WO2002038189A1 - Absolute lymphocyte recovery and cancer survival - Google Patents

Abstract

The invention relates to methods of (i) predicting cancer patient survival and (ii) determining drug efficacy and/or improvement in a cancer therapy. Methods are based on the absolute lymphocyte count of a blood sample obtained from a cancer patient within 15 days of autologous hematopoietic stem cell transplant. Methods involve treating a cancer patient with a drug, and obtaining an absolute lymphocyte count on a blood sample taken from the cancer patient within 15 days following an autologous hematopoietic stem cell transplant. A lymphocyte count of at least 200 lymphocytes/νL is indicative of cancer patient survival of at least 24 months, and is further indicative of the efficacy of the drug and an improved cancer therapy.

Description

ABSOLUTE LYMPHOCYTE RECOVERY AND CANCER SURVIVAL

BACKGROUND

1. Technical Field The invention relates to methods and materials involved in predicting cancer survival or predicting the effectiveness of a cancer drug.

2. Background fnformation

Autologous stem cell transplant (ASCT), compared to conventional chemotherapy, improves survival in both previously untreated multiple myeloma (MM) and relapsed, chemotherapy-sensitive, aggressive non-Hodgkin's lymphoma (NHL) patients. 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. (1995) New EnglJMed 333:1540). The high relapse rates post- ASCT have been attributed to the inability of high dose therapy to eradicate minimal residual disease. The lower relapse rates seen in allogeneic stem cell transplantation have been related to early absolute lymphocyte count (ALC) recovery as a manifestation of early graft- versus-tumor effect (Kersey et al. (1987) New Engl JMed 317:416; Marmont et al. (1991) Blood 78:2120). Post-allogeneic bone marrow transplant (post-ABMT) studies have demonstrated that early ALC recovery is associated with prolonged survival (Prowles et al. (1998) Blood 91 : 3481). hi ASCT, however, the relationship of ALC recovery with clinical outcomes in cancer patients is previously undescribed. Prior art prognostic factors that physicians use to assess MM patients treated with ASCT include β-2M, plasma cell labeling index, cytogenetic analysis, lactate dehydrogenase, bone marrow (BM) plasma cell percentage, clinical status prior to transplantation, and C-reactive protein. (Rajkumar et al. (1999) Bone Marrow Transplantation 23:1261; Rajkumar et al. ( 1999) Bone Marrow Transplantation 23:1261 . A more accurate predictive factor for the survival of patients following cancer treatment and for the efficacy of a given treatment is needed.

SUMMARY 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.

In some embodiments, an ALC of a patient that is > 200 lymphocyte/μL predicts survival of the cancer patient for at least 24 months. In other embodiments, an ALC of a patient that is > 500 lymphocyte/μL predicts survival of the cancer patient for at least 24 months. In yet other embodiments, an ALC that is > 500 lymphocytes/μL predicts survival of the cancer patient for at least 33 months. In further embodiments, an ALC that is > 500 lymphocytes/μL in a cancer patient predicts survival of the cancer patient for at least 36 months. In additional embodiments, an ALC that is > 500 lymphocytes/μL predicts survival of the cancer patient for at least 42 months. In yet other embodiments, an ALC that is > 500 lymphocytes/μL 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/μL indicates that the candidate drug is effective in the cancer patient. The present invention also provides a composition comprising a sample of "day

15" blood of an ASCT cancer patient, wherein the sample comprises > 200 lymphocytes/μL (e.g., ^300,400, or 500 lymphocytes/μL).

In a preferred embodiment, 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/μL measured in a blood sample 15 days following ASCT are at least 30 to 40 cells/μL, preferably 45 to 60 cells/μL, more preferably 60 to 80 cells/μL, and still more preferably 75 to 100 cells/μL. In preferred embodiments, the NK cell count comprises > 30, 40, 45, 60, 75, 80, or 100 or higher cells/μL. These NK cell counts are indicative of patient survival for at least 24 months. Preferably, 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.

In a preferred embodiment, the blood is peripheral blood (PB).

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/μL is indicative of an improvement in said cancer therapy. The lymphocyte count can be >^300, 400, or 500 lymphocytes/μL. In preferred embodiments, between patient treatment and the measurement of

ALC, 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/μL is indicative of the efficacy of the drag, or improvement of the cancer therapy. The lymphocyte count can be > 300, 400, or 500 lymphocytes/μL. 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.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

Figure 1 is a graph comparing the overall survival (OS) of MM patients with ALC > 500 cells/μL (median 33 months) and MM patients with ALC < 500 cells/μL (median 12 months), p < 0.0001.

Figure 2 is a graph comparing the progression free survival (PFS) of MM patients with ALC > 500 cells/μL (median: 16 months) and MM patients with ALC < 500 cells/μL (median: 8 months), p < 0.0001.

Figure 3 is a graph comparing the OS survival of NHL patients with ALC > 500 cells/μL (not reached) and NHL patients with ALC < 500 cells/μL (median: 6 months), p < 0.0001.

Figure 4 is a graph comparing the PFS of NHL patients with ALC > 500 cells/μL (not reached) and NHL patients with ALC < 500 cells/μL. (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/μL (not reached) with Hodgkin's disease patients having ALC < 500 cells/μL (29 months), p < 0.0001.

Figure 7 is a graph comparing the PFS of Hodgkin's disease patients having ALC > 500 cells/μL (not reached) with Hodgkin's disease patients having ALC < 500 cells/μL (14 months), p < 0.0001.

Figure 8 is a graph comparing the OS of patients having ALC > 500 cells/μL (median: 55 months) with the OS of patients with ALC < 500 cells/μL (median: 13 months) for all 386 patients, p < 0.0001.

Figure 9 is a graph comparing the PFS of patients having ALC ≥ 500 cells/μL (median: 40 months) with the PFS of patients with ALC < 500 cells/μL (median: 7 months) for all 386 patients, p < 0.0001.

DETAILED DESCRIPTION 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/μL at anytime before and including day 15 following ASCT indicates survival of the cancer patient for at least 24 months following ASCT.

Definitions

As used herein, the term "lymphocyte" includes the following cell types: B-cells, T-helper cells, T-suppressor cells, and NK cells. The different lymphocytes can be distinguished based on the cell type specific expression of particular molecular markers. 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. For example, 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). The slide is generally exposed to the antibodies overnight, then subjected to a series of washes using an appropriate buffer solution such as PBS.

Next, 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).

An example of 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.

As used herein, "absolute lymphocyte count" (ALC) 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 (μL). 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. 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.

As used herein, "autologous" as it relates to transplantation, 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. This is in contrast to an "allogenic" transplant, which 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.

As used herein, "autologous stem cell transplant" (ASCT) refers to a medical procedure in which a sample of a patient's own stem cells are removed from the patient and subsequently transplanted back to the same patient. The patient's stem cells are taken through a process called "harvesting." Once harvested, the stem cells may be frozen until needed. The frozen stem cells are thawed and returned to the body (i.e. transplanted) by blood transfusion after the patient has received high doses of chemotherapy, radiation therapy, or both. Prior to the transplant, the stem cells may be treated with anticancer drugs or antibodies (called purging) to reduce the number of cancerous cells that may be present as much as possible. ASCT can be performed with stem cells harvested from bone marrow (BM) or peripheral blood (PB).

As used herein, "day 15" refers to a 15 day period of time where day 1 is the day following completion of an ASCT. Thus, 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. For example, 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. Generally, 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.

As used herein, "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. "Neoplasm, " as used herein 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).

As used herein, "high dose therapy" (HDT) 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. Examples of 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, BCΝU, and NP-16); and treatment with BEAC (BCΝU, etoposide, ARA-C, and cyclophosphamide).

As used herein, "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. In certain embodiments of the present invention, an ALC of >200 lymphocytes/μL post-ASCT is indicative of a cancer survival of at least 24 months.

As used herein, "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. As used herein, 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. Examples of "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. See, Bungart et al. (1990) Br J Haematol 16: 174; Terella et al. (1993) Bone Marrow Transplant 11 :271; Molineux et al. (1991) Blood 85:275; Grzegorzewski et al. (1994) Blood 83:377; Laterveer et al. (1995) Blood 85:2269; Jackson et al. (1995) Blood 85:2371; and Lyman et al. (1994) Blood 83:2795. In one embodiment, 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/μL is predictive of survival of the cancer patient for at least 24 months post-ASCT. In further embodiments, 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 "day 15" post-ASCT ALC is determined, and an ALC >200 lymphocytes/μL indicates (i) an improvement in the cancer therapy and/or (ii) that the candidate drug is effective for treating cancer.

