Bone Marrow Transplantation (2000) 25, 345–350
2000 Macmillan Publishers Ltd All rights reserved 0268–3369/00 $15.00
www.nature.com/bmt
Editorial
Mini-allografts: ongoing trials in humans
AM Carella1, R Champlin2, S Slavin3, P McSweeney4 and R Storb4
1
San Martino Hospital, Genoa, Italy; 2MD Anderson Cancer Center, Houston, TX, USA; 3Hadassah University Hospital, Jerusalem,
Israel; and 4Fred Hutchinson Cancer Research Center, Seattle, WA, USA
Summary:
Conventional allogeneic stem cell transplantation is a
valuable approach to therapy for many hematologic
malignancies. However, high-dose conditioning regimens designed both to control the malignancy and to
prevent graft rejection are associated with a high incidence of acute and long-term side-effects. This has largely precluded the use of allografting for patients older
than 55 years or for younger patients with certain preexisting organ damage. In order to manage the sideeffects, transplants have traditionally been delivered in
highly specialized hospital wards or intensive care settings. Thus, an important goal is to develop safer allografting procedures that can be extended to older
patients or patients with pre-existing organ dysfunction
who are currently excluded from consideration for
transplant. Recent observations have shown that donor
lymphocyte infusions (DLI) can eradicate some malignancies that relapse after conventional allografting.
These observations confirmed earlier evidence in favor
of a graft-versus-leukemia effect based on the association of graft-versus-host disease (GVHD) with a lower
likelihood of relapse of malignancy after allografting.
Given the potential efficacy of DLI as the sole modality
for eradication of malignancy, new strategies for allografting can incorporate the concept of less intensive conditioning therapy which is given with the sole aim of
facilitating allogeneic engraftment. Recent pre-clinical
studies in a canine model have shown that conditioning
regimens for allografting can be markedly reduced in
intensity yet still achieve the goal of engraftment. This
review briefly summarizes the initial translational clinical studies, using a minimally myelosuppressive-conditioning regimen based on low dose total body
irradiation (TBI) or fludarabine alone or in combination with other drugs followed by a short course of
immunosuppression with post-grafting cyclosporine and
methotrexate or mycophenolate mofetil. Bone Marrow
Transplantation (2000) 25, 345–350.
Keywords: allografting; nonmyeloablative regimens;
graft-versus-tumor
Correspondence: Prof A Carella, Dept Haematology, Ospedale San
Martino, Largo Rosanno Brazzi, 10, 15142 Genova, Italy
Received 1 September 1999; accepted 9 November 1999
Until the late 1970s it was thought that the mechanism of
cure of a malignancy was due entirely to the conditioning
therapy used before transplantation, and that the transplant
itself was merely a supportive measure designed to allow
the patient to receive myeloablative treatment without
experiencing permanent aplasia.1,2 However, the desirable
effects of conditioning regimens are offset by their high
incidence of acute and long-term side-effects which are
responsible for considerable transplant-related mortality. It
is now known that the results of allogeneic hematopoietic
stem cell transplantation (allo-SCT) are partly attributable
to an immune effect mediated by donor lymphocytes,
recognized as the graft-versus-leukemia (GVL) effect.
There are major differences among malignancies in their
susceptibility to GVL effects at least as judged by their
response to DLI in the case of relapse after transplantation.
Among the major leukemias treated with allo-SCT, CML
is most sensitive, AML is intermediate and ALL is least
affected by GVL. Indolent lymphoid malignancies (low
grade lymphoma, CLL and myeloma) also appear to be susceptible to graft-versus-tumor effects.3–6 A major question
is whether graft-versus-tumor effects are active against
solid tumors. Pilot studies in breast cancer have reported
antitumor responses in patients with graft-versus-host disease (GVHD) suggesting a graft-versus-adenocarcinoma
effect.7–9 Anecdotal data also suggest graft-versus-tumor
effects against melanoma and potentially other malignancies. Further studies are required to determine whether
a clinically meaningful graft-versus-tumor effect will occur
in order to justify the added morbidity related to allo-SCT.
