WO2001070243A2

WO2001070243A2 - A method for treating early breast cancer

Info

Publication number: WO2001070243A2
Application number: PCT/US2001/009275
Authority: WO
Inventors: Bonnie J. Mills
Original assignee: Nexell Therapeutics Inc.
Priority date: 2000-03-23
Filing date: 2001-03-19
Publication date: 2001-09-27
Also published as: WO2001070243A3

Abstract

Methods for treating early stage breast cancer are disclosed. Generally, the methods disclosed use high dose chemotherapy in conjunction with hematopoietic stem cell transplants. Specifically, the methods disclosed use high dose chemotherapy together with selected hematopoietic stem cell transplants.

Description

A METHOD FOR TREATING EARLY STAGE BREAST CANCER USING HIGH DOSE CHEMOTHERAPY AND SELECTED STEM CELL TRANSPLANTS

FIELD OF THE INVENTION

The present invention is generally directed at a treatment for cancer, specifically breast cancer. More specifically the present invention is directed at a method of using high dose chemotherapy in conjunction with hematopoietic progenitor cell transplants for treating early stage breast cancer.

BACKGROUND OF THE INVENTION

Cancer treatment has progressed dramatically in the past six decades. Prior to 1940 most cancer treatments included surgical extraction and massive, near fatal doses of radiation. However, in 1941 Huggins initiated the chemotherapy era when he demonstrated that the synthetic estrogen diethylstilbestrol could dramatically reduce disseminated prostate cancer symptoms. Since that time, numerous new classes of anti-neoplastic agents have been developed including alkylating agents, androgen inhibitors, antibiotic derivatives, antiestrogens, antimetabolic agents, cytotoxic agents, hormones, immune modulators and steroids. The most commonly used anti-neoplastic agents are antibiotic derivatives/cytotoxic agents such as anthracycline antibiotics including doxorubicin (a.k.a. Adriamycin) and antimetabolic agents including fluorinated pyrimidines (Fluorouracil) and antimitotic agents such as paclitaxel (Taxol). These classes of anti-neoplastic agents kill cancer cells by interfering with basic cellular functions associated with replication. Anthracycline antibiotics intercalate between nucleic acid bases inhibiting deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) synthesis and reduce nucleic acid polymerase activity. Fluorinated pyrimidines interfere with DNA syntheses, and to a lesser extent RNA syntheses, by blocking the methylation of deoxyuridylic acid thus preventing its conversion to thymidylic acid. Antimitotic agents can either interfere with mitotic spindle development or stabilize the spindle to such an extent that mitosis cannot be initiated. (Taxol is an example of the latter.) Radiation kills cancer cells by fragmenting DNA reducing the precise genetic code to non-sense and oxidizing essential cellular enzymes and macromolecules. However, regardless of the specific pharmacology involved, the aforementioned chemotherapeutic agents and radiation all target actively dividing cells. Most adult mammalian cells are terminally differentiated spending the majority of their life cycle in a quiescent phase referred to as G₀/Gι. The G₀/Gι phases are characterized by the absence of DNA replication and cell division. In contrast, tumor cells rapidly divide making them ideal targets for chemotherapeutic agents that interfere with cell division. It is this difference between G₀/Gι phase cells and tumor cells that anti-neoplastic regimens generally exploit. (An exception to this generalization is bifunctional alkylating agents such as L- phenylalanine mustard that are also active against resting stage tumor cells.) However, there are essential organ systems that contain continuously replicating cells that are also susceptible to anti-neoplastic agents. Perhaps the most important function of continuously replicating normal cells is hematopoiesis.

Hematopoietic sites, especially the bone marrow, are responsible for peripheral blood cell replenishment. Blood cell replenishment depends on the presence of undifferentiated hematopoietic cells termed stem cells. The most primitive blood cell (hematopoietic stem cell) is pluripotent, capable of self renewal and differentiation. The process of differentiation results in a population of hematopoietic progenitor cells that continue to differentiate giving rise to the peripheral blood's mature blood cells including erythrocytes, lymphocytes, monocytes, granulocytes and thrombocytes. These mature blood cells have a relatively short life span and must be replenished regularly. When the hematopoietic stem and progenitor cells responsible for blood cell replenishment are destroyed, peripheral blood cell levels rapidly diminish resulting in significant impairment to the immune system, coagulation mechanisms and oxygen transport. The extent of chemotherapeutic induced hematopoietic cell death is dose dependent.

Higher doses of chemotherapeutic agents will result in greater numbers of hematopoietic stem and progenitor cells killed (myeloablation) and hence greater periods of bone marrow aplasia.

As with all diseases, different forms of cancer require specific therapeutic regimens. Generally speaking, cancer treatment regimens are determined based on numerous factors. Unlike antibiotic therapy, anti-neoplastic agents are generally not selected by actually testing cancer cells for sensitivity against a given agent, or panel of agents. Experience has demonstrated that sensitivity testing has little predictive value. However, in certain cases determining to which drugs a cancer is likely to be resistant can assist in making therapeutic decisions. Consequently, therapeutic regimens are chosen based on actual clinical experience in treating specific tumor types.

