Following the delivery of high-dose chemotherapy to patients for the treatment of cancer, infusion of stem cells is necessary to ensure recovery of bone marrow function and production of red blood cells, white blood cells and platelets. Historically, stem cells were harvested from bone marrow, but more recently, many cancer centers have adopted the practice of collecting stem cells from peripheral blood.
Techniques of Stem Cell Collection/Harvesting
The collection of stem cells from bone marrow has been safely performed for over 30 years. A bone marrow harvest is relatively simple and typically occurs in the operating room. During a bone marrow harvest, patients receive general anesthesia and then a surgeon inserts a large needle directly into the bone marrow cavity of bones of the lower back after the area has been sterilized. Bone marrow is aspirated or sucked out of the bones by inserting the needle into the bone multiple times. A typical bone marrow harvest takes about two hours and involves the removal of one liter of bone marrow containing the stem cells. The major side effect of this procedure is discomfort at the site of the bone marrow harvest. Infrequent complications include bleeding, infection and nerve damage.
Peripheral Blood Stem Cell Harvesting
The collection of stem cells from the blood is slightly more complicated than collection from bone marrow and has been performed safely for over a decade. Collecting stem cells from the peripheral blood may also have several clinical advantages compared to collecting them from bone marrow.
Stem cells normally circulate in the blood in very small quantities and can be collected from the blood through a small catheter inserted into a patient’s vein. The number of circulating stem cells in the blood is increased in patients whose bone marrow is recovering from chemotherapy. Cytokines (blood cell growth factors) administered to patients after myelosuppressive chemotherapy can also cause a 100-fold increase in the number of stem cells circulating in the blood. Injection of cytokines stimulates increased production of immature and mature bone marrow stem cells and their release into the blood where they can be collected. Cytokines can also be administered without chemotherapy and cause a substantial increase in the number of circulating blood stem cells for collection. The process of delivering a cytokine or growth factor with or without myelosuppressive chemotherapy for the purpose of collecting stem cells is referred to as “stem cell mobilization”. Two cytokines, Neupogen® and Leukine™, stimulate the bone marrow’s production of stem cells and are approved by the Food and Drug Administration for use in patients to increase the number of circulating stem cells and several others are in development.
During stem cell mobilization, patients receive an injection of a cytokine and are evaluated daily. The process of actually collecting the stem cells from the blood is called apheresis and this begins when there are sufficient stem cells circulating in the blood for collection. Stem cells are collected with an apheresis machine from the blood flowing through a catheter inserted into a vein. Blood flows from a vein through the catheter into the apheresis machine, which separates the stem cells from the rest of the blood and then returns the blood to the patient’s body. Apheresis is performed for several days until enough stem cells have been collected to support treatment with high-dose chemotherapy. Most donors have sufficient stem cells collected with 2-4 days of apheresis
Stem cells can be reliably identified and accurately measured because they have a specific marker or label on the stem cell surface. This marker is referred to as theCD34 antigen. Measuring the number of CD34 antigen-positive stem cells is important because doctors can accurately predict how fast the bone marrow recovers after high-dose chemotherapy administration based on the number of CD34 positive stem cells infused. Daily measurement of the CD34+ peripheral blood stem cell content is also useful for determining the number of days to perform apheresis.
An optimal number of stem cells to support rapid bone marrow recovery and blood cell production after treatment with high-dose chemotherapy is approximately 5 million CD34+ cells/kg patient weight. Infusion of over 5 million cells/kg results in the majority of patients recovering bone marrow blood cell production in only 10-21 days. The minimal number of allogeneic stem cells necessary to ensure safe recovery of bone marrow blood cell production is currently unknown.
Bone Marrow or Peripheral Blood Stem Cells?
Stem cells collected from blood are associated with more rapid bone marrow recovery and greater ease of collection than stem cells collected from bone marrow. In the early 1990′s, doctors began evaluating whether a growth factor ( Neupogen®) administered to normal donors could mobilize enough stem cells into the blood for collection and support of an allogeneic stem cell transplant. Comparisons evaluating stem cells collected from bone marrow and stem cells collected from peripheral blood have been performed in patients undergoing allogeneic stem cell transplant. Patients infused with stem cells collected from blood have faster recovery of bone marrow blood cell production; fewer red blood cell and platelet transfusions; and shorter admissions to the hospital than patients treated with allogeneic stem cells collected from bone marrow.
In patients undergoing allogeneic stem cell transplant, Neupogen®-mobilized stem cells also appear to provide more rapid bone marrow recovery compared to stem cells collected from bone marrow. The main side effect stem cell donors experience is mild bone pain from the Neupogen injection. Additionally, Neupogen® mobilization of blood stem cells also results in many more T-lymphocytes being collected. The increased numbers of T-lymphocytes could make graft-versus-host disease worse or have an anti-cancer effect. In order to determine the best source of stem cells for allogeneic stem cell transplant, clinical trials are currently being performed that directly compare stem cells collected from blood or bone marrow.
