After the effects of radiation on hematopoiesis became evident during World War II, Jacobson and colleagues reported that mice survived an otherwise lethal exposure to TBI if the spleen was shielded (Fig. 102.1).² Radiation protection was also conferred by infusion of bone marrow.³ A runting syndrome developed after recovery of hematopoiesis when the infused marrow was from a donor of a different mouse strain.⁴ This syndrome was due to GVHD, a complication that was soon recognized to limit the use of allogeneic marrow transplantation in humans. In further studies in mice, methotrexate and 6-mercaptopurine were found to be effective in inducing immune tolerance or ameliorating the graft-versus-host (GVH) reaction.⁵

The dog served as a random-bred large animal model for studies of principles and techniques of bone marrow transplantation applicable to humans. The dog was the first random-bred species in which it was demonstrated that the results of in vitro histocompatibility typing could predict the outcome of marrow transplantation.⁶ Littermates genotypically identical for the major histocompatibility complex (MHC) survived longer after marrow transplantation than did their MHC-nonidentical siblings. However, despite the MHC genotypic identity, GVHD was still potentially severe in many but not all dogs. This indicated that other factors identified as minor histocompatibility antigens (mHC) were involved in the development of GVHD. Pharmacologic immunosuppression with a calcineurin inhibitor
(i.e., cyclosporine or tacrolimus) or methotrexate for prevention of GVHD improved survival after allogeneic marrow grafting.⁷, ⁸ It was then established that methotrexate and cyclosporine in combination were more effective than either used alone.⁹ The efficacy of a calcineurin inhibitor and methotrexate combined for GVHD prevention was subsequently confirmed in clinical trials and remains the standard in many transplant programs.

Bone marrow was the first commonly used source of HSC for transplantation. Bone marrow transplantation from human leukocyte antigen (HLA)-identical sibling donors was first successfully used by two groups in 1968 to treat patients with immunologic deficiencies.¹⁰, ¹¹ After extensive preclinical studies of GVHD, Thomas et al. then reported the successful transplantation of marrow from a HLA-identical sibling donor for aplastic anemia in 1972.¹² Five years later, the same group reported their experience in 100 patients with end-stage leukemia treated with allogeneic marrow transplantation.¹³ Allogeneic HCT from an HLA-matched related or unrelated donor is now considered standard therapy for many malignant and nonmalignant hematologic diseases.

The CD34 antigen is a cell surface type 1 transmembrane protein which is highly O-glycosylated and expressed primarily on hematopoietic progenitor cells and vascular endothelium from many tissues.²⁹, ³⁰, ³¹ It is also expressed on stromal cell precursors identified by the STRO-1 antibody. Cell surface expression of CD34 is developmentally regulated in hematopoiesis and is inversely related to the stage of differentiation such that CD34 expression is absent beyond the committed progenitor stage. The functional significance of CD34 expression on hematopoietic progenitor cells, stromal cells, and developing blood vessels is unknown, except that CD34 on vascular endothelial cells binds to L-selectin.³² The CD34 antigen is expressed on 1% to 5% of normal human adult marrow cells, up to 1% of mobilized peripheral blood cells, and by 2% to 10% of normal fetal liver and marrow cells.²⁹, ³³ Approximately 90% to 95% of the CD34-positive cells express antigens indicating commitment to the lymphoid or myeloid lineages.³⁴, ³⁵ Purified populations of HSC can be obtained with strategies that lineage-deplete a CD34-positive population of cells using monoclonal antibodies specific for DR, CD33, CD38, CD71, and B and T cell markers (Table 102.2). Other work suggests that human HSC are Thy-1^low, c-KIT^low, Rhodamine^{123 low}, and CD133-positive.³⁶, ³⁷, ³⁸, ³⁹, ⁴⁰, ⁴¹ In vivo preclinical models for studying populations of purified human HSCs based on the aforementioned characteristics include transplantation into SCID-Hu mice or into fetal sheep.⁴⁰, ⁴², ⁴³

Enriched populations of autologous CD34-positive marrow cells or blood cells, in both animal and human studies, have been shown to protect from myeloablative doses of radiation or chemotherapy.²², ⁴⁴, ⁴⁵ Conversely, the CD34-negative subset of the marrow was not protective.⁴⁶, ⁴⁷ Complete and stable donor hematopoietic chimerism has also been shown in humans after transplantation with allogeneic CD34-enriched cells from the peripheral blood.²⁴, ⁴⁸ Thus, CD34-positive hematopoietic cells are capable of long-term stable reconstitution of multiple hematopoietic lineages.

