Allogeneic Hematopoietic Cell Transplantation Using Fludarabine, Melphalan and Bortezomib...

REVIEW ARTICLE doi:10.1016/j.ymthe.2005.09.011

Allogeneic Hematopoietic Cell Transplantation FollowingNonmyeloablative Conditioning as Treatment for

Hematologic Malignancies and Inherited Blood Disorders

Frederic Baron,1,2 Rainer Storb,1,3,*

1Fred Hutchinson Cancer Research Center and 3University of Washington, Seattle, WA 98109, USA2University of Liege, B-4000 Liege, Belgium

*To whom correspondence and reprint requests should be addressed at the Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North,

D1-100, P.O. Box 19024, Seattle, WA 98109-1024, USA. Fax: +1 206 667 6124. E-mail: [email protected].

Available online 8 November 2005

26

Allogeneic hematopoietic cell transplantation (HCT) after myeloablative conditioning regimens hasbeen an effective treatment for many patients with hematologic malignancies or inherited blooddisorders. Unfortunately, such regimens have been associated with significant toxicity, limiting theiruse to otherwise healthy, relatively young patients. In an attempt to extend treatment by allogeneicHCT to older patients and those with comorbid conditions, several groups of investigators havedeveloped reduced-intensity or truly nonmyeloablative conditioning regimens, lacking such toxicity.Analogous to conventional regimens, reduced-intensity regimens both eliminated host-versus-graft(rejection) reactions and produced major anti-tumor effects. In contrast, nonmyeloablativeregimens have relied on optimizing both pre-and posttransplant immunosuppression to overcomehost-versus-graft reactions, while anti-tumor responses have depended mainly on immune-mediated graft-versus-tumor effects. In this review, we define reduced-intensity and trulynonmyeloablative regimens, describe the preclinical development and clinical application of a verylow intensity nonmyeloablative regimen, and review results with reduced-intensity regimens inpatients with hematologic malignancies or inherited blood disorders.

Key Words: hematopoietic cell transplantation, nonmyeloablative conditioning,mixed chimerism, graft-versus-tumor effects, graft-versus-host disease

ContentsIntroduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27Reduced Intensity Versus Nonmyeloablative Conditioning Regimens . . . . . . . . . . . . . . . . . . . . . . . . . . 27Two-Gray-TBI-Based Low-Intensity Nonmyeloablative Regimen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

Preclinical Development in a Canine Model. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30TBI dose de-escalation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30Breaking tolerance in dogs with mixed chimerism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30HCT after nonmyeloablative conditioning as treatment for inherited canine blood disorder . . . . . . . . . 31

Clinical Translation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31Engraftment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31GVHD and GVT effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33Transplant-related toxicity and infections after nonmyeloablative versus myeloablative conditioning . . . 33Results of nonablative conditioning in specific diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35Nonmalignant diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Nonmyeloablative Conditioning for Cord Blood or HLA Haploidentical HCT . . . . . . . . . . . . . . . . . . . . 36HCT After Reduced-Intensity or Non-TBI-Based Nonmyeloablative Conditioning . . . . . . . . . . . . . . . . . . . 37

Engraftment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37GVHD and GVT Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37Results of Reduced-Intensity or Non-TBI-Based Nonmyeloablative Conditioning in Specific Diseases . . . . . . 37

Hematologic malignancies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37Nonmalignant diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

MOLECULAR THERAPY Vol. 13, No. 1, January 2006

Copyright C The American Society of Gene Therapy

1525-0016/$30.00

REVIEW ARTICLEdoi:10.1016/j.ymthe.2005.09.011

Combination of Gene Therapy with Nonmyeloablative Conditioning . . . . . . . . . . . . . . . . . . . . . . . . . . 38Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

IG. 1. Commonly used conditioning regimens in relation to their immuno-

ppressive and myelosuppressive properties. Please note that this classifica-

on is not based on direct experimentation and is thus hypothetical. TBI, total

ody irradiation; F, fludarabine; Cy, cyclophosphamide; Cy 120, cyclo-

hosphamide 120 mg/kg; Cy 200, cyclophosphamide 200 mg/kg; M,

elphalan, M 140; melphalan 140 mg/m2; M 180; melphalan 180 mg/m2;

lag-Ida, fludarabine/cytosine arabinoside/idarubicin; TT, thiotepa; ATG, anti-

ymocyte globulin; Ale, alemtuzumab; Bu8, busulfan 8 mg/kg; Bu16,

usulfan 16 mg/kg. Adapted from [19] by permission of the publisher.

INTRODUCTION

Allogeneic hematopoietic cell transplantation (HCT) hasbeen an effective treatment for many patients withhematological malignancies or inherited blood disorders[1]. Conventional (myeloablative) allogeneic HCT hasrelied upon administration of supralethal doses of totalbody irradiation (TBI) and/or chemotherapy to (1)overcome immunologically mediated host-versus-graft(rejection) reactions and (2) destroy underlying diseasesincluding malignancies. Given their intensity, myeloa-blative conditioning regimens have been associated withsignificant toxicity, which has limited their use tootherwise healthy, relatively young patients.

The antileukemic potential of allogeneic HCT hasbeen attributed not only to high-dose chemotherapyand TBI, but also to graft-versus-tumor (GVT) effects[2,3], thought to be mediated primarily by donor T cellsand possibly also NK cells contained in the grafts [4–6].The demonstrated dramatically higher risk of relapse inpatients given T-cell-depleted grafts compared topatients given unmanipulated grafts [4,5] has led severalgroups of investigators to explore the curative potentialof donor lymphocyte infusions (DLI) in patients whohave relapsed hematologic malignancies after allogeneicHCT [7,8]. The induction of durable complete remissionby DLI in a number of patients with either acute orchronic leukemia [7], lymphoma [9], or multiple mye-loma [10] has demonstrated that GVT effects are capableof eradicating hematological malignancies, even in theabsence of preceding chemotherapy. To extend the useof allogeneic HCT to include older patients and thosewith comorbid conditions, reduced-intensity [11–14] ortruly nonmyeloablative [15–17] conditioning regimenshave been introduced, in which the allografts havetaken on most or all of the task of tumor eradicationthrough immunological GVT effects [18].

This review will first define reduced-intensity andnonmyeloablative regimens, next review the preclinicaldevelopment and clinical translation of a truly non-myeloablative regimen, and finally review the resultswith reduced-intensity approaches.

