Prophylactic donor lymphocyte infusions after moderately ablative chemotherapy and stem cell transplantation for hematological malignancies: High remission rate among poor prognosis patients at the expense of graft-versus-host disease

Abstract

We investigated the use of 'prophylactic' donor lymphocyte infusions (DLI) containing 1 x 107 CD3+ cells, given at 30, 60 and 90 days post-allogeneic blood and marrow transplantation (BMT), following conditioning with fludarabine 30 mg/m(2)/4 days and melphalan 70 mg/m(2)/2 days. GVHD prophylaxis consisted of cyclosporin A (CsA) 2 mg/kg daily with early tapering by day 60. Our goals were the rapid achievement of chimerism and disease control, providing an immunological platform for DLIs to treat refractory patients with hematological malignancies. Twelve heavily pre-treated patients with life expectancy less than 6 months were studied; none were in remission. Diagnoses were AML (n = 4), MDS (n = 1), ALL (n = 3), CML (n = 3) and multiple myeloma (n = 1). Response rate was 75%. Three patients are alive at a median of 450 days (range, 450-540). Two patients are in remission of CML in blast crisis and AML for more than 14 months. Median survival is 116 days (range, 25-648). Six patients received 12 DLIs; three patients developed acute GVHD after the first infusion and were excluded from further DLIs, but no GVHD occurred among patients receiving subsequent DLIs. One patient with CML in blast crisis went into CR after the first DLI. The overall incidence of acute GVHD was 70%. Primary causes of death were infections (n = 3), acute GVHD (n = 3), chronic GVHD (n = 1) and disease relapse (n = 2). We observed high response and chimerism rates at the expense of an excessive incidence of GVHD. DLI given at day +30 post BMT caused GVHD in 50% of the patients, and its role in this setting remains unclear.

Full text

Bone Marrow Transplantation (2001) 27, 73–78
2001 Nature Publishing Group All rights reserved 0268–3369/01 $15.00
www.nature.com/bmt
Prophylactic donor lymphocyte infusions after moderately ablative
chemotherapy and stem cell transplantation for hematological
malignancies: high remission rate among poor prognosis patients at
the expense of graft-versus-host disease
M de Lima, M Bonamino, Z Vasconcelos, M Colares, H Diamond, I Zalcberg, R Tavares,
D Lerner, R Byington, L Bouzas, J da Matta, C Andrade, L Carvalho, V Pires, B Barone,
C Maciel and D Tabak
Bone Marrow Transplantation Unit, Instituto Nacional de Cancer, Rio de Janeiro, Brazil
Summary:
We investigated the use of ‘prophylactic’ donor lympho-
cyte infusions (DLI) containing 1 ⴛ10
7
CD3
ⴙ
cells,
given at 30, 60 and 90 days post-allogeneic blood and
marrow transplantation (BMT), following conditioning
with fludarabine 30 mg/m
2
/4 days and melphalan 70
mg/m
2
/2 days. GVHD prophylaxis consisted of cyclospo-
rin A (CsA) 2 mg/kg daily with early tapering by day
60. Our goals were the rapid achievement of chimerism
and disease control, providing an immunological plat-
form for DLIs to treat refractory patients with hematol-
ogical malignancies. Twelve heavily pre-treated patients
with life expectancy less than 6 months were studied;
none were in remission. Diagnoses were AML (nⴝ4),
MDS (nⴝ1), ALL (nⴝ3), CML (nⴝ3) and multiple
myeloma (nⴝ1). Response rate was 75%. Three
patients are alive at a median of 450 days (range, 450–
540). Two patients are in remission of CML in blast
crisis and AML for more than 14 months. Median sur-
vival is 116 days (range, 25–648). Six patients received
12 DLIs; three patients developed acute GVHD after
the first infusion and were excluded from further DLIs,
but no GVHD occurred among patients receiving sub-
sequent DLIs. One patient with CML in blast crisis went
into CR after the first DLI. The overall incidence of
acute GVHD was 70%. Primary causes of death were
infections (nⴝ3), acute GVHD (nⴝ3), chronic GVHD
(nⴝ1) and disease relapse (nⴝ2). We observed high
response and chimerism rates at the expense of an
excessive incidence of GVHD. DLI given at day ⴙ30
post BMT caused GVHD in 50% of the patients, and
its role in this setting remains unclear. Bone Marrow
Transplantation (2001) 27, 73–78.