In the developing human embryo, the production of hematopoietic cells shifts to the liver from the yolk sac after 6 weeks of gestation. At 16 weeks of gestation, the most active site of hematopoiesis is the fetal liver. At the end of gestation, essentially all hematopoietic production derives from the marrow with only small contributions from the liver and spleen. Umbilical cord blood is enriched for HSC. The number of progenitors in one cord blood unit were comparable to the number of progentiors from adult marrow that had been reported to achieve successful engraftment.⁴⁹

Historically, the bone marrow served as the routine collection site for HSC.⁵⁰ Marrow is a clinically reliable and easily accessible source of long-term reconstituting cells. The presence of HSC in peripheral blood was first documented in preclinical studies.⁵¹, ⁵² In early clinical studies, transplantation of autologous peripheral blood HSC resulted in reconstitution of hematopoiesis following myeloablative chemotherapy or chemoradiotherapy; however, obtaining sufficient HSC required a prolonged period of collection.⁵³ To overcome this problem, granulocyte-colony-stimulating factor (G-CSF) can be administered to mobilize HSC from the marrow to the peripheral blood. Collections from G-CSF-mobilized peripheral blood yielded similar or greater numbers of HSC than that harvested from marrow.⁵⁴ Combining chemotherapy and G-CSF administration for mobilization resulted in higher yield of CD34-positive cells than G-CSF alone.⁵⁵ Plerixafor is a small molecule that reversibly inhibits chemokine stromal cell-derived factor-1α binding to its cognate receptor CXC chemokine receptor 4. In a randomized clinical trial, G-CSF with plerixafor was more effective than G-CSF alone for collection of stem cells in patients with myeloma.⁵⁶ In this study, a total of 54% of plerixafor-treated patients collected a target number of 6.0 × 10⁶ CD34⁺ cells/kg after one apheresis, whereas 56% of placebo-treated patients required 4 daily aphereses to achieve this target. Other cytokines have been demonstrated to mobilize HSC into peripheral blood, including stem cell factor, granulocyte/macrophage-CSF, inter-leukin-6 (IL-6), IL-8, and flt-3 ligand.⁵⁷, ⁵⁸ While most normal donors receive only G-CSF for mobilization of HSC for allogeneic transplant, mobilization strategies including plerixafor or chemotherapy are now routinely used for the collection of autologous HSC from the peripheral blood of patients with hematologic malignancy.⁵⁹, ⁶⁰, ⁶¹ Plerixafor is not indicated for use in patients with acute leukemia.

Monoclonal antibodies specific to the adhesion molecule VLA-4 (very late antigen-4 or α4β1 integrin) and VCAM-1 (vascular cell adhesion molecule-1) can also mobilize hematopoietic progenitors in nonhuman primates.⁶², ⁶³ An essential step contributing to the release of hematopoietic progenitors from the marrow may be the cleavage of VCAM-1 expressed on stromal cells by neutrophil proteinases following the administration of G-CSF.⁶⁴ Hematopoietic progenitors reversibly downregulate VLA-4 expression and adhere significantly less to stroma and fibronectin during mobilization.⁶⁵ VLA-4 integrin expression is restored after progenitors are removed from the in vivo mobilizing milieu, which may account for their homing properties after transplantation.

Autologous marrow or mobilized peripheral blood stem cells are the grafts of choice for many patients. Autologous stem cell support is most commonly derived from “mobilized” peripheral blood stem cells instead of marrow because of faster hematopoietic recovery in the recipient.⁶⁰ Peripheral blood stem cell transplants have decreased the time required for hospitalization. Autologous stem cell support after myeloablative therapy has been successful for treatment of acute myelogenous leukemia (AML), non-Hodgkin lymphoma, and Hodgkin lymphoma.⁷⁶, ⁷⁷, ⁷⁸, ⁷⁹ Disease-free survival was prolonged in patients with multiple myeloma receiving a single autologous or two sequential autologous transplants.⁸⁰, ⁸¹ Significant transplant-related complications encountered most frequently include infections and organ toxicity of the liver or lung, caused in large part by the high-dose myeloablative cytotoxic therapy. A GVHD-like syndrome (also known as the engraftment syndrome or pseudo-GVHD) of the skin and gastrointestinal tract has been described, but it is generally not frequent or severe.⁸², ⁸³

