Methods for diagnosis, prognosis and methods of treatment

Description of equivalent WO 2009025847 A2

METHODS FOR DIAGNOSIS, PROGNOSIS AND METHODS OF TREATMENT

0002[0001] This application claims the benefit of the filing date of U.S. serial number 60/957,160 filed August 21, 2007 and U.S. serial number 61/048,920 filed April 29, 2008 and each of these provisional applications are hereby expressly incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

0004[0002] Many conditions are characterized by disruptions in cellular pathways that lead, for example, to aberrant control of cellular processes, or to uncontrolled growth and proliferation of cells. These disruptions are often caused by changes in the activity of molecules participating in cellular pathways. For example, specific signaling pathway alterations have been described for many cancers. Despite the increasing evidence that disruption in cellular pathways mediate the detrimental transformation, the precise molecular events underlying these transformations have not been elucidated. As a result, therapeutics may not be effective in treating conditions involving cellular pathways that are not well understood. Thus, the successful diagnosis of a condition and use of therapies will require knowledge of the cellular events that are responsible for the condition pathology. [0003] In addition, patients suffering from different conditions follow heterogeneous clinical courses. For instance, tremendous clinical variability among remissions is also observed in cancer patients, even those that occur after one course of therapy. Some leukemia patients survive for prolonged periods without definitive therapy, while others die rapidly despite aggressive treatment. Patients who are resistant to therapy have very short survival times, regardless of when the resistance occurs. While various staging systems have been developed to address this clinical heterogeneity, they cannot accurately predict whether an early or intermediate stage patient will experience an indolent or aggressive course of disease.

0005[0004] Accordingly, there is a need for a reliable indicator of an individual predicted disease course to help clinicians to identify those patients that will respond to treatment, patients that progress to a more advanced state of the disease and patients with emerging resistance to treatment. SUMMARY OF THE INVENTION

0006[0005] Other objects, features and advantages of the methods and compositions described herein will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

0007[0006] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. [0007] As disclosed herein is 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. In some embodiments the change in activation level of an activatable element is an increase in the activation level of an activatable element. In some embodiments the activatable element is a protein subject to phosphorylation or dephosphorylation. [0008] In some embodiments of the methods, 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 In some embodiments, the inhibitor is a kinase or phosphatase inhibitor, such as adaphostin, AG 490, AG 825, AG 957, AG 1024, aloisme, aloisine A, alsterpaullone, aminogenistein, API-2, apigenm, arctigenin, AY-22989, BAY 61-3606, bisindolylmaleimide IX, chelerythnne, 10- [4'-(N,N-Diethylamino)butyl]-2-chlorophenoxazine hydrochloride, dasatinib, 2-Dimethylamino-4,5,6,7-tetrabromo-lH- benzimidazole, 5,7-Dimethoxy-3-(4-pyπdinyl)quinoline dihydrochloride, edelfosme, ellagic acid, enzastaunn, ER 27319 maleate, erlotinib, ET18OCH3, fasudil, flavopindol, gefitmib, GW 5074, H-7, H-8, H-89, HA-100, HA-1004, HA-1077, HA-1100, hydroxyfasudil, mdirubm-3'-oxime, 5-Iodotubercidm, kenpaullone, KN-62, KY12420, LFM- A13, lavendustin A, luteolm, LY-294002, LY294002, mallotoxin, ML-9, NSC-154020, NSC-226080, NSC- 231634, NSC-664704, NSC-680410, NU6102, olomoucine, oxmdole I, PD-153035, PD-98059, PD-169316, phloretm, phloridzin, piceatannol, picropodophyllin, PKI, PPl, PP2, purvalanol A, quercetin, R406, R788, rapamune, rapamycin, Ro 31-8220, roscovitme, rottleπn, SB202190, SB203580, sirolimus, sorafenib, SL327, SP600125, staurospoπne, STI-571, SU-11274, SU1498, SU4312, SU6656, 4,5,6,7-Tetrabromotπazole, TG101348, Tπciπbme, Tyrphostm AG 490, Tyrphostin AG 825, Tyrphostin AG 957, Tyrphostin AG 1024, Tyrphostin SU1498, U0126, VX-509, VX-667, VX-680, W-7, wortmannm, XL-019, XL-147, XL- 184, XL-228, XL-281, XL-518, XL- 647, XL-765, XL-820, XL-844, XL-880, Y-27632, ZD-1839, ZM-252868, ZM-447439, H₂O₂, siRNA, miRNA, Canthaπdm, (-)-p-Bromotetramisole, Microcystin LR, Sodium Orthovanadate, Sodium Pervanadate, Vanadyl sulfate, Sodium oxodiperoxo(l,10-phenanthroline)vanadate, bis(maltolato)oxovanadium(IV), Sodium Mo lybdate, Sodium Perm olybdate, Sodium Tartrate, Imidazole, Sodium Fluoride, β-Glycerophosphate, Sodium Pyrophosphate Decahydrate, Calyculin A, Discodermia calyx, bpV(phen), mpV(pic), DMHV, Cypermethπn, Dephostatm, Okadaic Acid, NIPP-I, N-(9,10-Dioxo-9,10-dihydro-phenanthren-2-yl)-2,2-dimethyl-propionamide, α-Bromo-4- hydroxyacetophenone, 4-Hydroxyphenacyl Br, α-Bromo-4-methoxyacetophenone, 4-Methoxyphenacyl Br, α- Bromo-4-(carboxymethoxy)acetoρhenone, 4-(Carboxymethoxy)phenacyl Br, and bis(4- Tnfluoromethylsulfonamidophenyl)- 1 ,4-diisopropylbenzene, phenyarsine oxide, Pyrrolidme Dithiocarbamate, or Aluminum fluoride In some embodiments the phosphatase inhibitor is H₂O₂ [0009] In some embodiments the cell is a hematopoietic-deπved cell In some embodiments, the hematopoietically denved cell is selected from the group consisting of pluπpotent hematopoietic stem cells, B- lymphocyte lineage progenitor or denved cells, T-lymphocyte lineage progenitor or derived cells, NK cell lmeage progenitor or denved cells, granulocyte lineage progemtor or denved cells, monocyte lmeage progemtor or denved cells, megakaryocyte lmeage progemtor or denved cells and erythroid lineage progemtor or denved cells In some embodiments, the hematopoietic denved cell is a B-lymphocyte lmeage progemtor and denved 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 beanng a mutatated or non mutated heavy chain of the B cell receptor, or a B cell expressing Zap70

0008[0010] In some embodiments, the classification mcludes classifying the cell as a cell that is correlated with a clinical outcome In some embodiments, the clinical outcome is the prognosis and/or diagnosis of a condition In some embodiments, 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 chrome 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 In some embodiments, the neoplastic or hematopoietic condition is non-B lmeage denved, 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 In some embodiments, the neoplastic or hematopoietic condition is a B-CeIl or B cell lmeage deπved disorder, such as Chronic Lymphocytic Leukemia (CLL), B lymphocyte lineage leukemia, B lymphocyte lineage lymphoma, Multiple Myeloma, acute lymphoblastic leukemia (ALL), B-cell pro-lymphocyhc 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 In some embodiments, the condition is CLL In some embodiments, 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 [0011] In some embodiments, the clinical outcome is the staging or gradmg of a neoplastic or hematopoietic condition Examples of staging in methods provided by the invention include aggressive, indolent, benign, refractory, Roman Numeral stagmg, TNM Staging, Rai staging, Bmet 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

0009[0012] In some embodiments of the methods of the invention, classifying the cell based on activation levels of activatable element mcludes 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 compπse 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, mduction therapy, maintenance therapy, watchful waiting, or holistic/alternative therapy

0010[0013] In some embodiments of the methods of the invention, the classifying of the cell based on activation level mcludes classifying the cell as a cell that is correlated with mimmal residual disease or emerging resistance [0014] In some embodiments of the mvention, 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-ohgomer formation, glycosylation level, phosphorylation level, acetylation level, methylation level, biotinylation level, glutamylation level, glycylation level, hydroxylation level, isomenzation level, prenylation level, mynstoylation 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 protem turnover, multi-protein complex level, oxidation level, multi-hpid complex, and biochemical changes in cell membrane In some embodiments, the activation level is a phosphorylation level In some embodiments, the activatable element is selected from the group consisting of proteins, carbohydrates, lipids, nucleic acids and metabolites In some embodiments, the activatable element is a protem In some embodiments, the activatable element is a change m metabolic state, temperature, or local ion concentration In embodiments where the activatable element is a protem, m some embodiments the protem is a protem subject to phosphorylation or dephosphorylation, such as kinases, phosphatases, adaptor/scaffold proteins, ubiquitination enzymes, adhesion molecules, contractile protems, cytoskeletal protems, heterotnmenc 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. In some embodiments, the protein is selected from the group consisting of PI3-Kinase (p85, pi 10a, pi 10b, pi 1Od), Jakl, Jak2, SOCs, Rac, Rho, Cdc42, Ras-GAP, Vav, Tiam, Sos, DbI, Nek, Gab, PRK, SHPl, and SHP2, SHIPl, SHIP2, sSHIP, PTEN, She, Grb2, PDKl, SGK, Aktl, Akt2, Akt3, TSC1,2, Rheb, mTor, 4EBP-1, p70S6Kinase, S6, LKB-I, AMPK, PFK, Acetyl-CoAa Carboxylase, DokS, Rafs, Mos, Tpl2, MEK1/2, MLK3, TAK, DLK, MKK3/6, MEKK 1,4, MLK3, ASKl, MKK4/7, SAPK/JNK1,2,3, p38s, Erkl/2, Syk, Btk, BLNK, LAT, ZAP70, Lck, CbI, SLP-76, PLCγi, PLCγ2, STATl, STAT 3, STAT 4, STAT 5, STAT 6, FAK, pl30CAS, PAKs, LIMKl/2, Hsp90, Hsp70, Hsp27, SMADs, ReI-A (p65- NFKB), CREB, Histone H2B, HATs, HDACs, PKR, Rb, Cyclin D, Cyclin E, Cyclin A, Cyclin B, P16, pl4Arf, p27KIP, p21CIP, Cdk4, Cdk6, Cdk7, Cdkl, Cdk2, Cdk9, Cdc25,A/B/C, AbI, E2F, FADD, TRADD, TRAF2, RIP, Myd88, BAD, Bcl-2, McI-I, BcI-XL, Caspase 2, Caspase 3, Caspase 6, Caspase 7, Caspase 8, Caspase 9, PARP, IAPs, Smac, Fodrin, Actin, Src, Lyn, Fyn, Lck, NDC, IKB, p65(RelA), IKKα, PKA, PKC«, PKCβ, PKCQ, PKCδ, CAMK, Elk, AFT, Myc, Egr-1, NFAT, ATF-2, Mdm2, p53, DNA-PK, Chkl, Chk2, ATM, ATR, βcatenin, CrkL, GSK3α, GSK3β, and FOXO. In some embodiments, the protein selected from the group consisting of Erk, Syk, Zap70, Lck, Btk, BLNK, CbI, PLCγ2, Akt, ReIA, p38, S6. In some embodiments the protein is S6. [0015] In some embodiments, 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, TIEl, TIE2, FAK, Jakl, Jak2, Jak3, Tyk2, Src, Lyn, Fyn, Lck, Fgr, Yes, Csk, AbI, Btk, ZAP70, Syk, IRAKs, cRaf, ARaf, BRAF, Mos, Lim kinase, ILK, TpI, ALK, TGFβ receptors, BMP receptors, MEKKs, ASK, MLKs, DLK, PAKs, Mek 1, Mek 2, MKK3/6, MKK4/7, ASKl.Cot, NDC, Bub, Myt 1, Weel, Casein kinases, PDKl, SGKl, SGK2, SGK3, Aktl, Akt2, Akt3, p90Rsks, p70S6Kinase,Prks, PKCs, PKAs, ROCK 1, ROCK 2, Auroras, CaMKs, MNKs, AMPKs, MELK, MARKs, Chkl, Chk2, LKB-I, MAPKAPKs, Piml, Pim2, Pim3, DCKs, Cdks, Jnks, Erks, DCKs, GSK3α, GSK3β, Cdks, CLKs, PKR, PI3-Kinase class 1, class 2, class 3, mTor,

0011SAPK/JNK1,2,3, p38s, PKR, DNA-PK, ATM, ATR, Receptor protein tyrosine phosphatases (RPTPs), LAR phosphatase, CD45, Non receptor tyrosine phosphatases (NPRTPs), SHPs, MAP kinase phosphatases (MKPs), Dual Specificity phosphatases (DUSPs), CDC25 phosphatases, Low molecular weight tyrosine phosphatase, Eyes absent (EYA) tyrosine phosphatases, Slingshot phosphatases (SSH), serine phosphatases, PP2A, PP2B, PP2C, PPl, PP5, inositol phosphatases, PTEN, SHIPs, myotubularins, phosphoinositide kinases, phospholipases, prostaglandin synthases, 5-lipoxygenase, sphingosine kinases, sphingomyelinases, adaptor/scaffold proteins, She, Grb2, BLNK, LAT, B cell adaptor for PI3-kinase (BCAP), SLAP, Dok, KSR, MyD88, Crk, CrkL, GAD, Nek, Grb2 associated binder (GAB), Fas associated death domain (FADD), TRADD, TRAF2, RIP, T-CeIl leukemia family, IL-2, IL-4, IL-8, IL-6, interferon γ, interferon α, suppressors of cytokine signaling (SOCs), CbI, SCF ubiquitination ligase complex, APC/C, adhesion molecules, integrins, Immunoglobulin-like adhesion molecules, selectins, cadherins, catenins, focal adhesion kinase, pl30CAS, fodrin, actin, paxillin, myosin, myosin binding proteins, tubulin, eg5/KSP, CENPs, β-adrenergic receptors, muscarinic receptors, adenylyl cyclase receptors, small molecular weight GTPases, H-Ras, K-Ras, N-Ras, Ran, Rac, Rho, Cdc42, Arfs, RABs, RHEB, Vav, Tiam, Sos, DbI, PRK, TSCl, 2, Ras-GAP, Arf-GAPs, Rho-GAPs, caspases, Caspase 2, Caspase 3, Caspase 6, Caspase 7, Caspase 8, Caspase 9, PARP, Bcl-2, McI-I, BcI-XL, Bcl-w, BcI-B, Al , Bax, Bak, Bok, Bik, Bad, Bid, Bim, Bmf, Hrk, Noxa, Puma, IAPs, XIAP, Smac, Cdk4, Cdk 6, Cdk 2, Cdkl, Cdk 7, Cyclin D, Cyclin E, Cyclin A, Cyclin B, Rb, pl6, pl4Arf, p27KIP, p21CIP, molecular chaperones, Hsp90s, Hsp70, Hsρ27, metabolic enzymes, Acetyl-CoAa Carboxylase, ATP citrate lyase, nitric oxide synthase, caveolins, endosomal sorting complex required for transport (ESCRT) proteins, vesicular protein sorting (Vsps), hydroxylases, prolyl-hydroxylases PHD-I, 2 and 3, asparagine hydroxylase FIH transferases, Pinl prolyl isomerase, topoisomerases, deacetylases, Histone deacetylases, sirtuins, histone acetylases, CBP/P300 family, MYST family, ATF2, DNA methyl transferases, Histone H3K4 demethylases, H3K27,

0012JHDM2A, UTX, VHL, WT-I, p53, Hdm, PTEN, ubiquitin proteases, urokinase-type plasminogen activator (uPA) and uPA receptor (uPAR) system, cathepsins, metalloproteinases, esterases, hydrolases, separase, potassium channels, sodium channels, , multi-drug resistance proteins, P-Gycoprotein, nucleoside transporters, , Ets, Elk, SMADs, ReI-A (p65-NFKB), CREB₁ NFAT, ATF-2, AFT, Myc, Fos, SpI, Egr-1, T-bet, β-catemn, HIFs, FOXOs, E2Fs, SRFs, TCFs, Egr-1, β-catemn, FOXO STATl, STAT 3, STAT 4, STAT 5, STAT 6, p53, WT-I, HMGA, pS6, 4EPB-1, eIF4E-binding protein, RNA polymerase, initiation factors, elongation factors [0016] In some embodiments, 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 In some embodiments, the cell is a hematopoietic deπved cell In some embodiments, the hematopoietically denved cell is selected from the group consisting of plunpotent hematopoietic stem cells, B- lymphocyte lineage progemtor or deπved cells, T-lymphocyte lineage progenitor or denved cells, NK cell lmeage progemtor or denved cells, granulocyte lineage progemtor or denved cells, monocyte lineage progemtor or denved cells, megakaryocyte lineage progenitor or denved cells and erythroid lmeage progemtor or denved cells In some embodiments, the hematopoietic denved cell is a B-lymphocyte lineage progenitor and denved 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 In some embodiments, the condition is a neoplastic or hematopoietic condition [0017] In some embodiments of the methods of the invention, the modulator to which the cell is subjected is an activator or an inhibitor In some embodiments, 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 In some embodiments, 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 In some embodiments, the cross-linker is an antibody, or molecular bmding entity In some embodiments, the cross-linker is an antibody, such as 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 In some embodiments, the cross-linker is a molecular bmding entity, such as an entity that acts upon or bmds the B cell receptor complex via carbohydrates or an epitope m the complex In some embodiments, the molecular bmding entity is a monovalent, bivalent, or multivalent bmding entity that is made more multivalent by attachment to a solid surface or tethered on a nanoparticle surface to mcrease the local valency of the epitope binding domain In some embodiments where 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 bmding of an antibody or molecular bmdmg entity to the cell and then causmg its crosslinking via mteraction of the cell with a solid surface that causes crosshnking of the BCR complex via antibody or molecular bmdmg entity In some embodiments, the crosslinker is selected from the group consisting of F(ab)2 IgM₅ , IgG, IgD, polyclonal BCR antibodies, monoclonal BCR antibodies, Fc receptor denved 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 In some embodiments, 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

0013[0018] In some embodiments, 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, arctigemn, AY-22989, BAY 61-3606, bisindolylmaleimide IX, chelerythnne, 10-[4'-(N,N-Diethylamino)butyl]-2-chlorophenoxazine hydrochloπde, dasatinib, 2-Dimethylamino-4,5,6,7-tetrabromo-lH-benzimidazole, 5,7-Dimethoxy-3-(4-pyridinyl)quinohne dihydrochloπde, edelfosine, ellagic acid, enzastauπn, ER 27319 maleate, erlotmib, ET18OCH3, fasudil, flavopindol, gefitinib, GW 5074, H-7, H-8, H-89, HA-100, HA-1004, HA-1077, HA-1100, hydroxyfasudil, indirubin-3'-oxime, 5-Iodotubercidin, kenpaullone, KN-62, KY12420, LFM-A13, lavendustm A, luteolin, LY- 294002, LY294002, mallotoxin, ML-9, NSC-154020, NSC-226080, NSC-231634, NSC-664704, NSC-680410, NU6102, olomoucine, oxindole I, PD-153035, PD-98059, PD-169316, phloretin, phlondzin, piceatannol, picropodophyllin, PKI, PPl, PP2, purvalanol A, quercetin, R406, R788, rapamune, rapamycin, Ro 31-8220, roscovitine, rottlenn, SB202190, SB203580, sirolimus, sorafemb, SL327, SP600125, staurosponne, STI-571, SU- 11274, SU1498, SU4312, SU6656, 4,5,6,7-Tetrabromotπazole, TG101348, Tπcinbnie, Tyrphostin AG 490, Tyrphostin AG 825, Tyrphostin AG 957, Tyrphostin AG 1024, Tyrphostin SU1498, U0126, VX-509, VX-667, VX- 680, W-7, wortmannin, XL-019, XL-147, XL-184, XL-228, XL-281, XL-518, XL-647, XL-765, XL-820, XL-844, XL-880, Y-27632, ZD-1839, ZM-252868, ZM-447439, H₂O₂, siRNA, miRNA, Canthaπdin, (-)-p-

0014Bromotetramisole, Microcystm LR, Sodium Orthovanadate, Sodium Pervanadate, Vanadyl sulfate, Sodium oxodiperoxo(l,10-phenanthrolme)vanadate, bis(maltolato)oxovanadium(IV), Sodium Molybdate, Sodium Perm olybdate, Sodium Tartrate, Imidazole, Sodium Fluoride, β-Glycerophosphate, Sodium Pyrophosphate Decahydrate, Calyculin A, Discodermia calyx, bpV(phen), mpV(pic), DMHV, Cypermethπn, Dephostata, Okadaic Acid, NIPP- l, N-(9,10-Dioxo-9,10-dihydro-phenanthren-2-yl)-2,2-dimethyl-propionamide, α-Bromo-4-hydroxyacetophenone, 4-Hydroxyphenacyl Br, α-Bromo-4-methoxyacetophenone, 4-Methoxyphenacyl Br, α-Bromo-4- (carboxymethoxy)acetophenone, 4-(Carboxymethoxy)phenacyl Br, and bis(4-Tπfluoromethylsulfonamidophenyl)- 1,4-dusopropylbenzene, phenyarsine oxide, Pyrrolidine Dithiocarbamate, or Aluminum fluoride In some embodiments the phosphatase inhibitor is H₂O₂ [0019] In some embodiments of the methods of the invention, the cell is subjected to a B cell receptor activator and a phosphatase inhibitor or kinase inhibitor, such as F(ab)₂IgM or biotinylated F(ab)₂IgM and a phosphatase inhibitor (e g H₂O₂)

0015[0020] In some embodiments, 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 tome signaling pathway in the cell from the activation level In some embodiments, a condition of an individual is determined based on tonic signaling status of a cell In some embodiments, the condition is a neoplastic and/or hematopoietic condition In some embodiments, the neoplastic or hematopoietic condition is selected from the group consistmg 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 macroglobulmemia, myelodysplastic disorders, myeloproliferative disorders, myelofibrosis, and atypical immune lymphoprohferations In some embodiments, the neoplastic or hematopoietic condition is non-B lineage denved, 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 In some embodiments, the neoplastic or hematopoietic condition is a B-CeIl or B cell lineage deπved disorder such as B-CeIl or B cell lineage denved disorder is selected from the group consisting of Chrome Lymphocytic Leukemia (CLL), B lymphocyte lineage leukemia, B lymphocyte lineage lymphoma, Multiple Myeloma, and plasma cell disorders, e g , amyloidosis and Waldenstrom's macroglobulinemia In some embodiments, the condition is CLL In some embodiments, the CLL is defined by a monoclonal B cell population that co-expresses CD5 with CD 19 and CD23 or CD5 with CD20 and CD23 and by surface immunoglobulin expression (0021] In some embodiments, the tonic signaling status of a cell is correlated with a clinical outcome such as prognosis or diagnosis of the condition In some embodiments, the clinical outcome is the staging or gradmg 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, EPSS score, WPSS score, limited stage, extensive stage, staging accordmg 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

0016[0022] In some embodiments, 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 [0023] In some embodiments, the correlation mcludes classifying the cell as minimal residual disease or emerging resistance The correlation may further mclude determining a method of treatment, such as chemotherapy, biological therapy, radiation therapy, bone marrow transplantation, Peπpheral 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 waitmg [0024] In some embodiments of this aspect, the invention provides a method of correlating an activation level of a B-lymphocyte lineage deπved cell with a neoplastic or hematopoietic condition m an individual by subjectmg the B- lymphocyte lmeage deπved cell from the individual to a modulator, determining the activation levels of a plurality of activatable elements that participate in a tome signaling pathway m the B-lymphocyte lineage deπved cell, and identifying a pattern of the activation levels of the plurality of activatable elements in the tonic signaling pathway m the cell that correlates with a cluneal outcome, such as the prediction of outcome for a particular treatments prognosis or diagnosis of a cetam condition (e g a neoplastic condition) In some embodiments, the B-lymphocyte lmeage progemtor or deπved 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 expressmg Zap70 In some embodiments, the correlation is determining a clinical outcome, such as prognosis or diagnosis of the condition

0017[0025] In some embodiments of the methods of the invention, the modulator to which the cell is subjected is an activator or an inhibitor In some embodiments, 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 In some embodiments, 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 In some embodiments, the cross-linker is an antibody, or molecular binding entity In some embodiments, the cross-linker is an antibody, such as 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. In some embodiments, 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. In some embodiments, 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. In some embodiments where 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-. In some embodiments, 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. In some embodiments, 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.

