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WO2002038189A1 - Recuperation totale de lymphocytes et survie au cancer - Google Patents

Recuperation totale de lymphocytes et survie au cancer Download PDF

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Publication number
WO2002038189A1
WO2002038189A1 PCT/US2001/046798 US0146798W WO0238189A1 WO 2002038189 A1 WO2002038189 A1 WO 2002038189A1 US 0146798 W US0146798 W US 0146798W WO 0238189 A1 WO0238189 A1 WO 0238189A1
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WIPO (PCT)
Prior art keywords
cells
patients
alc
cancer
patient
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PCT/US2001/046798
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English (en)
Inventor
Luis F. Porrata
Svetomir N. Markovic
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Mayo Foundation For Medical Education And Research
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Priority to AU2002225959A priority Critical patent/AU2002225959A1/en
Publication of WO2002038189A1 publication Critical patent/WO2002038189A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the invention relates to methods and materials involved in predicting cancer survival or predicting the effectiveness of a cancer drug.
  • Autologous stem cell transplant compared to conventional chemotherapy, improves survival in both previously untreated multiple myeloma (MM) and relapsed, chemotherapy-sensitive, aggressive non-Hodgkin's lymphoma (NHL) patients.
  • MM multiple myeloma
  • NHS non-Hodgkin's lymphoma
  • the French Myeloma Group found an estimated 5-year survival rate of 52 % in the ASCT group and 12 % in the conventional-chemotherapy group (Attal et al. (1996) New England J. Med. 335: 91J.
  • the PARMA trial showed superiority of transplantation over salvage chemotherapy in treatment of relapsed chemotherapy sensitive, aggressive NHL.
  • the overall survival rates were 53 % for the transplanted group and 32 % from the salvage-chemotherapy group at 5 years (Phillip et al.
  • the invention relates to predicting survival of cancer patients or predicting the effectiveness of a drug for treating a cancer condition. More specifically, an ALC determined after ASCT, is used to predict survival and effectiveness of an anticancer drug.
  • the invention involves determining the ALC of a cancer patient at 5 to 15 days after hematopoietic stem cell transplant.
  • the ALC can be used as a prognostic factor for survival.
  • the ALC also can be used to determine if a candidate drug is effective for treating cancer.
  • the cancer can be MM, NHL, Hodgkin's lymphoma, acute myelogenous leukemia (AML), or metastatic breast cancer.
  • the invention provides a method of predicting survival of a cancer patient.
  • the method involves obtaining a blood sample from the cancer patient at 5 to 15 days post- ASCT; determining the ALC of the blood sample; and then correlating the ALC of the blood sample with a prediction of survival of the cancer patient subsequent to ASCT.
  • an ALC of a patient that is > 200 lymphocyte/ ⁇ L predicts survival of the cancer patient for at least 24 months. In other embodiments, an ALC of a patient that is > 500 lymphocyte/ ⁇ L predicts survival of the cancer patient for at least 24 months. In yet other embodiments, an ALC that is > 500 lymphocytes/ ⁇ L predicts survival of the cancer patient for at least 33 months. In further embodiments, an ALC that is > 500 lymphocytes/ ⁇ L in a cancer patient predicts survival of the cancer patient for at least 36 months. In additional embodiments, an ALC that is > 500 lymphocytes/ ⁇ L predicts survival of the cancer patient for at least 42 months. In yet other embodiments, an ALC that is > 500 lymphocytes/ ⁇ L predicts survival of the cancer patient for at least 60 months.
  • the invention also provides a method of determining the efficacy of a candidate drug in a cancer patient.
  • the drug can be a hematopoietic growth factor, such as flt-3 ligand or GM-CSF.
  • the method involves administering the candidate drug to the cancer patient before isolation of hematopoietic stem cells from the cancer patient; then administering cancer therapy that includes ASCT to the cancer patient.
  • the method further includes obtaining a blood sample from the cancer patient at 5-15 days post- ASCT; and then determining the ALC of the blood sample from the cancer patient.
  • An ALC > 200 lymphocytes/ ⁇ L indicates that the candidate drug is effective in the cancer patient.
  • the present invention also provides a composition comprising a sample of "day
  • the sample comprises > 200 lymphocytes/ ⁇ L (e.g., ⁇ 300,400, or 500 lymphocytes/ ⁇ L).
  • the lymphocytes which are measured in lieu of an ALC are natural killer (NK) cells (and do not include B-or T-cells).
  • the number of NK cells/ ⁇ L measured in a blood sample 15 days following ASCT are at least 30 to 40 cells/ ⁇ L, preferably 45 to 60 cells/ ⁇ L, more preferably 60 to 80 cells/ ⁇ L, and still more preferably 75 to 100 cells/ ⁇ L.
  • the NK cell count comprises > 30, 40, 45, 60, 75, 80, or 100 or higher cells/ ⁇ L.
  • These NK cell counts are indicative of patient survival for at least 24 months.
  • the number of NK cells measured on day 15 following ASCT that is indicative of patient survival for at least 24 months is at least 15 % of the ALC, preferably at least 20 % of the ALC.
  • the blood is peripheral blood (PB).
  • PB peripheral blood
  • the present invention further provides a method of improving a cancer therapy.
  • the method includes treating a cancer patient in need of said therapy with a drag, (e.g., a hematopoietic growth factor or a flt-3 ligand) and obtaining an ALC on a "day 15" blood sample post- ASCT, wherein a cell count > ⁇ 200 lymphocytes/ ⁇ L is indicative of an improvement in said cancer therapy.
  • the lymphocyte count can be > ⁇ 300, 400, or 500 lymphocytes/ ⁇ L.
  • the invention comprises administering the drag, removing a blood sample comprising stem cells for transplant, subjecting the cancer patient to high dose cancer therapy, administering the harvested stem cells to the patient, and obtaining an ALC from a "day 15" blood sample wherein a lymphocyte count of > 200 lymphocytes/ ⁇ L is indicative of the efficacy of the drag, or improvement of the cancer therapy.
  • the lymphocyte count can be > 300, 400, or 500 lymphocytes/ ⁇ L.
  • the invention provides a strong indicator of cancer patient survival, and provides methods and compositions for determining the survival of a cancer patient, the efficacy of cancer drags, and potential improvements in cancer therapies based on a measurement of the ALC obtained 15 days following an ASCT.
  • Figure 1 is a graph comparing the overall survival (OS) of MM patients with ALC > 500 cells/ ⁇ L (median 33 months) and MM patients with ALC ⁇ 500 cells/ ⁇ L (median 12 months), p ⁇ 0.0001.
  • Figure 2 is a graph comparing the progression free survival (PFS) of MM patients with ALC > 500 cells/ ⁇ L (median: 16 months) and MM patients with ALC ⁇ 500 cells/ ⁇ L (median: 8 months), p ⁇ 0.0001.
  • PFS progression free survival
  • Figure 3 is a graph comparing the OS survival of NHL patients with ALC > 500 cells/ ⁇ L (not reached) and NHL patients with ALC ⁇ 500 cells/ ⁇ L (median: 6 months), p ⁇ 0.0001.
  • Figure 4 is a graph comparing the PFS of NHL patients with ALC > 500 cells/ ⁇ L (not reached) and NHL patients with ALC ⁇ 500 cells/ ⁇ L. (median: 4 months), p ⁇ 0.0001.
  • Figure 5 is a graph comparing the OS (median: 60 months) and PFS (median: 40 months) of patients having Hodgkin's disease.
  • Figure 6 is a graph comparing the OS of Hodgkin's disease patients having ALC > 500 cells/ ⁇ L (not reached) with Hodgkin's disease patients having ALC ⁇ 500 cells/ ⁇ L (29 months), p ⁇ 0.0001.
  • Figure 7 is a graph comparing the PFS of Hodgkin's disease patients having ALC > 500 cells/ ⁇ L (not reached) with Hodgkin's disease patients having ALC ⁇ 500 cells/ ⁇ L (14 months), p ⁇ 0.0001.
  • Figure 8 is a graph comparing the OS of patients having ALC > 500 cells/ ⁇ L (median: 55 months) with the OS of patients with ALC ⁇ 500 cells/ ⁇ L (median: 13 months) for all 386 patients, p ⁇ 0.0001.
  • Figure 9 is a graph comparing the PFS of patients having ALC ⁇ 500 cells/ ⁇ L (median: 40 months) with the PFS of patients with ALC ⁇ 500 cells/ ⁇ L (median: 7 months) for all 386 patients, p ⁇ 0.0001.
  • the invention provides methods and materials related to predicting survival of a cancer patient or predicting the effectiveness of a drag for treating a cancer condition. More specifically, an ALC determined post- ASCT, is used to predict survival and effectiveness of an anticancer drag.
  • the present invention is based on the discovery that the total number of lymphocytes, i.e. absolute lymphocyte count (ALC), present in a blood sample taken from a cancer patient anytime before, and including, day 15 following ASCT is a powerful prognostic indicator of cancer patient survival. More specifically, an ALC of at least 200 lymphocytes/ ⁇ L at anytime before and including day 15 following ASCT indicates survival of the cancer patient for at least 24 months following ASCT.
  • ALC absolute lymphocyte count
  • lymphocyte includes the following cell types: B-cells, T-helper cells, T-suppressor cells, and NK cells.
  • B-cells produce immunoglobulins, and bear at least one of the cell surface markers CD 19 and CD20; these can be detected using CD 19 and CD20 specific antibodies.
  • T-cells are involved in the modulation of the immune response and in the regulation of erythropoiesis; they bear one or more of the following cell surface markers: CD3, CD4, and/or CD8. Cytotoxic T-cells express CD8, whereas helper T-cells express CD4.
  • CD3, CD4, and CD8 can be detected using anti-CD3, anti-CD4, and anti-CD8 antibodies, respectively.
  • NK cells represent the body's first line of defense against malignancy. NK cells are directly cytotoxic to any foreign cells, and do not require the mediation of complement to effect their lysis. NK cells bear on their surface one or more of the markers CD 16 and or CD56, which can be detected using anti-CD 16 and/or anti-CD56 antibodies, respectively.
  • B-, T-, and NK cells can be detected using antibodies to the specific cell surface markers possessed by each cell type.
  • a blood sample can be taken from a patient, from which a suitable amount is placed on a glass microscope slide. The blood is then smeared onto the slide to create a thin film. The slide is then placed in any fixative known to those of skill in the art, such as paraformaldehyde, neutral-buffered formalin, glutaraldehyde, Bouin's solution, mercuric chloride, or zinc formalin.
  • the slide is then exposed to various cell type-specific antibodies, each antibody recognizing a particular cell surface marker.
  • the cell surface marker-specific antibodies are in an appropriate buffer solution such as phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the slide is generally exposed to the antibodies overnight, then subjected to a series of washes using an appropriate buffer solution such as PBS.
  • the slide is exposed to a second antibody that recognizes and binds to the cell surface marker-specific antibody.