Marrow is obtained by multiple aspirations from the posterior iliac crest under general or epidural anesthesia.¹²³ The anterior iliac crest (or the sternum) may also be harvested if larger quantities of marrow are required. The target volume of marrow for transplant is 10 to 15 ml/kg of recipient or donor weight, whichever is the smaller individual. Marrow is collected with heparinized syringes through large bore needles and placed into small amounts of culture medium. The collected marrow must be filtered prior to intravenous transfusion into the recipient to remove small particles or clots. If the patient and donor are ABO-incompatible and there are high anti-A or -B titers, the marrow can be red blood cell-depleted or plasma-depleted, depending on whether it is a major or minor ABO mismatch.¹²⁴ In some cases of a major ABO mismatch, plasmapheresis of the recipient may be effective in reducing the high anti-A or anti-B titers so that RBC depletion of marrow is not required. Marrow is infused immediately after harvesting, but delays of 24 hours may occur without adverse consequences. Such delays are common when marrow is shipped to a transplant program after harvest from an unrelated donor. In an analysis of marrow harvest characteristics of 1,549 donors, the median total nucleated cell count from the marrow was 2.5 (range 0.3 to 12.0) × 10⁸/kg recipient weight.¹²⁵ Life-threatening complications from marrow harvesting, usually related to the administration of anesthesia, were reported in 0.27% to 0.4% of the donors.¹²⁶

HSC circulate in the peripheral blood but the concentration is very low and it requires multiple aphereses to collect adequate
numbers. The number of aphereses may be reduced to one or two sessions when HSC are mobilized to the peripheral blood after the administration of cytokines alone or in combination with chemotherapy or plerixafor. An effective mobilization strategy of autologous HSC for patients with malignancy is chemotherapy in conjunction with G-CSF, 6 µg/kg/day.⁶¹ After chemotherapy, patients are apheresed when the total white blood cell count has recovered to 1,000/µl or the CD34-positive cell count in the peripheral blood is at least >10/µl. For patients not requiring chemotherapy or normal allogeneic donors, mobilization is with G-CSF alone (5 to 16 µg/kg) by daily subcutaneous injections for 5 to 8 days.⁴⁸, ¹²⁷, ¹²⁸ These doses of G-CSF are generally well tolerated, with common side effects of bone pains, myalgias, and flu-like symptoms that are managed with acetaminophen or low-dose narcotics. Plerixafor in combination with G-CSF was effective for increasing the yields of circulating CD34⁺ progenitor cells and is indicated for patients with lymphoma or multiple myeloma who are being collected for autologous HCT.⁵⁶ The recommended dose is 0.24 mg/kg/day administered subcutaneously 11 hours before the apheresis procedure. The maximum dose is 40 mg/day. Common side effects included diarrhea, nausea, fatigue, headaches, and arthralgias. Apheresis was performed as early as day 4 after the start of G-CSF using a continuous blood flow separation technique. Apheresed products may be cryopreserved in 5% dimethylsulfoxide (DMSO) for use after thawing on the day of transplant. A more rapid sustained hematopoietic recovery of both neutrophil and platelet counts occurs with increasing numbers of CD34⁺ cells in the hematopoietic cell graft (up to 5 × 106/kg). Some investigators consider 2.5 × 10⁶/kg of recipient weight a minimum dose of CD34⁺ cells from the peripheral blood to achieve complete autologous recovery. Platelet recovery is more rapid at higher CD34⁺ cell doses.⁶⁰, ⁶¹ Since the cell dose used in the autologous transplant setting yields consistent and prompt engraftment, it is also considered an appropriate target for collection of allogeneic HSC from the peripheral blood. Donors of peripheral blood avoid general anesthesia and other complications of marrow harvesting. If peripheral veins are inadequate, a large bore double lumen catheter for vascular access may be required. In a large analysis of safety from the NMDP (n = 2,408 donors), it was concluded that collection of peripheral blood stem cells was safe but that nearly all patients will experience bone pain and 1 in 4 will have headache, nausea, or citrate toxicity.¹²⁹ Serious and unexpected toxicities were experienced by 0.6% of the donors, but complete recovery was universal.

