Introduction

Allogeneic hematopoietic cell transplantation (HCT) has been an effective treatment for many patients with hematological malignancies or inherited blood disorders¹. Conventional (myeloablative) allogeneic HCT has relied upon administration of supralethal doses of total body irradiation (TBI) and/or chemotherapy to (1) overcome immunologically mediated host-versus-graft (rejection) reactions and (2) destroy underlying diseases including malignancies. Given their intensity, myeloablative conditioning regimens have been associated with significant toxicity, which has limited their use to otherwise healthy, relatively young patients.

The antileukemic potential of allogeneic HCT has been attributed not only to high-dose chemotherapy and TBI, but also to graft-versus-tumor (GVT) effects^2,3, thought to be mediated primarily by donor T cells and possibly also NK cells contained in the grafts^4,5,6. The demonstrated dramatically higher risk of relapse in patients given T-cell-depleted grafts compared to patients given unmanipulated grafts^4,5 has led several groups of investigators to explore the curative potential of donor lymphocyte infusions (DLI) in patients who have relapsed hematologic malignancies after allogeneic HCT^7,8. The induction of durable complete remission by DLI in a number of patients with either acute or chronic leukemia⁷, lymphoma⁹, or multiple myeloma¹⁰ has demonstrated that GVT effects are capable of eradicating hematological malignancies, even in the absence of preceding chemotherapy. To extend the use of allogeneic HCT to include older patients and those with comorbid conditions, reduced-intensity^11,12,13,14 or truly nonmyeloablative^15,16,17 conditioning regimens have been introduced, in which the allografts have taken on most or all of the task of tumor eradication through immunological GVT effects¹⁸.

This review will first define reduced-intensity and nonmyeloablative regimens, next review the preclinical development and clinical translation of a truly nonmyeloablative regimen, and finally review the results with reduced-intensity approaches.

Reduced intensity versus nonmyeloablative conditioning regimens

Many of the reduced-intensity conditioning regimens have not met the criteria of nonmyeloablative conditioning as first proposed by Champlin et al.¹⁹, which include: (1) no eradication of host hematopoiesis, (2) prompt hematologic recovery (<4 weeks) without transplant, and (3) initial presence of mixed chimerism (i.e., coexistence of hematopoietic cells of host and donor origin) upon engraftment. Reduced-intensity conditioning regimens both eliminate host-versus-graft reactions (graft rejection) and produce major anti-tumor effects (Fig. 1). Most reduced-intensity conditioning regimens have combined fludarabine (a highly suppressive purine analog) with relatively high doses of busulfan (8 mg/kg)¹¹ or melphalan (140 to 180 mg/m²)^12,13 (see Table 1). In 1997, Giralt et al. reported on HLA-identical related grafts transplantation after conditioning with fludarabine 120 mg/m², cytarabine 8 g/m², and idarubicin 36 mg/m²²⁰. Initial engraftment was greater than 90% and nonrelapse mortality around 20%. The same group subsequently reported on a more intense regimen combining fludarabine (120–125 mg/m²) and melphalan (140–180 mg/m²)^12,21. Nonrelapse mortality at 100 days was 37% in a group of patients with high-risk hematological malignancies¹². Slavin et al. developed another protocol combining fludarabine (180 mg/m²), busulfan (8 mg/kg), and anti-thymocyte globulin (ATG)²². This regimen allowed the achievement of full donor chimerism in the majority of the patients with a low nonrelapse mortality in a group of relatively young patients. Kottaridis et al. added alemtuzumab (a humanized antibody recognizing CD52 antigen expressed on T cells, B cells, and NK cells, 100 mg/m²) to melphalan (140 mg/m²) and fludarabine (150 mg/m²)^13,23. This regimen allowed engraftment with low incidences of graft-versus-host disease (GVHD; an immune-mediated life-threatening complication of HCT) and nonrelapse mortality in HLA-matched related and unrelated recipients^13,23.

Figure 1.

Commonly used conditioning regimens in relation to their immunosuppressive and myelosuppressive properties. Please note that this classification is not based on direct experimentation and is thus hypothetical. TBI, total body irradiation; F, fludarabine; Cy, cyclophosphamide; Cy 120, cyclophosphamide 120 mg/kg; Cy 200, cyclophosphamide 200 mg/kg; M, melphalan, M 140; melphalan 140 mg/m²; M 180; melphalan 180 mg/m²; Flag-Ida, fludarabine/cytosine arabinoside/idarubicin; TT, thiotepa; ATG, anti-thymocyte globulin; Ale, alemtuzumab; Bu8, busulfan 8 mg/kg; Bu16, busulfan 16 mg/kg. Adapted from¹⁹ by permission of the publisher.

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Table 1 - Results with reduced-intensity or nonmyeloablative conditioning regimens.

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Typical complications of high-dose therapy such as nausea, vomiting, pancytopenias, mucositis, and new-onset alopecia have been observed with most of those regimens, and sinusoidal obstructive syndrome has also been seen, although less frequently than after myeloablative conditioning^{11,12,13,24,25,26}.

In contrast, a nonmyeloablative regimen, such as the one using 2 Gy of TBI either alone or combined with three doses of fludarabine (30 mg/m²/dose), has relied on optimal pre-and posttransplant immunosuppression to overcome host-versus-graft reactions, allowing allogeneic engraftment^27,28, which, in turn, resulted in GVT effects^16,29. Such a regimen has had few toxicities, has produced only mild myelosuppression (Fig. 2), and has been associated with a low 100-day incidence of nonrelapse mortality, even in elderly patients and those with comorbid conditions¹⁶.

Figure 2.

