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236 Blood, Vol. 59, No. 2 (February), 1982
Marrow Transplantation With or Without Donor Buffy Coat Cellsfor 65 Transfused Aplastic Anemia Patients
By Rainer Storb, Kristine C. Doney, E. D. Thomas, Fred Appelbaum, C. Dean Buckner, Reginald A. CIift, H. Joachim
Deeg, Brian W. Goodell. Robert Hackman, John A. Hansen, Jean Sanders, Keith Sullivan,
Paul L. Weiden, and Robert P. Witherspoon
Sixty-five multiply transfused patients with severe aplastic
anemia were given cyclophosphamide followed by grafts
from HLA-identical siblings. The effect of the administra-
tion of viable donor buffy coat cells following the marrow
inoculum was evaluated with regard to graft rejection and
survival. Results in 43 patients so treated are presented
along with those in 22 concurrent patients given marrow
alone. Most patients given buffy coat had positive in vitro
tests of sensitization indicating a high risk for graft rejec-
tion. while all but one of the patients given marrow alone
had negative tests. Thirty of the 43 (70%) patients given
marrow and buffy coat are alive between 10 and 61 mo
T RANSPLANTATION ofmarrow from an HLA-
identical sibling after immunosuppression with
high doses of cyclophosphamide (CY) has been used
over the past decade to treat patients with severe
aplastic anemia. Initial results before 1976 showed
long-term survival with normal hemopoiesis ranging
from 27% to 45%I-IO Most of the mortality occurred
early, within 6 mo of marrow transplantation. Most
commonly, death was related to rejection of the
marrow graft, a complication seen in 30%-60% of the
transplants. Nearly all patients had had multiple
transfusions before transplantation. Clinical results in
untransfused patients” and experimental data in
DLA-identical littermate 23 and H-2-compati-
ble mice’4”5 strongly suggested that marrow graft
rejection was due largely to sensitization of recipients
to non-HLA donor antigens by transfusions.
After 1976, attempts to reduce the incidence of
rejection and mortality in multiply transfused patients
included the use of “high-dose” immunosuppressive
From the Fred Hutchinson Cancer Research Center and the
Division of Oncology, Department of Medicine, University of
Washington, Seattle, Wash.
Supported in part by Grants CA 15704, CA 18029, CA 0523/,
CA 30924, and CA 18221 awarded by the National Cancer Insti-
tute, and HL 1 7265 awarded by the National Heart, Lung and
Blood Institute, DHHS. K.C.D., RH.. and F.A. are supported in
part by a Junior Faculty Clinical Fellowship from the American
Cancer Society. HiD. is a Fellow of the Leukemia Society of
America. ED. T. is a recipient ofResearch Career Award Al 02425
from the National Institute ofAllergy and Infectious Diseases.
Submitted April /4. 1981; accepted September 21. 1981.
Address reprint requests to R. Storb. M.D.. Division of Oncolo-
gy. Fred Hutchinson Cancer Research Center, I I 24 Columbia
Street, Seattle, Wash. 98104.
© I 982 by Grune & Stratton, Inc.
0006-4971/82/5902-0005$2.0O/0
(median 36) after grafting; 4 died after graft rejection and
6 with acute or chronic graft-versus-host disease (GVHD).
Eleven of the 22 (50%) patients given marrow alone are
alive between 29 and 65 mo (median 52); 7 died after graft
rejection and 3 with GVHD. The addition of buffy coat cell
infusions to the marrow inoculum reduced the risk of
rejection and increased survival in the currently reported
transfused patients when compared to patients grafted
before 1 976. However. there was an increased risk of
chronic GVHD. Recipients of marrow from female donors
survived slightly better (73%) than recipients of male
marrow (58%).
conditioning regimens consisting of chemotherapy,
antithymocyte globulin (ATG), and/or total body
irradiation (TBI) in addition to CY. However,
although the engraftment rates with these high-dose
regimens were significantly higher, this was not asso-
ciated with improved survival.’62#{176}
We reported that graft rejection was less likely
when larger numbers of marrow cells were infused.2’
The donor’s peripheral blood is a potential source of
additional pluripotent hemopoietic stem cells and/or
lymphoid cells capable of overcoming rejection. Circu-
lating stem cells have been shown to exist in mice,22
guinea pigs,23 dogs,2�26 and nonhuman primates.27Also, peripheral blood and thoracic duct lymphocytes
enhance allogeneic marrow engraftment in vivo in
mice28 and dogs29’3#{176}and erythropoiesis in vitro in dogs3’
and humans.32 Therefore, we began a study to evaluate
the effects of viable donor buffy coat cells in addition
to the marrow inoculum on graft rejection and survival
of multiply transfused patients conditioned with CY.