REDUCED INTENSITY VERSUS NONMYELOABLATIVE

CONDITIONING REGIMENS

Many of the reduced-intensity conditioning regimenshave not met the criteria of nonmyeloablative condi-tioning as first proposed by Champlin et al. [19], whichinclude: (1) no eradication of host hematopoiesis, (2)



prompt hematologic recovery (b4 weeks) without trans-plant, and (3) initial presence of mixed chimerism (i.e.,coexistence of hematopoietic cells of host and donororigin) upon engraftment. Reduced-intensity condition-ing regimens both eliminate host-versus-graft reactions(graft rejection) and produce major anti-tumor effects(Fig. 1). Most reduced-intensity conditioning regimenshave combined fludarabine (a highly suppressive purineanalog) with relatively high doses of busulfan (8 mg/kg)[11] or melphalan (140 to 180 mg/m2) [12,13] (see Table1). In 1997, Giralt et al. reported on HLA-identicalrelated grafts transplantation after conditioning withfludarabine 120 mg/m2, cytarabine 8 g/m2, and idarubi-cin 36 mg/m2 [20]. Initial engraftment was greater than90% and nonrelapse mortality around 20%. The samegroup subsequently reported on a more intense regimencombining fludarabine (120–125 mg/m2) and melphalan(140–180 mg/m2) [12,21]. Nonrelapse mortality at 100days was 37% in a group of patients with high-riskhematological malignancies [12]. Slavin et al. developedanother protocol combining fludarabine (180 mg/m2),

F

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ti

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p

m

F

th

b

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TABLE 1: Results with reduced-intensity or nonmyeloablative conditioning regimens

Center Preparative regimen

Postgraft

immuno-suppression

N of patients

(median agein years) Disease

GVHD NRM

(time aftertransplant) Outcome

Acute(grades II–IV) Chronic

Reduced-intensity regimensM. D. Anderson [12] Fludarabine

25 mg/m2/day

(or 2-CDA 12 mg/m2) �5 days

Melphalan

140–180 mg/m2

FK506 + MTX 86 (52) Hematological

malignancies

49% 68% 37%

(100 days)

�2-year

OS 28%

�2-yearDFS 23%

United KingdomConsortium [13]

Fludarabine30 mg/m2/day �5 days

Melphalan

140 mg/m2

Alemtuzumab

20 mg/day � 5 days

CSP 88 (48) Non-Hodgkinlymphoma

15%a 7%a 11b–38%c

(3 years)�3-yearOS 55%

King’s CollegeHospital [105]

Fludarabine30 mg/m2/day �5 days

Busulfan (po)

4 mg/kg/day �2 days

Alemtuzumab

20 mg/day � 5 days

CSP 62 (53) Myelodysplasticsyndromes

NR NR 7%(100 days)

15% (1 year)

�1-yearOS 74%

�1-year

DFS 62%

EBMT [103] Various Various 229 (52) Multiple myeloma 31% 50% 26%(2 years)

�3-yearOS 41%

�3-year

DFS 21%Hadassah–Hebrew

University [11]

Fludarabine

30 mg/m2/day �6 days

Busulfan (po)4 mg/kg/day � 2 days

ATG 5–10 mg/kg/day �4 days

CSP F MTX 24 (35) Chronic myeloid

leukemia

in first chronic phase

75%d 55% 3 pts (days

116, 499,

and 726)

�5-year

DFS 85%

Nonmyeloablative regimens

National Institutes

of Health [17]

Fludarabine

25 mg/m2/day �5 daysCyclophosphamide

60 mg/kg/day � 2 days

CSP 15 (50) Hematological + solid

malignancies

10/15 pts 1

after DLI

NR 2 pts (days 59

and 205)

�8/15 pts

survived

between121 and

409

(median,200) days

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M. D. Anderson [15] Fludarabine

25 mg/m2/day � 5 days

or Fludarabine

30 mg/m2/day � 3 daysCyclophosphamide

1 g/m2/day �2 days or 750 mg/m2/day �3 daysF Rituximab

FK506 + MTX 20 (51) Indolent

lymphomas

20% 64% 2 (at 45 and

before 300

days)

�2-year

DFS 84%

FHCRCe [76] TBI 2 Gy

F Fludarabine30 mg/m2/day � 3 days

CSP + MMF 122 (57) Acute myeloid

leukemia

40% 42%f 3%

(100 days)19% (2 years)

�2-year

OS 42%�2-year

PFS 36%


F Fludarabine30 mg/m2/day � 3 days

CSP + MMF 24 (58) Chronic myeloid

leukemia

50% 32%f 21% (4 years) �2-year

OS 70%(CP1)

�2-yrsr

OS 56%

(NCP1)FHCRCe [80] TBI 2 Gy

F Fludarabine


CSP + MMF 33 (53) Mantle cell

lymphoma

57% 64%f 24% (2 years) �2-year

OS 65%

�2-yearPFS 60%


F Fludarabine


CSP + MMF 64 (56) Chronic

lymphocytic

leukemia

55% 50%f 22% (2 years) �2-year

OS 60%

�2-yearDFS 52%

NRM, nonrelapse mortality; ATG, anti-thymocyte globulin; CSP, cyclosporin; FK506, tacrolimus; MTX, methotrexate; OS, overall survival; DFS, disease-free survival; PFS, progression-free survival; NR, not reported; CP1, first chronic

phase.a Before donor lymphocyte infusions given in 36 of 88 (41%) patients.b In patients with low-grade NHL.c In patients with high-grade NHL.d Grades I–IV.e The clinical trials were carried out jointly by a group of collaborators located at the Fred Hutchinson Cancer Research Center, University of Washington, Children’s Hospital and Regional Medical Center, and Veterans Administration

Medical Center, all in Seattle, WA, USA; Stanford University, Palo Alto, CA, USA; City of Hope National Medical Center, Duarte, CA, USA; University of Leipzig, Leipzig, Germany; University of Colorado, Denver, CO, USA; University of

Torino, Turin, Italy; University of Arizona, Tucson, AZ, USA; Baylor University, Dallas, TX, USA; University of Utah, Salt Lake City, UT, USA; Oregon Health and Sciences University, Portland, OR, USA; and, more recently, the Medical

College of Wisconsin, Milwaukee, WI, USA; Emory University, Atlanta, GA, USA; the Rocky Mountain Cancer Center, Denver, CO; the University of Koln, Cologne, Germany; and the Rigshospitalet, Copenhagen, Denmark.f Extensive chronic GVHD.

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busulfan (8 mg/kg), and anti-thymocyte globulin (ATG)[22]. This regimen allowed the achievement of fulldonor chimerism in the majority of the patients witha low nonrelapse mortality in a group of relativelyyoung patients. Kottaridis et al. added alemtuzumab (ahumanized antibody recognizing CD52 antigen ex-pressed 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 lowincidences of graft-versus-host disease (GVHD; animmune-mediated life-threatening complication ofHCT) and nonrelapse mortality in HLA-matched relatedand unrelated recipients [13,23].

