgM
  • H 2 O 2 H 2 O 2
  • F(ab) 2 IgM and H 2 O 2 reveals: (i) different kinetics (ii) subpopulation heterogeneity and (iii) differences between CLL patients as shown in FIG. 10 .
  • FIG. 12 shows different kinetics of Syk and Erk phosphorylation by F(ab) 2 IgM and H 2 O 2 . Peak phosphorylation of Syk and BLNK occurred after 5 min of activation, whereas peak phosphorylation of Erk after 30 min.
  • FIG. 13 shows moderate activation of Syk/Zap70 and Erk by F(ab) 2 IgM and H 2 O 2 .
  • FIG. 14 shows the kinetics of signaling by F(ab) 2 IgM and H 2 O 2 .
  • FIG. 21 shows minimal activation of Syk/Zap70 and Erk by F(ab)2IgM alone over time.
  • FIG. 22 shows minimal activation of Syk/Zap70 and Erk in response to F(ab)2IgM alone over time.
  • FIG. 23 shows an F(ab)2 time course of CD20+/CD5+ population, without H 2 O 2 .
  • FIGS. 15 and 16 show different levels of BLNK phosphorylation in response to an external stimulus in cells with different levels of ZAP70.
  • FIGS. 17-21 show the kinetics of phosphorylation of PLC ⁇ 2, S6 and Cblin the CD20+/CD5+ cell population of CLL samples in response to B cell receptor crosslinking with and without peroxide.
  • FIG. 24 shows the kinetics of H 2 O 2 treatment in CD20+/CD5+ population of CLL samples.
  • FIG. 25 shows the kinetics of H 2 O 2 treatment in CD20+/CD5+ population of CLL samples.
  • PBMC peripheral blood mononuclear cells
  • Paraformaldehyde-fixed, methanol-permeabilized cells were rehydrated by addition of 2 ml of PBS, gentle resuspension, and then centrifugation. The cells were washed and stained as in Example I except phosphospecific alexa (Ax) dye Ax488 and Ax647 (Molecular Probes) or R-PE-conjugated Abs were also used. Staining cocktails comprised fluor conjugated antibodies specific for p-ERK1/2(T202/Y204), and p-Syk(Y352)/Zap70(Y319), p-Lck/p-Lyn, p-BLNK, p-PLC ⁇ 2 (PLCr2) or p-S6. Detection, selection and gating of cell subsets was as described in Example I. Heat maps were generated using MeV (MultiExperiment Viewer) software ( FIGS. 26 , 27 & 28 ).
  • FIGS. 26 , 27 and 28 demonstrate that B-cells from various CLL patients display distinguishable patterns of activatable elements as visualized by a heat map. Modulators of phosphorylation further define additional patterns of activatable elements that allow identification, classification and grouping of cryptic or aberrant hematopoetic populations.
  • patient samples are indicated at the top of the heat map. Each column represents a single patient.
  • CLL indicates that the sample was obtained from a patient diagnosed with CLL.
  • CON indicates that the sample was obtained from a control patient.
  • FIG. 28 illustrates the identification of several patient clustering groups comprised of similar or distinct levels of p-PLC ⁇ 2, p-SyK/Zap-70, p-BLNK and p-Lck. Some patient clusterings become apparent upon modulation as illustrated by the boxed regions.
  • treatment with H 2 O 2 reveals a patient clustering defined by the levels of p-PLC ⁇ 2, p-SyK/Zap-70, p-BLNK and p-Lck (bottom right boxed area) that are similar to those of the four control patients (bottom center box).
  • Treatment with H 2 O 2 further reveals a patient clustering that is distinct from the controls (9 patients to the left of bottom boxed area).
  • Modulation with H 2 O 2 and BCR crosslinking defines another patient clustering comprised of levels of p-BLNK, p-Syk and p-PLC ⁇ 2 (top left boxed area) that are similar to the control patients (top center box) and an aberrant population of responders (10 patients to the right of top boxed area).
  • rituximab Fludarabine, a purine analog, inhibits DNA synthesis by interfering with ribonucleotide reductase and DNA polymerase.
  • Rituximab is a chimeric CD20 specific antibody and has mechanistically been shown to bind complement, induce antibody-dependent cellular cytotoxicity (ADCC) and, in some situations, rituximab binding to CD20 inhibits proliferation and induces cellular apoptosis (for a discussion of apoptosis see U.S. Ser. No. 61/085,789).
  • Cellular apoptosis in response to therapeutic agents can be measured by multiparameter flow cytometry using fluorophore-conjugated antibodies that recognize intracellular protein components or nodes of the apoptotic machinery.
  • nodes may include, but are not limited to, Caspase 3, Caspase 8, Cytochrome C, Poly ADP ribose polymerase (PARP), Bcl-2, Bcl-X, p-Chk2, p-BAD.
  • Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD. FMK) in order to reveal caspase-dependent and/or independent pathways.
  • OMe Benzyloxycarbonyl-Val-Ala-Asp
  • FMK fluoromethylketone
  • Cells are thawed at 37° C. in water bath until partially thawed ( ⁇ 50%) and are then added to RPMI/1% FBS at room temperature. Cells are centrifuged at 900 RPM for 10 minutes, the supernatants are decanted and pellets are resuspended in 25 milliliters of media. This step is repeated. An aliquot of cells is counted with a hemocytometer using trypan blue staining. Cells are resuspended in RPMI/1% or 10%/FBS at the desired concentration. After an incubation at 37° C. for 2 hr., cells are aliquoted at a concentration of 1 ⁇ 10 6 cells per well of a 96-well plate.
  • Cells are incubated with Fludarabine (at concentrations of 0-50 ⁇ M), Rituximab (at concentrations from 0-500 ⁇ M) and/or Staurosporine, alone or in combinations, in the absence or presence of ZVAD for various times.
  • Post-incubation with drug cells are processed for staining with cocktails of fluorochrome conjugated antibodies including CD3, CD5, CD19, CD20, CD3, CD5, CD19, CD20 (extracellular markers) and fluorochrome conjugated antibodies to the nodes/markers of apoptosis described above.
  • Cells are analyzed by flow cytometry. The details of cell processing and flow cytometry analysis are given in U.S. Ser. No. 61/085,789.

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