Methods for diagnosis, prognosis and methods of treatment

• US 20100009364A1
• Filed: 07/10/2009
• Published: 01/14/2010
• Est. Priority Date: 07/10/2008
• Status: Active Grant

• Current Assignee: Nodality Inc.
• Original Assignee: Nodality Inc.
• Inventors: Rosen, David B., Cohen, Aileen, Cesano, Alessandra, Fantl, Wendy J., Putta, Santosh K., Nolan, Garry, Evensen, Erik
• Granted Patent: US 8,227,202 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: G01N 2333/70589 CD45
• CPC Class Codes: G01N 2800/22 Haematology
• CPC Class Codes: G01N 2800/52 Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
• CPC Class Codes: G01N 33/5041 involving analysis of members of signalling pathways
• CPC Class Codes: G01N 33/5044 involving specific cell types
• CPC Class Codes: G01N 33/5055 involving macrophages
• CPC Class Codes: G01N 33/5073 Stem cells
• CPC Class Codes: G01N 33/57426 leukemia
• CPC Class Codes: G01N 33/6893 related to diseases not provided for elsewhere

Abstract

The present invention provides an approach for the determination of the activation states of a plurality of proteins in single cells. This approach permits the rapid detection of heterogeneity in a complex cell population based on activation states, expression markers and other criteria, and the identification of cellular subsets that exhibit correlated changes in activation within the cell population. Moreover, this approach allows the correlation of cellular activities or properties. In addition, the use of modulators of cellular activation allows for characterization of pathways and cell populations. Several exemplary diseases that can be analyzed using the invention include AML, MDS, and MPN.

Claims

1. A method of diagnosing, prognosing, or determining progression of acute leukemia, myelodysplastic syndrome or myeloproliferative neoplasms in an individual, said method comprising:

A] classifying one or more hematopoietic cells associated with acute leukemia, myelodysplastic syndrome or myeloproliferative neoplasms in said individual by a method comprising;

a) subjecting a cell population comprising said one or more hematopoietic cells from said individual to a plurality of modulators in plurality of cultures,

b) characterizing a plurality of pathways in one or more cells from said plurality of cultures by determining an activation level of at least one activatable element within a plurality of pathways, wherein

i) at least one of the pathways being characterized in at least one of the plurality of cultures is an apoptosis or a DNA damage pathway,

ii) the modulators activate or inhibit one or more of said plurality of pathways being characterized, and

c) classifying said one or more hematopoietic cells based on said pathways characterization; and

B] making a decision regarding diagnosis, prognosis or progression of acute leukemia, myelodysplastic syndrome or myeloproliferative neoplasms in said individual, wherein said decision is based on said classification of said cells.

2. The method of claim 1 wherein said acute leukemia is acute myeloid leukemia.

3. The method of claim 1 wherein the pathways are selected from the group consisting of apoptosis, cell cycle, signaling, or DNA damage pathways.

4. The method of claim 1 further comprising determining the levels of a cytokine receptor, growth factor receptor and/or a drug transporter in said one or more cells.

5. The method of claim 4 wherein said cytokine receptor, growth factor receptor or drug transporter are selected from the group consisting of MDR1, ABCG2, MRP, P-Glycoprotein, CXCR4, FLT3, and c-kit.

7. The method of claim 1 wherein the individual has a predefined clinical parameter.

8. The method of claim 7 wherein the predefined clinical parameter is selected from the group consisting of age, de novo acute myeloid leukemia patient, secondary acute myeloid leukemia patient, or a biochemical/molecular marker.

9. The method of claim 7 wherein said decision is based on said classification of said cells in combination with said predefined clinical parameter.

10. The method of claim 1 wherein said modulators are independently selected from the group consisting of growth factor, mitogen, cytokine, chemokine, adhesion molecule modulator, hormone, small molecule, polynucleotide, antibody, natural compound, lactone, chemotherapeutic agent, immune modulator, carbohydrate, protease, ion, reactive oxygen species, and radiation.