Typical complications of high-dose therapy such asnausea, vomiting, pancytopenias, mucositis, and new-onset alopecia have been observed with most of thoseregimens, and sinusoidal obstructive syndrome has alsobeen seen, although less frequently than after myeloa-blative conditioning [11–13,24–26].

In contrast, a nonmyeloablative regimen, such asthe one using 2 Gy of TBI either alone or combinedwith three doses of fludarabine (30 mg/m2/dose), hasrelied on optimal pre-and posttransplant immunosup-pression to overcome host-versus-graft reactions, allow-ing allogeneic engraftment [27,28], which, in turn,resulted in GVT effects [16,29]. Such a regimen hashad few toxicities, has produced only mild myelosup-pression (Fig. 2), and has been associated with a low100-day incidence of nonrelapse mortality, even inelderly patients and those with comorbid conditions[16].

TWO-GRAY-TBI-BASED LOW-INTENSITY

NONMYELOABLATIVE REGIMEN

Preclinical Development in a Canine ModelTBI dose de-escalation. A close relationship between TBIdose and rate of sustained engraftment of dog leukocyteantigen (DLA) identical marrow has been demonstratedin a preclinical canine model (Table 2). A TBI dose of 9.2Gy was sufficiently immunosuppressive to permitengraftment of DLA-identical littermate marrow in95% of dogs, in the absence of postgrafting immuno-suppression [30]. When the TBI dose was decreased to4.5 Gy, only 41% of dogs achieved sustained engraft-ment, while 59% eventually rejected their grafts [31].Since both host-versus-graft (rejection) and graft-versus-host reactions are mediated by T cells after DLA-identical HCT, it was hypothesized that optimizingposttransplant immunosuppression might not onlyprevent GVHD, but also increase the engraftment rate.Indeed, 7 of 7 dogs given 4.5 Gy TBI and postgraftingcyclosporin (CSP) achieved sustained engraftment [31].When the TBI dose was further decreased to 2 Gy,postgrafting immunosuppression either with CSP aloneor with a combination of CSP and methotrexate resulted

30

in graft rejection with autologous recovery in 4 of 4dogs and 3 of 5 dogs, respectively [27]. However, stablemixed donor/host hematopoietic chimerism wasachieved in 11 of 12 dogs given postgrafting immuno-suppression with a combination of mycophenolatemofetil (MMF) and CSP, as well as in 6 of 7 dogs givensirolimus (rapamycin) combined with CSP [27]. Whenthe TBI dose was further decreased to 1 Gy, all dogsgiven either of the two drug combinations eventuallyexperienced graft rejection, demonstrating a delicatebalance between host-versus-graft and graft-versus-hostreactions [27,32].

It was unclear whether the graft rejections observed indogs given less than 2 Gy TBI were due to a lack ofcreation of marrow space to which transplanted hema-topoietic stem cells could home or to insufficient pre-HCT host immunosuppression. To address this question,six dogs were conditioned with 4.5 Gy irradiationtargeted to the cervical, thoracic, and upper abdominallymph node chain and administered postgrafting immu-nosuppression with MMF and CSP [28]. Each dog showedinitial evidence of mixed chimerism. Two dogs rejectedtheir grafts by weeks 8 and 18 after HCT, respectively; onedog died with allogeneic engraftment from GVHD; andthree remained mixed stable donor/host hematopoieticchimeras with follow-up of 57 to 97 weeks. Evidence ofmixed donor/host hematopoietic chimerism in lymphnode and bone marrow spaces that were shielded fromirradiation was consistent with the notion that allogeneicgrafts could create their own marrow space throughsubclinical graft-versus-host reactions. Further experi-mental observations supported the contention that theprimary role of pre-HCT TBI in establishing mixedchimerism was to provide host immunosuppression.First, successful allografts were accomplished in dogsgiven 1 Gy TBI conditioning who had been ’’sensitized’’against donor peripheral blood mononuclear cells(PBMC) in the presence of T cell costimulatory blockadewith CTLA4-Ig [33]. Second, sustained engraftment ofDLA-identical marrow was achieved in dogs given selec-tive T cell ablation with a bismuth-213-labeled (a emitter)anti-T-cell receptor-ah monoclonal antibody, and post-grafting MMF/CSP [34]. Further, engraftment of posi-tively selected CD34+ hematopoietic cells from DLA-identical littermates has been achieved without condi-tioning in dogs with X-linked severe combined immu-nodeficiency disorders (SCID-X1) [35]. Other attempts atdecreasing host immunity before 1 Gy TBI have not metwith success [36,37] (Table 2).

Breaking tolerance in dogs with mixed chimerism. Stablemixed hematopoietic chimerism represents a state ofmutual host–donor tolerance [38]. Although it has beenspeculated that low levels of stable donor chimerismmight be sufficient to treat autoimmune diseases or toprevent rejection of donor solid organ grafts [39,40], high



FIG. 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/Al,

and platelet counts remained above 40,000/Al in the majority of patients,

demonstrating that the conditioning was truly nonmyeloablative. Reproduced

from [50] by permission of the publisher.


levels of donor hematopoiesis might be required toachieve normal hemoglobin levels in patients withthalassemia (Fig. 3A) or sickle cell disease [38,41] andwere found to be required to prevent hemolysis inpyruvate kinase-deficient dogs [42] (see below). In addi-tion, a stable mixed hematopoietic chimerism state isunlikely to be curative for patients with hematologicmalignancies [38,43,44]. These observations led Georgeset al. to investigate whether DLI could convert stablemixed hematopoietic chimerism to full donor chimerismin dogs given DLA-identical grafts after nonmyeloablativeconditioning [45]. Surprisingly, nonsensitized DLI failedto accomplish this task. However, lymphocyte infusionsfrom donors sensitized to the recipient’s minor histo-compatibility antigens by skin grafts ( ’’sensitized DLI’’ )converted mixed chimerism to full donor chimerism ineight of eight dogs studied [45]. The authors hypothe-sized that suppressor [46] or regulatory T cells [47] instable chimeras prevented primary allorecognition andsubsequent sensitization of newly infused T cells fromnonsensitized donors, but did not interfere with thecytotoxic action of already sensitized donor T cells.



HCT after nonmyeloablative conditioning as treatmentfor inherited canine blood disorders. Pyruvate kinasedeficiency in Basenji dogs causes severe hemolyticanemia with hematocrits ranging from 18 to 27%[42,48]. Zaucha et al. explored the efficacy of marrowtransplantation from DLA-identical littermates afterconditioning with 2 Gy TBI and postgrafting immuno-suppression combining MMF and CSP in five affecteddogs [42]. One dog died of liver failure (due to ironoverload) on day 27 with 60% donor engraftment, twodogs experienced nonfatal graft rejection with recur-rence of hemolytic anemia, and two dogs achievedsustained engraftment with 12 and 85% donor chimer-ism levels, respectively. Whereas the dog with the highdegree of donor chimerism had virtually completecorrection of hemolysis and resolution of marrowfibrosis, the dog with the low donor chimerism levelhad persistent clinical symptoms of hemolysis, suggest-ing that clinical responses correlated with donor chi-merism levels. Confirming this hypothesis, infusion ofsensitized donor lymphocytes in pyruvate kinase-defi-cient dogs with low-level donor chimerism and persis-tence of hemolytic anemia after nonmyeloablativeconditioning increased the donor hematopoietic cellcontributions and induced remissions of hemolyticanemia (Fig. 3B) [48].