  • the second antibody is in an appropriate buffer solution. If all of the cell surface marker-specific antibodies were raised in the same animal species, then only one secondary antibody is needed to bind to all of the different cell surface marker-specific antibodies. Therefore, B-, T- and NK cells can all be detected in one step providing one with the ability to count the total number of lymphocytes in a sample. If however, the cell surface marker-specific antibodies were raised in different species (i.e., goat, rabbit, donkey, horse, mouse, rat, etc.), then species-specific secondary antibodies are required to detect the cell surface marker-specific antibodies. This would allow for the differential detection of the different types of lymphocytes (Janeway et al. (1999) fmmunobiology 4 th Ed. Elsevier Science Ltd/Garland Publishing).
  • a cell-surface marker-specific antibody that recognizes the cell surface marker CD3 is an anti-CD3 IgG antibody. If the anti-CD3 IgG antibody was raised in a rabbit, then the second antibody can be anti-rabbit IgG.
  • Antibodies, as described herein may be obtained from any commercial source known in the art including, but not limited to Calbiochem (La Jolla, CA) or LifeTechnologies (Rockville, MD). Following exposure to the second antibody, the slide is again subjected to a series of washes and then a detection procedure is performed. The detection procedure involves detecting the presence of the second antibody. The detection procedure is based on the principle that a particular lymphocyte can be identified by identifying a cell specific surface marker.
  • the cell specific surface marker can be identified by a cell surface marker-specific antibody, which, in turn, is detected by a second antibody that is conjugated to a detectable moiety.
  • the detectable moiety can be any known in the art including, for example, a fluorescent molecule or an enzyme. Detection of the secondary antibody indicates the presence of the cell surface marker-specific antibody. Detection of the cell surface marker-specific antibody indicates the presence of the cell surface marker and thus the particular type of lymphocyte.
  • absolute lymphocyte count refers to the total number of lymphocytes per unit of whole blood or blood cells.
  • a unit can be, for example, a liter (L), milliliter (mL), or microliter ( ⁇ L).
  • ALC can be determined using a cell counting method that permits identification and quantitation of the total number of lymphocytes in a cell sample.
  • the cell counting method can include, without limitation, fluorescent automated cell sorting (FACS), immunolabeling, and hematoxalin and eosin (H & E) staining, as well as any clinical instrument capable of accurately counting the number of lymphocytes in a blood sample.
  • FACS fluorescent automated cell sorting
  • H & E hematoxalin and eosin stain
  • any clinical instrument capable of accurately counting the number of lymphocytes in a blood sample.
  • Such an instrument is, for example, the Beckman Coulter GEN S Cell system.
  • Immunolabeled cell samples can be counted manually by one of skill in the art using a microscope at a magnification sufficient to permit visualization of immunolabeled cells versus non-immunolabeled cells.
  • Immunolabeling refers to detection of a cell surface marker that is characteristic of a lymphocyte, such as the labeled antibody staining for cell surface markers described above.
  • H & E stained cell samples also can be counted manually by one of skill in the art using a microscope at a magnification sufficient to permit visualization of the physical appearance of a lymphocyte.
  • autologous refers to a graft in which the donor and recipient is the same individual, i an autologous transplant, cells taken from a patient are returned to the same patient.
  • allogenic refers to a graft in which the donor and recipient are genetically non-identical individuals from the same species.
  • Xenogenic in contrast, refers to a graft in which the donor and recipient are of different species.
  • ASCT autologous stem cell transplant
  • BM bone marrow
  • PB peripheral blood
  • day 15 refers to a 15 day period of time where day 1 is the day following completion of an ASCT.
  • a "day 15" blood sample can be obtained anytime within the 360 hours after the completion of an ASCT (i.e. post-ASCT) but not more than 384 hours after the completion of the ASCT.
  • a "day 15" sample can be obtained 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days after completion of ASCT. Samples obtained anytime between 3 to 15 days, 5 to 15 days, or 8 to 15 days following completion of ASCT are particularly useful.
  • an ASCT takes about 24 hours, during which time stem cells are administered to the patient. Completion of the stem cell transplant occurs at that time when all of the stem cells intended for transplant have been administered.
  • cancer refers to a neoplasm. Cancers that may be treatable by ASCT include, without limitation, MM, NHL, breast cancer, systemic amyloidosis, testicular cancer, and ovarian cancer.
  • Negative cancer refers to a medically accepted definition of "neoplasm” available in numerous pathology texts. For example, a “neoplasm” is defined as an "abnormal mass of tissue, the growth of which exceeds and is uncoordinated with that of the normal tissues and persists in the same excessive manner after cessation of the stimuli which evoked the change" (Cotran et al. (1999) Robbins: Pathologic Basis of Disease, 6 Ed. W. B. Saunders Co, Philadelphia, PA).
  • high dose therapy refers to treatment with chemotherapy drags known to those of skill in the art, and/or treatment with radiation in an effort to kill cancerous cells.
  • high dose therapy include, but are not limited to, treatments with melphalan coupled with total body irradiation (TBI); treatment with melphalan or cyclophosphamide, either together or separately, and either alone or in combination with TBI; treatment with CBN (cyclophosphamide, BC ⁇ U, and NP-16); and treatment with BEAC (BC ⁇ U, etoposide, ARA-C, and cyclophosphamide).
  • patient survival refers to the period of time between the completion ASCT and the day the cancer patient dies. Morbidity due to factors other than the cancer for which the patient received ASCT does not affect "patient survival" as used herein.
  • an ALC of >200 lymphocytes/ ⁇ L post-ASCT is indicative of a cancer survival of at least 24 months.
  • hematopoietic growth factor refers to a factor that (i) stimulates an increase in proliferation of stem cells and/or progenitor cells and/or (ii) stimulates the migration of stem cells and/or progenitor cells from the BM into the peripheral circulation.
  • a substance is said to be a "hematopoietic growth factor” if the substance induces, either directly or indirectly, an increase, by at least 5 %, in peripheral blood stem cells (PBSCs) usually found in low numbers in PB.
  • PBSCs peripheral blood stem cells
  • the term “stimulated” refers to a state in which the total number of immune cells in the circulating blood increases by at least 5 % following administration of an "hematopoietic growth factor" by a pharmaceutically acceptable route.
  • the hematopoietic growth factor can be administered with an adjuvant and/or other accessory substances, separately or in combinations as desired.
  • hematopoietic growth factors include, but are not limited to, granulocyte colony-stimulating factor (G-CSF); granulocyte/macrophage colony-stimulating factor (GM-CSF); c-kit ligand (stem cell factor (SCF)); interleukin-2, 7, 8, and 12 (IL-2, IL-7, IL-8, IL-12); and flt-3 ligand.
  • G-CSF granulocyte colony-stimulating factor
  • GM-CSF granulocyte/macrophage colony-stimulating factor
  • SCF stem cell factor
  • interleukin-2, 7, 8, and 12 IL-2, IL-7, IL-8, IL-12
  • flt-3 ligand See, Bungart et al. (1990) Br J Haematol 16: 174;
  • the present invention involves predicting the survival of a cancer patient by obtaining a "day 15" post-ASCT blood sample from the cancer patient, then determining the ALC.
  • An ALC of > 200 lymphocytes/ ⁇ L is predictive of survival of the cancer patient for at least 24 months post-ASCT.
  • the present invention involves a method of determining the efficacy of a candidate drag that includes treating a patient with the candidate drug, for example, an immunostimulatory drug that can mobilize the movement of BM stem cells into the peripheral circulation.
  • a candidate drug for example, an immunostimulatory drug that can mobilize the movement of BM stem cells into the peripheral circulation.
  • a "day 15" post-ASCT ALC is determined, and an ALC >200 lymphocytes/ ⁇ L indicates (i) an improvement in the cancer therapy and/or (ii) that the candidate drug is effective for treating cancer.
  • ALC is a measure of the total number of lymphocytes per unit of blood, wherein a unit is any volume of blood.
  • ALC is measured as the number of mature lymphocytes per ⁇ L of blood, and includes the cumulative numbers of B-cells, T-cells, and NK cells.
  • Stem cells, lymphocyte precursor cells, or lymphocyte progenitor cells typically are not included in the ALC.
  • Stem cells may be differentiated from "lymphocytes" as used herein, in that stem cells express the cell surface marker CD34, whereas mature lymphocytes do not.
  • lymphocytes express specific cell surface markers as described above (B-cells: CD20 and/or CD 19; T-cells: CD3, CD4, and/or CD8; NK cells: CD16 and/or CD56), whereas stem cells do not.
  • the ALC is obtained using an automated system for counting blood cells in a sample.
  • a system is the Gen-S Cell (Beckman-Coulter, Miami, FL).
  • Cell counting systems useful in the present invention are based on a principle by which unstained, unlabeled cells are sorted and counted based on morphological characteristics including, without limitation, size, shape, nuclear size, and nuclear shape.
  • the Gen-S Cell system identifies and counts cell types based on three general criteria: volume, conductivity, and scatter (see U. S. Pat. No. 5,125,737).
  • the blood sample may be treated, before analysis, with appropriate reagents coupled with physical agitation to lyse red blood cells (RBCs) thereby leaving only white blood cells (WBCs) for analysis.
  • RBCs red blood cells
  • WBCs white blood cells
  • the Gen-S Cell uses a process of DC impedance by which the cells are collided with light to physically measure the volume that the entire cell displaces in an isotonic diluent. Therefore, cell size can be accurately determined regardless of the orientation of the cell in the light path. Cells are further collided with alternating current in the radio frequency range that can permeate cell membranes such that information pertaining to cell size, internal structure, including chemical composition and nuclear structure can be determined.
  • a cell can be collided with a laser beam which, upon contacting the cell, scatters and spreads out in all directions, generating median angle light scatter signals, which can be collected to yield information regarding cellular granularity, nuclear lobularity, and cell surface structure.
  • a system can count and differentiate RBCs from WBCs based on the presence or absence of a nucleus, and can count and differentiate the different types of WBCs based on the ratio of nuclear to cytoplasmic volume, lobularity of the nucleus, and granularity of the cytoplasm.
  • One of skill in the art may refer to any one of a number of hematology or histological texts or atlases, for example Wheater et al. (1987) Functional Histology 2nd Ed. Churchill Livingstone, to determine the physical appearance of a lymphocyte which is most widely accepted and/or taught in the art, and as described below.
  • Lymphocytes are WBCs formed in lymphatic tissue throughout the human body (e.g., lymph nodes, spleen, thymus, tonsils, Peyer's Patches, and bone marrow). In normal adults, lymphocytes comprise approximately 22 to 28 % of the total number of leukocytes in the circulating blood. Lymphocytes range in size from 7 to 1 0 ⁇ m. When stained with H & E, the nucleus is deeply colored (purple-blue) and is composed of dense aggregates of chromatin within a sharply defined nuclear membrane. The nucleus is generally round in single-lobed and is generally eccentrically located within a small amount of light blue staining cytoplasm surrounding it.
