1. No category

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1993 81: 849-855
Acute graft-versus-host disease prophylaxis with methotrexate and
cyclosporine after busulfan and cyclophosphamide in patients with
hematologic malignancies
A von Bueltzingsloewen, R Belanger, C Perreault, Y Bonny, DC Roy, Y Lalonde, J Boileau, J
Kassis, R Lavallee and M Lacombe
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Acute Graft-Versus-Host Disease Prophylaxis With Methotrexate
and Cyclosporine After Busulfan and Cyclophosphamide
in Patients With Hematologic Malignancies
By Anne von Bueltzingsloewen, Robert Belanger, Claude Perreault, Yvette Bonny, Denis-Claude Roy,
Yves Lalonde, Jacques Boileau, Jeannine Kassis, Rene Lavallee, Michel Lacombe, and Martin Gyger
The combination of t w o powerful immunosuppressive
agents, methotrexate (MTX) and cyclosporine (CSP), has
resulted in a significant decrease in the morbidity and mortality after allogeneic bone marrow transplantation (BMT).
However, the additive toxicities from ablative preparative
regimens may lead t o suboptimal use of this combined immunoprophylaxis. We evaluated the efficacy and feasibility
of administering MTX/CSP with busulfan (4 mg/kg/d for 4
days) and cyclophosphamide (50 mg/kg/d for 4 days)
(BuCy4) in 101 consecutive patients with hematologic malignancies categorized into high- and low-risk groups receiving HLA-matched marrow grafts. Postgrafting immunosuppression consisted of MTX short course (15 mg/m2
on day 1 and 10 mg/m2 on days 3, 6, and 11) and intravenous CSP (1.5 mg/kg every 12 hours). Eighty-three patients (82. l%) received 100%of MTX calculated dose and
8 7 (86.1 %) achieved a CSP therapeutic level (250 t o 600
ng/mL) within a median of 1 6 days. Seventy-three patients
(72.2%) received optimal immunosuppressive therapy
comprising a full MTX course and achieving CSP therapeutic concentrations. The Kaplan-Meier estimated incidence
of grade II t o IV acute graft-versus-host disease (GVHD)
was 9.2% for all patients and 5.5% in patients receiving
optimal GVHD prophylaxis. Eighty-nine patients (88.2%)
survived r l 0 0 days posttransplant and 4 3 (48.3%) developed chronic GVHD, the majority of which were de novo
(31 of 43). The estimated incidence of relapse was 28.9%
for all patients and 14.8% in the low-risk group, with a median follow-up of 24.5 months. High-risk features and the
absence of chronic GVHD were significantly associatedwith
relapse (P = .002 and .036, respectively) in multivariate
analyses. Projected disease-free survival at 2 years was
52.3% for all patients and 65.2% in low-risk patients. Disease-free survival was significantly improved in optimally
treated patients (P = .03)
due to a lower incidence of early
deaths from acute GVHD and infectious episodes. In conclusion, optimal delivery of MTX/CSP in association with
BuCy4 resulted in a near complete abrogation of acute
GVHD in HLA-matched transplants and a significantly improved disease-free survival.
0 7 993 by The American Society of Hematology.
I
DFS was not increased due to a high rate of treatment-related
mortality. Retrospectively, this was mostly related to a high
incidence of acute GVHD rather than to the toxicity of the
regimen itself. A recent study by Geller et a126with BuCy4
accounting on more effective GVHD prophylaxis demonstrated a decreased regimen-related toxicity and improved
DFS.
We report herein the first study evaluating the efficacy and
feasibility of administering the MTX/CSP acute GVHD prophylactic regimen in combination with BuCy4. Results show
that the majority of our patients were delivered optimal
GVHD prophylaxis leading to a low incidence of acute
GVHD and minimal treatment-related mortality.
NTENSIVE chemotherapy or chemoradiotherapy followed by allogeneic bone marrow transplantation (BMT)
has emerged as a major therapeutic approach in demonstrating that long, durable disease-free survival (DFS) can be
achieved in an increasing number of patients with hematologic malignancies.I-’ However, the success of allogeneic BMT
is still impeded by a high incidence of either treatment-related
morbidity and mortality most often associated with acute
graft-versus-host disease (GVHD), or relapse of malignant
disease.’ The treatment of established acute GVHD is often
of no avail,” leading thus to the use of an array of immunosuppressive agents aimed rather at its prevention.
Methotrexate (MTX) and cyclosporine (CSP), among several other drugs such as methylprednisolone, cyclophosphamide, and antithymocyte globulin, have been widely used as
immunosuppressive agents to prevent acute G V H D . I I - ~ ~
Prospective randomized studies comparing MTX alone with
CSP alone have shown both drugs to be equally effective in
reducing the incidence of acute GVHD and improving overall
s u r ~ i v a l . l ~Moreover,
-’~
several trials have reported that the
combination of these two agents significantly lowers its incidence when compared with either drug alone.D22However,
there is some concern over a potentially enhanced organ toxicity and incidence of relapse in relationship with this combined prophylactic regimen, especially in patients with acute
myeloblastic l e ~ k e m i a . ~ ’In, ~addition,
~ , ~ ~ the cumulative
toxicities of MTX/CSP and preparative regimen may lead
to suboptimal use of this combined immunoprophylaxis.