Neutrophil and platelet count changes after HLA-matched related (n = 85) or unrelated (n = 35) HCT following conditioning with 2 Gy TBI with or without fludarabine (90 mg/m²) (n = 120)⁵⁰. The graphs show the median, 25th percentile, and 75th percentile. Neutrophil counts stayed above 500/l, and platelet counts remained above 40,000/l in the majority of patients, demonstrating that the conditioning was truly nonmyeloablative. Reproduced from⁵⁰ by permission of the publisher.

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Two-gray-TBI-based low-intensity nonmyeloablative regimen

Preclinical development in a canine model

TBI dose de-escalation

A close relationship between TBI dose and rate of sustained engraftment of dog leukocyte antigen (DLA) identical marrow has been demonstrated in a preclinical canine model (Table 2). A TBI dose of 9.2 Gy was sufficiently immunosuppressive to permit engraftment of DLA-identical littermate marrow in 95% of dogs, in the absence of postgrafting immunosuppression³⁰. When the TBI dose was decreased to 4.5 Gy, only 41% of dogs achieved sustained engraftment, while 59% eventually rejected their grafts³¹. Since both host-versus-graft (rejection) and graft-versus-host reactions are mediated by T cells after DLA-identical HCT, it was hypothesized that optimizing posttransplant immunosuppression might not only prevent GVHD, but also increase the engraftment rate. Indeed, 7 of 7 dogs given 4.5 Gy TBI and postgrafting cyclosporin (CSP) achieved sustained engraftment³¹. When the TBI dose was further decreased to 2 Gy, postgrafting immunosuppression either with CSP alone or with a combination of CSP and methotrexate resulted in graft rejection with autologous recovery in 4 of 4 dogs and 3 of 5 dogs, respectively²⁷. However, stable mixed donor/host hematopoietic chimerism was achieved in 11 of 12 dogs given postgrafting immunosuppression with a combination of mycophenolate mofetil (MMF) and CSP, as well as in 6 of 7 dogs given sirolimus (rapamycin) combined with CSP²⁷. When the TBI dose was further decreased to 1 Gy, all dogs given either of the two drug combinations eventually experienced graft rejection, demonstrating a delicate balance between host-versus-graft and graft-versus-host reactions^27,32.

Table 2 - Effects of TBI dose and postgrafting immunosuppression on engraftment of DLA-identical grafts.

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It was unclear whether the graft rejections observed in dogs given less than 2 Gy TBI were due to a lack of creation of marrow space to which transplanted hematopoietic stem cells could home or to insufficient pre-HCT host immunosuppression. To address this question, six dogs were conditioned with 4.5 Gy irradiation targeted to the cervical, thoracic, and upper abdominal lymph node chain and administered postgrafting immunosuppression with MMF and CSP²⁸. Each dog showed initial evidence of mixed chimerism. Two dogs rejected their grafts by weeks 8 and 18 after HCT, respectively; one dog died with allogeneic engraftment from GVHD; and three remained mixed stable donor/host hematopoietic chimeras with follow-up of 57 to 97 weeks. Evidence of mixed donor/host hematopoietic chimerism in lymph node and bone marrow spaces that were shielded from irradiation was consistent with the notion that allogeneic grafts could create their own marrow space through subclinical graft-versus-host reactions. Further experimental observations supported the contention that the primary role of pre-HCT TBI in establishing mixed chimerism was to provide host immunosuppression. First, successful allografts were accomplished in dogs given 1 Gy TBI conditioning who had been "sensitized" against donor peripheral blood mononuclear cells (PBMC) in the presence of T cell costimulatory blockade with CTLA4-Ig³³. Second, sustained engraftment of DLA-identical marrow was achieved in dogs given selective T cell ablation with a bismuth-213-labeled ( emitter) anti-T-cell receptor- monoclonal antibody, and postgrafting MMF/CSP³⁴. Further, engraftment of positively selected CD34⁺hematopoietic cells from DLA-identical littermates has been achieved without conditioning in dogs with X-linked severe combined immunodeficiency disorders (SCID-X1)³⁵. Other attempts at decreasing host immunity before 1 Gy TBI have not met with success^36,37 (Table 2).

Breaking tolerance in dogs with mixed chimerism

Stable mixed hematopoietic chimerism represents a state of mutual host–donor tolerance³⁸. Although it has been speculated that low levels of stable donor chimerism might be sufficient to treat autoimmune diseases or to prevent rejection of donor solid organ grafts^39,40, high levels of donor hematopoiesis might be required to achieve normal hemoglobin levels in patients with thalassemia (Fig. 3A) or sickle cell disease^38,41 and were found to be required to prevent hemolysis in pyruvate kinase-deficient dogs⁴² (see below). In addition, a stable mixed hematopoietic chimerism state is unlikely to be curative for patients with hematologic malignancies^38,43,44. These observations led Georges et al. to investigate whether DLI could convert stable mixed hematopoietic chimerism to full donor chimerism in dogs given DLA-identical grafts after nonmyeloablative conditioning⁴⁵. Surprisingly, nonsensitized DLI failed to accomplish this task. However, lymphocyte infusions from donors sensitized to the recipient's minor histocompatibility antigens by skin grafts ("sensitized DLI") converted mixed chimerism to full donor chimerism in eight of eight dogs studied⁴⁵. The authors hypothesized that suppressor⁴⁶ or regulatory T cells⁴⁷ in stable chimeras prevented primary allorecognition and subsequent sensitization of newly infused T cells from nonsensitized donors, but did not interfere with the cytotoxic action of already sensitized donor T cells.

Figure 3.

(A) Correlation between Hb level and donor chimerism levels in 26 patients with thalassemia major given allogeneic HCT after myeloablative conditioning reported by Andreani et al.⁴¹. Relatively high levels of donor chimerism were needed to achieve normal Hb values. (B) Donor chimerism levels in the granulocyte (continuous black line) and mononuclear (broken black line) fractions of the peripheral blood, hematocrit (continuous gray line), and reticulocyte counts (broken gray line) across time after transplantation in a dog with pyruvate kinase deficiency and hemolytic anemia⁴⁸. Early after nonmyeloablative HCT, the levels of donor chimerism in the myeloid compartment (granulocyte fraction) decreased with a recurrence of hemolytic anemia. The dog received two infusions of donor lymphocytes () and subsequently had an increase in donor chimerism in the myeloid compartment and resolution of hemolytic anemia. ((B) was adapted from⁴⁸ by permission of the publisher.)