  • the volume of nucleus to cytoplasm in a lymphocyte is approximately 1 : 1.2, as can be measured by a variety of data analysis software programs, including but not limited to NIH Image (available from the National Institutes of Health, Bethesda, MD), or Scion Image (Scion Corp., Frederick, MD).
  • Lymphocytes may be differentiated from RBCs in that RBCs have no nucleus, whereas lymphocytes have a round eccentrically located nucleus. Lymphocytes can be differentiated from neutrophils in that a neutrophil has a nucleus that possesses 2 to 5 lobes, while a lymphocyte nucleus is not lobed.
  • Lymphocytes can be differentiated from basophils in that basophils have basophilic cytoplasmic granules, while lymphocytes do not have cytoplasmic granules. Lymphocytes can be differentiated from eosinophils in that eosinophils have eosinophilic cytoplasmic granules, while lymphocytes do not have cytoplasmic granules. Lymphocytes can be differentiated from monocytes in that monocytes are 16 to 20 ⁇ m in diameter, while lymphocytes are 7 to 10 ⁇ m in diameter.
  • the ALC may be derived by immunolabeling the lymphocytes with antibodies specific for lymphocyte cell surface markers, and subsequently counting the immunolabeled cells using an instrument such as a fluorescence activated cell sorter (FACS).
  • FACS fluorescence activated cell sorter
  • B-cells can be labeled with fluorescently labeled antibodies specific for the adhesion molecules CD20 and/or CD19; T-cells may be labeled with one or more fluorescently labeled antibodies specific for CD3, CD4, and/or CD8; and NK cells may be labeled with one or more fluorescently labeled antibodies specific for CD 16 and/or CD56.
  • the cell surface marker specific antibodies may be labeled with the same fluorophore, such as Cy-5, fluoroscein, and Texas Red, etc.
  • FACS Fluorescence Activated Cell Sorting
  • the blood smear is dried and subsequently fixed using any fixative known to those of skill in the art including, but not limited to, neutral buffered formalin, formaldehyde, paraformaldehyde, glutaraldehyde, Bouin's solution, mercuric chloride, or zinc formalin.
  • the slides are then immersed in a solution of Harris Hematoxylin, rinsed in water, immersed in a solution of Eosin, rinsed in water, and dehydrated in ascending alcohol solutions.
  • the tissue sections are then cleared in xylenes and coverslips are mounted over the smear using Permount or other suitable organic mounting medium.
  • the blood smear may then be examined under a microscope at low and/or high power.
  • nuclei and other basophilic structures stain blue
  • cytoplasm and other acidophilic structures stain light to dark red (Sheehan et al. (1987) Theory and Practice of Histotechnology, 2nd Edition, Battelle Memorial Institute, Columbus, OH).
  • One of skill in the art may then manually count the number of lymphocytes present in the blood smear based on the lymphocytic morphological criteria accepted in the art.
  • an individual receives his or her own stem cells by infusion.
  • the stem cells are taken from the patient and preserved, for example by cryopreservation at temperatures ⁇ - 85 °C.
  • the individual is then subjected to a tumor debulking procedure.
  • an otherwise lethal debulking regimen e.g. HDT
  • chemotherapy or radiotherapy that severely damages or destroys the patient's bone marrow.
  • the patient's immune cells are reconstituted by stem cells present in the transplant.
  • Stem cells capable of reconstituting a cancer patient's immune system can be obtained from the patient's peripheral circulation following mobilization of such cells from the BM. Immobilization can be accomplished by treatment of the patient with granulocyte colony stimulating factor (G-CSF) or other appropriate factors that induce movement of stem cells from the BM into the peripheral circulation, including GM-CSF and flt-3 ligand. Following mobilization, stem cells can be collected from PB by any appropriate cell apheresis technique, for example through use of a commercially available blood cell collection device as exemplified by the CS 3000. RTM. Plus blood cell collection device marketed by the Fenwal Division of Baxter Healthcare Corporation.
  • G-CSF granulocyte colony stimulating factor
  • PBSC peripheral blood stem cells
  • the ASCT process begins with an eligibility analysis to determine if a patient is healthy enough to tolerate the transplant. Once a patient is determined to be eligible for an autologous transplant, they may or may not receive a preparative regimen of chemotherapy to reduce the amount of tumor present. The preparative regimen may be given over 1 to 3 months before the transplant by the patient's referring physician. After this regimen, the patient may undergo a pre-transplant workup to evaluate heart, liver, kidney, and lung function, as well as evaluate current disease status. The next steps in the process will be done at the transplant center, or other qualified institution. A central venous catheter, may be placed and the patient's stem cells may be collected by either a BM harvest or stem cell apheresis procedures.
  • the patient can be admitted to the BM transplant unit to begin the high- dose chemotherapy.
  • the high-dose chemotherapy may be administered intravenously through a central venous catheter over several days depending on the specific treatment protocol. High-doses of drugs to reduce nausea and vomiting may be given during this time.
  • the patients' stem cells will be reinfused intravenously (i.e. transplanted) over about 30 to 50 minutes and daily growth factor infusion also may be given.
  • the patient's immune system will be affected by the high-dose chemotherapy and will require protective isolation precautions. This is the beginning of the immunosuppressive phase of the transplant process.
  • the patient may receive many treatments, such as intravenous fluids; RBC and platelet transfusions; medications to control nausea, vomiting and diarrhea; antibiotics to prevent or treat infections; and blood tests to monitor blood counts.
  • treatments such as intravenous fluids; RBC and platelet transfusions; medications to control nausea, vomiting and diarrhea; antibiotics to prevent or treat infections; and blood tests to monitor blood counts.
  • the patient While the patient is immunosuppressed, they may be required to stay in the hospital or they may be released from the hospital as an outpatient and be closely monitored during extensive clinic appointments. This phase will last approximately 8 to 15 days after the stem cell reinfusion and will end when the reinfused stem cells begin to engraft. Undergoing an inpatient or outpatient stem cell transplant will depend on the condition of the patient and treatment protocol. If the patient stayed in the hospital for the entire transplant process, they will be discharged once engraftment begins.
  • Engraftment refers to a process whereby the transplanted stem cells begin to differentiate into mature blood cells. All patients may be evaluated in the outpatient clinic daily for 1 to 2 weeks after engraftment. Recovery from an ASCT is much quicker than an allogeneic stem cell transplant. Generally, patients return to about their normal level of energy and activity within the first 4 to 6 months after their transplant.
  • the present invention relates to a composition
  • a composition comprising a sample of "day 15" blood from a cancer patient following ASCT, wherein the blood sample has an ALC of > 200 lymphocytes/ ⁇ L, preferably an ALC of >500 lymphocytes/ ⁇ L.
  • a cancer patient who is eligible for ASCT may have PBSCs harvested by methods described herein, for example, by apheresis. The patient may then be given HDT, followed by re-infusion of the harvested stem cells.
  • a blood sample is taken from the cancer patient and an ALC is determined using one or more methods described hereinabove.
  • PBSCs Before harvesting PBSCs patients may be treated with one or more substances that stimulate the mobilization of stem cells from the BM to the peripheral circulation as described below. Administration of the substance will continue until the harvest is complete.
  • the PB will be assayed for hematopoietic progenitors before and during the administration of the stem cell stimulating substance.
  • Hematopoietic progenitor cells may be measured by any technique known in the art, including, for example, FACS counting using CD34 as a stem-cell specific marker.
  • the apheresis may be started, and will performed no more than four times per week.
  • the stem cell collection may be performed by any method known in the art, for example, the stem cells may be collected by a blood cell separator (for example, the CS 3,000; Fenwal Laboratories, Deerfield, Illinois). A total blood volume between 9.5 and 10 L per apheresis may be processed at a flow rate of 50 to 70 mL/min. Following the collection, a cell count may be performed on an aliquot of the total product to determine the number of stem cells. The apheresis product is subsequently centrifuged at 400 g for 10 minute, and the plasma is removed, yielding a total volume of approximately 100 mL.
  • a blood cell separator for example, the CS 3,000; Fenwal Laboratories, Deerfield, Illinois.
  • a total blood volume between 9.5 and 10 L per apheresis may be processed at a flow rate of 50 to 70 mL/min.
  • a cell count may be performed on an aliquot of the total product to determine the number of stem cells
  • the cell suspension is then mixed with 100 mL minimal essential medium (MEM-S; Life Technologies, Rockville, MD) supplemented with 20 % dimethylsulfoxide (DMSO).
  • MEM-S minimal essential medium
  • DMSO dimethylsulfoxide
  • a total of 100 mL may then be transferred to freezing bags (such as those manufactured by Delmed, Canton, MA) and frozen to -100 °C using a computer controlled cryopreservation device (such as the Cryoson-BN-6; Cryoson Germany GmbH, FRG).
  • the cells may then be transferred into liquid nitrogen and stored at -196 °C until the transplant.
  • Cancer patients are then treated with HDT.
  • the patients are infused with the harvested stem cells using techniques known in the art. Briefly, the stem cell aliquots are thawed, loaded into one or more sterile syringes, and slowly injected, intravenously, over a period of between 30 and 45 minutes.
  • 10 mL of blood is collected from the cancer patient. The blood is collected in rubber-stopped tubes containing EDTA, or other medically acceptable anti-coagulant.
  • the "day 15" blood may be collected from any route of entry to the circulatory system known in the art.
  • the blood sample will then be analyzed by the methods described herein to determine the ALC.
  • the cancer patient may be treated with one or more substances that stimulate the migration of stem cells from the patient's BM to the peripheral venous and arterial circulation.
  • substances include, but are not limited to G-CSF, GM-CSF, c-kit ligand, IL-2, IL-7, IL-8, IL-12, and flt-3 ligand. Whether the administration of one or more of these compounds can stimulate stem cell migration may be determined by assessing the numbers of CD34+ cells/kg body weight.
  • a target stem cell yield may be measured as the number of CD34+ cells/kg because the number of CD34+ cells infused correlates with the speed of hematopoietic recovery following ASCT (To et al. (1997) Blood 89:2233/ The accepted threshold for rapid hematopoietic recovery is generally considered to be 2 x 10 6 CD34+ cells/kg, whereas optimal stem cell yields are considered to be > 5 xlO 6 CD34+ cells/kg as this results in faster hematopoietic recovery (Bensinger et al. (1994) Br JHaematol 87:825 ⁇ Despite various methods of PBSC mobilization, sometimes, adequate numbers of PBSCs for ASCT are not collected from some patients.
  • BM harvest or a second attempt at PBSC mobilization can be performed.
  • these patients are excluded from proceeding to autografting.
  • G-CSF alone results in inadequate PBSC harvests in many cancer patients.
  • Numerous animal studies, however, have shown a synergistic effect of G-CSF or GM-CSF with Flt- 3 ligand on the mobilization of stem cells (Sudo et al. (1997) Blood 89:3186). Transplantation of these stem cells into lethally irradiated mice resulted in long-term multi-lineage hematopoietic reconstitution.