Conventional chemotherapy for cancer generally uses two or more drugs. There are several advantages to this approach. Each drug has a different mechanism of action; consequently, tumor cell resistance is less likely to develop. Secondly, anti-neoplastic agents are highly toxic and the maximum drug dosage is limited by their respective toxicity to a target organ (dose limiting toxicity). Therefore, by combining drugs having different dose limiting toxicities, a higher total concentration of effective anti-neoplastic agent can be administered. In an effort to ameliorate myeloablative side effects, conventional, or standard-dose, techniques use sub-myeloablation concentrations of anti-neoplastic agents and rest patients between drug cycles until peripheral blood cell counts return to acceptable levels.

However, not all forms of cancer respond well to standard-dose therapy. Consequently, oncologists began using high dose chemotherapy (HDCT) to treat certain malignancies. High dose chemotherapy has proven to be an effective alternative to standard dose therapy for relapsed non-Hodgkin's lymphoma, multiple myeloma and relapsed or resistant Hodgkin's Disease.

Most forms of HDCT utilize combinations of anti-neoplastic agents where myeloablation is an anticipated toxicity. Consequently, patients receiving HDCT require some form of hematopoietic stem and progenitor cell rescue (i.e., stem cell transplant). Bone marrow or hematopoietic stem and progenitor cell transplantation is used in combination with HDCT to rescue the patient from life-threatening bone marrow ablation.

There are generally two types of bone marrow transplants: allogeneic and autologous. Allogeneic transplants involve use of cells donated by a person other than the recipient. Allogeneic transplants ideally occur between matched pairs with identity (histocompatability) at three human lymphocyte antigen loci (HLAs): HLA-A, B and DR loci (2 alleles each). If a sibling is available, a five of six-antigen match is acceptable; in fact it is often preferred to a six of six match from an unrelated donor. In rare cases an identical twin is available resulting in a perfect, or syngeneic, match. The fewer the HLA-antigens that match, the greater the chances of severe graft-versus-host-disease (GVHD) and other complications. Graft-versus-host-disease can occur in any allogeneic transplant (except syngeneic transplants) when mature T-lymphocytes from the donor react with the recipient's tissues. If the GVHD is severe enough, it can result in severe long term disability or death. Conversely, a strong host reaction may result in graft rejection requiring a second transplant procedure and in some instances leading to long term aplasia and death. The selective removal of T-cells from donor blood or marrow can substantially reduce the potential for GVHD. However, it has been established that donor T-cells can have a beneficial anti-leukemia effect, therefore, caution must be taken when purging strategies are employed.

Due to an overall unfavorable donor/recipient ratio and the morbidity and mortality associated with allogeneic transplant, autologous transplants have become the preferred hematopoietic stem and progenitor source for many stem cell transplant procedures. In autologous transplant, hematopoietic stem and progenitor cells are recovered from the recipient prior to myeloablation treatment. After the treatment is completed, the patients' stem cells are reinfused. Recently, autologous transplant products have been obtained from the peripheral blood rather than the more painful and risky process of bone marrow aspiration. In this procedure a patient is treated with an anti-neoplastic such as cyclophosphamide and/or a cytokine which stimulates progenitor cell proliferation. This procedure results in a migration of stem cells from the bone marrow into the peripheral blood where they can be harvested by leukapheresis. One potential draw back to autologous transplantation is the potential presence of cancer cells in the leukapheresis product; this is especially true for hematological cancers and metastatic disease. Therefore, it has become a preferred practice to purge autologous transplants (autograft) prior to use. Purging is a process of removing, or killing tumor cells that may be present in leukapheresis product. Some purging techniques have used high doses of anti-neoplastic agents such as 4-hydroxyperoxycyclophosphamide (4-HC) to pre-treat autografts. However, highly toxic agents like 4-HC can also kill non-cancerous cells including stem cells. Moreover, non-cancer cell burden factors such as red cell content can significantly reduce the purging process efficiency. Consequently less toxic and more efficient means of purging autografts were developed. Rapid progress in cell surface antigen characterization has lead to the development of physical methods for removing specific cells from a mixed population (stem cell selection). In cancer therapy much attention has been directed towards the leukocyte antigen designated CD (Cluster of Differentiation) 34 which is found on pluripotent hematopoietic progenitor cells including stem cells. Monoclonal antibodies which react specifically with the CD34 antigen can be used to remove cells from a mixed population by physical separation means. One particularly efficient approach for performing this process employs a monoclonal antibody

CD34 negative cancer cells. One example of the aforementioned device is depicted in United States Patent number 5,536,475, which is hereby incorporated by reference. Autografts prepared in this fashion are now being used in experimental HDCT treatments for advanced or high risk breast cancer. However, the benefits and risks associated with this technique as compared to non-selected autografts in advanced or high risk forms of cancer have not been definitively established.