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HCT after nonmyeloablative conditioning as treatment for inherited canine blood disorders

Pyruvate kinase deficiency in Basenji dogs causes severe hemolytic anemia with hematocrits ranging from 18 to 27%^42,48. Zaucha et al. explored the efficacy of marrow transplantation from DLA-identical littermates after conditioning with 2 Gy TBI and postgrafting immunosuppression combining MMF and CSP in five affected dogs⁴². One dog died of liver failure (due to iron overload) on day 27 with 60% donor engraftment, two dogs experienced nonfatal graft rejection with recurrence of hemolytic anemia, and two dogs achieved sustained engraftment with 12 and 85% donor chimerism levels, respectively. Whereas the dog with the high degree of donor chimerism had virtually complete correction of hemolysis and resolution of marrow fibrosis, the dog with the low donor chimerism level had persistent clinical symptoms of hemolysis, suggesting that clinical responses correlated with donor chimerism levels. Confirming this hypothesis, infusion of sensitized donor lymphocytes in pyruvate kinase-deficient dogs with low-level donor chimerism and persistence of hemolytic anemia after nonmyeloablative conditioning increased the donor hematopoietic cell contributions and induced remissions of hemolytic anemia (Fig. 3B)⁴⁸.

Clinical translation

To date, the nonmyeloablative regimen has been used in more than 800 patients with hematological diseases who were ineligible for conventional allogeneic HCT because of age and/or concomitant diseases or extensive preceding therapies such as failed high-dose autologous or allogeneic HCT. The regimen was remarkably well tolerated, with the majority of eligible patients receiving their transplants in the outpatient setting. The first clinical results with this regimen are described in the following sections.

Engraftment

HCT from HLA-identical sibling: The initial clinical transplant regimen consisted of 2 Gy TBI given on day 0, followed by postgrafting immunosuppression with MMF given at 15 mg/kg bid for 28 days and CSP given at full dose until day 35 or 56¹⁶. The stem cell source was G-CSF-mobilized (G) PBMC. The hematological changes were much milder than usually observed after myeloablative or reduced-intensity conditioning (Fig. 2)⁴⁹. While most patients rapidly achieved full donor granulocyte chimerism (defined as 95% cells of donor origin), most remained mixed donor/host T cell chimeras for up to 180 days after HCT (Fig. 4A)⁵⁰. Patients who had received myelosuppressive chemotherapy before HCT had higher donor T cell chimerism levels compared to those who did not. Nine of the first 44 patients (20%) given this regimen had nonfatal graft rejections¹⁶. To reduce the risk of graft rejection, fludarabine 30 mg/m²/day 3 days was added to the 2 Gy TBI, and the rejection rate decreased to 3%⁵¹.

Figure 4.

(A) Engraftment kinetics after HCT with nonmyeloablative conditioning consisting of 2 Gy TBI with or without fludarabine (90 mg/m²). Median percentages of donor chimerism among peripheral blood cell subsets in 108 patients with sustained engraftment are shown⁵⁰. (B) Cumulative incidence of grades II–IV acute GVHD according to day 14 donor T cell chimerism levels after nonmyeloablative conditioning⁵⁰. (C) Peripheral blood donor T cell, CD4⁺T cell, CD8⁺T cell, and NK cell chimerism levels, and BCR/ABL bone marrow positive cells (assessed by FISH), in a patient with chronic myeloid leukemia in first chronic phase given unrelated G-PBMC after 2 Gy TBI and fludarabine. The patient had low T cell and NK cell chimerism levels early after HCT, predicting high risk of subsequent graft rejection. He received pentostatin (4 mg/m²) on day 43 followed by donor lymphocyte infusion 2 days later⁵⁹. This resulted in a significant increase in donor chimerism level among all subpopulations, and the patient is currently surviving in molecular remission with sustained graft > 300 days after HCT⁵³. ((A) was reproduced from⁵⁰ by permission of the publisher.)

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HCT from HLA-matched unrelated donor: The same fludarabine and 2 Gy TBI regimen was used to condition patients with 10-HLA-antigen-matched unrelated donors⁵². Compared to HLA-identical sibling recipients, the postgrafting immunosuppression with MMF was extended from 28 to 40 days with taper to day 96, and CSP was given for 100 days with taper through day 180. Twenty-seven percent of patients did not develop neutropenia (<500/l)⁵². Durable engraftment was observed in 85% of G-PBMC (n = 71) and 56% of marrow recipients (n = 18)⁵². Based on this observation, all subsequent unrelated recipients were given G-PBMC grafts. Among unrelated G-PBMC recipients, graft rejections were more frequently observed in patients with chronic myeloid leukemia⁵³ and in patients given G-PBMC containing less than 6.8 10⁶ CD34⁺cells/kg⁵⁴. Further, suboptimal postgrafting immunosuppression with MMF was suggested by pharmacokinetic studies showing that the t_1/2 of mycophenolic acid, the active metabolite of MMF, was 3 h, and its binding to IMPDH II was rapidly reversible⁵². Indeed, increasing administration of MMF from 15 mg/kg bid to 15 mg/kg tid increased the rate of durable engraftment from 85 to 95% among G-PBMC recipients (98/103 patients) (P = 0.004)⁵⁵.