The success of DLI in inducing remissions in patients in
relapse after allo-SCT suggests that long-term disease control
may be feasible without high-dose induction therapy.10–18
Thus the emphasis has now switched to attempting to
develop safer allografting procedures that can be extended
to older patients and patients with pre-existing organ dysfunction who would not be eligible for conventional alloSCT. This approach reduces drug toxicity and may also
reduce the risk for acute GVHD, since the clinical manifestations of this complication may partly result from the toxicity of the preparative regimen and subsequent cytokine
release in addition to the alloreactivity of the graft.19
Non-myeloablative regimens followed by transfusion of
allogeneic progenitor cells have been investigated in a number of centers as a way of harnessing the graft-versus-tumor
effect. Initial experiences confirmed that such an approach
is feasible in patients with hematologic neoplasia and gen-
Mini-allografts: ongoing trials in humans
AM Carella et al
346
etic diseases. It is hoped that the incorporation of more
effective but less toxic agents in conditioning regimens will
reduce procedure-related morbidity and mortality and ultimately improve long-term outcomes for patients undergoing allo-SCT.
The general concept
The basic observation which serves as the rationale for nonmyeloablative stem cell transplantation originates from the
demonstration that adoptive transfer of alloreactive donor
lymphocytes can eradicate resistant malignant host cells
that escaped maximally tolerated doses of chemoradiotherapy, an observation that has provided an option for cure of
patients with a large variety of hematologic malignancies10,11 especially CML.10–17 The observation that nonmyeloablative stem cell transplantation regimens based on
low-dose total body irradiation (TBI)20 or fludarabine combined with cytotoxic agents have resulted in engraftment
of allogeneic cells in hematologic malignancies, raises the
possibility that such conditioning might even be useful in
achieving a graft-versus-tumor effect.21,22 Fludarabine or
low-dose TBI are crucial to these less intensive regimens.
Fludarabine is an antileukemic and effective immunosuppressive agent as recently established at the MD Anderson
Hospital in Houston.23 Nonmyeloablative chemotherapy
using fludarabine combined with cytotoxic drugs clearly
provides enough immunosuppression to allow engraftment
of allogeneic blood progenitor cells.24 As recently
reported,24 these nonmyeloablative approaches can be
roughly divided into three categories: (1) reduced-intensity
allo-SCT regimens; (2) pre-SCT host immunosuppression
combined with post-SCT immunosuppression directed at
both host and donor immune cells; and (3) high-intensity
auto-SCT followed by an immunosuppressive cytotoxic
allo-SCT regimen alone. In all these settings, allo-SCT is
the platform for subsequent adoptive immunotherapy of the
underlying malignancies using donor lymphocytes.
Reduced-intensity allotransplantation regimens
Focusing on myelotoxic therapy
Purine analog containing non-myeloablative chemotherapy
is sufficiently immunosuppressive to allow engraftment of
HLA compatible hematopoietic progenitor cells, and
extended remissions were observed in some patients with
CML21 or recurrent AML.25 Giralt et al26 reported a study
combining melphalan (180 mg/m2) and either fludarabine
(125 mg/m2) or cladribine (60 mg/m2) for treatment of
advanced acute leukemia; patients with refractory or relapse
usually recurred rapidly, but 56% of patients with chemotherapy-sensitive disease remained in continuous remission
beyond 1 year. Indolent lymphoid malignancies also appear
to be amenable to this strategy. Khouri et al27 treated 15
heavily pretreated patients with CLL or lymphoma using a
nonmyeloablative regimen of fludarabine/cyclophosphamide or fludarabine/cytarabine/cisplatin. All patients
had failed to respond or relapsed after primary chemoBone Marrow Transplantation
therapy. Nine patients had CLL in relapse after prior fludarabine treatment and six had lymphoma. Eleven of the 15
patients had durable engraftment, with 50–100% donor
cells at 1 month post transplant, typically converting to
100% over the next 2 months spontaneously or after
infusion of additional donor lymphocytes. Hematopoietic
recovery was prompt and, with the exception of a patient
with hepatitis C infection, no patient had non-hematologic
toxicity of greater than grade 2. The patients failing to
engraft recovered endogenous hematopoiesis promptly and
had no serious adverse effects. All 11 patients with
engraftment have responded and eight have achieved complete remission. Maximal responses were slow to develop
and gradually occur over a period of several months to 1
year.
