Methods for diagnosis, prognosis and methods of treatment
This invention is directed to methods and compositions for diagnosis, prognosis and for determining methods of treatment. The physiological status of cells present in a sample (e.g. clinical sample) can be used in diagnosis or prognosis of a condition (e.g. Chronic Lymphocytic Leukemia), in patient selection for therapy, to monitor treatment and to modify or optimize therapeutic regimens. The physiological status of a cell can be determined by comparing the intracellular status of one or more activation elements (e.g. the phosphorylation status of a signaling molecule) in a cell (e.g. a cancer cell) to that of another cell (e.g. a normal cell). The physiological status of a cell can be further classified by adding one or more modulators (e.g. an inhibitor or activator) to the cell in question. In some embodiments, the invention is directed to methods of determining a phenotypic profile of a population of cells.
1. A method of determining a status of a B-cell receptor signaling pathway in a primary chronic lymphoid leukemia (CLL) cell comprising:
contacting said CLL cell with a B-cell receptor modulator which is selected from the group consisting of Rituxan, a cross-linker of a B-cell receptor complex, a B-cell co-receptor complex, F(ab)2 IgM, IgG, IgD, polyclonal BCR antibodies, monoclonal BCR antibodies, Fc receptor derived binding elements and/or combinations thereof;
determining an activation level of an activatable element in a B-cell receptor signaling pathway in said CLL cell on a single cell basis, said activatable element comprising p-Erk ½; and
determining the status of the B-cell receptor signaling pathway in said CLL cell from said activation level of p-Erk ½.
2. The method of claim 1 , wherein the B-cell receptor modulator is F(ab)2 IgM.
3. The method of claim 1 , wherein the activation level of said activatable element is compared to a normal cell contacted with said modulator.
4. The method of claim 1 , further comprising contacting said cell with a kinase or phosphatase inhibitor.
5. The method of claim 1 , further comprising determining an activation level of a plurality of activatable elements.
6. The method of claim 4 , wherein said kinase or phosphatase inhibitor is selected from the group consisting of adaphostin, aloisine A, alsterpaullone, aminogenistein, API-2, apigenin, arctigenin, AY-22989, BAY 61-3606, bisindolylmaleimide IX, chelerythrine, 10-[4′-(N,N-Diethylamino)butyl]-2-chlorophenoxazine hydrochloride, dasatinib, 2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole, 5,7-Dimethoxy-3-(4-pyridinyl)quinoline dihydrochloride, edelfosine, ellagic acid, enzastaurin, ER 27319 maleate, erlotinib, ET18OCH3, fasudil, flavopiridol, 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, PP1, PP2, purvalanol A, quercetin, R406, R788, rapamune, rapamycin, Ro 31-8220, roscovitine, rottlerin, SB202190, SB203580, sirolimus, sorafenib, SL327, SP600125, staurosporine, STI-571, SU-11274, 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, H 2 O 2 , siRNA, miRNA, Cantharidin, (−)-p-Bromotetramisole, Microcystin LR, Sodium Orthovanadate, Sodium Pervanadate, Vanadyl sulfate, Sodium oxodiperoxo(1,10-phenanthroline)vanadate, bis(maltolato)oxovanadium(IV), Sodium Molybdate, Sodium Permolybdate, Sodium Tartrate, Imidazole, Sodium Fluoride, β-Glycerophosphate, Sodium Pyrophosphate Decahydrate, Calyculin A, Discodermia calyx, bpV(phen), mpV(pic), DMHV, Cypermethrin, Dephostatin, Okadaic Acid, NIPP-1, 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, bis(4-Trifluoromethylsulfonamidophenyl)-1,4-diisopropylbenzene, phenyarsine oxide, Pyrrolidine Dithiocarbamate, and aluminum fluoride.
7. The method of claim 1 , wherein the activation level is selected from the group consisting of glycosylation level, phosphorylation level, acetylation level, methylation level, biotinylation level, glutamylation level, glycylation level, hydroxylation level, isomerization level, prenylation level, myristoylation level, lipoylation level, phosphopantetheinylation level, sulfation level, ISGylation level, nitrosylation level, palmitoylation level, SUMOylation level, ubiquitination level, neddylation level, citrullination level, deamidation level, disulfide bond formation level, proteolytic cleavage level, translocation level, changes in protein turnover, multi-protein complex level, oxidation level, multi-lipid complex level, and biochemical changes in cell membrane.
8. The method of claim 1 , wherein said activatable element further comprises a protein selected from the group consisting of Akt1, Akt2, Akt3, SAPK/JNK1,2,3, p38s, Syk, ZAP70, Btk, BLNK, Lck, PLCγ, PLC1γ 2 , STAT1, STAT 3, STAT 4, STAT 5, STAT 6, CREB, Lyn, p-S6, Cbl, NF-κB, GSK3β, CARMA/Bc110 and Tcl-1.
9. The method of claim 4 , wherein the kinase or phosphatase inhibitor is R788.
10. A method of determining a status of a B-cell receptor signaling pathway of a primary CLL cell, comprising:
subjecting said CLL cell to a first modulator comprising H 2 O 2 ;
subjecting the cell to at least a second modulator comprising F(ab)2 IgM;
determining an activation level of a phosphorylated protein, on a single cell basis, that participates in a B-cell receptor signaling pathway in said cell, said phosphorylated protein comprising p-Erk ½; and
determining the status of the B-cell receptor signaling pathway in said CLL cell from said activation level of p-Erk ½.
11. A method of determining a status of a B-cell receptor signaling pathway in a primary CLL cell comprising:
contacting the CLL cell with a modulator which is a B-cell receptor crosslinker selected from the group consisting of F(ab)2 IgM, IgG, IgD, polyclonal BCR antibodies, or monoclonal BCR antibodies, and Fc receptor derived binding elements;
determining an activation level of an activatable element, on a single cell basis, that participates in a tonic signaling pathway and a B-cell receptor signaling pathway in said CLL cell, said activatable element comprising p-Erk ½; and
determining the status of the B-cell receptor signaling pathway in said CLL cell from said activation level of p-Erk ½.
12. The method of claim 11 , wherein the B-cell receptor crosslinker comprises F(ab)2 IgM.
13. The method of claim 11 , further comprising providing a kinase or phosphatase inhibitor.
14. The method of claim 13 , wherein the kinase or phosphatase inhibitor comprises R788.
15. The method of claim 1 further comprising detecting a surface marker on the cell.
16. The method of claim 15 wherein the cell surface marker is selected from the group consisting of CD45, CD5, CD19, CD20, CD22, CD23, CD27, CD37, CD40, CD52, CD79, CD38, CD96, major histocompatability antigen (MHC) Classl, MHC Class 2, and combinations thereof.
17. The method of claim 1 wherein the activation level of the activatable element is determined by a method comprising:
(i) contacting the cell with a detectable binding element that specifically binds to the activated form of the activatable element; and
(ii) detecting the binding element.
18. The method of claim 17 wherein the detecting is performed using a flow cytometer or a mass spectrometer.