Absolute Lymphocyte Count

ALC is a measure of the total number of lymphocytes per unit of blood, wherein a unit is any volume of blood. Preferably, ALC is measured as the number of mature lymphocytes per μL 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. Moreover, 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.

In a preferred embodiment, the ALC is obtained using an automated system for counting blood cells in a sample. Such 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. For example, 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. 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. Thus, such 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 μm. 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 μm in diameter, while lymphocytes are 7 to 10 μm in diameter. Alternatively, 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). For example, 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. The principle of FACS is that individual cells, held within a thin stream of fluid, are passed through one or more laser beams, one cell at a time, causing light to scatter and the fluorescent dyes to emit light at various predetermined frequencies. Photomultiplier tubes convert the light to electrical signals allowing for quantitation of the number of cells bearing the fluorophore. Thus, since all the lymphocyte sub-types are labeled with the same fluorophore, quantification of the number of fluorophore bearing cell will yield an ALC. FACS and quantitation is further described in U. S. Patent No. 4,499,052. Alternatively, one may obtain a "day 15" post-ASCT blood sample, place a known volume of the sample onto a glass microscope slide, smear the blood sample onto the slide so as to create a thin film of blood, and stain the blood sample using standard histological stains such as, for example H & E stain. Briefly, 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. In blood smears that have been stained using H & E, nuclei and other basophilic structures stain blue, whereas 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.

Autologous Stem Cell Transplantation

In ASCT with human patients, an individual receives his or her own stem cells by infusion. Generally, 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. This permits an otherwise lethal debulking regimen (e.g. HDT) to be employed, i.e., chemotherapy or radiotherapy that severely damages or destroys the patient's bone marrow. Following the debulking procedure, 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. Methods for performing apheresis with the CS 3000.RTM. Plus machine are described in Willams et al. (1990) Bone Marrow Transplantation 5:129-33 and Hillyer et al. (1993) Transfusion 33:316-21. Stem cells collected from the peripheral blood are termed herein "peripheral blood stem cells" (PBSC).

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. Once the stem cells are collected and cryopreserved, 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. Approximately 48 hours after the chemotherapy is completed 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. During this time, 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. During this time, 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. 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.

'Day 15" post-ASCT Blood Samples

The present invention relates to 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/μL, preferably an ALC of >500 lymphocytes/μL. 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. Within fifteen days following the transplant, a blood sample is taken from the cancer patient and an ALC is determined using one or more methods described hereinabove.

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. At a WBC of greater that 10 cells/nL, 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. The cell suspension is then mixed with 100 mL minimal essential medium (MEM-S; Life Technologies, Rockville, MD) supplemented with 20 % dimethylsulfoxide (DMSO). 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 Deutschland 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. At 24 to 48 hours following 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. Within fifteen days following the transplant, 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.

In a further embodiment, 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. Such 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 10⁶ CD34+ cells/kg, whereas optimal stem cell yields are considered to be > 5 xlO⁶ CD34+ cells/kg as this results in faster hematopoietic recovery (Bensinger et al. (1994) Br JHaematol 87:825λ Despite various methods of PBSC mobilization, sometimes, adequate numbers of PBSCs for ASCT are not collected from some patients. In these patients, BM harvest or a second attempt at PBSC mobilization can be performed. Alternatively, these patients are excluded from proceeding to autografting. While there are a number of cytokines alone or in combination that can be used to mobilize PBSCs in adequate numbers, the use of 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. The combination of Flt-3 ligand and GM- CSF results in improved mobilization of PBSCs in patients with breast cancer, ovarian cancer, and lymphoma (Chao et al. (1999) Blood). In a randomized study of 71 NHL or ovarian cancer patients using (i) G-CSF alone at standard dose of 10 μg/kg/day, (ii) G- CSF (10 μg/kg/day) + Flt-3 ligand (50 μg/kg/day for the first 3 days), or (iii) GM-CSF (5 μg/kg/day) + Flt-3 ligand (50 μg/kg/day for the first 3 days), more patients in the Flt-3 ligand groups reached the minimum target of 1 x 10⁶ CD34+ cells/kg and the threshold of 2-3 x 10⁶ CD34+ cells/kg. A target of 2 x 10⁶ 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 10⁶ 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 xlO⁶ CD34+ cells/kg in < 3 apheresis and 75 % in < 4 apheresis. In the Flt-3 + G-CSF arm, approximately

48 % achieved the overall goal of 2.5 xlO⁶ CD34+ cells/kg in < 3 or 4 apheresis, while in the G-CSF alone arm, approximately 42 % and 46 % achieved the overall goal of 2.5 xlO⁶ CD34+ cells/kg in < 3 and < 4 apheresis, respectively (Stiff et al. (1999) Blood 93:1858;. Overall, Flt-3 ligand in combination with GM-CSF or G-CSF was well tolerated and side effects consisted of local braising and pain at the injection site, mild myalgias, arthralgias, fever, and headache. These side effects are similar to those noted with GM-CSF or G-CSF alone.

Accordingly, 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 μg/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 μg/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 μg/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 2xl0⁶ 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. In a further embodiment, 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. In a preferred embodiment, the immune cells are NK cells. In a still further preferred embodiment, 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 lxlO⁵ to lxlO⁷ units/m², 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. In a preferred embodiment, 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/μL measured in a blood sample obtained within 15 days following ASCT are between at least 30 to 40 cells/μL, preferably 45-60 cells/μL, more preferably 60 to 80 cells/μL, and still more preferably 75-100 cells/μL. In preferred embodiments, the NK cell count comprises 30, 40, 45, 60, 75, 80, or 100 NK cells/μL. o

These NK cell counts are indicative of patient survival of at least 24 months.

Preferably, 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.

Measuring Survival

In one embodiment, 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/μL, preferably, > 500 lymphocytes/μL 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. Similar to the methods described above, 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. Within 15 days following ASCT, a sample of the patient's blood may be taken to obtain an ALC.

In one embodiment, 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 μg/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 μg/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 μg/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 2xl0⁶ CD34+ stem cells/kg.

In a further embodiment, 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. In a preferred embodiment, the immune cells are NK cells. In a further preferred embodiment, 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 lxlO⁵ to lxlO⁷ units/m², 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.

Within fifteen days following the ASCT, where day 1 is the day immediately following the completion of the stem cell transplant, a blood sample can be taken from the patient and used to obtain an ALC. In one embodiment of the present invention, the ALC of the blood sample may be used as a prognostic indicator of the patients' survival. In a further embodiment of the invention, an ALC of > 200 lymphocytes/μL is indicative of survival of the cancer patient for at least 24 months following ASCT. In other preferred embodiments, a "day 15" ALC of > 300 lymphocytes/μL, preferably > 400 lymphocytes/μL, and preferably > 500 lymphocytes/μL, is indicative of survival of the cancer patient for at least 24 months following ASCT. In a preferred embodiment, 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/μL measured in a blood sample obtained within 15 days following ASCT are between at least 30 to 40 cells/μL, preferably 45 to 60 cells/μL, more preferably 60 to 80 cells/μL, and still more preferably 75 to 100 cells/μL. In preferred embodiments, the NK cell count comprises > 30, 40, 45, 60, 75, 80, or 100 NK cells/μL. These NK cell counts are indicative of patient survival for at least 24 months. Preferably, 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.

Without being bound by any one theory, a potential explanation for the survival advantage associated with ALC recovery post-ASCT i_{n cancer pa}tients is the possibility that early immune-reconstitution may have a protective effect against residual disease progression. This is analogous to the graft versus tumor effect in autologous bone marrow transplant recipients where the donor immune system is responsible for eradication of residual disease in the host. While reappearance of neutrophils and platelets is often considered the endpoint of hematologic recovery after ASCT, immunological reconstitution is a gradual process that may not be completed until months to years post-transplantation. The relative and absolute numbers of circulating mature B cells expressing CD 19 and/or CD20 are decreased between 3 to 18 months after ASCT (see Bengtsson et al. (1989) LeukRes 13:791; Pan-ado et al. (1997) Hematol Cell Ther 39:301; Storek and Saxon (1992) Bone Marrow Transplantation 9:395λ Deficient B-cell functional recovery is attributed to both decreased T-cell help and intrinsic B-cell defects (Witherspoon et al. (1982) Blood 59:844; Nadler et al. (1984) Lancet 2:427). T cell-dependent antibody responses to recall antigens (e.g. tetanus or diphtheria toxoid) can be elicited late (>1 year) but not early post-transplant (Baumgartner et al. (1988) E/oo 71:1211; Ljungman et α/. (1989) J fnfect Dis 159:610). 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). The relative and absolute numbers of 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)

Transplantation 46: 57; Sugita et al. (1994) Transplantation 57:1465). Miller et al. (1991) Blood 77:1845 showed that the frequency of mitogen responsive T-cells in PB, including cytokine-secreting helper T-cells, IL-2 responding T-cells, and cytotoxic T- cells remain low for up to five years after ASCT. Even the addition of exogenous IL-2 was shown to partially compensate for abnormal T-cell proliferative responses (Cayeux et al. (1989) Blood 74:2278). The first year post-ASCT is a critical time in which most MM or NHL relapses occur (Lowenberg et al. (1990) J Clin Oncol 8:287).