Clinical TranslationTo date, the nonmyeloablative regimen has been used inmore than 800 patients with hematological diseases whowere ineligible for conventional allogeneic HCT becauseof age and/or concomitant diseases or extensive preced-ing therapies such as failed high-dose autologous orallogeneic HCT. The regimen was remarkably welltolerated, with the majority of eligible patients receivingtheir transplants in the outpatient setting. The firstclinical results with this regimen are described in thefollowing sections.

Engraftment. HCT from HLA-identical sibling: The initialclinical transplant regimen consisted of 2 Gy TBI givenon day 0, followed by postgrafting immunosuppressionwith MMF given at 15 mg/kg bid for 28 days and CSPgiven at full dose until day 35 or 56 [16]. The stem cellsource was G-CSF-mobilized (G) PBMC. The hematolog-ical changes were much milder than usually observedafter myeloablative or reduced-intensity conditioning(Fig. 2) [49]. While most patients rapidly achieved fulldonor granulocyte chimerism (defined as z95% cells ofdonor origin), most remained mixed donor/host T cellchimeras for up to 180 days after HCT (Fig. 4A) [50].Patients who had received myelosuppressive chemother-apy before HCT had higher donor T cell chimerism levelscompared to those who did not. Nine of the first 44patients (20%) given this regimen had nonfatal graftrejections [16]. To reduce the risk of graft rejection,

31

TABLE 2: Effects of TBI dose and postgrafting immunosuppression on engraftment of DLA-identical grafts

Reference

Conditioning

[TBI dose (Gy)]/other

Stem cell

source

Postgrafting

immunosuppression

No. of dogs with stable engraftment

(%)a/No. of dogs transplanted

[30] 9.2 Marrow None 20/21 (95%)

[115] 8.0 Marrow None 4/5 (80%)

[115] 7.0 Marrow None 3/5 (60%)[115] 6.0 Marrow None 12/23 (52)

[115] 4.5 Marrow None 16/39 (41%)

[31] 4.5 Marrow CSPb 7/7 (100%)

[27] 2.0 Marrow CSPb 0/4 (0%)[27] 2.0 Marrow MTXc + CSPb 2/5 (40%)

[27] 2.0 Marrow MMFd + CSPb 11/12 (92%)

[27] 2.0 Marrow Rapae + CSPb 6/7 (86%)[27,32] 1.0 Marrow MMFd + CSPb or Rapae + CSPb 0/11 (0%)

[37] 1.0/FTY720f Marrow MMFd + CSPb 0/5 (0%)

[36] 1.0/ATGg Marrow MMFd + CSPb 1/5 (20%)

TBI, total body irradiation.a Mixed or full chimerism.b Cyclosporin, 15 mg/kg bid po, days �1 to 35.c Methotrexate, 0.4 mg/kg iv on days 1, 3, 6, and 11.d Mycophenolate mofetil, 10 mg/kg bid sc, days 0 to 27.e Rapamycin (sirolimus), 0.05 mg/kg bid sc, days 0 to 27.f FTY720, 5 mg/kg/day, days �5 and �4.g Anti-thymocyte globulins, 3.5 to 5.0 total dose administered from day �12 to day �7.


fludarabine 30 mg/m2/day � 3 days was added to the 2Gy TBI, and the rejection rate decreased to 3% [51].

HCT from HLA-matched unrelated donor: The samefludarabine and 2 Gy TBI regimen was used to conditionpatients with 10-HLA-antigen-matched unrelated donors[52]. Compared to HLA-identical sibling recipients, thepostgrafting immunosuppression with MMF wasextended from 28 to 40 days with taper to day 96, andCSP was given for 100 days with taper through day 180.Twenty-seven percent of patients did not develop neu-tropenia (b500/Al) [52]. Durable engraftment wasobserved in 85% of G-PBMC (n = 71) and 56% of marrowrecipients (n = 18) [52]. Based on this observation, allsubsequent unrelated recipients were given G-PBMCgrafts. Among unrelated G-PBMC recipients, graft rejec-tions were more frequently observed in patients withchronic myeloid leukemia [53] and in patients given G-PBMC containing less than 6.8 � 106 CD34+ cells/kg [54].Further, suboptimal postgrafting immunosuppressionwith MMF was suggested by pharmacokinetic studiesshowing that the t1/2 of mycophenolic acid, the activemetabolite of MMF, was 3 h, and its binding to IMPDH IIwas rapidly reversible [52]. Indeed, increasing adminis-tration of MMF from 15 mg/kg bid to 15 mg/kg tidincreased 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 HCToutcomes: Given that mixed chimerism had been asso-ciated with an increased risk of graft rejection, lowerincidence of acute GVHD, and increasing risk of relapseafter myeloablative conditioning [43,56], we thought to

32

analyze the relationship between kinetics of donorengraftment and HCT outcomes among 157 patientswith hematologic malignancies given HCT after non-myeloablative conditioning [50,57]. Day 14 donor chi-merism levels b50% among T cells ( P = 0.0007) and NKcells ( P = 0.003) predicted graft rejection [57]. Whenchimerism levels were modeled as a continuous linearvariable, high T cell chimerism levels on day 14 wereassociated with an increased probability of grades II–IVacute GVHD ( P = 0.02; Fig. 4B), while high donor T cell( P = 0.002) and NK cell ( P = 0.002) chimerism levels fromdays 14–42 were associated with decreased risk of relapse[57]. Further, high levels of donor NK cell chimerismearly after HCT correlated with better progression-freesurvival ( P = 0.02) [50].

Prevention of graft rejection in patients with lowdonor T-cell chimerism: Based on the observations thatlow donor T cell chimerism levels were associated withgraft rejection, and that success among patients givenDLI for low or falling donor T cell chimerism was seenonly when pre-DLI T cell chimerism levels were N40%[58], Sandmaier et al. evaluated the safety and efficacy ofthe immunosuppressive drug pentostatin (4 mg/m2)given 2 days before DLI to reverse pending graftrejection [59]. Preliminary results in eight patients,treated 54–339 days after HCT, have been analyzed. Tcell chimerism levels before pentostatin and DLI rangedfrom 5 to 34%. After pentostatin and DLI, four of eightpatients had increases in donor T cell chimerism levelsto 63–100% (Fig. 4C), while four patients had levelsremaining at 5–25%. These preliminary results suggestedthat immunosuppression with pentostatin followed by



FIG. 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 anemia [48]. 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 (A) and

subsequently had an increase in donor chimerism in the myeloid compart-

ment and resolution of hemolytic anemia. ((B) was adapted from [48] by

permission of the publisher.)