The strategy of a nonablative preparative regimen was
also applied to multiple myeloma to harness a graft-versusmyeloma effect while reducing regimen-related toxicities.
The same team in Houston explored a regimen of melphalan (140 mg/m2) and fludarabine (30 mg/m2 for 4 days).
This appears to be a promising strategy since seven of 13
patients with far advanced myeloma have achieved complete remissions. In conclusion, according to the Houston
experience, the immune graft-versus-tumor effect is responsible for much of the benefit of allo-SCT. Use of less
toxic, nonmyeloablative preparative regimens produced
engraftment and generated graft-versus-tumor effects. This
approach allowed the use of allo-SCT for older patients
and those with comorbidities which precluded high-dose
chemoradiotherapy. A regimen comparable to the one
reported from Houston and consisting of cyclophosphamide/fludarabine was used with success by investigators in Bethesda.28
Investigators in Boston used CY 150 to 200 mg/kg along
with ATG and thymic irradiation before HLA-identical sibling BMT in 21 patients with advanced, refractory hematologic malignancies. Grade II–IV GVHD was seen in only
one of 21 patients not receiving DLI. Prophylactic DLI was
given to patients in whom no GVHD was present by day
35. Seven patients were alive and free of disease progression 105–548 (median 445) days after transplantation.
Durable chimerism has been seen in about 90% of patients
receiving HLA-identical and mismatched transplants with
this protocol, and lasting mixed chimerism has been demonstrated .1.5 years in an extensively mismatched transplant recipient.29 The sustained remissions obtained in this
group of patients with advanced and refractory disease suggest that this is a promising approach to achieving disease
eradication, possibly with less GVHD than is seen with
conventional transplants.
Focusing on immunosuppressive therapy pretransplantation
Investigators at the Hadassah University Hospital in Jerusalem focused on induction of a window of immunosuppression (step 1) followed by induction of host-versus-graft tolerance accompanied by GVL effects mediated by donor
lymphocytes infused with mobilized blood stem cells (step
2) or DLI given later as an outpatient procedure (step 3),
reasoning that the same approach may offer the prospect
Mini-allografts: ongoing trials in humans
AM Carella et al
of safer treatment of malignant and nonmalignant diseases
in all age groups with minimal and controllable early and
late procedure-related toxicity and minimal mortality.30,31
Intensive pre-transplant immunosuppressive therapy was
accomplished with a combination of fludarabine 30
mg/m2/day for 6 days, busulfan 4 mg/kg/day for 2 days and
anti-T lymphocyte globulin (ATG, Fresenius) 5–10
mg/kg/day for 4 days. Following the conditioning, each
patient received one or two infusions of G-CSF-mobilized
blood stem cell collections. Low-dose cyclosporine (CsA)
(3 mg/kg/day) was used as the sole GVHD prophylaxis for
,100 days.
The most recent report included the cumulative experience with nonablative preparative regimen in 77 patients,
of whom 63 patients had a variety of hematologic malignancies including CML, AML, ALL, resistant lymphomas
and multiple myeloma and 14 had non-malignant diseases.
The results of this study indicated that cure of otherwise
lethal leukemia became feasible while minimizing early and
late procedure-related toxicities and mortality, thus
enabling safer cure for infants and children without impairment of growth and development. Similarly, nonmyeloablative SCT was well tolerated by elderly individuals, who
were excluded until recently from BMT programs due to
increased risk of procedure-related toxicity and mortality.