Generally, breast cancer is staged anatomically based on tumor size, number of positive nodes and presence or absence of metastases. Stage 0 is a noninvasive in situ lump either localized to the lobules or within a duct; Stage I tumors measure less than 2 cm and have not spread beyond the breast; Stage II tumors are larger than 2 cm and/or the cancer has spread to local lymph nodes; Stage IIIA tumors are either greater than 5 cm in diameter and or have spread to lymph nodes that have begun to aggregate or adhere to adjacent tissues; in Stage IIIB the cancer has spread to tissues including the skin, chest wall, or internal mammary lymph nodes; in Stage IV, cancers have metastasized, that is spread to tissues distant from the breast. Recently, cancer treatment using HDCT with autologous transplants has received significant attention. Many studies have been conducted to compare the relative benefits and risks of this approach. Currently, results remain inconclusive. The majority of patients receiving HDCT for breast cancer have had advanced forms of the disease (stage IV); relapse remains the most common cause of death among patients in this group. There is a critical need to develop methods that significantly increase relapse-free survival and overall survival rates for patients with all stages of breast cancer. Early detection methods which allow diagnosis and treatment of earlier stages of disease provide an opportunity to develop effective methods of treating early stage breast cancer (primarily Stage II and Stage Ilia) that may prevent development of metastatic disease thus significantly increase relapse-free survival rates as well as overall survival.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method for treating early stage breast cancer that increases relapse-free survival. It is another object of the present invention to provide a method of treating early stage breast cancer using high dose chemotherapy and stem cell rescue using selected autologous stem cell transplants.

The present invention is a method for treating early stage breast cancer using high dose chemotherapy (HDCT) in conjunction with stem cell transplant. Stem cells sources of the present invention include, but are not limited to, bone marrow aspirates, and leukapheresed peripheral blood. Stem cells, as used in the present invention, include CD34 positive cells capable of self renewal and differentiation, also known as hematopoietic progenitor cells including, but not limited to, totipotent hematopoietic stem cells, pluripotent hematopoietic stem cells, Colony Forming Unit (CFU)-granulocyte, erythroid, monocyte, megakaryocyte (CFU-GEMM) and CFU-granulocyte-monocytes/macrophage (CFU-GM). High dose chemotherapy as used in the present invention includes methods of treating cancer using anti- neoplastic agents, either individually, or in combination, that have a myeloablation effect necessitating stem cell rescue. Generally, the method for treating early stage breast cancer is composed of first obtaining a hematopoietic stem cell containing sample from an early stage breast cancer patient. Next, the sample is enriched for hematopoietic stem cells using a selection process so as to produce a selected stem cell transplant. Then a high dose chemotherapeutic regimen is administered to an early stage breast cancer patient and thereafter the early stage breast cancer patient is infused with the selected stem cell transplant.

In another embodiment of the present invention the method for treating early stage breast cancer includes the steps of administrating a hematopoietic stem cell mobilization regimen to an early stage breast cancer patient before obtaining a hematopoietic stem cell containing sample from the early stage breast cancer patient. Next, the sample is enriched for hematopoietic stem cells using a selection process so as to produce a selected stem cell transplant. This is followed by administering a high dose chemotherapeutic regimen to the early stage breast cancer patient and thereafter infusing the early stage breast cancer patient with the selected stem cell transplant. Then, as a final measure, recombinant human granulocyte colony stimulating factor is administered to the early stage breast cancer patient. Chemotherapeutic agents suitable for use with the present invention include, but are not limited to, antibiotic derivatives, antimetabolites, cytotoxic agents, and nitrogen mustard derivatives. In one embodiment of the present invention early stage breast cancer patients (Stage II and IIIA) are administered myeloablation cytoreduction therapy (a treatment that reduces the overall tumor burden in patients that also destroys a significant number of stem cells). After cytoreduction myeloablation therapy is complete, (or between cycles of myeloablation treatment in specific treatment regimens) selected autologous transplants are administered to patients to decrease the period of bone marrow suppression.

In one embodiment of the present invention stem cell mobilization is performed (stem cell migration from the bone marrow into the peripheral blood) using, but not limited to granulocyte colony stimulating factor (G-CSF) such as, but not limited to, filgrastim (Neupogen®, Amgen, Inc., Thousand Oaks, CA), granulocyte-monocyte/macrophage-colony stimulating factor (GM-CSF), and cytokines such as, but not limited to stem cell factor PIXY 321. In accordance with one embodiment of the present invention recombinant human G-CSF (rhG-CSF) is administered until peripheral blood CD34 positive cell counts of greater than or equal to 20 cells/μL are reached. Following successful stem cell mobilization, stems cells are harvested by leukapheresis and then selected using immunomagnetic separation. In one embodiment of the present invention stem cell selection is performed using the Isolex® 300t Magnetic Cell Separator System (Nexell Therapeutics Inc., Irvine, CA). After selection, the purified stem cells are transplanted into the patient followed by administration of rhG-CSF.

Additional objects and advantages of the present invention and methods of construction of same will become readily apparent to those skilled in the art from the following detailed description, wherein only the preferred embodiments are shown and described, simply by way of illustration of the best mode contemplated of carrying out the invention. As will be realized, the invention is capable of modification in various respects, all without departing from the invention. Accordingly, the following detailed description and examples are to be regarded as illustrative in nature, and not as restrictive.