DLI might effectively prevent graft rejection in patientswith low levels of donor chimerism after nonmyeloa-blative conditioning.

GVHD and GVT effects. In animal models, the intensityof the preparative regimens has been shown to con-tribute to GVHD, presumably by inducing tissuedamage and the elaboration of a cytokine storm [60].Further, mixed donor–host hematopoietic chimerismhas been associated with a decreased risk of GVHDboth in animal models and in humans [38,43,44,50,61].Thus, one might expect less GVHD after nonmyeloa-blative conditioning. To test this hypothesis, Mielcareket al. retrospectively compared GVHD among concur-rent age-matched recipients of related or unrelatedgrafts given after either nonmyeloablative (n = 44) or



myeloablative (n = 52) conditioning [62]. The cumu-lative incidence of grades II–IV acute GVHD was lowerafter nonmyeloablative conditioning (64% versus 85%;P = 0.001), but there were no differences in chronicGVHD (73% vs 71%; P = 0.96). Nonmyeloablativetransplantation was associated with delayed ( P b

0.001) and less frequent ( P = 0.06) initiation of steroidtreatment for GVHD. There was a suggestion thatimmunosuppressive therapy for GVHD was discontin-ued earlier in nonmyeloablative recipients ( P = 0.25).Furthermore, nonmyeloablative patients experiencedless 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 GVHDand GVT responses after myeloablative conditioning[2,8,63,64]. We investigated whether such a relationshipexisted for patients given nonmyeloablative condition-ing in 322 patients given grafts from HLA-matchedrelated (n = 192) or unrelated (n = 130) donors (Fig. 5A)[29]. Fifty-seven percent of patients with measurabledisease 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 withincreased probability of achieving remission, but therewas a trend for a higher probability of remission inpatients with extensive chronic GVHD ( P = 0.07).Further, grades II and III–IV acute GVHD had nosignificant impact on relapse/progression but wereassociated with an increased nonrelapse mortality anddecreased progression-free survival. Conversely, exten-sive chronic GVHD was associated with decreasedrelapse/progression ( P = 0.006) and better progression-free survival ( P = 0.003) (Fig. 5B).

Transplant-related toxicity and infections afternonmyeloablative versus myeloablative conditioning.Transplant-related toxicity and infections have beenfrequent complications of allogeneic HCT and havebeen attributed to both the intensity of the condition-ing and graft-versus-host reactions. Several retrospec-tive studies compared transplant-related toxicity andinfections after HCT following nonmyeloablative ver-sus myeloablative conditioning to determine the rela-tive contributions of conditioning intensity to thesecomplications.

Transplant-related toxicity: By definition, the hemato-logical changes after nonmyeloablative conditioning weremuch milder than seen after myeloablative conditioning.Twenty-three and 63% of the nonmyeloablative recipi-ents versus 100 and 96% of the myeloablative recipients,respectively, required platelet and red blood cell trans-fusions [49]. Liver, kidney, and lung toxicities weresignificantly reduced with nonmyeloablative condition-ing. The cumulative incidence of bilirubin N4 mg/dl was26% at 200 days in nonmyeloablative recipients versus

33


48% in myeloablative recipients [65]. The 100-dayincidence of dialysis was 3% in nonmyeloablative reci-pients versus 12% in myeloablative recipients [66]. The120-day incidence of idiopathic pneumonia syndromewas 2.2% in nonmyeloablative recipients versus 8.4% inmyeloablative recipients [67]. Finally, the risk for experi-encing decreased pulmonary function (FEV1) was signifi-cantly lower for nonmyeloablative than for myeloablativepatients (odds ratio 0.3, P = 0.01) [68].

Sorror et al. analyzed transplantation-related toxicityfollowing HLA-matched unrelated HCT in 134 concur-rent patients given either nonmyeloablative (n = 60) or

FIG. 4. (A) Engraftment kinetics after HCT with nonmyeloablative condition

ing 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 shown [50]. (B) Cumulative incidence

of grades II–IV acute GVHD according to day 14 donor T cell chimerism level

after nonmyeloablative conditioning [50]. (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 afte

HCT, predicting high risk of subsequent graft rejection. He received

pentostatin (4 mg/m2) on day 43 followed by donor lymphocyte infusion 2

days later [59]. This resulted in a significant increase in donor chimerism leve

among all subpopulations, and the patient is currently surviving in molecula

remission with sustained graft N300 days after HCT [53]. ((A) was reproduced

from [50] by permission of the publisher.)

34

myeloablative (n = 74) conditioning using the NationalCancer Institute Common Toxicity Criteria grading [69].Even though patients given nonmyeloablative condition-ing were older, had advanced disease more often, hadmore extensive prior therapies, and had more comorbi-dities at HCT, they experienced significantly less gastro-intestinal, hepatic, and hemorrhagic grades III–IVtoxicity compared to patients concurrently transplantedwith myeloablative conditioning. The 1-year nonrelapsemortality was 20% in nonmyeloablative recipients versus32% in myeloablative recipients ( P = 0.04). Comparableresults were reported by Diaconescu et al. in patientsgiven grafts from related donors [70].

More recently, we have developed an HCT-specificcomorbidity index based on retrospective review ofcomorbidities among 1055 patients given allogeneicHCT at the FHCRC between 1997 and 2003 after non-myeloablative (n = 294) or myeloablative (n = 761)conditioning [71]. Comparing nonmyeloablative andmyeloablative conditioning, respectively, 2-year nonre-lapse mortalities were 5% versus 10% ( P = 0.4) andoverall survival 85% versus 75% ( P = 0.1) in patients withscores of 0–1, nonrelapse mortalities were 17% versus27% ( P = 0.04) and overall survival 61% versus 59% ( P =0.2) in patients with scores of 2–3, and nonrelapsemortalities were 33% versus 54% ( P = 0.03) and overallsurvival 43% versus 30% ( P = 0.006) in patients withscores of z4 [72]. These data suggested that comorbidityscoring was an important tool for assessing patients toconditioning regimens.