  • a target of 2 x 10 6 CD34+ cells/kg was reached in 63 % of patients treated with G-CSF, 74 % of patients treated with G-CSF + Flt-3 ligand, and 92 % of patients treated with GM-CSF + Flt-3 ligand.
  • a target of 3 x 10 6 CD34+ cells/kg was associated with 42 %, 61 %, and 79 % success, respectively.
  • Approximately 50 % of patients in the Flt-3 + GM-CSF arm achieved the overall goal of 2.5 xlO 6 CD34+ cells/kg in ⁇ 3 apheresis and 75 % in ⁇ 4 apheresis.
  • approximately 50 % of patients in the Flt-3 + G-CSF arm achieved the overall goal of 2.5 xlO 6 CD34+ cells/kg in ⁇ 3 apheresis and 75 % in ⁇ 4 apheresis.
  • approximately xlO 6 CD34+ cells/kg achieved the overall
  • cancer patients treated according to the present invention may be treated with a combination of GM-CSF and Flt-3 ligand before PBSC harvest to enhance the mobilization of stem cells from the BM to the peripheral circulation.
  • Flt-3 ligand binds to its receptor, a member of the tyrosine kinase receptor family type III. This receptor appears to be selectively expressed on hematopoietic stem cells and progenitor cells.
  • GM-CSF may be administered at a concentration of at least 5 ⁇ g/kg/day by subcutaneous injection until the stem cell collection is complete.
  • Flt-3 ligand may be administered at a concentration of at least 50 ⁇ g/kg/day for at least the first three days of stem cell harvesting.
  • Flt3 -ligand also may be administered at a concentration of at least 50 ⁇ g/kg/day for the entire duration of stem cell collection.
  • PBSCs are collected until the blood samples taken from the cancer patient reach a concentration of at least 2xl0 6 CD34+ stem cells/kg.
  • Mobilization of stem cells following treatment with GM-CSF and Flt-3 ligand may be evaluated by determining the numbers of CD34+ cells present following treatment. The number of CD34+ cells can be determined, for example, using FACS analysis.
  • the number of cells from a blood sample obtained from a cancer patient treated with GM-CSF and Flt-3 ligand is labeled with CD34-specific antibodies conjugated to fluorescent or other labeling moieties.
  • the cancer therapy may include treatment of the patient, post-ASCT, with one or more substances that stimulate proliferation, maturation, and/or differentiation of immune cells.
  • the immune cells are NK cells.
  • the substance is interferon.
  • Patients may be treated with low doses of interferon post-ASCT, wherein the concentration of interferon administered to the patient is between about lxlO 5 to lxlO 7 units/m 2 , and wherein the patient is administered interferon from the day of the transplant to up to 21 days following the transplant.
  • Interferon may be administered by any pharmaceutically acceptable route known in the art, including intravenous injection, intra-arterial injection, or oral administration in the form of a tablet, capsule, or syrup.
  • the lymphocytes that are measured in lieu of an ALC obtained from a cancer patient treated with a combination of GM-CSF and Flt-3 ligand prior to stem cell harvest, HDT, and transplant are NK cells (and do not include B-or T- cells).
  • the number of NK cells/ ⁇ L measured in a blood sample obtained within 15 days following ASCT are between at least 30 to 40 cells/ ⁇ L, preferably 45-60 cells/ ⁇ L, more preferably 60 to 80 cells/ ⁇ L, and still more preferably 75-100 cells/ ⁇ L.
  • the NK cell count comprises 30, 40, 45, 60, 75, 80, or 100 NK cells/ ⁇ L.
  • NK cell counts are indicative of patient survival of at least 24 months.
  • the number of NK cells measured on "day 15" following ASCT is at least 15 % of the ALC, preferably at least 20 % of the ALC.
  • the present invention relates to a method of predicting survival of a cancer patient comprising obtaining a "day 15" post-ASCT ALC as an indicator of survival of the cancer for at least 24 months, i a further embodiment, the invention provides a method of predicting the survival of a cancer patient comprising obtaining a "day 15" post-ASCT ALC.
  • the ALC can be > 200 lymphocytes/ ⁇ L, preferably, > 500 lymphocytes/ ⁇ L and is an indicator of survival of the cancer patient for at least 24 months following the ASCT.
  • the present invention relates to a method of assessing the survival of a cancer patient by determining the ALC at "day 15" post-ASCT.
  • a cancer patient may be treated with one or more substances that enhance the mobilization of stem cells from the patient's BM to the peripheral circulation. Mobilization can be determined by techniques (e.g. FACS) that involve counting the number of CD34+ cells before, during, and/or after treatment with the mobilizing substance.
  • the patient's stem cells may then be harvested, the patient subjected to HDT, and the stem cells transplanted back to the patient following HDT treatment.
  • a sample of the patient's blood may be taken to obtain an ALC.
  • the patient is treated, before and/or during stem cell collection, with one or more substances that stimulate the migration of stem cells from the BM to the peripheral circulation.
  • these substances include, without limitation, GM- CSF and Flt-3 ligand.
  • GM-CSF may be administered at a concentration of at least 5 ⁇ g/kg/day by subcutaneous injection until the stem cell collection is complete.
  • Flt-3 ligand may be administered at a concentration of at least 50 ⁇ g/kg/day for at least the first three days of stem cell harvesting.
  • Flt3-ligand also may be administered at a concentration of at least 50 ⁇ g/kg/day for the entire duration of stem cell collection.
  • PBSCs are collected until the blood samples taken from the cancer patient reach a concentration of at least 2xl0 6 CD34+ stem cells/kg.
  • the cancer therapy may include treatment, post-ASCT, with one or more substances that stimulate proliferation and/or maturation and/or differentiation of immune cells.
  • the immune cells are NK cells.
  • the substance is interferon.
  • Patients may be treated with low doses of interferon post-ASCT, wherein the concentration of interferon administered to the patient is between about lxlO 5 to lxlO 7 units/m 2 , and wherein the patient is administered interferon from the day of the transplant to up to 21 days following the transplant.
  • Interferon may be administered by any pharmaceutically acceptable route known in the art, including intravenous injection, intra-arterial injection, or oral administration in the form of a tablet, capsule, or syrup.
  • a blood sample can be taken from the patient and used to obtain an ALC.
  • the ALC of the blood sample may be used as a prognostic indicator of the patients' survival.
  • an ALC of > 200 lymphocytes/ ⁇ L is indicative of survival of the cancer patient for at least 24 months following ASCT.
  • a "day 15" ALC of > 300 lymphocytes/ ⁇ L, preferably > 400 lymphocytes/ ⁇ L, and preferably > 500 lymphocytes/ ⁇ L is indicative of survival of the cancer patient for at least 24 months following ASCT.
  • the lymphocytes that are measured in lieu of an ALC are NK cells (and do not include B-or T-cells).
  • the number of NK cells/ ⁇ L measured in a blood sample obtained within 15 days following ASCT are between at least 30 to 40 cells/ ⁇ L, preferably 45 to 60 cells/ ⁇ L, more preferably 60 to 80 cells/ ⁇ L, and still more preferably 75 to 100 cells/ ⁇ L.
  • the NK cell count comprises > 30, 40, 45, 60, 75, 80, or 100 NK cells/ ⁇ L.
  • These NK cell counts are indicative of patient survival for at least 24 months.
  • the number of NK cells measured at "day 15" following ASCT, which is indicative of patient survival for at least 24 months following ASCT is at least 15 % of the ALC and preferably at least 20 % of the ALC.
  • T cell-independent B cell antibody responses e.g. to pneumococcal polysaccharide recover within 1 to 2 years post transplant (Witherspoon et al. (1981) Blood 58:360).
  • Post-ASCT studies have demonstrated defects of in vivo B-cell function with normal serum levels of IgM returning at 6 months, IgG at 12 to 18 months, and IgA after 2 years (see Pechazzini et al. (1989) Blood 74:2230; Korholz et al. (1996) Bone Marrow Transplantation 18:1123).
  • T-cells subset demonstrating immuno- recovery are also delayed post-ASCT, for example, immuno-recovery of CD3+ cells, 3 to 5 months (Parrado et al. (1997) Hematol Cell Ther 39:301; Korholz et al. (1996) Bone Marrow Transplantation 18:1123); CD4+ cells, one year or more (Parrado et al. (1997) Hematol Cell Ther 39:301); and CD8+ cells, 3 to 18 months (Olsen et al. (1998)
  • NK cells recover normal absolute and relative numbers within one month after transplant (Talmadge et al. (1996) Transplantation 17:101 and Bosly et al. (1987) Exp Hematol 15:1048).
  • NK cells normally comprise 5 to 8 % of human PB lymphocytes and mo ⁇ hologically resemble large granular lymphocytes (Timonen et al. (1981) JExp Med 153:569).
  • Mechanisms of NK cell function/cytotoxicity include (i) spontaneous antibody-independent non-MHC- restricted cytotoxicity and (2) antibody-dependent cellular cytotoxicity (Trincheri (1989) Adv fmmunol 47: 187).
  • the present invention provides a method of determining the efficacy of an anti-cancer drag in a cancer patient.
  • the method comprises the steps of treating a cancer patient with the drag, harvesting stem cells from the patient, providing the patient with HDT, infusing the harvested stem cells back to the patient, and obtaining a "day 15" post-ASCT ALC, wherein an ALC > 200 lymphocytes/ ⁇ L is indicative of the efficacy of the drag, hi preferred embodiments, the ALC is >300 lymphocytes/ ⁇ L, preferably > 400 lymphocytes/ ⁇ L, and preferably > 500 lymphocytes/ ⁇ L, and is, thus indicative of the efficacy of the drag.
  • the anti-cancer drag may be selected from the group of commonly used drags in cancer therapy including, without limitation: Aldesleukin, Altretamine, Asparaginase, Azathioprine, Becaplermin, Bexarotene, BGC vaccine, Bleomycin, Busulfan, Butabarbital Sodium, Capecitibine, Carboplatin, Carmustine, CCNU, Chlorambucil, Cisplatin, Cladribine, Corticosteroids, Cyclophosphamide, Cyterabine, dacarbazine, Dactinomycin, Daunomycin, 2'-Deoxycoformycin, Dexamethasone, Docetaxel, Doxorubicin, Epirabicin, Epeotin alfa, Estramustine, Ethambutol, Etoposide, Filgrastim, Floxuridine, Fludarabine, Fluorouracil, Ganciclovir, Gemcitabine, Hydroxyurea, Idara
  • the drag is Flt-3 ligand and/or GM-CSF.
  • a cancer patient may be treated with Flt-3 ligand, in combination with other drags, including GM- CSF, before and/or during the harvest of stem cells for ASCT to stimulate the migration of stem cells from the BM to the peripheral circulation.
  • Flt-3 ligand has been shown to increase ⁇ K cells in vitro and in animals (Mckenna et al. 2000) Blood 95:3489). McKenna et al. showed that when mice are treated with Flt-3 ligand, immature B cells, ⁇ K cells, and dendritic cells are expanded in vivo.