DETAILED DESCRIPTION OF THE INVENTION The use of high dose chemotherapy (HDCT) as a treatment for breast cancer remains experimental. Clinical trials have been conducted comparing conventional, low dose chemotherapy with high dose chemotherapy and autologous stem cell transplants for metastatic breast cancer (Stage IV) and high risk Stage II/III cancers. Published results are inconclusive and HDCT for advanced breast cancer remains controversial. High dose chemotherapy is usually myeloablation; consequently stem cell rescue must be used to restore the patients blood cell producing tissues as quickly as possible. Failure to do so often results in prolonged susceptibility to life threatening opportunistic infections, anemia and thrombocytopenia (thrombocytes are blood cells essential for proper coagulation). Due to the morbidity and mortality associated with allogeneic transplant, autologous transplants have become preferred for breast cancer HDCT.

The most common cause of death associated with breast cancer following initial disease remission is cancer reoccurance. The period of disease free survival varies significantly depending on the therapeutic regimen used, disease stage prior to initiating therapy and the tumors' susceptibility to the therapy. Cancer reoccurrence can theoretically result from incomplete treatment of the primary tumor, residual populations of resistant tumors, or the reintroduction of tumors present in the autologous transplant. In an effort to minimize the latter, purging techniques have been developed to eliminate tumor cell contamination. Chemotherapeutic agents such as 4-hydroxyperoxycyclophosphamide (4-HC) have been used with varying degrees of success.

The primary draw back to chemotherapeutic purging techniques is that the drug can also damage or kill stem cells present in the transplant. Consequently, stem cell selection technologies based on physical separation methods have been developed. One of the most successful methods for stem cell selection employs immunomagnetic methods based on the use of mouse monoclonal antibodies directed against stem cell surface antigens such as CD34. Because CD34 is found primarily on stem cells and progenitor cells, and only rarely on cancer cells, it is an ideal surface antigen for stem cell selection. Autologous transplants depleted of cancer cells using physical separation techniques are referred to as "selected" products.

In the present invention HDCT and autologous selected stem cell transplants were used on early stage breast cancer patients (high risk stage II and IIIA). Contrary to the inconclusive results seen in treating advanced breast cancer patients, early results using HDCT with selected autologous stem cell transplants showed a statistically significant increase in disease free survival (absence of cancer reoccurrence) when compared to patients receiving HDCT and non-selected autologous stem cell transplants (autologous stem cell transplants which have not undergone any form of cancer cell purging prior to administration).

Breast cancer patients with advanced or high risk breast cancer were enrolled in randomized clinical trials between 1993 and 1997. The primary purpose of these trials was to determine whether selected stem cells would engraft (survive long enough to restore a patient's mature peripheral blood cell population) comparably to non-selected stem cells, and to assess toxicity of transplanting selected cells relative to using non-selected stem cells. Stem cells were harvested from the peripheral blood using leukapheresis following stem cell mobilization. Stem cell mobilization was conducted using chemotherapy followed by recombinant human granulocyte colony stimulating factor (rhG-CSF) or using rhG-CSF alone. Other stem cell mobilization factors (not used in these studies) include, but are not limited to, granulocyte- monocyte/macrophage-colony stimulating factor (GM-CSF), and cytokines such as, but not limited to, stem cell factors flt-3 and PIXY 321.

Chemotherapy used for mobilization was consistent with the institutional protocols for the individual study centers. These chemotherapy mobilization regimens included, but were not limited to, ifosfamide 4 g/m², cisplatin 50mg/m², etoposide 500 mg/m²; cyclophosphamide 4g/m , placitaxel 170 mg/m ; cyclophosphamide 5g/m ; cyclophosphamide 3g/m , epirubicin 100mg/m²; and cyclophosphamide 2g/m², etoposide 450 mg/m². Recombinant hG-CSF (Neupogen®, Amgen, Inc., Thousand Oaks, CA) was administered subcutaneously 24 to 48 hours after chemotherapy at concentrations ranging from 5 μg/kg/day to lOμg/kg/day. When rhG-CSF was given alone, 10 μg/kg/day was administered subcutaneously. Peripheral blood levels of CD34+ cells were monitored using flow cytometry until a minimum concentration of 20 CD34+ cells per μL was reached.

When peripheral blood CD34+ cell concentrations reached the minimum threshold and white blood cell (WBC) counts were greater than or equal to 1000 cells/μL, leukapheresis was initiated using a CS3000® Plus Blood Separator (Fenwal Division, Baxter Healthcare Corp., Round Lake, IL), or another suitable blood collection system. Methods used to collect blood from patients were consistent with those known to persons of ordinary skill in the art. Specifically, a large bore multi-lumen catheter is inserted into an appropriate vein and 10 to 12 liters of whole blood was processed per day. This process was continued on sequential days with a target collection of 5.0 x 10⁶ CD34+ cells/kg of body mass.