Keywords: non-ablative chemotherapy; bone marrow
transplantation; lymphocytes
Correspondence: Dr M de Lima at his current address: Department of
Blood and Marrow Transplantation, University of Texas MD Anderson
Cancer Center, 1515 Holcombe Blvd, Box 24, Houston TX 77030–
4095, USA
Received 9 June 2000; accepted 12 October 2000
The therapeutic benefit of allogeneic BMT is in part related
to an immunological graft-versus-leukemia (GVL) effect
that frequently evolves in the context of graft-versus-host
disease (GVHD). The ability of donor lymphocytes to
induce remission in patients relapsing after allogeneic trans-
plantation illustrates the potency of this effect.
1
Estab-
lishing donor–recipient tolerance with less toxic regimens
may provide the basis for further immunological manipu-
lations in order to maximize the GVL effect. However, rap-
idly evolving diseases may not be amenable to this strategy,
considering that the immune-mediated elimination of
malignant cells may take weeks or months to occur. This
fact suggests the need for strategies to reinforce the
immune-mediated phenomena in the post-transplant period.
Groups in Jerusalem and Houston pioneered the use of
sub-lethal doses of fludarabine-based conditioning regi-
mens. These regimens have been shown to be less toxic
and to provide enough immunosuppression to prevent graft
rejection and establish stable mixed or complete chimer-
ism.
2,3
The combination of melphalan and fludarabine has
enabled allogeneic stem cell engraftment in the majority
of patients treated, at least in the setting of HLA-identical
transplantation. Patients with refractory relapses of
advanced leukemias appear to benefit the least.
3
Mixed hematopoietic chimerism is the product of
reciprocal graft–host tolerance. A rapidly achieved state of
mutual tolerance may enable the use of donor lymphocytes
in the post-transplant period, minimizing the development
of GVHD. In order to separate GVL from GVHD, investi-
gators have used lower doses of lymphocytes.
4
CsA is the most used drug for the prevention of GVHD.
However, its inhibitory and modulatory actions may abro-
gate the GVL effect. Patients receiving CsA 5 mg/kg/day
and methotrexate had a greater risk of relapse when com-
pared to patients treated with CsA 1 mg/kg/day.
5
Further-
more, shortening the length of CsA taper post BMT has
been associated with decreased risk of disease relapse in
high-risk patients.
6
We postulated that by reducing the intensity of the con-
ditioning regimen using fludarabine and melphalan (‘mini
BMT’), with CsA 2 mg/kg as GVHD prophylaxis with
early tapering, it would be possible to achieve rapidly
mixed or full chimerism with disease cytoreduction. This,
in turn, would allow us to give prophylactic DLIs in a con-

Mini-BMT and prophylactic DLJs
M de Lima
et al
74
Bone Marrow Transplantation
text of decreased burden of malignant cells. This strategy
was designed to maximize the GVL effect against refrac-
tory and largely incurable, rapid evolving diseases.
Patients and methods
Eligibility criteria
Patients of any age were eligible for the study if they had
hematological malignancies relapsing after allogeneic
BMT, CML in blast crisis, or a medical condition that
would preclude inclusion in ‘standard’ BMT protocols.
Relapses post allogeneic BMT should not be responsive to
DLI and have no acute or extensive chronic GVHD. Orig-
inal BMT donors had to be willing to undergo G-CSF
primed PBSC collection and all patients should have an
HLA-identical sibling donor. The protocol was approved
by the Institutional Review Board of the Instituto Nacional
de Cancer. All donors and patients (or the persons legally
responsible for them) signed written informed consent.
Patient characteristics
From March 1998 to August 1999, 12 patients with a life
expectancy of less than 6 months were enrolled. Median
age was 34 years (range, 8–53). Patients were divided into
two groups: patients relapsing post allogeneic BMT (group
1, n⫽7) and patients without prior transplant (group 2,
n⫽5).