Studies of immuno-reconstitution post-ASCT have shown that 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 moφhologically 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). Divine et al. (1999) Br JHaematol 105 :349 showed complete recovery of absolute NK cell counts within six weeks post-ASCT. Our own data demonstrate normal absolute NK cell counts at two weeks post-ASCT (Porrata et al. (2000) Proc ASC0 19:61a). Talmadge et al. (1997) Bone Marrow Transplantation 19:161 demonstrated normal NK cell activity early post-ASCT. These studies confirm that recovery of NK cell activity after ASCT occurs sooner than B or T-cell recovery.

Determining Drug Efficacy

In one embodiment, 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/μL is indicative of the efficacy of the drag, hi preferred embodiments, the ALC is >300 lymphocytes/μL, preferably > 400 lymphocytes/μL, and preferably > 500 lymphocytes/μL, and is, thus indicative of the efficacy of the drag. In one embodiment, 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, Idarabicin, Ifosfamide, Interferon, Irinotecan, Isoniszid, L- asparaginase, Mechlorethamine, Melphalan, Mercaptopraine, Methotrexate, Methyl- CCNU, Mithramycin, Mitomycin-C, Mitotane, Mitoxantrone, Nitrogen Mustard, Nitrosoureas, Paclitaxel, Procarbazine, Saquinavir, Streptozocin, Tamoxifen, Temozolamide, Teniposide, Thiotepa, Topotecan, Trimetrexate, Valrabicin, Vinblastine, Nincristine, and Ninorelbine. In a preferred embodiment, 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 ΝK 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, ΝK cells, and dendritic cells are expanded in vivo. In addition, Flt-3 ligand has also induced tumor regression in animal models of melanoma and lymphoma (Lynch et al. (1991) Nat Med 3:625).

Administration of drags to treat a cancer patient may be performed according to the guidelines for dosage and administration for a particular drag, or combination of drags known to those of skill in the art, and/or available though publications such as the Physician's Desk Reference (53^rd Ed. (1999) Medical Economics Company, Inc., Montvale, ΝJ). For example, GM-CSF may be administered at a concentration of at least 5 μg/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 μg/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 μg/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 2xl0⁶ CD34+ stem cells/kg.

In a further embodiment, 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. In a preferred embodiment, the immune cells are NK cells. In a further preferred embodiment, 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 lxlO⁵ to lx 10⁷ units/m², 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.

Within fifteen days after ASCT, 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/μL is indicative of the efficacy of the drags of (a), (b), and/or (c).

In a preferred embodiment, 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/μL determined for a blood sample obtained within 15 days following ASCT, indicative of drag efficacy, can be at least 30 to 40 cells/μL, preferably 45 to 60 cells/ μL, more preferably 60 to 80 cells/μL, and still more preferably 75 to cells/μL. In preferred embodiments, the NK cell count comprises 30, 40, 45, 60, 75, 80, or 100 cells/μL. Preferably, 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

In a further embodiment, 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/μL is indicative of an improvement in the cancer therapy. In a further preferred embodiment, an ALC of ≥ 500 lymphocytes/μL 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.

In one embodiment, 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. For example, 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. In a further embodiment, Flt-3 ligand and GM-CSF are administered alone before harvest of PBSCs in an effort to improve cancer therapy. Subsequently, 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.

In a further embodiment, 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. In a preferred embodiment, the immune cells are NK cells. In a further preferred embodiment, 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 0⁵ to lxlO⁷ units of activity/m², 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.

Within fifteen days following the transplant of harvested stem cells to the cancer patient, a sample of blood is taken from the patient and the ALC is determined. An ALC of : 200 lymphocytes/μL is indicative of an improvement in the cancer therapy. In preferred embodiments, the ALC is > 300 lymphocytes/μL, preferably ≥ 400 lymphocytes/μL, and preferably > 500 lymphocytes/μL, and is, thus, indicative of an improvement in cancer therapy.

In a preferred embodiment, 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/μL 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/μL, preferably 45 to 60 cells/μL, more preferably 60 to 80 cells/μL, and still more preferably 75 to 100 cells/μL. In preferred embodiments, the NK cell count comprises >; 30, 40, 45, 60, 75, 80, or 100 cells/μL.

Preferably, 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.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

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. The eligibility criteria for ASCT included adequate cardiac, hepatic, pulmonary, and renal functions; Eastern Cooperative Group (ECOG) performance status of 0, 1, or 2; and no active infection. Patients were excluded if they did not meet the eligible criteria or have other concomitant illness that would preclude ASCT.

Data from transplant patients were collected prospectively and entered into a computerized database. Transplant patients were continuously assessed for their response to therapy, relapse, and survival. All patients gave written, informed consent allowing utilization of their medical records for medical research. Approval for the retrospective review of these records was obtained from the Mayo Clinic Institutional Review Board and was in accordance with US federal regulations and the Declaration of Helsinki.

Example 2 - Conditioning Regimens Conditioning regimens for MM patients were the following. One hundred and one patients were treated with melphalan (140 mg/m²) and total body irradiation (TBI) (12 Gy). Eleven patients were treated with melphalan (140 mg/m²), cyclophosphamide (60 mg/m²), and TBI (12 Gy). Eleven patients were treated with melphalan (200 mg/m²). 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/m²).

Conditioning regimens for NHL patients were the following. Seven patients were treated with cyclophosphamide (1.5 g/m²), carmustine (BCNU) (300 mg/m²), and etoposide (125 mg/m²). Thirty-one patients were treated with cyclophosphamide (60 mg/m²) and TBI (12 Gy). Sixty-six patients were treated with BCNU (300 mg/m²), etoposide (NP-16: 100 mg/m²), cytarabine (ARA-C: 100 mg/m²), 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/m²), BCΝU (300 mg/m²), NP-16 (125 mg/m²) (CBV). Fifteen patients were treated with BCΝU (300 mg/m²), etoposide (100 mg/m²), ARA-C (100 mg/m²), 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/m²) 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/m²/d), carboplatin (200 mg/m²/d), and thiotepa (125 mg/m²/d) (STAMP N).

All patients had stem cells re-infusion after high dose therapy. Hematologic engraftment was defined at the first day of an absolute neutrophil count (AΝC) > 500 cells/μL.

Example 3 - Hematological Engraftment Neutrophil engraftment was taken to be the first day of three consecutive days in which ANC > 500 cells/μL was achieved. Platelet engraftment was defined as a platelet count > 20 x 10⁹/L, independent of transfusion support. ALC threshold (immunologic engraftment) was determined at 500 cells/μL at day 15 after ASCT.

Example 4 - Prognostic Factors For MM patients, prognostic factors included age (> 50 years), β2-microglobulin (β2-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/μL), absolute lymphocyte count (ALC) at day 15 post-ASCT, stem cell source (BM vs PBSC), platelet count at day 15 post-ASCT (> 20 x 10⁹/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).

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). In addition, the number of pre-transplant treatments, stem cell source (BM versus PBSC), ANC at day 15 post-ASCT (> 500 cells/μL), ALC at day 15 post-ASCT (> 500 cells/μL), platelet count at day 15 (> 20 x 10⁹/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. Data did not show any correlation between specific cut points and outcome for these variables. Prognostic factors for Hodgkin's lymphoma patients were selected from the Hasenclever index (Hasenclever et al. (1998) New EnglJ Med 339(21):1506-1514).

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 at diagnosis included: age (> 45 years old); adjuvant radiation therapy; albumin (< 4 g/dL); alkaline phosphatase (> normal for age/sex); B-symptoms (weight loss > 10 % of body weight in 6 months, unexplained fever of > 38 °C, or night sweats); disease stage at diagnosis (< IN); gender; hemoglobin (< 10.5 g/dL); histology; LDH (> normal for age/sex); leukocytosis (while blood cell (WBC) count >15 x 10⁹/L); lymphopenia (< 600 cells/μL); presence of mediastinal mass; sedimentation rate; and splenectomy. 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 10⁹/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, while 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. 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. In patients with MM, 21 received BM stem cells, while 105 received PBSC. In patients with NHL, 49 received BM stem cells, 53 received PBSC, and two received both. In patients with Hodgkin's lymphoma, 57 received unmobilized BM stem cells, 21 received PBSC mobilized with G-CSF, three received PBSC mobilized with GM-CSF, and one received stem cells from both sources (BM and PBSC). In patients with AML, 25 received unmobilized BM stem cells, 20 received PBSC mobilized with ARA-C and G-CSF, and three patients received stem cells from both sources. Fourteen patients with metastatic breast cancer participated in a Phase II stem cell mobilization study with Flt-3 ligand. Eleven patients received Flt-3 ligand in combination with G-CSF or GM-CSF.