Hence, the combination of MTX/CSP and cyclophosphamide with total body irradiation (CY-TBI) in leukemic patients resulted in a truncated MTX administration in a Seattle
study.” Santos et alZ5first reported the results of allogeneic
BMT with busulfan, 4 mg/kg/d X 4 followed by cyclophosphamide, 50 mg/kg/d X 4 (BuCy4). Although this study suggested that BuCy4 had a high tumoricidal potential, overall
Hood, Vol 81, No 3 (February 1). 1993: pp 849-855
MATERIALS AND METHODS
Patient accrual and characteristics. This study group consisted
of 101 consecutive patients with hematologic malignancies referred
to our BMT unit from January 1987 through June 30,199 1. Analysis
was conducted after December 3 1, 1991. Median age was 34 years,
with a range of 1 to 50 years. Patient characteristics and type of
hematologic malignancy are summarized in Table 1. Within each
disease category, patients were stratified into low- and high-risk groups
From the Unite de Transplantation de Moelle Osseuse et Dipartement d’Hkmatologie, H6pital Maisonneuve-Rosemont, Universitk de
Montrial, Montrial, Quibec, Canada.
Submitted May 4, 1992; accepted October 7, 1992.
Address reprint requests to Martin Gyger, MD, FRCPC, Department of Hematology, Maisonneuve-Rosemont Hospital, 5415 de
I’Assomption Blvd, Montreal, Quebec, H I T 2M4 Canada.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C.section 1734 solely to
indicate this fact.
0 1993 by The American Society of Hematology.
0006-4971/93/8103-0027$3.00/0
849
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VON BUELTZINGSLOEWEN ET AL
850
Table 1. Patient Characteristics
Disease
No. of patients
Low-risk/high-risk
CML
MDS
Total
ANLL
ALL
26
19/7
22
15/7
7
14
5
8
10
4
1
20
17
0
7
8
16 (15.8)
51 (50.5)
34 (33.7)
8
12
6
5
13
4
1
21
16
0
8
14 (13.9)
54 (53.5)
33 (32.6)
7
8
4
7
6
4
3
14
8
8
8
2/9
4/8
9/7
19
3
4
18
2
2
28
5
21
15
38
3216 0/15
101
66/35
(65.3/34.7)
Recipient age (yr)
<20
220 and 1 4 0
240
Donor age (yr)
<20
220 and <40
240
Donor --* recipient sex
M+M
M-F
F-M
F-F
Donor parity
Yes/no
AB0 compatibility
Matched
Minor incompatibility
Major incompatibility
CMV serology
Donor-recipient negative
Donor and/or recipient
positive
9
7
32 (31.7)
24 (23.8)
24 (23.7)
21 (20.8)
19/26
2
9
1
5
74 (73.2)
14 (13.9)
13 (12.9)
10
14
5
34 (33.7)
12
24
10
67 (66.3)
8
Numbers in parentheses indicate percentages
according to pretransplant characteristics potentially linked to recurrency of disease post-BMT. High-risk criteria were defined as follows: chronic myelogenous leukemia (CML): acceleration phase,
second chronic phase; acute nonlymphocytic leukemia (ANLL): secondary to chemo/radiotherapy, >second complete remission (CR,),
early relapse; acute lymphoblastic leukemia (ALL): rCR,, early relapse. In patients with myelodysplastic syndrome (MDS), those with
excess blasts, excess blasts in transformation, and secondary refractory
anemia were categorized as high risk. Low-risk criteria were defined
as follows: CML in first chronic phase, de novo ANLL in first complete
remission (CR,), ALL in CR, , and primary refractory anemia with
or without ring sideroblasts. All donor/recipient pairs were HLA genotypically identical, as determined by serological HLA-A, -B, -C,
and -Dr typing and results of mixed leukocyte culture.
Preparationfor transplant. All patients received a total dose of
16 mg/kg busulfan (administered orally on days -10, -9, -8, and -7)
and 900 mg phenytoin 1 day before busulfan initiation, followed by
300 mg daily for 6 days to prevent seizures. Cyclophosphamide (50
mg/kg) was administered intravenously (IV) on days -5, -4, -3, and
-2, followed by marrow infusion on day 0.25Doses of busulfan were
administered according to ideal body weight (or actual weight if less)
and those of cyclophosphamide according to actual weight. Patients
with ALL received testicular irradiation, 4 Gy/d for 2 consecutive
days (days -3 and -2), whereas those with M-4, M-5 ANLL, and ALL
received two intrathecal injections of MTX during initiation of systemic therapy ( I O mg/m2, maximum 15 mg), with leucovorin rescue
on days -6 and -2. Those patients were also administered a total of
six intrathecal injections of MTX on days 32, 46, 60, 74, 88, and
102 after grafting, as central nervous system prophylaxis. Engraftment
was evaluated directly by sex chromosomes and red blood cell antigens. Indirect evidence for engraftment included increasing marrow
cellularity and increasing peripheral blood counts.
GVHD prophylaxis. MTX was administered at a dose of 15 mg/
m2 of body surface area IV on day 1 and 10 mg/m2 on days 3, 6,
and 11 after grafting.” There was no dose reduction schedule for
MTX; however, the drug was withheld in patients with aspartate
aminotransferase (AST) 2500 U/L and/or a creatinine level 2177
pmol/L. CSP was begun on the day before transplantation and was
infused IV over 2 hours at a dose of I .5 mg/kg (ideal weight or actual
weight if less) every 12 hours until the patient recovered from conditioning regimen-induced gastrointestinal toxicity. Thereafter, the
patient orally received 5 mg/kg CSP every 12 hours. The full dose
of CSP was administered until day 50, after which it was decreased
by 10% every 2 weeks. If the serum creatinine level increased 50%
above the day 0 baseline value, the dose was reduced by 50%. The
dose was further reduced by 50% if the serum creatinine level doubled
above the day 0 baseline value. The drug was withheld if the creatinine
level exceeded 177 pmol/L. Patients requiring 250% dose reduction
received IV methylprednisolone or oral prednisone 0.5 mg/kg every
I2 hours until resumption of full dose of CSP. If steroids were continued for more than 14 days, the drug was slowly tapered at a rate
of I O mg per week. CSP trough levels, as measured by radioimmunoassay using a specific monoclonal antibody without any significant
cross-reactivity with CSP metabolites (cyclo-trac, sp-whole blood;
Incstar Corporation, Stillwater, MN) were monitored three times
weekly. Levels of 250 to 600 ng/mL were considered therapeutic.
