Functional immunohistochemical cell cycle analysis as a prognostic indicator for cancer

Abstract

The present invention relates to a method of determining the presence of a retinoblastoma protein (Rb)-mediated cancerous or pre-cancerous condition in a sample of fixed cells that involves exposing the sample to an antibody that recognizes phosphorylation of serine on the retinoblastoma protein (pS-Rb), and identifying cells in the sample that are recognized by the antibody to pS-Rb, where the recognition of pS-Rb in the sample determines the presence of an Rb-mediated cancerous or pre-cancerous condition. The method also involves identifying the presence of specific cell cycle markers and positive and negative cell cycle regulators to assess the stage of disease progression in a cancer patient having an (Rb)-mediated cancer. The present invention also relates to methods of assessing the efficacy of cancer therapy for an Rb-mediated cancer, determining the mechanism of cell cycle progression in an Rb-mediated cancer, and identifying cells predisposed to developing an Rb-mediated cancerous condition.

What is claimed: 
     
       1. A method of determining the presence of a retinoblastoma protein (Rb)-mediated hematopoietic cancerous or pre-cancerous condition, said method comprising:
 providing a patient biopsy sample as a histologic section comprising fixed hematopoietic cells; 
 exposing the patient sample to (i) a first antibody that recognizes phosphorylation of serine on the retinoblastoma protein (pS-Rb) and indicates that the cell bound by said first antibody has entered mid-G1 phase of cell cycle progression, and (ii) a second antibody that recognizes a cell surface or cytoplasmic marker for neoplastic hematopoietic cells; and 
 identifying hematopoietic cells of the patient sample that are recognized by both the first and second antibodies, wherein recognition of hematopoietic cells of the patient biopsy sample by both first and second antibodies determines the presence of an Rb-mediated hematopoietic cancerous or pre-cancerous condition. 
 
     
     
       2. The method according to  claim 1  further comprising:
 providing a second patient biopsy sample as a histologic section comprising fixed hematopoietic cells, the second patient biopsy sample having been obtained from the same patient after a time delay; 
 performing said exposing and said identifying on the second patient biopsy sample; and 
 determining whether the second patient biopsy sample, relative to the first patient biopsy sample, contains a change in the percentage of hematopoietic cells that are recognized by both the first and second antibodies, 
 wherein an increase indicates advancement of the Rb-mediated hematopoietic cancerous or pre-cancerous condition, and a decrease indicates remission of the Rb-mediated hematopoietic cancerous or pre-cancerous condition. 
 
     
     
       3. The method according to  claim 1  further comprising:
 exposing the patient biopsy sample to an antibody that recognizes a nuclear cell cycling protein marker; and 
 identifying cells in the patient biopsy sample that are recognized by the antibody that recognizes the cell cycling protein marker. 
 
     
     
       4. The method according to  claim 3 , wherein the nuclear cell cycling protein marker is Ki-67 or skp-2. 
     
     
       5. The method according to  claim 1 , wherein the phosphorylated serine residue of the Rb protein is selected from the group consisting of serine-608, serine-780, serine-795, and serine-807/811. 
     
     
       6. The method according to  claim 1 , wherein the patient biopsy sample is selected from the group consisting of a solid tumor containing a substantially homogenous population of cells; a heterogeneous population of non-neoplastic and neoplastic cells; and bone marrow. 
     
     
       7. The method according to  claim 6 , wherein the patient biopsy sample is from a solid tumor containing a substantially homogenous population of cells. 
     
     
       8. The method according to  claim 6 , wherein the patient biopsy sample is from a patient tissue containing a heterogeneous population of non-neoplastic and neoplastic cells. 
     
     
       9. The method according to  claim 1 , wherein the Rb-mediated hematopoietic cancerous or pre-cancerous condition is selected from the group consisting of a lymphoma/lymphoid leukemia, a B cell neoplasm, a T cell neoplasm, a plasma cell neoplasm, monoclonal gammopathy of undetermined significance (MGUS), multiple myeloma (MM), a natural killer cell (NK) neoplasm, and Hodgkin lymphoma. 
     