11. The method of claim 1 wherein said modulators are independently selected from the group consisting of FLT3L, GM-CSF, SCF, G-CSF, SDF1a, LPS, PMA, Thapsigargin, IFNg, IFNa, IL-27, IL-3, IL-6, IL-10, ZVAD, H₂O₂, Staurosporine, Etoposide, Mylotarg, Daunorubicin, and AraC.

12. The method of claim 1 wherein said activation level is based on the activation state selected from the group consisting of extracellular protease exposure, novel hetero-oligomer formation, glycosylation state, phosphorylation state, acetylation state, methylation state, biotinylation state, glutamylation state, glycylation state, hydroxylation state, isomerization state, prenylation state, myristoylation state, lipoylation state, phosphopantetheinylation state, sulfation state, ISGylation state, nitrosylation state, palmitoylation state, SUMOylation state, ubiquitination state, neddylation state, citrullination state, deamidation state, disulfide bond formation state, proteolytic cleavage state, translocation state, changes in protein turnover, multi-protein complex state, oxidation state, multi-lipid complex, and biochemical changes in cell membrane.

13. The method of claim 12 wherein said activation state is a phosphorylation state.

14. The method of claim 1 wherein said activatable element is selected from the group consisting of proteins, carbohydrates, lipids, nucleic acids and metabolites.

15. The method of claim 14 wherein said activatable element is a protein capable of being to phosphorylated and/or dephosphorylated.

16. The method of claim 14 wherein said activatable element is a protein selected from the group consisting of p-Slp-76, p-Plcg2, p-Stat3, p-Stat5, p-Stat1, p-Stat6, p-Creb, cleaved Parp, p-Chk2, p65/Rel-A , p-Akt, p-S6, p-ERK, Cleaved Caspase 8, Cleaved Caspase 3, Cytoplasmic Cytochrome C, and p38.

17. The method of claim 1, wherein said method further comprises determining the presence or absence of one or more cell surface markers, intracellular markers, or combination thereof.

18. The method of claim 17 wherein said cell surface markers and said intracellular markers are independently selected from the group consisting of proteins, carbohydrates, lipids, nucleic acids and metabolites.

19. The method of claim 17 wherein said determining of the presence or absence of one or more cell surface markers or intracellular markers comprises determining the presence or absence of an epitope in both activated and non-activated forms of said cell surface markers or said intracellular markers.

20. The method of claim 17 wherein diagnosing, prognosing or determining progression of acute leukemia, myelodysplastic syndrome or myeloproliferative neoplasms in said individual is based on both the activation levels of said activatable element and the presence or absence of said one or more cell surface markers, intracellular markers, or combination thereof.

21. The method of claim 1 wherein said activation level is determined by a process comprising the binding of a binding element which is specific to a particular activation state of the particular activatable element.

22. The method of claim 21, wherein said binding element comprises an antibody.

23. The method of claim 1, wherein the step of determining the activation level comprises the use of flow cytometry, immunofluorescence, confocal microscopy, immunohistochemistry, 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, ELISA, and label-free cellular assays to determine the activation level of one or more intracellular activatable element in single cells.

24. The method of claim 1 further comprising predicting a response to a treatment or choosing a treatment for acute myeloid leukemia, myelodysplastic syndrome or myeloproliferative neoplasms in said individual.

25. The method of claim 24 further comprising determining whether the apoptosis, cell cycle, signaling, or DNA damage pathways are functional in said individual based on the activation levels of said activatable elements, wherein a pathway is functional if it is permissive for a response to said treatment, wherein if the apoptosis, cell cycle, signaling, and DNA damage pathways are functional the individual can respond to treatment, and wherein if at least one of the pathways is not functional the individual can not respond to treatment.

26. The method of claim 24 further comprising determining whether the apoptosis, cell cycle, signaling, or DNA damage pathways are functional in said individual based on the activation levels of said activatable elements, wherein a pathway is functional if it is permissive for a response to said treatment, wherein if the apoptosis and DNA damage pathways are functional the individual can respond to treatment.

27. The method of claim 24 wherein said treatment is a chemotherapy agent.

28. The method of claim 27 wherein said chemotherapy agent is selected from the group consisting of cytarabine (ara-C), daunorubicin, idarubicin, etoposide, mitoxantrone and 6-thioguanine.