CSP after day 5 was steadily increased by 25% of initial dose until
therapeutic levels were reached, and reduced by 25% in patients with
toxic serum trough levels. Acute and chronic GVHD were graded
according to criteria proposed by the Seattle
Primary therapy
of established grade I1 to 1V acute GVHD consisted initially of methylprednisolone, 2.5 mg IV every 12 hours for 4 consecutive days,
after which the patient’s status was reevaluated. Progressive chronic
GVHD was treated with alternate day CSP/prednisone, whereas patients with quiescent and de novo GVHD received prednisone l mg/
kg/d for 1 month.
Bone murroM’donors All donations were performed under general anesthesia and patients were discharged between 1 and 2 days
after marrow harvest. No complication of the marrow harvest procedure occurred.
Supportive cure. All patients were treated in single patient’s room
equipped with high-efficiency particulate air filtration (HEPA-filtered).
To reduce CSP nephrotoxicity, patients received daily glucoheptanate
magnesium supplements. Also, a non-nephrotoxic broad spectrum
antibiotic (ceftazidime)was initiated if fever developed during aplasia.
Amphotericin B (0.6 mg/kg) was administered if fever persisted for
5 consecutive days despite the initiation of ceftazidime. Seropositive
patients for herpes simplex virus (HSV) received acyclovir 250 mg/
mz every 12 hours IV from day 0 until day 2 1 posttransplantation.
Patients with cytomegalovirus (CMV) seropositivity or with a CMVseropositive donor received IV acyclovir, 500 mg/m2 every 8 hours,
from day -5 to day +30. CMV-seronegative blood products were
used in all seronegative recipients. All blood products were irradiated
(25 Cy). Patients with clinical and enzymatic suspicion of veno-occlusive disease (VOD)29were treated with IV methylprednisolone,
0.5 mg/kg every 12 hours, strict fluid restriction and a combination
of furosemide and spironolactone. Trimethoprim-sulfamethoxazol
was administered until 6 months after transplant for Pneumocystis
carinii prophylaxis.
Statistical methods. Event times were measured from date of
transplantation to date of relapse, death, or last follow-up examination. DFS was defined as time until relapse or death, whichever
occurred first. The Fisher’s exact probability test” or likelihood ratio
test” were used to test independent pretransplantion risk factors for
significant association with acute/chronic GVHD and relapse. These
risk factors were type of hematologic malignancy, categorization into
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MTX
+ CSP AFTER BUSULFAN AND CYCLOPHOSPHAMIDE
high- or low-risk group, donor/recipient age, A B 0 compatibility, do-
nor/recipient sex combinations, donor parity, CMV status, percent
of calculated MTX dose administered, achievement of a CSP therapeutic serum trough level, steroid administration, acute GVHD (for
chronic GVHD and relapse), and chronic GVHD (for relapse). A
logistic regression model3*was performed to determine possible multivariate relation between risk factors and events such as relapse and
acute and chronic GVHD. The same tests were used to evaluate
whether variables such as hematologic malignancy, categorization
into high- and low-risk groups, and recipient age could have influenced
compliance to MTX and CSP. All tests were performed at the nominal
5% level of significance and were two-sided. Cumulative incidence
curves for grade I1 to IV acute GVHD, chronic GVHD, and relapse
were generated with Kaplan-Meier estimates taking into account time
of death and time to follow-up (treated as censored values). Only
patients who survived 2 100 days posttransplant were included in
the analyses of chronic GVHD. The probability of DFS was estimated
by the method of Kaplan-Meier. Comparisons of acute GVHD and
relapse cumulativeincidenceand DFS curves between different groups
of patients were performed through the Wilcoxon test.
RESULTS
Engraftment and early toxicity. Ninety-seven patients
showed evidence of engraftment. Of the remaining four patients, three died too early to evaluate the fate of the graft
and one died in BM failure with an absolute neutrophil count
(ANC) (0. I X 109/Lon day 29 in a severe septicemic status.
Two patients died before achieving a platelet count 220 X
109/L.The median time to reach an ANC 20.5 X 109/L for
3 consecutive days was 21 days (range, 11 to 37 days) and
24 days for a platelet count 220 X 109/L (range, 12 to 82
days). Mucosal, hepatic, and renal toxicities were most frequently observed. All but three patients had mild to severe
oral mucositis. Hepatic dysfunction with elevated bilirubin
levels and/or increased AST occurred in 86 patients. Eighteen
patients presented with clinical symptoms of VOD, which
resolved in all. Serum creatinine levels increased in 69 patients; however, dialysis was never required. Hemorrhagic
cystitis was seen in 18 patients, three developed seizures during busulfan and one died of early neurologic toxicity. Mild
to severe idiopathic interstitial pneumonitis was observed in
eight patients, leading to death in one. Methylprednisolone
(0.5 mg/kg every 12 hours) was initiated at a median of 22
days posttransplant (range, 4 to 47 days) in 63 patients. The
major reason for steroid introduction was CSP reduction or
discontinuation or persistence of CSP subtherapeutic serum
trough levels (n = 56). VOD and idiopathic interstitial pneumonitis were responsible for steroid initiation in five patients
and one patient, respectively. In one patient, MTX had to
be discontinued after day +4 for hepatic dysfunction and
was replaced with steroids. Steroids were maintained until
CSP could be reintroduced at full dosage, which occurred
within I to 4 weeks after their initiation. Only 35 patients
were treated with amphotericin B for a median of 9 days
(range, 2 to 42 days).