Patients in group 1 had received and failed a median of
1 DLI (range, 1–4) at a median of 102 days prior to entry
(range, 58–730). Median remission duration post first BMT
was 147 days (range, 50–524) and median time between the
two transplants was 338 days (range, 198–784). Preparative
regimens for the first BMT included busulfan and cyclo-
phosphamide (n⫽5) and cyclophosphamide, vepeside and
total body irradiation (n⫽2). Patient UPN 5 did not
receive the second transplant due to fungal sepsis and Pneu-
mocystis carinii pneumonia which developed after the
second dose of fludarabine. Patient characteristics are
summarized in Table 1.
Preparative regimen and GVHD prophylaxis
The preparative regimen consisted of fludarabine 30 mg/m
2
i.v. on days ⫺5to⫺2 and melphalan 70 mg/m
2
i.v. on
days ⫺2 and ⫺1. GVHD prophylaxis consisted of CsA
administered at 2 mg/kg daily by continuous i.v. infusion
from day ⫺1. Doses were adjusted to maintain whole blood
steady state levels at 100–200 ng/ml (initial six patients),
and at 200–400 ng/ml (last six patients) by fluorescence
polarization immunoassay (Cyclosporine Monoclonal
Whole Blood, Abbott Laboratories, Chicago, IL, USA). By
keeping the levels in the lower therapeutic range we
intended to maximize the GVL effect; the target level was
changed when severe acute GVHD occurred among the first
six patients. CsA was to be tapered from day ⫹30, by
approximately 30% per week, provided no GVHD was
present. If a diagnosis of GVHD was made, the discontinu-
ation schedule would follow the discretion of the attending
physician. The protocol called for interruption of CsA on
day ⫹60. Patients without evidence of GVHD by day ⫹30
received DLIs with 1 ⫻10
7
CD3
⫹
cells/kg recipient body
weight, on days ⫹30, ⫹60 and ⫹90. The presence of
GVHD at any of these time points would exclude the
patient from further DLIs.
Supportive care
All patients were treated in HEPA-filtered rooms. High-
dose acyclovir was given for herpes viruses prophylaxis,
and trimethoprim-sulfamethoxazole or pentamidine were
used for Pneumocystis carinii prevention. All patients
received prophylactic fluconazole. Immunohistochemical
assays for cytomegalovirus pp65 antigenemia were perfor-
med weekly from engraftment. Patients developing any
level of antigenemia received ganciclovir.
Donor characteristics, peripheral blood stem cells and
donor lymphocyte collections
All recipient–donor pairs were HLA-matched siblings and
all second transplants used the same donor. The preferred
source of stem cells was the peripheral blood, but one donor
refused G-CSF mobilization and donated bone marrow
instead. All PBSC donors received G-CSF (Amgen, Thou-
sand Oaks, CA, USA) 10
␮
g/kg/day s.c. in two daily doses
for 5 days. PBSC were collected on days 4 and 5 during a
large volume leukapheresis using a COBE spectra apheresis
system (Cobe Laboratories, Lakewood, CO, USA) or
Haemonetics MCS plus (Haemonetics Corporation,
Braintree, MA, USA). The target number of mononuclear
cells was ⬎5⫻10
8
/kg recipient body weight and of
CD34
⫹
cells was ⬎3⫻10
6
/kg. All PBSC products were
infused fresh, unmanipulated, with the exception of PBSC
donated to patient UPN 6, who received a frozen product
due to donor logistic problems.
At post-transplant days ⫹30, ⫹60 and ⫹90 donors were
to undergo leukapheresis. The percentage of CD3
⫹
cells in
the product was determined using a fluorescein isothiocyan-
ate-conjugated CD3-specific monoclonal antibody with a
FACScan cell analyser (Becton Dickinson, San Jose, CA,
USA). Bone marrow was collected following standard pro-
cedures.
Assessment of chimerism
Engraftment was confirmed by polymerase chain reaction
amplification of variable number of tandem repeat (VNTR)
loci (33.6, 33.1, 33.4, H-ras and pYNZ22). Chimerism was
also assessed by cytogenetic analysis on unstimulated bone
marrow, using conventional methods for sex mismatched
donor–recipient pairs.