Results in 43 patients so treated are presented, along
with those in 22 concurrently transplanted patients
given marrow alone. Most patients given additional
donor buffy coat cells were shown by positive in vitro
tests of sensitization3336 to be at high risk for graft
rejection, while all but one of the patients given
marrow alone had negative in vitro tests.
MATERIALS AND METHODS
Tables I and 2 show some of the characteristics of the 65 patients
before and after transplantation.
All patients had multiple transfusions of red blood cells, platelets,
or both. The numbers of preceding transfusions probably represent
underestimates since they do not include data on six patients with a
mean history of 40 mo who were reported to have had “many”
transfusions; also, in some patients exact numbers were unknown,
e.g., >100. Nine of the patients (unique patient numbers [UPNsJ
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MARROW GRAFTS FOR A PLASTIC ANEMIA 237
Table 1 . Descriptive Data on 65 Transfused Patients With
Aplastic Anemia and Their Marrow Donors
Datum Value
Patient
Age (yr) 3-53 (median 20)
Sex (F/M) 26/39
Possible cause of aplastic anemia
(no. of patients)
Unknown 46
Drug or chemical-induced 6
Hepatitis 6
Pregnancy 3
Fanconi’s syn&ome 2
Preleukemia 2
Duration of aplastic anemia (mo) 0.25- i08 (median 2)
Preceding transfusions (no. of units)
Red blood cells 0-63 (median 8)
Platelets (and granulocytes) 0-573 (median 25)
Refractory to random donor platelets
(no. of patients) 22
History of (no. of patients)
Infection 34
Hemorrhage 51
Androgen treatment 37
Prednisone treatment 37
Donor
Age (yr) i -49 (median 20)
Sex (F/M) 22/43
Transplant data
HLA-A, B. and D identical All pairs
ABO incompatible
Minor (for example, 0 -‘ A) 1 i pairs
Major (for example, A -‘ 0) 8 pairs
Protective environment (no. of patients) 23
Granulocyte transfusions (no. of patients) 31
Prednisone for acute GVHD (no. of 14
patients)
ATG for acute GVHD (no. of patients) i 0
547, 561, 630, 637, 747, 753, 765, 1080, 1254) had had pregnancies;
none had miscarriages or abortions.
The HLA identity of sibling marrow donors and recipients was
determined by serologic histocompatibility typing and mixed leuko-
cyte culture (MLC).’3 The response in MLC of patient cells to
sibling cells was compared to their response to pooled freshly drawn
cells from two unrelated donors or cryopreserved cells from three
unrelated donors and was then expressed as a ratio, the relative
response.33’M On the basis of data in 21 2 healthy, H LA-identical
siblings, a positive relative response was defined as >2.6% and a
negative one as <2.6% The average relative response in this control
group was 0.0%, with 2 standard deviations -2.6%. We interpreted
a positive relative response to be an expression of transfusion-
induced sensitization of the patient against the marrow donor.
A second assay that appeared capable of detecting recipient
sensitization against the donor involved release of 5’Cr from donor
target cells.�’35 Two separate assays of 5Cr release were employed:
direct cell-mediated lympholysis and antibody-dependent lympholy-
sis mediated by random effector lymphocytes. A51Cr release of 3.5%
or greater with a difference between experimental and control
release significant at p < 0.05 by the t test was considered evidence
for sensitization.
Details on the methods of assessing allogeneic engraftment by
erythrocyte antigen and isoenzyme (electrophoretic) phenotype
markers3’37 and karyotype analyses, including quinacrine dihydro-
chloride banding of marrow and peripheral blood cells, have been
described.3�
Details on the immunosuppressive conditioning regimen for trans-
plantation consisting of CY, 50 mg/kg body weight on each of 4
successive days, marrow infusion, ATG, and supportive care after
grafting have all been � The number of marrow cellsinfused is shown in Table 2.