Acute GVHD. Eighty-three patients (82.1%) received
100%of the MTX calculated dose and 87 (86.1%) achieved
a therapeutic CSP serum trough level. The mean CSP dose
administered to achieve a therapeutic level was 143%of initial
dose and the median time to reach this therapeutic level was
16 days (range, 3 to 49 days). Seventy-three patients (72.2%)
85 1
had an optimal immunosuppressive prophylaxis with administration of 100% of the MTX calculated dose and
achievement of a therapeutic CSP serum trough level. One
to three MTX doses were omitted for hepatic, mucosal, and
renal toxicity in 12, three, and two patients, respectively. One
patient died at day +8 posttransplant before receiving the
last MTX dose. Increased creatinine levels led to CSP reduction in 35 patients or discontinuation in 34 at a median
of 21 days posttransplant (range, 3 to 47 days). Initial diagnosis, risk factors for relapse, and recipient age did not affect
either compliance to MTX or achievement of CSP therapeutic
concentrations. The Kaplan-Meier estimated incidence of
acute grade I1 to IV GVHD was 9.2% for all patients (Fig 1).
Initial diagnosis, risk of relapse, donor/recipient age, donor/
recipient sex combination, donor parity, CMV status, A B 0
compatibility, steroid intake, compliance to MTX, and CSP
subtherapeutic serum trough levels were not significantly associated with the onset of acute GVHD in univariate and
multivariate analyses. However, the cumulative incidence of
acute GVHD was significantly lower in patients with optimal
immunosuppressive compliance when compared with the
suboptimal group (P = .02). We attempted to determine
whether time to achieve a therapeutic CSP level had an influence on the incidence of acute GVHD. We observed that
reaching a therapeutic CSP level by day 16 had no influence
on the incidence of acute GVHD (P = S7); in contrast, the
occurrence of acute GVHD was significantly more frequent
in patients with subtherapeutic CSP levels by day 20 ( P =
.02).
Chronic GVHD. Eighty-eight patients (87.1%) survived
100 days posttransplant and, hence, were at risk for chronic
GVHD. Forty-three patients (48.3%) developed chronic
GVHD (31 de novo, 10 quiescent, and 2 progressive). The
Kaplan-Meier estimated incidence of chronic GVHD was
50.6% for all patients (Fig 2). Univariate analysis of risk factors
known to be associated with chronic GVHD showed CML
diagnosis ( P = .005) and donor/recipient age ( P = .006/P =
.002) to be significantly associated with chronic GVHD.
Multivariate logistic regression analysis showed only CML
diagnosis ( P = .OO 1) to be significantlyassociated with chronic
GVHD. Steroid intake, compliance to MTX, and achieve-
n=
0
5
10 15
20 25 30 35 40 45 50
55 60 65 70 75
101
(9.2%)
80 85 90 95 100
Days after Transplantation
Fig 1. Kaplan-Meier estimated incidence of acute grade II to IV
GVHD in 101 patients administered HLA-matched marrow grafts
receiving MTX/CSP immunoprophylaxisin combinationwith BuCy4.
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852
VON BUELTZINGSLOEWEN ET AL
DISCUSSION
4
0
n=88
5
10
IS
20
25
30
35
40
45
(50.6%)
50
55
60
Months after Transplantation
Fig 2. Kaplan-Meier estimated incidence of chronic GVHD in 101
patients administered HLA-matched marrow grafts receiving MTX/
CSP immunoprophylaxis in combination with BuCy4.
ment of CSP subtherapeutic levels did not significantly alter
the onset of chronic GVHD. All living patients with chronic
GVHD have Karnofsky scores 290 except for three with
scores 270.
Leukemic recurrence. The estimated relapse rate at 2
years was 28.9% for all patients and 14.8% in those with lowrisk features. The median follow-up was 24.5 months (range,
6 to 58 months). Leukemic recurrence was seen in 18 patients
(ANLL, 5 of 26; ALL, 5 of 22; CML, 4 of 38; MDS, 4 of 15)
at a median of 12 months (range, 3 to 28 months). Thirteen
of 18 (72.2%) relapses occurred in high-risk patients. Univariate analyses showed relapse to be significantly associated
with high-risk categorization (P= .003). In multivariate logistic regression analyses, high-risk features and absence of
chronic GVHD were found to be significantly associated with
relapse (P= .002 and P = .036, respectively). Three patients
are alive in second complete remission, one after a second
BMT and two after standard rescue chemotherapy. One CML
patient in hematologic relapse remains Philadelphia chromosome-positive after interferon a2b.
Survival and cause of death. Projected DFS at 2 years
was 52.3% for all patients and 65.2% in those with low-risk
features. DFS was significantly improved in low-risk patients
( P = .007) (Fig 3 ) and in those receiving optimal GVHD
prophylaxis ( P = .03). Early posttransplant survival at 3
months in patients with optimal/suboptimal GVHD prophylaxis was 94.6% and 69. I%, respectively. This was attributable to a lower incidence of early deaths from acute GVHD
and infectious episodes. Thirty-five patients died posttransplant. Complications related to acute and chronic GVHD
were responsible for death in five and four patients, respectively; the death rate from these complications in patients
given optimal GVHD prophylaxis was 2% in both. Fourteen
patients died of relapse. Six patients experienced fatal infection ( 1 septicemia, 1 hepatic and cerebral mycosis, 2 severe
colitis, 2 viral encephalitis). Idiopathic interstitial pneumonitis
was responsible for death in one patient. Finally, five patients
presented miscellaneous lethal complications ( 1 hemorrhage,
1 toxic encephalopathy, 1 CNS lymphoma, 1 accident, 1 suicide).