Physicians at The Fred Hutchinson Cancer Center, City of Hope and Stanford University performed a randomized clinical trial comparing allogeneic bone marrow transplantation to peripheral blood stem cell transplantation in patients with leukemia and lymphoma. The results of this study were presented at the 1999 American Society of Hematology Annual Meeting. The study had a planned accrual of 200 patients, but was terminated after entry of only 138 patients because of improved survival in the patients with advanced leukemia and lymphoma receiving peripheral blood stem cells compared to bone marrow transplants.
Patients receiving peripheral blood stem cells experienced more rapid recovery from treatment than patients receiving bone marrow transplants. White blood cell counts recovered 5 days earlier and platelets recovered 8 days earlier. There were more deaths in patients receiving bone marrow due to lung complications, infections and cancer recurrence. This occurred predominantly in patients with more advanced cancers. There was no difference in the incidence of acute graft-versus-host disease and there was an increase in the incidence of chronic graft-versus-host disease of approximately 10% in patients receiving peripheral blood stem cells. The follow-up period for this study is too short to make definite conclusions about the incidence and severity of chronic graft versus host disease.
The physicians concluded that allogeneic peripheral blood stem cells were superior to bone marrow stem cells. They are continuing to enroll good-risk patients on the study in order to clarify whether or not this benefit is limited only to patients with advanced cancer. However, there were no suggestions that allogeneic peripheral blood stem cells produced inferior results in patients with less advanced cancer.
Stem Cell Processing
A typical stem cell collection is unmodified and contains red blood cells, immune cells and stem cells when it is processed. The stem cell collection however, can be modified with the intent of improving treatment of cancer. With allogeneic stem cell transplantation, T-lymphocytes are an immune cell present in the stem cell collection and are responsible for causing graft-versus-host disease in the patient after infusion of the stem cells.
In the 1980′s, many cancer centers developed techniques for the removal of T-lymphocytes from bone marrow stem cell collections in order to reduce the severity of graft-versus-host disease. This was mainly accomplished by mixing the stem cells with monoclonal antibodies that recognized T-lymphocytes. This process was referred to as T-cell depletion and the removal of T-lymphocytes did decrease the incidence of graft-versus-host disease in patients undergoing allogeneic stem cell transplant. Unfortunately, removal of T-lymphocytes also caused an increased risk of graft failure and doctors learned that some T-lymphocytes are necessary for bone marrow engraftment.
Techniques for removal of specific T-lymphocytes from stem cell collections are now available. This process is very appealing because specific groups of T-lymphocytes can be removed or even added back based on the number of cells necessary to achieve the desired effect in the patient. For example, the cells responsible for graft-versus-host disease could be removed and those necessary for engraftment could be infused into the patient.
Scientists have also discovered that stem cells have certain markers (antigens) on their surface that distinguish them from other cells. One of the main antigens on stem cells is the CD34 antigen and positive selection is one technique that has been developed for the separation of stem cells from other cells. CD34 selection uses a device that binds the CD34 positive stem cells and removes them from the other cells in the stem cell collection. CD34 positive selection devices are capable of removing large numbers of non-specific T-lymphocytes from the stem cell product. Unfortunately, they also remove 25%-50% of the stem cells, immune cells and other cells. Many cancer centers are using CD34 positive selection devices and other techniques of stem cell processing in an attempt to change the cell content of stem cell collections in order to improve the safety and potential benefit of allogeneic stem cell transplant.
Cellular Therapy and Enhancement of Immunity after Stem Cell Transplant
Allogeneic stem cell transplants are more effective in preventing cancer recurrences than autologous transplants because the donor cells recognize the cancer as foreign and kill cancer cells immunologically. Despite this graft-versus-leukemia reaction, many patients still experience a cancer recurrence. Several different approaches that attempt to enhance this graft-versus-leukemia effect are currently being evaluated.
Donor White Blood Cell Infusions: In patients who do not have graft-versus-host disease, infusions of white blood cells from the donor are being evaluated to prevent or treat cancer recurrences that occur after allogeneic stem cell transplant. In some studies, donor white cells are combined with a biologic response modifier such as interleukin-2 to further enhance the graft-versus-leukemia reaction.