0018[0026] In some embodiments of the methods of the invention, 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. In some embodiments, 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-lH-benzimidazole, 5,7-Dimethoxy-3-(4-pyridinyl)quinoline dihydrochloride, edelfosine, ellagic acid, enzastaurin, ER 27319 maleate, erlotinib, ET18OCH3, fasudil, flavopiridol, gefitinib, GW 5074, H-7, H-8, H-89, HA-100, HA-1004, HA-1077, HA-1100, hydroxyfasudil, indirubin-3'-oxime, 5- Iodotubercidin, kenpaullone, KN-62, KY12420, LFM-A13, lavendustin A, luteolin, LY-294002, LY294002, mallotoxin, ML-9, NSC-154020, NSC-226080, NSC-231634, NSC-664704, NSC-680410, NU6102, olomoucine, oxindole I, PD-153035, PD-98059, PD-169316, phloretin, phloridzin, piceatannol, picropodophyllin, PKI, PPl, PP2, purvalanol A, quercetin, R406, R788, rapamune, rapamycin, Ro 31-8220, roscovitine, rottlerin, SB202190, SB203580, sirolimus, sorafenib, SL327, SP600125, staurosporine, STI-571, SU-11274, SU1498, SU4312, SU6656, 4,5,6,7-Tetrabromotriazole, TG101348, Triciribine, Tyrphostin AG 490, Tyrphostin AG 825, Tyrphostin AG 957, Tyrphostin AG 1024, Tyrphostin SU1498, U0126, VX-509, VX-667, VX-680, W-7, wortmannin, XL-019, XL-147, XL-184, XL-228, XL-281, XL-518, XL-647, XL-765, XL-820, XL-844, XL-880, Y-27632, ZD-1839, ZM-252868, ZM-447439, H₂O₂, siRNA, miRNA, Cantharidin, (-)-p-Bromotetramisole, Microcystin LR, Sodium Orthovanadate, Sodium Pervanadate, Vanadyl sulfate, Sodium oxodiperoxo(l,10-phenanthroline) vanadate, bis(maltolato)oxovanadium(IV), Sodium Molybdate, Sodium Perm olybdate, Sodium Tartrate, Imidazole, Sodium Fluoride, β-Glycerophosphate, Sodium Pyrophosphate Decahydrate, Calyculin A, Discodermia calyx, bpV(phen), mpV(pic), DMHV, Cypermethrin, Dephostatin, Okadaic Acid, NIPP-I, N-(9,10-Dioxo-9,10-dihydro-phenanthren-2- yl)-2,2-dimethyl-proρionamide, α-Bromo-4-hydroxyacetophenone, 4-Hydroxyphenacyl Br, α-Bromo-4- methoxyacetophenone, 4-Methoxyphenacyl Br, α-Bromo-4-(carboxymethoxy)acetophenone, 4- (Carboxymethoxy)phenacyl Br, and bis(4-Trifluoromethylsulfonamidoρhenyl)- 1 ,4-diisopropylbenzene, phenyarsine oxide, Pyrrolidine Dithiocarbamate, or Aluminum fluoride. In some embodiments the phosphatase inhibitor is H₂O₂. [0027] In some embodiments of the methods of the invention, 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)₂IgM or biotinylated F(ab)₂IgM and a phosphatase inhibitor (e g H₂O₂)

0019[0028] In some embodiments of the invention, 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-ohgomer formation, glycosylation level, phosphorylation level, acetylation level, methylation level, biotinylation level, glutamylation level, glycylation level, hydroxylation level, lsomenzation level, prenylation level, mynstoylation level, lipoylation level, phosphopantetheinylation level, sulfation level, ISGylation level, mtrosylation level, palmitoylation level, SUMOylation level, ubiquitination level, neddylation level, citrullination level, deamidation level, disulfide bond formation level, proteolytic cleavage level, translocation level, changes m protein turnover, multi-protein complex level, oxidation level, multi-hpid complex, and biochemical changes in cell membrane In some embodiments, the activation level is a phosphorylation level In some embodiments, the activatable element is selected from the group consisting of proteins, carbohydrates, lipids, nucleic acids and metabolites In some embodiments, the activatable element is a protem In some embodiments, the activatable element is a change m metabolic state, temperature, or local ion concentration In embodiments where the activatable element is a protem, in some embodiments the protem is a protem subject to phosphorylation or dephosphorylation, such as kinases, phosphatases, adaptor/scaffold proteins, ubiquitination enzymes, adhesion molecules, contractile proteins, cytoskeletal proteins, heterotnmeπc G proteins, small molecular weight GTPases, guanine nucleotide exchange factors, GTPase activatmg proteins, caspases and proteins involved m apoptosis (e g PARP), ion channels, molecular transporters, molecular chaperones, metabolic enzymes, vesicular transport proteins, hydroxylases, lsomerases, transferases, deacetylases, methylases, demethylases, proteases, esterases, hydrolases, DNA bmdmg proteins or transcription factors In some embodiments, the protein is selected from the group consisting of PI3-Kinase (p85, pllOa, pllOb, pllOd), Jakl, Jak2, SOCs, Rac, Rho, Cdc42, Ras-GAP, Vav, Tiam, Sos, DbI, Nek, Gab, PRK, SHPl, and SHP2, SHIPl, SHIP2, sSHIP, PTEN, She, Grb2, PDKl, SGK, Aktl, Akt2, Akt3, TSC1.2, Rheb, mTor, 4EBP-1, p70S6Kmase, S6, LKB-I, AMPK, PFK, Acetyl-CoAa Carboxylase, DokS, Rafs, Mos, Tpl2, MEK 1/2, MLK3, TAK, DLK, MKK3/6, MEKK1.4, MLK3, ASKl, MKK4/7, SAPK/JNK1,2,3, p38s, Erkl/2, Syk, Btk, BLNK, LAT, ZAP70, Lck, CbI, SLP-76, PLCγi, PLCγ2, STATl, STAT 3, STAT 4, STAT 5, STAT 6, FAK, pl30CAS, PAKs, LIMKl/2, Hsp90, Hsp70, Hsp27, SMADs, ReI-A (p65- NFKB), CREB, Histone H2B, HATs, HDACs, PKR, Rb, Cyclin D, Cyclm E, Cyclin A, Cyclin B, P16, pl4Arf, p27KIP, p21CIP, Cdk4, Cdk6, Cdk7, Cdkl, Cdk2, Cdk9, Cdc25,A/B/C, AbI, E2F, FADD, TRADD, TRAF2, RIP, Myd88, BAD, Bcl-2, McI-I, BcI-XL, Caspase 2, Caspase 3, Caspase 6, Caspase 7, Caspase 8, Caspase 9, PARP, IAPs, Smac, Fodrm, Actm, Src, Lyn, Fyn, Lck, NIK, IKB, p65(RelA), KKa, PKA, PKCa, PKCβ, PKC0, PKCδ, CAMK, Elk, AFT, Myc, Egr-1, NFAT, ATF-2, Mdm2, p53, DNA-PK, CbM, Chk2, ATM, ATR, βcatenin, CrkL, GSK3α, GSK3β, and FOXO In some embodiments, the protem selected from the group consisting of Erk, Syk, Zap70, Lck, Btk, BLNK, CbI, PLCγ2, Akt, ReIA, p38, S6 In some embodiments the protem is S6

0020[0029] In some embodiments, the protem 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, TIEl, TIE2, FAK, Jakl, Jak2, Jak3, Tyk2, Src, Lyn, Fyn, Lck, Fgr, Yes, Csk, AbI, Btk, ZAP70, Syk, IRAKs, cRaf, ARaf, BRAF, Mos, Lim kinase, ILK, TpI, ALK, TGFβ receptors, BMP receptors, MEKKs, ASK, MLKs, DLK, PAKs, Mek 1, Mek 2, MKK3/6, MKK4/7, ASKl.Cot, NDC, Bub, Myt 1, Weel, Casern kinases, PDKl, SGKl, SGK2, SGK3, Aktl, Akt2, Akt3, p90Rsks, p70S6Kinase,Prks, PKCs, PKAs, ROCK 1, ROCK 2, Auroras, CaMKs, MNKs, AMPKs, MELK, MARKs, Chkl, Chk2, LKB-I, MAPKAPKs, Piml, Pim2, Pim3, IKKs, Cdks, Jnks, Erks, IKKs, GSK3α, GSK3β, Cdks, CLKs, PKR, PI3-Kinase class 1, class 2, class 3, mTor, SAPK/JNK1,2,3, p38s, PKR, DNA-PK, ATM, ATR, Receptor protein tyrosine phosphatases (RPTPs), LAR phosphatase, CD45, Non receptor tyrosine phosphatases (NPRTPs), SHPs, MAP kinase phosphatases (MKPs), Dual Specificity phosphatases (DUSPs), CDC25 phosphatases, Low molecular weight tyrosine phosphatase, Eyes absent (EYA) tyrosine phosphatases, Slingshot phosphatases (SSH), serine phosphatases, PP2A, PP2B, PP2C, PPl, PP5, inositol phosphatases, PTEN, SHIPs, myotubulaπns, phosphoinositide kinases, phosphokpases, prostaglandin synthases, 5-hpoxygenase, sphingosine kinases, sphingomyelinases, adaptor/scaffold proteins, She, Grb2, BLNK, LAT, B cell adaptor for PI3-kinase (BCAP), SLAP, Dok, KSR, MyD88, Crk, CrkL, GAD, Nek, Grb2 associated binder (GAB), Fas associated death domain (FADD), TRADD, TRAF2, RIP, T-CeIl leukemia family, IL-2, IL-4, IL-8, IL-6, interferon γ, interferon α, suppressors of cytokine signaling (SOCs), CbI, SCF ubiquitination hgase complex, APC/C, adhesion molecules, lntegrins, Immunoglobulin-hke adhesion molecules, selectins, cadhenns, catenins, focal adhesion kinase, ρl30CAS, fodπn, actin, paxillin, myosin, myosin binding proteins, tubulin, eg5/KSP, CENPs, β-adrenergic receptors, muscarinic receptors, adenylyl cyclase receptors, small molecular weight GTPases, H-Ras, K-Ras, N-Ras, Ran, Rac, Rho, Cdc42, Arfs, RABs, RHEB, Vav, Tiam, Sos, DbI, PRK, TSC 1,2, Ras-GAP, Arf-GAPs, Rho-GAPs, caspases, Caspase 2, Caspase 3, Caspase 6, Caspase 7, Caspase 8, Caspase 9,

0021PARP, Bcl-2, McI-I, BcI-XL, Bcl-w, BcI-B, Al, Bax, Bak, Bok, Bik, Bad, Bid, Bun, Bmf, Hrk, Noxa, Puma, IAPs, XIAP, Smac, Cdk4, Cdk 6, Cdk 2, Cdkl, Cdk 7, Cyclin D, Cyclin E, Cyclin A, Cyclin B, Rb, pl6, pl4Arf, p27KIP, p21CIP, molecular chaperones, Hsp90s, Hsp70, Hsp27, metabolic enzymes, Acetyl-CoAa Carboxylase, ATP citrate lyase, nitric oxide synthase, caveolins, endosomal sorting complex required for transport (ESCRT) proteins, vesicular protein sorting (Vsps), hydroxylases, prolyl-hydroxylases PHD-I, 2 and 3, asparagine hydroxylase FIH transferases, Pinl prolyl isomerase, topoisomerases, deacetylases, Histone deacetylases, sirtums, histone acetylases, CBP/P300 family, MYST family, ATF2, DNA methyl transferases, Histone H3K4 demethylases, H3K27, JHDM2A, UTX, VHL, WT-I, p53, Hdm, PTEN, ubiquitin proteases, urokinase-type plasminogen activator (uPA) and uPA receptor (uPAR) system, cathepsms, metalloprotemases, esterases, hydrolases, separase, potassium channels, sodium channels, , multi-drug resistance proteins, P-Gycoprotein, nucleoside transporters, , Ets, Elk,

0022SMADs, ReI-A (p65-NFKB), CREB, NFAT, ATF-2, AFT, Myc, Fos, SpI, Egr-1, T-bet, β-catenin, HIFs, FOXOs, E2Fs, SRFs, TCFs, Egr-1, β^'-catenin, FOXO STATl, STAT 3, STAT 4, STAT 5, STAT 6, p53, WT-I, HMGA, pS6, 4EPB-1, eIF4E-binding protein, RNA polymerase, initiation factors, elongation factors [0030] In addition to determining the activation level of an activatable protein, m some embodiments the methods for classifying a cell further compnse determining the level of an additional intracellular marker and/or a cell surface marker In some embodiments the methods for classifying a cell compnse 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 In some embodiments the cell surface marker is CD45, CD5, CD 19, CD20, CD22, CD23, CD27, CD37, CD40, CD52, CD79, CD38, CD96, major histocompatability antigen (MHC) Class 1 or MHC Class 2

0023[0031] In some embodiments the methods of the invention for prognosis, diagnosis, or determination of treatment further compnse determining the level of an additionalserum marker In some embodiments the serum marker compnses a protein In some embodiments the serum marker is a cytokine, growth factor, chemokine, soluble receptor, small compound, or phamaceutical drug In some embodiments the serum marker compnses a component or product of a pathogen or parasite In some embodiment the serum marker is selected from a group consisting of beta-2-microglobulin, calcitonin, thymidine kinase and ferritin

0024[0032] In some embodiments, the invention provides a method of correlating an activation level of B-lymphocyte lineage denved cells with a neoplastic or hematopoietic condition in an individual by subjecting the B-lymphocyte lineage denved cell from the individual to a modulator, determining the activation levels of a plurality of activatable elements in the B-lymphocyte lmeage 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 In some embodiments, the activatable element is selected from the group consisting of elements selected from the group consisting of Erk, Syk, Zap70, Lck, Btk, BLNK, CbI, PLCγ2, Akt, ReIA, p38, S6 In some embodiments, the activatable element is selected from the group consisting of CbI, PLOγ2, and S6 In some embodiments, the activatable element is S6 In some embodiments, the B-lymphocyte lineage progemtor or denved 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 In some embodiments, the invention provides methods for correlating and/or classifying an activation state of a CLL cell with a clinical outcome m an individual by subjecting the CLL cell from the individual to a modulator, where the CLL cell expresses a B-CeIl 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, wherem the presence of the alteration is indicative of a cluneal outcome [0033] In some embodiments the method compπses identifying a pattern of said activation levels of said pluralilty of activatable elements m said cell, wherein said pattern is correlated to a disease or condition [0034] In some embodiments, the correlation is determining a cluneal outcome, such as prognosis or determination of treatment of the condition In some embodiments, the neoplastic or hematopoietic condition is selected from the group consisting of Non-Hodgkin Lymphoma, Hodgkin or other lymphomas, acute or chrome leukemias, polycythemias, thrombocythemias, multiple myeloma and plasma cell disorders, such as amyloidosis and

0025Waldenstrom's macroglobulinemia, myelodysplastic disorders, myeloproliferative disorders, myelofibrosis, or atypical immune lymphoproliferations In some embodiments, the neoplastic or hematopoietic condition is non-B lineage denved, such as acute myeloid leukemia (AML), Chrome 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 In some embodiments, the neoplastic or hematopoietic condition is a B-CeIl or B cell lmeage denved disorder, such as B- CeIl or B cell lmeage denved disorder is selected from the group consisting of Chronic Lymphocytic Leukemia (CLL), B lymphocyte lmeage leukemia, B lymphocyte lmeage lymphoma, Multiple Myeloma, or plasma cell disorders, e g , amyloidosis and Waldenstrom's macroglobulinemias In some embodiments, the condition is CLL In some embodiments the CLL is defined by a monoclonal B cell population that co-expresses CD5 with CD 19 and CD23 or CD5 with CD20 and CD23 and dim surface immunoglobulin expression

0026[0035] In some embodiments, the clinical outcome is the staging or gradmg 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 accordmg 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 [0036] In some embodiments, 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.

0027[0037] In some embodiments, 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. In additional embodiments the correlation may further include determination of the appropriate dosage or timing of a given treatment.

0028[0038] In some embodiments of the methods of the invention, the modulator to which the cell is subjected is an activator or an inhibitor. In some embodiments, 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. In some embodiments the modulator is an antibody e.g. anti- CD20 (Rituxan), anti-CD22 (epratuzumab), anti-CD23

0029(lumiliximab) or anti-CD52 (Alemtuzumab), that recognize antigens on the cell surface. In some embodiments, 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. In some embodiments, the cross-linker is an antibody, or molecular binding entity. In some embodiments, the cross-linker is an antibody, such as 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. In some embodiments, 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. In some embodiments, 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. In some embodiments where 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 crosslihking via interaction of the cell with a solid surface that causes crosslinking of the BCR complex via antibody or molecular binding entity. In some embodiments, 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. In some embodiments, 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.

0030[0039] In some embodiments of the methods of the invention, 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. In some embodiments, 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-chlorophenoxa2ine hydrochloride, dasatinib, 2- Dimethylamino-4,5,6,7-tetrabromo-lH-benzimidazole, 5,7-Dimethoxy-3-(4-pyridinyl)quinoline dihydrochloride, edelfosine, ellagic acid, enzastaunn, ER 27319 maleate, erlotinib, ET18OCH3, fasudil, flavopindol, gefituub, GW 5074, H-7, H-8, H-89, HA-100, HA-1004, HA-1077, HA-1100, hydroxyfasudil, indιrabin-3'-oxime, 5- Iodotubercidin, kenpaullone, KN-62, KY12420, LFM-A13, lavendustin A, luteolin, LY-294002, LY294002, mallotoxin, ML-9, NSC-154020, NSC-226080, NSC-231634, NSC-664704, NSC-680410, NU6102, olomoucine, oxindole I, PD-153035, PD-98059, PD-169316, phloretin, phlondzin, piceatannol, picropodophyllm, PKI, PPl, PP2, purvalanol A, quercetin, R406, R788, rapamune, rapamycin, Ro 31-8220, roscovitine, rottleπn, SB202190, SB203580, sirolimus, sorafemb, SL327, SP600125, staurosporme, STI-571, SU-11274, SU1498, SU4312, SU6656, 4,5,6,7-Tetrabromotπazole, TG101348, Tπciπbine, Tyφhostin AG 490, Tyrphostin AG 825, Tyrphostin AG 957, Tyrphostin AG 1024, Tyiphostin SU1498, U0126, VX-509, VX-667, VX-680, W-7, wortmannin, XL-019, XL-147, XL-184, XL-228, XL-281, XL-518, XL-647, XL-765, XL-820, XL-844, XL-880, Y-27632, ZD-1839, ZM-252868, ZM-447439, H₂O₂, siRNA, miRNA, Canthaπdin, (-)-p-Bromotetramisole, Microcystin LR, Sodium Orthovanadate, Sodium Pervanadate, Vanadyl sulfate, Sodium oxodiperoxo(l,10-phenanthroline)vanadate, bis(maltolato)oxovanadium(IV), Sodium Molybdate, Sodium Perm olybdate, Sodium Tartrate, Imidazole, Sodium Fluoride, β-Glycerophosphate, Sodium Pyrophosphate Decahydrate, Calyculin A, Discodermia calyx, bpV(ρhen), mpV(pic), DMHV, Cypermethπn, Dephostatin, Okadaic Acid, NIPP-I, N-(9,10-Dioxo-9,10-dihydro-phenanthren-2- yl)-2,2-dimethyl-propionamide, α-Bromo-4-hydroxyacetophenone, 4-Hydroxyphenacyl Br, α-Bromo-4- methoxyacetophenone, 4-Methoxyphenacyl Br, α-Bromo-4-(carboxymethoxy)acetophenone, 4- (Carboxymethoxy)phenacyl Br, and bis(4-Tnfluoromethylsulfonamidophenyl)-l,4-dnsopropylbenzene, phenyarsme oxide, Pyrrolidine Dithiocarbamate, or Aluminum fluoride In some embodiments the phosphatase inhibitor is H₂O₂

0031[0040] In some embodiments of the methods of the invention, 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)₂IgM or biotinylated F(ab)₂IgM and H₂O₂ [0041] In some embodiments of the invention, the activation level of the activatable element m the cell is selected from the group consistmg 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, lsomeπzation level, prenylation level, myπstoylation level, lipoylation level, phosphopantetheinylation level, sulfation level, ISGylation level, mtrosylation level, palmitoylation level, SUMOylation level, ubiquitmation 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-hpid complex, and biochemical changes m cell membrane In some embodiments, the activation level is a phosphorylation level In some embodiments, the activatable element is selected from the group consistmg of proteins, carbohydrates, lipids, nucleic acids and metabolites In some embodiments, the activatable element is a protem In some embodiments, the activatable element is a change m metabolic state, temperature, or local ion concentration In embodiments where the activatable element is a protem, in some embodiments the protem is a protem subject to phosphorylation or dephosphorylation, such as kinases, phosphatases, adaptor/scaffold proteins, ubiquitmation enzymes, adhesion molecules, contractile proteins, cytoskeletal proteins, heterotπmenc G proteins, small molecular weight GTPases, guanine nucleotide exchange factors, GTPase activatmg proteins, caspases and proteins involved in apoptosis (e g PARP), ion channels, molecular transporters, molecular chaperones, metabolic enzymes, vesicular transport proteins, hydroxylases, lsomerases, transferases, deacetylases, methylases, demethylases, proteases, esterases, hydrolases, DNA bmdmg proteins or transcription factors [0042] In another aspect, 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 m 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 In some embodiments, the inhibitor is an inhibitor of a cellular factor or a plurality of factors that participates in a signaling cascade in the cell In some embodiments, the inhibitor is a phosphatase or kinase inhibitor Examples of kinase inhibitors include adaphostin, AG 490, AG 825, AG 957, AG 1024, aloisine, aloisine A, alsterpaullone, aminogenistein, API-2, apigemn, arctigenin, AY-22989, BAY 61-3606, bisindolylmaleimide IX, chelerythnne, 10- [4'-(N,N-Diethylamino)butyl]-2-chlorophenoxazine hydrochloride, dasatimb, 2-Dimethylamino-4,5,6,7-tetrabromo-lH- benzimidazole, 5,7-Dimethoxy-3-(4-pyridinyl)quinoline dihydrochloπde, edelfosine, ellagic acid, enzastaunn, ER 27319 maleate, erlotimb, ET18OCH3, fasudil, flavopiπdol, gefitinib, GW 5074, H-7, H-8, H-89, HA-100, HA- 1004, HA-1077, HA-1100, hydroxyfasudil, indirubin-3'-oxime, 5-Iodotubercidin, kenpaullone, KN-62, KY12420, LFM-A13, lavendustin A, luteolin, LY-294002, LY294002, mallotoxin, ML-9, NSC-154020, NSC-226080, NSC- 231634, NSC-664704, NSC-680410, NU6102, olomoucme, oxmdole I, PD-153035, PD-98059, PD 169316, phloretin, phloπdzui, piceatannol, picropodophyllin, PKI, PPl, PP2, purvalanol A, quercetm, R406, R788, rapamune, rapamycin, Ro 31-8220, roscovitme, rottlenn, SB202190, SB203580, sirohmus, sorafenib, SL327, SP600125, staurosporine, STI-571, SU-11274, SU1498, SU4312, SU6656, 4,5,6,7-Tetrabromotriazole, TG101348, Tπciπbme, Tyrphostin AG 490, Tyrphostin AG 825, Tyrphostin AG 957, Tyrphostin AG 1024, Tyrphostin SU1498, U0126, VX-509, VX-667, VX-680, W-7, wortmanmn, XL-019, XL-147, XL-184, XL-228, XL-281, XL-518, XL- 647, XL-765, XL-820, XL-844, XL-880, Y-27632, ZD-1839, ZM-252868, ZM-447439, siRNA, miRNA Examples of phosphatase inhibitors include, but are not limited to H₂O₂, siRNA, miRNA, Canthandin, (-)-p- Bromotetramisole, Microcystin LR, Sodium Orthovanadate, Sodium Pervanadate, Vanadyl sulfate, Sodium oxodiperoxo(l,10-phenanthrolme)vanadate, bis(maltolato)oxovanadium(IV), Sodium Molybdate, Sodium Perm olybdate, Sodium Tartrate, Imidazole, Sodium Fluoπde, β-Glycerophosphate, Sodium Pyrophosphate Decahydrate, Calyculin A, Discodermia calyx, bpV(phen), mpV(pic), DMHV, Cypermethnn, Dephostatin, Okadaic Acid, NIPP- l, N-(9,10-Dioxo-9,10-dihydro-phenanthren-2-yl)-2,2-dimethyl-propionamide, α-Bromo-4-hydroxyacetophenone, 4-Hydroxyphenacyl Br, α-Bromo-4-methoxyacetophenone, 4-Methoxyphenacyl Br, α-Bromo-4- (carboxymethoxy)acetophenone, 4-(Carboxymethoxy)phenacyl Br, and bis(4-Tnfluoromethylsulfonamidophenyl)- 1,4-dnsopropylbenzene, phenyarsme oxide, Pyrrolidine Dithiocarbamate, and Aluminum fluoπde In some embodiments, the phosphatase inhibitor is H₂O₂

0032[0043] In some embodiments, the modulator is an activator or an inhibitor In some embodiments, the modulators are mdependently 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 In some embodiments, at least one modulator is a B cell receptor modulator In some embodiments, 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 [0044] In some embodiments, the modulator is PMA, BAFF, April, SDFIa, SCF, CD40L, IGF-I, Imiquimod, polyCpG, fludarabine, cyclophosphamide, chlorambucil IL-7, IL-6, lL-10, IL-27, IL-4, IL-2, IL-3, thapsigargin and/or a combination thereof [0045] In some embodiments, the activatable element is a protein In some embodiments, the protein is selected from the group consistmg of Aktl, Akt2, Akt3, SAPK/JNK1,2,3, p38s, Erkl/2, Syk, ZAP70, Btk, BLNK, Lck, PLCγ, PLCiγ2, STATl, STAT 3, STAT 4, STAT 5, STAT 6, CREB, Lyn, p-S6, CbI, NF-κB, GSK3β, CARMA/BcllO and Tcl-1 [0046] In another aspect, the mvention 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, H₂O₂, PMA, BAFF, Apnl, SDFIa, CD40L, IGF-I, Imiquimod, polyCpG, fludarabine, cyclophosphamide, chlorambucil, IL-7, IL-6, IL-IO, 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 mcrease in the activation of the activatable element