29. The method of claim 24 wherein said treatment is allogeneic stem cell transplant or autologous stem cell transplant.

30. The method of claim 1 wherein the individual is under 60 years old and wherein said plurality of distinct modulators and said activatable elements are selected from the modulators and activatable elements listed in table 6.

31. The method of claim 1 wherein the individual is over 60 years old and wherein said plurality of distinct modulators and said activatable elements are selected from the modulators and activatable elements listed in table 7.

32. The method of claim 1 wherein said individual is a secondary acute myeloid leukemia patient and wherein said plurality of distinct modulators and said activatable elements are selected from the modulators and activatable elements listed in table 8 and table 9.

33. The method of claim 1 wherein said individual is a de novo acute myeloid leukemia patient and wherein said plurality of distinct modulators and said activatable elements are selected from the modulators and activatable elements listed in table 10 and table 11.

34. The method of claim 1 wherein said individual has a wild type FLT3 and wherein said plurality of modulators and said activatable elements are selected from the modulators and activatable elements listed in table 13.

6. The method of 4 wherein the levels of said cytokine receptor and/or said drug transporter in combination with said cell classification and the clinical parameter are indicative of the diagnosis, prognosis or progression of acute myeloid leukemia, myelodysplastic syndrome or myeloproliferative neoplasms.

35. A method of predicting a response to a treatment or choosing a treatment for acute leukemia, myelodysplastic syndrome or myeloproliferative neoplasms in an individual, said method comprising:

(1) classifying one or more hematopoietic cells associated with acute leukemia, myelodysplastic syndrome or myeloproliferative neoplasms in said individual by a method comprising;

a) subjecting a cell population comprising said one or more more hematopoietic cells from said individual to at least three distinct modulators in separate cultures, wherein;

i) a first modulator is a growth factor or a mitogen

ii) a second modulator is a cytokine

iii) a third modulator is a modulator that slows or stops the growth of cells, and/or induces apoptosis of cells, and/or is an inhibitor of a cellular function

b) determining an activation level of at least one activatable element in one or more cells from each of said separate cultures, wherein;

i) a first activatable element is an activatable element within the PI3K/AKT, or MAPK pathways and the activation level is measured in response to said growth factor or mitogen,

ii) a second activatable element is an activatable element within the STAT pathway and the activation level is measured in response to said cytokine

iii) a third activatable element is an activatable element within an apoptosis pathway and the activation level is measured in response to said modulator that slows or stops the growth of cells and/or induces apoptosis of cells, or the third activatable element is an activatable element within a phospholipase C pathway and the activation level is measured in response to said inhibitor, or the third activatable element is a phosphatase and the activation level is measured in response to said inhibitor, and

c) classifying said one or more hematopoeitic cells based on said activation levels of said activatable elements; and

(2) making a decision regarding a response to a treatment or a selection of treatment for acute leukemia, myelodysplastic syndrome or myeloproliferative neoplasms in said individual based on said classification of said one or more hematopoeitic cells.

36. The method of claim 35 wherein said acute leukemia is acute myeloid leukemia.

37. The method of claim 35 wherein the individual has a predefined clinical parameter.

38. The method of claim 37 wherein the predefined clinical parameter is selected from the group consisting of age, de novo acute myeloid leukemia patient, secondary acute myeloid leukemia patient, or a biochemical/molecular marker.

39. The method of claim 35 wherein activation levels higher than a threshold level of the activatable element within the STAT pathway in response to said cytokine is indicative that individual can not respond to treatment.

40. The method of claim 39 wherein said activatable element within the STAT pathway is selected from the group consisting of p-Stat3, p-Stat5, p-Stat1, and p-Stat6 and said cytokine is selected from the group consisting of IFNg, IFNa, IL-27, IL-3, IL-6, IL-10, and G-CSF.

41. The method of claim 40 wherein said activatable element within the STAT pathway is Stat 1 and said cytokine is IL-27 or G-CSF.

42. The method of claim 35 wherein activation levels higher than a threshold level of the activatable element within the PI3K/AKT, or MAPK pathway in response to said growth factor or mitogen is indicative that individual can not respond to treatment.