The success of allogeneic BMT in hematologic malignancies is intimately related to the conditioning regimen's tumoricidal potential and to the prevention of morbid and often
lethal complications associated with the occurrence of acute
GVHD.9 When tested in a prospective trial in patients with
ANLL and CML, MTX/CSP combination, in association
with the CY-TBI preparative regimen, reduced the overall
incidence of acute GVHD to 33%, compared with 54% for
those receiving CSP alone.20Similar findings were reported
by others after CY-TBI.Z2,23.33
However, these encouraging
results were offset by an increased incidence of leukemic recurrences, mostly in patients with ANLL.2'-24.
MTX/CSP has been administered in 35 patients in association with a shortened BuCy regimen (cyclophosphamide
60 mg/kg/d for 2 days) with significant hepatic
This is the first study using the combined immunoprophylaxis
MTX/CSP with the highly tumoricidal BuCy4 preparative
regimen, as originally reported by Santos et
Of major
concern was the possibility that regimen-related toxicity could
lead to unacceptable MTX/CSP dose reductions, thereby
impairing the efficacy of this drug combination in preventing
acute GVHD. Indeed, in an initial study with CY-TBI," only
58% of the patients received 100% of MTX calculated dose.
In our study, 82.1% of the patients received 100% of the
MTX calculated dose and 86.1% achieved a therapeutic CSP
serum trough level, with a mean of 143% of projected dose.
Seventy-three patients had an optimal immunosuppressive
prophylaxis receiving full MTX course and achieving a therapeutic CSP level. This combined immunoprophylaxis resulted in a Kaplan-Meier estimated incidence of grade I1 to
IV acute GVHD of only 9.2% for all patients, with an overall
mortality related to acute GVHD of 5%. The 5.5% KaplanMeier estimated probability of acute GVHD in patients with
optimal prophylaxis was comparable or lower to the results
obtained after T-cell d e p l e t i ~ n . ~ It
' . ~is~noteworthy that neither full MTX course nor achievement of a CSP therapeutic
100
l%
:...I.
.-
.......
!.....I
n =66
I
U l i l
(65.2%)
I
, , , I
u
LIJ
v)
f
.-i..I.....I........
t
8 1
.E
20
0
iL.1 ..I
n
L .....1...
-...
I..
=
35
(31.6%)
..........."I..............
.
p = 0.007
5
10
15
20
25
30
35
40
45
50
55
60
Months after Transplantation
Fig 3. Kaplan-Meier DFS in 101 patients administered HLAmatched marrow grafts for hematologic malignancy with low-risk
features (top curve) or high-risk features (bottom curve) receiving
MTX/CSP immunoprophylaxis in combination with BuCy4. Comparisons between both groups were performed with the Wilcoxon
test.
From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
MTX
+ CSP AFTER
853
BUSULFAN AND CYCLOPHOSPHAMIDE
level, when analyzed independently, were significantly associated with a decreased incidence of acute GVHD. Thus,
in this study, full MTX delivery with achievement of a CSP
therapeutic trough level proved to be essential in preventing
acute GVHD. During the early posttransplant period, MTX
probably plays a major role in inducing tolerance against
donor lymphocyte proliferating in response to host alloantigens. The marginal contribution of CSP in preventing acute
GVHD within that specific period has been emphasized in
a recent study from Seattle using MTX/CSP in which CSP
was reduced by 50%during the first 15 days of transplantation
without any adverse effects in the incidence, time of onset,
and severity of acute GVHD.38 This is in accordance with
our study in which occurrence of acute GVHD was not influenced by achieving a therapeutic CSP trough level by day
16. Yee et al,39who studied the relation between CSP concentrations and acute GVHD, did not find any correlation
during the first week posttransplant. However they observed
that after the first week low CSP trough levels for a given
week were significantly associated with the risk of developing
acute GVHD during the following week. The significantly
reduced incidence of acute GVHD observed in our patients
achieving CSP therapeutic concentration by day 20 corroborates these findings. These observations suggest that CSP
may have a marginal impact on GVHD prevention during
the early posttransplant period, contrasting with its important
role once MTX is ceased. Results from studies using shortcourse MTX alone to prevent acute GVHD confirmed the
role played by CSP.40
The compliance to MTX/CSP observed in this study is
superior to the one reported by the Seattle group, mostly
with regards to percent of MTX calculated dose administration.*' Several factors are likely to explain this discrepancy.
First, the Seattle team's guidelines for altering the projected
MTX dose may be more rigid. In our study, MTX was omitted only in patients with AST 2500 U/L and/or in patients
with a creatinine value of 177 pmol/L. All other patients
were supposed to receive 100% of the calculated MTX projected dose, although three patients had one dose omitted for
mucosal toxicity. Also, to limit renal toxicity, CSP was monitored on a regular basis, three times weekly, with a 50%dose
reduction when creatinine levels were increased 50% above
the baseline value (compared with a creatinine level doubling
in the Seattle group). This precocious lowering of CSP may
have helped in sparing renal function. Furthermore, all
nephrotoxic antibiotics were avoided if possible. None of the
patients received aminoglycosides as first line broad spectrum
antibiotics. Amphotericin B was administered to only 34.6%
of our patients for a median of 9 days (range, 2 to 42 days).
Finally, 63 patients received oral or intravenous steroids for
various reasons (CSP dose reductions, VOD, pulmonary toxicity). In contrast to the Seattle data:' the addition of steroids
had no significant adverse effect on the cumulative incidence
of acute or chronic GVHD. However, steroids may have
contributed to the high compliance to MTX/CSP administration through inhibition of inflammatory acute phase reactants such as tumor necrosis factor a (TNFa), interleukin-1
(IL-l), IL-6, and IL-8.42 This is in agreement with results
from a recent Seattle study in which the percentage of MTX
calculated dose administered was significantly higher in patients receiving steroid^.^' Steroid inhibition of early phase
inflammatory cytokines may have also contributed to the
low grade of regimen-related hepatic toxicity.