0033[0047] In some embodiments the cell population is a hematopoietic-deπved cell population In some embodiments, the hematopoietically deπved cell population is selected from the group consisting of plunpotent hematopoietic stem cells, B-lymphocyte lmeage progenitor or deπved cells, T-lymphocyte lineage progenitor or denved cells, NK cell lineage progenitor or deπved cells, granulocyte lineage progenitor or denved cells, monocyte lmeage progenitor or deπved cells, megakaryocyte lmeage progenitor or denved cells and erythroid lmeage progemtor or deπved cells In some embodiments, the hematopoietic denved cell population is a B-lymphocyte lmeage progemtor and denved 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

0034[0048] In some embodiments, the classification mcludes classifying the cell population as a cell population that is correlated with a clinical outcome In some embodiments, the clinical outcome is the predicted respose to a specific therapy, or the prognosis and/or diagnosis of a condition In some embodiments, the cluneal outcome is the presence or absence of a neoplastic or a hematopoietic condition, such as Non-Hodgkin Lymphoma, Hodgkin or other lymphomas, acute or chrome leukemias, polycythemias, thrombocythemias, multiple myeloma or plasma cell disorders, e g , amyloidosis and Waldenstrom's macroglobulmemia, myelodysplastic disorders, myeloproliferative disorders, myelofibrosis, or atypical immune lymphoprohferations In some embodiments, the neoplastic or hematopoietic condition is non-B lineage denved, such as acute myeloid leukemia (AML), Chrome 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 lymphoprohferations In some embodiments, the neoplastic or hematopoietic condition is a B-CeIl or B cell lmeage deπved disorder, such as Chrome Lymphocytic Leukemia (CLL), B lymphocyte lineage leukemia, B lymphocyte lmeage lymphoma, Multiple Myeloma, or plasma cell disorders, e g , amyloidosis or Waldenstrom's macroglobulmemia In some embodiments, the condition is CLL In some embodiments, 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

0035[0049] In some embodiments, the clinical outcome is the staging or grading of a neoplastic or hematopoietic condition Examples of staging in methods provided by the mvention mclude aggressive, indolent, benign, refractory, Roman Numeral staging, TNM Staging, Rai staging, Bmet staging, WHO classification, FAB classification, IPSS score, WPSS score, limited stage, extensive stage, staging according to cellular markers such as ZAP70, Ig V_H mutation status and CD38, occult, including information that may inform on time to progression, progression free survival, overall survival, or event-free survival

0036[0050] In some embodiments of the methods of the invention, 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, Penpheral 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

0037[0051] In some embodiments of the methods of the invention, 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

0038BRIEF DESCRIPTION OF THE DRAWINGS [0052] The novel features of the invention are set forth with particularity m the appended claims A better understanding of the features and advantages of the present mvention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, m which the principles of the invention are utilized, and the accompanying drawings of which

0039[0053] Figure 1 depicts histograms showing the activation of p-Erk and p-Syk/pZap70 in Ramos cells following F(ab)₂IgM stimulation

0040[0054] Figure 2 depicts histograms showmg the phosphorylation of BLNK, CbI, PLCγ2, Lck, and p38 m Ramos

0041Cell lines following treatment with F(ab)₂lgM

0042[0055] Figure 3 depicts histograms showing increased phosphorylation of Erk m Pheresed CLL Samples and m the Ramos cell lines following PMA activation [0056] Figure 4 depicts a histogram showmg the activation of Erk in Pheresed CLL Samples and Ramos cells following activation with mcreasmg amounts of F(ab)₂IgM for 15 Mm

0043[0057] Figure 5 depicts contour plots showing increased phosphorylation of Erk and Syk/Zap 70 in CLL Samples following treatment with PMA or F(ab)₂IgM

0044[0058] Figure 6 depicts contour plots showing phosphorylation of Erk and Syk/Zap70 in CLL samples following treatment with H₂O₂ alone or in combination with F(ab)₂IgM

0045[0059] Figure 7 depicts histograms showmg the phosphorylation of Erk and Syk/Zap70 m healthy B cells following treatment with H₂O₂

0046[0060] Figure 8 depicts contour plots showing the phosphorylation of Erk and Syk/ Zap70 m CLL cells upon treatment with H₂O₂ and F(ab)₂IgM [0061] Figure 9 depicts contour plots showing Erk and Syk in CLL samples following activation with PMA or

0047F(ab)₂IgM

0048[0062] Figure 10 depicts contour plots showing the phosphorylation of Erk and Syk/ Zap70 following treatment with H₂O₂ alone or in combination with F(ab)₂IgM

0049[0063] Figure 11 depicts contour plots comparing CLL samples with low and high Frequency ZAP70 the phosphorylation or Erk and Syk/ Zap70 following treatment with H₂O₂ alone or in combination with F(ab)₂IgM [0064] Figure 12 depicts contour plots showing Syk/Zap70 and Erk phosphorylation after treatment F(ab)₂IgM and H₂O₂ at different times in CLL samples

0050[0065] Figure 13 depicts contour plots showing Syk/Zap70 and Erk phosphorylation after treatment with F(ab)₂IgM and H₂O₂ in CLL samples [0066] Figure 14 depicts histograms showing the kinetics of signaling mediated by F(ab)₂IgM and H₂O₂ m CLL [0067] Figure 15 depicts contour plots showing the influence of %ZAP70 on BLNK phosphorylation following treatment with H₂O₂ alone or in combination with F(ab)₂IgM

0051[0068] Figure 16 depicts histograms showing the influence of ZAP70 status on BLNK phosphorylation [0069] Figure 17 depicts histograms showing the kinetics of phosphorylation of pPLCγ2, prpS6, and pCbl after B- Cell Receptor crosslinking by F(ab)₂IgM Alone in CD20+/CD5+ population of CLL samples

0052[0070] Figure 18 depicts histograms showing the kinetics of phosphorylation of pPLCγ2, ρrpS6, and pCbl after B- CeIl Receptor crosslinking by F(ab)₂IgM and H₂O₂ m CD20+/CD5+ population in CLL samples [0071] Figure 19 depicts histograms showing the kinetics of phosphorylation of pPLCγ2, prpS6, and pCbl after B- CeIl Receptor crosslinking by F(ab)₂IgM Alone m CD20+/CD5+ population in CLL samples [0072] Figure 20 depicts histograms showing the kinetics of phosphorylation of pPLCγ2, prpS6, and pCbl after B- CeIl Receptor crosslinking by F(ab)₂lgM and H₂O₂ in CD20+/CD5+ population in CLL samples [0073] Figure 21 depicts contour plots showing the phosphorylation of Syk/Zap 70 and Erk by F(ab)₂IgM alone over time [0074] Figure 22 depicts contour plots showing the phosphorylation of Syk/Zap 70 and Erk m response to F(ab)₂IgM alone over time

0053[0075] Figure 23 depicts histograms showing phosphorylation of Syk, Erk and BLNK in response to F(ab)₂IgM over time in the CD20+/CD5+ population of CLL samples

0054[0076] Figure 24 depicts histograms showing the kinetics of phosphorylation of pPLCγ2, prpS6, and pCbl after H₂O₂ treatment m CD20+/CD5+ population of CLL samples [0077] Figure 25 depicts histograms showing the kinetics of phosphorylation of pPLCγ2, prpS6, and pCbl after H₂O₂ treatment in CD20+/CD5+ population of CLL Samples

0055[0078] Figure 26 depicts a heat- map showing intracellular responses of CLL patient peripheral blood samples to BCR and/or H₂O₂ stimulation CLL indicates that the sample was taken from a patient diagnosed with CLL CON mdicates 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 nght of the scale) represents the greatest increase (i e + 3 0 Log Change) Black (located m 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 m phosphorylation status (i e - 3 0 Log Change) These levels of phosphorylation are deπved from an equation which calculates the log 10-fold increase, or decrease, in median fluorescence intensity (MFI), of a stimulated sample divided by the MFI of an unstimulated sample The rows of the heatmap indicate the identity of the phospho-protem that was analyzed For example, the label "ρ-Blnk/H₂O₂/ F(ab)2 IgM" mdicates that the phosphorylation status of phosphorylated BLNK was measured in response to hydrogen peroxide (H₂O₂) and the Fab fragment that recognizes IgM ( F(ab)2 IgM) "US" mdicates that the sample was unstimulated [0079] Figure 27 depicts the lower portion of the heat map shown in Figure 26 White framed boxes show 2 distinct clusters of CLL patient samples in which signaling increases in response to H₂O₂ treatment (left hand side) or signaling decreases in response to H₂Ch treatment (nghthand side)

0056[0080] Figure 28 depicts the lower portion of the heat map shown in Figure 26 further illustrating the two patient clusters, 10/22 patients (top left white framed box) and 11/22 patients (bottom nght white framed box), that are distinguished by H₂O₂ treatment The cartoon on the nght of the figure depicts the B cell receptor signaling pathway

0057DETAILED DESCRIPTION OF THE INVENTION [0081] The present mvention incorporates information disclosed in other applications and texts The following patent and other publications are hereby incorporated by reference m their entireties Haskell et al, Cancer Treatment, 5^th Ed , W B Saunders and Co , 2001 , Alberts et al , The Cell, 4^th Ed , Garland Science, 2002, Vogelstein and Kinzler, The Genetic Basis of Human Cancer, 2d Ed , McGraw Hill, 2002, Michael, Biochemical Pathways, John Wiley and Sons, 1999, Weinberg, The Biology of Cancer, 2007, Immunobiology, Janeway et al 7^th Ed , Garland, and Leroith and Bondy, Growth Factors and Cytokines in Health and Disease, A Multi Volume Treatise, Volumes IA and IB, Growth Factors, 1996 Patents and applications that are also incorporated by reference include U S Patent No 7,381535 and 7,393,656 and U S Serial Numbers (USSN) 10/193,462, 11/655,785, 11/655,789, 11/655,821, 11/338,957, 61/048,886, 61/048,920, 61/048,657, 61/079,766, 61/155,362, 61/079,579, 61/079,537, 61/079,551, 61/087,555 and 61/085,789 Some commercial reagents, protocols, software and instruments that are useful in some embodiments of the present invention are available at the Becton Dickinson Website http //www bdbiosciences com/features/products/, and the Beckman Coulter website, http //www beckmancoulter com/Default asp"?bhfv=7 Relevant articles include High-content single-cell drug screening with phosphospecific flow cytometry, Krutzik et al , Nature Chemical Biology, 23 December (2007), Irish et al , FLt3 ligand Y591 duplication and Bcl-2 over expression are detected in acute myeloid leukemia cells with high levels of phosphorylated wild-type p53, Neoplasia, (2007), Irish et al Mapping normal and cancer cell signaling networks towards single-cell proteomics, Nature (2006) 6 146-155, and Irish et al , Smgle cell profiling of potentiated phospho-protein networks in cancer cells, Cell, (2004) 118, 1-20, Schulz, K R , et al , Single-cell phospho-protem analysis by flow cytometry, Curr Protoc Immunol, (2007) 78 8 8 17 1-20, Krutzik, P O , et al , Coordmate analysis of murine immune cell surface markers and intracellular phosphoproteins by flow cytometry, J Immunol (2005) 175(4) 2357-65, Krutzik, P O , et al , Characterization of the murine immunological signaling network with phosphospecific flow cytometry, J Immunol (2005) 175(4) 2366-73, Shulz et al , Current Protocols in Immunology (2007) 78 8 17 1-20, Stelzer et al Use of Multiparameter Flow Cytometry and Immunophenotyping for the Diagnosis and Classfication of Acute Myeloid Leukemia, Immunophenotyping, Wiley, 2000, and Krutzik, P O and Nolan, G P , Intracellular phospho-protein staining techniques for flow cytometry monitoring single cell signaling events, Cytometry A (2003) 55(2) 61-70, Hanahan D .Weinberg, The Hallmarks of Cancer, CELL (2000) 100 57-70, Krutzik et al, High content smgle cell drug screening with phophosphospecific flow cytometry, Nat Chem Biol (2008) 4 132-42 Experimental and process protocols and other helpful information can be found at http /proteomices Stanford edu The articles and other references cited below are also incorporated by reference in their entireties for all purposes Introduction

0058[0082] In some embodiments, this mvention is directed to methods and compositions for diagnosis, prognosis and to methods of treatment In some embodiments, the physiological status of cells present in a sample (e g clinical sample) is used, e g , in diagnosis or prognosis of a condition, patient selection for therapy, to momtor treatment, modify therapeutic regimens, and to further optimize the selection of therapeutic agents, which may be administered as one or a combination of agents Hence, therapeutic regimens can be individualized and tailored according to the data obtained pnor to, and at different times over the course of treatment, thereby providing a regimen that is individually appropπate [0083] In some embodiments, the present mvention is directed to methods for classifying a sample deπved from an individual having or suspected of havmg a condition, e g , a neoplastic or a hematopoietic condition The mvention allows for identification of prognostically and therapeutically relevant subgroups of conditions and prediction of the cluneal course of an individual The methods of the mvention provide tools useful in the treatment of an individual afflicted with a condition, including but not limited to methods of choosmg a therapy for an individual, methods of predictmg response to a therapy for an individual, methods of determining the efficacy of a therapy in an individual, methods for assigning a nsk group, methods of predicting an increased πsk of relapse, methods of predicting an mcreased πsk of developing secondary complications, and methods of determining the prognosis for an individual The present mvention 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 aidmg the clinician m managing the patient and evaluatmg the modality of treatment to be used [0084] In some embodiments, the mvention 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 m an individual In some embodiments the modulators may themselves be used directly within individuals as therapeutic agents In some embodiments the activation of an activateable agent may be used as an indicator to predict course of the condition, identify nsk group, predict an mcreased πsk of developing secondary complications, and determine the prognosis for an individual

0059[0085] In some embodiments, the mvention 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 m 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 subjectmg a cell from the individual to a modulator and an inhibitor, determining the activation level of an activatable element m the cell, and determining the presence or absence of the condition based on the activation level upon treatment with a modulator and an inhibitor [0086] In some embodiments, 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 m the activation of the activatable element In some embodiments the change is an increase In some embodiments the change is a decrease [0087] In some embodiments, the mvention is directed to methods of determining tome signaling status of a cell by subjectmg the cell to a modulator, determining the activation level of an activatable element that participates in a tome signaling pathway in the cell, and determining the status of a tome signaling pathway m the cell from the activation level Tome signaling in a cell may have functional consequences, for instance, to maintain certain differentiated cellular properties or functions In some embodiments of the invention, the status of a tome signaling pathway is used to correlate the status to differences in populations

0060[0088] In some embodiments, the invention is directed to methods of determining a phenotypic profile of a population of cells by exposmg 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 m 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 m each separate culture [0089] In some embodiments, 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, H₂O₂, PMA, BAFF, Apπl, SDFIa, CD40L, IGF-I, Imiquimod, polyCpG, IL-7, IL-6, IL-IO, IL-27, IL-4, IL-2, IL-3, thapsigargin and/or a combination thereof, determining the presence or absence of an mcrease 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 [0090] In some embodiments, the invention is directed to methods of correlating and/or classifying an activation state of a CLL cell with a clinical outcome m an individual by subjecting the CLL cell from the individual to a modulator, where the CLL cell expresses a B-CeIl 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

0061[0091] In some embodiments 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 In some embodiments the change is an increase In some embodiments the change is a decrease

0062[0092] In some embodiments the method of classifying a cell further comprises determining the level of an intracellular marker, cell surface marker or any combination thereof For example 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 In addition 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, determmg treatments effects and/or choosmg a treatment

0063[0093] One or more cell surface markers may also be used m the method of the invention in addition to intracellular markers (e g phospho-proteins) In some embodiments, the method compnses 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 In some embodiments the cell surface marker is a human leukocyte differentiation antigen In some embodiments the human leukocyte differentiation antigen is selected from the list CDl, CD2, CD3, CD4, CD5, CD8, CDlO, CD14, CD19, CD20, CD22, CD23, CD40, CD52, CDlOO, CD280, CD281, CD282, CD283, CD284, and CD289 In some embodiments the human leukocyte differentiation antigen is selected from the list comprising CDl though CD300 In some embodiments the cell surface marker is any cell surface receptor or hgand Examples of cell surface hgands and receptors include, but are not limited to, members of the TNF superfamily, lnterleukins, hormones, neurotransmitters, interferons, growth factor.s chemokines, mtegπns, toll receptor hgands, prostaglandins, or leukotπene families Other examples of cell surface markers mclude, 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 In some embodiments the marker is CD45, CD5, CD14, CD19, CD20, CD22, CD23, CD27, CD37, CD40, CD52, CD79, CD38, CD96, major histocompatability antigen (MHC) Class 1 or MHC Class 2 In some embodiments the cell surface marker is membrane bound IgD In some embodiments the cell surface marker is membrane bound IgG In some embodiments, 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 In some embodiments, the method of classifying a cell compnses determining the level of cell surface IgM on said cell In some embodiments, the method comprises determining the level of cell surface IgD on said cell In some embodiments, the method composes determining the level of a BCR on said cell In some embodiment 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 [0094] One or more intracellular markers may be used in the method of the mvention The levels of these markers can be determined before they are secreted and are referred to as "captured" Examples of captured intracellular markers include, but are not limited to, TNF superfamily members, lnterleukins, hormones, neurotransmitters, interferons, growth factors, chemokines, integπns, prostaglandins, leukotnnes and receptors for all of the above Examples of intracellular markers also mclude, but are not limited to, metalloproteases Examples of intracellular markers also mclude, but are not limited to, proteins mvolved m programmed cell death and proliferation Examples of intracellular markers also mclude, but are not limited to viruses, pathogens, parasites and components or products thereof In some embodiments, the method of classifying a cell further comprises determining the level of an intracellular pathogen or component of a pathogen In some embodiments the intracellular pathogen is HIV In some embodiments the intracellular pathogen is EBV In some embodiments the intracellular component of a pathogen is a nucleic acid sequence derived from said pathogen In some embodiments the intracellular component of a pathogen is a pathogen deπved polypeptide [0095J The method of the invention may comprise determining the level of one or more serum markers In some embodiments the serum marker is a marker of a condition In some embodiments the serum marker is a marker of inflammation In some embodiments the serum marker is a soluble cytokine, TNF superfamily member, interleukin, hormone, neurotransmitter, interferon, growth factor, chemokine, integπn, prostaglandin, leukotnene or any soluble receptor thereof In some embodiments the serum marker is a marker of a specific disease or condition In some embodiments the serum marker is a cancer marker In some embodiments the serum marker is a leukemia marker In some embodiments the serum marker is beta-2 -microglobulin, calcitonin, CD20, CD23, CD52, IL6, IL2R, ICAM-I, CD14, IgG, thymidine kinase or ferritin In some embodiments 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 [00961 The subject mvention also provides kits for use in determining the physiological status of cells in a sample, the kit comprising one or more specific binding elements for signaling molecules, and may additionally compnse one or more therapeutic agents The kit may further compose a software package for data analysis of the physiological status, which may include reference profiles for comparison with the test profile

0064Methods

0065[0097] In some embodiments, the mvention 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, mcludmg methods to classify a cell accordmg 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 tune, treatment with drugs or other modalities The physiological status of the cells provided in a sample (e g clinical sample) may be classified accordmg 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 [0098] 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, mtestme, skin, cervix, prostate, and stomach For example, a tissue sample can comprise a region of functionally related cells or adjacent cells Such samples can compnse complex populations of cells, which can be assayed as a population, or separated mto sub-populations Such cellular and acellular samples can be separated by centπfugation, elutπation, density gradient separation, apheresis, affinity selection, panning, FACS, centrifugation with Hypaque, etc By using antibodies specific for markers identified with particular cell types, a relatively homogeneous population of cells may be obtained Alternatively, a heterogeneous cell population can be used Cells can also be separated by usmg filters For example, whole blood can also be applied to filters that are engmeered 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 μm, as disclosed m U S Patent Application No 09/790,673 Other devices can separate tumor cells from the bloodstream, see Demirci U, Toner M , Direct etch method for microfluidic channel and nanoheight post-fabπcation by picoliter droplets, Applied Physics Letters 2006, 88 (5), 053117, and Iπmia D, Geba D, Toner M , Universal microfluidic gradient generator, Analytical Chemistry 2006, 78 3472-3477 Once a sample is obtained, it can be used directly, frozen, or maintained in appropriate culture medium for short peπods of time Methods to isolate one or more cells for use accordmg to the methods of this invention are performed accordmg to standard techniques and protocols well-established in the art [0099] Suitable cells mclude those cell types associated m a wide variety of disease conditions, even while in a non-diseased state Accordmgly, suitable eukaryotic cell types mclude, 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 mcludmg mononuclear leukocytes, stem cells such as haemopoetic, neural, skin, lung, kidney, liver and myocyte stem cells (for use m screening for differentiation and de-differentiation factors), osteoclasts, chondrocytes and other connective tissue cells, keratinocytes, melanocytes, liver cells, kidney cells, and adipocytes Suitable cells also mclude primary disease state cells, such as primary tumor cells Suitable cells also include known research cells, mcludmg, but not limited to, Jurkat T cells, NIH3T3 cells, CHO, COS, etc See the ATCC cell lme catalog, hereby expressly incorporated by reference [00100] In some embodiments, 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. When serum is present in the media it could be present at a level ranging from 0.0001 % to 100%. In some embodiments serum is present in the media at a level ranging from .0001% to 90%. In some embodiments serum is present in the media at a level ranging from 0.01% to 30%. In some embodiments serum is present in the media at 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 %. In some embodiments, serum is present in the media at any suitable level.

0066[00101] In some embodiments, the cell is a hematopoietic cell. Examples of 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.

0067[00102] In some embodiments, 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. [00103] 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.

0068[00104] In some embodiments, 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. In some embodiments the change is a decrease. In some embodiments the change is an increase. In some embodiments of the invention, 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. 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 a modulator. In some embodiments of the invention, 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.

0069[00105] 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. In some embodiments, the clinical outcome is the prognosis and/or diagnosis of a condition. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 In some embodiments, the condition is CLL In some embodiments, the clinical outcome is the staging or grading of a neoplastic or hematopoietic condition Examples of staging include, but are not limited to, aggressive, mdolent, bemgn, 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 [00106] The classification of a cell accordmg 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 In some embodiments, 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 [00107] The classification of a cell accordmg to the status of an activatable element can compnse classifying the cell as a cell that is correlated with minimal residual disease or emergmg resistance

0070[00108] The classification of a cell accordmg to the status of an activatable element can compnse selectmg a method of treatment Example of methods of treatments include, but are not limited to, chemotherapy, biological therapy, radiation therapy, bone marrow transplantation, Penpheral stem cell transplantation, umbilical cord blood transplantation, autologous stem cell transplantation, allogeneic stem cell transplantation, syngeneic stem cell transplantation, surgery, mduction therapy, maintenance therapy, watchful waiting, and holistic/alternative therapy [00109] Modulators mclude 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 mclude 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 protem fragments, either alone or m 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) Examples of modulators include, but are not limited to, F(ab)2 IgM, Rituxan, alemtuzumab, fludarabine, cyclophosphamide, chlorambucil, anti CD22 (epratuzumab), anti CD23 (lumiliximab), H₂O₂, PMA, BAFF, Apπl, SDFIa, CD40L, IGF-I, 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 US 61/085,789 which is hereby incorporated by reference m its entirety [00110] In some embodiments, the modulator is an activator In some embodiments the modulator is an inhibitor In some embodiments, the mvention provides methods for classifying a cell by contacting the cell with an inhibitor, determining the presence or absence of a change m 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 accordmg to the activation level of a plurality of activatable elements after the cell have been subjected to an inhibitor In some embodiments, the inhibitor is an inhibitor of a cellular factor or a plurality of factors that participates in a signaling cascade in the cell In some embodiments, the inhibitor is a kinase or phosphatase inhibitor Examples of kinase inhibitors mclude 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-chlorophenoxa.:ine hydrochloride, dasatinib, 2- Dimethylamino-4,5,6,7-tetrabromo-lH-benzimidazole, 5,7-Dimethoxy-3-(4-ρyridinyl)quinoline dihydrochloride, edelfosine, ellagic acid, enzastaurin, ER 27319 maleate, erlotinib, ET18OCH3, fasudil, flavopiridol, gefitinib, GW 5074, H-7, H-8, H-89, HA-100, HA-1004, HA-1077, HA-1100, hydroxyfasudil, indirubin-3'-oxime, 5-

0071Iodotubercidin, kenpaullone, KN-62, KY12420, LFM-A13, lavendustin A, luteolin, LY-294002, LY294002, mallotoxin, ML-9, NSC-154020, NSC-226080, NSC-231634, NSC-664704, NSC-680410, NU6102, olomoucine, oxindole I, PD-153035, PD-98059, PD 169316, phloretin, phloridzin, piceatannol, picropodophyllin, PKI, PPl, PP2, purvalanol A, quercetin, R406, R788, rapamune, rapamycin, Ro 31-8220, roscovitine, rottlerin, SB202190, SB203580, sirolimus, sorafenib, SL327, SP600125, staurosporine, STI-571, SU-11274, SU1498, SU4312, SU6656, 4,5,6,7-Tetrabromotriazole, TG101348, Triciribine, Tyrphostin AG 490, Tyrphostin AG 825, Tyrphostin AG 957, Tyrphostin AG 1024, Tyiphostin SU1498, U0126, VX-509, VX-667, VX-680, W-7, wortmannin, XL-019, XL-147, XL-184, XL-228, XL-281, XL-518, XL-647, XL-765, XL-820, XL-844, XL-880, Y-27632, ZD-1839, ZM-252868, ZM-447439, Examples of phosphatase inhibitors include, but are not limited to H₂O₂, siRNA, miRNA, Cantharidin, (-)-p-Bromotetramisole, Microcystin LR, Sodium Orthovanadate, Sodium Pervanadate, Vanadyl sulfate, Sodium oxodiperoxo(l,10-phenanthroline)vanadate, bis(maltolato)oxovanadium(IV), Sodium Molybdate, Sodium Perm olybdate, Sodium Tartrate, Imidazole, Sodium Fluoride, β-Glycerophosphate, Sodium Pyrophosphate Decahydrate, Calyculin A, Discodermia calyx, bpV(phen), mpV(pic), DMHV, Cypermethrin, Dephostatin, Okadaic Acid, NIPP- l, N-(9,10-Dioxo-9,10-dihydro-phenanthren-2-yl)-2,2-dimethyl-propionamide, α-Bromo-4-hydroxyacetophenone, 4-Hydroxyphenacyl Br, α-Bromo-4-methoxyacetophenone, 4-Methoxyphenacyl Br, α-Bromo-4-

0072(carboxymethoxy)acetoρhenone, 4-(Carboxymethoxy)phenacyl Br, and bis(4-Trifluoromethylsulfonamidophenyl)- 1 ,4-diisopropylbenzene, phenyarsine oxide, Pyrrolidine Dithiocarbamate, and Aluminum fluoride. In some embodiments, the phosphatase inhibitor is H₂O₂. [00111] In some embodiments, 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. In some embodiments, the activation level of a plurality of activatable elements in the cell is determined. The inhibitor can be an inhibitor as described herein. In some embodiments, the inhibitor is a phosphatase inhibitor. In some embodiments, the inhibitor is H₂O₂. The modulator can be any modulator described herein. In some embodiments, the modulator is a B cell receptor modulator. In some embodiments, 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. In some embodiments, 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.