Stem cell selection was performed aseptically using a semi-automated or fully automated immunomagnetic cell selection system (Isolex® 300/300*^' Magnetic Cell Separator System, Nexell Therapeutics Inc., Irvine, CA). Leukapheresed blood products were processed individually or were pooled. The cells were washed to remove platelets and resuspended in an isotonic buffer at physiological pH. Approximately 2.5 mg of a mouse monoclonal antibody specific for the cell surface antigen CD34 (α34-MoAb) was added to the cell suspension and incubated for 15 minutes at approximately 22° C (room temperature). The cell suspension was then washed in Ca⁺² and Mg⁺² free Dulbecco's phosphate buffered saline containing 1% human serum albumin and 0.2% sodium citrate using methods known to those skilled in the art. Next, paramagnetic beads coated with sheep anti-mouse antibodies (Dynabeads® M450 paramagnetic beads, Dynal a.s., Oslo, Norway) were added to the cell suspension and incubated for an additional 30 minutes at room temperature. Stem cells that reacted with the α34-MoAb (target cells) bind, or rosette, to the antibody coated paramagnetic beads and are then immobilized in a magnetic field. Non-target cells are washed away and target cells are released from the paramagnetic beads using a suitable releasing reagent, such as, but not limited to, a non-enzymatic octapeptide (PR34+® Nexell Therapeutic Inc., Irvine, CA) and the target cells are collected. Collected target cells are then enumerated, tested for viability and characterized prior to cryopreservation using methods known in the art until the patient was ready for transplantation. High dose chemotherapy regimens differed based on the institutional standards and included but were not limited to, cyclophosphamine 6 g/m², thiotepa 500 mg/m², carboplatin

800 mg/m²; cyclophosphamine 6 g/m², thiotepa 600 mg/m², carboplatin 800 mg/m²; cyclophosphamine 6 g/m², etoposide 625 mg/m², carboplatin 2 g/m²; busulfan 12 mg/kg, melphalan 100 mg/m², thiotepa 500 mg/m²; cyclophosphamine 120 mg/kg, mitoxantrone 45

7 7 7 mg/m , melphalan 140 mg/m ; adriamycin 165 mg/m , etoposide 60 mg/kg, cyclophosphamine 100 mg/kg; thiotepa 750 mg/m², mitoxantrone 40 mg/m², carboplatin 999mg/m²; and thiotepa 675 mg/m², cyclophosphamine 7.5 g/m². Each HDCT regimen was administered to the early stage breast cancer patient using methods known to those of ordinary skill in the art. Twenty-four to 72 hours following HDCT administration, selected stem cell transplants were infused into the patients. The dose of CD34+ cells infused per transplant was approximately between 1.1-16.7 x 10⁶ cells/kg of body mass. Following infusion, rhG-CSF was administered and patients absolute neutrophil counts (ANC) were monitored daily until a minimum peripheral blood concentration of 500 neutrophils/μL was reached on each of three consecutive days. Patients were also administered supportive therapy consisting of antiemetics, anti-infectives, and other drugs and blood products as clinically indicated to those of ordinary skill in the art.

Patients receiving HDCT as in accordance with the teachings of the present invention initially received post treatment follow-up including, but not limited to, daily red blood cell and thrombocyte counts, differential white blood cell counts, and weekly blood chemistries. After the patient was stabilized and discharged, follow-up was continued for up to two years at six month intervals, or as indicated to those of ordinary skill in the art of breast cancer treatment.

EXAMPLES

Example 1

Pt. ID: 1239

P Initial: SKS

Sex: Female

Race: Caucasian

DOB: 11/23/46

Baseline Wt.: 70.9 Kg

Initial Diagnosis 5/12/97

Disease: Breast CA - Stage IIA Surgical Treatment: Right Lumpectomy 5/12/97

Axillary Dissection 5/20/97

Medical Treatment: Adriamycin & Cytoxan x 3 cycles 7/1/97 — > 8/12/97 Karnofsky Performance Status at Time of Randomization: Not available Disease Status at Time of Randomization: Complete remission Mobilization Chemotherapy: GCSF 780 mcg/d 9/12/97 → 9/16/97

Date of successful mobilization to > 20 CD34 cells/μl: 9/17/97 Randomization: Test

Cytoreductive therapy:

Cytoxan 5100 mg/d 9/22/97 → 9/23/97 Carboplatin 850 mg/d 9/23/97 → 9/26/97 Etoposide 210 mg/d 9/22/97 → 9/26/97 CD34 Cell Transplant:

Date: 9/29/97

Dose: 1.9 x 10⁶ Selected CD34 cells/kg Engraftment summary:

Date patient reached ANC >500/μl: 10/09/97 - D10 post transplant Date patient reached platelet count >20K μl: 10/12/97 - D 13 post transplant Last Follow-up:

Date: 11/30/99

Days post transplant: 792 Patient Status: Alive Disease Status: Complete remission

Example 2

Pt. ID: 1046

Pt. Initial: SDB

Sex: Female

Race: Caucasian

DOB: 7/2/55

Baseline Wt.: 81 Kg

Initial Diagnosis: 4/17/96

Disease: Breast CA - Stage IIB Surgical Treatment: Right modified radical mastectomy 4/29/96 Medical Treatment: Adriamycin & Cytoxan x 4 cycles 5/23/96 -> 7/24/96 Karnofsky Performance Status at Time of Randomization: 100% Disease Status at Time of Randomization: Complete remission Mobilization Chemotherapy: GCSF 780 mcg/d 8/31/96 → 9/3/96