     
       10. The method according to  claim 1 , wherein the hematopoietic cancerous or precancerous condition is selected from the group consisting of acute myelogenous leukemia (AML), acute promyelocytic leukemia (APL); a myeloproliferative disorder (MPD); chronic myelogenous leukemia (CML); polycythemia vera; essential thrombocythemia; idiopathic myelofibrosis; myelodysplastic syndromes; myelodysplastic/myeloproliferative diseases; acute lymphoblastic leukemias (ALL); precursor B-lymphoblastic leukemia/lymphoma; precursor T lymphoblastic leukemia/lymphoma; chronic lymphocytic leukemia (CLL); histiocytic disorders and mastocytosis. 
     
     
       11. The method according to  claim 1 , wherein the cell surface marker for neoplastic hematopoietic cells is selected from the group consisting of CD20, CD3, immunoglobulinκ light chain, immunoglobulinλ light chain, CD138, CD30, CD31, CD43, CD56, CD68, CD117, and CD34. 
     
     
       12. The method according to  claim 11 , wherein the cell surface marker is CD20 and the neoplastic cell is a cancerous or pre-cancerous B-cell or popcorn cell of nodular lymphocyte predominant Hodgkin lymphoma. 
     
     
       13. The method according to  claim 11 , wherein the cell surface marker is CD3 and the neoplastic cell is a cancerous or precancerous T-cell. 
     
     
       14. The method according to  claim 11 , wherein the cell surface marker is CD138 and the neoplastic hematopoietic cell is a cancerous or precancerous plasma cell. 
     
     
       15. The method according to  claim 11 , wherein the cell surface marker is CD31 and the neoplastic cell is a cancerous or precancerous plasma cell or associated with AML or CML. 
     
     
       16. The method according to  claim 11 , wherein the cell surface marker is CD43 and the neoplastic cell is a cancerous or precancerous B-cell or a myeloid neoplasm. 
     
     
       17. The method according to  claim 11 , wherein the cell surface marker is CD56 and the neoplastic cell is associated with multiple myeloma or myeloid leukemia. 
     
     
       18. The method according to  claim 11 , wherein the cell surface marker is CD68 and the neoplastic cell is a myeloid cell. 
     
     
       19. The method according to  claim 11 , wherein the cell surface marker is CD117 and the neoplastic cell is a myeloid cell or mast cell. 
     
     
       20. The method according to  claim 11 , wherein the cell surface marker is CD34 and the neoplastic cell is a B, T, or myeloid cell. 
     
     
       21. The method according to  claim 3  further comprising:
 providing a second patient biopsy sample as a histologic section comprising fixed cells, the second patient biopsy sample having been obtained from the same patient after a time delay; 
 performing each said exposing and each said identifying on the second patient biopsy sample; and 
 determining whether there exists, following the time delay, an increase in the percentage of cells in the second patient biopsy sample, relative to the first patient biopsy sample, that are identified during each said identifying, wherein an increase indicates advancement of the Rb-mediated cancerous or pre-cancerous condition, and a decrease indicates remission or a stable phase of the Rb-mediated cancerous or pre-cancerous condition. 
 
     
     
       22. A method of assessing the efficacy of cancer therapy for a retinoblastoma protein (Rb)-mediated cancer, said method comprising:
 performing the method of  claim 2  on the first and second patient biopsy samples, wherein the first and second biopsy samples are obtained from the patient before and after, respectively, administration of cancer therapy to the patient, and wherein a decrease in the percentage of identified cells in the second biopsy sample relative to the first biopsy sample indicates the efficacy of the cancer therapy. 
 