The overall estimated relapse rate at 2 years posttransplant
was 28.9%; however, despite a low acute GVHD incidence,
the projected relapse rate in low-risk patients was only 14.8%.
Two factors are likely to explain this phenomenon. Since its
original description, BuCy has been widely recognized as a
highly effective tumoricidal regimen.2532634345
Moreover, the
high incidence of chronic GVHD may have precluded disease
recurrence through the hitherto recognized GVHD linked
antileukemic effect.* Indeed, 48.3% of our patients developed
chronic GVHD. Fortunately, 72.1% of these were de novo
chronic GVHD occurring at the very end of CSP tapering
and responding well to steroid therapy. We have no obvious
explanation for this high incidence of de novo chronic GVHD
and, in contrast to the Seattle study, steroids delivery was
not significantly associated with chronic GVHD.4' Unexpectedly, CML was found to be significantly related to the
incidence of chronic GVHD. It is unclear whether this finding
can be explained by the disease itself or by the age of the
patients with CML. Indeed, 50%of the patients over 40 years
of age were in the CML group. Disease recurrence was the
major cause of treatment failure in this study. The most statistically significant factor associated with relapse was highrisk pretransplant characteristics, emphasizing the need for
improved tumoricidal preparative regimens or innovative
posttransplant therapeutic strategies.
The combination of MTX/CSP proved highly effective in
preventing acute GVHD in this study. Achievement of optimal GVHD prophylaxis, both in the early posttransplant
period through a full course of MTX administration and in
the late posttransplant period with therapeutic CSP levels,
resulted in a low incidence of acute GVHD, minimal treatment-related mortality, and significantly improved DFS.
ACKNOWLEDGMENT
We are indebted to the inpatient and outpatient physicians and
nurses of the Maisonneuve-RosemontHospital, to Nicole TremblayFennel1 for her excellent secretarial assistance, and to Dr James 0.
Armitage for critical reading of the manuscript.
REFERENCES
1. Thomas ED, Buckner CD, Clift RA, Fefer A, Johnson F, Nei-
man P, Sale G, Sanders J, Singer J, Shulman H, Storb R, Weiden P:
Marrow transplantation for acute nonlymphoblastic leukemia in first
remission. N Engl J Med 301597, 1979
2. McGlave PB, Haake RJ, Bostrom BC, Brunning R, Hurd DD,
Kim TH, Nesbit ME, Vercellotti GM, Weisdorf D, Woods WG,
Ramsay KC, Kersey JH: Allogeneic bone marrow transplantation
for acute nonlymphocytic leukemia in first remission. Blood 72: 1512,
1988
3. Blume KG, Forman SJ, Snyder DS, Nademanee AP, ODonnell
MR, Fahey JL, Krance RA, Sniecinski IJ, Stock AD, Findley DO,
Lipsett JA, Schmidt GM, Nathwani MB, Hill LR, Metter GE: Allogeneic bone marrow transplantation for acute lymphoblastic leukemia during first complete remission. Transplantation 43:389, 1987
4. Vernant JP, Marit G , Maraninchi D, Guyotat D, Kuentz M,
Cordonnier C, Reiffers J: Allogeneic bone marrow transplantation
From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
854
in adults with acute lymphoblastic leukemia in first complete remission. J Clin Oncol 6:227, 1988
5. Doney KC, Buckner CD, Thomas ED, Sanders J, Clift RA,
Hansen JA, Sale GE, Singer J, Storb R: Allogeneic bone marrow
transplantation for chronic granulocytic leukemia. Exp Hematol 9:
966, 1981
6. Goldman JM, Apperley JF, Jones L, Marcus R, Goolden AWG,
Batchelor R, Hale G, Waldman H, Reid CD, Hows J, Gordon-Smith
E, Catowsky D, Galton DAG: Bone marrow transplantation for patients with chronic myeloid leukemia. N Engl J Med 314:202, 1986
7. Appelbaum FR, Storb R, Ramberg RE, Shulman HM, Buckner
CD, Clift RA, Deeg HJ, Fefer A, Sanders J, Self S, Singer J, Stewart
P, Sullivan K, Witherspoon R, Thomas ED: Treatment of preleukemic syndromes with marrow transplantation. Blood 69:92, 1987
8. Belanger R, Gyger M, Perreault C, Bonny Y, St-Louis J: Bone
marrow transplantation for myelodysplastic syndromes. Br J Haematol 69:29, 1988
9. Storb R: Critical issues in bone marrow transplantation. Transplant Proc 19:2774, 1987
IO. Sullivan KM, Deeg HJ, Sanders J, Klosterman A, Amos D,
Shulman H, Sale G, Martin P, Witherspoon R, Appelbaum F, Doney
K, Stewart P, Meyers J, McDonald GB, Weiden P, Fefer A, Buckner
CD, Storb R, Thomas ED: Hyper-acutegraft-v-hostdisease in patients
not given immunosuppression after allogeneic marrow transplantation. Blood 67:1172, 1986
I I . Lazarus HM, Coccia PF, Herzig RH, Graham-Pole J, Gross
S, Strandjord S, Gordon E, Cheung N-KV, Warkentin PI, Spitzer
TR, Warm SE: Incidence of acute graft-versus-host disease with and
without methotrexate prophylaxis in allogeneic bone marrow transplant patients. Blood 64:215, 1984
12. Ramsay NKC, Kersey JH, Robison LL, McGlave PB, Woods
WG, Krivit W, Kim TH, Goldman AI, Nesbit ME Jr: A randomized
study of the prevention of acute graft-versus-host disease. N Engl J
Med 306:392, 1982
13. Powles RL, Clink HM, Spence D, Morgenstern G, Watson
JG, Selby PJ, Woods M, Barrett A, Jameson B, Sloane J, Lawler SD,
Kay HEM, Lawson D, McElwain TJ, Alexander P: Cyclosporin A
to prevent graft-versus-host disease in man after allogeneic bone
marrow transplantation. Lancet 1:327, 1980