In 23 patients, chronic graft-versus-host disease (GVHD) was
treated by prednisone, initially I mg/kg/day and later I mg/
kg/q.o.d., either alone or in combination with either azathioprine,
procarbazine or CY, all at 1.5 mg/kg/day.’#{176}
Two groups of patients were studied. Group I consisted of 43
patients who were given viable donor buffy coat in addition to the
marrow inoculum. Twenty-nine of the 43 had positive in vitro tests
of sensitization before transplantation and were thus considered at
high risk of graft rejection (Table 3). Group 2 included 22 patients
who were not given buffy coat. Only I of the 22 had positive in vitro
tests (Table 4). The uneven distribution of patients between the two
groups was due to the policy that, between March 1976 and
November 1978, patients with negative in vitro tests were given
marrow only and patients with positive in vitro tests were given
marrow and additional buffy coat. At that time, data were analyzed
and it was found that three of 23 evaluable patients ( I 3%) given
added buffy coat had rejected and 17 of 24 (71%) were surviving,
while six of 20 (30%) not given buffy coat had rejected and 10 of the
20 (50%) were surviving. While intriguing, these differences were
not statistically significant. However, a comparison with the 60
previously transplanted transfused patients2’3 given the same CY
regimen and marrow but no buffy coat showed that the current
patients given added buffy coat had both a significantly reduced
rejection rate (p =0.03, two-tailed continuity-corrected chi-square
test) and increased survival (p =0.02). Results among the 20
patients not given added buffy coat did not differ from those
previously transplanted. Next, a multivariant analysis of data on all
the I 04 transplanted transfused patients given the CY regimen was
carried out using previously published techniques2’ but including
buffy coat infusion as an additional variable. The binary logistic
regression analysis confirmed the results published earlier,2’ with
buffy coat infusion emerging as an additional factor significantly
associated with a lower rejection rate. On the basis of these observa-
tions, the decision was made to give additional donor buffy coat to
all patients with multiple transfusions irrespective of in vitro test
results. Patients UPN 561 and 983 represent protocol violations.
Buffy coat was obtained from the marrow donors using either a
continuous flow centrifuge or a blood bag technique similar to the
one used for platelet collection and infused on days 1-3 or 1-5 after
marrow grafting. The numbers of white blood cells and mononu-
clear cells infused are shown in Table 2.
The marrow transplant protocols and consent forms for the
current study were approved by the Human Subjects Review
Committees of the Fred Hutchinson Cancer Research Center or the
University of Washington School of Medicine.
RESULTS
Tables 1-4 summarize the marrow transplant data.
Survival
Thirty of the 43 transfused patients (70%) in group
I given marrow and viable donor buffy coat are
currently alive between 304 and 1844 (median I 100)
days after transplantion. Figure 1 shows their survival
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238 STORB ET AL.
Table 2. Quantitative Data on 65 Transfused Patients With Ap lastic Anemia Treated b y Marrow Grafts
Datum Range Median Mean ± SD
Admission data
Granulocytes (/cu mm) 0-2,300 i85 404 ± 539
Hematocrit (%)t 13-27.5 22 22 ± 4
Reticulocytes (%; corrected for hemato-
cdt) 0-4.5 0.i 0.4 ± 0.8
Platelets (/cu mm)t 800-45,000 9,000 8.900 ± 9.000Marrow cellularity (% normaI)�
Overall 0-iOO iO i5 ± 16
Myeloid 0-200 iO 15 ± 27
Erythroid 0-100 8 13 ± i8
Megakaryocytes 0-35 0 4 ± 7
Hospital stay, days
Regular ward (42 patients) 28- 1 1 3 37 47 ± 22
Protective environment
(22patients)� 27-113 57 52 ± 20
Number of marrow cells infused
(x 10/kg body weight)
Allpatients i.5-i4.3 3.5 4.1 ± 2.3
Patients in group 1 II 1 .6- i4.3 3.3 3.8 ± 2.1
Patients in group 211 1 .5- i 4.0 3.6 4.5 ± 2.7
Number of buffy coat cells
infused ( x 1 08/kg body weight)
Totalleukocytes i.i-3i.7 3.4 7.i ± 8.6
Mononuclearcells 0.8-14.2 2.6 3.5 ± 3.0
Day of granulocyte count
rise after graftingil 6-28 i4 i5 ± 5
Latest blood counts in
surviving patients:
Daysaftergrafting 145-1651 950 935 ± 424
Leukocytes(/cumm) 2,350-16,600 7,450 7,100 ± 2,700
Granulocytes (/cu mm) i .400- 13,800 4,000 4,300 ± 2,200
Hematocrit (%) 32-52 43 42.0 ± 4.5
Platelets(/cumm) 98,000-730,000 229,000 249,000± 115,000
Patients fulfilled the criteria of severe aplastic anemia as defined by the International Aplastic Anemia Study ‘#{176}
tBefore transfusion.�Estimated on the basis of marrow aspirate, particle preparation, and marrow biopsy except in four patients who had only marrow aspirates.