Patients unable to mobilize adequate peripheral CD34 cells (2 x 10⁶/kg) underwent BM harvest.

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.

Among 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.

Among Hodgkin's disease patients, 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.

Example 7 — Response and Survival For MM 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.

For NHL patients, 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) peφendicular 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 peφendicular 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.

For AML patients, CR was defined as (a) normal BM moφhology 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/μL and platelets > 100,000/μL 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) peφendicular 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 peφendicular 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.

For metastatic breast cancer patients, CR was defined as no evidence of disease, while PR was defined as a reduction of at least 50 % of the product of peφendicular diameters. Progression of disease was defined as a greater than 25 % increase in the product of peφendicular diameters or new lesions.

Overall survival (OS) time was measured from the date of transplantation to the date of death or last follow-up. Progression-free survival (PFS) was defined as time from transplantation to disease progression, relapse, or death. Leukemia- free survival (LFS) 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. For analysis involving the entire cohort, 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/ μL was based on the median of ALC for the cohort group. The cut-point of 500 cells/μL yielded the greatest differential in survival as determined from χ² values analyzed at different cut-points (e.g. 200 to 900 cells/μL) from log-rank tests. The χ and Fisher's exact tests were used to determine relationshrps between nominal variables including: ALC > 500 cells/μL, ANC > 500 cells/μL, and platelet count > 20 x 10⁹/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/μL) versus low (< 500 cells/μL) 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.

In the MM group, 63 patients had ALC > 500 cells/μL with a mean value of 1100 ± 600 cells/μL (95 % Cl, 938-1240 cells/μL) and a median of 870 cells/μL (range 500- 3100 cells/μL). Of these, six patients had ALC > 2000 cells/μL. For the 63 patients with ALC < 500 cells/μL, the mean value was 278 ± 117 cells/μL (95 % Cl, 250-307 cells/μL) and the median value was 292 cells/μL (range 50-472 cells/μL). Three patients had ALC < 100 cells/μL.

All NHL patients had intermediate-grade histology. Fifty-six patients had ALC > 500 cells/μL with a mean value of 857 ± 219 cells/μL (95 % Cl, 799-917 cells) and a median value of 635 cells/μL (range 510-4440 cells/μL). Of these, one patient had ALC of 4440 cells/μL. For the 48 patients with ALC < 500 cells/μL, the mean value was 240 ± 130 cells/μL (95 % Cl, 203-279 cells/μL) and the median value was 230 cells/μL (range 20-490 cells/μL). Four NHL patients had ALC < 100 cells/μL.

In the group of patients with Hodgkin's disease, 41 had ALC > 500 cells/μL on day 15 with a median ALC of 710 cells/μL (range 500-2500 cells/μL), and 41 had ALC < 500 cells/μL with a median ALC of 240 cells/μL (range 80-480 cells/μL). The median ANC and platelet count at day 15 post-ASCT were 1.1 x 10⁹/L (range 0.04-9.5 x 10⁹/L) and 31.5 x 10⁹/L (range 3-276 x 10⁹/L), respectively. Seventy-three patients achieved an ANC > 500 cells/μL at day 15, and 74 patients achieved an untransfused platelet count > 20 x 10⁹/L at day 15 post-ASCT.

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). Six patients received radiation as the initial therapy after diagnosis; 25 patients received ABND (adriamycin, bleomycin, vinblastin, and dacarbazine); 12 patients received MOPP (nitrogen mustard, vinblastin, prednisone, procarbazine); 4 patients received BCNPP (BCΝU, cytoxan, vincristine, procarbazine, and prednisone); 2 patients received Stanford N (doxorabicin, vinblastine, vincristine, bleomycin, mustard, VP-16, and prednisone); 32 patients received hybrid chemotherapy combinations (28 MOPP/ABN and 4 COPP/ABND); and one patient participated in the CCG-5932 study and received a combination chemotherapy consisting of cyclophosphamide, vincristine, vinblastine, procarbazine, prednisone, adriamycin, and bleomycin. Sixteen patients relapsed within the field of radiation. Sixty-four patients received salvage chemotherapy.

In the AML group, 23 patients had ALC > 500 cells/μL on day 15 with a median ALC of 760 cells/μL (range 520-1980 cells/μL), and 22 patients had ALC < 500 cells/μL with a median ALC of 125 cells/μL (range 10-410 cells/μL). The median AΝC and platelet count at day 15 post-ASCT were 0.6 x 10⁹/L (range 0.05-5.4 x 10⁹/L) and 20 x 10⁹/L (range 3-64 x 10⁹/L), respectively. Twenty-nine patients achieved an AΝC > 500 cells/μL at day 15, and 24 patients achieved untransfused platelet count > 20 x 10⁹/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). Of the 45 AML patients, 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).

Collectively, for the 386 patients with MM, NHL, Hodgkin's lymphoma, AML, and metastatic breast cancer, 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 had a median age of 49 years (range 12-75) at transplantation. Of the 386 patients, only five (all AML patients) received a purged stem cell product, while none received CD34-selected stem cells. Of the 386 patients, 204 had ALC > 500 cells/μL with a median of 740 cells/μL (range 500-4400 cells/μL), and 182 had ALC < 500 cells/μL with a median of 240 cells/μL (range 10-490 cells/μL). Base line characteristics of all 386 patients are summarized in Table 6.

Of the 118 patients in which CD34 counts were available (110 PB, and 8 BM), the median CD34 count was 3.8 x 10⁶/kg (range: 0.8 - 85 x 10⁶/kg). The MNC count, available in 220 patients (113 PB and 107 BM), had a median of 7.2 x 10⁸/kg (range: 0.5- 22 x 10⁸/kg). Of the 48 patients in which NCC was available (47 PB and 1 BM), the median nucleated cell count was 2.5 x 10⁸/kg (0.7-4.4 x 10⁸/kg).

Table 1. Characteristics of multiple myeloma patients (n=126)

Multiple myeloma (N=126)

Characteristics Age (yr)^* 55 (range, 33-71) Sex (%) Male 65 Female 35

Disease status at transplant (%) Plateau response 13.5 Relapsed on chemotherapy 33.3 Relapsed off chemotherapy 38.1 Primary chemo-refractory 15.1 Prognostic factors for MM (%) 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 l0⁹/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

Years express as the median of the group. Table 2. Base-line characteristics of non-Hodgkin's lymphoma patients (n = 104) non-Hodgkin's lymphoma (N=104) Characteristics

Age (yr)^* 51 (range, 14-73) Sex (%)

Male 60.6

Female 39.4 Histology (%) [REAL classification]

Diffuse large cell 50

Follicular Large cell 28

Peripheral T-cell 8

Anaplastic T-cell 6

Angiocentric T cell 3

Angiocentric B cell 1

NK cell 1 Disease status at transplant (%) First relapse 7.7

Progression 0.9

Partial response 68.3

Complete response 23.1 Prognostic factors for NHL (%) Age>60 32

LDH>normal for age/sex 57.7

Performance status>2 7.7

Extra-nodal sites>2 6.7

Stage III/IV disease 66 Stem cell source (%)

BM 47.1

PBSC 50.9

Both 2

Platelets > 20 x 10⁹/l (%) 71.1

ALC at day 15 post-ASCT (%)

>500 cells/ul 53.8

<500 cells/ul 46.2 ANC at day 15 post-ASCT (%) >500 cells/ul 84

<500 cells/ul 16 Years express as the median of the group Table 3. Characteristics of Hodgkin's disease patients at diagnosis (n = 82)

No. of Patients

Characteristic (N = 82) % of Patients

Sex Male 51 62

Female 31 38

Age, years

Median 28

Range 11-56

Age, years

>45 7 9

<45 75 91

Alkaline phosphatase U/L (adjusted for age/age) Abnormal 29 ^* 35

Normal 53 65

Ann Arbor stage

I 6 7

II 32 39

III 26 32

IV 18 22

Histology

Nodular sclerosis 68 83

Mixed cellularity 4 5 Lymphocyte predominant 4 5 Lymphocyte depleted 3 4 Interfollicular 1 1