Nonmyeloablative SCT resulted in durable engraftment in
all 11 patients receiving bone marrow or blood stem cell
allografts from matched unrelated donors with relatively
low incidence of GVHD.32 Likewise, this less intensive
conditioning regimen was well-tolerated by recipients of
second stem cell allografts after failing a primary procedure
of autologous bone marrow or blood SCT, suggesting that
this regimen may be able to cure patients with resistant
disease who have failed maximal tolerated doses of chemoradiotherapy. Moreover, replacement of host with donor
immunohematopoietic cells and eradication of malignant or
genetically abnormal stem cells and their products may be
accomplished with no support of blood products, with no
episodes of septic fever, while avoiding a nadir of aplasia,
in 10–25% of the patients, respectively, in contrast to the
pattern of recovery of hematopoiesis following standard
myeloablative conditioning, hence possible on an outpatient basis.
Allografting in an ambulatory care setting using
immunosuppressive, minimally myelosuppressive
conditioning
Canine studies have been used in Seattle for several decades to study immunosuppressive regimens for achieving
engraftment across minor and major histocompatibility barriers and for the prevention of GVHD.33 An advantage of
this model is the ready availability of dog lymphocyte antigen (DLA)-identical littermates as a model for HLA-identical transplants in humans. The following allografting studies deal exclusively with the use of DLA-identical
marrow grafts.
347
Marrow toxicity of TBI
TBI delivered in a single fraction at a dose rate of 7 cGy
per minute in dogs given intensive supportive care but no
stem cell grafts invariably caused lethal marrow failure at
<400 cGy whereas at doses ,200 cGy dogs spontaneously
recovered after a period of myelosuppression.34
Enhancement of engraftment with post-graft
immunosuppressive agents
Single dose TBI at 920 cGy was sufficiently immunosuppressive to allow stable engraftment of marrow from 95%
of DLA-identical littermate donors in the absence of postgraft immunosuppression.35 At 450 cGy, only 41% of dogs
had stable engraftment. Additional experiments evaluated
whether immunosuppressive agents given after transplant
could facilitate engraftment. CsA alone was effective at a
dose of 450 cGy with stable engraftment in seven of seven
dogs,36 and the combination of CsA and mycophenolate
mofetil (MMF) allowed for stable engraftment in 10 of 11
dogs after a non-myeloablative dose of only 200 cGy TBI.37
Engraftment was achieved as stable mixed chimerism with
the donor component comprising 45–80% of hematopoietic
cells. Dogs were followed for up to several years and continued to show stable donor engraftment. At 100 cGy TBI,
MMF and CsA did not allow for stable engraftment.
Mechanism of low dose TBI
To evaluate the mechanism by which low dose TBI contributed to engraftment six dogs were given marrow grafts after
450 cGy lymph node irradiation at 200 cGy/min and then
post grafting MMF and CsA.20 The radiation was given to
target cervical, thoracic and upper abdominal lymph nodes
while shielding the remainder of the dogs including most
marrow spaces with lead blocks. Each dog showed evidence of initial mixed chimerism. Two dogs rejected their
grafts, one died with full engraftment from GVHD, and
three remained stable mixed chimeras with follow-up of 1–
2 years. Marrow samples from unirradiated bones showed
chimerism from as early as 4 weeks post transplant that
persisted for the period of observation. These results supported the hypothesis that marrow grafts could create their
own space and that myelosuppressive therapy was not
necessary to establish allogeneic engraftment. These observations, together with results of additional preclinical studies involving second T cell activation signal blockade,38
suggest that in the future TBI may be replaced with
immunosuppressive agents that lack the undesirable sideeffects of ionizing radiation.
Clinical studies
Nonmyeloablative allografting in patients with T cell
immunodeficiencies
The investigators in Seattle hypothesized that patients with
T cell immunodeficiencies would not require pre-transplant
conditioning therapy and that marrow grafts would be able
Bone Marrow Transplantation
Mini-allografts: ongoing trials in humans
AM Carella et al
348
to create their own space. Post grafting, GVH and HVG
reactions would be controlled solely with the use of CsA
and MMF. Results of SCT from HLA-identical sibling
donors in two patients with severe T cell immunodeficiency
syndromes support the hypothesis.39 In both cases stable
donor engraftment was established with varying proportions of myeloid and lymphoid engraftment. Corrections
of the profound underlying immunodeficiencies were
accomplished although as yet not completely. GVHD
occurred early in both patients and was controlled with
immunosuppressive drugs. One patient had chronic GVHD
and died of sepsis and the other patient survives at 19
months with sustained engraftment, off immunosuppression
and leading a normal life.
