Following the delivery of high-dose chemotherapy 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, however many cancer centers have recently adopted the practice of collecting stem cells from peripheral blood. Autologous stem cells must be collected, or harvested, from a patient prior to treatment with high-dose chemotherapy. The harvested stem cells are then frozen and can be stored for many years. Stem cells can also be processed in ways that remove cancer cells and/or attempt to activate immune cells in the stem cell collection for the purpose of treating the cancer.
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. A surgeon then inserts a large needle directly into the bone marrow cavity of bones of the lower back, which 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. This procedure 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. The main advantage of peripheral blood stem cells over bone marrow is that enough peripheral blood cells can be collected to support several courses of high-dose chemotherapy. This may have significant advantages for treatment of several blood cancers as well as solid tumors such as breast cancer.
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. Once released into the blood, stem cells 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. Several other cytokines 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—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, which is 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.
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 the CD34 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 in 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 nine to10 days. The minimal number of stem cells necessary to ensure safe recovery of bone marrow blood cell production is currently unknown. Patients who do not have enough stem cells harvested can undergo stem cell mobilization a second or third time. In the majority of cases, patients will and have enough stem cells to perform a transplant. If peripheral blood stem cells are harvested early in the disease course, sufficient stem cells can be collected to support multiple treatment courses.
Bone Marrow or Peripheral Blood Stem Cells?
Today, virtually all autologous stem cell transplants are performed with peripheral blood stem cells collected after mobilization with chemotherapy and Neupogen® or with Neupogen® alone. This is because peripheral blood stem cells are easier to harvest and result in more rapid recovery of blood cell counts.
Stem Cell Processing
A typical stem cell collection is unmodified and contains red blood cells, immune cells and stem cells when it is processed and stored. The stem cell collection, however, can be modified with the intent of improving treatment of cancer. Doctors have known for many years that stem cell collections from some patients also contain cancer cells. Many doctors believe that removal of the cancer cells from the stem cell collection could improve a patient’s chance of cure with high-dose chemotherapy and autologous stem cell transplant. Any method of removing cancer cells from the stem cell collection requires that enough cancer cells are removed to make a difference, while other cells important to the bone marrow or immune recovery of the patient remain.
Purging: Cancer cells can be removed from the bone marrow or peripheral blood stem cell collection by several techniques, each of which utilizes monoclonal antibodies that recognize and adhere to antigens on the cancer cells. Once the antibody attaches to the cancer cells, there are a number of ways these cells are eliminated from the stem cell product. In one such effective technique, the antibody is attached to high-density microparticles containing the heavy metal nickel. After stem cells are mixed with the high-density microparticles, the attached cells rapidly settle to the bottom of the disposable container due to the greater weight. They then can be separated and discarded, preserving the stem cells and leaving the lighter fraction depleted of virtually all the targeted cancer cells.
CD34 Selection: Mechanical techniques for removing cancer cells from stem cell collections began development in the early 1990s. Mechanical techniques were designed to remove or select only the stem cells from the stem cell collection. It was reasoned that it would be easier to remove a few stem cells necessary to support high-dose chemotherapy then attempt to kill or remove all the cancer cells in a stem cell collection. Once the stem cells were removed, the remaining cells, including the cancer cells, could be discarded.
In order to only remove stem cells, scientists had to first be able to reliably identify the stem cells. Once the stem cells could be identified, techniques could then be developed to remove the stem cells from the other cells in the stem cell collection. Scientists 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. Positive selection is one technique developed for the separation of stem cells from other cells. This method 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 have been evaluated in clinical trials. Although CD34 selection devices are capable of removing large numbers of cancer cells from the stem cell product, they also remove many stem cells and immune cells.
Ex Vivo Expansion
For the past two decades, many doctors have been working on ways to get small quantities of bone marrow 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 complications of collecting stem cells from bone marrow or blood could be avoided. 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. This culture system has an added advantage of not supporting the growth of the cancer cells. Thus, one could take small numbers of stem cells that contained cancer cells, place these cells in a culture system with the appropriate hormones and produce a significant number of stem cells that do not contain cancer and are suitable for transplantation.
Doctors at three U.S. medical centers have reported the first autologous transplants using expanded stem cells in the journal Blood. With local anesthesia, they obtained small samples of bone marrow from 19 patients with breast cancer and placed these cells in an expansion system for 12 days. These 19 patients received high-dose chemotherapy with cyclophosphamide, Paraplatin® and Thioplex® followed by the infusion of the expanded cells. The average time to recovery of blood counts was similar to that observed following bone marrow infusion, but was slower than has been observed following autologous peripheral blood stem cell infusion. However, this technique is associated with the infusion of more mature and functional white blood cells, which may be of added benefit to the patient in the first week after transplantation in preventing infection. One patient had cancer cells in the bone marrow before treatment, but cancer cells were not detected in the expanded stem cells that were infused after high-dose chemotherapy.
This clinical trial clearly demonstrates the potential for using expanded bone marrow stem cells for autologous transplantation. Currently, it is not clear who would preferentially benefit from this technique and who would be better served having a blood stem cell transplant. This would be a valuable technique if it could be successfully performed in patients who had damaged bone marrow from chemotherapy or radiation therapy and had insufficient stem cells to perform an autologous transplant. Patients with cancer in the bone marrow would benefit if cancer cells could be consistently eliminated by the culture technique. This technique can also be used to expand umbilical cord blood where the numbers of stem cells obtained from this source are inadequate for allogeneic transplantation into adults. Thus, this clinical trial could be a very important development in the field of transplantation and just the beginning of research in this area.