Research involving adult haematopoietic stem cells has progressed at a rapid pace, culminating in the clinical use of stem cells to treat human diseases. Adult haematopoietic stem cells have served to correct genetic blood disorders, e.g. immunodeficiency diseases and haemoglobinopathies, have been used to restore the acquired loss of marrow function such as in aplastic anaemia and have been used to eradicate malignant diseases such as leukaemias and lymphomas, whereby the infused stem cells serve to rescue the patient from chemoradiation therapy-induced marrow aplasia. These studies also discovered the concept of adoptive immunotherapy, an immunological reaction of donor lymphocytes contained in the graft resulting in killing recipient tumour cells, a phenomenon called graft-versus-tumour effect.
The advances in the field of haematopoietic cell transplantation (HCT) make this review timely. To appreciate the enthusiasm of researchers, clinicians and patients, it is important to reflect on how the field has developed and to document the efforts by numerous investigators who have worked to bring the experimental field of HCT to a clinical reality. This report will highlight the hallmark discoveries that have brought this field from one that was declared dead in the 1960s to the amazing clinical results obtained today. It will conclude with a look to future research in the field of allogeneic HCT.
Major research efforts on how to prevent or repair radiation effects were set in motion by the observations on radiation damage made in victims of the first atomic bomb explosions in Japan. Over the course of these studies, Leon Jacobson et al.1 at the University of Chicago (Chicago, IL, USA) made the pivotal observation in 1949 that mice could be protected from the lethal effects of X-irradiation on marrow by shielding their spleens with lead.1 A similar protective effect was achieved when shielding the femur. Two years later, Egon Lorenz et al.2 at the Oak Ridge National Laboratories in Tennessee (USA) observed that protection against irradiation in both mice and guinea pigs could also be accomplished by infusing marrow cells intravenously.2 Initially many researchers, including Jacobson, thought that the protection was achieved by some humoral factor present in the spleen or marrow that led to endogenous marrow recovery.1–3 However, by the mid-1950s, this ‘humoral hypothesis’ was rejected and the ‘cellular hypothesis’ was firmly established. A number of investigators, using various blood genetic markers, showed that the radiation protection was a result of colonization of the recipient marrow by donor cells.4–8
This discovery was greeted with excitement by haematologists, immunologists and radiation biologists because of its implications not only for cell biology, but also for therapy for patients with life-threatening haematological disorders. The principle of clinical marrow transplantation was as follows: high doses of chemotherapy or radiation therapy would be used both to eradicate the diseased marrow and to suppress the patient’s immune system so that a marrow graft from a healthy donor would be accepted. The marrow graft, in turn, would repopulate the empty marrow spaces. Within 1 year of the pivotal publications in mice and rats, Thomas et al.9 in Cooperstown (NY, USA) published a report in the New England Journal of Medicine showing that marrow could be safely infused intravenously into human patients with leukaemia and engraft, even though, in the end, the patients were not cured of leukaemia.9 In the following year, Mathé et al.10 in Paris attempted the dramatic rescue, by marrow transplantation, of six nuclear reactor workers who were victims of an irradiation accident. Four of the six workers survived although persistence of donor cells was only partial and transient.10 In 1965, Mathé et al.11 were the first to describe a successful allogeneic marrow graft in a patient suffering from leukaemia. The patient was given total body irradiation (TBI) and then ‘rescued’ by marrow infusions from six relatives. The marrow from one of the relatives engrafted. The physicians were able to show donor cell chimerism and antileukaemic effects. Unfortunately, although the leukaemia was kept at bay, the patient eventually died from a complication, which was subsequently called graft-versus-host disease (GVHD). Mathé coined the termed graft-versus-leukaemia effect, consistent with findings in leukaemic mice reported by Barnes et al.12 from the Harwell National Laboratories (Oxford, UK) about a decade earlier.