43. The method of claim 42 wherein said activatable element within the PI3K/AKT, or MAPK pathway is selected from the group consisting of p-Akt, p-ERK, p38 and pS6 and said growth factor or mitogen is selected from the group consisting of FLT3L, SCF, G-CSF, SCF, G-CSF, SDF1a, LPS, PMA, Thapsigargin.

44. The method of claim 35 wherein activation levels higher than a threshold level of the activatable element within the phospholipase C pathway in response to said inhibitor is indicative that individual can respond to treatment.

45. The method of claim 44 wherein said activatable element within the phospholipase C pathway is selected from the group consisting of p-Slp-76, and Plcg2 and said inhibitor is H₂O₂.

46. The method of claim 35 wherein activation levels higher than a threshold of an activatable element within the apoptosis pathway in response to a modulator that slows or stops the growth of cells and/or induces apoptosis of cells is indicative that the individual can respond to treatment.

47. The method of claim 46 wherein said activatable element within the apoptosis pathway is selected from the group consisting of Parp+, Cleaved Caspase 3, Cleaved Caspase 8, and Cytochrome C, and said modulator that slows or stops the growth of cells and/or induces apoptosis of cells is selected from the group consisting of Staurosporine, Etoposide, Mylotarg, Daunorubicin, and AraC.

48. The method of claim 35 further comprising determining an activation level of an activatable element within a DNA damage pathway or a cell cycle pathway in response to a modulator that slows or stops the growth of cells and/or induces apoptosis of cells.

49. The method of claim 48 wherein said activatable element within a DNA damage pathway is selected from the group consisting of Chk1, Chk2, ATR, ATM, and 14-3-3 and said modulator that slows or stops the growth of cells and/or induces apoptosis of cells is selected from the group consisting of Staurosporine, Etoposide, Mylotarg, Daunorubicin, and AraC.

50. The method of claim 48 wherein activation levels higher than a threshold of the activatable element within a DNA damage pathway and activation levels lower than a threshold of the activatable element within the apoptosis pathway in response to a modulator that slows or stops the growth of cells and/or induces apoptosis of cells is indicative of a communication breakdown between the DNA damage response pathway and the apoptotic machinery and that the individual can not respond to treatment.

51. The method of claim 48 wherein said activatable element within a cell cycle pathway is selected from the group consisting of Cdc25, p53, CyclinA-Cdk2, CyclinE-Cdk2, CyclinB-Cdk1, p21, p-Histone H3 and Gadd45, and said modulator that slows or stops the growth of cells and/or induces apoptosis of cells is selected from the group consisting of Staurosporine, Etoposide, Mylotarg, Daunorubicin, and AraC.

52. The method of claim 35 further comprising determining the levels of a drug transporter, growth factor receptor and/or a cytokine receptor.

53. The method of claim 52 wherein said cytokine receptor, growth factor receptor or drug transporter are selected from the group consisting of MDR1, ABCG2, MRP, P-Glycoprotein, CXCR4, FLT3, and c-kit.

54. The method of claim 52 wherein levels higher than a threshold of said drug transporter, growth factor receptor and/or said cytokine receptor is indicative that the individual can not respond to treatment.

55. The method of claim 35 further comprising determining the activation levels of an activatable element within the Akt pathway in response to an inhibitor, wherein activation levels higher that a threshold of said activatable element within the Akt pathway in response to said inhibitor is indicative that the individual can not respond to treatment.

56. The method of claim 35 wherein activation levels higher than a threshold of the activatable element in the PI3K/AKT pathway in response to said growth factor is indicative that the individual can not respond to treatment.

57. The method of claim 56 wherein said activatable element in the PI3K/Akt pathway is Akt and said growth factor is FLT3L.

58. The method of claim 35 wherein activation levels higher than a threshold of said activatable element in the apoptosis pathway in response to said modulator that slows or stops the growth of cells and/or induces apoptosis of cells is indicative that the individual can respond to treatment.