Umbilical cord blood cells are now being routinely collected and cryopreserved for storage in a cord blood bank.¹³⁰, ¹³¹ Directed-donor banking of cord blood for siblings in a current good tissue practices environment has also now been reported.¹³² After the separation of the placenta, umbilical cord blood cells are collected into a closed system which utilizes a sterile donor blood collection set. The placenta and umbilical cord can be suspended on a frame and the blood drained as a “standard gravity phlebotomy” into CPD (citrate, phosphate, dextrose) anticoagulant. The median volume of umbilical cord blood collected in one study of 44 patients was 100 ml (range 42 to 282 ml).¹⁰⁴ The median number of total nucleated cells per kilogram of recipient weight for banked umbilical cord blood is 2.5 × 10⁷ (range 1 to 33) and corresponds to a CD34⁺ cell dose of 1.5 × 10⁵ per kilogram of recipient weight.¹¹⁰

Myeloablative conditioning regimens are sufficiently intense that recovery of hematopoiesis would not be expected without the support of transplanted hematopoietic progenitor cells. The ideal myeloablative conditioning regimen should fulfill the following criteria: (1) eliminate or reduce the tumor load; (2) suppress the host immune system to prevent graft rejection (not applicable to hematopoietic support with autologous cells); and (3) have tolerable nonhematopoietic toxicity. The first conditioning regimens consisted of TBI, alone or in combination with cyclophosphamide (CY).⁵⁰ TBI is an effective antineoplastic modality that is both cell cycle nonspecific and immunosuppressive. CY is a chemotherapeutic agent with immunosuppressive properties that has few nonhematopoietic toxicities that are similar to TBI, and therefore can be used in combination. Other conditioning regimens which have since been developed include: (1) the replacement of CY with other chemotherapeutic agents (e.g., etoposide, Ara-C, and melphalan) in combination with TBI.¹³⁸, ¹³⁹; (2) other chemotherapeutic agents in combination with both CY and TBI¹⁴⁰, ¹⁴¹; (3) chemotherapeutic agents used in combination with CY but without TBI, including busulfan (BU)/CY or carmustine-cyclophosphamide-etoposide (BCV)¹⁴², ¹⁴³; and (4) replacement of CY with fludarabine and used in combination with BU or melphalan.¹⁴⁴, ¹⁴⁵ In one study, BCV was associated with a significant incidence of transplant-related complications and mortality.¹⁴⁶ Other high-dose cytotoxic regimens have been used, especially with autologous stem cell support.¹⁴⁷, ¹⁴⁸

Most preparative regimens used for the treatment of malignant diseases have not been tested in Phase III studies, so it is generally unclear if any one regimen represents an improvement over those previously used. Two Phase III studies in allogeneic marrow transplantation have compared differing intensities of TBI
(1,200 cGy versus 1,575 cGy).¹⁴⁹, ¹⁵⁰ The relapse rate was reduced in the group of patients receiving the higher dose of TBI, but was associated with an increase in complications from regimen-related toxicity and GVHD which negated any improvement in disease-free survival compared to the group receiving the lower dose of TBI. In a more recent study, conditioning with TBI 800 cGy and fludarabine was compared with TBI 1,200 cGy and CY and there was no difference in overall survival, relapse, or treatment-related mortality.¹⁵¹ The combination of BU/CY was determined to be an acceptable alternative to CY/TBI for patients with leukemia in several studies.¹⁵², ¹⁵³, ¹⁵⁴ However in one study, patients in the BU/CY group had an increased risk of sinusoidal obstruction syndrome (SOS) of the liver and other transplant-related complications.¹⁵⁵ Since this last study, it has been demonstrated that targeting of busulfan levels in the plasma may decrease the risk of relapse and severe regimen-related toxicities, contributing to an improved disease-free survival (Fig. 102.4).¹⁵⁶ Monitoring levels of metabolites from cyclophosphamide may also be important to decrease the risk of liver toxicity and nonrelapse mortality.¹⁵⁷, ¹⁵⁸

In patients already profoundly immunosuppressed with SCID syndrome, engraftment of allogeneic stem cells from matched related siblings may occur without conditioning therapy.¹⁵⁹ High-dose CY in combination with antithymocyte globulin (ATG) as a preparative regimen for patients with aplastic anemia was associated with graft rejection in less than 5% of cases.⁹⁰ The actuarial survival rate was 88% at 6 years after transplantation (Fig. 102.3). For transplantation of patients with thalassemia or sickle cell disease, a myeloablative conditioning regimen, usually consisting of the combination of BU and CY, was thought to be necessary to prevent a high incidence of graft rejection, since most patients with these disorders had received multiple blood transfusions and were potentially sensitized to donor-derived mHC.¹⁶⁰, ¹⁶¹ However, reduced intensity conditioning regimens have now been shown to successfully overcome the risk of graft rejection associated with these hematologic disorders.¹⁶²