Correlation between engraftment kinetics and HCT outcomes: Given that mixed chimerism had been associated with an increased risk of graft rejection, lower incidence of acute GVHD, and increasing risk of relapse after myeloablative conditioning^43,56, we thought to analyze the relationship between kinetics of donor engraftment and HCT outcomes among 157 patients with hematologic malignancies given HCT after nonmyeloablative conditioning^50,57. Day 14 donor chimerism levels <50% among T cells (P = 0.0007) and NK cells (P = 0.003) predicted graft rejection⁵⁷. When chimerism levels were modeled as a continuous linear variable, high T cell chimerism levels on day 14 were associated with an increased probability of grades II–IV acute GVHD (P = 0.02; Fig. 4B), while high donor T cell (P = 0.002) and NK cell (P = 0.002) chimerism levels from days 14–42 were associated with decreased risk of relapse⁵⁷. Further, high levels of donor NK cell chimerism early after HCT correlated with better progression-free survival (P = 0.02)⁵⁰.

Prevention of graft rejection in patients with low donor T-cell chimerism: Based on the observations that low donor T cell chimerism levels were associated with graft rejection, and that success among patients given DLI for low or falling donor T cell chimerism was seen only when pre-DLI T cell chimerism levels were >40%⁵⁸, Sandmaier et al. evaluated the safety and efficacy of the immunosuppressive drug pentostatin (4 mg/m²) given 2 days before DLI to reverse pending graft rejection⁵⁹. Preliminary results in eight patients, treated 54–339 days after HCT, have been analyzed. T cell chimerism levels before pentostatin and DLI ranged from 5 to 34%. After pentostatin and DLI, four of eight patients had increases in donor T cell chimerism levels to 63–100% (Fig. 4C), while four patients had levels remaining at 5–25%. These preliminary results suggested that immunosuppression with pentostatin followed by DLI might effectively prevent graft rejection in patients with low levels of donor chimerism after nonmyeloablative conditioning.

GVHD and GVT effects

In animal models, the intensity of the preparative regimens has been shown to contribute to GVHD, presumably by inducing tissue damage and the elaboration of a cytokine storm⁶⁰. Further, mixed donor–host hematopoietic chimerism has been associated with a decreased risk of GVHD both in animal models and in humans^{38,43,44,50,61}. Thus, one might expect less GVHD after nonmyeloablative conditioning. To test this hypothesis, Mielcarek et al. retrospectively compared GVHD among concurrent age-matched recipients of related or unrelated grafts given after either nonmyeloablative (n = 44) or myeloablative (n = 52) conditioning⁶². The cumulative incidence of grades II–IV acute GVHD was lower after nonmyeloablative conditioning (64% versus 85%; P = 0.001), but there were no differences in chronic GVHD (73% vs 71%; P = 0.96). Nonmyeloablative transplantation was associated with delayed (P < 0.001) and less frequent (P = 0.06) initiation of steroid treatment for GVHD. There was a suggestion that immunosuppressive therapy for GVHD was discontinued earlier in nonmyeloablative recipients (P = 0.25). Furthermore, nonmyeloablative patients experienced less 15-month mortality from GVHD (24% vs 35%, P = 0.27) and better 1-year overall survival (68% vs 50%; P = 0.04).

There has been a close relationship between GVHD and GVT responses after myeloablative conditioning^2,8,63,64. We investigated whether such a relationship existed for patients given nonmyeloablative conditioning in 322 patients given grafts from HLA-matched related (n = 192) or unrelated (n = 130) donors (Fig. 5A)²⁹. Fifty-seven percent of patients with measurable disease at HCT achieved complete (44%) or partial (13%) remission 27 to 963 days (median 144 days) after HCT. Acute GVHD of any grade was not associated with increased probability of achieving remission, but there was a trend for a higher probability of remission in patients with extensive chronic GVHD (P = 0.07). Further, grades II and III–IV acute GVHD had no significant impact on relapse/progression but were associated with an increased nonrelapse mortality and decreased progression-free survival. Conversely, extensive chronic GVHD was associated with decreased relapse/progression (P = 0.006) and better progression-free survival (P = 0.003) (Fig. 5B).

Figure 5.

(A) Schedule of the study evaluating graft-versus-tumor effects in 322 patients receiving allogeneic HCT after nonmyeloablative conditioning²⁹. TBI, total body irradiation; MMF, mycophenolate mofetil; CSP, cyclosporin; G-PBMC, G-CSF-mobilized peripheral blood mononuclear cells; BM, bone marrow. (B) Semi-landmark plot illustrating better progression-free survival due to lower risk of relapse in patients with extensive chronic GVHD²⁹. ((B) was reproduced from²⁹ by permission of the publisher.)

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Transplant-related toxicity and infections after nonmyeloablative versus myeloablative conditioning

Transplant-related toxicity and infections have been frequent complications of allogeneic HCT and have been attributed to both the intensity of the conditioning and graft-versus-host reactions. Several retrospective studies compared transplant-related toxicity and infections after HCT following nonmyeloablative versus myeloablative conditioning to determine the relative contributions of conditioning intensity to these complications.

Transplant-related toxicity: By definition, the hematological changes after nonmyeloablative conditioning were much milder than seen after myeloablative conditioning. Twenty-three and 63% of the nonmyeloablative recipients versus 100 and 96% of the myeloablative recipients, respectively, required platelet and red blood cell transfusions⁴⁹. Liver, kidney, and lung toxicities were significantly reduced with nonmyeloablative conditioning. The cumulative incidence of bilirubin >4 mg/dl was 26% at 200 days in nonmyeloablative recipients versus 48% in myeloablative recipients⁶⁵. The 100-day incidence of dialysis was 3% in nonmyeloablative recipients versus 12% in myeloablative recipients⁶⁶. The 120-day incidence of idiopathic pneumonia syndrome was 2.2% in nonmyeloablative recipients versus 8.4% in myeloablative recipients⁶⁷. Finally, the risk for experiencing decreased pulmonary function (FEV1) was significantly lower for nonmyeloablative than for myeloablative patients (odds ratio 0.3, P = 0.01)⁶⁸.