Outpatient allografting in patients with malignancy
High-intensity autotransplantation followed by
immunosuppressive cytotoxic allotransplantation
regimen
Based on the efficacy and reduced toxicity of nonmyeloablative regimens, investigators in Genoa designed a combined
protocol consisting of autografting followed by mini-allo-
en
pi
1
Re
ci
D
on
or
t
The safety and efficacy of the canine pre-clinical studies
helped develop a conceptual scheme of studies in patients
with hematologic malignancy (Figure 1). Immunosuppression is divided into two components, one directed at host
cells before the transplant, and the other at both donor and
host cells after the transplant to provide simultaneous control of both GVH and HVG reactions. The goal is to establish bidirectional graft–host tolerance as manifested by
stable mixed donor–host hematopoietic chimerism. This
would establish an immunologic platform for the subsequent administration of DLI given specifically to eradicate the underlying malignancy.
The goal of these studies was to evaluate whether allogeneic hematopoietic cell engraftment could be established
in patients with malignancies using peripheral blood stem
cell grafts from HLA-identical donors using an immunosuppressive conditioning regimen of 200 cGy TBI pretransplant and short-term post-graft immunosuppression
with MMF and CsA.40 CsA was given at 6.25 mg/kg p.o.
twice daily from day −1 to day +35 or day +56 and targeted
to blood levels at the upper end of the therapeutic range,
with supratherapeutic levels tolerated in the absence of CsA
toxicities. MMF was given at a dose of 15 mg p.o. twice
daily from day 0 to day +27 and discontinued without tapering. Eligibility for the study required a contraindication to
the use of conventional allografting because of age, prior
high-dose therapy, or organ dysfunction. Forty-six patients
with a median age of 56 years (range 31–72) were treated.
Diagnoses were AML (n = 11), CML (n = 8), CLL (n =
8), multiple myeloma (n = 8), Hodgkin’s disease (n = 4),
non-Hodgkin’s lymphoma (n = 3), acute lymphoblastic leukemia (n = 1), myelodysplastic syndrome (n = 1), breast
cancer (n = 1), amyloidosis (n = 1). Thirty-two patients
were eligible for outpatient allografting while the remainder
were already hospitalized before transplant. Amongst those
patients transplanted in an outpatient setting, the median
number of days of subsequent hospitalization during the
initial 60 days post transplant was 0 (range 0–26). Transplants were very well tolerated with mild myelosuppression, no development of mucositis and no additional alopecia. The only significant regimen-induced toxicities were
reversible hepatoxicity in three patients. In two of these,
predisposing factors were liver cirrhosis in one patient and
concomitant amphotericin therapy in another. Of the fortysix patients treated, non-fatal graft rejection occurred in
nine (16%) patients. Four patients were not evaluable for
sustained engraftment because of death from either transplant complications (n = 1) or early disease progression (n
= 3). Spontaneous acute GVHD requiring treatment
occurred in 36% of patients. Transplant-related deaths
occurred in three patients (6.5%).
With median follow-up of 160 (range 30–450) days, significant disease responses have been observed in the
majority of patients with sustained engraftment after transplant. This has included molecular remissions in four CML
patients and in two CLL patients and disappearance of paraprotein in four myeloma patients. Many of the patients are
still being monitored for disease responses. Responses have
frequently been gradual in onset occurring over a period of
4–12 months. This is an important difference from conventional allografting in which detection of disease post transplant usually represents failure of the treatment approach,
and is potentially important in the development of optimal
DLI strategies for use after nonmyeloablative transplants.