In 1970, Mortimer Bortin, a researcher from Milwaukee, summarized the outcomes of all human marrow grafts which had been reported between 1957 and 1967.13 In all, 200 patients were transplanted, 73 with aplastic anaemia, 115 with haematological malignancies and 12 with immunodeficiency diseases. None of these patients survived long term: 125 died from graft failure and 47 from GVHD, while others died from infections or recurrence of their underlying malignancies.
All of these transplants were performed before the discovery of histocompatibility matching and before the introduction of post-grafting immunosuppressive drugs. The clinical work was based directly on experimental work in inbred mice, for which histocompatibility matching is not an absolute requirement. The prominent experimental haematologist Dirk van Bekkum from the Radiobiological Laboratory TNO (Rijswijk, The Netherlands) stated in 1967, ‘these failures have occurred mainly because the clinical applications were undertaken too soon, most of them before even the minimum basic knowledge required to bridge the gap between mouse and patient had been obtained’.3 Clinical allogeneic HCT was declared a complete failure and a dead end, and prominent immunologists stated that the genetic barrier between individuals could never be crossed. van Bekkum and de Vries, in their landmark book Radiation Chimaeras, wrote that ‘these factors have caused many investigators to abandon the idea that bone marrow transplantation can ever become a valuable asset to clinical medicine’.3
Renewed efforts in preclinical animal models
Although most researchers left the field, pronouncing it a dead end, a small number of investigators persevered and continued working with experimental animals to understand and overcome the hurdles encountered in humans treated with allogeneic HCT. van Bekkum’s group at the Rijswijk Institute (Rijswijk, The Netherlands) chose a primate model, George Santos at Johns Hopkins University in Baltimore (MD, USA) chose rats and the Seattle group at the University of Washington (Seattle, WA, USA) chose random-bred dogs as experimental animals. The reasons for choosing the dog as a study animal were that, aside from humans, only dogs have that rare combination of unusual phenotypic diversity and a widespread, well-mixed gene pool. Additionally, dogs have many spontaneous haematological diseases in common with humans, such as non-Hodgkin lymphoma, severe congenital haemolytic anaemia and many others that could serve as pre-clinical models. In addition to determining the best way to administer TBI, new drugs were introduced that had myeloablative or immunosuppressive qualities comparable to radiation, such as cyclophosphamide and busulfan. These agents helped to improve graft acceptance and provided for tumour cell killing in a manner that compared well with TBI. Based on the mouse histocompatibility system identified 10 years earlier,14 a histocompatibility typing system for dogs was developed and results showing the great importance of in vitro histocompatibility matching for transplantation outcomes were reported for the first time in 1968. One report showed that dogs immunosuppressed by high-dose TBI and given grafts from dog leucocyte antigen (DLA)-matched littermates survived significantly longer than their DLA-mismatched counterparts.15 Another report showed similar findings with unrelated grafts, even though the histocompatibility typing techniques at that time were still quite primitive and the complexity of the genetic coding region for major histocompatibility antigens was not yet fully appreciated.16 Although serious GVHD was first described in major histocompatibility complex (H-2)-mismatched mice and in unrelated, randomly selected monkeys,3 the canine studies first drew attention to the fact that fatal GVHD could also occur after transplantation across minor histocompatibility barriers. This pivotal observation prompted the search for effective drug regimens delivered after transplantation to control the detrimental effects of GVHD. The first drug with the most promise in this regard was the folic acid antagonist methotrexate, which was used clinically during the 1970s.17 Further studies in dogs showed that transfusion-induced sensitization to minor histocompatibility antigens resulted in rejection of DLA-identical grafts.18,19 Continued experiments in dogs eventually led to understanding the mechanisms of transfusion-induced sensitization and identifying ways of preventing and overcoming it with the result that the risk of graft rejection was markedly reduced. This led to significant improvements in transplantation outcomes for human patients with severe aplastic anaemia.20 Also, the mechanisms of graft host tolerance without the need for lifelong immunosuppressive drugs were elucidated.21 It was found that immunosuppression could generally be discontinued after a period of 6 months and donor-derived T-lymphocytes were identified that down-regulated immune reactions of effector donor T-cells against host antigen. Transplantable stem cells from peripheral blood were found in the canine and primate transplantation models.22,23 Importantly, studies in dogs with spontaneous non-Hodgkin lymphoma showed that these cancers could be cured in part owing to graft-versus-tumour effects.24