Infections: Junghanss et al. compared the incidence ofposttransplant infections in 56 nonmyeloablative recip-ients with that in 112 matched controls given myeloa-blative conditioning [73,74]. The 30- and 100-dayincidences of bacteremia were 9 and 27% in the non-myeloablative group versus 27 ( P = 0.01) and 41% ( P =0.07) in the myeloablative group, respectively. Invasiveaspergillosis occurred at a similar rate ( P = 0.30). Theonset of CMV disease was significantly delayed amongnonmyeloablative compared to myeloablative patients


Copyright C The American Society of Gene Therap

-

s

,

r

l

r

y

FIG. 5. (A) Schedule of the study evaluating graft-versus-tumor effects in 322

patients receiving allogeneic HCT after nonmyeloablative conditioning [29].

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 GVHD [29]. ((B)

was reproduced from [29] by permission of the publisher.)


(medians of 130 versus 52 days; P = 0.02). However, the 1-year probability of CMV disease for high-risk CMVpatients was similar in the two groups ( P = 0.87).

Results of nonablative conditioning in specificdiseases. Hematologic malignancies: Results of nonmye-loablative conditioning in patients with hematologicmalignancies have been reviewed elsewhere [51,75].Encouraging results were observed in patients with acutemyeloid leukemia in first or second complete remission(2-year overall survival of 45 and 51%, respectively) [76],as well as in patients with myelodysplastic syndromewith b5% blasts at HCT (2-year overall survival of 55%)[77], chronic myeloid leukemia (2-year overall survivalof 70% for patients in first chronic phase) [78], chroniclymphocytic leukemia (2-year overall survival of 60%)[79], or indolent or chemotherapy-sensitive aggressivenon-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 completeremission, chemotherapy-insensitive high-grade non-Hodgkin lymphoma or multiple myeloma, or advancedmyelodysplastic syndromes) have been less favorable.

Tandem autologous/allogeneic HCT: To allow olderpatients with aggressive chemosensitive disease to bene-fit from both high-dose chemotherapy and GVT effects,protocols have been developed that first use high-doseconditioning and autologous HCT, which can beadministered with overall mortality rates of less than5%, followed 1 to 3 months later by allogeneic HCTafter nonmyeloablative conditioning (tandem autolo-gous/allogeneic HCT) [81]. Maloney et al. investigatedthe safety of such an approach in patients with multiplemyeloma [82]. Patients were first given cytoreductiveautologous HCT after 200 mg/m2 melphalan, followed40 to 229 (median 62) days later by allogeneic HCT after2 Gy TBI. Fifty-four patients, 29–71 (median 52) years ofage, were included in the study. The 100-day mortalitiesafter autologous and allogeneic HCT were 2 and 2%,respectively. The 2-year overall and progression-freesurvivals were 78 and 55%, respectively. Georges et al.investigated a similar approach in 10 patients withchemorefractory multiple myeloma and HLA-matchedunrelated donors [83]. With a median follow-up of 25months, 8 patients were alive, including 6 patients incomplete remission, 1 with stable disease, and 1 withprogressive disease.

Nonmalignant diseases. Paroxysmal nocturnal hemoglo-binuria: Paroxysmal nocturnal hemoglobinuria (PNH) is arare clonal disorder caused by a somatic mutation of theX-linked phosphatidylinositol glycan class A gene.Hegenbart et al. treated seven adult patients with high-risk paroxysmal nocturnal hemoglobinuria by allogeneicHCT following conditioning with fludarabine and 2 GyTBI [84]. Two patients were given G-PBMC from HLA-matched related donors, and five were given G-PBMCfrom unrelated donors. Patients were deemed ineligiblefor conventional HCT because of Budd–Chiari syndrome(n = 2), life-threatening hemolysis or infections (n = 4),and/or Karnofsky score V80 (n = 4). The median numberof red blood cell transfusions before HCT was 14. Allseven patients achieved sustained engraftment andcomplete remission of PNH. Three patients died ofpancreatitis, infection, or bleeding after a liver biopsyfor chronic GVHD, while the remaining four patientswere alive 13 to 38 months after HCT, with 100% donorchimerism.

Sickle cell disease and h-thalassemia: Allogeneic HCThas remained the only curative treatment for sickle celldisease and h-thalassemia [41,85]. Although long-termsurvival has been excellent (80 to 90%) in good-riskpatients with HLA-identical sibling donors, the procedurehas been associated with significant long-term toxicitiesrelated to the myeloablative conditioning (such as

35


infertility with gonadal failure or secondary malignan-cies), evincing the interest of using nonmyeloablativeconditioning.

Iannone et al. [86] and Horan et al. [87] investigatedthe feasibility of allogeneic HCT after nonmyeloablativeconditioning in 11 patients with sickle cell disease (n = 9)or h-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 hadevidence of donor chimerism (range, 25–100%). How-ever, all but 1 patient lost their grafts after discontinua-tion of postgrafting immunosuppression. The patientwith stable engraftment was still doing well 27 monthsafter HCT with full donor T cell chimerism. One of the 10patients with graft rejection died after a second HCT,while the remaining 9 patients were alive with recurrentdisease. These results showed that it is difficult to achievesustained donor engraftment in patients with hemoglo-binopathies, perhaps because recipients have been sensi-tized to minor histocompatibility antigens of theirdonors by preceding blood transfusions or perhapsbecause h-thalassemic and sickle cell marrows weredifficult to eradicate.

Primary immunodeficiencies: Despite encouragingresults with gene therapy in patients with SCID-X1 [88]and SCID due to adenosine deaminase deficiency [89],allogeneic HCT remains the treatment of choice [90].This is especially true given the increasingly recognizedrisks of insertional mutagenesis associated with currenttechniques of gene therapy [91,92]. However, the sub-stantial risks for mortality and morbidity associated withmyeloablative conditioning regimens have precludedtransplants for all but healthy young patients withoutcomorbid conditions.

Woolfrey et al. reported data from 13 patients withSCID (n = 2) or other primary immunodeficiency syn-dromes (n = 11) given marrow (n = 7), G-PBMC (n = 5), or

FIG. 6. Donor chimerism levels in four representative patients given graft

from HLA-matched related donors for T cell deficiency (n = 3) or a graf

from an HLA-matched unrelated donor for severe combined immunodeficiency

(n = 1) after nonmyeloablative conditioning [94].

36

s

t

cord blood (n = 1) transplantation from HLA-matchedrelated (n = 7) or unrelated (n = 6) donors [93]. Patientswere deemed not to be candidates for conventional HCTbecause of comorbidities or ongoing infections. Twopatients without T cell function who had related donorsdid not receive pre-HCT conditioning, while 11 patientswere conditioned with 2 Gy TBI with (n = 8) or without (n =3) fludarabine (90 mg/m2). Postgrafting immunosuppres-sion consisted of MMF and CSP in all patients. Theregimen was not marrow suppressive, and all patientshad evidence of donor T cell engraftment (T cell chimer-ism levels ranging from 5 to 100%). B cell donor chimerismlevels in the two SCID patients were 50 and 99%,respectively. Day 100 transplant-related mortality was0%. Grades II, III, and IV acute GVHD were seen in 9, 1,and 0 patients, respectively, while 7 patients developedchronic GVHD, which contributed to death in 3 patients.Two patients with low donor chimerism were givensecond HCT. One of them died of transplant-relatedmortality, while the other achieved full donor chimerism.Chimerism levels among various blood cell subpopula-tions in 4 representative patients with stable engraftment[94] are shown in Fig. 6.