Sorror et al. analyzed transplantation-related toxicity following HLA-matched unrelated HCT in 134 concurrent patients given either nonmyeloablative (n = 60) or myeloablative (n = 74) conditioning using the National Cancer Institute Common Toxicity Criteria grading⁶⁹. Even though patients given nonmyeloablative conditioning were older, had advanced disease more often, had more extensive prior therapies, and had more comorbidities at HCT, they experienced significantly less gastrointestinal, hepatic, and hemorrhagic grades III–IV toxicity compared to patients concurrently transplanted with myeloablative conditioning. The 1-year nonrelapse mortality was 20% in nonmyeloablative recipients versus 32% in myeloablative recipients (P = 0.04). Comparable results were reported by Diaconescu et al. in patients given grafts from related donors⁷⁰.

More recently, we have developed an HCT-specific comorbidity index based on retrospective review of comorbidities among 1055 patients given allogeneic HCT at the FHCRC between 1997 and 2003 after nonmyeloablative (n = 294) or myeloablative (n = 761) conditioning⁷¹. Comparing nonmyeloablative and myeloablative conditioning, respectively, 2-year nonrelapse mortalities were 5% versus 10% (P = 0.4) and overall survival 85% versus 75% (P = 0.1) in patients with scores of 0–1, nonrelapse mortalities were 17% versus 27% (P = 0.04) and overall survival 61% versus 59% (P = 0.2) in patients with scores of 2–3, and nonrelapse mortalities were 33% versus 54% (P = 0.03) and overall survival 43% versus 30% (P = 0.006) in patients with scores of 4⁷². These data suggested that comorbidity scoring was an important tool for assessing patients to conditioning regimens.

Infections: Junghanss et al. compared the incidence of posttransplant infections in 56 nonmyeloablative recipients with that in 112 matched controls given myeloablative conditioning^73,74. The 30- and 100-day incidences of bacteremia were 9 and 27% in the nonmyeloablative group versus 27 (P = 0.01) and 41% (P = 0.07) in the myeloablative group, respectively. Invasive aspergillosis occurred at a similar rate (P = 0.30). The onset of CMV disease was significantly delayed among nonmyeloablative compared to myeloablative patients (medians of 130 versus 52 days; P = 0.02). However, the 1-year probability of CMV disease for high-risk CMV patients was similar in the two groups (P = 0.87).

Results of nonablative conditioning in specific diseases

Hematologic malignancies: Results of nonmyeloablative conditioning in patients with hematologic malignancies have been reviewed elsewhere^51,75. Encouraging results were observed in patients with acute myeloid leukemia in first or second complete remission (2-year overall survival of 45 and 51%, respectively)⁷⁶, as well as in patients with myelodysplastic syndrome with < 5% blasts at HCT (2-year overall survival of 55%)⁷⁷, chronic myeloid leukemia (2-year overall survival of 70% for patients in first chronic phase)⁷⁸, chronic lymphocytic leukemia (2-year overall survival of 60%)⁷⁹, or indolent or chemotherapy-sensitive aggressive non-Hodgkin lymphoma (2-year overall survival of 65% in patients with mantle cell lymphoma)⁸⁰ (Table 1). Conversely, results in patients with advanced aggressive diseases (such as acute leukemias not in complete remission, chemotherapy-insensitive high-grade non-Hodgkin lymphoma or multiple myeloma, or advanced myelodysplastic syndromes) have been less favorable.

Tandem autologous/allogeneic HCT: To allow older patients with aggressive chemosensitive disease to benefit from both high-dose chemotherapy and GVT effects, protocols have been developed that first use high-dose conditioning and autologous HCT, which can be administered with overall mortality rates of less than 5%, followed 1 to 3 months later by allogeneic HCT after nonmyeloablative conditioning (tandem autologous/allogeneic HCT)⁸¹. Maloney et al. investigated the safety of such an approach in patients with multiple myeloma⁸². Patients were first given cytoreductive autologous HCT after 200 mg/m² melphalan, followed 40 to 229 (median 62) days later by allogeneic HCT after 2 Gy TBI. Fifty-four patients, 29–71 (median 52) years of age, were included in the study. The 100-day mortalities after autologous and allogeneic HCT were 2 and 2%, respectively. The 2-year overall and progression-free survivals were 78 and 55%, respectively. Georges et al. investigated a similar approach in 10 patients with chemorefractory multiple myeloma and HLA-matched unrelated donors⁸³. With a median follow-up of 25 months, 8 patients were alive, including 6 patients in complete remission, 1 with stable disease, and 1 with progressive disease.

Nonmalignant diseases

Paroxysmal nocturnal hemoglobinuria: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare clonal disorder caused by a somatic mutation of the X-linked phosphatidylinositol glycan class A gene. Hegenbart et al. treated seven adult patients with high-risk paroxysmal nocturnal hemoglobinuria by allogeneic HCT following conditioning with fludarabine and 2 Gy TBI⁸⁴. Two patients were given G-PBMC from HLA-matched related donors, and five were given G-PBMC from unrelated donors. Patients were deemed ineligible for conventional HCT because of Budd–Chiari syndrome (n = 2), life-threatening hemolysis or infections (n = 4), and/or Karnofsky score 80 (n = 4). The median number of red blood cell transfusions before HCT was 14. All seven patients achieved sustained engraftment and complete remission of PNH. Three patients died of pancreatitis, infection, or bleeding after a liver biopsy for chronic GVHD, while the remaining four patients were alive 13 to 38 months after HCT, with 100% donor chimerism.