  • Flt-3 ligand has also induced tumor regression in animal models of melanoma and lymphoma (Lynch et al. (1991) Nat Med 3:625).
  • GM-CSF may be administered at a concentration of at least 5 ⁇ g/kg/day by subcutaneous injection until the stem cell collection is complete.
  • Flt-3 ligand may be administered at a concentration of at least 50 ⁇ g/kg/day for at least the first three days of stem cell harvesting.
  • Flt3-ligand also may be administered at a concentration of at least 50 ⁇ g/kg/day for the entire duration of stem cell collection.
  • PBSCs are collected until the blood samples taken from the cancer patient reach a concentration of at least 2xl0 6 CD34+ stem cells/kg.
  • the cancer therapy may include treatment, post-ASCT, with one or more substances that stimulate the proliferation and/or maturation and/or differentiation of immune cells.
  • the immune cells are NK cells.
  • the substance is interferon.
  • Patients may be treated with low doses of interferon post-ASCT, wherein the concentration of interferon administered to the patient is between about lxlO 5 to lx 10 7 units/m 2 , and wherein the patient is administered interferon from the day of the transplant to up to 21 days following the transplant.
  • Interferon may be administered by any pharmaceutically acceptable route known in the art, including intravenous injection, intra-arterial injection, or oral administration in the form of a tablet, capsule, or syrup.
  • a blood sample is taken from the cancer patient and an ALC is determined.
  • the patient can be treated with: (a) one or more of the cancer drugs described herein, (b) GM-CSF and or Flt-3 ligand, and or (c) interferon as described herein.
  • An ALC of ⁇ 200 lymphocytes/ ⁇ L is indicative of the efficacy of the drags of (a), (b), and/or (c).
  • the lymphocytes measured in lieu of an ALC, wherein the patient has been treated with one or more of the drags of (a), (b), or (c) above, can be NK cells (and do not include B- or T-cells).
  • the number of NK cells/ ⁇ L determined for a blood sample obtained within 15 days following ASCT, indicative of drag efficacy, can be at least 30 to 40 cells/ ⁇ L, preferably 45 to 60 cells/ ⁇ L, more preferably 60 to 80 cells/ ⁇ L, and still more preferably 75 to cells/ ⁇ L.
  • the NK cell count comprises 30, 40, 45, 60, 75, 80, or 100 cells/ ⁇ L.
  • the number of NK cells measured on "day 15" following ASCT, which is indicative of drug efficacy is at least 15 % of the ALC, preferably at least 20 % of the ALC. Improving Cancer Therapy
  • the present invention provides a method of improving a cancer therapy.
  • the method comprises the steps of (i) treating a cancer patient in need of therapy with one or more drugs that may mobilize stem cells from the BM to the peripheral circulation, (2) harvesting stem cells from the patient for use in ASCT, (3) providing the patient with HDT, (4) infusing the patient with the harvested stem cells and (4) obtaining a blood sample within 15 days following ASCT to determine the ALC, wherein an ALC of ⁇ 200 lymphocytes/ ⁇ L is indicative of an improvement in the cancer therapy.
  • an ALC of ⁇ 500 lymphocytes/ ⁇ L is indicative of an improvement in the cancer therapy.
  • An improvement is indicated where a specific population of cancer patients, when treated, has a longer survival time compared to another specific population of untreated cancer patients.
  • the cancer patient may be provided with a cancer therapy that includes the administration of one or more anti-cancer drugs as described above.
  • the anti-cancer drag may be administered separately from, or in conjunction with, one or more substances that stimulate the mobilization of stem cells from the BM to the peripheral circulation.
  • one or more of the anti-cancer drugs described herein may be administered to the cancer patient in concert with substances such as Flt-3 ligand and GM-CSF, G-CSF, c-kit ligand, interferon, IL-2, IL-7, IL-8, IL-12, or interferon.
  • Flt-3 ligand and GM-CSF are administered alone before harvest of PBSCs in an effort to improve cancer therapy.
  • one or more of the anti-cancer drags described herein may be administered as part of the HDT.
  • Administration of the anti-cancer drug may or may not be coupled with further administration of one or more substances that stimulate the mobilization of stem cells from the BM to the peripheral circulation, including, but not limited to Flt-3 ligand and/or GM-CSF.
  • the cancer therapy may include treatment, post-ASCT, with one or more substances that stimulate the proliferation and/or maturation and/or differentiation of immune cells.
  • the immune cells are NK cells.
  • the substance is interferon.
  • Patients may be treated with low doses of the substance post-ASCT, wherein the concentration of the substance administered to the patient is between about lxl 0 5 to lxlO 7 units of activity/m 2 , and wherein the substance is administered to the patient from the day of the transplant to up to 21 days following the transplant.
  • the substance may be administered by any pharmaceutically acceptable route known in the art, including intravenous injection, intra-arterial injection, or oral administration in the form of a tablet, capsule, or syrup.
  • Interferon is one such substance and may be administered in the dosage range and mode as described above.
  • an ALC of : 200 lymphocytes/ ⁇ L is indicative of an improvement in the cancer therapy.
  • the ALC is > 300 lymphocytes/ ⁇ L, preferably ⁇ 400 lymphocytes/ ⁇ L, and preferably > 500 lymphocytes/ ⁇ L, and is, thus, indicative of an improvement in cancer therapy.
  • the lymphocytes that are measured in lieu of an ALC can be NK cells (and do not include B- or T-cells).
  • the number of NK cells/ ⁇ L measured in a blood sample obtained within 15 days following ASCT that is indicative of an improvement in cancer therapy is between at least 30 to 40 cells/ ⁇ L, preferably 45 to 60 cells/ ⁇ L, more preferably 60 to 80 cells/ ⁇ L, and still more preferably 75 to 100 cells/ ⁇ L.
  • the NK cell count comprises >; 30, 40, 45, 60, 75, 80, or 100 cells/ ⁇ L.
  • the number of NK cells measured at "day 15" following ASCT that is indicative of an improvement in cancer therapy is at least 15 % of the ALC, preferably at least 20 % of the ALC.
  • Example 1- Patient Populations Three hundred and eighty-six patients underwent ASCT at the Mayo Clinic, Rochester, Minnesota. Consecutive patients were selected to minimize selection bias. Of the 386 patients, 126 had multiple myeloma (MM), 104 had non-Hodgkin's lymphoma (NHL), 82 had Hodgkin's disease, 45 had acute myelogenous leukemia (AML), and 29 had metastatic breast cancer. Patients who had Hodgkin's disease were those who relapsed after conventional chemotherapy or those who demonstrated an incomplete response to conventional chemotherapy before ASCT.
  • MM myeloma
  • NHL non-Hodgkin's lymphoma
  • AML acute myelogenous leuk
  • Example 2 - Conditioning Regimens Conditioning regimens for MM patients were the following. One hundred and one patients were treated with melphalan (140 mg/m 2 ) and total body irradiation (TBI) (12 Gy). Eleven patients were treated with melphalan (140 mg/m 2 ), cyclophosphamide (60 mg/m 2 ), and TBI (12 Gy). Eleven patients were treated with melphalan (200 mg/m 2 ). Two patients were treated with cyclophosphamide (60 mg/m ) and TBI (12 Gy). One patient was treated with busulfan (16 mg/kg) and cyclophosphamide (60 mg/m 2 ).
  • NHL patients were treated with cyclophosphamide (1.5 g/m 2 ), carmustine (BCNU) (300 mg/m 2 ), and etoposide (125 mg/m 2 ). Thirty-one patients were treated with cyclophosphamide (60 mg/m 2 ) and TBI (12 Gy). Sixty-six patients were treated with BCNU (300 mg/m 2 ), etoposide (NP-16: 100 mg/m 2 ), cytarabine (ARA-C: 100 mg/m 2 ), and cyclophosphamide (35 mg/kg).
  • Conditioning regimens for patients with Hodgkin's disease included the following. Fifty-nine patients were treated with cyclophosphamide (1.5 g/m 2 ), BC ⁇ U (300 mg/m 2 ), NP-16 (125 mg/m 2 ) (CBV). Fifteen patients were treated with BC ⁇ U (300 mg/m 2 ), etoposide (100 mg/m 2 ), ARA-C (100 mg/m 2 ), and melphalan (140 mg/kg) (BEAM). Seven patients were treated with cyclophosphamide (60 mg/m ) and TBI (12 Gy). One patient was treated with NP-16 (60 mg/kg) and TBI (12 Gy). All patients had stem cells re-infusion after high dose therapy.
  • Conditioning regimens for AML patients included the following. Thirty-three patients were treated with cyclophosphamide (60 mg/m 2 ) and TBI (13.2 Gy). Eight patients were treated with busulfan (1 mg/kg) and cyclophosphamide (60 mg/kg). Four patients received busulfan (1 mg/kg) and cyclophosphamide (50 mg/kg).
  • Conditioning regimen for patients with metastatic breast cancer included treatment with cyclophosphamide (1.5 gm/m 2 /d), carboplatin (200 mg/m 2 /d), and thiotepa (125 mg/m 2 /d) (STAMP N).
  • Example 3 Hematological Engraftment Neutrophil engraftment was taken to be the first day of three consecutive days in which ANC > 500 cells/ ⁇ L was achieved. Platelet engraftment was defined as a platelet count > 20 x 10 9 /L, independent of transfusion support. ALC threshold (immunologic engraftment) was determined at 500 cells/ ⁇ L at day 15 after ASCT.
  • prognostic factors included age (> 50 years), ⁇ 2-microglobulin ( ⁇ 2-M, > 2.7 mg/dL), C-reactive protein (CRP, > 0.8 mg/dL), circulating plasma cells, lactate dehydrogenase (LDH, > normal for age/sex), plasma cell-labeling index (PCLI, ⁇ 1 %), bone marrow plasma cell percentage (> 40 %), cytogenetic analysis, ANC at day 15 post-ASCT (> 500 cells/ ⁇ L), absolute lymphocyte count (ALC) at day 15 post-ASCT, stem cell source (BM vs PBSC), platelet count at day 15 post-ASCT (> 20 x 10 9 /L), number of pre-transplant chemotherapy regimens (continuous variable), and clinical status prior to transplant (primary refractory, plateau response, relapse off chemotherapy, and relapse on chemotherapy).
  • age > 50 years
  • ⁇ 2-microglobulin > 2.7 mg/dL
  • CRP C-reactive protein
  • Prognostic factors for NHL patients included age (> 60), LDH (> normal for age/sex), performance status (PS, ECOG > 2), extra-nodal sites (> 2), and stage (international age-adjusted prognostic index as described in The International Non-
  • Hodgkin's Lymphoma Prognostic Factors Project A predictive model for aggressive non- Hodgkin's lymphoma (1993) NEnglJMed 329:987-994 and Ansell et al. (1991) J Clin Oncol 15:2296-2301).