The rebirth of clinical transplantation
The second half of the 1960s saw the development of high-intensity conditioning regimens for transplantation. These included maximally tolerated doses of TBI and of chemotherapeutic agents such as cyclophosphamide or busulfan. The regimens were aimed at eliminating the underlying disease burden, such as leukaemia, and at suppressing the patients’ immune systems so that the foreign marrow grafts would be accepted. Based on the canine studies, in vitro histocompatibility matching of donor–recipient pairs was recognized to be of fundamental importance to reduce the risks of both graft rejection and GVHD. However, even when donor and recipient were well matched, GVHD remained a problem unless post-grafting immunosuppression was given with the drug methotrexate, which dampened the speed with which donor lymphocytes replicated in response to recipient antigens. Rapid advances in the understanding of the complexity of the major human histocompatibility complex – called HLA (human leucocyte antigen) – helped to improve histocompatibility matching of human donor recipient pairs.
By 1968, clinical trials resumed with reports on the first successful transplants for patients with immunodeficiency disorders. However, during the first 7 or 8 years, most clinical studies were aimed at patients with advanced and refractory haematological malignancies who were in poor general condition and at patients with severe aplastic anaemia. These patients presented enormous challenges in supportive care. Given their underlying diseases and the profound pancytopenia caused by the conditioning regimens, patients required transfusions of blood products and antimicrobial therapy to prevent or treat bacterial and fungal infections. Antiviral therapies did not, as yet, exist. Therefore, in addition to discoveries made about the biology of marrow transplantation, these clinical trials stimulated progress in infectious diseases and transfusion research.
The initial clinical studies showed that GVHD still occurred in many patients, even though donors were HLA matched and patients received methotrexate immunosuppression after transplantation.17 This finding confirmed earlier observations in dogs and stimulated further research in pre-clinical model systems. Major improvements in GVHD prevention and patient survival were made when methotrexate was combined with calcineurin inhibitors such as cyclosporin or tacrolimus.25–29 These drug combinations remain the most widely used method for GVHD prevention to date.
Early studies in patients with severe aplastic anaemia given marrow grafts from HLA-identical siblings after conditioning with cyclophosphamide showed 45% long-term survival.30–32 A major impediment to greater success was graft rejection from transfusion-induced sensitization to minor histocompatibility antigens as predicted from canine studies. Subsequent studies in the laboratory identified dendritic cells in the transfusion product to be key in transfusion-induced sensitization. Therefore, removing white cells from red blood cell and platelet transfusions reduced the incidence of graft rejection. Further studies resulted in a conditioning regimen consisting of alternating cyclophosphamide and antithymocyte globulin, which greatly reduced the risk of graft rejection.33,34 Finally, irradiating blood products with 2000 cGy in vitro almost completely averted sensitization to minor histocompatibility antigens.35 Therefore, graft rejection in transplantation for aplastic anaemia has largely been overcome and current survivals with HLA-identical sibling grafts approach 100%.36
In 1975, the Seattle Transplantation Group presented two major findings that held the promise of further improving allogeneic HCT results in patients with leukaemia.17 One was that patients in a good general condition had better long-term outcomes than those who were in a poor medical condition. The other was that patients with a great leukaemic cell burden had a high disease recurrence after transplantation of approximately 75%. Taken together, these findings suggested that transplantation should be carried out earlier in the patients’ disease course while they were in good general condition and their leukaemia burden was low. Subsequent transplantations in patients with acute leukaemia, who were in chemotherapy-induced morphological remission or had chronic myelocytic leukaemia during the first chronic phase, showed vastly improved outcomes.37,38
In the 1980s, the transplant group at Johns Hopkins University substituted the chemotherapeutic agent busulfan for TBI and obtained results in patients with acute myelocytic leukaemia that compared well with those in patients given TBI.39 Subsequent prospective, randomized studies compared the two conditioning regimens and confirmed that outcomes after busulfan/cyclophosphamide were equal to those after cyclophosphamide/TBI in patients with myeloid leukaemias.40 Targeting busulfan pharmacokinetically to certain blood levels further refined this regimen by avoiding excess toxicities. The regimen has now become the standard for patients with myeloid leukaemias.