Date of Successful Mobilization to >20 CD34 cells/μl: 9/3/96 Randomization: Test

Cytoreductive Therapy:

Cyclophosphamide 4830 mg/d 9/4/96 → 9/5/96

Carboplatin 800 mg/d 9/5/96 → 9/8/96

Etoposide 200 mg/d 9/4/96 → 9/9/96

CD34 Cell Transplant:

Date: 9/11/96

Dose: 2.84 x 10⁶ Selected CD34 cells/kg Engraftment Summary:

Date patient reached ANC >500/μl: 9/22/96 - Dl 1 post transplant

Date patient reached platelet count >20K/μl: 9/22/96 - D 11 post transplant Last Follow-up:

Date: 11/8/99

Days post transplant: 1152

Patient status: Alive

Disease Status: Complete remission Example 3

Pt. ID: 1294

Pt. Initial: DHF

Sex: Female

Race: Caucasian

DOB: 3/9/60

Baseline W : 70 Kg

Initial Diagnosis: 5/3/96

Disease: Breast CA - Stage IIIA Surgical Treatment: Lumpectomy 5/20/96

Modified radical mastectomy with axillary node dissection 6/8/96

Medical Treatment: Cytoxan Adriamycin/fluorouracil x 4 cycles 7/96 — » 9/96

Karnofsky Performance Status at Time of Randomization: 90%

Disease Status at Time of Randomization: Complete remission Mobilization Chemotherapy:

Ifosfamide 3600 mg/d 10/14/96 → 10/15/96

Etoposide 900 mg/d 10/14/96 → 10/15/96

Cisplatin 90 mg/d 10/15/96 → 10/16/96

GCSF 5 mcg/Kg/d 10/17/96 → unknown Date of successful mobilization to > 20 CD34 cells/μl: 10/28/96

Randomization: Test

Cytoreductive therapy:

Thiotepa 220 mg/d 11/7/96 → 11/11/96

Cyclophosphamide 2625 mg/d 11/7/96 → 11/10/96 Carboplatin 350 mg/d 11/8/96 → 11/10/96

CD34 Cell Transplant:

Date: 11/13/96

Dose: 2.32 x 10⁶ Selected CD34 cells/kg

Engraftment summary: Date patient reached ANC >500/μl: 11/24/96 - Dl 1 post transplant

Date patient reached platelet count >20K/μl: 11/23/96 - D 10 post transplant

Last Follow-up 24 mos.:

Date: 1/13/99

Days post transplant: 791 Patient Status: Alive Disease Status: Complete remission

Example 4

Study: 92004 302103

Site: 17

Pt. ID: 1156

Pt. Initial: RSM

Sex: Female

Race: Caucasian

DOB: 6/10/52

Baseline Wt.: 73.6 Kg

Initial Diagnosis: 2/6/96

Disease: Breast C A - Stage IIIA

Surgical Treatment: Right modified radical mastectomy with lymph node dissection 2/23/96

Medical Treatment: Adriamycin & Cytoxan x 3 cycles 3/28/96 → 5/30/96 Karnofsky Performance Status at Time of Randomization: 100% Disease Status at Time of Randomization: Complete remission Mobilization Chemotherapy:

Cytoxan 4 gm/m²/d 7/18/96

Taxol 170 mg/m²/d 7/17/96

GCSF 10 mcg/kg/d 7/19/96 → 7/30/96

Date of successful mobilization to > 20 CD34 cells/μl: 7/30/96 Randomization: Test

Cytoreductive therapy:

Busulfan 232 mg/d 8/13/96 → 8/15/96

Melphalan 90 mg/d 8/16/96 → 8/17/96

Thiotepa 450 mg/d 8/18/96 → 8/19/96

CD34 Cell Transplant: Date: 8/21/96 Dose: 3.14 x 10° Selected CD34 cells/kg

Engraftment summary:

Date patient reached ANC >500/μl: 8/31/96 - Dl 0 post transplant Date patient reached platelet count >20K/μl: 9/2/96 - D 12 post transplant

Last Follow-up:

Date: 10/13/98

Days post transplant: 783 Patient Status: Alive

Disease Status: Complete remission

Contrary to the inconclusive results seen with treating advanced breast cancer patients using HDCT and selected stem cell transplants, the use of selected stem cell transplants in conjunction with HDCT in early stage (primarily Stage II and IIIA) breast cancer demonstrates surprisingly superior clinical results than early stage (primarily Stage II and IIIA) patients receiving HDCT and non-selected stem cell transplants. Preliminary analysis of patient histories indicate that there is a statistically significant difference in the predicted disease free interval (time to relapse) between early stage breast cancer patients treated in accordance with the teaching of the present invention and early stage patients treated with high dose chemotherapy and non-selected transplants. Generally, the estimated median time from transplant to relapse was not reached with a median follow up of 707 days in patients receiving treatments as disclosed in the present invention versus an estimated median of 807 days to relapse for patients receiving non-selected stem cell transplants in conjunction with HDCT with a median follow-up of 674 days. Therefore, the present invention represents a significant and unexpected advance in the treatment of early stage breast cancer.