Not otherwise specified 2 2

Adjuvant radiation Yes 41 50

No 41 50

Albumin g/dl (N = 53) <4 20 38

>4 33 62

B symptoms Yes 45 55

No 37 45

Bone marrow involvement 5 6

Bulky disease (>10 cm) Mediastinum 56 68

Abdomen 1 1

None 25 31

Duration of response after initial chemotherapy <12 months 43 52

>12 months 39 48 Diagnosis to AHSCT, months

Median 26

Range 5-330

>18months 61 74

< or = 18 months 21 26

Extranodal involvement

Bone 1

Liver 1

Lung 9

Lung + bone 1

Lung + liver 1

Hemoglobin g/dl (N=53)

<10.5 13 25

>10.5 40 75

Lymphocyte count x 10⁹/1 (N=53)

<600 cells/ l 10 19

>600 cells/ tl 43 81

White blood cell count x 10⁹/1 (N=53)

>15 5 9

< or =15 48 91

Lactate dehydrogenase U/l

(adjusted for sex/age) (N=53)

Abnormal 14 26

Normal 39 74

Number of chemotherapy regimens before ASCT

1 11 13

2 42 51

3 25 32

4 2 2

5 2 2

Initial chemotherapy failure 11 13

Sedimentation rate (N=53)

(adjusted for sex/age)

Abnormal 22 42

Normal 31 58

Splenectomy

Yes 14 17

No 68 83 Abbreviation: ASCT,- Autologous Stem Cell Transplantation Table 4. Characteristics of Hodgkin's lymphoma patients at transplantation (n = 82)

No of Patients Characteristic (N=82) % of Patients

Age, years

Median 32

Range 12-57

Disease status

C Coommpplleettee rreemmiissssiioonn 2^nd CR CRu 11 13.4

3^rdCR/CRu 2 2.4

4^thCR/CRu 2 2.4

Resistant disease

Primary resistant 1 1.2 1^st relapse 6 7.4 2^nd relapse 1 1.2 Sensitive disease

11^sstt PPRR 3366 43.9

2^nd PR 9 10.9

3^rd PR 1 1.2

4^th PR 1 1.2

1^st relapse untreated 5 6.2 22^nndα rreellaappssee uunnttrreeaatteedd 55 6.2 3^rd relapse untreated 2 2.4 Ann Arbor Stage

I 19 23

II 18 22

1155 18

IN 30 37

B symptoms 3 4

Bone marrow involvement 4 5

Bulky disease (> 10 cm) MMeeddiiaassttiinnuumm 1133 16

Abdomen 2 1

Pelvis 1 1

Consolidation radiation

Yes 32 39

NNoo 5500 61

ECOG performance status

0 53 65

1 28 34

2 1 1

Extra nodal disease

Bone 4 Liver

Lung

Thyroid

Breast + pleural space

Lung + adrenal

Lung + bone

Lung + liver Stem cell source

BM 55 67

PBSC 27 33 Year of transplantation

< or =1994 42 51

>1994 40 49

Abbreviations: BM, bone marrow; ECOG, Eastern Cooperative Oncology Group; PBSC, peripheral blood stem cell.

Table 5. Characteristics of acute myelogenous leukemia patients at transplantation (N = 45) Characteristics Number of Patients Percentage

Sex

Female 32 71

Male 13 29

FAB classification MO 4 9

Ml 7 16

M2 10 22

M3 6 13

M4 11 24 M5 5 11

M6 2 5

Cytogenetics (N = 38)

Favorable 10 26

Intermediate 14 37 Unfavorable 14 37

Clinical Status before transplantation

First CR 13 29

First relapse PR 1 2

First relapse untreated 10 22 Second CR 20 45

Second PR 1 2

ECOG performance status

Before transplantation

0 35 78 1 10 22

Stem cells

CD34 20 45

Mononuclear cells 25 55

Stem cell source BM 25 55

PBSC 20 45

WBC at diagnosis (x 10⁹/1)

>10.5 19 42

< or = 10.5 26 58 Table 6. Characteristics of ASCT Patients

Characteristics Number of patients (N = 386)

Sex Female 177

Male 209 Malignancy

Multiple myeloma 126 Non-Hodgkin' s lymphoma 104

Hodgkin's disease 82

Acute myeloid leukemia 45

Metastatic breast cancer 29 Stem Cell Source Bone Marrow 160

Peripheral Blood 223

Both 3 Stem Cell Infused

CD34 118 Mononuclear cells 220

Nucleated cells 48 ALC at day 15 post-transplantation

> 500 cells/μl 204 < 500 cells/μl 182 ANC at day 15 post-transplantation

> 500 cells/μl 311 < 500 cells/μl 75

Platelet count at day 15 post-transplantation

> 20,000 cells/μl 321 < 20,000 cells/μl 65

Example 10 — Relationships Among Prognostic Factors Examined In the MM patient group, there was no association between ALC and the number of pre- transplant chemotherapy regimen (p = 0.76), the type of conditioning regimen (p = 0.7852), or the post- transplant ANC (p = 0.17). Furthermore, no association was observed between OS, or ALC, and CD34 cell count (N = 57, p = 0.41), MNC count (N = 122, p = 0.86), or NCC (N = 48, p = 0.78) values. The CD34 cell count, MNC count, and NCC were continuous variables in the analysis. ALC was not significantly related to other prognostic factors tested with the exception of a correlation between ALC and LDH (p = 0.03). ALC was not significantly related to the clinical status in the MM group prior to transplant (p = 0.01). In contrast, both relapse on therapy and relapse off therapy correlated with ALC in MM patient (p = 0.014 and p = 0.017, respectively).

In the NHL patient group, there was no association between the ALC and the number of pre-transplant chemotherapy regimens (p = 0.84), the type of conditioning regimen (p = 0.2223), or the post- transplant ANC (p = 0.54). No correlation was observed between OS, or ALC, and CD34 cell counts (N = 57, p = 0.41), MNC count (N = 122, p = 0.86), and NCC (N = 48, p = 0.78) values. The CD34, MNC count, and NCC were continuous variables in the analysis. ALC was not related to the other prognostic factors tested with the exception of a correlation between ALC and LDH (p = 0.03).

In the group of patients with Hodgkin's disease, there was no association between ALC and the type of conditioning regimen (p = 0.34), the number of pre-transplant chemotherapy regimens (p = 0.14), and the number of pre-transplant treatment cycles (p = 0.26). No correlation was observed between ALC recovery and ANC at day 15 post- ASCT (p = 0.07), platelet count at day 15 post-ASCT (p = 0.45), growth factor post- ASCT (G-CSF or GM-CSF) (p = 0.54), or stem cell source (BM or PBSC) (p = 0.15). In the 27 patients in whom CD34 counts were available (23 PBSC, and 4 BM), the median number of CD34 positive cell infused was 2.9 x 10⁶/kg (range: 2.0-6.2 x 10⁶/kg). The MNC count, available in 55 patients (2 PBSC and 53 BM), had a median of 2.1 x 10⁸/kg (range: 1.1-8.6 x 10⁸/kg). CD34 cell count (N = 27, p = 0.38) and MNC count (N = 55, p = 0.44) were not significant predictors of ALC recovery.

In the group of patients with AML, there was no association between ALC and the type of conditioning regimen (p = 0.92), the number of pre-transplant chemotherapy regimens (p = 0.74), and the number of pre-transplant treatment cycles (p = 0.50). Furthermore, no correlation was observed between ALC and CD34 cell count (N = 20, p = 0.33) or ALC and MNC count (N = 25, p = 0.47). Of the 20 patients in which CD34 counts were available (19 PBSC, and 1 BM), the median number of CD34 positive cells infused was 4.7 x 10⁶/kg (range: 2.1-6.2 x 10⁶/kg). MNC counts, available in 25 patients (1 PBSC and 24 BM), had a median of 2.0 x 10⁸/kg (range: 0.5-9.2 x 10⁸/kg). There was no association between ALC and ANC at day 15 post-ASCT (p = 0.54), platelet count at day 15 post- ASCT (p = 0.77), or stem cell source (BM or PBSC) (p = 0.14). In summary, there was no association between ALC at day 15 after ASCT and the number of chemotherapy regimens (p = 0.18) or between ALC at day 15 after ASCT and the number of the treatment cycles (p = 0.35) before transplantation. No correlation was identified between the number of chemotherapy regimens, or the number of treatment cycles before transplantation, and ALC at day 15 after ASCT for each disease in the study. In addition, there was no association between ALC recovery at day 15 after ASCT and (i) ANC at day 15 (median: 1.3 x 10⁹/L; range: 0.02-18.8 x 10⁹/L) after transplantation (p = 0.52), (ii) platelet count at day 15 (median: 35 x 10⁹/L; range: 3-315 x 10⁹/L) after transplantation (p = 0.58), (iii) growth factor or no growth factor after transplantation (p = 0.08), (iv) G-CSF versus GM-CSF after ASCT (p = 0.35), or (v) stem cell source (BM or PB) (p = 0.09). CD34 cell count (N = 110, p = 0.41), MNC count (N = 220, p = 0.57), and NCC (N = 48, p = 0.52) were not significant predictors of ALC recovery.