  • the number of pre-transplant treatments stem cell source (BM versus PBSC), ANC at day 15 post-ASCT (> 500 cells/ ⁇ L), ALC at day 15 post-ASCT (> 500 cells/ ⁇ L), platelet count at day 15 (> 20 x 10 9 /L), chemo-sensitive disease status (defined as CR or PR), and CR status prior to transplantation were also included.
  • CD34 dose, MNC counts, and NCC were used as continuous variables.
  • Prognostic factors were determined at the time of diagnosis of Hodgkin's disease as well as before and after ASCT (Carella et al. (1991) Bone Marrow Transplantation 8:99-103; Nademanee et al. (1999) Biology of Blood and Marrow Transplantation 5:292-298; and Lazarus et al. (1999) J Clin Oncol 17(2)534-545).
  • Prognostic factors determined before ASCT included: B-symptoms; bulky disease; clinical status (complete remission (CR), partial remission (PR), relapse untreated (RU), or relapse resistant (RR)); CR status alone; disease stage (I/II versus III/IN); duration of response after initial chemotherapy (> 12 months); extranodal disease; failure of initial chemotherapy; interval from diagnosis to ASCT (> 18 months); performance status (ECOG ⁇ l); prior number of chemotherapy regimens; and relapse within the field of previous radiation.
  • Prognostic factors determined after transplantation included: ALC at day 15; ANC at day 15; conditioning regimen; platelet count at day 15; post-transplant consolidation radiation therapy; stem cell numbers (CD34 dose or mononuclear cell (MNC) count); and stem cell source (BM versus PB).
  • Prognostic factors for patients with AML included those determined at the time of diagnosis as well as those determined before and after transplantation (Mehta et al. (1995) Bone Marrow Transplantation 16(4):499-506; Keating et al. (1996) Bone Marrow Transplantation 17(6):993-1001; Harousseau et al. (1997) Blood 90(8):2978-86; and Ferrant et al. (1997) Blood 90(8):2931-2938).
  • Prognostic factors at diagnosis included: cytogenetic abnormalities (favorable, intermediate, unfavorable); French- American- British (FAB) classification (M3 versus other); gender; and WBC count (> 10.5 x 10 9 /L).
  • Prognostic factors determined before transplantation included: age (> 45 years old); clinical status (CR, PR, or RU); CR status alone; duration of CR after induction chemotherapy (> 12 months); interval from CRl or CR2 to transplantation (> 4 months); and performance status prior to transplantation (ECOG >1).
  • CRl is the first CR after first chemotherapy regimen
  • CR2 is the second CR after relapsing from first chemotherapy regimen and achieving CR after the second chemotherapy regimen.
  • Prognostic factors determined after transplantation included: ALC at day 15 post-ASCT, ANC at day 15 post-ASCT, conditioning regimens (TBI + chemotherapy versus chemotherapy alone), platelet count at day 15 post-ASCT, and stem cell source (BM versus PB).
  • Favorable cytogenetic abnormalities included translocation from chromosome 8 to 12 [t(8;12)], translation from chromosome 15 to 17 [t(15;17)], and inversion and translation of chromosome 16 [inv(16)/t(16;16)j.
  • Cytogenetic abnormalities of intermediate prognosis included normal karyotype and missing Y chromosome; all others as well as complex chromosomal changes were classified as unfavorable (Keating et al. (1996) Bone Marrow Transplantation 17(6):993-1001).
  • Example 5 Stem Cell Source
  • the stem cell source for ASCT included BM or PB.
  • BM or PB One hundred and sixty patients received unmobilized BM stem cells, while 203 received PBSC mobilized with granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony- stimulating factor (GM-CSF), or combinations of both.
  • G-CSF granulocyte colony-stimulating factor
  • GM-CSF granulocyte-macrophage colony- stimulating factor
  • Example 6 Supportive Care Three hundred and forty-one patients received post-transplant growth factors, while forty-five patients did not receive post-transplant growth factors. Of the 341 patients that received growth factors after transplant, 292 received G-CSF and 49 received GM-CSF. Different prophylactic antibiotics, antifungal and antiviral medications, and transfusion requirements were used as per BM transplant supportive care guidelines during the transplant phase.
  • AML patients 42 received post-transplant growth factors, while three did not. Of the 42 patients that received post-transplant growth factors, 30 received G-CSF and 12 received GM-CSF. Different prophylactic antibiotics, antifungal and antiviral medications, and transfusion requirements were used as per BM transplant supportive care guidelines during the transplant phase.
  • 59 received post-transplant growth factors, while twenty-three patients did not. Of the 59 patients that received post-transplant growth factors, 33 received G-CSF and 26 patients received GM-CSF. Different prophylactic antibiotics, antifungal and antiviral medications, and transfusion requirements were used as per BM transplant supportive care guidelines during the transplant phase. Similar supportive care regimens were used for NHL patients, MM patients, and metastatic breast cancer patients.
  • CR was defined as a lack of detectable monoclonal protein in serum and urine by immunofixation.
  • PR was defined as a reduction of serum monoclonal protein and 24-hour urinary light-chain excretion by at least 50%, accompanied by a similar reduction of soft tissue plasmacytomas, if present.
  • Disease progression was defined as a 50 % increase in the serum monoclonal protein or 24-hour urinary monoclonal protein excretion over the lowest remission level.
  • An increase in the size or number of lytic bony lesions or soft tissue plasmacytomas constituted progression. In those with CR, any detectable monoclonal protein by immunofixation constituted progression.
  • CR was defined as complete regression of all measurable or evaluable disease including radiologically demonstrable disease, BM involvement or PB involvement.
  • PR was defined as a reduction of 50 % in the sum of the products of (a) longest diameter and (b) pe ⁇ endicular diameter of all measurable lesions as well as a > 30 % decrease in hepatomegaly or splenomegaly, measured from the costal margin, if there was previous known liver or spleen involvement.
  • Disease progression was defined as > 25 % increase in the sum of the products of (a) the longest diameter and (b) its pe ⁇ endicular diameter of all measurable lesion(s) from pre-study measurement, appearance of new lesions, or greater than 2 cm increase in spleen or liver size due to lymphoma.
  • CR was defined as (a) normal BM mo ⁇ hology with 20 % cellularity and fewer than 5 % blasts, (b) resolution of previously abnormal cytogenetics, (c) no evidence of extramedullary leukemia, and (d) an ANC > 1500/ ⁇ L and platelets > 100,000/ ⁇ L for at least four weeks.
  • Patients with regenerated PB values with > 5 % but ⁇ 25 % myeloblasts were considered to be in PR, as were patients fulfilling the BM criteria of CR but do not shown full recovery of PB platelet and/or WBC counts.
  • Patients with Hodgkin's disease or NHL were staged according to the Ann Arbor system (Carbone et al. (1971) Cancer Res 31:1860-1861).
  • CR was defined as complete regression of all measurable or evaluable disease including radiologically demonstrable disease, BM involvement, and PB involvement. Uncertain complete remission (CRu) was defined as persistent radiologic abnormalities without clinical evidence of Hodgkin's disease.
  • PR was defined as a reduction of > 50 % in the sum of the products of (a) the longest diameter and (b) pe ⁇ endicular diameter of all measurable lesions as well as > 30 % decrease in hepatomegaly or splenomegaly, measured from the costal margin, if there was previous known liver or spleen involvement.
  • Disease progression was defined as (a) > 25 % increase in the sum of the products of (i) the longest diameter and (ii) its pe ⁇ endicular diameter of all measurable lesion(s) from pre-study measurements, (b) appearance of new lesions, or (c) greater than 2 cm increase in spleen or liver size due to lymphoma.
  • Initial chemotherapy failure was defined as failure to achieve a PR or CR after initial treatment with combination chemotherapy, using one or more regimens.
  • CR was defined as no evidence of disease
  • PR was defined as a reduction of at least 50 % of the product of pe ⁇ endicular diameters.
  • Progression of disease was defined as a greater than 25 % increase in the product of pe ⁇ endicular diameters or new lesions.
  • OS time was measured from the date of transplantation to the date of death or last follow-up.
  • Progression-free survival was defined as time from transplantation to disease progression, relapse, or death.
  • Leukemia- free survival was defined as time from transplantation to disease progression, relapse, or death.
  • Example 8 Statistical Analysis OS and PFS were analyzed using the method described by Kaplan and Meier (Kaplan et al. (1958) JAm StatAssoc 53:457-481). Differences between survival curves were tested for statistical significance using the two-tailed log-rank test.
  • the Cox proportional hazards model (Cox (1972) JR Stat Soc 34:187-202) was used to assess ALC as a prognostic factor for post-transplant OS and PFS rates as well as to adjust for other known prognostic factors.
  • the Cox model was stratified on disease type, i.e. MM versus NHL.
  • the validity of the proportional hazards model was confirmed using the techniques of Grambsch and Therneau (Grambsch et al. (1994) Biometrika 81:515-26).
  • the cut-off of ALC > 500 cells/ ⁇ L was based on the median of ALC for the cohort group.
  • the cut-point of 500 cells/ ⁇ L yielded the greatest differential in survival as determined from ⁇ 2 values analyzed at different cut-points (e.g. 200 to 900 cells/ ⁇ L) from log-rank tests.
  • the ⁇ and Fisher's exact tests were used to determine relationshrps between nominal variables including: ALC > 500 cells/ ⁇ L, ANC > 500 cells/ ⁇ L, and platelet count > 20 x 10 9 /L at day 15 after ASCT; growth factor versus no growth factor after transplantation; G-CSF versus GM-CSF after transplantation; and stem cell source (BM versus PB).
  • Wilcoxon rank-sum test was used to compare continuous variables including CD34 cell dose, MNC count, and nucleated cell count (NCC); prior number of chemotherapy regimens; and prior number of treatment cycles before transplantation. All p-values represented are two-sided and statistical significance was declared at p ⁇ 0.05. Risk ratios reported were for risks associated with patients having high (> 500 cells/ ⁇ L) versus low ( ⁇ 500 cells/ ⁇ L) ALC values.
  • Example 9 Patient Characteristics Base-line characteristics of the 126 MM patients at the time of transplantation, 104 NHL patients at the time of transplantation, 82 patients with Hodgkin's disease, at diagnosis and at transplantation, 45 AML patients at transplantation are summarized in Tables 1-5, respectively.
  • 63 patients had ALC > 500 cells/ ⁇ L with a mean value of 1100 ⁇ 600 cells/ ⁇ L (95 % Cl, 938-1240 cells/ ⁇ L) and a median of 870 cells/ ⁇ L (range 500- 3100 cells/ ⁇ L). Of these, six patients had ALC > 2000 cells/ ⁇ L. For the 63 patients with ALC ⁇ 500 cells/ ⁇ L, the mean value was 278 ⁇ 117 cells/ ⁇ L (95 % Cl, 250-307 cells/ ⁇ L) and the median value was 292 cells/ ⁇ L (range 50-472 cells/ ⁇ L). Three patients had ALC ⁇ 100 cells/ ⁇ L.