Study endpoints, definitions of response and statistical
considerations
This was a pilot study designed to treat 20 patients. Primary
study objectives were: (1) to achieve stable mixed or com-
plete chimerism; (2) to determine remission rate; and (3) to
assess the incidence of GVHD with this strategy. Should

Mini-BMT and prophylactic DLJs
M de Lima
et al
75
Table 1 Patient characteristics
UPN Sex/Age Diagnosis Diagnosis/Stage at Co-morbid Previous Treatment administered prior to
mini-BMT conditions
a
allogeneic mini-BMT
BMT
1 F/10 AML relapse 2 PS3 yes DLI – vincristine/hydrea
2 F/8 ALL relapse 3 HCV hepatitis/lungs: yes DLI – vincristine/6-
bronchiolitis mercaptopurine/methotrexate
3 F/9 AML relapse 2 fungal infection yes DLI – hydrea
4 M/35 CML accelerated phase 2 yes DLI ⫹/⫺interferon and cytarabine
6 F/38 MDS refractory disease yes Supportive care
7 M/10 AML relapse 1 yes DLI⫹/⫺IL-2 hydrea
8 M/51 MM refractory relapse HTLV-I infection no Alkylating agents and interferon
post autologous BMT
9 F/17 ALL relapse 1 HCV hepatitis/treated no Relapse while on maintenance
herpetic encephalitis chemotherapy
10 M/49 CML blast crisis no Interferon – hydrea
11 M/44 CML 2nd blast crisis no Cytarabine/daunorubicin (3⫹7)
12 F/53 AML primary refractory fungal infection no Cytarabine/daunorubicin (3⫹7), then
high-dose cytarabine
BMT ⫽blood and marrow transplantation; AML ⫽acute myelogenous leukemia; ALL ⫽acute lymphocytic leukemia; CML ⫽chronic myelogenous
leukemia; MDS ⫽myelodysplastic syndrome; MM ⫽multiple myeloma; PS ⫽performance status; HTLV-I ⫽human T lymphotropic virus -I; HCV
⫽hepatitis virus C. Conditioning regimens: Cy/VP16/TBI ⫽cyclophosphamide, vepeside and total body irradiation; BuCy ⫽busulfan and cyclophos-
phamide.
PS ⫽Zubrod performance status.
7
a
All donor–recipient pairs had at least one person seropositive for cytomegalovirus (CMV).
severe acute GVHD occur in more than 50% of evaluable
cases, the trial was to be interrupted.
Neutrophil recovery was defined as the first of 3 consecu-
tive days that the absolute neutrophil count exceeded 0.5
⫻10
9
/l and platelet recovery was defined as the first of 3
consecutive days that the platelet count exceeded 20 ⫻
10
9
/l, with platelet transfusion independence. CR was
defined as ⬍5% blasts in the bone marrow with a granulo-
cyte count of ⬎1.0 ⫻10
9
/l and platelets ⬎100 ⫻10
9
/l with
reconstitution of donor hematopoiesis as documented by
cytogenetics or VNTR. Remission with partial hematologic
recovery (HPR) was defined as above, except for platelets
⬍100 ⫻10
9
/l. Molecular remissions were not required for
the definition of CR. Bone marrow aspirations for response
and chimerism assessment were performed 3 to 4 weeks
after transplant, monthly thereafter for 6 months and every
3 months afterwards.
We used the criteria of Bearman to grade regimen-related
toxicity.
8
Infections were not scored as regimen-related tox-
icity. GVHD diagnosis required histological confirmation.
Acute and chronic GVHD grading followed standard cri-
teria.
9,10
Cumulative actuarial probabilities of overall sur-
vival were calculated according to the Kaplan–Meier
method.
11
Results
Hematological response and survival
Five of nine evaluable patients (55%) achieved a complete
response (CR) and two (20%) had a HPR. All patients had
fast disappearance of peripheral blood blasts with the
chemotherapy, including patient UPN 9, who had 95 ⫻10
9
ALL blast cells/l at the start of fludarabine. None had evi-
dence of peripheral blood leukemic cells by day ⫹7.
Bone Marrow Transplantation
Among the complete responders, two patients are cur-
rently in remission more than 14 months post treatment,
two patients have relapsed (5 and 11 months post treatment)
and one patient died in CR from chronic GVHD. Patient
UPN 7 achieved a longer remission after mini-BMT than
after the first transplant. Two patients died in HPR and two
patients did not respond. Response could not be assessed
in three patients. Three patients are alive at a median time
of 450 days (range, 450–540) post transplant. Median sur-
vival is 116 days (range, 25–648). Figure 1 depicts the
Kaplan–Meier survival curve for the group. Tables 2 and
3 summarize responses, remission duration and outcomes.