Lymphocytes are white blood cells that are part of the body’s immune system and are capable of destroying cancer cells. Doctors have been trying for several years to use lymphocytes reactive specifically against cancer cells as a form of treatment. For many reasons, this has been a difficult goal to achieve. First, billions of lymphocytes are needed in order to have a therapeutic effect because it takes several lymphocytes to kill a single cancer cell. Thus, in order for lymphocyte infusions to be practical therapy, extremely large numbers of specific immune lymphocytes need to be produced. Getting lymphocytes to grow and multiply in culture systems outside the body has been difficult. Second, the lymphocytes grown in culture have to be specifically reactive to the cancer cell that has to be killed. Lymphocytes normally attack and kill a variety of foreign invaders, but each lymphocyte is specific and only kills one target and no other. Third, the immune lymphocytes must survive and not be destroyed when infused into a patient with cancer.
White blood cells can be collected in large numbers from the original, healthy donor of stem cells by apheresis and then infused back into the relapsed patient. This procedure, known as Donor Leukocyte Infusion, or DLI, is a rapidly growing practice that, strikingly, has resulted in 70% to 75% complete remission rates in chronic leukemia patients. Unfortunately, the standard DLI procedure is associated with very high rates of severe and potentially fatal GvHD.
Unfortunately, the use of donor lymphocytes can also be associated with the development of graft-versus-host disease. Several recent studies, however, suggest that the risk for developing graft-versus-host disease may be decreased if a specific type of lymphocyte, the CD8 lymphocyte, is removed. Until now, there has not been an effective and efficient way to remove, or deplete, these CD8 cells from the other donor lymphocytes. Just recently, European researchers presenting at the 2000 European Group for Blood and Marrow Transplantation meeting in Austria reported the use of a new technique to deplete the CD8 lymphocytes from the donor cells that are to be infused into the patient.
Researchers treated 9 patients who experienced a recurrence of leukemia after undergoing high-dose therapy and an allogeneic stem cell transplantation. The researchers collected lymphocytes from the respective donors. They were able to remove 98 to 100% of the CD8 lymphocytes from the donor lymphocyte samples, while still retaining 75% of the other lymphocytes needed to treat the recurrent leukemia. The donor CD8-depleted lymphocytes were then infused into the corresponding patients. The results showed that all patients with CML had a complete response to treatment, one patient with CLL had a partial response to treatment and the other five patients had a stabilization, or halt in progression, of their disease. Only one of the nine patients developed graft-versus-host disease, a number much lower than would usually occur if CD8 lymphocytes were not depleted from the infusion.
These researchers concluded that the depletion of CD8 lymphocytes from the other donor lymphocytes by high-density microsphere separation appears to be effective. Furthermore, the CD8-depleted donor lymphocyte infusion appeared to decrease the incidence of graft-versus-host disease, while preserving the therapy’s anti-leukemia effect.
Recently, doctors in Holland have been able to grow and expand lymphocytes outside the body that kill leukemia cells without damaging normal cells. They have now infused these lymphocytes into a patient with leukemia who had relapsed after an allogeneic bone marrow transplant. Following infusion of the lymphocytes, this patient achieved a complete disappearance of leukemia. This may represent the first time expanded T-lymphocytes have been shown to have a beneficial anti-cancer effect when infused into a patient. This observation is important because the technique can potentially be used against a variety of cancers and offer a less toxic and more specific approach to cancer therapy.
Ex Vivo Expansion
During an allogeneic bone marrow or blood stem cell transplant, it is necessary to harvest or collect a relatively small number of “stem cells” to repopulate the bone marrow after it has been destroyed by radiation therapy and/or high-dose chemotherapy. In some instances, it is difficult to collect enough stem cells to provide rapid and safe recovery of bone marrow function. In the past, bone marrow was obtained by several hundred needle punctures from the hip bones under general anesthesia. More recently, stem cells have been obtained from the peripheral blood through a procedure called apheresis. Collection of blood stem cells requires one or more apheresis procedures to obtain enough stem cells for a transplant, but is relatively safe and does not require general anesthesia. However, apheresis requires access to two large veins, which is not always possible, especially in small patients, without the placement of special catheters. Some stem cell donors, especially umbilical cord blood, contain insufficient stem cells for optimal recovery without stem cell transplant.
For the past two decades, doctors have been working on ways to get small quantities of bone marrow or stem cells to grow in a culture system outside the body. If small quantities of stem cells could be expanded in a culture system as they are in the body, then the problems of collecting stem cells from bone marrow or blood or of transplanting insufficient numbers of cells could be avoided. The procedure for growing stem cells outside the body is called ex-vivo expansion. Over the years, doctors have discovered the hormones that tell stem cells to divide and multiply. They can now add these hormones to a sterile culture system outside the body. Thus, one could place small numbers of stem cells in a culture system with the appropriate hormones and produce a lot of “stem cells” suitable for transplantation. The results of clinical trials now suggest that small quantities of stem cells collected from umbilical cord blood can be ex-vivo expanded and facilitate stem cell transplantation.