It is apparent that while a preferred embodiment of the invention has been shown and described, various modifications and changes may be made without departing from the true spirit and scope of the invention.

Claims

What is claimed is:

1. A selected autologous hematopoietic stem cell suspension in combination with high dose chemotherapy for use as a medicament for treating early stage breast cancer.

2. A selected autologous hematopoietic stem cell suspension in combination with high dose chemotherapy for use as a medicament for extending the time until relapse in cancer patents.

3. The medicament of claims 1 or 2 wherein said hematopoietic stem cell suspension includes bone marrow aspirates, peripheral blood and leukapheresed peripheral blood.

4. The medicament of claim 3 wherein said hematopoietic stem cell suspension is selected using an antibody-base selection apparatus.

5. The medicament of claim 4 wherein said antibody-based selection aparatus is the Nexell Therapeutics Isolex® 300/ Magnetic Cell Separator System.

6. The medicament of claim 3 wherein said hematopoietic stem cell suspension is mobilized in the donor using a compound selected from the group consisting ifosfamide 4 g/m², cisplatin 50mg/m², etoposide 500 mg/m²; cyclophosphamide 4g/m², placitaxel 170 mg/m²; cyclophosphamide 5g/m²; cyclophosphamide 3g/m², epirubicin 100mg/m²;

7 7 7 cyclophosphamide 4g/m , etoposide lg/m ; and cyclophosphamide 2g/m , etoposide 450 mg/m².

7. The medicament of claim 3 wherein said hematopoietic stem cell suspension is mobilized in the donor using a compound selected from the group consisting of granulocyte colony stimulating factor, granulocyte-monocyte/macrophage-colony stimulating factor, stem cell factor PIXY 321 and stem cell factor flt-3.

8. The medicament of claim 7 wherein said granulocyte colony stimulating factor is recombinant human granulocyte colony stimulating factor at concentrations ranging from 5 μg/kg/day to lOμg/kg/day.

9. The medicament of claims 1 or 2 wherein said high does chemotherapy is selected from the group consisting of cyclophosphamine 6 g/m², thiotepa 500 mg/m², carboplatin 800 mg/m²;

7 7 7 cyclophosphamine 6 g/m , thiotepa 600 mg/m , carboplatin 800 mg/m ; cyclophosphamine 6 g/m², etoposide 625 mg/m², carboplatin 2 g/m²; busulfan 12 mg/kg, melphalan 100 mg/m², thiotepa 500 mg/m²; cyclophosphamine 120 mg/kg, mitoxantrone 45 mg/m², melphalan 140 mg/m²; adriamycin 165 mg/m², etoposide 60 mg/kg, cyclophosphamine 100 mg/kg; thiotepa 700 mg/m², mitoxantrone 40 mg/m², carboplatin 999mg/m²; and thiotepa 675 mg/m², cyclophosphamine 7.5 g/m².

10. The medicament of claim 2 wherein said cancer patients are early stage breast cancer patients.

11. The medicament of claims 1 or 10 wherein said early stage breast cancer patients are Stage II or Stage Ilia.

12. A method of treating an early stage breast cancer patient comprising the steps of:

(a) obtaining a hematopoietic stem cell containing sample from said early stage breast cancer patient;

(b) enriching said sample for said hematopoietic stem cells using a selection process so as to produce a selected stem cell transplant;

(c) administering a high dose chemotherapeutic regimen to said early stage breast cancer patient ; and

(d) infusing said early stage breast cancer patient with said selected stem cell transplant.

13. The method of treating an early stage breast cancer patient of claim 12 wherein in said hematopoietic stem cell sample is selected from the group consisting of bone marrow aspirates, peripheral blood and leukapheresed peripheral blood.

14. The method of treating an early stage breast cancer patient of claim 12 wherein said selection process comprises the Nexell Therapeutics Isolex® 300z Magnetic Cell Separator System.

15. The method of treating an early stage breast cancer patient of claim 12 wherein said high does chemotherapy and regimen is selected from the group consisting of cyclophosphamine 6 g/m², thiotepa 500 mg/m², carboplatin 800 mg/m²; cyclophosphamine 6 g/m², thiotepa 600 mg/m², carboplatin 800 mg/m²; cyclophosphamine 6 g/m², etoposide 625 mg/m², carboplatin 2 g/m²; busulfan 12 mg/kg, melphalan 100 mg/m², thiotepa 500 mg/m²; cyclophosphamine 120 mg/kg, mitoxantrone 45 mg/m², melphalan 140 mg/m²; adriamycin 165 mg/m², etoposide 60 mg/kg, cyclophosphamine 100 mg/kg; thiotepa 700 mg/m², mitoxantrone 40 mg/m², carboplatin 999mg/m²; and thiotepa 675 mg/m², cyclophosphamine 7.5 g/m².

16. The method of treating an early stage breast cancer patient of claim 12 further comprising the step of administrating a hematopoietic stem cell mobilization regimen to said early stage breast cancer patient prior to obtaining said hematopoietic stem cell containing sample.