59. The method of claim 58 wherein said activatable element within the apoptosis pathway is Parp+ and said modulator that slows or stops the growth of cells and/or induces apoptosis of cells is selected from the group consisting of Staurosporine, Etoposide, Mylotarg, Daunorubicin, and AraC.

60. The method of claim 35 wherein said cytokine is selected from the group consisting of G-CSF, IFNg, IFNa, IL-27, IL-3, IL-6, and IL-10.

61. The method of claim 35 wherein said growth factor or mitogen is selected from the group consisting of FLT3L, SCF, G-CSF, SCF, G-CSF, SDF1a, LPS, PMA, and Thapsigargin.

62. The method of claim 35 wherein said modulator that slows or stops the growth of cells and/or induces apoptosis of cells is selected from the group consisting of Staurosporine, Etoposide, Mylotarg, Daunorubicin, and AraC.

63. The method of claim 35 wherein said inhibitor is selected from the group consisting of AG 490, AG 825, AG 957, AG 1024, aloisine A, alsterpaullone, aminogenistein, API-2, apigenin, arctigenin, AY-22989, BAY 61-3606, Azacitidine 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, decitibine, 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, 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, ZM447439, H₂O₂, 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 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-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, and bis(4-Trifluoromethylsulfonamidophenyl)-1,4-diisopropylbenzene, phenyarsine oxide, Pyrrolidine Dithiocarbamate, and Aluminum fluoride.

64. The method of claim 35 wherein said activation level is determined by a process comprising the binding of a binding element which is specific to a particular activation state of the particular activatable element.

65. The method of claim 64 wherein said binding element comprises an antibody.

66. The method of claim 35 wherein the step of determining the activation level comprises the use of flow cytometry, immunofluorescence, confocal microscopy, immunohistochemistry, 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, ELISA, and label-free cellular assays to determine the activation level of one or more intracellular activatable element in single cells.

67. A kit comprising

a) at least two modulators selected from the group consisting of Staurosporine, Etoposide, Mylotarg, Daunorubicin, AraC, G-CSF, IFNg, IFNa, IL-27, IL-3, IL-6, IL-10, FLT3L, SCF, G-CSF, SCF, G-CSF, SDF1a, LPS, PMA, Thapsigargin and H₂O₂;

b) at least three binding elements specific to a particular activation state of the activatable element selected from the group consisting of p-Slp-76, p-Plcg2, p-Stat3, p-Stat5, p-Stat1, p-Stat6, p-Creb, Parp+, Chk2, p-65/Rel-A, p-Akt, p-S6, p-Erk, Cleaved Caspase 3, Cleaved Caspase 8, Cytoplasmic Cytochrome C, and p38; and

c) instructions for diagnosis, prognosis, determining acute myeloid leukemia progression, predicting response to a treatment and/or choosing a treatment for acute myeloid leukemia, myelodysplastic syndrome or myeloproliferative neoplasms in an individual.

68. The kit of claim 67 further comprising a binding element specific for a cytokine receptor, growth factor receptor or drug transporter are selected from the group consisting of MDR1, ABCG2, MRP, P-Glycoprotein, CXCR4, FLT3, and c-kit.

69. The method of claim 67 wherein said binding element is an antibody.

70. A method of drug screening, said method comprising:

A] classifying one or more hematopoietic cells associated with acute leukemia, myelodysplastic syndrome or myeloproliferative neoplasms in said individual by a method comprising;

a) subjecting a cell population comprising said one or more hematopoietic cells from said individual to a test compound and a plurality of modulators in plurality of cultures,

b) characterizing a plurality of pathways in one or more cells from said plurality of cultures by determining an activation level of at least one activatable element within a plurality of pathways, wherein

i) at least one of the pathways being characterized in at least one of the plurality of cultures is an apoptosis or a DNA damage pathway,

ii) the modulators activate or inhibit one or more of said plurality of pathways being characterized, and

c) classifying said one or more hematopoietic cells based on said pathways characterization; and

B] making a decision regarding the test compound and its therapeutic potential for the treatment of acute leukemia, myelodysplastic syndrome or myeloproliferative neoplasms, wherein said decision is based on said classification of said cells.