Nonmyeloablative Conditioning for Cord Blood orHLA Haploidentical HCTGiven that HLA-matched donors can be found for only 50–80% of patients, depending of their ethnic group, therehas been a considerable interest in extending the use ofnonmyeloablative conditioning to cord blood or HLA-haploidentical HCT. Due to greater degrees of histoin-compatibility, the use of such alternative donors has beenassociated with increased risks of both graft rejection andGVHD. Barker et al. investigated the feasibility of unre-lated cord blood transplantation after nonmyeloablativeconditioning consisting of fludarabine (200 mg/m2),cyclophosphamide (50 mg/kg), and 2 Gy TBI [95,96]. Datafrom 51 patients (median age 50 years) with hematologicmalignancies given 1 (n = 13) or 2 (n = 38) unrelated cordblood units have been recently analyzed [95,96]. Eightpatients not given myelosuppressive chemotherapy in the6 months preceding HCT were also given ATG. Postgraft-ing immunosuppression consisted of MMF and CSP. Cordblood units were predominantly 1-or 2-HLA-antigenmismatched with the recipient. Five of 51 patients hadeither failure of engraftment (n = 4) or graft rejection (n =1), while 46 had sustained primary engraftment. Themedian chimerism level was 100% (range 72–100%) atday 100. The cumulative incidence of grades II–IV acuteGVHD was 61%, while chronic GVHD was seen in 36% ofpatients. One-year probability of progression-free survivalwas 48%.

O’Donnell et al. investigated the feasibility of unma-nipulated haploidentical marrow HCT after nonmyeloa-blative conditioning combining fludarabine (150 mg/m2), cyclophosphamide (29 mg/kg), and 2 Gy TBI in 10




patients with advanced hematologic malignancies [97].Postgrafting immunosuppression consisted of MMF,tacrolimus, and cyclophosphamide, the latter given as asingle dose of 50 mg/kg on day 3 after HCT. Two patientshad graft rejection, while 8 achieved sustained donorengraftment with chimerism levels ranging from 93 to100% at day 180. Grades II, III, and IV acute GVHD wereseen in 3, 3, and 0 patients, respectively. With a medianfollow-up of 284 days, 6 of 10 patients were still alive atthe time of the report, while 4 had died from diseaseprogression (n = 3) or GVHD (n = 1).

HCT AFTER REDUCED-INTENSITY OR

NON-TBI-BASED NONMYELOABLATIVE

CONDITIONING

In contrast to the nonmyeloablative approach describedabove, which relied almost exclusively on GVT effects fortumor eradication, reduced-intensity conditioning regi-mens have combined drugs with demonstrated activityagainst the targeted malignancies with the hope ofdisease control while allowing GVT effects to occur. Themyelosuppressive and immunosuppressive abilities havevaried considerably from one regimen to another (Fig. 1).In addition, while many studies have been performed inpatients unable to tolerate myeloablative conditioningbecause of age, comorbidity, or previous high-dose HCT,other studies have included younger patients who wouldhave been eligible for conventional high-dose allogeneicHCT with standard eligibility criteria, precluding mean-ingful comparisons among reduced-intensity regimens.

EngraftmentThe kinetics of donor engraftment varied considerablyfrom one reduced-intensity regimen to another. Forexample, Childs et al. studied 36 patients conditionedwith fludarabine (125 mg/m2) and cyclophosphamide(120 mg/kg) and given postgrafting immunosuppressionwith CSP [17,98]. Neutrophils decreased to b100/Al in allpatients and recovered to N500/Al at a median of 11 daysafter HCT. Median T cell and granulocyte chimerismlevels were 92 and 38%, respectively, at day 30, and 100and 84%, respectively, at day 100 after HCT.

Ueno et al. analyzed engraftment kinetics in patientstransplanted after conditioning with fludarabine (150mg/m2) and melphalan (140 mg/m2) [21]. Neutrophilsdecreased to b100/Al in all patients and recovered toN500/Al at a median of 12 days after HCT. On days 30 and100, all patients had 100% donor T cell and granulocytechimerism levels.

GVHD and GVT EffectsCouriel et al. reported that patients given grafts fromHLA-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 regi-mens (n = 63) [99].

Perez-Simon et al. analyzed the impact of GVHD onoutcome in 86 recipients of HLA-identical grafts fromsibling donors after conditioning with fludarabine (150mg/m2) plus melphalan (140 mg/m2) or fludarabine (150mg/m2) plus busulfan (10 mg/kg). Patients who devel-oped grades III–IV acute GVHD had significantly worseprogression-free survival ( P = 0.006). In contrast, chronicGVHD was associated with better progression-free sur-vival ( P b 0.0001) in time-dependent analyses [100].

Some reduced-intensity conditioning regimens haveused in vivo T cell depletion of the grafts (with either ATGor alemtuzumab) to decrease the incidence of acute andchronic GVHD. While these strategies achieved theirgoals [13,101], delayed immune reconstitution andincreased incidences of both infections and diseaserelapses were observed [102,103].

Results of Reduced-Intensity or Non-TBI-BasedNonmyeloablative Conditioning in Specific DiseasesHematologic malignancies. Results of reduced-intensityconditioning in patients with hematologic malignancieshave been reviewed elsewhere [51,75]. As shown in Table1 and as observed with nonmyeloablative conditioning,encouraging results were observed in patients with acutemyeloid leukemia in complete remission [14,104], as wellas in patients with myelodysplastic syndrome [105],chronic myeloid leukemia [11], chronic lymphocyticleukemia [25], indolent or chemotherapy-sensitiveaggressive non-Hodgkin lymphoma [13,15,26], or che-motherapy-sensitive multiple myeloma [103]. Con-versely, results in patients with advanced aggressivediseases (such as acute leukemias not in completeremission or chemotherapy-insensitive high-grade non-Hodgkin lymphoma or multiple myeloma) have been lessfavorable.

Nonmalignant diseases. Hemoglobinopathies: Jacob-sohn et al. treated four patients (ages 4 to 22 years) withh-thalassemia (n = 1) or sickle cell disease (n = 3) byallogeneic G-PBMC transplantation following condition-ing with fludarabine (180 mg/m2), intermediate-dose ofiv busulfan (6.4 mg/kg), and ATG [106]. The regimen wasmarrow suppressive with a median duration of neutro-penia of 18 days. One patient had sustained engraftmentbut died of chronic GVHD on day 377 after HCT. Thethree others had graft rejection. Two of them were alivewith recurrent disease, while the other died of infectionon day 780.