Example 11 - Survival

At the end of the study period, 54 deaths had occurred among the 126 MM patients; 54 deaths had occurred among the 104 NHL patients; 34 deaths had occurred among the 82 patients with Hodgkin's disease; 20 deaths had occurred among the 45 AML patients; and 18 deaths had occurred among the 29 metastatic breast cancer patients.

In the MM group, 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. There was no significant difference in supportive care, which included antibiotics, growth factors, or transfusion requirements. No correlation was identified between the year of transplantation and transplant related mortality (p < 0.12). None of the patients developed autologous graft versus host disease. The median follow-up time for all patients was 12.5 months with a maximum of 123 months.

In the NHL group, 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.

In the group of patients with Hodgkin's disease, 26 patients died from recurrent or progressive disease, while 8 died of transplant related complications (transplant-related mortality was 10 % (8/82)). Four patients died of post-transplant secondary acute leukemia. One patient died of pulmonary hemorrhage in the setting of post-transplant secondary myelodysplastic syndrome, while one patient died of diffuse alveolar hemorrhage after transplantation. One patient died of acute respiratory distress syndrome, and one patient died of septic shock due to Streptococcus viridans. Within 100 days following post-ASCT, 62 patients achieved CR/CRu, 6 patients achieved PR, 3 patients achieved stable disease, and 11 patients had evidence of progression of disease. There were 7 deaths in the 41 patients with ALC > 500 cells/μL, and 27 deaths in the 41 patients with ALC < 500 cells/μL. Of the transplant-related mortality, one patient had ALC > 500 cells/μL, and seven patients had ALC < 500 cells/μL. Twenty-seven patients had developed progressive Hodgkin's disease but remained alive (22 patients with ALC > 500 cells/μL and 5 patients with ALC < 500 cells/μL). Twenty-one patients remained alive without progression of disease (14 patients with ALC > 500 cells/μL and 7 patients with ALC < 500 cells/μL). None of the patients developed clinically evident autologous GVHD. All patients were followed with a median follow-up of 22 months and a maximum of 159 months. In the AML group, 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/μL, and one patient had ALC < 500 cells/μL. There were six deaths in the 23 patients with ALC > 500 cells/μL and 14 deaths in the 22 patients with ALC < 500 cells/μL. Three patients had a relapse of leukemia but remained alive (one patient with ALC > 500 cells/ μL and two patients with ALC < 500 cells/μL). Twenty-two patients did not have relapse leukemia (sixteen patients with ALC > 500 cells/μL and six patients with ALC < 500 cells/μL). None ofthe patients developed clinically evident autologous GVHD. All patients were followed with a median follow-up of 14 months and a maximum of 129 months. The median OS and LFS were significantly greater for patients with ALC > 500 cells/μL versus ALC < 500 cells/μL (not reached versus 10 months, p < 0.0009; 105 versus 9 months, p < 0.0008, respectively). For the group of patients with metastatic breast cancer, 17 patients died with progression. Of patients who were alive at the end ofthe study, a few had experienced cancer progression while most did not show progression. Patients were followed for a minimum of 7 months or until death. The median follow-up was 15 months.

Collectively, 170 patients died from recurrent or progressive disease. The transplant-related mortality was 9 % (36/387). Ofthe 36 transplant-related mortality cases, 20 patients had ALC > 500 cells/μL and 16 had ALC < 500 cells/μL. There were 68 deaths in the 204 patients with ALC > 500 cells/μL, and 102 deaths in the 182 patients with ALC < 500 cells/μL. Fifty-eight patients had developed progressive disease but remained alive (43 patients with ALC > 500 cells/μL and 15 patients with ALC < 500 cells/μL). One hundred and twenty-two patients remained alive without progression of disease (93 patients with ALC > 500 cells/μL and 29 patients with ALC < 500 cells/μL). None ofthe patients developed clinically evident autologous GNHD. Table 7 summarizes the OS and PFS for each disease in the study. Table 7. Median Overall and Progression-Free Survival For Each Malignancy According to Absolute Lymphocyte Count

Disease Absolute Overall Progression- Lymphocyte Survival Free Survival Count (months) (months)

Multiple > 500 cells/μL 46 < 0.0001 17 < 0.0001 Myeloma (N = 63) (N = 126) < 500 cells/μL 12 8 (N = 63)

non-Hodgkin's > 500 cells/μL N O.0001 NR O.OOOl lymphoma (N = 56)

(N = 104) < 500 cells/μL 6 4 (N = 48)

Hodgkin's > 500 cells/μL NR 0.0001 NR 0.0001 Disease (N = 41)

(N = 82) < 500 cells/μL 29 14 (N = 41)

Acute Myeloid > 500 cells/μL NR <0.0009 105 .0008 Leukemia (N = 23) (N = 45) < 500 cells/μL 10 9 (N = 22)

Metastatic > 500 cells/μL NR <0.0006 24 .0015 Breast Cancer (N = 20) (N = 29) < 500 cells/μL 14 7 (N = 9)

Abbreviation: NR, not reached

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.

For MM patients, age, β 2M, CRP, and ANC were not predictive of OS or PFS based on univariate analysis (see Table 8). In contrast, ALC, stem cell source, circulating plasma cells, platelet count > 20 x 10⁹/L, PCLI, LDH, the number of pre-transplant chemotherapy regimen, clinical status prior to transplantation, and abnormal cytogenetics were significant predictors of OS and PFS. The relationship between the patients' clinical status prior to transplantation also was examined. Table 8. Univariate Comparisons for Multiple : Myeloma i Patients: Overall and PFS Rates

Overall Survival PFS

Prognostic Factors at Relative Relative Time of Transplant Risk 95 % Cl P Risk 95 % Cl P

Abnormal Cytogenetics .39 .23-.68 .001 .44 .27-.70 <0007 vs normal

Age>50 years 1.03 .6-1.8 .91 1.04 .65-1.67 .86 vs <50 years

ALC 500 cells/μl .24 .14-.42 <.0001 .34 .21-.55 <.0001 vs < 500 cells/ μl ANC > 500 cells/μl .40 12-1.33. 14 .37 .13-1.04 .06 vs < 500 celh/μl B-2M > 2.7mg/l .58 .33-1.01 .054 .7 .44-1.11 .13 vs ≤ 2.7 mg/1 Bone marrow

Plasma cell >40% 49 .3-.8 .007 .55 .35-.87 .01 vs < 40% Circulating

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 l0⁹/1 .18 .09-.36 <.0001 .3 .16-.57 .00024 vs ≤ 20 x l0⁹/l # of pre-transplant chemotherapy regimens 1.58 1.05-2.38 .028 1.7 1.18-2.44 .004 Primary refractory

Disease NA^' 2.94 .63-13.7 .17 vs plateau/response

Relapse off Therapy 1.16^** .5-2.7 .73 5.99 1.43-25.1 .014 vs plateau/response Relapse on Therapy 3.08^** 1.4-6.76 .005 13.94 3.3-58.5 .0003 vs plateau/response Stem cell source

PBSC vs BM .45 .26-.78 .004. 48 .29- 9 .004

NA = non-applicable; *level absorbed into model since plateau/response level omitted; **versus primary refractory level

In multivariate analysis in which all prognostic factors were compared, however, only ALC was found to be the most effective independent prognostic predictor for OS (RR = 0.176, p < 0.0001) and PFS (RR = 0.275, p < 0.0006). In contrast, stem cell source, circulating plasma cells, platelet count > 20 x 10 IL, PCLI, LDH, the number of pre-treatment chemotherapy regimen, clinical status prior to transplantation, and abnormal cytogenetics, identified as significant prognostic predictors by univariate analysis, were determined to be not significant prognostic predictors. Clinical status prior to transplantation was omitted from the multivariate analysis on OS.