Chimerism
Group 1: Four patients achieved 100% bone marrow chim-
erism by day ⫹30; one of them has had stable complete
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0 6004002000
Time (days)
Cumulative proportion surviving
Survival
Figure 1 Overall survival. Kaplan–Meier estimate for survival is shown.
Three patients are alive, two of them in remission ⬎14 months post treat-
ment.

Mini-BMT and prophylactic DLJs
M de Lima
et al
76
Bone Marrow Transplantation
Table 2 Outcomes post mini-BMT
UPN Response Remission Chimerism
a
GVHD Time to DLI post Disease status
(days) DC
c
CsA Mini-BMT
(days) (No.)
Pre Post (day ⫹30) Acute Chronic Pre-DLI Post DLI
1 CR 152 Predominant recipient 100% donor no no 75 yes/3
e
CR CR
2 CR 121 Predominant recipient 100% donor skin, gut, Gd. III
g
skin, lungs, 17 yes/1 CR CR
extensive
3 HPR 51 Predominant recipient 100% donor skin, liver, gut, Gd. IV NE 21 no NA NA
4 NR 0 80% recipient
d
80% recipient no no 49 yes/3
b
NR NR
b
6 NE 0 84% recipient
d
NE NE NE NE no NA NA
7 CR 420⫹Predominant recipient 100% donor skin, gut, Gd. III skin, liver ND no NA NA
8 NR 0 NA Predominant recipient skin, gut, Gd. II
g
skin, limited ND yes/1 NR NR
9 CR 369 NA 100% donor no no 80 yes/3 CR CR
10 CR 420⫹NA Predominant donor
f
skin, gut, Gd. II
g
gut, liver 55 yes/1 Chronic CR
phase
11 HPR 70 NA 100% donor skin, gut, Gd. III NE ND no NA NA
12 NE 0 NA NE gut, skin, liver, Gd. IV NE NE no NA NA
NA ⫽not applicable; CR ⫽hematological complete response; HPR ⫽hematological partial response; NE ⫽not evaluable; NR ⫽no response; ND
⫽not done; CG ⫽cytogenetics; CsA ⫽cyclosporin A.
a
VNTR, unless indicated;
b
CML in accelerated phase after DLI No. 4 off protocol with interferon ⫹hydrea;
c
discontinue;
d
cytogenetics;
e
received one
more DLI off protocol;
f
converted to 100% donor after DLI;
g
developed acute GVHD after first DLI.
Table 3 Survival
UPN Survival Chimerism duration Causes of death Comments
duration (days)
(days)
1 650 complete 130 Disease progression relapsed 5 months post mini-BMT; received further DLIs
off protocol
2 141 complete 121 chronic GVHD – bronchiolitis died in CR
obliterans and respiratory failure
3 71 complete 51 acute GVHD; HUS/TTP; fungal hypoplastic marrow, in HPR
sepsis
4 540⫹mixed, predominantly receptor accelerated phase CML, on interferon; received further 1
DLI off protocol
6 29 pre-treatment pattern no engraftment; multi-organism
sepsis
7 450⫹complete 420⫹In CR ⬎14 months; in treatment for tuberculosis, with
chronic GVHD
8 116 mixed, predominantly encephalitis with GVHD and HTLV-1 seropositivity
donor post DLI 50 pancytopenia
9 523 complete 340 disease relapse relapsed, 11 months post mini-BMT
10 450⫹complete 420⫹in molecular remission, 14 months post mini-BMT, with
chronic GVHD
11 105 complete 70 GVHD; colecistitis and sepsis died in HPR
12 37 NE acute GVHD – fungal sepsis hypoplastic marrow
HUS/TTP ⫽hemolytic-uremic syndrome/thrombotic thrombocytopenic purpura; NE ⫽not evaluable; NR ⫽no response; ⫹⫽alive.
chimerism for more than 14 months. Patient UPN 4 showed
no change in chimerism before and after treatment, main-
taining approximately 20% donor cells, suggesting ‘auto-
logous’ recovery.
Group 2: Two patients achieved 100% donor chimerism
by day ⫹30; two patients converted to a predominantly
donor pattern after the first DLI, with the development of
grade II acute GVHD in both cases (patients UPN 10 and
8). The latter achieved 100% donor chimerism with hema-
tological and molecular remission of CML in blast crisis.