§Until recently. the protective environment required a 50-day stay after transplantation. Excluded UPN 697 (1 2 days).
I� Marrow doses not significantly different between the two groups of patients (two-sided Mann-Whitney-U test).#{182}Therewas no difference in the speed of granulocyte recovery between patients given marrow only and patients given marrow and buffy coat. The
analysis was restricted to patients with successful first grafts.
curve plotted according to the method of Kaplan and
Meier4’ with a projected 2-4.5 yr survival of 70%.
Eleven of 22 transfused patients in group 2 (50%)
given marrow only are alive between 880 and 1971
(median 1560) days after transplantation. Their long-
term survival is 50% (Fig. I).
For comparison, Fig. 1 also shows an actuarial
survival curve for 60 transfused patients who received
HLA-identical sibling marrow grafts after CY (no
donor buffy coat) between October 1 970 and Novem-
her l975.’� Their long-term survival is 43%. Twenty-
one of 56 evaluable patients (excluding 4 early deaths)
rejected (38%), and 1 7 of these died.
Also shown in Fig. 1 is an actuarial survival curve of
39 untransfused patients given HLA-identical marrow
grafts after CY” (and unpublished observations).
Thirty-two of the 39 (82%) are currently alive, and
their long-term survival is projected to be 80%.
Marrow graft rejection was a rare complication
among the 39.
The survival data of patients in groups I and 2 were
compared by log-rank statistics,42� and differences
were not found to be significant (p = 0.17), presum-
ably related to small numbers. Survival of patients in
group 1,however, was significantly (p = 0.009) better
than that of transfused patients grafted before 1976,
while that of patients in group 2 was not significantly
improved.
Graft Rejection and Second Grafts
Six patients in group 1 (14%) and 7 in group 2
(32%) showed marrow graft rejection following initial
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697 23
615 30
621 19
565 16
Table 3. Transfused Patients Given CY Followed by Infusion of Marrow and Viable Donor Buffy Coat (Group 1)
UniquePatient Age
Test ofSensitization
Sustained
1St
Clinical GVHD ( 1 at raft) Posttrans-plantation
SurvivalAcuteNumber (yr) RR/Cr Graft (Grade) Chronic Outcome (Karnofsky Performance Score) (Days)t
-/+
-1+-/+
716
593
611
645660
630686
693707
732
736
734
742
747
770
837863
866850
9491080
9841010
49 -/+
15 -/+
25
12
19 -/+
37 -1+
15
612 -1+
925 -/+
39 -1+
20
27 -1+
16 -/+
15 -1+
19 NE/+
21 -/+
28 -/+
17
3753 -1+
37
NE NE
No 0
Ni 0
No 0
Yes 0 No
Yes 0 No
Yes 0 No
Yes 0 No
Yes 0 Yes-Rxt
Yes 0 Yes-resolved
Yes 0 Yes-Rx
Yes I Yes-resolved
Yes IV Yes
Yes 0 No
Yes II Yes-resolved
Yes 0 Yes-RxYes 0 Yes-resolved
Yes IV No
Yes II Yes
Yes I Yes-resolved
Yes III Yes-Rx
Yes 0 No
Yes I Yes-resolved
Yes 0 Yes-Rx
Yes 0 No
Yes IV No
No 0 No
Yes 0 No
Yes 0 No
Yes II Yes-resolved
Yes I Yes-Rx
Yes 0 No
Yes Ill No
No 0 No
6
63
30106
25
>1,848
>1,776
>1,649
>1,619
>1,587
>1,520
>1.510
208
>1,419
>1,418
>1,418
>1.385
80535
>1,158
>1,111
>1,098
>1,096>909
>621
65
>776
>1,277
>1,207
>1,049
>956
>920
>89463
>791
99
286
>706
110
>349>339
>318
>304
1005 9 -/- No 0
1027 25 -/ND Yes IV No
1042 30 -/ND Yes 0 No
1043 22 -/ND Yes 0 Yes-Rx1065 15 -/ND Yes IV No
1232 29 -1+ Yes III No
1241 31 -1+ Yes III Yes-Rx
1254 30 NE/ND Yes III No
1263 19 -I- Yes 0 Yes-Rx
RR, relative response in MLC; “CI’ release from donor t�get cells. For details see Materials and Methods. NE, not evaluable; ND, not done.