Figure 1 is a graph comparing the OS of MM patients with ALC > 500cells/μL and MM patients with ALC < 500 cells/μL. MM patients with ALC > 500cells/μL had a median OS of 33 months, while MM patients with ALC < 500 cells/μL had a median OS of 12 months (p < 0.0001). Figure 2 is a graph comparing the PFS of MM patients with ALC > 500cells/μL and MM patients with ALC < 500 cells/μL. MM patients with ALC > 500cells/μL had a median PFS of 16 months, while MM of patients with ALC of < 500 cells/μL had a median PFS of 8 months (p < 0.0001, respectively). For MM patients who received PBSC graft, ALC remained an effective prognostic predictor for OS (RR = 0.36, p < 0.0001) and PFS (RR =0.46, p < 0.0001). For MM patients who received PBSC, the median OS for patients with ALC > 500cells/μL was 46 months, while the median OS for patients with ALC < 500 cells/μL was 12 months (p < 0.0001). Similarly, for MM patients who received PBSC, the median PFS for patients with ALC > 500cells/μL was 17 months, while the median PFS for patients with ALC < 500 cells/μL was 8 months, (p < 0.0001). For patients who received BM graft, ALC was not associated with better survival (OS, p = 0.27, and PFS, p = 0.86). There was correlation for neither OS nor PFS in patients that received the BM graft and higher ALC recovery (p < 0.27, andp < 0.86, respectively).

For NHL patients, ALC, ANC, and PS were identified as significant predictors for OS and PFS by univariate analyses (see Table 9). The two patients who received both BM and PBSC were excluded because they did not contribute any additional significant predictive ability. From multivariate analysis in which all known prognostic factors were compared, however, only ALC was identified as a significant independent prognostic predictor for OS (RR = 0.08, p < 0.0001) and PFS (RR = 0.09, p < 0.0001). The other significant prognostic predictors identified in the univariate analysis, including ANC and PS, were not determined to be significant prognostic predictors of OS and PFS by multivariate analysis. ALC also was a prognostic factor for patients who received PBSC or BM graft. For patients who received PBSC grafts, ALC remained an effective prognostic predictor for OS (RR = 0.25, p 0.0001) and PFS (RR =0.35, p < 0.0001). Similarly, for patients who received BM grafts, ALC remained an effective prognostic predictor of OS (RR = 0.11, p < 0.0001) and PFS (RR = 0.08, p< 0.0001). Figure 3 is a graph comparing the OS survival of NHL patients with ALC > 500 cells/μL and NHL patients with ALC < 500 cells/μL. NHL patients with ALC < 500 cells/μL had a median OS time of 6 months, while most NHL patients with ALC > 500 cells/μL remained alive at the end ofthe study period, ie., the median OS could not be determined (p < 0.0001). Figure 4 is a graph comparing the PFS of NHL patients with ALC > 500 cells/μL and NHL patients with ALC < 500 cells/μL. NHL patients with ALC < 500 cells/μL had a median PFS time of 4 months, while most NHL patients with ALC ≥ 500 cells/μL 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/μL showed significantly better median OS than those with ALC < 500 cells/ μL, regardless of type of stem cells used (PBSC or BM). Similarly, NHL patients with ALC > 500 cells/μL showed significantly better median OS than those with ALC < 500 cells/μL, 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/μL was 6 months, while the median OS time of those with ALC > 500 cells/μiL could not be determined as a large number of patients were still alive at the end ofthe study (p < 0.0001). For NHL patients who received PBSC grafts, the PFS of those with ALC < 500 cells/μL was 3.6 months, while the PFS of those with ALC > 500 cells/μL could not be determined as progression had not been reached for a large number of patients (p < 0.0001). For NHL patients who received BM grafts, the median OS time of those with ALC < 500 cells/μL was 5.7, while the median OS time of those with ALC > 500 cells/μL could not be determined as most patients were still alive at the end ofthe study period (p < 0.0001). For NHL patients who received BM grafts, the median PFS time of those with ALC < 500 cells/μL was 4 months, while the PFS of those with ALC > 500 cells/μL could not be determined as progression had not reached for a large number of patients (p < 0.0001).

Table 9. Univariate Analysis for non-Hodgkin's lymphoma Patients: Overall and PFS rates

Overall Survival PFS

Prognostic Factors at Relative Relative

Time of Transplant Risk 95%CI P Risk 95%CI P

Age > 60 1.05 .57-1.94 .87 .94 .54-1.66 .84 vs < 60

ALC > 500 cells/ Λ .07 .03-.16 <.0001 .09 .04-.18 <.0001 vs < 500 cells/μl

ANC > 500 cells/ μl .43 .21-.88 .02 .49 .25-.95 .034 vs < 500 cells/μl

Chemo-sensitive

Disease (CR + PR) .39 .095-1.62 .2 .73 .26-2.02 .55

CR status prior to

Transplantation .71 .34-1.47 .35 .66 .33-1.32 .24

Extra-nodal sites > 2 2.66 .95-7.48 .06 2.14 .77-5.958 .14 vs <2

LDH = normal .59 .33-1.06 .077 .81 .47-1.39 .44 for age/sex vs > normal

Performance status≥ 2 2.6 1.09-6.16 .03 2.43 1.03-5.71 .04 vs < 2

# of pre-transplant chemotherapy regimens 1.11 .78-1.58 .57 .96 .67-1.37 .82

Stage III/IV 1.59 .8-3.1 .17 1.2 .68-2.15 .53

Stem cell source

BM vsPBSC .85 .46-1.57 .61 .84 .48-1.47 .55

Platelets > 20 x l0⁹/l .67 .35-1.26 .21 .89 .48-1.63 .70 vs ≤ 20 x l0⁹/l

CR = complete remission; PR = partial remission

Results of multivariate analyses demonstrating that ALC is an independent prognostic factor for OS and PFS in both MM and NHL patients are shown in Table 10.

Table 10. Multivariate Analysis for Multiple Myeloma and non-Hodgkin's lymphoma Patients:

Overall and PFS Rates

Multiple mveloma non-Hodgkin's lvmphoma

Prognostic Factors OS PFS OS PFS

RR 95 %CI p RR 95 %CI p RR 95%CI p RR 95%CI p

ALC > 500 cells/μl. .176 .075-.414 .0001 .275 .13-.98 .0006 .08 .04-.17 .0001.09 .04-19 .0001 vs < 500 cells/ /l

Ciculating

Plasma cells .27 .11-.65 .0035 .33 .15-.75 .008

PCLI > 1% .3 .11-.79 .014 vs < 1%

Platelet > 20 x l0⁹/l .18 .06-.5 .001 vs < 20 l0⁹/l

Relapse on therapy 8.45 1.64-43.58 .011 vs plateau/response

Likelihood-ratio P < 0.0001

OS, overall survival; PFS, progression-free survival; RR, relative risk

For patients with Hodgkin's disease, univariate analysis of available data, obtained from 53 patients, corresponding to albumin, hemoglobin, WBC count, lymphocyte count, LDH, and sedimentation rate at diagnosis showed that none of these variables were significant prognostic predictors for OS and PFS. In contrast, ALC at day 15 post-ASCT, bulky disease before transplantation, ECOG performance status before transplantation, and splenectomy were significant prognostic predictors for both OS and PFS. The year of transplantation was only a significant prognostic predictor for PFS. In the multivariate analysis, ALC at day 15 post-ASCT was found to be the strongest independent prognostic predictor for both OS (RR = 0.46, p < 0.0001) and PFS (RR = 0.43, 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/μL with Hodgkin's disease patients having ALC < 500 cells/μL. The median OS of patients with ALC > 500 cells/μL (not reached) was significantly greater than the median OS of patients with ALC < 500 cells/μL (29 months), p < 0.0001. Figure 7 is a graph comparing the PFS of Hodgkin's disease patients having ALC > 500 cells/μL with Hodgkin's disease patients having ALC < 500 cells/μL. The median PFS of patients with ALC > 500 cells/μL (not reached) was significantly greater than the median PFS of patients with ALC < 500 cells/μL (14 months), p < 0.0001. In addition, 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.

Table 11. Univariate and Multivariate Analyses for Overall and PFS Rates

Abbreviations: ECOG, Eastern Cooperative Group; OS, overall survival; PFS, progression-free survival; RR, relative risk

For the AML patients, univariate analysis ofthe available cytogenetics data obtained from 38 patients at diagnosis showed that inteπnediate and unfavorable cytogenetics were significant prognostic predictors for OS. In addition, ALC at day 15 and CR status before transplantation were also significant prognostic predictor for OS and LFS. In multivariate analysis, however, ALC at day 15 post-ASCT was found to be the only significant prognostic predictor for both OS and LFS (OS, RR =0.46, p < 0.0047 and LFS, RR = 0.53, p < 0.0076). Table 12 summarizes the significant prognostic predictors for the univariate and multivariate analyses.