14. Weiden PL, Doney K, Storb R, Thomas ED: Anti-human
thymocyte globulin for prophylaxis of graft-versus-host disease: A
randomized trial in patients with leukemia treated with HLA-identical
sibling matched grafts. Transplantation 27:227, 1979
15. Santos GW, Tutschka PJ, Brookmeyer R, Saral R, Beschomer
WE, Bias WB, Braine HG, Bums WH, Farmer ER, Hess AD, Kaizer
H, Mellits D, Sensenbrenner L, Stuart R, Yeager A: Cyclosporine
plus methylprednisolone versus cyclophosphamide plus methylprednisolone as prophylaxis for graft-versus-host disease: A randomized
double blind study in patients undergoing allogeneic marrow transplantation. Clin Transplant l:21, 1987
16. Forman SJ, Blume KG, Krance RA, Miner PJ, Metler GE,
Hill LR, ODonnell MR, Nademanee AP, Snyder DJ: A prospective
randomized study of acute graft-versus-host disease in 107 patients
with leukemia-Methotrexate/prednisone versus cyclosporine/prednisone. Transplant Proc 2 1:2605, 1987
17. Deeg HJ, Storb R, Thomas ED, Flournoy M, Kennedy MS,
Banaji M, Appelbaum FR, Bensiger WI, Buckner CD, Clift RA, Doney K, Fefer A, McGuffin R, Sanders JE, Singer J, Stewart P, Sullivan
KM, Witherspoon R P Cyclosporine as prophylaxis for graft-versushost disease: A randomized study in patients undergoing marrow
transplantation for acute nonlymphoblastic leukemia. Blood 65: 1325,
1985
18. Storb R, Deeg HJ, Thomas ED, Appelbaum FR, Buckner
CD, Cheever MA, Clift RA, Doney KC, Flournoy N, Kennedy MS,
VON BUELTZINGSLOEWEN ET AL
Loughran TP, McGuffin RW, Sale GE, Sanders JE, Singer JW, Stewart PS, Sullivan KM, Witherspoon RP: Marrow transplantation for
chronic myelocytic leukemia: A controlled trial of cyclosporine versus
methotrexate for prophylaxis of graft-versus-host disease. Blood 66:
698, 1985
19. Irle C, Deeg HJ, Buckner CD, Kennedy M, Clift R, Storb R,
Appelbaum FR, Beatty P, Bensinger W, Doney K, Cheever M, Fefer
A, Greenberg P, Hill R, Martin P, McGuffin R, Sanders J, Stewart
P, Sullivan K, Witherspoon R, Thomas ED: Marrow transplantation
for leukemia following fractionated total body irradiation: A comparative trial of methotrexate and cyclosporine. Leuk Res 9:1255,
1985
20. Storb R, Deeg HJ, Whitehead J, Appelbaum F, Beatty P, Bensinger W, Buckner CD, Clift R, Doney K, Farewell V, Hansen J,
Hill R, Lum L, Martin P, McGuffin R, Sanders J, Stewart P, Sullivan
K, Witherspoon R, Yee G, Thomas ED: Methotrexate and cyclosporine compared with cyclosporine alone for prophylaxis of acute
graft-versus-host disease after marrow transplantation for leukemia.
N Engl J Med 314:729, 1986
2 1. Storb R, Deeg HJ, Pepe M, Appelbaum F, Anasetti C, Beatty
P, Bensinger W, Berenson R, Buckner CD, Clift R, Doney K, Longton
G , Hansen J, Hill R, Loughran T, Martin P, Singer J, Sanders J,
Stewart P, Sullivan K, Witherspoon R, Thomas E D Methotrexate
and cyclosporine versus cyclosporine alone for prophylaxis of graftversus-host disease in patients given HLA-identical marrow grafts
for leukemia: Long-term follow-up of a controlled trial. Blood 73:
1729, 1989
22. Tollemar J, Ringdkn 0, Heimdahl A, Loonqvist B, Sundberg
B: Decreased incidence and severity of graft-versus-host disease in
HLA matched and mismatched marrow recipients of cyclosporine
and methotrexate. Transplant Proc 20:470, 1988 (suppl 3 )
23. Aschan J, RingdCn 0, Sundberg B, Gaharton G, Ljungman
P, Winiarski J: Methotrexate combined with cyclosporin A decreases
graft-versus-host disease, but increases leukemic relapse compared
to monotherapy. Bone Marrow Transplant 7: I 13, I99 1
24. Mehatchard JM, Fyles GM, Mirden MD, Curtis JE, Lockwocd
G, Tpitchler D, Mesner HA: Acute graft-versus-host disease and relapse rate in allogeneic bone marrow transplant recipients receiving
prophylaxis with cyclosporine A and methotrexate in comparison to
methotrexate and prednisone. Exp Hematol 16:517, 1988 (abstr)
25. Santos GW, Tutschka PJ, Brookmeyer R, Saral R, Beschomer
WE, Bias WB, Braine HG, Bums WH, Elfenbein GJ, Kaizer H, Mellits
D, Sensenbrenner LL, Stuart RK, Yeager AM: Marrow transplantation for acute nonlymphocytic leukemia after treatment with busulfan and cyclophosphamide. N Engl J Med 309:1347, 1983
26. Geller RB, Saral R, Piantadosi S, Zahurak M, Vogelsang GB,
Wingard JR, Ambinder RF, Beschomer WB, Braine HG, Bums WH,
Hess AD, Jones RJ, May WS, Rowley SD, Wagner JE, Yeager AM,
Santos GW: Allogeneic bone marrow transplantation after high-dose
busulfan and cyclophosphamide in patients with acute nonlymphocytic leukemia. Blood 73:2209, 1989
27. Sullivan KM: Acute and chronic graft-versus-host disease in
man. Int J Cell Cloning 4:42, 1986 (suppl 1)
28. Thomas ED, Storb R, Clift RA, Fefer A, Johnson FL, Neiman
PE, Lerner KG, Glucksberg H, Buckner CD: Bone-marrow transplantation. N Engl J Med 292395, 1975
29. Rollins BJ: Hepatic veno-occlusive disease. Am J Med 81:
297, 1986
30. Siege1 S: Non-Parametric Statistics for the Behavioral Sciences
(ed 2). New York, NY, McGraw-Hill, 1988
3 I . Zar HJ: BiostatisticalAnalysis. Englewood Cliffs, NJ, PrenticeHall, Biological Sciences Series, 1974
32. Koch GG, Edwards S: Logistic regression, in Johnson NL,
Ketz S (eds): Encyclopedia of Statistical Sciences, vol 5. New York,
NY, Wiley, 1985
From www.bloodjournal.org by guest on November 14, 2014. For personal use only.