0073[00112] The cross-linker can be a molecular binding entity. In some embodiments, the molecular binding entity acts upon or binds the B cell receptor complex via carbohydrates or an epitope in the complex. In some embodiments, 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. [00113] The cross-linking of the B cell receptor complex or the B-cell co-receptor complex can compπse binding of an antibody or molecular binding entity to the cell and then causing its crosshnking via interaction of the cell with a solid surface that causes crosslinking of the BCR complex via antibody or molecular bmding entity [00114] The crosslinker can be F(ab)2 IgM, IgG, IgD, polyclonal BCR antibodies, monoclonal BCR antibodies, Fc receptor deπved binding elements and/or a combination thereof The Ig can be deπved from a species selected from the group consisting of mouse, goat, rabbit, pig, rat, horse, cow, shark, chicken, llama or human The Ig or bmdmg element can be fully human or partially human and can be generated by any suitable method known in the art In some embodiments, the crosslinker is F(ab)2 IgM, Polyclonal IgM antibodies, Monoclonal IgM antibodies, Biotinylated F(ab)2 IgCM, Biotrnylated Polyclonal IgM antibodies, Biotinylated Monoclonal IgM antibodies and/or a combmation thereof

0074[00115] In some embodiments, the methods of the mvention provides for the use of more than one modulator In some embodiments, the methods of the invention utilize a B cell receptor activator and a phosphatase inhibitor In some embodiments, the methods of the invention utilize F(ab)2IgM or biotinylated F(ab)2IgM and H₂O₂ [00116] In some embodiments, the methods of the mvention provides for methods of classifying a cell population by exposing the cell population m 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 m 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 herem In some embodiments, the modulator is selected from the group consisting of F(ab)2 IgM, H₂O₂, PMA, BAFF, Apnl, SDFIa, CD40L, IGF-I, Imiquimod, polyCpG, IL-7, IL-6, IL-10, IL-27, IL-4, IL-2, IL-3, thapsigargin and a combmation thereof hi some embodiments of the invention, the status of an activatable element is determined by contacting the cell population with a bmding 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 bmdmg elements, where each bmdmg element is specific for an activation state of an activatable element [00117] In some embodiments, the methods of the mvention 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 (lumihximab), Campath, H₂O₂, PMA, BAFF, Apnl, SDFIa, CD40L, IGF-I, Imiquimod, polyCpG, fludarabine, cyclophosphamide, chlorambucil, IL-7, IL-6, IL-IO, IL-27, IL-4, IL-2, IL-3, thapsigargin and/or a combmation thereof, and determining the status of an activatable element in the cell population In some embodiments, the status of a plurality of activatable elements in the cell population is determined hi some embodiments of the invention, the status of an activatable element is determined by contacting the cell population with a bmdmg 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 bmdmg elements, where each bmding element is specific for an activation state of an activatable element

0075[00118] hi some embodiments, the methods of the mvention provide for determining a phenotypic profile of a population of cells by exposmg the population of cells in separate cultures to a plurality of modulators, wherem at least one of the modulators is an inhibitor, determining the presence or absence of a change m activation level of an activatable element m 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 In some embodiments the change is a decrease In some embodiments the change is an increase In some embodiments, the modulator is selected from the group consisting of F(ab)2 IgM, Rituxan, Alemtuzumab, anti CD22 (epratuzumab), anti-CD23 (lumiliximab), Campath, H₂O₂, PMA, BAFF, April, SDFIa, CD40L, IGF-I, Imiquimod, polyCpG, fludarabine, cyclophosphamide, chlorambucil, IL-7, IL-6, IL-IO, IL-27, IL-4, IL-2, IL-3, thapsigargin and combination thereof In some embodiments, the status of a plurality of activatable elements m the cell population is determined In some embodiments, the phenotypic profile of a population of cells is used to classify the population as described herein In some embodiments, the presence or absence of an increase m 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 In some embodiments, the status of a plurality of activatable elements is determined by contacting the cell population with a plurality of bindmg elements, where each binding element is specific for an activation state of an activatable element [00119] In some embodiments, the mvention provides a method for classifying a B-lymphocyte progenitor or denved cell as descnbed herein by contacting the cell with a modulator, determining the presence or absence of a change in activation level of an activatable element m 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, the presence or absence of a change m 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 In some embodiments, 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 In some embodiments, 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 bmdmg elements, where each bindmg elements is specific for an activation state of an activatable element In some embodiments, the method for classifying a B-lymphocyte progenitor or denved cell further compnses determining the level of at least one cell- surface marker In some embodiments, the method for classifying a B-lymphocyte progenitor or denved cell futher composes determining the level of at least one intracellular marker, for example a captured intracellular cytokine In some embodiments, the B-lymphocyte progenitor or derived cell is associated with a condition such a neoplastic or hematopoetic condition Thus, in some embodiments, the mvention provides methods for classifying a B- lymphocyte progenitor or denved cell associated with a condition (e g neoplastic or hematopoetic condition) by contacting the cell with a modulator, determining the presence or absence of a change in activation level of one or more activatable elements m the cell, and classifying the cell based on the presence or absence of the change in the activation of the one or more activatable elements In some embodiments the change is a decrease In some embodiments the change is an mcrease

0076[00120] In some embodiments, the mvention provides methods for correlating and/or classifying an activation state of a CLL cell with a cluneal outcome in an individual by subjectmg the CLL cell from the individual to a modulator, wherein the CLL cell expresses B-CeIl 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 m signaling proximal to the BCR, wherein the presence of the alteration is indicative of a clinical outcome In some embodiments, the activation levels of a plurality of activatable elements are determined by contacting the cell with a plurality of bmdmg elements, where each bindmg element is specific for an activation state of an activatable element The clinical outcome can be any clinical outcome descnbed herein [00121] In some embodiments, the methods of the mvention provide methods for determining tonic signaling status of a cell by subjectmg 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 tome signaling pathway in the cell from the activation level In some embodiments, the status of a plurality of activatable elements m the cell population is determined In some embodiments, the activation level of an activatable element is determined by contacting the cell with a bmdmg element that is specific for an activation state of the activatable element In some embodiments, the activation level of a plurality of activatable elements is determined by contacting the cell with a plurality of bmdmg elements, where each binding element is specific for an activation state of an activatable element In some embodiments, the tonic signaling is a cellular receptor tome signaling In some embodiments, the tome signaling is a T-cell receptor (TCR) tome signaling In some embodiments, the tome signaling is a BCR tome signaling In some embodiments, the tome signaling status m the cell is used to classify the cell as descnbed herem [00122] Patterns and profiles of one or more activatable elements are detected using the methods known in the art mcludmg those descnbed herem In some embodiments, patterns and profiles of activatable elements that are cellular components of a cellular pathway are detected usmg the methods descnbed herem In some embodiments, patterns and profiles of activatable elements that are cellular components of a signaling pathway are detected using the methods descnbed herem In some embodiments, patterns and profiles of activatable elements that are cellular components of a tome signaling pathway are detected usmg the methods descnbed herem For example, patterns and profiles of one or more phosphorylated polypeptide are detected usmg methods known m art including those descnbed herem

0077[00123] In some embodiments of the methods descnbed herem, cells (e g normal non-transformed 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) can be used, e g , m assigning a nsk group, predictmg an mcreased nsk of relapse, predictmg an mcreased nsk of developmg secondary complications, choosing a therapy for an individual, predictmg response to a therapy for an individual, determining the efficacy of a therapy m an individual, and/or determining the prognosis for an individual That is that cells other than cells associated with a condition (e g cancer cells) are in fact reflective of the condition process For instance, in the case of cancer, infiltrating immune cells can determine the outcome of the disease In another aspect, a combination of information from a cancer cell plus responding immune cells m the bloodof a cancer patient can be used for diagnosis or prognosis of the cancer

0078Conditions

0079[00124] The methods of the mvention are applicable to any condition m an individual mvolvmg, indicated by, and/or ansing from, in whole or m part, altered physiological status m a cell The term "physiological status" includes mechanical, physical, and biochemical functions in a cell In some embodiments, the physiological status of a cell is determined by measuring charactenstics of cellular components of a cellular pathway Cellular pathways are well known m the art In some embodiments 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 mvolvmg or charactenzed 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

0080[00125] In certain embodiments of the mvention, the condition is a neoplastic or hematopoietic condition In some embodiments, the neoplastic or hematopoietic condition is selected from the group consisting of Non-Hodgkm Lymphoma, Hodgkin or other lymphomas, acute or chrome leukemias, polycythemias, thrombocythemias, multiple myeloma and plasma cell disorders, mcludmg amyloidosis and Waldenstrom's macroglobuhnemia, myelodysplastic disorders, myeloproliferative disorders, myelofibrosis, and atypical immune lymphoproliferations [00126] In some embodiments, the neoplastic or hematopoietic condition is non-B lineage deπved Examples of non- B lmeage deπved neoplastic or hematopoietic condition include, but are not limited to, Acute myeloid leukemia (AML), Chrome 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

0081[00127] In some embodiments, the neoplastic or hematopoietic condition is a B-CeIl or B cell lmeage deπved disorder Examples of B-CeIl or B cell lmeage deπved neoplastic or hematopoietic condition mclude but are not limited to Chronic Lymphocytic Leukemia (CLL), B lymphocyte lmeage leukemia, B lymphocyte lineage lymphoma, Multiple Myeloma, and plasma cell disorders, including amyloidosis and Waldenstrom's macroglobuhnemia

0082[00128] In some embodiments, the condition is CLL In some embodiments, CLL is defined by a monoclonal B cell population that co-expresses CD5 with CD 19 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 CD 19 and CD23 or CD5 with CD20 and CD23 and dim surface immunoglobulin expression [00129] Other conditions within the scope of the present mvention include, but are not limited to, cancers such as gliomas, lung cancer, colon cancer and prostate cancer Specific signaling pathway alterations have been descπbed for many cancers, including loss of PTEN and resultmg activation of Akt signaling m prostate cancer (Whang Y E Proc Natl Acad Sci USA Apr 28, 1998,95(9) 5246-50), increased IGF-I expression in prostate cancer (Schaefer et al , Science October 9 1998, 282 199a), EGFR over expression and resulting ERK activation m glioma cancer (Thomas C Y Int J Cancer Mar 10, 2003, 104(1) 19-27), expression of HER2 m breast cancers (Menard et al

0083Oncogene Sep 29 2003, 22(42) 6570-8), and APC mutation and activated Wnt signaling in colon cancer (Bienz M Curr Opm Genet Dev 1999 October, 9(5) 595-603)

0084[00130] Diseases other than cancer mvolvmg altered physiological status are also encompassed by the present invention For example, it has been shown that diabetes mvolves 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) Similarly, cardiovascular disease has been shown to involve hypertrophy of the cardiac cells mvolvmg multiple pathways such as the PKC family (Malhotra A MoI Cell Biochem 2001 September,225 (1-) 97- 107) Inflammatory diseases, such as rheumatoid arthπtis, 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 mvention is not limited to diseases presently known to involve altered cellular function, but mcludes diseases subsequently shown to mvolve physiological alterations or anomalies

0085[00131] In some embodiments, the present invention is directed to methods for classifying one or more cells m a sample deπved from an individual having or suspected of havmg condition In some embodiments, the mvention allows for identification of prognostically and therapeutically relevant subgroups of the conditions and prediction of the clinical course of an individual In some embodiments, the mvention provides method of classifying a cell accordmg to the activation level of one or more activatable element m a cell from an individual havmg or suspected of havmg condition In some embodiments, the classification mcludes 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 stagmg or gradmg of a condition In some embodiments, the classifying of the cell mcludes classifying the cell as a cell that is correlated to a patient response to a treatment In some embodiments, the classifying of the cell mcludes classifying the cell as a cell that is correlated with minimal residual disease or emerging resistance Activatable elements

0086[00132] 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)

0087[00133] As will be appreciated by those in the art, a wide variety of activation events can find use in the present invention In general, 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 bmdmg 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 m 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") [00134] The activation state of an individual activatable element is either in the on or off state As an illustrative example, and without intending to be limited to any theory, an individual phosphorylatable site on a protem can activate or deactivate the protem The terms "on" and "off," when applied to an activatable element that is a part of a cellular constituent, are used here to descπbe the state of the activatable element, and not the overall state of the cellular constituent of which it is a part Typically, a cell possesses a plurality of a particular protem 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 bmdmg element that recogmzes a specific activation state, only those activatable elements in the specific activation state recognized by the bmdmg element, representmg 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 m a single cell is the "activation level" for that activatable element in that cell [00135] 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 In some embodiments, 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 Other measurements, such as cellular levels (e g , expression levels) of biomolecules that may not contain activatable elements, may also be used to classify cells m addition to activation levels of activatable elements, it will be appreciated that these levels also will follow a distribution, similar to activatable elements Thus, 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 mto a class Once the activation level of intracellular activatable elements of individual single cells is known they can be placed mto 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. [00136] 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 m the classification of cells encompassed here [00137] In some embodiments, 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 m 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.

0088[00138] Additional elements may also be used to classify a cell, such as the expression level of extracellular or intracellular markers, nuclear antigens, enzymatic activity, protem expression and localization, cell cycle analysis, chromosomal analysis, cell volume, and morphological characteristics like granularity and size of nucleus or other distinguishing characteristics. For example, 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 lor MHC Class 2

0089[00139] Alternatively, 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. [00140] In some embodiments, 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 In some embodiments, a cell is classified according to the activation level of a plurality of activatable elements. In some embodiments, a hematopoietic cell is classified according to the activation levels of a plurality of activatable elements. In some embodiments, the activation level of one or more activatable elements of a hematopoietic cell is correlated with a condition. In some embodiments, the activation level of one or more activatable elements of a hematopoietic cell is correlated with a neoplastic or hematopoietic condition as described herein Examples of hematopoietic cells include but are not limited to pluπpotent hematopoietic stem cells, myeloid progenitors, B-lymphocyte lineage progenitor or derived cells, T-lymphocyte lineage progemtor or derived cells, NK cell lmeage progenitor or denved cells, granulocyte lineage progenitor or denved cells, monocyte lineage progenitor or derived cells, megakaryocyte lmeage progenitor or derived cells and erythroid lmeage progenitor or denved cells In some embodiments, the hematopoietic cell is a B-lymphocyte lineage progenitor or derived cell as described herem. [00141] In some embodiments, the activation level of one or more activatable elements in single cells within the sample is determined. Cellular constituents that may mclude 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 protem 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 Upon activation, 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 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 For example, a protein may have multiple activatable elements, and the particular activation states of these elements may overall determine the activation state of the protem, the state of a smgle 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 [00142] In some embodiments, 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

0090[00143] Activation states of activatable elements may result from chemical additions or modifications of biomolecules and mclude biochemical processes such as glycosylation, phosphorylation, acetylation, methylation, biotinylation, glutamylation, glycylation, hydroxylation, isomenzation, prenylation, myπstoylation, hpoylation, phosphopantetheinylation, sulfation, ISGylation, mtrosylation, palmitoylation, SUMOylation, ubiquitination, neddylation, citrullination, amidation, and disulfide bond formation, disulfide bond reduction Other possible chemical additions or modifications of biomolecules include the formation of protein carbonyls, direct modifications of protem side chains, such as o-tyrosine, chloro-, mtrotyrosine, and dityrosme, and protem adducts derived from reactions with carbohydrate and lipid deπvatives Other modifications may be non-covalent, such as binding of a hgand or binding of an allosteπc modulator.

0091[00144] Examples of proteins that may mclude activatable elements mclude, but are not limited to kinases, phosphatases, lipid signaling molecules, adaptor/scaffold proteins, cytokines, cytokine regulators, ubiquitination enzymes, adhesion molecules, cytoskeletal/contractile proteins, heterotnmeπc G proteins, small molecular weight GTPases, guanine nucleotide exchange factors, GTPase activating proteins, caspases, proteins involved m apoptosis (e g PARP), cell cycle regulators, molecular chaperones, metabolic enzymes, vesicular transport proteins, hydroxylases, lsomerases, deacetylases, methylases, demethylases, tumor suppressor genes, proteases, ion channels, molecular transporters, transcription factors/DNA binding factors, regulators of transcription, and regulators of translation Examples of activatable elements, activation states and methods of determining the activation level of activatable elements are described m US Publication Number 20060073474 entitled "Methods and compositions for detectmg the activation state of multiple proteins in smgle cells" and US 7,393,656 entitled "Methods and compositions for πsk stratification" the content of which are incorporate here by reference

0092[00145] In some embodiments, the protem 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, TIEl, TIE2, FAK, Jakl, Jak2, Jak3, Tyk2, Src, Lyn, Fyn, Lck, Fgr, Yes, Csk, AbI, Btk, ZAP70, Syk, IRAKs, cRaf, ARaf, BRAF, Mos, Lim kinase, ILK, TpI, ALK, TGFβ receptors, BMP receptors, MEKKs, ASK, MLKs, DLK, PAKs, Mek 1, Mek 2, MKK3/6, MKK4/7, ASKl, Cot, NIK, Bub, Myt 1, Weel, Casern kinases, PDKl, SGKl, SGK2, SGK3, Aktl, Akt2, Akt3, p90Rsks, p70S6Kinase,Prks, PKCs, PKAs, ROCK 1, ROCK 2, Auroras, CaMKs, MNKs, AMPKs, MELK, MARKs, Chkl, Chk2, LKB-I, MAPKAPKs, Piml, Pim2, Pim3, IKKs, Cdks, Jnks, Erks, IKKs, GSK3α, GSK3β, Cdks, CLKs, PKR, PI3-Kinase class 1, class 2, class 3, mTor, SAPK/JNK1 ,2,3, p38s, PKR, DNA-PK, ATM, ATR, Receptor protein tyrosine phosphatases (RPTPs), LAR phosphatase, CD45, Non receptor tyrosine phosphatases (NPRTPs), SHPs, MAP kinase phosphatases (MKPs), Dual Specificity phosphatases (DUSPs), CDC25 phosphatases, Low molecular weight tyrosine phosphatase, Eyes absent (EYA) tyrosine phosphatases, Slingshot phosphatases (SSH), serine phosphatases, PP2A, PP2B, PP2C, PPl, PP5, inositol phosphatases, PTEN, SHIPs, myotubulaπns, phosphoinositide kinases, phospholipases, prostaglandin synthases, 5 -lipoxygenase, sphingosine kinases, sphingomyelinases, adaptor/scaffold proteins, She, Grb2, BLNK, LAT, B cell adaptor for PI3-kinase (BCAP), SLAP, Dok, KSR, MyD88, Crk, CrkL, GAD, Nek, Grb2 associated bmder (GAB), Fas associated death domain (FADD), TRADD, TRAF2, RIP, T-CeIl leukemia family, IL-2, IL-4, IL-8, IL-6, interferon γ, interferon α, suppressors of cytokine signaling (SOCs), CbI, SCF ubiquitmation hgase complex, APC/C, adhesion molecules, lntegπns, Immunoglobulin-hke adhesion molecules, selectms, cadhenns, catenins, focal adhesion kinase, pl30CAS, fodπn, actm, paxillin, myosin, myosin bmdmg proteins, tubulin, eg5/KSP, CENPs, β-adrenergic receptors, muscarinic receptors, adenylyl cyclase receptors, small molecular weight GTPases, H-Ras, K-Ras, N-Ras, Ran, Rac, Rho, Cdc42, Arfs, RABs, RHEB, Vav, Tiam, Sos, DbI, PRK, TSC 1,2, Ras-GAP, Arf-GAPs, Rho-GAPs, caspases, Caspase 2, Caspase 3, Caspase 6, Caspase 7, Caspase 8, Caspase 9, PARP, Bcl-2, McI-I, BcI-XL, Bcl-w, BcI-B, Al, Bax, Bak, Bok, Bik, Bad, Bid, Bun, Bmf, Hrk, Noxa, Puma, IAPs, XIAP, Smac, Cdk4, Cdk 6, Cdk 2, Cdkl, Cdk 7, Cyclin D, Cyclin E, Cyclm A, Cyclin B, Rb, pl6, pl4Arf, p27KIP, p21CIP, molecular chaperones, Hsp90s, Hsp70, Hsp27, metabolic enzymes, Acetyl-CoAa Carboxylase, ATP citrate lyase, nitric oxide synthase, caveolms, endosomal sorting complex required for transport (ESCRT) proteins, vesicular protem sorting (Vsps), hydroxylases, prolyl-hydroxylases PHD-I, 2 and 3, asparagine hydroxylase FIH transferases, PmI prolyl isomerase, topoisomerases, deacetylases, Histone deacetylases, sirtuins, histone acetylases, CBP/P300 family, MYST family, ATF2, DNA methyl transferases, Histone H3K4 demethylases, H3K27, JHDM2A, UTX, VHL, WT-I, p53, Hdm, PTEN, ubiquitm proteases, urokinase-type plasminogen activator (uPA) and uPA receptor (uPAR) system, cathepsins, metalloproteinases, esterases, hydrolases, separase, potassium channels, sodium channels, , multi-drug resistance proteins, P-Gycoprotein, nucleoside transporters, , Ets, Elk, SMADs, ReI-A (p65-NFKB), CREB, NFAT, ATF-2, AFT, Myc, Fos, SpI, Egr-1, T-bet, β-catenm, HIFs, FOXOs, E2Fs, SRFs, TCFs, Egr-1, FOXO STATl, STAT 3, STAT 4, STAT 5, STAT 6, p53, WT-I, HMGA, pS6, 4EPB-1, eIF4E-binding protein, RNA polymerase, initiation factors, elongation factors [00146] In some embodiments, the classification of a cell according to activation level of an activatable element, e g , m a cellular pathway compπses classifying the cell as a cell that is correlated with a clinical outcome In some embodiments, the cluneal outcome is the prognosis and/or diagnosis of a condition In some embodiments, the clinical outcome is the presence or absence of a neoplastic or a hematopoietic condition In some embodiments, the clinical outcome is the staging or grading of a neoplastic or hematopoietic condition Examples of staging include, but are not limited to, aggressive, mdolent, benign, refractory, Roman Numeral staging, TNM Staging, Rai staging, Binet stagmg, WHO classification, FAB classification, IPSS score, WPSS score, limited stage, extensive stage, staging accordmg to cellular markers such as ZAP70, IgV_H mutational status and CD38, occult, mcludmg information that may inform on time to progression, progression free survival, overall survival, or event-free survival [00147] In some embodiments, 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 In some embodiments, 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

0093[00148] In some embodiments, 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 compπses classifying the cell as a cell that is correlated with minimal residual disease or emerging resistance

0094[00149] In some embodiments, methods and compositions are provided for the classification of a cell accordmg to the activation level of an activatable element, e g , in a cellular pathway wherem the classification compπses selecting a method of treatment Example of methods 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

0095[00150] Generally, the methods of the invention involve determining the activation levels of an activatable element m a plurality of single cells in a sample

0096A. Signaling Pathways [00151] In some embodiments, the methods of the mvention are employed to determine the status of an activatable element in a signaling pathway In some embodiments, a cell is classified, as descπbed herein, accordmg to the activation level of one or more activatable elements m one or more signaling pathways Signaling pathways and their members have been extensively descπbed See (Hunter T Cell (2000) 100(1) 13-27) Exemplary signaling pathways include the following pathways and their members The MAP kinase pathway mcludmg Ras, Raf, MEK, ERK and elk, the PI3K/Akt pathway mcludmg PI-3-kinase, PDKl , Akt and Bad, the NF-κB pathway including

0097IKKs, IkB and NF-KB and the Wnt pathway mcludmg frizzled receptors, beta-catemn, APC and other co-factors and TCF (see Cell Signaling Technology, Inc 2002 Catolog pages 231-279 and Hunter T , supra ) In some embodiments of the invention, the correlated activatable elements bemg assayed (or the signaling proteins being examined) are members of the MAP kinase, Akt, NFkB, WNT, STAT and/or PKC signaling pathways The methods of the mvention also comprise the methods, signaling pathways and signaling molecules disclosed in US 61/085,789 which is hereby incorporated by reference in its entirety