Sickle cell disease and -thalassemia: Allogeneic HCT has remained the only curative treatment for sickle cell disease and -thalassemia^41,85. Although long-term survival has been excellent (80 to 90%) in good-risk patients with HLA-identical sibling donors, the procedure has been associated with significant long-term toxicities related to the myeloablative conditioning (such as infertility with gonadal failure or secondary malignancies), evincing the interest of using nonmyeloablative conditioning.

Iannone et al.⁸⁶ and Horan et al.⁸⁷ investigated the feasibility of allogeneic HCT after nonmyeloablative conditioning in 11 patients with sickle cell disease (n = 9) or -thalassemia (n = 2). Patients were 3 to 30 years of age. Stem cell sources were marrow (n = 9) or G-PBMC (n = 2) from HLA-identical donors. Ten of eleven patients had evidence of donor chimerism (range, 25–100%). However, all but 1 patient lost their grafts after discontinuation of postgrafting immunosuppression. The patient with stable engraftment was still doing well 27 months after HCT with full donor T cell chimerism. One of the 10 patients with graft rejection died after a second HCT, while the remaining 9 patients were alive with recurrent disease. These results showed that it is difficult to achieve sustained donor engraftment in patients with hemoglobinopathies, perhaps because recipients have been sensitized to minor histocompatibility antigens of their donors by preceding blood transfusions or perhaps because -thalassemic and sickle cell marrows were difficult to eradicate.

Primary immunodeficiencies: Despite encouraging results with gene therapy in patients with SCID-X1⁸⁸ and SCID due to adenosine deaminase deficiency⁸⁹, allogeneic HCT remains the treatment of choice⁹⁰. This is especially true given the increasingly recognized risks of insertional mutagenesis associated with current techniques of gene therapy^91,92. However, the substantial risks for mortality and morbidity associated with myeloablative conditioning regimens have precluded transplants for all but healthy young patients without comorbid conditions.

Woolfrey et al. reported data from 13 patients with SCID (n = 2) or other primary immunodeficiency syndromes (n = 11) given marrow (n = 7), G-PBMC (n = 5), or cord blood (n = 1) transplantation from HLA-matched related (n = 7) or unrelated (n = 6) donors⁹³. Patients were deemed not to be candidates for conventional HCT because of comorbidities or ongoing infections. Two patients without T cell function who had related donors did not receive pre-HCT conditioning, while 11 patients were conditioned with 2 Gy TBI with (n = 8) or without (n = 3) fludarabine (90 mg/m²). Postgrafting immunosuppression consisted of MMF and CSP in all patients. The regimen was not marrow suppressive, and all patients had evidence of donor T cell engraftment (T cell chimerism levels ranging from 5 to 100%). B cell donor chimerism levels in the two SCID patients were 50 and 99%, respectively. Day 100 transplant-related mortality was 0%. Grades II, III, and IV acute GVHD were seen in 9, 1, and 0 patients, respectively, while 7 patients developed chronic GVHD, which contributed to death in 3 patients. Two patients with low donor chimerism were given second HCT. One of them died of transplant-related mortality, while the other achieved full donor chimerism. Chimerism levels among various blood cell subpopulations in 4 representative patients with stable engraftment⁹⁴ are shown in Fig. 6.

Figure 6.

Donor chimerism levels in four representative patients given grafts from HLA-matched related donors for T cell deficiency (n = 3) or a graft from an HLA-matched unrelated donor for severe combined immunodeficiency (n = 1) after nonmyeloablative conditioning⁹⁴.

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Nonmyeloablative conditioning for cord blood or HLA haploidentical HCT

Given that HLA-matched donors can be found for only 50–80% of patients, depending of their ethnic group, there has been a considerable interest in extending the use of nonmyeloablative conditioning to cord blood or HLA-haploidentical HCT. Due to greater degrees of histoincompatibility, the use of such alternative donors has been associated with increased risks of both graft rejection and GVHD. Barker et al. investigated the feasibility of unrelated cord blood transplantation after nonmyeloablative conditioning consisting of fludarabine (200 mg/m²), cyclophosphamide (50 mg/kg), and 2 Gy TBI^95,96. Data from 51 patients (median age 50 years) with hematologic malignancies given 1 (n = 13) or 2 (n = 38) unrelated cord blood units have been recently analyzed^95,96. Eight patients not given myelosuppressive chemotherapy in the 6 months preceding HCT were also given ATG. Postgrafting immunosuppression consisted of MMF and CSP. Cord blood units were predominantly 1-or 2-HLA-antigen mismatched with the recipient. Five of 51 patients had either failure of engraftment (n = 4) or graft rejection (n = 1), while 46 had sustained primary engraftment. The median chimerism level was 100% (range 72–100%) at day 100. The cumulative incidence of grades II–IV acute GVHD was 61%, while chronic GVHD was seen in 36% of patients. One-year probability of progression-free survival was 48%.

O'Donnell et al. investigated the feasibility of unmanipulated haploidentical marrow HCT after nonmyeloablative conditioning combining fludarabine (150 mg/m²), cyclophosphamide (29 mg/kg), and 2 Gy TBI in 10 patients with advanced hematologic malignancies⁹⁷. Postgrafting immunosuppression consisted of MMF, tacrolimus, and cyclophosphamide, the latter given as a single dose of 50 mg/kg on day 3 after HCT. Two patients had graft rejection, while 8 achieved sustained donor engraftment with chimerism levels ranging from 93 to 100% at day 180. Grades II, III, and IV acute GVHD were seen in 3, 3, and 0 patients, respectively. With a median follow-up of 284 days, 6 of 10 patients were still alive at the time of the report, while 4 had died from disease progression (n = 3) or GVHD (n = 1).