Primary immunodeficiencies: Horwitz et al. gaveCD34-selected grafts from HLA-identical siblings afterconditioning with fludarabine (125 mg/m2), cyclophos-phamide (120 mg/kg), and ATG in 10 patients withchronic granulomatous disease [107]. Postgrafting immu-

37


nosuppression consisted of CSP. The regimen was mar-row suppressive with a median duration of neutropeniaof 10 days. After a median follow-up of 17 months, 6patients were alive with sustained engraftment andresolution of preexisting granulomatous lesion, 1 patientwas alive with graft rejection and autologous reconstitu-tion, and 3 patients died of infection, GVHD, orhemorrhagic cystitis developing after a second HCT givenfor graft failure.

Rao et al. analyzed outcomes of 33 patients withprimary immunodeficiencies given bone marrows fromHLA-matched (n = 21) or mismatched (one of sixantigens, n = 11, or two of six antigens, n = 1) unrelateddonors after reduced-intensity conditioning consisting offludarabine (150 mg/m2), melphalan (140 mg/m2), andalemtuzumab or ATG [108]. Postgrafting immunosup-pression was CSP alone. Diagnoses were SCID (n = 6),Wiscott–Aldrich syndrome (n = 4), T cell deficiencies (n =14), CD40 ligand deficiency (n = 4), and phagocytedisorders (n = 5). Median age at HCT was 5.9 years. Thisstudy was not restricted to patients ineligible for myeloa-blative conditioning. The regimen was marrow suppres-sive with a median duration of neutropenia of 13 days.After a median follow-up of 40 months, 2 of 33 patientshave died (1 from infection during the conditioningregimen, the other from chronic GVHD), while 31patients remained alive. At 1 year after HCT, 17 of 31patients had 100% donor chimerism, 10 were mixedchimeras with a high donor contribution, 2 were mixedchimeras with a low donor contribution, and 2 weremixed chimeras with a very low donor contribution.Interestingly, both children with very low donor chimer-ism had received one-HLA-antigen-mismatched grafts.All children with full donor chimerism, mixed chimerismwith high donor contributions, or mixed chimerism withlow donor contributions were free of disease, while the 2children with very low donor contributions wererestarted on prophylactic medications. At 12 monthsafter HCT, 66, 65, 59, and 41% of patients had age-relatednormal levels of T cells, CD4+ T cells, B cells, andresponses to stimulation with phytohemagglutinin,respectively, consistent with relatively slow immunereconstitution probably due in part to the in vivodepletion of the graft by alemtuzumab or ATG.

The same group recently reported an update of theirexperience with reduced-intensity conditioning in 81children (1 of them was given two HCT from twodifferent donors) with immunodeficiency [109]. Donorswere HLA-matched unrelated donors (n = 40), HLA-mismatched unrelated donors (n = 21), HLA-matchedsibling donors (n = 11), and other matched family donors(n = 10). Seventy-one patients received bone marrow, 10G-PBMC, and 1 umbilical cord blood. The use of G-PBMCwas associated with higher donor chimerism levels, butalso with more GVHD. At the time of the analyses, 68patients (84%) were alive, with no significant difference

38

between the donor types or between SCID and otherdiseases.

Shenoy et al. recently reported data from 16 patientswith various nonmalignant hematologic disorders whowere conditioned with fludarabine (150 mg/m2), mel-phalan (70–140 mg/m2), and alemtuzumab (48 mg totaldose) given 3 weeks before HCT [110]. Two patients diedfrom infections before engraftment, while the remainingpatients achieved mixed (n = 2) or full (n = 12) donor Tcell 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 patientshad stable, improved, or completely resolved disease.Remarkably, CD4+ T cell and B cell counts recovered to50% of normal values by 3 months and were completelynormal by 6–9 months after HCT. These results con-trasted with the previously observed delayed immunereconstitution in patients given alemtuzumab the weekbefore HCT.

COMBINATION OF GENE THERAPY WITH

NONMYELOABLATIVE CONDITIONING

Gene therapy approaches might be used in the future toimprove the efficacy of allogeneic HCT with nonmyeloa-blative conditioning. First, infusions of tumor (includingminor histocompatibility antigens restricted to hemato-poietic cells)-specific donor cytotoxic T cells have beenproposed as a way to increase GVT effects withoutinducing GVHD [111]. However, this approach has beenlimited by technical difficulties in generating suchspecific cytotoxic T cells in vitro and by immune evasionmechanisms developed by tumor cells. Several preclinicalstudies 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 forsignaling domains of costimulatory molecules) mightovercome these limitations (recently reviewed in [112]).Another approach might consist of transferring a chemo-therapy resistance gene (such as the O6-methylguanine-DNA methyltransferase gene that confers resistance toBCNU) into donor stem cells, allowing posttransplantselection of donor cells by chemotherapy (BCNU ortemozolomide) administration [113].

Conversely, nonmyeloablative conditioning has beensuccessfully used to promote engraftment of gene-modi-fied autologous stem cells. Those regimens have generallyused nonmyeloablative doses of either TBI or busulfan,given their potential to target nondividing hematopoieticstem cells [89,114]. For example, Aiuti et al. transplantedautologous adenosine deaminase (ADA)-transduced stemcells into two children with severe combined immuno-deficiency due to ADA deficiency after nonmyeloablativeconditioning with busulfan (4 mg/kg) [89]. Both childrenachieved sustained engraftment of transduced stem cells,resulting in increased T cell, B cell, and NK cell counts,




and improved immune functions. Finally, nonmyeloa-blative immunosuppressive conditioning might be usedin the future to promote long-term tolerance to trans-genes after infusion of genetically modified stem cells.

CONCLUSIONS

Nonmyeloablative and reduced-intensity conditioningallowed engraftment of allogeneic hematopoietic cellsand the development of GVT effects. Remarkably, aminimally toxic regimen of 2 Gy TBI with or withoutfludarabine followed by postgrafting immunosuppres-sion with MMF and CSP ensured engraftment ratessimilar to those after myeloablative conditioning formost patients with hematologic disorders. However,nonfatal graft rejections with autologous reconstitutionand recurrence of anemia have been the rule in patientswith hemoglobinopathies.

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

ACKNOWLEDGMENTS

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 pres l’ULg, and the Leon Fredericq Fund.

RECEIVED FOR PUBLICATION AUGUST 10, 2005; REVISED SEPTEMBER 21,

2005; ACCEPTED SEPTEMBER 21, 2005.

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