0098[00152] In some embodiments, the methods of the mvention are employed to determine the status of a signaling protein in a signaling pathway known m the art mcludmg those descnbed herem Exemplary types of signaling proteins within the scope of the present mvention 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)) Kmases and protem binding domains, for example, have been well descπbed (see, e g , Cell Signaling Technology, Inc , 2002 Catalogue "The Human Protem Kmases" and "Protem Interaction Domains" pgs 254-279) [00153] Exemplary signaling proteins mclude, but are not limited to, kmases, HER receptors, PDGF receptors, Kit receptor, FGF receptors, Eph receptors, Trk receptors, IGF receptors, Insulin receptor, Met receptor, Ret, VEGF receptors, TIEl, TIE2, FAK, Jakl, Jak2, Jak3, Tyk2, Src, Lyn, Fyn, Lck, Fgr, Yes, Csk, AbI, Btk, ZAP70, Syk, IRAKs, cRaf, ARaf, BRAF, Mos, Lim kinase, ILK, TpI, ALK, TGFβ receptors, BMP receptors, MEKKs, ASK, MLKs, DLK, PAKs, Mek 1, Mek 2, MKK3/6, MKK4/7, ASKl,Cot, NIK, Bub, Myt 1, Weel, Casern kinases, PDKl, SGKl, SGK2, SGK3, Aktl, Akt2, Akt3, p90Rsks, p70S6Kinase,Prks, PKCs, PKAs, ROCK 1, ROCK 2, Auroras, CaMKs, MNKs, AMPKs, MELK, MARKs, Chkl, Chk2, LKB-I, MAPKAPKs, Piml, Pim2, Pim3, IKKs, Cdks, Jnks, Erks, IKKs, GSK3α, GSK3β, Cdks, CLKs, PKR, PI3-Kinase class 1, class 2, class 3, mTor, SAPK/JNK1,2,3, p38s, PKR, DNA-PK, ATM, ATR, phosphatases, Receptor protein tyrosine phosphatases (RPTPs), LAR phosphatase, CD45, Non receptor tyrosine phosphatases (NPRTPs), SHPs, MAP kinase phosphatases (MKPs), Dual Specificity phosphatases (DUSPs), CDC25 phosphatases, low molecular weight tyrosine phosphatase, Eyes absent (EYA) tyrosine phosphatases, Slingshot phosphatases (SSH), serine phosphatases, PP2A, PP2B, PP2C, PP 1 , PP5, inositol phosphatases, PTEN, SHIPs, myotubularins, lipid signaling, phosphoinositide kinases, phospholipases, prostaglandin synthases, 5 -lipoxygenase, sphingosine kinases, sphingomyelinases, adaptor/scaffold proteins, She, Grb2, BLNK, LAT, B cell adaptor for PI3-kinase (BCAP), SLAP, Dok, KSR, MyD88, Crk, CrkL, GAD, Nek, Grb2 associated binder (GAB), Fas associated death domain (FADD), TRADD, TRAF2, RIP, T-CeIl leukemia family, cytokines, IL-2, IL-4, IL-8, IL-6, interferon γ, interferon α, cytokine regulators, suppressors of cytokine signaling (SOCs), ubiquitination enzymes, CbI, SCF ubiquitination ligase complex, APC/C, adhesion molecules, integrins, Immunoglobulin-like adhesion molecules, selectins, cadherins, catenins, focal adhesion kinase, pl30CAS, cytoskeletal/contractile proteins, fodrin, actin, paxillin, myosin, myosin binding proteins, tubulin, eg5/KSP, CENPs, heterotrimeric G proteins, β-adrenergic receptors, muscarinic receptors, adenylyl cyclase receptors, small molecular weight GTPases, H-Ras, K-Ras, N-Ras, Ran, Rac, Rho, Cdc42, Arfs, RABs, RHEB, guanine nucleotide exchange factors, Vav, Tiam, Sos, DbI, PRK, TSC1,2, GTPase activating proteins, Ras-GAP, Arf-GAPs, Rho-GAPs, caspases, Caspase 2, Caspase 3, Caspase 6, Caspase 7, Caspase 8, Caspase 9, PARP, proteins involved in apoptosis, Bcl-2, McI-I, BcI-XL, Bcl-w, BcI-B, Al, Bax, Bak, Bok, Bik, Bad, Bid, Bim, Bmf, Hrk, Noxa, Puma, IAPs, XIAP, Smac, cell cycle regulators, Cdk4, Cdk 6, Cdk 2, Cdkl, Cdk 7, Cyclin D, Cyclin E, Cyclin A, Cyclin B, Rb, pl6, pl4Arf, p27KIP, p21CIP, molecular chaperones, Hsp90s, Hsp70, Hsp27, metabolic enzymes, Acetyl-CoAa Carboxylase, ATP citrate lyase, nitric oxide synthase, vesicular transport proteins, caveolins, endosomal sorting complex required for transport (ESCRT) proteins, vesicular protein sorting (Vsps), hydroxylases, prolyl-hydroxylases PHD-1 , 2 and 3, asparagine hydroxylase FIH transferases, isomerases, Pinl prolyl isomerase, topoisomerases, deacetylases, Histone deacetylases, sirtuins, acetylases, histone acetylases, CBP/P300 family, MYST family, ATF2, methylases, DNA methyl transferases, demethylases, Histone H3K4 demethylases, H3K27, JHDM2A, UTX, tumor suppressor genes, VHL, WT-I, p53, Hdm, PTEN, proteases, ubiquitin proteases, urokinase-type plasminogen activator (uPA) and uPA receptor (uPAR) system, cathepsins, metalloproteinases, esterases, hydrolases, separase, ion channels, potassium channels, sodium channels, molecular transporters, multi-drug resistance proteins, P-Gycoprotein, nucleoside transporters, transcription factors/ DNA binding proteins, Ets, Elk, SMADs, ReI-A (p65-NFKB), CREB, NFAT, ATF-2, AFT, Myc, Fos, Sp 1 , Egr- 1 , T-bet, HIFs, FOXOs, E2Fs, SRFs, TCFs, Egr- 1 , β-catenin, FOXO STATl , STAT 3, STAT 4, STAT 5, STAT 6, p53, WT-I, HMGA, regulators of translation, pS6, 4EPB-1, eIF4E-binding protein, regulators of transcription, RNA polymerase, initiation factors, and elongation factors.

0099[00154] In some embodiments the protein is selected from the group consisting of PI3-Kinase (p85, pi 10a, pi 10b, pi 1Od), Jakl, Jak2, SOCs, Rac, Rho, Cdc42, Ras-GAP, Vav, Tiam, Sos, DbI, Nek, Gab, PRK, SHPl, and SHP2, SHIPl, SHIP2, sSHIP, PTEN, She, Grb2, PDKl, SGK, Aktl, Akt2, Akt3, TSC1,2, Rheb, mTor, 4EBP-1, p70S6Kinase, S6, LKB-I, AMPK, PFK, Acetyl-CoAa Carboxylase, DokS, Rafs, Mos, Tpl2, MEK1/2, MLK3, TAK, DLK, MKK3/6, MEKK1.4, MLK3, ASKl, MKK4/7, SAPK/JNK1,2,3, p38s, Erkl/2, Syk, Btk, BLNK, LAT, ZAP70, Lck, CbI, SLP-76, PLCγi, PLCγ2, STATl, STAT 3, STAT 4, STAT 5, STAT 6, FAK, pl30CAS, PAKs, LIMKl/2, Hsp90, Hsp70, Hsp27, SMADs, ReI-A (p65-NFKB), CREB, Histone H2B, HATs, HDACs, PKR, Rb, Cyclin D, Cyclin E, Cyclin A, Cyclin B, P16, pl4Arf, p27KIP, p21CIP, Cdk4, Cdk6, Cdk7, Cdkl, Cdk2, Cdk9, Cdc25,A/B/C, AbI, E2F, FADD, TRADD, TRAF2, RIP, Myd88, BAD, Bcl-2, McI-I, BcI-XL, Caspase 2, Caspase 3, Caspase 6, Caspase 7, Caspase 8, Caspase 9, PARP, IAPs, Smac, Fodrin, Actin, Src, Lyn, Fyn, Lck, NIK, IKB, p65(RelA), IKKα, PKA, PKC«, PKCβ, PKCΘ, PKCδ, CAMK, Elk, AFT, Myc, Egr-1, NFAT, ATF-2, Mdm2, p53, DNA-PK, Chkl, Chk2, ATM, ATR, βcatenin, CrkL, GSK3α, GSK3β, and FOXO

0100[00155] MAP kinase pathway In some embodiments, the methods of the mvention are employed to determine the status of an activatable element in the MAP kinase pathway Without intending to be limited to any theory, 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 mcludes many protein components In many cell types, activation of this pathway promotes cell division

0101[00156] 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 bmd to the phosphotyrosines of the activated receptor GRB2 bmds 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 bmd 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 senne/threonme-selective protein kinase RAF kinase phosphorylates and activates MEK, another serine/threonine kinase MEK phosphorylates and activates mitogen-activated protein kinase (MAPK)

0102[00157] Technically, RAF, MEK and MAPK are all mitogen-activated kinases, as is MNK MAPK was originally called "extracellular signal-regulated kinases" (ERKs) and microtubule-associated protem kinase (MAPK) One of the first proteins known to be phosphorylated by ERK was a microtubule-associated protem Many additional targets for phosphorylation by MAPK have been found and the protem was re-named "mitogen-activated protem kinase" (MAPK) The seπes of kinases from RAF to MEK to MAPK is an example of a protem kinase cascade Such series of kinases provide opportunities for feedback regulation and signal amplification RAS is activated m a wide range of cancers (see Cell Signaling Technology, Inc Catolog, supra at pages 231-279 and Hunter T, supra and references therein)

0103[00158] Pl$K/ AkI pathway In some embodiments, the methods of the mvention are employed to determine the status of an activatable element m a PI3K/Akt pathway Without intendmg to be limited to any theory, the PI3K/Akt pathway plays a role m 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 m 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 pomt for a constellation of convergmg upstream signaling pathways, which involve stimulation of receptor tyrosine kinases such as IGF-IR, 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 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 These phosphorylation events, in turn, mediate the effects of Akt on cell growth, proliferation, protection from pro-apoptotic stimuli, and stimulation of neoangiogenesis 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 oncogemc 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) [00159] NF- KB pathway In some embodiments, the methods of the invention are employed to determine the status of an activatable element in a NF-κB pathway Without intendmg to be limited to any theory, the NF-κB pathway is involved m regulating many aspects of cellular activity, in stress, injury and especially in pathways of the immune response Some examples are the response to and mduction of IL-2, the induction of TAPl and MHC molecules by NF-κB, 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-I and ICAM-I) Moreover, NF-κB 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-κB 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 encodmg NF-κB transcription factors In most cancer cells NF-KB is constitutive Iy active and resides m the nucleus In some cases, this may be due to chrome stimulation of the IKK pathway, while in others the gene encodmg IkBa may be defective Such continuous nuclear NF-KB activity not only protects cancer cells from apoptotic cell death, but may even enhance then- growth activity Designing anti-tumor agents to block NF-KB activity or to mcrease their sensitivity to conventional chemotherapy may have great therapeutic value [00160] WNT pathway In some embodiments, the methods of the mvention are employed to determine the status of an activatable element m a WNT pathway Without intending to be limited to any theory, the Wnt signaling pathway describes a complex network of protems 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 senes 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 m a change in the amount of β-catenin that reaches the nucleus Dishevelled (DSH) 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 axm, GSK-3, and the protein APC The axin/GSK-3/APC complex normally promotes the proteolytic degradation of the β-catemn intracellular signaling molecule After this "β-catemn destruction complex" is inhibited, a pool of cytoplasmic β-catemn stabilizes, and some β-catemn is able to enter the nucleus and interact with TCF/LEF family transcription factors to promote specific gene expression

0104[00161] PKC pathway In some embodiments, the methods of the mvention 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 then- structure, biochemical properties, tissue distribution, subcellular localization, and substrate specificity They are classified as conventional (α, βl, β2, γ), novel (δ, ε, η, θ, μ), and atypical (ζ, λ) isozymes Conventional PKC isozymes are Ca2+-dependent, while novel and atypical isozymes do not require Ca2+ for their activation All PKC isozymes, with the exception of ζ and λ, are activated by diacylglycerol (DAG) PKC isozymes negatively or positively regulate cπtical cell cycle transitions, including cell cycle entry and exit and the Gl 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 pituitaπes, thyroid cancer tissue, leukemic cells, and lung cancer cells Down regulation of PKCα is reported m 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 m 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 m diseases such as cardiovascular disease and diabetes [00162] In some embodiments of the invention, the methods descπbed 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 Examples of hematopoietic cells include but are not limited to plunpotent hematopoietic stem cells, B-lymphocyte lineage progemtor or deπved cells, T-lymphocyte lmeage progenitor or deπved cells, NK cell lineage progenitor or derived cells, granulocyte lmeage progenitor or denved cells, monocyte lineage progemtor or deπved cells, megakaryocyte lineage progenitor or derived cells and erythroid lmeage progemtor or deπved cells [00163] In some embodiments, the classification of a cell according to the status of an activatable element in a signaling pathway compnses classifying the cell as a cell that is correlated with a clinical outcome In some embodiments, the clinical outcome is the prognosis and/or diagnosis of a condition In some embodiments, the clinical outcome is the presence or absence of a neoplastic or a hematopoietic condition In some embodiments, the clinical outcome is the staging or grading of a neoplastic or hematopoietic condition Examples of staging include, but are not limited to, aggressive, indolent, benign, refractory, Roman Numeral staging, TNM Staging, Rai staging, Bmet 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, mcludmg information that may inform on time to progression, progression free survival, overall survival, or event-free survival

0105[00164] In some embodiments, 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 compnses classifying a cell as a cell that is correlated to a patient response to a treatment In some embodiments, 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

0106[00165] In some embodiments, methods and compositions are provided for the classification of a cell according to the status of an activatable element in a signaling pathway wherem the classification compnses classifying the cell as a cell that is correlated with minimal residual disease or emerging resistance

0107[00166] In some embodiments, methods and compositions are provided for the classification of a cell according to the status of an activatable element m a signaling pathway wherem the classification compnses selecting a method of treatment Example of methods of treatments include, but are not limited to, chemotherapy, biological therapy, radiation therapy, bone marrow transplantation, Penpheral 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 waitmg, and holistic/alternative therapy [00167] The mvention is not limited to presently elucidated signaling pathways and signal transduction proteins, and encompasses signaling pathways and proteins subsequently identified

0108B. B-CeIl Receptor Pathway [00168] In some embodiments, the methods and compositions of the invention may be employed to examine and profile the status of any activatable element in B-CeIl Receptor (BCR) signaling, or collections of such activatable elements in a B-lymphocyte lineage progemtor or deπved cell In some embodiments, the physiological status of one or more B-lymphocyte lineage progemtor or deπved cell is determined by examining and profiling the status of one or more activatable element m BCR signaling In some embodiments, a B-lymphocyte lmeage progenitor or deπved cell is classified, as descnbed herein, accordmg to the activation level of one or more activatable elements in BCR signaling Examples of B-lymphocyte lmeage deπved cell mclude, but are not limited to, B-lymphocyte lmeage 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 In some embodiments, the B-lymphocyte lineage progenitor or deπved cell is a cell associated with a condition as descπbed herein

0109[00169] Without intending to be limited to any theory, BCR cross-linking triggers phosphorylation of tyrosines within the ITAM motif domains of Igα and Igβ by Src family member tyrosine kinases (e g , Lyn, Lck, BIk, Fyn) The phosphorylated ITAMs of Igαβ 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-I) and compartmentalizes the BCR m lipid rafts with coreceptors CD19 and CD21 Following Syk and Btk activation, the enzymes phospholipase-C γ2 (PLC γ 2) and PI3K propagate BCR signaling PLC γ2 activation generates calcium flux, inositol- 1,4,5-tπphosphate, and diacylglycerol, and results m activation of protein kinase C and NF-κB Syk interacts with PLC γ 2 via adapters, whereas Btk can interact directly, and each is required for PLC γ 2 activity following BCR cross-linking Both Syk and Btk can activate PI3K following BCR cross-linking Activation of PI3K enables Akt-mediated survival signaling, and PI3K is required for BCR-mediated survival during B cell development PLC γ 2 and PI3K also initiate kinase cascades that result in phosphorylation of the MAPK family protems ERK 1/2 and p38 Activation of the Ras-Raf-ERKl/2 signaling cascade is considered a central event m BCR signaling, and decreased Ras activation due to RasGRPl and RasGRP3 loss m mouse impairs B cell proliferation In contrast, p38 is a stress response protein that interacts with p53 and regulates cell cycle checkpoints Differential activation of ERKl/2 and p38 might enable the BCR to dnve 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

0110[00170] Efficient activation of BCR signaling depends on generation of H₂C>₂ and mactivation of negative regulatory protem tyrosine phosphatases (PTPs) Following BCR cross-linking, recruitment and activation of calcium-dependent NADPH oxidases (NOX) protems, such as NOX5, enables production of H₂O₂ and lowers the signaling threshold for the BCR BCR-induced H₂O₂ transiently inactivates membrane proximal PTPs, including SHP-I, via reversible oxidation of the catalytic cysteine to sulfemc acid Elegant work reconstituting the BCR signaling pathway in insect cells has suggested a model of redox feedback loops where H₂O₂ inactivates PTPs and enables amplification of early signaling events, such as Syk phosphorylation and ITAM bmdmg Recent work characterized endogenously generated H₂O₂ as the primary redox species generated by BCR signaling and mdicated that NOX-dependent production OfH₂O₂ was cπtical to initiate a wave of BCR signaling m mouse A20 B cells [00171] In some embodiments, the invention provides a method for classifying a B-lymphocyte lineage progenitor or denved cell upon treatment with a modulator and/or inhibitor Examples of B-lymphocyte lineage progenitor or deπved cells mclude, 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 expressmg Zap70 [00172] In some embodiments, the classification mcludes classifying the cell accordmg to the status of an activatable element in a BCR pathway as a cell that is correlated with a clinical outcome In some embodiments, the invention provides methods for classifying a B-lymphocyte lineage progemtor or denved cell based on an alteration in signaling proximal to the BCR In some embodiments, the cluneal outcome is the prognosis and/or diagnosis of a condition In some embodiments, the clinical outcome is the presence or absence of a neoplastic or a hematopoietic condition, such as Chrome Lymphocytic Leukemia (CLL), B lymphocyte lineage leukemia, B lymphocyte lineage lymphoma, Multiple Myeloma, or plasma cell disorders, e g , amyloidosis or Waldenstrom's macroglobulinemia In some embodiments, the condition is CLL In some embodiments, 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 In some embodiments CLL is defined by a monoclonal B cell population that co-expresses CD5 with CD 19 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 CD 19 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 denved cell Non-limiting examples of such markers include CD45, CD5, CD14, CD19, CD20, CD22, CD23, CD27, CD37, CD40, CD52, CD79, CD38, CD96, major histocompatability antigen (MHC) Class 1 and MHC Class 2 [00173] In some embodiments, the clinical outcome is the staging or grading of a neoplastic or hematopoietic condition Examples of staging m methods provided by the invention mclude aggressive, indolent, bemgn, refractory, Roman Numeral stagmg, 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 [00174] In some embodiments of the methods of the invention, the classifying of the B-lymphocyte lmeage progemtor or denved 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 compnse 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

0111[00175] In some embodiments of the methods of the invention, the classifying of the B-lymphocyte lineage progemtor or denved cells based on activation of an activatable element m BCR pathway includes classifying the cell as a cell that is correlated with minimal residual disease or emerging resistance

0112C. Tonic Signaling

0113[00176] In some embodiments, the methods and compositions of the invention may be employed to determine the status of a tome 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 descnbed herein, according to the status of one or more activatable elements in a tome signaling pathway The term "tonic signaling" includes antigen-independent signaling, mdependent basal signaling and non-induced or hgand-independent signaling [00177] Without intending to be limited to any theory, recent evidence supports the notion that in most signal transduction systems regulated by cellular receptors some basal level of signaling occurs continuously in a hgand- mdependent manner, although the flux through such systems may vary considerably The basal tone or the steady state level of signaling in unstimulated cells is the result of equihbnum of positive and negative regulators within a signaling pathway Thus, the balanced actions of positive and negative regulators of signal transduction set the steady state equihbnum Receptor stimulation then perturbs the equihbnum state in vanous ways to initiate cellular responses The steady state level of signaling in the unstimulated state may itself have functional consequences, for instance, to mamtain certain differentiated cellular properties or functions [00178] In some embodiments, the invention provides for methods of determining tonic signaling status of a cell In some embodiments, the tonic signaling is a cellular receptor tonic signaling In some embodiments, 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 Examples of hematopoietic cells include but are not limited to pluπpotent hematopoietic stem cells, B-lymphocyte lineage progenitor or denved cells, T-lymphocyte lmeage progenitor or denved cells, NK cell lineage progenitor or derived cells, granulocyte lineage progemtor or denved cells, monocyte lmeage progemtor or denved cells, megakaryocyte lmeage progenitor or denved cells and erythroid lmeage progenitor or denved cells [00179] In some embodiments, the classification of a cell according to the status of an activatable element m a tome signaling pathway compnses classifying the cell as a cell that is correlated with a cluneal outcome In some embodiments, the clinical outcome is the prognosis and/or diagnosis of a condition In some embodiments, the cluneal outcome is the presence or absence of a neoplastic or a hematopoietic condition Examples of neoplastic or hematopoietic conditions include, but are not limited to, such as Chrome Lymphocytic Leukemia (CLL), B lymphocyte lmeage leukemia, B lymphocyte lmeage lymphoma, Multiple Myeloma, or plasma cell disorders, e g , amyloidosis or Waldenstrom's macroglobulinemia In some embodiments, the condition is CLL In some embodiments, CLL is defined by a monoclonal B cell population that co-expresses CD5 with CD 19 and CD23 or CD5 with CD20 and CD23 and by surface immunoglobulin expression

0114[00180] In some embodiments, the clinical outcome is the staging or gradmg of a neoplastic or hematopoietic condition Examples of staging mclude, but are not limited to, aggressive, mdolent, benign, refractory, Roman Numeral staging, TNM Staging, Rai staging, Bmet staging, WHO classification, FAB classification, IPSS score, WPSS score, limited stage, extensive stage, staging accordmg 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 [00181] In some embodiments, 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 tome signaling The presence of the alteration is indicative of a cluneal outcome, where the clinical outcome is as descnbed herein

0115[00182] In some embodiments, methods and compositions are provided for the classification of a cell accordmg to the status of an activatable element m a tonic signaling pathway wherein the classification compnses classifying a cell as a cell that is correlated to a patient response to a treatment In some embodiments, the patient response is selected from the group consistmg of complete response, partial response, nodular partial response, no response, progressive disease, stable disease and adverse reaction

0116[00183] In some embodiments, methods and compositions are provided for the classification of a cell accordmg to the status of an activatable element in a tome signaling pathway wherein the classification compnses classifying the cell as a cell that is correlated with mimmal residual disease or emergmg resistance [00184] In some embodiments, methods and compositions are provided for the classification of a cell accordmg to the status of an activatable element m a tonic signaling pathway wherein the classification compnses selectmg a method of treatment Example of methods of treatments mclude, but are not limited to, chemotherapy, biological therapy, radiation therapy, bone marrow transplantation, Penpheral stem cell transplantation, umbilical cord blood transplantation, autologous stem cell transplantation, allogeneic stem cell transplantation, syngeneic stem cell transplantation, surgery, mduction therapy, maintenance therapy, watchful waiting, and holistic/alternative therapy Binding Element

0117[00185] In some embodiments of the invention, 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. The term "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. [00186] In some embodiments, 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. Thus "amino acid", or "peptide residue", as used herein include both naturally occurring and synthetic amino acids. For example, homo-phenylalanine, citrulline and noreleucine are considered amino acids for the purposes of the invention. The side chains may be in either the (R) or the (S) configuration. In some embodiments, the amino acids are in the (S) or L-confϊguration. If non-naturally occurring side chains are used, 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:(l-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.

0118[00187] 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. For example, as demonstrated (see, e.g., the Examples) and described herein, 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. Hence, in some embodiments the expression and phosphorylation of one or more polypeptides are detected and quantified using methods of the present invention. In some embodiments, 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. As used herein, 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. Preferably, the corresponding and specific antigen is a specific form of an activatable element. Also preferably, the binding of the activation state-specific antibody is indicative of a specific activation state of a specific activatable element. [00188] In some embodiments, 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. [00189] The term "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. Examples of 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. The term

0119"antibody" comprises monoclonal and polyclonal antibodies. Antibodies can be antagonists, agonists, neutralizing, inhibitory, or stimulatory.