Table 12. Univariate Analysis and Multivariate Analysis: Overall and LFS Rates

Likelihood-ratio^ < 0.0007

Abbreviations: CR, complete remission; OS, overall survival; LFS, leukemia-free survival; RR, relative risk

For patients with metastatic breast cancer, the OS and PFS were significantly longer in patients with ALC < 500 cells/μL versus those with ALC < 500 cells/μL (p < 0.001). The estimated 1-year overall survival for those with ALC < 500 cells/μL was 50 % (95 % Cl: 28 %-88 %) versus 91% for those with ALC > 500 cells/μL (95 % Cl: 75 % -100 %). The estimated 1-year PFS for those with ALC < 500 cells/μL was 17 % (95% Cl: 5 % - 59 %) versus 56 % for those with ALC > 500 cells/μL (95 % Cl: 36 % - 88 %).

Collectively, patients with ALC > 500 cells/μL had significantly greater OS and PFS time than patients with ALC < 500 cells/μL. Figure 8 is a graph comparing the OS of patients having ALC > 500 cells/μL (median: 55 months) with the OS of patients with ALC < 500 cells/μL (median: 13 months) for all 386 patients, p < 0.0001. Figure 9 is a graph comparing the PFS of patients having ALC > 500 cells/μL (median: 40 months) with the PFS of patients with ALC < 500 cells/μL (median: 7 months) for all 386 patients, ρ < 0.0001. In conclusion, ALC > 500 cells/μL on day 15 after ASCT was the strongest predictor of clinical outcome in patients with MM, NHL, Hodgkin's disease, AML, and metastatic breast cancer. ALC > 500 cells/μL on day 15 after ASCT correlated with better OS and PFS. This indicates that immunologic recovery after ASCT is a significant factor in the control of minimal residual disease. Furthermore, the antitumor effect mediated by rapid immune recovery suggests the use of autologous immunocompetant stem cells in transplants. The observed clinical benefit of early ALC recovery may be analogous to the GVT effect seen in the allogeneic transplant setting.

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. Before stem cell aphoresis, patients are started on a cancer drug regimen. 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, and interferon. Briefly, patients are treated with drugs such as Flt-3 ligand and GM-CSF at concentrations of at least 50 μg/kg/day and 5 μL/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. At a WBC count of greater than 10 cells/nL, 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. 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.

To count the number of stem cells present in the apheresis product, cells in the apheresis samples may be exposed to antibodies that specifically recognize stem cells. Briefly, 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. Alternatively, 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.

Subsequent to apheresis, the patient is treated with a HDT conditioning regimen. The patients can be treated with melphalan (140 mg/m²) and TBI (12 Gy); melphalan (140 mg/m²), cyclophosphamide (60 mg/m²) and TBI; or cyclophosphamide (60 mg/m ) and TBI. The patients also can be treated with busulfan (16 mg/kg) and cyclophosphamide (60 mg/m²); cyclophosphamide (1.5 g/m²), BCNU (300 mg/m²), and etoposide (125 mg/m²); or BCNU (300 mg/m²), etoposide (100 mg/m²), ARA-C (100 mg/m²), and cyclophosphamide (33 mg/kg). Patients may also be treated with antibiotics and/or antiemetic drugs as warranted by the patient's physician.

Following HDT, i.e. approximately 24 to 48 hours after the completion of HDT, 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. Alternatively, the stem cells may be infused through a peripheral vein such as the median cubital vein. Following stem cell transplant, the patient is additionally treated with between 1 x 10⁵ and 1 x 10⁷ units/m² of interferon. Patients are treated with interferon from the day that they receive the transplant to 21 days following stem cell transplant.

To evaluate drug efficiency, within fifteen days following transplantation ofthe stem cells, 10 mL of PB is collected and an ALC is determined using the Beckman Coulter Gen-S Cell according to the manufacturers instructions. An ALC of > 200 lymphocytes/μL, and preferably 300, 400, or 500 lymphocytes/μL is indicative ofthe efficacy ofthe drug in treating cancer. An ALC of less than 200 lymphocytes/μL indicates that the drugs used in the stem cell mobilization treatment were not maximally effective. In this case, 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.

In a randomized study, Flt-3 ligand in combination with G-CSF or GM-CSF, or G-CSF alone, is used for PBSC mobilization. Of 32 patients with breast cancer, 14 patients received Flt-3 ligand and G-CSF or GM-CSF, and 18 patients received G-CSF alone. Not only did Flt-3 ligand increased PBSC mobilization, but in the 14 patients who received Flt-3 ligand, 11 (79 %) had an ALC greater than 500 lymphocytes/μL at day 15 post-ASCT. In contrast, in the 18 patients who did not receive Flt-3 ligand as part ofthe mobilization, only 8 of 18 patients (44 %) achieved an ALC of greater than 500 lymphocytes/μL at day 15 post-ASCT and 10 (56 %) had an ALC less than 500 lymphocytes/μL. Thus, the combination Flt-3 ligand + GM-CSF for mobilization of PBSC in patients with NHL, HD, MM, and amyloidosis, or any other cancer treatable by ASCT, may result in higher CD34+ yields leading to a decreased number of apheresis per patient and faster hematopoietic recovery post-ASCT. In addition, the combination of 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/μL, preferably >_300, 400, or 500 lymphocytes/μL.

Accordingly, 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. Briefly, patients are treated with Flt-3 ligand and GM-CSF at concentrations of at least 50 μg/kg/day and 5 μg/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. At a WBC count of greater than 10 cells/nL, 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, Deerfϊeld, 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. 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. 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.

Within fifteen days following the transplantation ofthe stem cells, 10 mL of peripheral blood is collected and an ALC obtained using the Beckman Coulter Gen-S Cell according to the manufacturers instructions. An ALC of at least 200 lymphocytes/μL and preferably at least 300, 400, or 500 lymphocytes/μL is indicative of an improvement in the cancer therapy. An ALC of less than 200 lymphocytes/μL indicates that efforts to improve the cancer therapy were not maximally effective. In this case, the dose of Flt-3 ligand and GM-CSF may be increased to up to 500 μg/kg/day and 50 μg/kg/day for Flt-3 ligand and GM-CSF respectively. Higher concentrations of interferon may also be used.

OTHER EMBODIMENTS It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope ofthe invention, which is defined by the scope ofthe appended claims. Other aspects, advantages, and modifications are within the scope ofthe following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of predicting survival of a cancer patient, said method comprising:

(a) obtaining a blood sample from said cancer patient at 5-15 days post- autologous hematopoietic stem cell transplant; (b) determining the absolute lymphocyte count of said blood sample; and

(c) correlating said absolute lymphocyte count of said blood sample with a prediction of survival of said cancer patient subsequent to said autologous hematopoietic stem cell transplant, wherein an absolute lymphocyte count that is > 200 lymphocyte/μL predicts survival of said cancer patient for at least 24 months.

2. The method of claim 1 , wherein said cancer is selected from the group consisting of multiple myeloma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute myelogenous leukemia, and metastatic breast cancer.

3. The method of claim 1 , wherein an absolute lymphocyte count that is > 500 lymphocyte/μL predicts survival of said cancer patient for at least 24 months.

4. The method of claim 1, wherein an absolute lymphocyte count that is > 500 lymphocytes/μL predicts survival of said cancer patient for at least 33 months.

5. The method of claim 1, wherein an absolute lymphocyte count that is > 500 lymphocytes/μL predicts survival of said cancer patient for at least 36 months.

6. The method of claim 1, wherein an absolute lymphocyte count that is > 500 lymphocytes/μL predicts survival of said cancer patient for at least 42 months.

7. The method of claim 1, wherein an absolute lymphocyte count that is > 500 lymphocytes/μL predicts survival of said cancer patient for at least 60 months.

8. A method of determining the efficacy of a candidate drug in a cancer patient, said method comprising:

(a) administering said candidate drug to said cancer patient prior to isolation of hematopoietic stem cells from said cancer patient;

(b) administering cancer therapy to said cancer patient, wherein said cancer therapy comprises autologous hematopoietic stem cell transplant; (c) obtaining a blood sample from said cancer patient at 8-15 days post- autologous hematopoietic stem cell transplant; and (d) determining the absolute lymphocyte count of said blood sample from said cancer patient, wherein an absolute lymphocyte count > 200 lymphocytes/μL indicates the efficacy of said candidate drug in said cancer patient.

9. The method of claim 8, wherein said drug comprises a hematopoietic growth factor.

10. The method of claim 8, wherein said drag is flt-3 ligand.

11. The method of claim 8, wherein said drug is GM-CSF.