17. The method of treating an early stage breast cancer patient of claim 12 further comprising the steps of administering recombinant human granulocyte colony stimulating factor after infusing said early stage breast cancer patient with,- said selected stem cell transplant.

18. The method of treating an early stage breast cancer patient of claim 16 wherein said stem cell mobilization regimen further comprises chemotherapy mobilization regimens

7 7 selected from, the group consisting of ifosfamide 4 g/m , cisplatin 50mg/m , etoposide 500 mg/m²; cyclophosphamide 4g/m², placitaxel 170 mg/m²; cyclophosphamide 5g/m²;

7 7 7 7 cyclophosphamide 3g/m , epirubicin lOOmg/m ; cyclophosphamide 4g/m , etoposide lg/m ;

7 7 and cyclophosphamide 2g/m , etoposide 450 mg/m .

19. The mobilization regimen of claim 18 further comprising administering a compound selected from the group consisting of granulocyte colony stimulating factor, granulocyte- monocyte/macrophage-colony stimulating factor, stem cell factor PIXY 321 and stem cell factor flt-3.

20. The mobilization regimen of claim 19 wherein said granulocyte colony stimulating factor is recombinant human granulocyte colony stimulating factor that is administered 24 to 48 hours after chemotherapy mobilization regimens have been administered at concentrations ranging from 5 μg/kg/day to lOμg/kg/day.

21. The mobilization regimen of claim 19 wherein said recombinant human granulocyte colony stimulating factor is administered subcutaneously at concentrations between approximately from 5 μg/kg/day to lOμg/kg/day.

22. A method of treating an early stage breast cancer patient comprising the steps of:

(a) administrating a hematopoietic stem cell mobilization regimen to said early stage breast cancer patient;

(b) obtaining a hematopoietic stem cell containing sample from said early stage breast cancer patient;

(c) enriching said sample for said hematopoietic stem cells using a selection process so as to produce a selected stem cell transplant;

(d) administering a high dose chemotherapeutic regimen to said early stage breast cancer patient;

(e) infusing said early stage breast cancer patient with said selected stem cell transplant; and

(f) administering recombinant human granulocyte colony stimulating factor to said early stage breast cancer patient.

23. The method of treating an early stage breast cancer patient of claim 22 wherein in said hematopoietic stem cell sample is selected from the group consisting of bone marrow aspirates and leukapheresed peripheral blood.

24. The method of treating an early stage breast cancer patient of claim 22 wherein said selection process comprises the Nexell Therapeutics Isolex® 300/ Magnetic Cell Separator System.

25. The method of treating an early stage breast cancer patient of claim 22 wherein said high does chemotherapy and regimen is selected from the group consisting of cyclophosphamine 6 g/m², thiotepa 500 mg/m², carboplatin 800 mg/m²; cyclophosphamine 6 g/m², thiotepa 600 mg/m², carboplatin 800 mg/m²; cyclophosphamine 6 g/m², etoposide 625 mg/m², carboplatin 2 g/m²; busulfan 12 mg/kg, melphalan 100 mg/m², thiotepa 500 mg/m²; cyclophosphamine 120 mg/kg, mitoxantrone 45 mg/m², melphalan 140 mg/m²; adriamycin 165 mg/m², etoposide 60 mg/kg, cyclophosphamine 100 mg/kg; thiotepa 700 mg/m², mitoxantrone 40 mg/m², carboplatin 999mg/m²; and thiotepa 675 mg/m², cyclophosphamine 7.5 g/m².

26. The method of treating an early stage breast cancer patient of claim 22 wherein said stem cell mobilization regimen further comprises a chemotherapy mobilization regimens selected from, the group consisting of ifosfamide 4 g/m², cisplatin 50mg/m², etoposide 500 mg/m²; cyclophosphamide 4g/m², placitaxel 170 mg/m²; cyclophosphamide 5g/m²; cyclophosphamide 3g/m², epirubicin 100mg/m²; cyclophosphamide 4g/m², etoposide lg/m²; and cyclophosphamide 2g/m², etoposide 450 mg/m².

27. The mobilization regimen of claim 26 further comprising administering a compound selected from the group consisting of granulocyte colony stimulating factor, granulocyte-monocyte/macrophage-colony stimulating factor, and stem cell factor PIXY 321.

28. The mobilization regimen of claim 27 wherein said granulocyte colony stimulating factor is recombinant human granulocyte colony stimulating factor that is administered 24 to 48 hours after chemotherapy mobilization regimens have been administered at concentrations ranging from 5 μg/kg/day to lOμg/kg/day.

29. The mobilization regimen of claim 27 wherein said recombinant human granulocyte colony stimulating factor is administered subcutaneously at concentrations between approximately from 5 μg/kg/day to lOμg/kg/day.

30. A method for extending the time until relapse in cancer patients comprising combining high dose chemotherapy and selected stem cell transplant.

31. The method of claim 30 wherein said cancer patients are early stage breast cancer patients.

32. The method of claim 31 wherein said early stage breast cancer patients are Stage II or Stage Ilia.

33. The method of claim 30 wherein said selected stem cell transplant is produced using the Nexell Therapeutics Isolex® 300t Magnetic Cell Separator System.