HCT after reduced-intensity or non-TBI-based nonmyeloablative conditioning

In contrast to the nonmyeloablative approach described above, which relied almost exclusively on GVT effects for tumor eradication, reduced-intensity conditioning regimens have combined drugs with demonstrated activity against the targeted malignancies with the hope of disease control while allowing GVT effects to occur. The myelosuppressive and immunosuppressive abilities have varied considerably from one regimen to another (Fig. 1). In addition, while many studies have been performed in patients unable to tolerate myeloablative conditioning because of age, comorbidity, or previous high-dose HCT, other studies have included younger patients who would have been eligible for conventional high-dose allogeneic HCT with standard eligibility criteria, precluding meaningful comparisons among reduced-intensity regimens.

Engraftment

The kinetics of donor engraftment varied considerably from one reduced-intensity regimen to another. For example, Childs et al. studied 36 patients conditioned with fludarabine (125 mg/m²) and cyclophosphamide (120 mg/kg) and given postgrafting immunosuppression with CSP^17,98. Neutrophils decreased to <100/l in all patients and recovered to >500/l at a median of 11 days after HCT. Median T cell and granulocyte chimerism levels were 92 and 38%, respectively, at day 30, and 100 and 84%, respectively, at day 100 after HCT.

Ueno et al. analyzed engraftment kinetics in patients transplanted after conditioning with fludarabine (150 mg/m²) and melphalan (140 mg/m²)²¹. Neutrophils decreased to <100/l in all patients and recovered to >500/l at a median of 12 days after HCT. On days 30 and 100, all patients had 100% donor T cell and granulocyte chimerism levels.

GVHD and GVT effects

Couriel et al. reported that patients given grafts from HLA-identical siblings after myeloablative regimens (n = 74) had higher incidences of grades II–IV acute (HR, 3.6; 95% CI, 1.5–8.8) and chronic (HR, 5.2; 95% CI, 1.2–23.2) GVHD than those given various nonmyeloablative regimens (n = 63)⁹⁹.

Perez-Simon et al. analyzed the impact of GVHD on outcome in 86 recipients of HLA-identical grafts from sibling donors after conditioning with fludarabine (150 mg/m²) plus melphalan (140 mg/m²) or fludarabine (150 mg/m²) plus busulfan (10 mg/kg). Patients who developed grades III–IV acute GVHD had significantly worse progression-free survival (P = 0.006). In contrast, chronic GVHD was associated with better progression-free survival (P < 0.0001) in time-dependent analyses¹⁰⁰.

Some reduced-intensity conditioning regimens have used in vivo T cell depletion of the grafts (with either ATG or alemtuzumab) to decrease the incidence of acute and chronic GVHD. While these strategies achieved their goals^13,101, delayed immune reconstitution and increased incidences of both infections and disease relapses were observed^102,103.

Results of reduced-intensity or non-TBI-based nonmyeloablative conditioning in specific diseases

Hematologic malignancies

Results of reduced-intensity conditioning in patients with hematologic malignancies have been reviewed elsewhere^51,75. As shown in Table 1 and as observed with nonmyeloablative conditioning, encouraging results were observed in patients with acute myeloid leukemia in complete remission^14,104, as well as in patients with myelodysplastic syndrome¹⁰⁵, chronic myeloid leukemia¹¹, chronic lymphocytic leukemia²⁵, indolent or chemotherapy-sensitive aggressive non-Hodgkin lymphoma^13,15,26, or chemotherapy-sensitive multiple myeloma¹⁰³. Conversely, results in patients with advanced aggressive diseases (such as acute leukemias not in complete remission or chemotherapy-insensitive high-grade non-Hodgkin lymphoma or multiple myeloma) have been less favorable.

Nonmalignant diseases

Hemoglobinopathies: Jacobsohn et al. treated four patients (ages 4 to 22 years) with -thalassemia (n = 1) or sickle cell disease (n = 3) by allogeneic G-PBMC transplantation following conditioning with fludarabine (180 mg/m²), intermediate-dose of iv busulfan (6.4 mg/kg), and ATG¹⁰⁶. The regimen was marrow suppressive with a median duration of neutropenia of 18 days. One patient had sustained engraftment but died of chronic GVHD on day 377 after HCT. The three others had graft rejection. Two of them were alive with recurrent disease, while the other died of infection on day 780.

Primary immunodeficiencies: Horwitz et al. gave CD34-selected grafts from HLA-identical siblings after conditioning with fludarabine (125 mg/m²), cyclophosphamide (120 mg/kg), and ATG in 10 patients with chronic granulomatous disease¹⁰⁷. Postgrafting immunosuppression consisted of CSP. The regimen was marrow suppressive with a median duration of neutropenia of 10 days. After a median follow-up of 17 months, 6 patients were alive with sustained engraftment and resolution of preexisting granulomatous lesion, 1 patient was alive with graft rejection and autologous reconstitution, and 3 patients died of infection, GVHD, or hemorrhagic cystitis developing after a second HCT given for graft failure.

Rao et al. analyzed outcomes of 33 patients with primary immunodeficiencies given bone marrows from HLA-matched (n = 21) or mismatched (one of six antigens, n = 11, or two of six antigens, n = 1) unrelated donors after reduced-intensity conditioning consisting of fludarabine (150 mg/m²), melphalan (140 mg/m²), and alemtuzumab or ATG¹⁰⁸. Postgrafting immunosuppression was CSP alone. Diagnoses were SCID (n = 6), Wiscott–Aldrich syndrome (n = 4), T cell deficiencies (n = 14), CD40 ligand deficiency (n = 4), and phagocyte disorders (n = 5). Median age at HCT was 5.9 years. This study was not restricted to patients ineligible for myeloablative conditioning. The regimen was marrow suppressive with a median duration of neutropenia of 13 days. After a median follow-up of 40 months, 2 of 33 patients have died (1 from infection during the conditioning regimen, the other from chronic GVHD), while 31 patients remained alive. At 1 year after HCT, 17 of 31 patients had 100% donor chimerism, 10 were mixed chimeras with a high donor contribution, 2 were mixed chimeras with a low donor contribution, and 2 were mixed chimeras with a very low donor contribution. Interestingly, both children with very low donor chimerism had received one-HLA-antigen-mismatched grafts. All children with full donor chimerism, mixed chimerism with high donor contributions, or mixed chimerism with low donor contributions were free of disease, while the 2 children with very low donor contributions were restarted on prophylactic medications. At 12 months after HCT, 66, 65, 59, and 41% of patients had age-related normal levels of T cells, CD4⁺T cells, B cells, and responses to stimulation with phytohemagglutinin, respectively, consistent with relatively slow immune reconstitution probably due in part to the in vivo depletion of the graft by alemtuzumab or ATG.