     
     
       23. A method of assessing the efficacy of cancer therapy for a retinoblastoma protein (Rb)-mediated cancer, said method comprising:
 performing the method of  claim 21  on the first and second patient biopsy samples, wherein the first and second biopsy samples are obtained from the patient before and after, respectively, administration of cancer therapy to the patient, and wherein a decrease in the percentage of identified cells in the second biopsy sample, relative to the first biopsy sample, indicates the efficacy of the cancer therapy. 
 
     
     
       24. A method of determining the presence of Rb-mediated multiple myeloma, said method comprising:
 providing a patient bone marrow sample as a histologic section comprising fixed hematopoietic cells; 
 exposing the patient bone marrow sample to a first antibody that recognizes phosphorylation of serine on the retinoblastoma protein (pS-Rb) and a second antibody that recognizes a cell surface or cytoplasmic marker for myeloma cells; and 
 identifying myeloma cells of the patient bone marrow sample that are recognized by both the first and second antibodies, wherein recognition of myeloma cells of the patient sample by both first and second antibodies determines the presence of an Rb-mediated multiple myeloma. 
 
     
     
       25. The method according to  claim 24  further comprising:
 providing a second patient bone marrow sample as a histologic section comprising fixed hematopoietic cells, the second patient sample having been obtained from the same patient after a time delay; 
 performing said exposing and said identifying on the second patient bone marrow sample; and 
 determining whether the second patient bone marrow sample, relative to the first patient bone marrow sample, contains a change in the percentage of myeloma cells that are recognized by both the first and second antibodies, 
 wherein an increase indicates the presence and advancement of the Rb-mediated multiple myeloma, and a decrease indicates a decline in the presence or remission of the Rb-mediated multiple myeloma. 
 
     
     
       26. The method according to  claim 24  further comprising:
 exposing the patient bone marrow sample to a third antibody that recognizes a nuclear cell cycling protein marker; and 
 identifying cells in the patient bone marrow sample that are recognized by the third antibody. 
 
     
     
       27. The method according to  claim 26 , wherein the nuclear cell cycling protein marker is Ki-67 or skp-2. 
     
     
       28. The method according to  claim 24 , wherein the phosphorylated serine residue of the Rb protein is selected from the group consisting of serine-608, serine-780, serine-795, and serine-807/811. 
     
     
       29. The method according to  claim 24 , wherein the cell surface marker for myeloma cells is selected from the group consisting of CD138 and CD56. 
     
     
       30. The method according to  claim 26  further comprising:
 providing a second patient bone marrow sample as a histologic section comprising fixed hematopoietic cells, the second patient bone marrow sample having been obtained from the same patient after a time delay; 
 performing each said exposing and each said identifying on the second patient bone marrow sample; and 
 determining whether there exists, following the time delay, an increase in the percentage of myeloma cells in the second patient bone marrow sample, relative to the first patient bone marrow sample, that are identified during each said identifying, wherein an increase indicates the presence and advancement of the Rb-mediated multiple myeloma, and a decrease indicates a decline in the presence or remission of the Rb-mediated multiple myeloma. 
 
     
     
       31. A method of assessing the efficacy of cancer therapy for a retinoblastoma protein (Rb)-mediated multiple myeloma, said method comprising:
 performing the method of  claim 25  on the first and second patient bone marrow samples, wherein the first and second bone marrow samples are obtained from the patient before and after, respectively, administration of cancer therapy to the patient, and wherein a decrease in the percentage of identified myeloma cells in the second sample relative to the first indicates a decline in the presence of myeloma cells and the efficacy of the cancer therapy. 
 
     
     
       32. A method of assessing the efficacy of cancer therapy for a retinoblastoma protein (Rb)-mediated multiple myeloma, said method comprising:
 performing the method of  claim 30  on the first and second patient bone marrow samples, wherein the first and second bone marrow samples are obtained from the patient before and after, respectively, administration of cancer therapy to the patient, and wherein a decrease in the percentage of identified myeloma cells in the second sample relative to the first indicates a decline in the presence of myeloma cells and the efficacy of the cancer therapy. 
 