0120[00190] The antibodies of the present invention may be nonhuman, chimeric, humanized, or fully human. For a description of the concepts of chimeric and humanized antibodies see Clark et al., 2000 and references cited therein (Clark, (2000) Immunol. Today 2L397-402). 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). In some embodiments, the antibodies of the present invention are humanized. By "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. Pat. No. 5,530,101; U.S. Pat. No. 5,585,089; U.S. Pat. No. 5,693,761; U.S. Pat. No. 5,693,762; U.S. Pat. No. 6,180,370; U.S. Pat. No. 5,859,205; U.S. Pat. No. 5,821,337; U.S. Pat. No. 6,054,297; U.S. Pat. No. 6,407,213). 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. Methods for humanizing non-human antibodies are well known in the art, and can be essentially performed following the method of Winter and co-workers (Jones et al., 1986, Nature 321 :522-525; Riechmann et al., 1988, Nature 332:323-329; Verhoeyen et al., 1988, Science, 239:1534-1536). Additional examples of 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). In an alternate embodiment, the antibodies of the present invention may be fully human, that is the sequences of the antibodies are completely or substantially human. A number of methods are known in the art for generating fully human antibodies, including the use of transgenic mice (Bruggemann et al.,

01211997, Curr Opin Biotechnol 8:455-458) or human antibody libraries coupled with selection methods (Griffiths et al.,

01221998, Curr Opin Biotechnol 9: 102-108).

0123[00191] Specifically included within the definition of "antibody" are aglycosylated antibodies. By "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. Alternatively, 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.

0124[00192] As pointed out above, activation state specific antibodies can be used to detect kinase activity, however additional means for determining kinase activation are provided by the present invention. For example, 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 (phospho- substrate antibodies) may be used to determine the presence of activated kinase in a sample.

0125[00193] In a further embodiment, an element activation profile is determined using a multiplicity of activation state antibodies that have been immobilized. Antibodies may be non-diffiαsibly bound to an insoluble support having isolated sample-receiving areas (e.g. a microliter 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. These are typically made of glass, plastic (e.g., polystyrene), polysaccharides, nylon or nitrocellulose, teflon™, etc. Microtiter plates and arrays are especially convenient because a large number of assays can be earned out simultaneously, using small amounts of reagents and samples. In some cases magnetic beads and the like are included.

0126[00194] 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 steπcally block either the ligand bmding site or activation sequence when the protein is bound to the support), direct binding to "sticky" or iomc supports, chemical cross-linking, the synthesis of the antibody on the surface, etc. Following binding of the antibody, excess unbound matenal is removed by washing. The sample receiving areas may then be blocked through mcubation with bovine serum albumin (BSA), casein or other innocuous protein or other moiety

0127[00195] The antigenicity of 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. In some embodiments, an activated isoform of an element possesses an epitope that is absent in a non- activated isoform of an element, or vice versa. In some embodiments, 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 mduced conformational change in an element which causes the element to present the same sequence in an antigenically distinguishable way. In some embodiments, 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. In some embodiments, the epitopes for the distinguishing antibodies are centered around the active site of the element, although as is known m the art, conformational changes in one area of an element may cause alterations in different areas of the element as well.

0128[00196] Many antibodies, many of which are commercially available (for example, see Cell Signaling Technology, www cellsignal.com, Millipore, eBioscience, Caltag, Santa Cruz Biotech, Abeam, BD Biosciences, Sigma and Anaspec) the contents which are incorporated herein by reference) have been produced which specifically bind to the phosphorylated isoform of a protem but do not specifically bind to a non-phosphorylated isoform of a protein. Many such antibodies have been produced for the study of signal transducing proteins which are reversibly phosphorylated. Particularly, many such antibodies have been produced which specifically bind to phosphorylated, activated iso forms of protein. Examples of 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, TIEl, TIE2, FAK, Jakl, Jak2, JaB, Tyk2, Src, Lyn, Fyn, Lck, Fgr, Yes, Csk, AbI, Btk, ZAP70, Syk, IRAKs, cRaf, ARaf, BRAF, Mos, Lim kinase, ILK, TpI, ALK, TGFβ receptors, BMP receptors, MEKKs, ASK, MLKs, DLK, PAKs, Mek 1, Mek 2, MKK3/6, MKK4/7, ASKl.Cot, NIK, Bub, Myt 1, Weel, Casein kinases, PDKl, SGKl, SGK2, SGK3, Aktl, Akt2, Akt3, p90Rsks, p70S6Kinase,Prks, PKCs, PKAs, ROCK 1 , ROCK 2, Auroras, CaMKs, MNKs, AMPKs, MELK,

0129MARKs, Chkl, Chk2, LKB-I, MAPKAPKs, Piml, Pim2, Pim3, IKKs, Cdks, Jnks, Erks, IKKs, GSK3α, GSK3β, Cdks, CLKs, PKR, PI3-Kinase class 1, class 2, class 3, mTor, SAPK/JNK1,2,3, p38s, PKR, DNA-PK, ATM, ATR, phosphatases, Receptor protem tyrosine phosphatases (RPTPs), LAR phosphatase, CD45, Non receptor tyrosine phosphatases (NPRTPs), SHPs, MAP kinase phosphatases (MKPs), Dual Specificity phosphatases (DUSPs), CDC25 phosphatases, Low molecular weight tyrosine phosphatase, Eyes absent (EYA) tyrosine phosphatases,

0130Slmgshot phosphatases (SSH), serine phosphatases, PP2A, PP2B, PP2C, PPl, PP5, inositol phosphatases, PTEN, SHIPs, myotubularins, lipid signaling, phosphoinositide kinases, phospholipases, prostaglandin synthases, 5- lipoxygenase, sphingosine kinases, sphingomyelinases, adaptor/scaffold proteins, She, Grb2, BLNK, LAT, B cell adaptor for PI3-kinase (BCAP), SLAP, Dok, KSR, MyD88, Crk, CrkL, GAD, Nek, Grb2 associated binder (GAB), Fas associated death domain (FADD), TRADD, TRAF2, RIP, T-CeIl leukemia family, cytokines, IL-2, IL-4, IL-8, IL-6, mterferon γ, interferon α, cytokine regulators, suppressors of cytokine signaling (SOCs), ubiquitination enzymes, CbI, SCF ubiquitination hgase complex, APC/C, adhesion molecules, integπns, Immunoglobulin-like adhesion molecules, selectms, cadhenns, catemns, focal adhesion kinase, pl30CAS, cytoskeletal/contractile proteins, fodπn, actm, paxilhn, myosin, myosm binding proteins, tubulin, eg5/KSP, CENPs, heterotπmenc G proteins, β-adrenergic receptors, muscarinic receptors, adenylyl cyclase receptors, small molecular weight GTPases, H-Ras, K-Ras, N-Ras, Ran, Rac, Rho, Cdc42, Arfs, RABs, RHEB, guanine nucleotide exchange factors, Vav, Tiam, Sos, DbI, PRK, TSC 1,2, GTPase activating proteins, Ras-GAP, Arf-GAPs, Rho-GAPs, caspases, Caspase 2,

0131Caspase 3, Caspase 6, Caspase 7, Caspase 8, Caspase 9, PARP, proteins mvolved in apoptosis, Bcl-2, McI-I, Bel- XL, Bcl-w, BcI-B, Al, Bax, Bak, Bok, Bik, Bad, Bid, Bim, Bmf, Hrk, Noxa, Puma, IAPs, XIAP, Smac, cell cycle regulators, Cdk4, Cdk 6, Cdk 2, Cdkl, Cdk 7, Cyclin D, Cyclm E, Cyclin A, Cyclin B, Rb, pl6, pl4Arf, p27KIP, p21CIP, molecular chaperones, Hsp90s, Hsp70, Hsp27, metabolic enzymes, Acetyl-CoAa Carboxylase, ATP citrate lyase, nitric oxide synthase, vesicular transport proteins, caveolms, endosomal sorting complex required for transport (ESCRT) proteins, vesicular protein sorting (Vsps), hydroxylases, prolyl-hydroxylases PHD-I, 2 and 3, asparagine hydroxylase FIH transferases, isomerases, PmI prolyl isomerase, topoisomerases, deacetylases, Histone deacetylases, sirtums, acetylases, histone acetylases, CBP/P300 family, MYST family, ATF2, methylases, DNA methyl transferases, demethylases, Histone H3K4 demethylases, H3K27, JHDM2A, UTX, tumor suppressor genes, VHL, WT-I, p53, Hdm, PTEN, proteases, ubiquitin proteases, urokinase-type plasmmogen activator (uPA) and uPA receptor (uPAR) system, cathepsins, metalloprotemases, esterases, hydrolases, separase, ion channels, potassium channels, sodium channels, molecular transporters, multi-drug resistance proteins, P-Gycoprotein, nucleoside transporters, transcription factors/ DNA bindmg proteins, Ets, Elk, SMADs, ReI-A (p65-NFKB), CREB, NFAT, ATF-2, AFT, Myc, Fos, SpI, Egr-1, T-bet, β-catenm, HIFs, FOXOs, E2Fs, SRFs, TCFs, Egr-1, β-catenin, FOXO STATl, STAT 3, STAT 4, STAT 5, STAT 6, p53, WT-I, HMGA, regulators of translation, pS6, 4EPB-1, eIF4E- bmdmg protem, regulators of transcription, RNA polymerase, initiation factors, elongation factors In some embodiments, the protein is S 6

0132[00197] In some embodiments, an epitope-recognizing fragment of an activation state antibody rather than the whole antibody is used In some embodiments, the epitope-recognizing fragment is immobilized In some embodiments, the antibody light chain that recognizes an epitope is used A recombinant nucleic acid encoding a light chain gene product that recogmzes an epitope may be used to produce such an antibody fragment by recombinant means well known in the art

0133[00198] Non-activation state antibodies may also be used in the present mvention In some embodiments, 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 In some embodiments, non-activation state antibodies bmd 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 descnbed herem, 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 [00199] In some embodiments, 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 descnbed 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. [00200] In alternative embodiments of the instant invention, 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-I-, 2-, 3- or 4- pyreneylalamne, D- or L-3-thieneylalamne, D- or L-(2-pyπdinyl)-alamne, D- or L-(3-pyridinyl)-alanine, D- or L-(2- pyrazinyl)-alanine, D- or L-(4-isopropyl)-phenylglycine, D-(trifluoromethyl)-phenylglycine, D-(tnfluoromethyl)- phenylalamne, D-p-fluorophenylalanine, D- or L-p-biphenylphenylalanine, D- or L-p- methoxybiphenylphenylalanine, D- or L-2-indole(alkyl)alanines, and D- or L-alkylalanines where alkyl may be substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl, sec-isotyl, iso-pentyl, and non-acidic ammo acids of C1-C20.

0134[00201] Acidic ammo acids can be substituted with non-carboxylate ammo acids while maintaining a negative charge, and derivatives or analogs thereof, such as the non-limiting examples of (phosphono)alanine, glycine, leucme, isoleucine, threonine, or serine; or sulfated (e g , --SO3H) threonine, serine, or tyrosine.

0135[00202] Other substitutions may include non-natural hydroxylated amino acids may made by combining "alkyl" with any natural amino acid. The term "alkyl" as used herem refers to a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isoptopyl, n-butyl, lsobutyl, t-butyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracisyl and the like. Alkyl includes heteroalkyl, with atoms of nitrogen, oxygen and sulfur In some embodiments, alkyl groups herem contain 1 to 12 carbon atoms. Basic ammo acids may be substituted with alkyl groups at any position of the naturally occurring amino acids lysine, arginine, ornithine, citrulhne, or (guanidino)-acetic acid, or other (guanidino)alkyl-acetic acids, where "alkyl" is define as above. Nitrile deπvatives (e.g., containing the CN-moiety in place of COOH) may also be substituted for asparagine or glutamine, and methionine sulfoxide may be substituted for methionine. Methods of preparation of such peptide derivatives are well known to one skilled in the art.

0136[00203] In addition, any amide linkage in any of the polypeptides may be replaced by a ketomethylene moiety. Such denvatives 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. [00204] Additional ammo 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 deπvatives. 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-chloromercunbenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-l,3-diazole. [00205] Histidyl residues may be denvatized 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 IM sodium cacodylate at pH 6 0 [00206] Lysinyl and amino terminal residues may be reacted with compounds such as succinic or other carboxylic acid anhydrides Denvatization with these agents is expected to have the effect of reversing the charge of the lysinyl residues

0137[00207] Other suitable reagents for denvatizmg alpha-amino-containing residues include compounds such as lmidoesters, e g , as methyl picolinimidate, pyπdoxal phosphate, pyπdoxal, chloroborohydπde, tπmtrobenzenesulfomc acid, O-methyhsourea, 2,4 pentanedione, and transammase-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 ninhydnn accordmg to known method steps Denvatization of 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 tetrarutromethane

0138[00208] N-acetylimidizol and tetramtromethane may be used to form O-acetyl tyrosyl species and 3-nitro denvatives, respectively Carboxyl side groups (aspartyl or glutamyl) may be selectively modified by reaction with carbodiimides (R'~N-C-N~R') such as l-cyclohexyl-3-(2-morpholmy- l-(4-ethyl) carbodiimide or l-ethyl-3-(4- azonia-4,4-dimethylpentyl) carbodiimide Furthermore aspartyl and glutamyl residues may be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions

0139[00209] 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 [00210] In some embodiments, the activation state-specific binding element is a peptide compπsmg a recognition structure that binds to a target structure on an activatable protem A variety of recogmtion structures are well known m 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 (200I) J Am Chem Soc 123 625-32, Houimel et al (2001) Int J Cancer 92 748-55, each incorporated herein by reference) Further, fluorophores can be attached to such antibodies for use m the methods of the present invention

0140[00211] A variety of recognitions structures are known in the art (e g , Cochran et al , (2001) J Am Chem Soc 123 625-32, Boer et al , (2002) Blood 100 467-73, each expressly incorporated herein by reference)) and can be produced using methods known m the art (see e g , Boer et al , (2002) Blood 100 467-73, Guahllo et al , (2002) MoI Cell Endocrinol 190 83-9, each expressly incorporated herem by reference)), including for example combmatonal chemistry methods for producing recogmtion structures such as polymers with affinity for a target structure on an activatable protem (see e g , Barn et al , (2001) J Comb Chem 3 534-41, Ju et al , (1999) Biotechnol 64 232-9, each expressly incorporated herem by reference) In another embodiment, the activation state-specific antibody is a protem that only bmds to an isoform of a specific activatable protein that is phosphorylated and does not bmd to the isoform of this activatable protem when it is not phosphorylated or non-phosphorylated In another embodiment the activation state-specific antibody is a protem that only binds to an isoform of an activatable protem that is intracellular and not extracellular, or vice versa In a some embodiment, the recogmtion 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 MoI Biol 312 85-94, each expressly incorporated herein by reference)

0141[00212] In some embodiments the binding element is a nucleic acid The term "nucleic acid" include nucleic acid analogs, for example, phosphoramide (Beaucage et al , (1993) Tetrahedron 49(10) 1925 and references therein, Letsmger, J (1970) Org Chem 35 3800, Spπnzl 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

0142110 4470, and Pauwels et al , (1986) Chemica Scπpta 26 141-9), phosphorothioate (Mag et al , (1991) Nucleic Acids Res 19 1437, and U S Pat No 5,644,048), phosphorodithioate (Bπu et al , (1989) J Am Chem Soc

0143111 2321, O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues A Practical Approach, Oxford University Press), and peptide nucleic acid backbones and linkages (see Egholm, (1992) J Am Chem Soc 114 1895, Meier et al , (1992) Chem Int Ed Engl 31 1008, Nielsen, (1993) Nature, 365 566, Carlsson et al , (1996) Nature 380 207, all of which are incorporated by reference) Other analog nucleic acids mclude those with positive backbones (Denpcy et al , (1995) Proc Natl Acad Sci USA 92 6097, non-ionic backbones (U S Pat Nos 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863, Kiedrowshi et al , Angew Chem Intl Ed English 30 423 (1991), Letsmger et al , (1988) J Am Chem Soc 110 4470, Letsinger et al , (1994) Nucleoside &

0144Nucleotide 13 1597, Chapters 2 and 3, ASC Symposium Senes 580, "Carbohydrate Modifications in Antisense Research", Ed Y S Sanghui and P Dan Cook, Mesmaeker et al , (1994) Bioorganic & Medicinal Chem Lett 4 395, Jeffs et al , (1994) J Biomolecular NMR 34 17, Tetrahedron Lett 37 743 (1996)) and non-πbose backbones, including those descπbed m U S Pat Nos 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Senes 580, "Carbohydrate Modifications m Antisense Research", Ed Y S Sanghui and P Dan Cook Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids (see Jenkins et al , (1995) Chem Soc Rev ppl69-176) Several nucleic acid analogs are descπbed m Rawls, C & E News Jun 2, 1997 page 35 All of these references are hereby expressly incorporated by reference These modifications of the πbose- phosphate backbone may be done to facilitate the addition of additional moieties such as labels, or to mcrease the stability and half-life of such molecules in physiological environments

0145[00213] As will be appreciated by those in the art, all of these nucleic acid analogs may find use m the present invention In addition, mixtures of naturally occurring nucleic acids and analogs can be made Alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made In some embodiments, peptide nucleic acids (PNA) which includes peptide nucleic acid analogs are used These backbones are substantially non-ionic under neutral conditions, m contrast to the highly charged phosphodiester backbone of naturally occurring nucleic acids

0146[00214] The nucleic acids may be smgle 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 hybπd, where the nucleic acid contains any combination of deoxyπbo- and πbo-nucleotides, and any combmation of bases, mcludmg uracil, adenine, thymine, cytosine, guanine, mosine, xathamne hypoxathanine, isocytosine, isoguanine, etc

0147[00215] In some embodiments, 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 generatmg new compounds, including random chemistry methods as well as enzymatic methods [00216] Alternatively, some embodiments utilize natural compounds, as binding elements, in the form of bactenal, fungal, plant and animal extracts that are available or readily produced

0148[00217] Additionally, natural or synthetically produced compounds are readily modified through conventional chemical, physical and biochemical means Known pharmacological agents may be subjected to directed or random chemical modifications, including enzymatic modifications, to produce bmdmg elements that may be used in the instant invention

0149[00218] In some embodiment the binding element is a small organic compound Bmdmg elements can be synthesized from a senes of substrates that can be chemically modified "Chemically modified" herem 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 (mcludmg arenes and heteroaryl), alcohols, ethers, amines, aldehydes, ketones, acids, esters, amides, cyclic compounds, heterocyclic compounds (mcludmg purines, pyπmidmes, benzodiazepine, beta-lactams, tetracylines, cephalosporins, and carbohydrates), steroids (mcludmg estrogens, androgens, cortisone, ecodysone, etc ), alkaloids (mcludmg ergots, vinca, curare, pyrollizdine, and mitomycmes), organometallic compounds, hetero-atom bearing compounds, amino acids, and nucleosides Chemical (mcludmg enzymatic) reactions may be done on the moieties to form new substrates or binding elements that can then be used m the present invention

0150[00219] In some embodiments the bmdmg element is a carbohydrate As used herein the term carbohydrate is meant to mclude any compound with the general formula (CFkO)_n Examples of carbohydrates are di-, hi- and oligosaccharides, as well polysaccharides such as glycogen, cellulose, and starches [00220] In some embodiments the bmdmg element is a lipid As used herem the term lipid herem is meant to mclude any water insoluble organic molecule that is soluble m nonpolar organic solvents Examples of lipids are steroids, such as cholesterol, and phospholipids such as sphingomeylin

0151[00221] Examples of activatable elements, activation states and methods of determining the activation level of activatable elements are described m US publication number 20060073474 entitled "Methods and compositions for detectmg the activation state of multiple proteins m single cells" and US publication number 20050112700 entitled "Methods and compositions for nsk stratification" the content of which are incorporate here by reference

0152A Labels

0153[00222] The methods and compositions of the instant invention provide bmdmg elements compπsing a label or tag By label is meant a molecule that can be directly (i e , a primary label) or indirectly (l 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 bmdmg molecules, etc Specific binding molecules include pairs, such as biotm and streptavidin, digoxin and antidigoxin etc Examples of labels mclude, but are not limited to, optical fluorescent and chromogemc dyes mcludmg labels, label enzymes and radioisotopes

0154[00223] In some embodiments, one or more binding elements are umquely label Using the example of two activation state specific antibodies, by "uniquely labeled" is meant that a first activation state antibody recogmzing a first activated element comprises a first label, and second activation state antibody recogmzmg a second activated element compπses a second label, wherein the first and second labels are detectable and distinguishable, making the first antibody and the second antibody uniquely labeled

0155[00224] In general, labels fall into four classes a) isotopic labels, which may be radioactive or heavy isotopes, b) magnetic, electrical, thermal labels, c) colored, optical labels mcludmg luminescent, phosphorous and fluorescent dyes or moieties; and d) binding partners. Labels can also include enzymes (horseradish peroxidase, etc.) and magnetic particles. In some embodiments, the detection label is a primary label. A primary label is one that can be directly detected, such as a fluorophore.

0156[00225] 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).

0157[00226] Suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue™, 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 1 J9; 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. Clontech Laboratories, Inc., 1020 East Meadow Circle, Palo Alto, Calif. 94303), luciferase (Ichiki, et al., J. Immunol.

0158150(12):5408-5417 (1993)), .beta.-galactosidase (Nolan, et al., Proc Natl Acad Sci USA 85(8):2603-2607 (April 1988)) and Renilla WO 92/15673; WO 95/07463; WO 98/14605; WO 98/26277; WO 99/49019; U.S. Pat. No. 5,292,658; U.S. Pat. No. 5,418,155; U.S. Pat. No. 5,683,888; U.S. Pat. No. 5,741,668; U.S. Pat. No. 5,777,079; U.S. Pat. No. 5,804,387; U.S. Pat. No. 5,874,304; U.S. Pat. No. 5,876,995; and U.S. Pat. No. 5,925,558). All of the above-cited references are expressly incorporated herein by reference.

0159[00227] In some embodiments, 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, 111.), Cy5, Cy5.5, Cy7 (Amersham Life Science, Pittsburgh, Pa.). 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. In some embodiments 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, Abeam and Sigma, the contents of which are incorporated herein by reference.