Engraftment
The median number of infused CD34
⫹
cells per kg recipi-
ent body weight was 3.9 ⫻10
6
(range, 1.25–7.84 ⫻10
6
).
Median time to an absolute neutrophil count ⬎0.5 ⫻10
9
/l
was 11 days (range, 10–23) (n⫽10), to platelet count ⬎20
⫻10
9
/l was 14 days (range, 12–23) (n⫽9) and to platelet
count ⬎100 ⫻10
9
/l was 31 days (range, 12–43) (n⫽6).
Two patients in group 1 had graft rejection (patient UPN
6 rejected a previous transplant with ablative condition-
ing regimen).

Mini-BMT and prophylactic DLJs
M de Lima
et al
77
GVHD and response
Three of five evaluable group 1 patients and four of five
in group 2 developed acute GVHD. Acute GVHD
developed in five of the seven responders (70%) and in
three of five patients achieving a CR. Eight patients sur-
vived more than 100 days and five of them developed
chronic GVHD (60%). Chronic GVHD was diagnosed in
two of the three patients whose responses lasted longer than
6 months.
Three patients had unintended sub-therapeutic levels of
CsA during the first month post BMT, and all developed
acute GVHD. CsA dose reduction was done due to throm-
bocytopenic purpura/hemolytic-uremic syndrome (TTP/
HUS) in two cases, and in one case was induced by drug
interaction. The median time to CsA withdrawal in 6 cases
was 52 days (range, 17–80).
Donor lymphocyte infusions
Six patients received 12 DLIs, with a median number of
CD3
⫹
, CD4
⫹
and CD8
⫹
cells of 1.04 ⫻10
7
(range, 1–1.3
⫻10
7
), 0.66 ⫻10
7
(range, 0.15–0.9 ⫻10
7
) and 0.32 ⫻
10
7
(range, 0.16–0.48 ⫻10
7
), respectively.
Three patients (50%) developed acute GVHD after the
first DLI, two of them while on therapeutic levels of CsA.
Patient UPN 8 also developed pancytopenia. No acute
GVHD developed among the three patients who completed
treatment with DLI numbers 2 and 3. Three patients
received the first infusion of donor lymphocytes in CR, two
patients showed no response and patient UPN 10 recovered
from transplant in accelerated phase CML that was con-
verted to complete hematologic and molecular remission
by the first DLI. Figure 2 depicts development of GVHD
and DLIs.
Toxicity, infections and causes of death
Three patients developed grade II mucositis, and two
patients had moderate veno-occlusive disease of the liver.
The most serious toxicity in this heavily pretreated popu-
lation, however, was severe TTP/HUS, observed in two
cases. Patients UPN 2 and 3 responded to major reductions
in CsA doses, but this led to the onset of acute GVHD in
both cases (fatal in case UPN 3).
CMV reactivation occurred in eight of 10 subjects that
engrafted, requiring prolonged and, in some cases, multiple
10 patients alive at day +30
6 without aGVHD
(received DLI No. 1)
3 developed aGVHD
(2, GD II and 1, GDIII)
received no further DLIs
all developed cGVHD no cGVHD
3 received DLIs Nos 1, 2 and 3
no acute GVHD
4 with aGVHD
(not eligible)
Figure 2 Donor lymphocyte infusions and development of GVHD.
Bone Marrow Transplantation
courses of ganciclovir. Six of the eight patients had GVHD.
However, no clinically apparent CMV disease occurred.
Fungal sepsis was observed as a terminal event in two other
cases and one patient developed pulmonary tuberculosis.
One subject developed BM aplasia following the first DLI.
Nine patients have died. Primary causes of death were
infections (n⫽3), acute GVHD (n⫽3), chronic GVHD
(n⫽1) and relapsed disease (n⫽2). Tables 2 and 3
summarize outcomes.
Considering both the high incidence and severity of
GVHD the study was prematurely closed after 12 patients
were enrolled.
Discussion
We studied a heterogeneous population that shared
advanced stage of disease and refractoriness to first-line
therapies. The combination of melphalan and fludarabine
was well tolerated, even among this heavily pretreated
group.