     
     
       33. A method of determining the presence of a retinoblastoma protein (Rb)-mediated hematopoietic cancerous or precancerous condition, said method comprising:
 providing a patient biopsy sample as a histologic section comprising fixed hematopoietic cells; 
 treating the histologic section with an agent that prevents dephosphorylation; 
 exposing the treated patient sample to (i) a first antibody that recognizes phosphorylation of serine on the retinoblastoma protein (pS-Rb) and indicates that the cell bound by said first antibody has entered mid-G1 phase of cell cycle progression, and (ii) a second antibody that recognizes a cell surface or cytoplasmic marker for neoplastic hematopoietic cells; and 
 identifying hematopoietic cells of the patient sample that are recognized by both the first and second antibodies, wherein recognition of hematopoietic cells of the patient biopsy sample by both first and second antibodies determines the presence of an Rb-mediated hematopoietic cancerous or pre-cancerous condition. 
 
     
     
       34. The method according to  claim 33 , wherein the agent that prevents dephosphorylation is calyculin A. 
     
     
       35. A method of determining the presence of a retinoblastoma protein (Rb)-mediated hematopoietic cancerous or pre-cancerous condition, said method comprising:
 providing a patient biopsy sample as a histologic section comprising fixed hematopoietic cells;   exposing the patient sample to (i) a first antibody that recognizes phosphorylation of serine on the retinoblastoma protein (pS-Rb) and indicates that the cell bound by said first antibody has entered mid-G1 phase of cell cycle progression, or a first antibody that recognizes Ki-67, and (ii) a second antibody that recognizes a cell surface or cytoplasmic marker for neoplastic hematopoietic cells selected from the group consisting of CD20, CD3, CD138, CD31, CD43, CD56, CD68, CD117, and CD34; and   identifying hematopoietic cells of the patient sample that are recognized by both the first and second antibodies, wherein recognition of hematopoietic cells of the patient biopsy sample by both first and second antibodies determines the presence of an Rb-mediated hematopoietic cancerous or pre-cancerous condition.    
     
     
       36. The method according to claim 35 further comprising:
 providing a second patient biopsy sample as a histologic section comprising fixed hematopoietic cells, the second patient biopsy sample having been obtained from the same patient after a time delay;   performing said exposing and said identifying on the second patient biopsy sample; and   determining whether the second patient biopsy sample, relative to the first patient biopsy sample, contains a change in the percentage of hematopoietic cells that are recognized by both the first and second antibodies,   wherein an increase indicates advancement of the Rb-mediated hematopoietic cancerous or pre-cancerous condition, and a decrease indicates remission of the Rb-mediated hematopoietic cancerous or pre-cancerous condition.    
     
     
       37. The method according to claim 35, wherein the patient biopsy sample is selected from the group consisting of a solid tumor containing a substantially homogenous population of cells; a heterogeneous population of non-neoplastic and neoplastic cells; and bone marrow.  
     
     
       38. The method according to claim 37, wherein the patient biopsy sample is from a solid tumor containing a substantially homogenous population of cells.  
     
     
       39. The method according to claim 37, wherein the patient biopsy sample is from a patient tissue containing a heterogeneous population of non-neoplastic and neoplastic cells.  
     
     
       40. The method according to claim 37, wherein the patient biopsy sample is from bone marrow.  
     
     
       41. The method according to claim 35, wherein the cell surface marker is CD20 and the neoplastic cell is associated with a B-cell lymphoma.  
     
     
       42. The method according to claim 35, wherein the cell surface marker is CD3 and the neoplastic cell is associated with a T-cell lymphoma.  
     
     
       43. The method according to claim 35, wherein the cell surface marker is CD138 and the neoplastic cell is associated with a plasma cell neoplasm.  
     