  • the median duration of response after initial chemotherapy prior to transplantation in patients with Hodgkin's disease was 10 months (range: 1-204 months).
  • the median time from diagnosis to ASCT was 26 months (range: 5-330).
  • AML group 23 patients had ALC > 500 cells/ ⁇ L on day 15 with a median ALC of 760 cells/ ⁇ L (range 520-1980 cells/ ⁇ L), and 22 patients had ALC ⁇ 500 cells/ ⁇ L with a median ALC of 125 cells/ ⁇ L (range 10-410 cells/ ⁇ L).
  • the median A ⁇ C and platelet count at day 15 post-ASCT were 0.6 x 10 9 /L (range 0.05-5.4 x 10 9 /L) and 20 x 10 9 /L (range 3-64 x 10 9 /L), respectively.
  • Twenty-nine patients achieved an A ⁇ C > 500 cells/ ⁇ L at day 15, and 24 patients achieved untransfused platelet count > 20 x 10 9 /L at day 15 post-ASCT.
  • the median duration of CR after induction chemotherapy before transplantation in AML patients was 9 months (1-88 months).
  • the median time from diagnosis to ASCT was 14 months (range 3-88 months).
  • the median duration from the time patients were classified as being in CR (CRl or CR2) to ASCT was 5 months (range 0.5-37 months).
  • 22 developed AML de novo, while three patients developed secondary AML from myelodysplastic syndrome. Two patients had central nervous system involvement, and four patients presented with extramedullary disease (gum swelling - one patient and lymph nodes - three patients).
  • the median number of chemotherapy regimens before transplantation was 2 (range 1-5), and the median number of treatment cycles before transplantation was 9 (range 1-20).
  • the 386 patients only five (all AML patients) received a purged stem cell product, while none received CD34-selected stem cells.
  • the median CD34 count was 3.8 x 10 6 /kg (range: 0.8 - 85 x 10 6 /kg).
  • the MNC count available in 220 patients (113 PB and 107 BM), had a median of 7.2 x 10 8 /kg (range: 0.5- 22 x 10 8 /kg).
  • the median nucleated cell count was 2.5 x 10 8 /kg (0.7-4.4 x 10 8 /kg).
  • Plasma cell labeling index > 1% 37 B 2-microglobulin (>2.7mg/l) 59.5 LDH> normal for age/sex 85.2 Cytogenetics Normal 64.2 Abnormal 35.8 C-reactive protein>0.8mg/dl 30.3 Stem cell source (%) BM 20 PBSC 80 Platelets > 20 x l0 9 /l (%) 81 ALC at day 15 post-ASCT (%) >500 cells/ul 50 ⁇ 500 cells/ul 50 ANC at day 15 post-ASCT (%) >500 cells/ul 95.7 ⁇ 500 cells/ul 4.3
  • Angiocentric B cell 1 Angiocentric B cell 1
  • BM bone marrow
  • ECOG Eastern Cooperative Oncology Group
  • PBSC peripheral blood stem cell
  • CD34 positive cell infused was 2.9 x 10 6 /kg (range: 2.0-6.2 x 10 6 /kg).
  • MNC counts available in 25 patients (1 PBSC and 24 BM), had a median of 2.0 x 10 8 /kg (range: 0.5-9.2 x 10 8 /kg).
  • recurrent or progressive myeloma was the cause of death in 35 patients. Seventeen patients died of transplant related complications including Staphylococcus aureus septicemia (2 patients), Aspergillus meningitis (1 patient), CMN pneumonitis (1 patient), PCP pneumonia (1 patient), Enterococcus bacteremia (1 patient), Streptococcus pneumonia (1 patient), Candida sepsis (1 patient), disseminated varicella zoster (1 patient), intra-cranial bleeding (3 patients), lung failure (acute respiratory distress syndrome, i.e. ARDS) (2 patients), renal failure (2 patients), and liver failure (1 patient). In addition, one patient committed suicide and one died of sudden cardiac death.
  • recurrent or progressive lymphoma was the cause of death in 45 patients. Eight patients died of transplant related complications including lung failure (ARDS) (3 patients), intra-cranial bleeding (1 patient), AML (2 patients), Candida sepsis (1 patient), and pneumonia (Staphylococcus aureus) (1 patient). In addition, one died in a motor vehicle accident. There was no significant difference in the supportive care, which included antibiotics, growth factors, or transfusion requirements. No correlation was identified between the year of transplantation and transplant related mortality (p ⁇ 0.8). None of the patients developed autologous GNHD. The median follow-up time for all patients was 12.5 months, with a maximum of 123 months.
  • transplant-related mortality 18 patients died from recurrent or progressive disease, while 2 died of transplant-related complications (transplant-related mortality was 4. One patient died of diffuse alveolar hemorrhage, and the others of acute respiratory distress syndrome. Of the transplant-related mortality, one patient had ALC > 500 cells/ ⁇ L, and one patient had ALC ⁇ 500 cells/ ⁇ L. There were six deaths in the 23 patients with ALC > 500 cells/ ⁇ L and 14 deaths in the 22 patients with ALC ⁇ 500 cells/ ⁇ L. Three patients had a relapse of leukemia but remained alive (one patient with ALC > 500 cells/ ⁇ L and two patients with ALC ⁇ 500 cells/ ⁇ L).
  • non-Hodgkin's > 500 cells/ ⁇ L N O.0001 NR O.OOOl lymphoma (N 56)
  • Example 12 Univariate and Multivariate Analysis Prognostic factors were tested to determine if they were predictive of OS and PFS, i.e., whether they would be effective prognostic predictor for OS and PFS.
  • Plasma cells 39 .23-.68 .0007 .46 .28-.77 .003 CRP > 0.8 mg/dl ,57 .31-1.04 .067 .65 .38-1.1 .11 vs ⁇ 0.8 mg/dl LDH normal for age/sex .45 .21-.95 .037 .45 .24-.86 .015 vs > normal PCLI 1% 34 .2-.6 .0001 .34 .21-.55 ⁇ .0001 vs ⁇ 1% Platelet> 20 x l0 9 /1 .18 .09-.36 ⁇ .0001 .3 .16-.57 .00024 vs ⁇ 20 x l0 9 /l # of pre-transplant chemotherapy regimens 1.58 1.05-2.38 .028 1.7 1.18-2.44 .004 Primary refractory
  • NA non-applicable; *level absorbed into model since plateau/response level omitted; **versus primary refractory level
  • Figure 1 is a graph comparing the OS of MM patients with ALC > 500cells/ ⁇ L and MM patients with ALC ⁇ 500 cells/ ⁇ L.
  • MM patients with ALC > 500cells/ ⁇ L had a median OS of 33 months, while MM patients with ALC ⁇ 500 cells/ ⁇ L had a median OS of 12 months (p ⁇ 0.0001).
  • Figure 2 is a graph comparing the PFS of MM patients with ALC > 500cells/ ⁇ L and MM patients with ALC ⁇ 500 cells/ ⁇ L.
  • MM patients with ALC > 500cells/ ⁇ L had a median PFS of 16 months, while MM of patients with ALC of ⁇ 500 cells/ ⁇ L had a median PFS of 8 months (p ⁇ 0.0001, respectively).
  • ALC also was a prognostic factor for patients who received PBSC or BM graft.
  • Figure 3 is a graph comparing the OS survival of NHL patients with ALC > 500 cells/ ⁇ L and NHL patients with ALC ⁇ 500 cells/ ⁇ L.
  • FIG. 4 is a graph comparing the PFS of NHL patients with ALC > 500 cells/ ⁇ L and NHL patients with ALC ⁇ 500 cells/ ⁇ L.
  • NHL patients with ALC ⁇ 500 cells/ ⁇ L had a median PFS time of 4 months, while most NHL patients with ALC ⁇ 500 cells/ ⁇ L remained alive at the end ofthe study period, ie., the median OS could not be determined (p ⁇ 0.0001).
  • NHL patients with ALC > 500 cells/ ⁇ L showed significantly better median OS than those with ALC ⁇ 500 cells/ ⁇ L, regardless of type of stem cells used (PBSC or BM). Similarly, NHL patients with ALC > 500 cells/ ⁇ L showed significantly better median OS than those with ALC ⁇ 500 cells/ ⁇ L, regardless of type of stem cells used (PBSC or BM). For NHL patients who received PBSC grafts, the median OS time of those with ALC ⁇ 500 cells/ ⁇ L was 6 months, while the median OS time of those with ALC > 500 cells/ ⁇ iL could not be determined as a large number of patients were still alive at the end ofthe study (p ⁇ 0.0001).
  • the PFS of those with ALC ⁇ 500 cells/ ⁇ L was 3.6 months, while the PFS of those with ALC > 500 cells/ ⁇ L could not be determined as progression had not been reached for a large number of patients (p ⁇ 0.0001).
  • the median OS time of those with ALC ⁇ 500 cells/ ⁇ L was 5.7, while the median OS time of those with ALC > 500 cells/ ⁇ L could not be determined as most patients were still alive at the end ofthe study period (p ⁇ 0.0001).
  • ALC > 500 cells/ ⁇ .07 .03-.16 ⁇ .0001 .09 .04-.18 ⁇ .0001 vs ⁇ 500 cells/ ⁇ l
  • ANC > 500 cells/ ⁇ l .43 .21-.88 .02 .49 .25-.95 .034 vs ⁇ 500 cells/ ⁇ l
  • ALC > 500 cells/ ⁇ l. .176 .075-.414 .0001 .275 .13-.98 .0006 .08 .04-.17 .0001.09 .04-19 .0001 vs ⁇ 500 cells/ /l
  • OS overall survival
  • PFS progression-free survival
  • RR relative risk
  • FIG. 5 is a graph comparing the OS (median: 60 months) and PFS (median: 40 months) of patients having Hodgkin's disease.
  • Figure 6 is a graph comparing the OS of Hodgkin's disease patients having ALC > 500 cells/ ⁇ L with Hodgkin's disease patients having ALC ⁇ 500 cells/ ⁇ L.
  • FIG. 7 is a graph comparing the PFS of Hodgkin's disease patients having ALC > 500 cells/ ⁇ L with Hodgkin's disease patients having ALC ⁇ 500 cells/ ⁇ L.
  • the median PFS of patients with ALC > 500 cells/ ⁇ L (not reached) was significantly greater than the median PFS of patients with ALC ⁇ 500 cells/ ⁇ L (14 months), p ⁇ 0.0001.
  • splenectomy was found to be a predictor for OS, PFS, and bulky disease.
  • Table 11 summarizes the significant prognostic factors in the univariate and multivariate analyses.
  • the lack of correlation between CD34 counts or MNC and day 15 ALC suggests that the repopulating lymphocytes post-AHSCT may be derived from sources other than transplanted stem cells, for example, mature lymphocytes present in the re-infused auto-graft.