tRx. still requiring therapy for chronic GVHD: prednisone alone or in combination with azathioprine, procarbazine. or CY.�#{176}�SurvivalasofJuly2O, 1981.
MARROW GRAFTS FOR A PLASTIC ANEMIA 239
777 18 -I-
818 11 -/-.
882 41 -/-
927 20 -/--
942 17 -/-
954 16 -/-
955 14 -I-
NE Died-CY-cardiotoxocity
No Died-pneumonia after successful 2nd graft
No Died-infection
No Died-GVHD and pneumonia after successful
2nd graft
Died-aspergillus sinusitis
Well (100%)
Well (100%)
Well (100%)
Well (100%)
Living (90%)
Living (80%)
Well (100%)
Died-GVHD and infection
Well (100%)
Living (90%)
Living (90%)
Well (100%)
Died-GVHD and infection
Died-GVHD and measles
Well (100%)
Living (80%)
Well (100%)
Well (100%)
Well (100%)
Well (100%)
Died-GVHD
Living (90%). Successful 2nd graft: chronic
GVHD-Rx
Well (100%)
Well (100%)
Well (100%)
Living (70%)
Well (100%)
Well (100%)
Died-GVHD and infection after successful
2nd graft
No Living (60%). Successful 2nd graft: chronic
GVHD-Rx
Died-GVHD and infection
Died-following Rx for leukemia
Living (90%)Died-GVHD and infection
Well (100%)
Living (90%)
Livin9 (80%)
Well (100%)
engraftment (Tables 3-5). Four patients in group 1 However, patients in group 1 showed a significantly
(9%) and all 7 in group 2 (32%) died as a direct or decreased (p = 0.02) incidence of rejection when
indirect consequence of rejection. compared to patients grafted before 1 976; the rejec-
The difference in rejection rates between patients in tion rate in patients of group 2 was not significantly
groups 1 and 2 did not reach statistical significance different from that of the previous patients.