     
       44. The method according to claim 35, wherein the cell surface marker is CD31 and the neoplastic cell is associated with a plasma cell neoplasm in extramedullary sites.  
     
     
       45. The method according to claim 35, wherein the cell surface marker is CD43 and the neoplastic cell is associated with myeloid leukemia.  
     
     
       46. The method according to claim 35, wherein the cell surface marker is CD56 and the neoplastic cell is associated with multiple myeloma or myeloid leukemia.  
     
     
       47. The method according to claim 35, wherein the cell surface marker is CD68 and the neoplastic cell is associated with myeloid leukemia.  
     
     
       48. The method according to claim 35, wherein the cell surface marker is CD117 and the neoplastic cell is associated with acute myelogenous leukemia.  
     
     
       49. The method according to claim 35, wherein the cell surface marker is CD34 and the neoplastic cell is associated with acute lymphoblastic leukemia.  
     
     
       50. The method according to claim 35, wherein said exposing is carried out using the first antibody that recognizes Ki-67 and the second antibody that recognizes CD138.  
     
     
       51. A method of assessing the efficacy of cancer therapy for a retinoblastoma protein (Rb)-mediated multiple myeloma, said method comprising:
 performing the method of claim 36 on the first and second patient biopsy samples, wherein the first and second biopsy samples are obtained from the patient before and after, respectively, administration of cancer therapy to the patient, and wherein a decrease in the percentage of identified myeloma cells in the second biopsy sample relative to the first biopsy sample indicates the efficacy of the cancer therapy.    
     
     
       52. A method of determining the presence of Rb-mediated multiple myeloma, said method comprising:
 providing a patient bone marrow sample as a histologic section comprising fixed hematopoietic cells;   exposing the patient bone marrow sample to (i) either a first antibody that recognizes phosphorylation of serine on the retinoblastoma protein (pS-Rb) or a first antibody that recognizes Ki-67, and (ii) a second antibody that recognizes a cell surface or cytoplasmic marker for myeloma cells; and   identifying myeloma cells of the patient bone marrow sample that are recognized by both the first and second antibodies, wherein recognition of myeloma cells of the patient sample by both first and second antibodies determines the presence of an Rb-mediated multiple myeloma.    
     
     
       53. The method according to claim 52 further comprising:
 providing a second patient bone marrow sample as a histologic section comprising fixed hematopoietic cells, the second patient sample having been obtained from the same patient after a time delay;   performing said exposing and said identifying on the second patient bone marrow sample; and   determining whether the second patient bone marrow sample, relative to the first patient bone marrow sample, contains a change in the percentage of myeloma cells that are recognized by both the first and second antibodies,   wherein an increase indicates the presence and advancement of the Rb-mediated multiple myeloma, and a decrease indicates a decline in the presence or remission of the Rb-mediated multiple myeloma.    
     
     
       54. The method according to claim 52, wherein said exposing is carried out using the first antibody that recognizes Ki-67.  
     
     
       55. The method according to claim 54, wherein the cell surface marker for myeloma cells is selected from the group consisting of CD138 and CD56.  
     
     
       56. The method according to claim 52, wherein the cell surface marker for myeloma cells is selected from the group consisting of CD138 and CD56.  
     
     
       57. A method of assessing the efficacy of cancer therapy for a retinoblastoma protein (Rb)-mediated multiple myeloma, said method comprising:
 performing the method of claim 52 on the first and second patient bone marrow samples, wherein the first and second bone marrow samples are obtained from the patient before and after, respectively, administration of cancer therapy to the patient, and wherein a decrease in the percentage of identified myeloma cells in the second sample relative to the first indicates a decline in the presence of myeloma cells and the efficacy of the cancer therapy.    
     
     
       58. The method according to claim 33, wherein the cell surface marker for neoplastic hematopoietic cells is selected from the group consisting of CD20, CD3, CD138, CD31, CD43, CD56, CD68, CD117, and CD34.