MTX
+ CSP AFTER BUSULFAN AND CYCLOPHOSPHAMIDE
33. MrsiC M, Labar B, BogdaniC V, Nemet D, PavletiC Z, PlavSiC
F, DobriC I, MaruSiC M, FrancetiC I, KaStelan A, KaleniC S, Vrtar
M, Markulin-GrgiC, Aurer I: Combination of cyclosporin and methotrexate for prophylaxis of acute graft-versus-host disease after allogeneic bone marrow transplantation for leukemia. Bone Marrow
Transplant 6: 137, 1990
34. Nevill TJ, Barnett MJ, Klingemann HG, Reece DE, Shepherd
JD, Phillips GL: Regimen-related toxicity of a busulfan-cyclophosphamide conditioning regimen in 70 patients undergoing allogeneic
bone marrow transplantation. J Clin Oncol 9:1224, 1991
35. Hale G, Cobbold S, Waldmann H: T-cell depletion with Campath- 1 in allogeneic bone marrow transplantation. Transplantation
45:753, 1988
36. Champlin R, Ho W, Gajewski J, Feig S, Burnison M, Holley
G, Greenberg P, Lee K, Schmid I, Giorgi J, Yam P, Petz L, Winston
D, Warner N, Reichert T: Selective depletion of CD8+ T lymphocytes
for prevention of graft-versus-hostdisease after allogeneic bone marrow transplantation. Blood 76:418, 1990
37. Marmont AM, Horowitz MM, Gale RP, Sobocinski K, Ash
RC, van Bekkum DW, Champlin RE, Dicke KA, Goldman JM,
Good RA, Herzig RH, Hong R, Masaoka T, Rimm AA, RingdCn
0,Speck B, Weiner RS, Bortin M M T-cell depletion ofHLA-identical
transplants in leukemia. Blood 78:2120, 1991
38. Stockschlaeder M, Storb R, Pepe M, Longton G, McDonald
G, Anasetti C , Appelbaum F, Doney K, Martin P, Sullivan K, Witherspoon R: A pilot study of low-dose cyclosporin for graft-versushost prophylaxis in marrow transplantation. Br J Haematol 80:49,
1992
39. Yee GC, Self SG, McGuire TR, Carlin J, Sanders JE, Deeg
HJ: Serum cyclosporine concentration and risk of acute graft-versushost disease after allogeneic marrow transplantation. N Engl J Med
319:65, 1988
855
40. Sullivan KM, Storb R, Buckner CD, Fefer A, Fisher L, Weiden
PL, Witherspoon RP, Appelbaum FR, Banaji M, Hansen J, Martin
P, Sanders JE, Singer J, Thomas E D Graft-versus-host disease as
adoptive immunotherapy in patients with advanced hematologic
neoplasms. N Engl J Med 3202328, 1989
4 1. Storb R, Pepe M, Anasetti C , Appelbaum FR, Beatty P, Doney
K, Martin P, Stewart P, Sullivan KM, Witherspoon R, Bensinger W,
Buckner CD, Clift R, Hansen J, Longton G, Loughran T, Petersen
FB, Singer J, Sanders J, Thomas ED: What role for prednisone in
prevention of acute graft-versus-host disease in patients undergoing
marrow transplants? Blood 76: 1037, 1990
42. Abbas AK, Lichtman AH, Pober JS: Cytokines in Cellular
and Molecular Immunology. Philadelphia, PA, Saunders, 199 I, p
225
43. Tutschka PJ, Copelan EA, Klein J P Bone marrow transplantation for leukemia following a new busulfan and cyclophosphamide
regimen. Blood 701382, 1987
44. Beelen DW, Quabeck K, Graeven U, Sayer HG, Mahmoud
HK, Schaefer UW: Acute toxicity and first clinical results of intensive
postinduction therapy using a modified busulfan and cyclophosphamide regimen with autologous bone marrow rescue in first remission
of acute myeloid leukemia. Blood 74: 1507, 1989
45. Copelan EA, Biggs JC, Thompson JM, Crilley P, Szer J, Klein
JP, Kapoor N, Avalos BR, Cunningham I, Atkinson K, Downs K,
Harmon GS, Daly MB, Brodsky I, Bulova SI, Tutschka PJ: Treatment
for acute myelocyticleukemia with allogeneic bone marrow transplantation following preparation with BuCy2. Blood 78:838, 1991
46. Horowitz MM, Gale RP, Sonde1 PM, Goldman JM, Kersey
J, Kolb HJ, Rimm AA, RingdCn 0, Rozman C, Speck B, Truitt RL,
Zwaan FE,Bortin MM: Graft-versus-leukemia reactions after bone
marrow transplantation. Blood 75555, 1990