Nonmyeloablative
HSCT
Hematopoietic
microsatellite
markers
Mixed
chimerism
2
DLI
Full
donor
chimerism
GVL
±
GVHD
Figure 1 Conceptual scheme for non-myeloablative stem cell allografting. In step 1 stable mixed hematopoietic chimerism is established with immunosuppressive and minimally toxic conditioning therapy, thus minimizing regimen toxicities and GVHD with the initial transplant. This allows for step 2,
the infusion of donor lymphocytes without rejection, resulting in eradication of host hematopoiesis including the graft-versus-leukemia reaction. Newer
strategies for minimizing GVHD with step 2 include the use of T cell dose escalation schemes, of CD8-depleted DLI, and the insertion of a suicide
(eg herpes simplex thymidine kinase) gene into the T cells that can be activated by drugs.
Bone Marrow Transplantation
Mini-allografts: ongoing trials in humans
AM Carella et al
grafting for patients with advanced hematologic neoplasia
and metastatic breast cancer.41,42 Thirty-two patients have
been treated: 25 have follow-ups of over 3 months, and
seven are within 45 days. Diagnoses in 25 evaluable
patients were high-risk Hodgkin’s disease (HD) (n = 10),
non-Hodgkin’s lymphoma (NHL) (n = 5), blastic (BP) or
accelerated (AP) CML (n = 3) , liver/bone metastatic breast
cancer (n = 4). Patients with RAEB-t (n = 2) and AML
(n = 1) received only mini-allografting. In preparation for
autografting, the patients underwent high-dose therapy on
protocols appropriate for the underlying disease. After
engraftment of autologous stem cells, all patients were conditioned for allografting with fludarabine 30 mg/m2/day ×
3 days with cyclophosphamide 300 mg/m2/day × 3 days,
designated the Flu-Cy protocol. Progenitor cells from HLAmatched donors were mobilized with G-CSF and infused
to the patients. GVHD prophylaxis comprised CsA and
methotrexate. After autografting, the results in the different
diseases were as follows: NHL and HD: three complete
remissions (CR), 11 partial remissions (PR) and one progressive disease; CML: AP: 1; BP: 2; breast cancer: all
patients showed a reduction of pain but no changes in metastatic disease. After mini-allografting, complete chimerism
was achieved in 15/25 (60%) patients and mixed chimerism
in seven (30%) patients. Three patients (BP-CML, RAEB-t
and AML) appear to have had autologous recovery. One
patient with HD in remission after autografting maintained
the remission after mini-allografting; two patients relapsed
and are now alive in partial remission. Eight patients with
NHL and HD, who obtained partial remission after autografting, achieved CR after mini-allografting and donor
lymphocyte infusion. One patient with HD died while in
CR of aspergillus infection in the brain and another patient
with HD died of progressive disease combined with liver
chronic GVHD. One patient (RAEB-t) achieved hematologic and cytogenetic remission and another patient (APCML) had hematologic and molecular remissions. Subsequently, the RAEB-t patient relapsed and she is now alive
on day +520 after mini-allografting; the AP-CML patient
is in continuous molecular remission at day +470. One
patient with breast cancer is alive in good partial remission
(50% reduction of bone metastasis) at day +600. Grade I–
II acute GVHD was the single major complication in nine
patients; grade III acute GVHD was observed in two
patients. In only two patients did the absolute neutrophil
count decrease to ,1.0 × 109/l. No patient required a sterile
room. Five patients died of progressive disease. To date,
19/25 evaluable patients are alive with a median followup from mini-allografting of 13 months (range 3–26). In
summary, immunosuppressive therapy with Flu-Cy allowed
engraftment of hematopoietic progenitor cells from HLAmatched sibling donors; only one patient died of the
procedure 120 days after mini-allografting.
Conclusions
While periods of follow-up are still short in the patients
described above, this new approach has dramatically
reduced the acute toxicity of allografting even among
patients who would otherwise have been excluded from
conventional allografting because of age or other high risk
factors. These new less toxic procedures should facilitate
future studies of adoptive immunotherapy for malignancies
and allow us to extend the use of this type of allografting
to the treatment of selected non-malignant diseases.
349
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