  • ECOG Eastern Cooperative Group
  • OS overall survival
  • PFS progression-free survival
  • RR relative risk
  • CR complete remission
  • OS overall survival
  • LFS leukemia-free survival
  • RR relative risk
  • the OS and PFS were significantly longer in patients with ALC ⁇ 500 cells/ ⁇ L versus those with ALC ⁇ 500 cells/ ⁇ L (p ⁇ 0.001).
  • the estimated 1-year overall survival for those with ALC ⁇ 500 cells/ ⁇ L was 50 % (95 % Cl: 28 %-88 %) versus 91% for those with ALC > 500 cells/ ⁇ L (95 % Cl: 75 % -100 %).
  • the estimated 1-year PFS for those with ALC ⁇ 500 cells/ ⁇ L was 17 % (95% Cl: 5 % - 59 %) versus 56 % for those with ALC > 500 cells/ ⁇ L (95 % Cl: 36 % - 88 %).
  • FIG 8 is a graph comparing the OS of patients having ALC > 500 cells/ ⁇ L (median: 55 months) with the OS of patients with ALC ⁇ 500 cells/ ⁇ L (median: 13 months) for all 386 patients, p ⁇ 0.0001.
  • Figure 9 is a graph comparing the PFS of patients having ALC > 500 cells/ ⁇ L (median: 40 months) with the PFS of patients with ALC ⁇ 500 cells/ ⁇ L (median: 7 months) for all 386 patients, ⁇ ⁇ 0.0001.
  • Example 13 Determining the Efficacy of a Drug in a Cancer Patient
  • the following example describes a method for determining the efficacy of a drug in a cancer patient by treating the cancer patient with a candidate drug, and obtaining an ALC from a blood sample taken from the patient within 15 days following ASCT.
  • Patients are selected for treatment based on whether they have a cancer treatable by ASCT. Examples of such cancers include, MM, NHL, breast cancer, testicular cancer, and systemic amyloidosis.
  • cancers include, MM, NHL, breast cancer, testicular cancer, and systemic amyloidosis.
  • drugs including, but not limited to, G-CSF, GM-CSF, Flt-3 ligand, c-kit ligand, IL-2, IL-7, IL-8, IL-12, and interferon.
  • patients are treated with drugs such as Flt-3 ligand and GM-CSF at concentrations of at least 50 ⁇ g/kg/day and 5 ⁇ L/kg/day for Flt-3 ligand and GM-CSF, respectively.
  • Patients are started on a regimen of GM-CSF administration by subcutaneous injection beginning the same day that stem cell collection is started.
  • GM-CSF is administered for the duration of stem cell collection.
  • Flt-3 ligand is administered by subcutaneous injection beginning on the day that stem cell collection begins and continues for at least the first three days of stem cell collection.
  • stem cell apheresis may be started, and is performed no more than four times per week.
  • Stem cells are collected using a blood cell separator (CS 3000; Fenwal Laboratories, Deerfield, IL). A total blood volume of 9.5 to 10 L per apheresis may be processed at a flow rate of 50 to 70 mL/minute. Following the collection, a cell count is performed on an aliquot of apheresis product to determine the number of stem cells. The apheresis product is subsequently centrifuged at 400 g for 10 minutes, and the plasma is removed, yielding a total volume of approximately 100 mL. The cell suspension is then mixed with 100 mL minimal essential medium (MEM-S; Life Technologies, Rockville, MD) supplemented with 20 % DMSO.
  • MEM-S minimal essential medium
  • a total of 100 mL is then transferred to freezing bags (Delmed, Canton, MA) and frozen to -100 °C using a computer controlled cryopreservation device (Cryoson-BN-6; Cryoson Deutschland GmbH, FRG).
  • the cells may then be transferred into liquid nitrogen and stored at -196 °C until the transplant.
  • cells in the apheresis samples may be exposed to antibodies that specifically recognize stem cells.
  • a sample ofthe apheresis product may be exposed to an anti-CD34 specific antibody (Calbiochem, La Jolla, CA) that recognizes and binds to stem cells.
  • the primary antibody may then be localized using species-specific secondary antibody conjugated to a detectable marker such as biotin or a fluorescent moiety.
  • CD-34+ cells may be counted manually using a microscope.
  • a CD-34+ labeled apheresis sample may be quantitated using FACS. Apheresis is continued until the number of stem cells in the apheresis product collected in three or fewer collections reaches 25 x 10 CD- 34+ cells/kg.
  • the patient is treated with a HDT conditioning regimen.
  • the patients can be treated with melphalan (140 mg/m 2 ) and TBI (12 Gy); melphalan (140 mg/m 2 ), cyclophosphamide (60 mg/m 2 ) and TBI; or cyclophosphamide (60 mg/m ) and TBI.
  • the patients also can be treated with busulfan (16 mg/kg) and cyclophosphamide (60 mg/m 2 ); cyclophosphamide (1.5 g/m 2 ), BCNU (300 mg/m 2 ), and etoposide (125 mg/m 2 ); or BCNU (300 mg/m 2 ), etoposide (100 mg/m 2 ), ARA-C (100 mg/m 2 ), and cyclophosphamide (33 mg/kg). Patients may also be treated with antibiotics and/or antiemetic drugs as warranted by the patient's physician.
  • the frozen aliquot ofthe patient's stem cells is thawed and administered to the patient via intravenous injection into either the superior vena cava or the inferior vena cava.
  • the stem cells may be infused through a peripheral vein such as the median cubital vein.
  • the patient is additionally treated with between 1 x 10 5 and 1 x 10 7 units/m 2 of interferon. Patients are treated with interferon from the day that they receive the transplant to 21 days following stem cell transplant.
  • an ALC is determined using the Beckman Coulter Gen-S Cell according to the manufacturers instructions.
  • An ALC of > 200 lymphocytes/ ⁇ L, and preferably 300, 400, or 500 lymphocytes/ ⁇ L is indicative ofthe efficacy ofthe drug in treating cancer.
  • An ALC of less than 200 lymphocytes/ ⁇ L indicates that the drugs used in the stem cell mobilization treatment were not maximally effective.
  • the dose of Flt-3 ligand and GM-CSF may be increased to up to 500 pg/kg/day and 50 pg/kg/day, respectively. Higher concentrations of interferon also may be used.
  • Flt-3 ligand in combination with G-CSF or GM-CSF, or G-CSF alone is used for PBSC mobilization.
  • G-CSF G-CSF
  • G-CSF G-CSF alone
  • Flt-3 ligand and GM-CSF may lead to increased ALC and NK cells resulting in a more immuno-competent peripheral stem cell graft leading to faster immunological engraftment, and may confer a protective effect against minimal residual disease.
  • Example 14 Improving Cancer Therapy This example describes a method for improving cancer therapy by treating a cancer patient with a drug, and determining the drug efficacy using an ALC determined at "day 15".
  • the protocol described in Example 13 may be employed to improve existing cancer therapies. For example, despite the survival advantage of ASCT in treating a variety of malignancies, post-ASCT relapse rates range from 40 % to 70 %.
  • the present invention provides a method for improving cancer therapy comprising treating a cancer patient with a drug and obtaining a "day 15" post-ASCT ALC of >_200 lymphocytes/ ⁇ L, preferably >_300, 400, or 500 lymphocytes/ ⁇ L.
  • patients are treated with a drug regimen comprising one or more drugs intended to improve the outcome ofthe cancer treatment.
  • Patients may be treated with one or more drugs including, but not limited to, G-CSF, GM-CSF, Flt-3 ligand, c-kit ligand, IL-2, IL-7, IL-8, IL-12, or interferon.
  • drugs including, but not limited to, G-CSF, GM-CSF, Flt-3 ligand, c-kit ligand, IL-2, IL-7, IL-8, IL-12, or interferon.
  • patients are treated with Flt-3 ligand and GM-CSF at concentrations of at least 50 ⁇ g/kg/day and 5 ⁇ g/kg/day for Flt-3 ligand and GM-CSF, respectively.
  • Patients are on a regimen of GM-CSF administration by subcutaneous injection beginning the same day that stem cell collection is started.
  • GM-CSF is administered for the duration of stem cell collection.
  • Flt-3 ligand is administered by subcutaneous injection beginning on the day that stem cell collection begins and continues for at least the first three days of stem cell collection.
  • the stem cell apheresis maybe started, and will be performed no more than four times per week.
  • Stem cells are collected using a blood cell separator (CS 3000; Fenwal Laboratories, Deerf ⁇ eld, IL).
  • CS 3000 blood cell separator
  • a total blood volume of 9.5 to 10 L per apheresis may be processed at a flow rate of 50 to 70 mL/minute.
  • a cell count is performed on an aliquot of apheresis product to determine the number of stem cells.
  • the apheresis product is subsequently centrifuged at 400 g for 10 minutes, and the plasma is removed, yielding a total volume of approximately 100 mL.
  • the cell suspension is then mixed with 100 mL minimal essential medium (MEM-S; Life Technologies, Rockville, MD) supplemented with 20 % DMSO.
  • a total of 100 mL is then transferred to freezing bags (Delmed, Canton, MA) and frozen to -100 °C using a computer controlled cryopreservation device (Cryoson-BN-6; Cryoson Germany GmbH, FRG).
  • the cells may then be transferred into liquid nitrogen and stored at -196 °C until the transplant.
  • the ASCT will then proceed as described in Example 13. Subsequent to the transplant, patients may be additionally treated with low doses of interferon, as described above, in a further effort to improve the cancer therapy.
  • an ALC obtained using the Beckman Coulter Gen-S Cell according to the manufacturers instructions.
  • An ALC of at least 200 lymphocytes/ ⁇ L and preferably at least 300, 400, or 500 lymphocytes/ ⁇ L is indicative of an improvement in the cancer therapy.
  • An ALC of less than 200 lymphocytes/ ⁇ L indicates that efforts to improve the cancer therapy were not maximally effective.
  • the dose of Flt-3 ligand and GM-CSF may be increased to up to 500 ⁇ g/kg/day and 50 ⁇ g/kg/day for Flt-3 ligand and GM-CSF respectively. Higher concentrations of interferon may also be used.

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Abstract

L'invention concerne des procédés permettant (i) de prédire la survie d'un patient atteint du cancer et (ii) de déterminer l'efficacité d'un médicament et/ou l'amélioration d'une thérapie anticancéreuse. Ces procédés se basent sur le nombre total de lymphocytes dans un échantillon de sang prélevé chez un patient atteint du cancer durant les 15 jours suivant une transplantation autologue de cellules souches hématopoïétiques. Ces procédés consistent à traiter un patient atteint du cancer avec un médicament, et à obtenir un nombre total de lymphocytes dans un échantillon de sang prélevé chez le patient atteint du cancer durant les 15 jours suivant une transplantation autologue de cellules souches hématopoïétiques. Un nombre de lymphocytes d'au moins 200 lymphocytes/νL indique une survie d'une durée d'au moins 24 moins d'un patient atteint du cancer, et indique également l'efficacité du médicament et une amélioration de la thérapie anticancéreuse.
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