0160[00228] In some embodiments, the fluorescent label is a GFP and, more preferably, a Renilla, Ptilosarcus, or Aequorea species of GFP. [00229] In some embodiments, 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. [00230] In some embodiments, the secondary label is a binding partner pair. For example, the label may be a hapten or antigen, which will bind its binding partner. For example, suitable binding partner pairs include, but are not limited to: antigens (such as proteins (including peptides) and small molecules) and antibodies (including fragments thereof (F Abs, etc.)); proteins and small molecules, including biotin/streptavidin; enzymes and substrates or inhibitors, other protein-protein interacting pairs, receptor-hgands, 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 lmino-biotin) and streptavidin, digeoxinin and Abs, and Prohnx™ reagents

0161[00231] In some embodiments, the binding partner pan- compπses an antigen and an antibody that will specifically bind to the antigen By "specifically bind" herein is meant that the partners bmd with specificity sufficient to differentiate between die 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 In some embodiments, the dissociation constants of the pair will be less than about 10^"4 to 10⁹ M ', with less than about 10 ⁵ to 10⁹ M ' being preferred and less than about 10⁷ to 10 ' M ' bemg particularly preferred [00232] In some embodiment, the secondary label is a chemically modifiable moiety In this embodiment, 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, ammo groups, carboxy groups, maleimide groups, oxo groups and thiol groups, with amino groups and thiol groups bemg particularly preferred For example, primary labels containing amino groups can be attached to secondary labels comprising amino groups, for example using linkers as are known m 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)

0162[00233] In some embodiments, multiple fluorescent labels are employed in the methods and compositions of the present invention In some embodiments, each lable is distmct and distinguishable from other labels [00234] As will be appreciated in the art antibody-label conjugation may be performed usmg standard procedures or by usmg protein-protein/protein-dye cross-linking kits from Molecular Probes (Eugene, Oreg ) [00235] In some embodiments, labeled antibodies are used for functional analysis of activatable proteins m cells In performing such analysis several areas of the expeπment are considered (1) identification of the proper combination of antibody cocktails for the stains (2), identification of the sequential procedure for die staining usmg 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 cπtical component in detecting differences For example, some cell lines have the ability to adapt to culture conditions and can yield heterogeneous responses [00236] In some embodiments, activation state-specific antibodies are labeled with quantum dots as disclosed by

0163Chattopadhyay, 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 mvitroeen com/products/qdot/ [00237] Quantum dot labeled antibodies can be used alone or they can be employed m conjunction with organic fluorochrome-conjugated antibodies to increase the total number of labels available As the number of labeled antibodies mcrease 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 Patent No 7,018850, entitled Salicylamide-Landianide Complexes for Use as Luminescent Markers Other methods of detecting fluorescence may also be used, e g , 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 [00238] In some embodiments, the activatable elements are labeled with tags suitable for Inductively Coupled Plasma Mass Spectrometer (ICP-MS) as disclosed m Tanner et al Spectrochimica Acta Part B Atomic Spectroscopy, 2007 Mar,62(3) 188-195,Ornatsky et al, mRNA Detection m Leukemia Cell lmes by Novel Metal- Tagged m situ Hybridization usmg 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

0164[00239] Alternatively, detection systems based on FRET, discussed in detail below, may be used FRET finds use in the instant invention, for example, m detecting activation states that involve clustering or multimeπzation wherem the proximity of two FRET labels is altered due to activation In some embodiments, at least two fluorescent labels are used which are members of a fluorescence resonance energy transfer (FRET) pair

0165[00240] 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 A Changes in the fluorescence emission spectrum compπsmg FRET pairs can be detected, mdicatmg changes in the number of that are in close proximity (i e , within 100 521 of each other) This will typically result from the bmdmg or dissociation of two molecules, one of which is labeled with a FRET donor and the other of which is labeled with a FRET acceptor, wherem such binding brings the FRET pair m close proximity Bmdmg of such molecules will result in an mcreased fluorescence emission of the acceptor and/or quenching of the fluorescence emission of the donor

0166[00241] FRET pairs (donor/acceptor) useful m the invention mclude, but are not limited to, EDANS/fluorescein, IAEDANS/fluorescem, fluorescein/tetramethylrhodamine, fluorescein/LC Red 640, fluorescein/Cy 5, fluorescein/Cy 5 5 and fluorescein/LC Red 705

0167[00242] In some embodiments when FRET is used, a fluorescent donor molecule and a non-fluorescent acceptor molecule ("quencher") may be employed In this application, 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 mclude, but are not limited to EDANS/DABCYL, Texas Red/D ABCYL, BODIPY/D ABCYL, Lucifer yellow/D ABCYL, coumaπn/D ABCYL and fluorescein/QSY 7 dye

0168[00243] The skilled artisan will appreciate that FRET and fluorescence quenching allow for monitoring of binding of labeled molecules over time, providing continuous information regarding the time course of binding reactions [00244] Preferably, changes m the degree of FRET are determined as a function of the change m the ratio of the amount of fluorescence from the donor and acceptor moieties, a process referred to as "ratioing " Changes m the absolute amount of substrate, excitation intensity, and turbidity or other background absorbances m the sample at the excitation wavelength affect the intensities of fluorescence from both the donor and acceptor approximately m parallel Therefore the ratio of the two emission intensities is a more robust and preferred measure of cleavage than either intensity alone

0169[00245] The ratio-metric fluorescent reporter system descnbed herein has significant advantages over existing reporters for protem integration analysis, as it allows sensitive detection and isolation of both expressmg and non- expressing single living cells In some embodiments, 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 Because the reporter readout is ratiometπc it is unique among reporter protein assays m 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 pπor 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 [00246] The methods and composition of the present mvention may also make use of label enzymes By label enzyme is meant an enzyme that may be reacted in the presence of a label enzyme substrate that produces a detectable product Suitable label enzymes for use m the present mvention include but are not limited to, horseradish peroxidase, alkaline phosphatase and glucose oxidase Methods for the use of such substrates are well known m 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 Other 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 descnbed m 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

0170[00247] ] By radioisotope is meant any radioactive molecule Suitable radioisotopes for use m the invention include, but are not limited to ¹⁴C, ³H, ³²P, ³³P, ³⁵S, ¹²⁵I, and ¹³¹I The use of radioisotopes as labels is well known in the art [00248] As mentioned, labels may be indirectly detected, that is, the tag is a partner of a binding pair By "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 mclude, 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 bmding protein (CBP)/calmodulin Other suitable binding pairs mclude 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 As will be appreciated by those in the art, binding pair partners may be used in applications other than for labeling, as is descnbed herem [00249] As will be appreciated by those in the art, a partner of one bindmg pair may also be a partner of another binding pair For example, an antigen (first moiety) may bind to a first antibody (second moiety) that may, in turn, be an antigen for a second antibody (third moiety) It will be further appreciated that such a circumstance allows indirect bindmg of a first moiety and a third moiety via an intermediary second moiety that is a binding pair partner to each [00250] As will be appreciated by those in the art, a partner of a bindmg pair may compnse a label, as described above It will further be appreciated that this allows for a tag to be indirectly labeled upon the bmding of a bindmg partner comprising a label Attaching a label to a tag that is a partner of a binding pair, as just descnbed, is referred to herein as "indirect labeling"

0171[00251] By "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 , MoI Cell Biol , 8 2159-2165 (1988)], the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibody substrates thereto [Evan et al , Molecular and Cellular Biology, 5 3610-3616 (1985)], and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody substrate [Paborsky et al , Protein Engineering, 3(6) 547-553 (1990)] In general, surface binding substrate molecules useful in the present mvention include, but are not limited to, polyhistidine structures (His-tags) that bmd mckel substrates, antigens that bind to surface substrates comprising antibody, haptens that bmd to avidin substrate (e g , biotin) and CBP that binds to surface substrate compnsmg calmodulin [00252] Production of antibody-embedded substrates is well known, see Slinkin et al , Bioconj Chem , 2 342-348 (1991), Torchilin et al , supra, Trubetskoy et al , Bioconj Chem 3 323-327 (1992), King et al , Cancer Res 54 6176-6185 (1994), and Wilbur et al , Bioconjugate Chem 5 220-235 (1994) (all of which are hereby expressly incorporated by reference), and attachment of or production of proteins with antigens is descnbed above Calmodulin-embedded substrates are commercially available, and production of proteins with CBP is descnbed in Simcox et al , Strategies 8 40-43 (1995), which is hereby incorporated by reference in its entirety

0172[00253] As will be appreciated by those in the art, tag-components of the invention can be made in vanous ways, dependmg largely upon the form of the tag Components of the invention and tags are preferably attached by a covalent bond [00254] The production of tag-polypeptides by recombinant means when the tag is also a polypeptide is descnbed 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, m 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 [00255] 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, carboxyhc 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 Similarly, a large number of haptenylation reagents are also known (Id ) [00256] Methods for labeling of proteins with radioisotopes are known in the art For example, such methods are found Ui Ohta et al , (1999) Molec Cell 3 535-541, which is hereby incorporated by reference in its entirety [00257] Production of proteins havmg tags by recombinant means is well known, and kits for producing such proteins are commercially available For example, such a kit and its use are descnbed in the QIAexpress Handbook from Qiagen by Joanne Crowe et al , hereby expressly incorporated by reference [00258] The functionalization of labels with chemically reactive groups such as thiols, amines, carboxyls, etc is generally known in the art In some embodiments, the tag is functionahzed to facilitate covalent attachment The covalent attachment of the tag may be either direct or via a linker In one embodiment, 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. Alternatively, the coupling moiety may have at least two functional groups, which are used to attach a functionalized component to a functionalized tag, for example. In an additional embodiment, the linker is a polymer. In this embodiment, covalent attachment is accomplished either directly, or through the use of coupling moieties from the component or tag to the polymer. In some embodiments, the covalent attachment is direct, that is, no linker is used. In this embodiment, 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. In some embodiments, 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. [00259] In some embodiments, the tag is functionalized to facilitate covalent attachment, as is generally outlined above. Thus, a wide variety of 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. Thus, for example, for direct linkage to a carboxylic acid group of a protein, amino modified or hydrazine modified tags will be used for coupling via carbodiimide chemistry, for example using 1- ethyl-3-(3-dimethylaminoproρyl)-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). In one embodiment, the carbodiimide is first attached to the tag, such as is commercially available for many of the tags described herein.

0173Alternative Activation State Indicators

0174[00260] 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. For example, phosphorylation of a substrate can be used to detect the activation of the kinase responsible for phosphorylating that substrate. Similarly, 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.

0175Modulators

0176[00261] In some embodiments, 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. [00262] Modulation can be performed in a variety of environments. In some embodiments, cells are exposed to a modulator immediately after collection. In some embodiments where there is a mixed population of cells, purification of cells is performed after modulation. In some embodiments, whole blood is collected to which a modulator is added. In some embodiments, cells are modulated after processing for single cells or purified fractions of single cells. As an illustrative example, whole blood can be collected and processed for an enriched fraction of lymphocytes that is then exposed to a modulator Modulation can mclude 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 [00263] In some embodiments, cells are cultured post collection in a suitable media before exposure to a modulator In some embodiments, the media is a growth media In some embodiments, the growth media is a complex media that may include serum In some embodiments, the growth media comprises serum In some embodiments, the serum is selected from the group consisting of fetal bovme serum, bovine serum, human serum, porcme serum, horse serum, and goat serum In some embodiments, the serum level ranges from 0 0001% to 30 % In some embodiments any suitable amount of serum is used In some embodiments, the growth media is a chemically defined minimal media and is without serum In some embodiments, cells are cultured in a differentiating media [00264] Modulators mclude chemical and biological entities, and physical or environmental stimuli Modulators can act extracellularly or intracellularly Chemical and biological modulators mclude 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 mclude complex and undefined biologic compositions that may compnse cellular or botanical extracts, cellular or glandular secretions, physiologic fluids such as serum, amniotic fluid, or venom Physical and environmental stimuli mclude electromagnetic, ultraviolet, infrared or particulate radiation, redox potential and pH, the presence or absences of nutrients, changes m 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 dependmg on the concentration and duration of exposure to the smgle cells or whether they are used m combination or sequentially with other modulators

0177Modulators 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 wherem the expressed gene product is the activatable element or is a modulator of the activatable element [00265] In some embodiments, modulators produce different activation states dependmg on the concentration of the modulator, duration of exposure or whether they are used in combmation or sequentially with other modulators [00266] In some embodiments 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) In some embodiments, the modulator is a physical stimuli such as heat, cold, UV radiation, and radiation Examples of modulators, include but are not limited to, F(ab)2 IgM, Rituxan, Alemtuzumab, anti CD22 (epratuzumab), anti-CD23 (lumihximab), Campath, H₂O₂, PMA, BAFF, Apnl, SDFIa, CD40L, IGF-I, Imiquimod, polyCpG, fludarabine, cyclophosphamide, chlorambucil, IL-7, IL-6, IL-IO, IL-27, IL-4, IL-2, IL-3, and thapsigargin

0178[00267] In some embodiments, 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, wherem 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

0179[00268] In some embodiments, the modulator is a B cell receptor modulator In some embodiments, 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 In some embodiments, cross-linker is an antibody or molecular bindmg 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 bindmg domain

0180[00269] In some embodiments, the cross-linker is a molecular binding entity In some embodiments, the molecular bmdmg entity acts upon or bmds the B cell receptor complex via carbohydrates or an epitope in the complex In some embodiments, 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 mcrease the local valency of the epitope bmdmg domain [00270] In some embodiments, the cross-linking of the B cell receptor complex or the B-cell co-receptor complex compnses bmdmg of an antibody or molecular bmdmg entity to the cell and then causing its crosslinkmg via mteraction of the cell with a solid surface that causes crosslinkmg of the BCR complex via antibody or molecular bmdmg entity [00271] In some embodiments, the crosslinker is F(ab)2 IgM, IgG, IgD, polyclonal BCR antibodies, monoclonal BCR antibodies, Fc receptor derived bmdmg elements and/or a combmation 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 In some embodiments, the crosslinker is F(ab)2 IgM, Polyclonal IgM antibodies, Monoclonal IgM antibodies, Biotmylated F(ab)2 IgCM, Biotinylated Polyclonal IgM antibodies, Biotinylated Monoclonal IgM antibodies and/or combmation thereof [00272] In some embodiments, the inhibitor is an inhibitor of a cellular factor or a plurality of factors that participates m a cellular pathway (e g signaling cascade) m the cell In some embodiments, the inhibitor is a kinase or phosphatase inhibitor Examples of kinase inhibitors mclude adaphostin, AG 490, AG 825, AG 957, AG 1024, aloisine, aloisine A, alsteipaullone, aminogenistein, API-2, apigenin, arctigenin, AY-22989, BAY 61-3606, bismdolylmaleimide IX, chelerythπne, 10-[4'-(N,N-Diethylamino)butyl]-2-chlorophenoxazine hydrochloride, dasatinib, 2- Dmiethylaimno-4,5,6,7-tetrabromo-lH-benzimidazole, 5,7-Dimethoxy-3-(4-pyndinyl)quinoline dihydrochloπde, edelfosme, ellagic acid, enzastaurm, ER 27319 maleate, erlotinib, ET18OCH3, fasudil, flavopiπdol, gefitimb, GW 5074, H-7, H-8, H-89, HA-100, HA-1004, HA-1077, HA-1100, hydroxyfasudil, indirubm-3'-oxime, 5- Iodotuberαdin, kenpaullone, KN-62, KY12420, LFM-A13, lavendustm A, luteolin, LY-294002, LY294002, mallotoxin, ML-9, NSC-154020, NSC-226080, NSC-231634, NSC-664704, NSC-680410, NU6102, olomoucine, oxmdole I, PD-153035, PD-98059, PD 169316, phloretin, phloπdzm, piceatannol, picropodophyllm, PKI, PPl, PP2, purvalanol A, quercetm, R406, R788, rapamune, rapamycin, Ro 31-8220, roscovitine, rottleπn, SB202190, SB203580, sirolimus, sorafemb, SL327, SP600125, staurospoπne, STI-571, SU-11274, SU1498, SU4312, SU6656, 4,5,6,7-Tetrabromotnazole, TG101348, Tπcinbme, Tyrphostm AG 490, Tyrphostin AG 825, Tyrphostin AG 957, Tyrphostin AG 1024, Tyrphostm SU1498, U0126, VX-509, VX-667, VX-680, W-7, wortmannin, XL-019, XL-147, XL-184, XL-228, XL-281, XL-518, XL-647, XL-765, XL-820, XL-844, XL-880, Y-27632, ZD-1839, ZM-252868, ZM-447439, siRNA, miRNA Examples of phosphatase inhibitors mclude, but are not limited to H₂O₂, siRNA, miRNA, Canthaπdin, (-)-ρ-Bromotetramisole, Microcystm LR, Sodium Orthovanadate, Sodium Pervanadate, Vanadyl sulfate, Sodium oxodiperoxo(l,10-phenanthroline) vanadate, bis(maltolato)oxovanadium(IV), Sodium Molybdate, Sodium Perm olybdate, Sodium Tartrate, Imidazole, Sodium Fluoπde, β-Glycerophosphate, Sodium Pyrophosphate Decahydrate, Calyculin A, Discodermia calyx, bpV(phen), mpV(pic), DMHV, Cypermethnn,

0181Dephostatin, Okadaic Acid, NIPP-I, N-(9,10-Dioxo-9,10-dihydro-phenanthren-2-yl)-2,2-dimethyl-propionamide, α- Bromo-4-hydroxyacetophenone, 4-Hydroxyphenacyl Br, α-Bromo-4-methoxyacetophenone, 4-Methoxyphenacyl Br, α-Bromo-4-(carboxymethoxy)acetophenone, 4-(Carboxymethoxy)phenacyl Br, and bis(4- Tnfluoromethylsulfonamidophenyl)-l,4-diisoρropylbenzene, phenyarsine oxide, Pyrrolidine Dithiocarbamate, and Aluminum fluoride In some embodiments, the phosphatase inhibitor is H₂O₂

0182[00273] In some embodiments H₂O₂ is administered as an inhibitor In some embodiments H₂O₂ is administered at between 0 01 and 50 mM In some embodiments H₂O₂ is administered at between 0 1 and 10 mM In some embodiments H₂O₂ is administered at between 1 and 10 mM In some embodiments H₂O₂ is administered at between 1 and 5 mM In some embodiments H₂O₂ 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, H₂O₂ is administered at 3 0 mM In certain embodiments, H₂O₂ is administered at 3 3 mM In some embodiments the duration of exposure OfH₂O₂ is between 0 01 and 360 minutes In some embodiments the duration of exposure OfH₂O₂ is between 0 1 and 240 minutes In some embodiments the duration of exposure OfH₂O₂ is between 0 5 and 180 minutes In some embodiments the duration of exposure OfH₂O₂ is between 0 and 120 minutes In some embodiments the duration of exposure to H₂O₂ is between 5 and 15 minutes In some embodiments the duration of exposure OfH₂O₂ 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 OfH₂O₂ is 10 minutes In some embodiments H₂O₂ is administered as an inhibitor with at least one other modulator In some embodiments H₂O₂ is administered as an inhibitor with F(ab)2 IgM or any suitable BCR agonist In some embodiments H₂O₂ is administered before administration of F(ab)2 IgM In some embodiments H₂O₂ is administered simultaneously with F(ab)2 IgM In some embodiments H₂O₂ is administered after F(ab)2 IgM [00274] In some embodiments, the activation level of an activatable element m 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 m 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 m 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 m a cell is determined after contacting the cell with two or more modulators [00275] In some embodiments, 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 In some embodiments, the modulators include F(ab)2 IgM, Rituxan, Alemtuzumab, anti CD22

0183(epratuzumab), anti-CD23 (lumiliximab), Campath, H₂O₂, PMA, BAFF, Apπl, SDFIa, CD40L, IGF-I, Imiquimod, polyCpG, fludarabine, cyclophosphamide, chlorambucil, IL-7, IL-6, IL-IO, IL-27, IL-4, IL-2, IL-3, thapsigardin and/or a combination thereof For instance a population of cells can be exposed to one or more, all or a combmation of the following combination of modulators (i) F(ab)2 IgM, (ii) Rituxan,, (in) Campath, (iv) H₂O₂ (v) PMA, (vi) BAFF, (vu) Apnl, (via) SDFIa, (ix) CD40L, (x) IGF-I, (xi) Imiquimod, (xπ) polyCpG, (xiu) fludarabine, (xiv)<img file="WO2009025847A2_D0001.tif" /> xv) chlorambucil, IL-7, (xvi) IL-6, (xvu)IL-lO, (xvm) IL-27, (xx) IL-4, (xx) IL-2, (xxi) IL-3, (xxn) Alemtuzumab, (xxin) anti CD22 (epratuzumab), (xxiv) anti-CD23 (lumiliximab), (xxv) thapsigargin, and (xxvi) F(ab)2 IgM and H₂O₂ In some embodiments, the phenotypic profile of the population of cells is used to classify the population as descnbed herein Detection

0184[00276] In practicing the methods of this invention, 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.

0185[00277] 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. 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.

0186[00278] In some embodiments, 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) are used to analyze cells on the basis of activatable element activation level, and can be detected as described below. Alternatively, non- binding elements systems as described above can be used in any system described herein. [00279] When using fluorescent labeled components in the methods and compositions of the present invention, it will recognized that different types of fluorescent monitoring systems, e.g., Cytometric measurement device systems, can be used to practice the invention. In some embodiments, 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. Taylor, D. L. &amp; Wang, Y.-L., San Diego: Academic Press (1989), pp. 219-243; Turro, N. J., Modern Molecular Photochemistry, Menlo Park: Benjamin/Cummings Publishing Col, Inc. (1978), pp. 296-361.

0187[00280] Fluorescence in a sample can be measured using a fluorimeter. In general, 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. In response, 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. According to one embodiment, 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. In general, known robotic systems and components can be used. [00281] Other methods of detecting fluorescence may also be used, e g , 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 In general, 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 photomultipher tubes to create a readable output, e g size, granularity, or fluorescent intensity [00282] The detectmg, sorting, or isolating step of the methods of the present mvention 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 A variety of FACS systems are known in the art and can be used m the methods of the invention (see e g , WO99/54494, filed Apr 16, 1999, U S Ser No 20010006787, filed JuI 5, 2001, each expressly incorporated herem by reference)

0188[00283] In some embodiments, a FACS cell sorter (e g a FACSVantage™ Cell Sorter, Becton Dickinson Immunocytometry Systems, San Jose, Calif) is used to sort and collect cells based on their activation profile (positive cells) m the presence or absence of a change m activation level in an activatable element in response to a modulator In some embodiments the change is a decrease In some embodiments the change is an mcrease [00284] In some embodiments, 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 In such an embodiment, 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 stenle collection vessels These cell-sorting procedures are descπbed in detail, for example, in the FACSVantage™ Training Manual, with particular reference to sections 3-11 to 3-28 and 10-1 to 10-17, which is hereby incorporated by reference in its entirety [00285] In another embodiment, positive cells can be sorted usmg magnetic separation of cells based on the presence of an isoform of an activatable element In such separation techniques, cells to be positively selected are first contacted with specific bindmg 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 The cell-binding element-particle complex can then be physically separated from non-positive or non-labeled cells, for example, using a magnetic field When using magnetically responsive particles, the positive or labeled cells can be retained m a container usmg a magnetic filed while the negative cells are removed These and similar separation procedures are descπbed, for example, in the Baxter Immunotherapy Isolex training manual which is hereby incorporated m its entirety [00286] In some embodiments, methods for the determination of a receptor element activation state profile for a smgle cell are provided The methods compπse 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 [00287] In some embodiment, cell analysis by flow cytometry on the basis of the activation level of at least two elements is combmed 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 smgle cells [00288] As will be appreciated, the present invention also provides for the ordering of element clustering events in signal transduction Particularly, 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 tune points to observe subpopulations arising out of the others

0189[00289] 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 smgle cell, the present invention allows the individual evaluation of cells to allow true differences to be identified m a significant way

0190[00290] Thus, the invention provides methods of distinguishing cellular subsets within a larger cellular population As outlined herein, these cellular subsets often exhibit altered biological characteristics (e g activation levels, altered response to modulators) as compared to other subsets within the population For example, as outlined herein, the methods of the invention allow the identification of subsets of cells from a population such as primary cell populations, e g penpheral blood mononuclear cells that exhibit altered responses (e g response associated with presence of a condition) as compared to other subsets In addition, this type of evaluation distinguishes between different activation states, altered responses to modulators, cell lineages, cell differentiation states, etc [00291] As will be appreciated, these methods provide for the identification of distinct signaling cascades for both artificial and stimulatory conditions m complex cell populations, such a peripheral blood mononuclear cells, or naive and memory lymphocytes

0191[00292] When necessary, cells are dispersed into a smgle cell suspension (e g by enzymatic digestion with a suitable protease, collagenase, dispase, etc, and the like) An appropπate 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 Convement buffers include HEPES 1 phosphate buffers, lactate buffers, etc The cells may be fixed, e g with 3% paraformaldehyde, and are usually peπneabihzed, e g with ice cold methanol, HEPES-buffered PBS containing 0 1% saponin, 3% BSA, covenng for 2 mm m acetone at -200C, and the like as known in the art and accordmg to the methods descπbed herem [00293] In some embodiments, one or more cells are contained in a well of a 96 well plate or other commercially available multi-well plate In an alternate embodiment, the reaction mixture or cells are m 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 [00294] 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 m a predetermined order or grouping under conditions appropnate for the activity that is assayed for Such conditions are descπbed here and known in the art Moreover, further guidance is provided below (see, e g , in the Examples) [00295] In some embodiments, the activation level of an activatable element is measured using Inductively Coupled Plasma Mass Spectrometer (ICP-MS) A binding element that has been labeled with a specific element binds to the activatable element When the cell is introduced into the ICP, it is atomized and lomzed The elemental composition of the cell, mcludmg 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.). [00296] As will be appreciated by one of skill in the art, the instant methods and compositions find use in a variety of other assay formats in addition to flow cytometry analysis. For example, 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. In addition, 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. In some embodiments, 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. [00297] In some embodiments, a chip comprises a multiplicity of the "second set binding elements," in this case generally unlabeled. Such a chip is contacted with sample, preferably cell extract, and 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. Preferably, each of the multiplicity of activation state-specific binding elements is uniquely labeled to facilitate detection. [00298] In some embodiments confocal microscopy can be used to detect activation profiles for individual cells.

0192Confocal 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. In some embodiments 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.

0193[00299] In some embodiments, the methods and compositions of the instant invention can be used in conjunction with an "In-CeIl Western Assay." In such 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., Nunc ™ 96 Microwell ™ 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. After incubation with the modulator cells are fixed and stained with labeled antibodies to the activation elements being investigated. Once the cells are labeled, 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 vl.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. [00300] In some embodiments, 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.

0194[00301] These instruments can fit in a sterile laminar flow or fume hood, or are enclosed, self-contained systems, for cell culture growth and transformation in multi-well plates or tubes and for hazardous operations. 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.