Peripheral blood was the preferable source of stem cells
considering that, at least for CML, the relapse rate seems
to be decreased when compared to bone marrow, and the
velocity of hematological reconstitution with PBSC may be
an advantage in this context.
12,13
Disease status pre-transplant is a major determinant of
response to therapy. In the MD Anderson Cancer Center
experience, 86 patients treated with purine analog-contain-
ing, non-ablative regimens had a disease-free survival at 2
years of 23.3 ⫾5%, but the subgroup of non-refractory
patients had a disease free survival of 46% at 2 years.
3
We
observed an overall response rate of 75%, with 55% CRs.
Three patients had responses lasting more than 6 months
and two patients diagnosed with CML in blast crisis and
AML are alive in unmaintained remission for more than
14 months.
Seven patients (70%) were complete chimeras by day
⫹30, but rapid achievement of mixed or complete chimer-
ism, within the limitations of the method used to detect it,
did not protect against GVHD. Seventy percent of the
patients achieving complete chimerism developed GVHD,
including three of five patients that were mixed chimeras
pre-mini-BMT.
Spitzer and colleagues
14
treated 21 patients with cyclo-
phosphamide 150–200 mg/kg, anti-thymocyte globulin
(ATG) pre- and post transplant and thymic irradiation. CsA
was used for GVHD prophylaxis. Ten patients received
prophylactic DLIs 5 to 6 weeks post BMT. GVHD occurred
in one of five patients who received one prophylactic DLI,
and in three of four patients treated with more than one
DLI.
All patients who developed GVHD post-prophylactic
DLI in our trial did so after the first infusion. Three patients
that went to the second infusion received the third, without
signs of acute or chronic GVHD. It is possible that fludar-
abine and melphalan induced more cytokine release than
the regimen studied by Spitzer et al, and for that reason,
more GVHD occurred following the first infusion. There
may also be a role for ATG and thymic irradiation in
decreasing the rate of GVHD, as proposed by pre-clinical

Mini-BMT and prophylactic DLJs
M de Lima
et al
78
Bone Marrow Transplantation
studies.
15,16
The absence of GVHD post DLIs given at days
⫹60 and ⫹90 suggests that postponing the infusion until
the damage induced by the conditioning is overcome, may
minimize the risk of severe GVHD. ‘Prophylactic’ DLIs
have been used after T cell-depleted marrow grafts, in
patients receiving myeloablative cyclophosphamide and
total body irradiation, and CsA given from day ⫺4. The
probability of acute GVHD grade II or greater was 100%
in 12 recipients of 10
7
donor T cells/kg on day 30. How-
ever, patients receiving lymphocytes at 2 ⫻10
6
cells/kg on
day 30 and 5 ⫻10
7
cells/kg on day 45 had a probability
of acute GVHD of 31.5% (15.5% post T cell add-back).
17
We assumed that it would be possible to potentiate the
GVL effect by minimizing the exposure to CsA and omit-
ting methotrexate. This assumption was based on the expec-
tation that GVHD incidence in the context of moderately
ablative preparative regimens is lower. However, four of
10 evaluable patients developed acute GVHD prior to DLI,
and another three after DLI, to a global incidence of 70%,
mainly due to severe acute GVHD. This rate was deemed
unacceptable and the study was interrupted. It is possible
that the use of more effective GVHD prophylaxis could
ultimately improve the results of our treatment strategy.
Future interventions may include the use of engineered
clones of cytotoxic T lymphocytes that recognize hemato-
poietic tissue-restricted antigens or leukemia-restricted anti-
gens, separating the anti-leukemia effect from GVHD.
18
In conclusion, we obtained a high response rate and achi-
eved a state of mixed or complete hematopoietic chimerism
among refractory patients at the expense of excessive inci-
dence of GVHD. DLIs given on day ⫹30 after a non-mye-
loablative regimen caused GVHD in 50% of the patients,
and their role in this setting remains unclear.
References
1 Kolb H, Schattenberg A, Goldman JM et al. Graft-versus-leu-
kemia effect of donor lymphocyte transfusions in marrow
grafted patients. Blood 1995; 86: 2041–2050.
2 Slavin S, Nagler A, Naparstek E et al. Nonmyeloablative stem
cell transplantation and cell therapy as an alternative to con-
ventional bone marrow transplantation with lethal cytoreduc-
tion for the treatment of malignant and nonmalignant hemato-
logic diseases. Blood 1998; 91: 756–763.