In 1981, busulfan/cyclophosphamide was used for the first successful transplant for thalassaemia major. The regimen is now also widely used for patients with sickle cell disease.41
Some transplant centres have focused on removing T-cells from the bone marrow as a means of preventing GVHD. Initial studies found high incidences of graft rejection, relapse of underlying malignancies and infections, but more recent studies showed that, while disease relapse has remained a problem in patients with chronic myeloid leukaemia, it seemed a lesser problem in patients with acute leukaemia. Other centres have used T-cell depletion with subsequent careful monitoring for minimal residual disease and have been treating patients in cases of disease recurrence with donor leucocyte infusions with the hope of initiating a graft-versus-leukaemia response without causing GVHD.42
The 1990s saw the first use of granulocyte colony-stimulating factor-mobilized peripheral blood stem cells.43–46 Randomized prospective studies showed that marrow and peripheral blood stem cells were equivalent as far as engraftment and overall survival were concerned. However, there seemed to be an increase in the incidence of chronic GVHD following the use of peripheral blood stem cells. For patients with non-malignant diseases, such as aplastic anaemia, marrow has remained the preferred source of stem cells in order to keep the rate of chronic GVHD relatively low, whereas most patients with haematological malignancies receive stem cells harvested from the peripheral blood.
Allogeneic graft-versus-tumour effects
As noted above, the graft-versus-tumour effect was first reported by Barnes et al. in 1956,12 who observed that leukaemic cells injected into irradiated mice were eradicated after allogeneic, but not after syngeneic, HCT. Mathé et al.11 coined the term ‘adoptive immunotherapy’ in 1965 when they observed disappearance of leukaemia in a patient after allogeneic HCT.11
By the end of the 1970s and the beginning of the 1980s, definitive findings of T-cell-mediated graft-versus-tumour effects in humans were reported, demonstrating that patients who developed GVHD were less likely to experience leukaemic relapse than those without GVHD.47–50 Moreover, patients who received grafts that were depleted of T-cells had very high relapse rates.51 Recurrence of leukaemia was also shown to be more common in patients receiving syngeneic sibling grafts than in those receiving HLA-identical sibling grafts. Further support for graft-versus-tumour effects came from the success of donor lymphocyte infusions in containing leukaemic relapse after allogeneic HCT.52,53 The infused donor lymphocytes become sensitized to leukaemic cell surface antigens, either polymorphic minor histocompatibility antigens or leukaemia-associated antigens, resulting in killing of the leukaemic cells. The most impressive results with donor lymphocyte infusions have been seen in patients with chronic myeloid leukaemia, perhaps because the host antigen-presenting dendritic cells are part of the malignant clone. Results are less impressive in patients with acute leukaemias, multiple myeloma or non-Hodgkin lymphoma.
Continued evolution of transplantation
During the 1980s and 1990s, the stem cell transplantation techniques were refined and alternative graft donors and sources were identified. Monoclonal antibodies specific for haematopoietic cells were developed and conjugated to antibiotics, toxins or radioisotopes to selectively target cancer cells for destruction. Prophylactic and therapeutic antibacterial, antifungal and antiviral drugs were developed to protect the immunocompromised recipient.