0195[00302] Flow cytometry or capillary electrophoresis formats can be used for individual capture of magnetic and other beads, particles, cells, and organisms. [00303] Flexible hardware and software allow instrument adaptability for multiple applications 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

0196[00304] In some embodiment, 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 In addition, any or all of the steps outlmed herein may be automated, thus, for example, the systems may be completely or partially automated [00305] As will be appreciated by those in the art, there are a wide variety of components which can be used, including, but not limited to, one or more robotic arms, plate handlers for the positioning of microplates, automated lid or cap handlers to remove and replace lids for wells on non-cross contamination plates, tip assemblies for sample distribution with disposable tips, washable tip assemblies for sample distribution, 96 well loading blocks, cooled reagent racks, microtiter plate pipette positions (optionally cooled), stacking towers for plates and tips, and computer systems

0197[00306] 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, dispensmg, 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 senal dilutions, and high capacity operation Additional examples of automation, automated sample collection and analysis are disclosed in US 61/048,657 which is hereby incorporated by reference in its entirety [00307] In some embodiments, chemically deπvatized 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 bmd 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 mvention

0198[00308] In some embodiments, 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 In some embodiments, the methods of the mvention mclude the use of a plate reader [00309] In some embodiments, 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 O⁰ C to 100° C [00310] In some embodiments, interchangeable pipet heads (single or multi-channel) with single or multiple magnetic probes, affinity probes, or pipetters robotically mampulate 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

0199[00311] In some embodiments, the instrumentation will include a detector, which can be a wide variety of different detectors, depending on the labels and assay In some embodiments, useful detectors include a microscope(s) with multiple channels of fluorescence, plate readers to provide fluorescent, ultraviolet and visible spectrophotometπc 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 [00312] In some embodiments, the robotic apparatus mcludes a central processmg unit which communicates with a memory and a set of input/output devices (e g , keyboard, mouse, momtor, printer, etc ) through a bus Again, as outlined below, this may be m addition to or in place of the CPU for the multiplexing devices of the invention The general interaction between a central processmg unit, a memory, input/output devices, and a bus is known m the art Thus, a variety of different procedures, depending on the experiments to be run, are stored in the CPU memory [00313] These robotic fluid handling systems can utilize any number of different reagents, mcludmg buffers, reagents, samples, washes, assay components such as label probes, etc

0200Analysis

0201[00314] Advances m flow cytometry have enabled the individual cell enumeration of up to thirteen simultaneous parameters (De Rosa et al , 2001) and are moving towards the study of genomic and proteomic data subsets (Krutzik and Nolan, 2003, Perez and Nolan, 2002) Likewise, advances in other techniques (e g microarrays) allow for the identification of multiple activatable elements As the number of parameters, epitopes, and samples have increased, the complexity of experiments and the challenges of data analysis have grown rapidly An additional layer of data complexity has been added by the development of stimulation panels which enable the study of activatable elements under a growing set of experimental conditions Methods for the analysis of multiple parameters are well known in the art In some embodiments flow cytometry applications require software for different phases of operation and analysis, see 61/079,579, 61/079,551, 61/079,537, 61/087,555, 61/085,789 which are hereby incorporated by reference in their entireties

0202[00315] In some embodiments where flow cytometry is used, flow cytometry experiments are arrayed and the results are approximated as fold changes using a heat map to facilitate evaluation Generally speaking, 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 m a set of flow cytometry samples is to overlay histograms of one parameter on the same plot Arrayed flow cytometry expeπments 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) [00316] In some embodiments where flow cytometry is used, pπor to analyzing of data the populations of interest and the method for characterizing these populations are determined 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 m a trial) In this more common case, cell populations are homogenous or lineage gated in such a way as to create distmct 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 cluneal 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 (u) "Inside-out" comparison of Parameters at the level of individual cells in a heterogeneous population An example of this would be the signal transduction state mappmg of mixed hematopoietic cells under certain conditions and subsequent comparison of computationally identified cell clusters with lineage specific markers This could be considered an inside-out approach to single cell studies as it does not presume the existence of specific populations pπor to classification A major drawback of this approach is that it creates populations which, at least initially, require multiple transient markers to enumerate and may never be accessible with a single cell surface epitope As a result, the biological significance of such populations can be difficult to determine The mam advantage of this unconventional approach is the unbiased tracking of cell populations without drawing potentially arbitrary distinctions between lmeages or cell types

0203[00317] Each of these techniques capitalizes on the ability of flow cytometry to deliver large amounts of multiparameter data at the single cell level For cells associated with 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 accordmg to historical techniques These techniques have included organ or tissue of oπgm, degree of differentiation, proliferation mdex, metastatic spread, and genetic or metabolic data regarding the patient

0204[00318] In some embodiments, the present invention uses variance mapping techniques for mappmg condition signaling space These methods represent a significant advance m 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) generally rely on the comparison of cells associated with a condition from each patient sample with a normal control, generally adjacent and theoretically untransformed tissue Alternatively, they rely on multiple clusterings and re-clustermgs to group and then further stratify patient samples accordmg to phenotype In contrast, variance mappmg of condition states compares condition samples first with themselves and then against the parent condition population As a result, activation states with the most diversity among conditions provide the core parameters m 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 [00319] In some embodiments, when variance mappmg is used to profile the signaling space of patient samples, conditions whose signaling response to modulators is similar are grouped together, regardless of tissue or cell type of origin Similarly, two conditions (e g two tumors) that are thought to be relatively alike based on lmeage markers or tissue of origin could have vastly different abilities to interpret environmental stimuli and would be profiled m two different groups [00320] When groups of signaling profiles have been identified it is frequently useful to determine whether other factors, such as cluneal responses, presence of gene mutations, and protem expression levels, are non-randomly distnbuted within the groups If expeπments or literature suggest such a hypothesis m an arrayed flow cytometry experiment, it can be judged with simple statistical tests, such as the Student's t-test and the X² test Similarly, if two variable factors within the experiment are thought to be related, the r² correlation coefficient from a linear regression is used to represent the degree of this relationship [00321] Examples of analysis for activatable elements are described in US publication number 20060073474 entitled "Methods and compositions for detecting the activation state of multiple protems in smgle cells" and US publication number 20050112700 entitled "Methods and compositions for πsk stratification" and US 61/085,789 the contents of which are incorporate here by reference

0205[00322] CLL serves as an example of the methods of the invention The data shown in Figures 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) In Figures 26, 27 &amp; 28 patient samples are indicated at the top of the heat map Each column represents a smgle patient CLL mdicates that the sample was obtained from a patient diagnosed with CLL CON mdicates 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 mcrease, or decrease, in mean fluorescence intensity (MFI), relative to the unstimulated control (0 mm)

0206[00323] 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 Thus, the heat map can define the presence or absence of an increase m the activation level of a plurality of activatable elements m a cell upon contacting said cell with an inhibitor or a modulator Labels to the πght of the heat map indicate the activatable element detected, e g a phospho-protem Labels to the πght also indicate the modulator or inhibitor treatment for that row "US" indicates unstimulated or untreated Figure 28 illustrates a pattern of activation levels of a plurality of activatable elements in a cell Figure 28 further illustrates the identification of patient clustering groups (i e clustering groups) A patient clustering group is comprised of samples from patients that display similar or distmct patterns of activation levels m one or more activatable elements m response to one or more modulators (e g , an inhibitor, or an inhibitor and another modulator) Figure 28 illustrates a clustering group compπsed of samples from patients in which the activation levels of p-PLCγ2, 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 H₂O₂ reveals a patient clustering group defined by the levels of p-PLCγ2, p-SyK/Zap-70, p-BLNK and p-Lck (Figure 28, bottom right boxed area) that are similar to those of the four control patients (Figure 28, bottom center box) Treatment with H₂O₂ further reveals a patient clustering group that is distinct from the controls (Figure 28, 9 patients to the left of bottom boxed area) Modulation with H₂O₂ 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-PLCγ2 (Figure 28, top left boxed area) that are similar to the control patients (top center box) In addition, modulation with H₂O₂ and BCR crosslinking further reveals another clustering group distmct from the controls (10 patients to the πght of top boxed area) [00324] Thus, also provided herein is a method of deπving a classification Deriving a classification mvolves defining a clustering group A clusteπng group is defined by determining the activation state of a plurality of activatable elements from a plurality of cells wherein each cell is denved from an individual with a known conditions and /or known cluneal outcome A clustenng 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 denved from a patient with an unknown condition and/or unknown clinical outcome may be classified depending upon which clusteπng group it is identified with This can further lead to diagnosis, prognosis, and/or evaluation or choice of treatment for the patient Kits

0207[00325] In some embodiments the invention provides kits Kits provided by the invention may comprise one or more of the state-specific bmdmg element descnbed herem, such as phospho-specific antibodies In some embodiments, the kit comprises one or more of the phospho-specific antibodies specific for the proteins selected from the group consisting of PI3-Kmase (p85, pi 10a, pi 10b, pi 1Od), Jakl, Jak2, SOCs, Rac, Rho, Cdc42, Ras- GAP, Vav, Tiam, Sos, DbI, Nek, Gab, PRK, SHPl, and SHP2, SHIPl, SHIP2, sSHIP, PTEN, She, Grb2, PDKl, SGK, Aktl, Akt2, Akt3, TSC1,2, Rheb, mTor, 4EBP-1, p70S6Kinase, S6, LKB-I, AMPK, PFK, Acetyl-CoAa Carboxylase, DokS, Rafs, Mos, Tpl2, MEK1/2, MLK3, TAK, DLK, MKK3/6, MEKK1.4, MLK3, ASKl, MKK4/7, SAPK/JNK1,2,3, p38s, Erkl/2, Syk, Btk, BLNK, LAT, ZAP70, Lck, CbI, SLP-76, PLCyi, PLCγ2, STATl, STAT 3, STAT 4, STAT 5, STAT 6, FAK, pl30CAS, PAKs, LIMK1/2, Hsp90, Hsp70, Hsp27, SMADs, ReI-A (p65- NFKB), CREB, Histone H2B, HATs, HDACs, PKR, Rb, Cyclin D, Cyclin E, Cyclin A, Cyclin B, P16, pHArf, P27KIP, p21CIP, Cdk4, Cdk6, Cdk7, Cdkl, Cdk2, Cdk9, Cdc25,A/B/C, AbI, E2F, FADD, TRADD, TRAF2, RIP, Myd88, BAD, Bcl-2, McI-I, BcI-XL, Caspase 2, Caspase 3, Caspase 6, Caspase 7, Caspase 8, Caspase 9, PARP, IAPs, Smac, Fodπn, Actm, Src, Lyn, Fyn, Lck, NIK, IKB, _P65(RelA), KKa, PKA, PKCa, PKCβ, PKCΘ, PKCδ, CAMK, Elk, AFT, Myc, Egr-1, NFAT, ATF-2, Mdm2, p53, DNA-PK, Chkl , Chk2, ATM, ATR, βcatenin, CrkL, GSK3α, GSK3β, and FOXO In some embodiments, the kit compnses one or more of the phospho-specific antibodies specific for the proteins selected from the group consisting of Erk, Syk, Zap70, Lck, Btk, BLNK, CbI, PLCγ2, Akt, ReIA, p38, S6 In some embodiments, the kit comprises one or more of the phospho-specific antibodies specific for the proteins selected from the group consisting of Aktl, Akt2, Akt3, SAPK/JNK1,2,3, p38s, Erkl/2, Syk, ZAP70, Btk, BLNK, Lck, PLCγ, PLCiγ 2, STATl, STAT 3, STAT 4, STAT 5, STAT 6, CREB, Lyn, P-S6, CbI, NF-KB, GSK3β, CARMA/BcllO and Tcl-1

0208[00326] Kits provided by the invention may comprise one or more of the modulators descnbed herem In some embodiments, the kit compnses one or more modulators selected from the group consistmg of F(ab)2 IgM, H₂O₂, PMA, BAFF, Apnl, SDFIa, CD40L, IGF-I, Imiquimod, polyCpG, IL-7, IL-6, IL-IO, IL-27, IL-4, IL-2, IL-3, thapsigargin and a combination thereof

0209[00327] 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 , polysacchandes and the like The kit may further mclude, 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

0210[00328] Such 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

0211[00329] Such kits may additionally compnse one or more therapeutic agents The kit may further compnse a software package for data analysis of the physiological status, which may include reference profiles for companson with the test profile [00330] Such kits may also include information, such as scientific literature references, package insert matenals, clinical tnal results, and/or summanes of these and the like, which mdicate or establish the activities and/or advantages of the composition, and/or which descnbe dosing, administration, side effects, drug interactions, or other information useful to the health care provider Such kits may also mclude instructions to access a database such as descπbed in USSN 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 m vivo models and studies based on human clinical trials Kits descπbed herein can be provided, marketed and/or promoted to health providers, including physicians, nurses, pharmacists, formulary officials, and the like Kits may also, m some embodiments, be marketed directly to the consumer

0212[00331] The following examples serve to more fully describe the manner of using the above-descπbed invention, as well as to set forth the best modes contemplated for carrying out various aspects of the invention It is understood that these examples in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes All references cited herein are expressly incorporated by reference m their entirety

EXAMPLES

0214Example 1: Signaling pathways in CLL samples

0215Signals propagated through the B cell receptor (BCR) guide the maturation and survival of B cells and might factor in the pathogenesis and progression of chrome lymphocytic leukemia (CLL) In this example, BCR signaling m CLL cells was investigated at the single-cell level using multiparametnc flow cytometry Concurrent analysis was performed usmg fluorochrome-conjugated antibodies specific for B-cell surface antigens and a panel of antibodies recognizing specific phospho-peptide epitopes within a selected group of intracellular signaling proteins CLL samples from patients (N=6) showed weak or minimal signaling activity at p72SYK/p70ZAP, Erkl/2, B-CeIl linker protein (BLNK) and phosρholipase-Cγ-2, (PLCγ2) when stimulated only at the BCR with anti-μ 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 protem required for signaling, or by enhanced inhibition mediated by phosphatases such as SHIP- 1 , SHIP-2 , SHP- 1 , or SHP-2 To determine whether phosphatases were preventing or dampemng BCR activation in CLL samples, CLL cells were treated with hydrogen peroxide (H₂O₂), a physiologic phosphatase inhibitor generated during BCR signaling that has been used previously to reveal dysregulated BCR signaling in follicular lymphoma (Sing et al , (2005) Cell, Reth (2002) Nat Immunol , Insh et al , (2006) Blood H₂O₂ treatment of CLL cells induced high-levels of phosphorylated p72SYK/p70ZAP, ERKl/2, BLNK, and PLCy2, m some patients independent of surface F(ab)2anti-μ ligation In contrast, other CLL-B cells were significantly less responsive to this treatment, even in the presence of F(ab)2anti-μ Exposure of blood B cells from healthy donors to H₂O₂ failed to elicit a substantial increase m phosphorylation of these same intracellular signaling proteins These studies reveal a previously unrecognized, constitutive high-level phosphatase activity in some CLL cells possibly contributing to the attenuated signaling observed m these cells following surface IgM ligation

0216Analysis of signaling pathways in CLL cells [00332] Ramos cells were maintained and cultured using methods known in the art Cells from CLL patients were obtained using methods known m the art

0217[00333] BCR cross-linking and preparation for staining Cells were thawed at 37⁰C m a water bath until partially thawed (-50%) Cells were thawed mto 5% FBS/RPMI at room temperature The cell suspension was added drop- wise into media Cells were centπfuged 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-centnfuged 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 37⁰C for 2 hours. The final cell concentration, including additives and reagents (e.g. stimulant, Fab fragment, H₂O₂, aqua, etc.) was 1.6 million/ml. Cells were aliquoted into wells. F(ab)₂lgM alone, 3.3 mM H₂O₂ alone orF(ab)₂IgM and H₂O₂ (H₂O₂ added within 30 seconds of F(ab)₂IgM) were added to the appropriate tubes and mixed by vortexing the samples. Cells were incubated with the different treatments for 15 minutes, unless a time course was performed. If 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).

0218[00334] Staining: Tubes were washed with 2 ml of 0.4% BSA PBS (ice cold) and centrifuged at 2000 RPM for 10 minutes at 4⁰C. 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. Cocktail

0219(i) CD3 pac blue = 160 μl (ii) CD4 APC = 80 μl (iii) CD8 PE Cy7 = 80 μl (iv) CD5 PE Cy5 = 400 μl (v) CD20 PerCP Cy5.5 = 800 μl

0220(vi) p-Erk = 800 μl (vii) p-BLNK = 800 μl (viii) p-Syk/ZAP70 = 800 μl b. Total antibody volume in the cocktail mix was 3920 μl. PBS was added to bring the total volume to 8.0 ml. Each well received 100 μl of this cocktail mix.

0221[00335] Analysis by Flow Cytometry: Between 10,000 and 100,000 un-gated events were collected for each sample on the BD LSR II flow cytometry machine. The fluorescent anti-bodies directed to extra-cellular markers (i.e. CD3, CD4, CD8, CD5 and CD20) were used to mark the cells and a hierarchical gating strategy was used to identify the B-CeIIs and mature B-CeIl population among the recorded events as described below: a. Forward and side scatter were first used to gate the Lymphocyte (Lymph) population. b. This "Lymph" population was then displayed on the CD20 and CD3 axes to gate for high CD3 expressing cells (CD3+) and high CD20 expressing cells (CD20+). c. The "CD20+" population was further gated to identify the mature B-CeIl (high CD5 expressing cells - CD5+) population by displaying them on the CD20 and CD5 axes. d. All the recorded events were also marked by the following intra-cellular markers p-Erk, p-

0222Syk/Zap70, and p-BLNK.

0223[00336] A probability density estimate of log₁₀ 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.

0224[00337] Results: Figure 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. After both stimulation with PMA (dotted line) and F(ab)₂ IgM (thin solid line) the p-Erk and p-Syk fluorescence is above the autofluorescence and background signal indicating activation of those proteins upon stimulation. Figure 2 shows that F(ab)₂ IgM (dotted line) also activates pBLNK, pCbl, ρPLCγ2, pLck, p38 in Ramos cells. Ramos cells (Ramos cell lines) are used as positive control throughout the experiments performed herein.

0225[00338] Cells from CLL patients were analyzed. Figure 3 shows that PMA (thin solid line) activates Erk in pheresed CLL samples. However, increasing amounts of amounts of F(ab)₂IgM for 15 min minimally activated signaling in CLL samples (Figure 4). [00339] CLL samples were divided into populations with low and high frequency of ZAP70. Zap70 can be a prognostic indicator used for CLL. However the clinical implications of CLL samples that contain ZAP70 are unclear. CLL samples show modest p-Erk (y-axis) and p-Syk (x-axis) phosphorylation when activated with PMA (Figure 5). These cells show weak activation when activated with F(ab)₂IgM as shown in Figure 5. [00340] CLL samples were treated with H₂O₂ or in combination with F(ab)₂IgM. H₂O₂ is a known inhibitor of phosphatases. Inhibition of phosphatases activates Erk and Syk as shown in Figure 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)₂IgM alone (Figure 4-5). F(ab)₂IgM and H₂O₂ reveals different kinetics subpopulation (heterogeneity) differences between CLL patients (Figure 6). In contrast, in normal B cells H₂O₂ blocks phosphatases, but is incapable of activating post BCR by itself (Figure 7). Without meaning to be limited to any theory, these results suggest that these CLLs have alterations in signaling proximal to the BCR. In CLL H₂O₂ 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 Figure 8.

0226[00341] Other CLL samples show modest and weak Erk and Syk phosphorylation when activated with PMA and F(ab)₂IgM, respectively (Figure 9). When these CLL samples are treated with H₂O₂, inhibition of phosphatases does not reveal strong tonic BCR signaling (Figure 10). Treatment with F(ab)₂IgM and H₂O₂ reveals: (i) different kinetics (ii) subpopulation heterogeneity and (iii) differences between CLL patients as shown in Figure 10. Kinetics of Signaling in CLL Specimens [00342] Cells were prepared and stained as described above. [00343] Figure 12 shows different kinetics of Syk and Erk phosphorylation by F(ab)₂IgM and H₂O₂. Peak phosphorylation of Syk and BLNK occurred after 5 min of activation, whereas peak phosphorylation of Erk after 30 min.

0227[00344] Figure 13 shows moderate activation of Syk/Zap70 and Erk by F(ab)₂IgM and H₂O₂. Figure 14 shows the kinetics of signaling by F(ab)₂IgM and H₂O₂. Figure 21 shows minimal activation of Syk/Zap70 and Erk by F(ab)2IgM alone over time. Figure 22 shows minimal activation of Syk/Zap70 and Erk in response to F(ab)2IgM alone over time. Figure 23 shows an F(ab)2 time course of CD20+/CD5+ population, without H₂O₂.

0228[00345] Figure 15 and 16 show different levels of BLNK phosphorylation in response to an external stimulus in cells with different levels of ZAP70.

0229[00346] Figures 17-21 show the kinetics of phosphorylation of PLCγ2, S6 and Cblin the CD20+/CD5+ cell population of CLL samples in response to B cell receptor crosslinking with and without peroxide. Figure 24 shows the kinetics OfH₂O₂ treatment in CD20+/CD5+ population of CLL samples. Figure 25 shows the kinetics OfH₂O₂ treatment in CD20+/CD5+ population of CLL samples. These results show that F(ab)₂IgM, alone mediates an increase in the phosphorylation of rpS6, in contrast to previously evaluated signaling molecules. H₂O₂ alone or in combination with F(ab)₂lgM attenuates this phosphorylation PLCγ2 phosphorylation increased in response to H₂O₂ alone or to the combination with F(ab)₂lgM CbI has a minimal response to all treatments Without being limited to any theory, these results suggest that a separate pathway emanates from the BCR that regulates prpS6 and is distmct from the pathway(s) that regulate Erk, Syk/Zap, BLNK and PLCγ2 with a distinct negative feedback loop [00347] In summary, these results show that for CLL patient samples PMA's activation of p-Erk is comparable m all patients regardless of ZAP70 expression and F(ab)₂IgM alone did not activate signaling In two out of six CLL specimens two subpopulations of cells with distinct signaling profiles were observed Finally, the kinetics of activation is different for Syk/ZAP70, BLNK and Erk Example 2: CLL Patients Display Distinguishable Patterns [00348] Isolation, storage, thawing, and equilibration of primary cells PBMC were isolated using density gradient separation (Ficoll-Paque Plus, Amersham Biosciences) In some embodiments, PBMC were pelleted by low-speed centnfugation, resuspended in medium composed of 90% FCS (HyClone) + 10% DMSO (Sigma- Aldπch), frozen slowly in the vapor phase of liquid nitrogen m multiple cryotubes, and stored in liquid nitrogen For signaling analysis of frozen samples, an individual cryotube was thawed into 5 ml of RMPI +1% serum, counted, pelleted, and resuspended at 3 3 x 10⁶ cells/ml Thawed PBMC were allowed to rest at 37°C in a CO₂ incubator for 2 h before stimulation

0230Modulation

0231[00349] At least half an hour before stimulation, 300 μl of medium containing 1 x 10⁶ PBMC was ahquoted into flow cytometry tubes (Falcon 2052, BD Biosciences) and allowed to rest at 37°C m a CO₂ incubator Cross-linking of B cell receptors was achieved usmg goat polyclonal anti-IgM and anti-IgG F(ab')₂ (BioSource International) When used, H₂O₂ was at 3 3 mM final concentration and was added as 2 μl of a 500-mM stock solution immediately after BCR cross-linking During signaling, cells were kept in a 37°C CO₂ incubator to allow signal transduction and phosphorylation Signaling was stopped after 10 nun by fixing the cells To determine basal levels of phosphorylation, unstimulated cells were maintained in parallel with stimulated cells and fixed at tune zero For fixation, paraformaldehyde (Electron Microscopy Services) was added to each tube of cells to a final concentration of 1 4% Cells were fixed for 5 mm at room temperature, pelleted, permeabilized by resuspension in 2 ml of methanol for 10 mm, and stored at 4^CC until being stained for flow cytometry Flow Cytometry [00350] Paraformaldehyde-fixed, methanol-permeabilized cells were rehydrated by addition of 2 ml of PBS, gentle resuspension, and then centnfugation 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-ERKl/2(T202/Y204), and p-Syk(Y352)/Zap70(Y319), p- Lck/p-Lyn, p-BLNK, ρ-PLCγ2 (PLCr2) or p-S6 Detection, selection and gating of cell subsets was as described in Example I Heat maps were generated using MeV (MultiExpenment Viewer) software (Figures 26, 27 &amp; 28) Results

0232[00351] The data shown in Figures 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 groupmg of cryptic or aberrant hematopoetic populations In Figures 26, 27 and 28 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) Labels to the right of the histogram indicate the phospho-protein stained and the modulator treatment for that row "US" indicates unstimulated Figure 28 illustrates the identification of several patient clustering groups comprised of similar or distinct levels of p-PLCγ2, p-SyK/Zap-70, p-BLNK and p-Lck Some patient clusterings become apparant upon modulation as illustrated by the boxed regions

0233[00352] In Figure 28, treatment with H₂C>₂ reveals a patient clustering defined by the levels of p-PLCγ2, 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 H₂O₂ further reveals a patient clustering that is distinct from the controls (9 patients to the left of bottom boxed area) Modulation with H₂O₂ and BCR crosslinking defines another patient clustering comprised of levels of p-BLNK, p-Syk and p-PLCγ2 (top left boxed area) that are similar to the control patients (top center box) and an abberant population of responders (10 patients to the nght of top boxed area)

0234Example 3: Evaluation of Apoptosis Pathways in CLL Patient Samples [00353] Current therapeutic approaches for CLL involve fludarabine-based regimens combined with monoclonal antibodies such as ntuximab 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, m some situations, ntuximab binding to CD20 inhibits proliferation and mduces cellular apoptosis (for a discussion of apoptosis see USSN 61/085,789)

0235[00354] Cellular apoptosis in response to therapeutic agents, including but not limited to, DNA damagmg agents such as Fludarabine or biological agents such as Rituximab, can be measured by multiparameter flow cytometry usmg fluorophore-conjugated antibodies that recognize intracellular protem components or nodes of the apoptotic machinery Such nodes may include, but are not limited to, Caspase 3, Caspase 8, Cytochrome C, Poly ADP πbose polymerase (PARP), Bcl-2, BcI-X, p-Chk2, p-BAD Further information may be gathered by treating cells with a pan-caspase inhibitor Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD FMK) m order to reveal caspase-dependent and/or mdependent pathways The profile of how the apoptotic proteins respond to treatment with a therapeutic agent can be used to inform clinical decisions

0236Experimental Procedure for measuring response to apoptosis of samples treated with Fludarabine and Rituximab [00355] Cells are thawed at 37⁰C m water bath until partially thawed (-50%) and are then added to RPMI/1% FBS at room temperature Cells are centπfuged 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 mcubation at 37°C for 2 hr , cells are ahquoted at a concentration of IxIO⁶ cells per well of a 96-well plate Cells are incubated with Fludarabine (at concentrations of 0- 50 μM), Rituximab (at concentrations from 0-500 μM) and/or Staurospoπne, alone or m combinations, in the absence or presence of ZVAD for various times Post- mcubation 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 descπbed above Cells are analyzed by flow cytometry The details of cell processing and flow cytometry analysis are given in USSN 61/085,789 [00356] While preferred embodiments of the present invention have been shown and descnbed herein, it will be obvious to those skilled m the art that such embodiments are provided by way of example only Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention It should be understood that various alternatives to the embodiments of the mvention described herein may be employed in practicing the invention It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and then- equivalents be covered thereby.