3 Giralt S, Khouri I, Braunschweig I et al. Melphalan and purine
analog containing preparative regimens. Less intensive con-
ditioning for patients with hematologic malignancies undergo-
ing allogeneic progenitor cell transplantation. Blood 1999; 94:
Abstr. 2518.
4 Mackinnon S, Papadopoulos EB, Carabasi MH et al. Adoptive
immunotherapy evaluating escalating doses of donor leuko-
cytes for relapse of chronic myelogenous leukemia after bone
marrow transplantation: separation of graft-versus-leukemia
responses from graft-versus-host disease. Blood 1995; 85:
1261–1269.
5 Bacigalupo A, van Lint MT, Occhini D et al. Increased risk of
leukemia relapse with high-dose cyclosporine after allogeneic
marrow transplantation for acute leukemia. Blood 1991; 77:
1423–1428.
6 Carlens S, Aschan J, Remberger M et al. Low dose cyclospor-
ine of short duration increases the risk of mild and moderate
graft-versus-host disease and reduces the risk of relapse in
HLA-identical sibling marrow transplantation recipients with
leukaemia. Bone Marrow Transplant 1999; 24: 629–635.
7 Zubrod CG, Scheniderman M, Frei E et al. Appraisal of
methods for the study of chemotherapy of cancer in man: com-
parative therapeutic trial of nitrogen mustard and trimethylene
thioposphoramide. J Chron Dis 1960; 11: 7–33.
8 Bearman SI, Appelbaum FR, Buckner CD et al. Regimen
related toxicities in patients undergoing bone marrow trans-
plantation. J Clin Oncol 1988; 6: 1562–1566.
9 Przepiorka D, Weisdorf D, Martin P et al. Consensus confer-
ence on acute GVHD grading. Bone Marrow Transplant 1995;
15: 825–828.
10 Shulman HM, Sullivan KM, Weiden PL et al. Chronic graft-
versus-host disease syndrome in man. A long term clinical
pathologic study of 20 Seattle patients. Am J Med 1980; 69:
204–208.
11 Kaplan E, Meier P. Non parametric estimation from incom-
plete observations. J Am Stat Assoc 1958; 53: 457–462.
12 Elmaagacli AH, Beelen DW, Opalka B et al. The risk of
residual molecular and cytogenetic disease in patients with
Philadelphia-chromosome positive first chronic phase chronic
myelogenous leukemia is reduced after transplantation of allo-
geneic peripheral blood stem cells compared with bone mar-
row. Blood 1999; 94: 384–389.
13 Przepiorka D, Ippolitti C, Khouri I et al. Allogeneic transplan-
tation for advanced leukemia: improved short-term outcome
with blood stem cell grafts and tacrolimus. Blood 1996; 62:
1806–1810.
14 Spitzer TR, McAfee S, Sackstein R et al. Mixed lymphohema-
topoietic chimerism following non-myeloablative conditioning
and HLA-matched allogeneic bone marrow transplantation:
the effects of chimerism and anti-tumor response of prophy-
lactic donor lymphocyte infusions. Blood 1999; 94 Abstr.
31340.
15 Sykes M, Sachs DH, Spitzer TR. Mixed chimerism followed
by DLI for lymphohematopoietic GVH reactions without
GVHD: from animal models to patients. Hematology 1999;
pp 405–412.
16 Pelot MR, Pearson DA, Swenson K et al. Lymphohematopo-
ietic graft-versus-host reactions can be induced without graft-
versus-host disease in murine mixed chimeras established with
a cyclophosphamide-based non-myeloablative conditioning
regimen. Biol Blood Marrow Transplant 1999; 5: 133–139.
17 Barrett AJ, Mavroudis D, Tisdale J et al. T cell-depleted bone
marrow transplantation and delayed T cell add-back to control
acute GVHD and conserve a graft-versus-leukemia effect.
Bone Marrow Transplant 1998; 6: 543–551.
18 Falkenburg JHF, Wafelman AR, Joosten P et al. Complete
remission of accelerated phase chronic myeloid leukemia by
treatment with leukemia-reactive cytotoxic T lymphocytes.
Blood 1999; 94: 1201–1208.