Because only approximately 35% of patients have HLA-identical siblings, alternative transplant donors have been identified, including extended family members who share one HLA haplotype and unrelated individuals who are HLA matched. Following up on a canine study published in 1977,54 the first successful transplant from an unrelated HLA-matched donor in a patient with leukaemia was reported in 1980.55 Subsequently, national unrelated donor registries were established, and currently more than 20 million HLA-typed unrelated volunteers are included in these registries. The likelihood of finding a suitably HLA-matched unrelated donor is approximately 80% for patients of Caucasian origin, although this percentage declines dramatically for patients from minority groups. Additional alternative sources of stem cells include unrelated cord blood.56–59 In addition, recent studies have reported successful transplantations from HLA-haploidentical related donors.60,61 As a result of these developments, a suitable donor can be found for virtually every patient.
To date, more than 1 million HCTs have been reported from more than 500 transplantation centres worldwide. Unfortunately, conventional HCT following high-intensity, marrow-ablative approaches is associated with dangerous toxicities and must be carried out in specialized intensive care wards. This restricts transplantation to relatively young patients in good medical condition. As a result, very few patients older than 60 years have received conventional transplantation. The age restriction is very unfortunate because the median age at diagnosis of patients with most candidate diseases (such as acute and chronic leukaemias, myelodysplastic syndromes, myelofibrosis, myelomas or lymphomas) ranges from 65 to 75 years, while the median age of patients treated by conventional, myeloablative allogeneic HCT generally ranges from 35 to 40 years.
To overcome the age restriction, less intensive conditioning programmes have been developed that rely less on chemoradiation therapy for tumour cell death and shift this task towards graft-versus-tumour effects.62 The reduced-intensity regimens have in part been directly translated from pre-clinical animal models into the clinic. The dramatically reduced incidence of conditioning regimen-related toxicities has allowed extending allogeneic HCT to include both elderly and medically infirm patients who were previously excluded from transplantation.
Most reduced-intensity conditioning regimens are downscaled versions of conventional regimens. A collaborative consortium of researchers based at the Fred Hutchinson Cancer Research Center (Seattle, WA, USA) and other academic centres has taken an alternative approach which combines conditioning with a drug, fludarabine (Fludara®, Sanofi-Aventis Ltd, Paris, France), and very low-dose TBI followed by post-grafting immunosuppression consisting of an inhibitor of de novo purine synthesis, mycophenolate mofetil, and a calcineurin inhibitor, either cyclosporin or tacrolimus. The regimen serves to both assist allogeneic engraftment and control GVHD. The approach has been used to treat patients with advanced haematological malignancies who were older than 60 years, had serious comorbidities or had already failed preceding high-intensity HCT. A report in 2013 highlighted the results among the first 1092 patients who were treated by either HLA-matched related or unrelated grafts using this approach.63 The median patient age was 56 years and 45% of patients had comorbidity scores of ≥ 3. The median follow-up was 5 years (range 0.6–12.7 years). Depending on the relapse risk of the underlying malignancies, on comorbidities and on GVHD, 45–75% of patients achieved lasting remissions and 5-year survivals ranged from 25% to 60%. The 5-year non-relapse mortality rate was 24% and the overall relapse mortality rate was 34.5%. Most non-relapse mortality resulted from GVHD. Serious comorbidities and grafts from unrelated donors were the most significant risk factors for GVHD-related mortality. Most disease relapses occurred early, while the patients’ immune system was compromised. Graft-versus-tumour effects were comparable after related and unrelated transplantations. Chronic GVHD, but not acute GVHD, was significantly associated with graft-versus-tumour effects; however, the potential benefit associated with chronic GVHD was offset by an increased risk of non-relapse mortality. Improved results could come from methods that control progression of malignancy during the early, immune-compromised, post-transplantation period and effectively prevent GVHD.