The same group recently reported an update of their experience with reduced-intensity conditioning in 81 children (1 of them was given two HCT from two different donors) with immunodeficiency¹⁰⁹. Donors were HLA-matched unrelated donors (n = 40), HLA-mismatched unrelated donors (n = 21), HLA-matched sibling donors (n = 11), and other matched family donors (n = 10). Seventy-one patients received bone marrow, 10 G-PBMC, and 1 umbilical cord blood. The use of G-PBMC was associated with higher donor chimerism levels, but also with more GVHD. At the time of the analyses, 68 patients (84%) were alive, with no significant difference between the donor types or between SCID and other diseases.

Shenoy et al. recently reported data from 16 patients with various nonmalignant hematologic disorders who were conditioned with fludarabine (150 mg/m²), melphalan (70–140 mg/m²), and alemtuzumab (48 mg total dose) given 3 weeks before HCT¹¹⁰. Two patients died from infections before engraftment, while the remaining patients achieved mixed (n = 2) or full (n = 12) donor T cell chimerism. Four patients developed acute GVHD (skin grades I–II). With a median follow-up of 281 days, the overall survival was 75%, and all evaluable patients had stable, improved, or completely resolved disease. Remarkably, CD4⁺ T cell and B cell counts recovered to 50% of normal values by 3 months and were completely normal by 6–9 months after HCT. These results contrasted with the previously observed delayed immune reconstitution in patients given alemtuzumab the week before HCT.

Combination of gene therapy with nonmyeloablative conditioning

Gene therapy approaches might be used in the future to improve the efficacy of allogeneic HCT with nonmyeloablative conditioning. First, infusions of tumor (including minor histocompatibility antigens restricted to hematopoietic cells)-specific donor cytotoxic T cells have been proposed as a way to increase GVT effects without inducing GVHD¹¹¹. However, this approach has been limited by technical difficulties in generating such specific cytotoxic T cells in vitro and by immune evasion mechanisms developed by tumor cells. Several preclinical studies have suggested that gene modifications of T cells (for example, by transfer of genes coding for tumor-specific T cell receptors with or without genes coding for signaling domains of costimulatory molecules) might overcome these limitations (recently reviewed in¹¹²). Another approach might consist of transferring a chemotherapy resistance gene (such as the O⁶-methylguanine-DNA methyltransferase gene that confers resistance to BCNU) into donor stem cells, allowing posttransplant selection of donor cells by chemotherapy (BCNU or temozolomide) administration¹¹³.

Conversely, nonmyeloablative conditioning has been successfully used to promote engraftment of gene-modified autologous stem cells. Those regimens have generally used nonmyeloablative doses of either TBI or busulfan, given their potential to target nondividing hematopoietic stem cells^89,114. For example, Aiuti et al. transplanted autologous adenosine deaminase (ADA)-transduced stem cells into two children with severe combined immunodeficiency due to ADA deficiency after nonmyeloablative conditioning with busulfan (4 mg/kg)⁸⁹. Both children achieved sustained engraftment of transduced stem cells, resulting in increased T cell, B cell, and NK cell counts, and improved immune functions. Finally, nonmyeloablative immunosuppressive conditioning might be used in the future to promote long-term tolerance to transgenes after infusion of genetically modified stem cells.

Conclusions

Nonmyeloablative and reduced-intensity conditioning allowed engraftment of allogeneic hematopoietic cells and the development of GVT effects. Remarkably, a minimally toxic regimen of 2 Gy TBI with or without fludarabine followed by postgrafting immunosuppression with MMF and CSP ensured engraftment rates similar to those after myeloablative conditioning for most patients with hematologic disorders. However, nonfatal graft rejections with autologous reconstitution and recurrence of anemia have been the rule in patients with hemoglobinopathies.

Ongoing efforts are directed at decreasing the acute GVHD incidence and at improving antitumoral efficacy of the regimens, especially for patients with "aggressive" diseases such as acute leukemia or high-grade lymphomas not in remission, by combining nonmyeloablative HCT with "disease-targeted" therapy, including imatinib, rituximab, or radiolabeled monoclonal antibodies. Finally, progress in the understanding of tumor antigens and tissue-specific polymorphic minor histocompatibility antigens might allow posttransplant infusion of (genetically modified or not) tumor-specific cytotoxic T cells, potentially increasing the anti-tumor efficacy of nonmyeloablative HCT without inducing GVHD^111,112.

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Acknowledgements

We thank Helen Crawford, Bonnie Larson, and Sue Carbonneau for help with manuscript preparation. We are grateful to Heather Hildebrant and Deborah Bassuk for data processing; the research nurses Steve Minor, Mary Hinds, and John Sedwick; and the medical nursing and clinical staffs for their dedicated care of the patients. We acknowledge Ted Gooley, Barry Storer, and Stacy Zellmer for help with the figures. This work was supported by Grants CA78902, CA18029, CA15704, and HL36444 from the National Institutes of Health (Bethesda, MD, USA). F.B. is research associate of the National Fund for Scientific Research Belgium and supported in part by postdoctoral grants from the Fulbright Commission, the Centre Anticancereux près l'ULg, and the Leon Fredericq Fund.