A subsequent study among 459 patients who were transplanted at the Fred Hutchinson Cancer Research Center with the same regimen confirmed that acute GVHD and graft-versus-tumour effects may be caused by different immunological mechanisms based on two key findings.64 First, low neutrophil nadirs within the first 3 weeks after transplantation significantly predicted increased risks of both acute GVHD and 5-year non-relapse mortality, but showed no association with the risk of relapse. Second, high lymphocyte counts immediately before transplantation predicted significantly reduced risks of relapse and overall mortality, but were not associated with the risks of GVHD or non-relapse mortality. As an illustration of these findings, the risk of grade III–IV acute GVHD rose from 3.5% among patients with neutrophil nadirs of ≥ 750/μl to 11% among patients with neutrophil nadirs < 250/μl. Corresponding figures for 5-year non-relapse mortality were 3% in the highest neutrophil nadir group and 25% in the lowest nadir group. The reasons for these findings are as yet not entirely clear. However, low neutrophil nadirs were associated with low neutrophil counts at the patients’ arrival at the transplant centre. Typically, low neutrophil counts result from extensive previous chemotherapy. Patients with chemotherapy-induced neutropenia generally receive broad-spectrum antibiotics that can impart unintended collateral damage to symbiotic microorganisms living in the gastrointestinal tract. This, in turn, can lead to dramatic alterations of the composition of gut microbial communities, which can persist for long periods of time. Perhaps both the neutropenia and the altered microbiota can lead to a proinflammatory state that induces production of cytokines. In this hypothetical cytokine-rich environment, skin and gut epithelia might conceivably up-regulate class II HLA expression and present minor histocompatibility antigens to donor T-cells, thereby amplifying GVHD manifestations in skin and gut. As for the role of high lymphocyte counts in the control of post-transplantation relapse, it could be argued that the lymphocyte numbers serve as a proxy for the number of host antigen-presenting cells. It could be that higher levels of antigen-presenting cells were associated with stronger graft-versus-tumour effects. Even though these findings are currently poorly explained, they are consistent with the possibility that graft-versus-tumour effects can be separated from acute GVHD and that these two entities are caused by different mechanisms.
In the future, reduced-intensity HCT might become an even more effective therapeutic tool. Better understanding of polymorphic minor histocompatibility antigens specific to haematopoietic cells might result in vaccines that could be used to direct donor immune cells towards haematopoietic targets including malignant cells, rather than inducing general GVHD.
Attempts at better targeting the pre-transplant radiation and replacing external beam TBI have included using a monoclonal antibody to the ubiquitous haematopoietic cell surface antigen, CD45, coupled to β-emitting radionuclides, such as iodine-131 or yttrium-90. However, β-emitters are less than ideal because of their long half-lives (which is 7.2 days in the case of iodine-131), low energy and long path lengths, which result in off-target effects. A far more promising approach has been successfully tested in the pre-clinical dog model by substituting an alpha-emitting radionuclide, astatine-211, for the β-emitters.65 Astatine-211 has a half-life of 7.2 hours, has very high energy (111 keV/μm) and has a path length of only 40–70 μm, which keeps off-target effects to a minimum.
The Johns Hopkins Transplantation Group used the basic fludarabine/2 Gy TBI conditioning regimen developed in Seattle and expanded it for use in HLA-haploidentical related recipients. In order to accomplish this, it added two small doses of cyclophosphamide before transplantation and two high doses of cyclophosphamide given 3 and 4 days after transplantation, followed by mycophenolate mofetil and tacrolimus.61 This regimen ensured both engraftment across a major histocompatibility barrier and control of GVHD, especially chronic GVHD. This regimen has opened up the use of HLA-haploidentical transplantation for older recipients and those with comorbidities. Initial results have been encouraging. However, relapse of the underlying malignancy has remained a problem. Some transplant centres now investigate adding donor natural killer cell infusions after transplantation in order to reduce the relapse rate.
During the past five decades, intensive investigations of HCT for treatment of malignant and non-malignant blood diseases have led this treatment modality from one that was thought to be plagued with insurmountable complications to the standard treatment for many diseases. The past several years have extended the application of HCT to include both young and elderly patients with a wide variety of haematological diseases. The donor pool has been expanded such that virtually every patient will have a donor. The ongoing research by numerous transplant teams throughout the world predicts continued progress towards developing novel and improved treatment modalities for an even wider application of the use of haematopoietic cell transplantation.