(two-tailed, continuity-corrected chi-square test). Rejection was diagnosed between days 19 and 82
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Table 4. Transfused Patients Given CV, Marrow. and No Viable Donor Buffy Coat Cells (Group 2)
Unique
Patient Age
Test of
Sensitization
Sustained
1 st
Clinical GVHD (1st Graft) Posttrans-plantation
SurvivalAcute
Number (yr) RR/”Cr Graft (Grade) Chronic Outcome (Karnofsky Performance Score) (DayS)t
610 6 -/- No 0 No Died-infection after 2nd graft 128
570 17 -/- Yes 0 No WeII(100%) >1,952
614 3 -I- No 0 No Died-septicemia after unsuccessful 2nd
graft
84
605 4 -I- Yes 0 No WeIl(100%) >1,804
632 19 -/- Yes IV No Died-GVHD and septicemia 41
564 20 - / - Yes 0 No Died-interstitial pneumonia 156
637 22 NE/- Yes 0 No WelI(100%) >1,696
626 21 NE/- Yes 0 No WeII(100%) >1,735
619 13 NE/- Yes 0 No WeII(100%) >1,769
547 23 -/- No 0 No Died-infection after unsuccessful 2nd graft 42
704 22 -I- Yes III Yes Died-GVHD and infection 637
691 9 -/- No 0 No Died-leukemia 261
733 28 -I- Yes II Yes Died-GVHD and infection 472
741 29 -I- Yes I No WeIl(100%) >1,388
753 2 1 - / - No 0 No Died-infection after unsuccessful 2nd graft 1 1 1
758 23 -I- Yes 0 No WeII(100%) >1,336
765 29 -I- Yes 0 No WeIl(100%) >1,319
771 23 -I- Yes 0 No WeIl(100%) >1,309
676 30 -/- Yes I Yes-Rx� WeII(90%) >1,561
923 22 -/- No 0 No Died-infection after unsuccessful 2nd graft 80
983 14 -I- Yes 0 No WeIl(100%) >823
561 20 +/+ No 0 No Died-infection shortly after 2nd graft 43
RR, relative response in MLC; t1Cr release from donor target cells. For details, see Materials and Methods. NE, not evaluable.
tSurvival as of July 20, 198 1.
tRx, still requiring therapy for chronic GVHD: prednisone alone or in combination with azathioprine, procarbazine, or CV.”
(median 43), as evidenced by progressively falling
peripheral blood counts and severely aplastic marrow.
Cytogenetic analyses of marrow and peripheral blood
cells in eight patients with markers showed mixtures of
donor and host cells early after transplantation. In the
YEARS
Fig. 1 . Kaplan-Meier product limit estimates of percentsurviving for patients with aplastic anemia treated by cyclophos-phamide and a marrow graft from an HLA-identical familymember. Shown are data in 43 transfused patients given marrow
and buffy coat (group 1 ) and 22 patients given marrow only (group2). For comparison. the survival curves of 39 untransfused and 60transfused patients (grafted between 1970 and 1976) givenmarrow only are shown. Day “0” is the day of marrow transplan-tation. Tick marks indicate surviving patients. Survivals are as ofJuly20. 1981.
240 STORB ET AL.
process of rejection, increasing proportions of cells
with host karyotypes were seen.
Rejection was more frequent in both male and
female recipients who were given marrow from male
donors than in those given grafts from female donors.
Ten of 42 evaluable recipients of male marrow
rejected (24%) compared to 2 of 22 given female
marrow (9%).
This difference was more pronounced in patients of
group 2 (46% of recipients given male marrow rejected
compared to 1 1 % of recipients given female marrow)
than those of group I (1 7% of recipients given male
marrow rejected, compared to 8% of recipients given
female marrow).
One of the rejecting patients (UPN 691) did not
receive a second transplant; she remained aplastic,
later developed acute myeloblastic leukemia, and died.
Retrospective chromosome analysis of pretransplant
marrow revealed abnormalities compatible with the
diagnosis of preleukemia. Cells examined showed
either 45,XX,-7 or 46,XX,-7+2l karyotypes. The
leukemic myeloblasts showed a 45,XX, - 7 karyotype.
One patient (UPN 621 ) received a second marrow
infusion without preconditioning on day 29 and died
on day 30 with infection. The remaining 1 1 patients
were given second marrow grafts 24-86 (median 51)
days after the first from either the same HLA-
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Table 6. Summary of Data on Patients in Groups I and 2
Patients Studied AliveSustained
Graft Rejection
Dead With
GVHD PlusInfection Rejection Miscellaneous
Marrow plus buffy coat
(group 1)
Marrow only
(group 2)
43�
22
30 (70)
1 1 (50)
Number of patients (%)
36 (86)t 6 (14)t
15 (68) 7 (32)
6 (14)t
3 (14)
4 (9)’
7 (32)t
1 -CV cardiotoxicity
1 -Aspergillus infection
1 -Leukemia
1 -Idiopathic interstitial
pneumonia
Percent based on patients who can be evaluated; excludes one early death (day 6) in group 1.
tone of these (UPN 69 1) died with leukemia after a prolonged period of aplastic anemia.
Table 6. Incidence of Acute and Chronic Graft-Versus-Host Disease Among Patients With Sustained First Grafts
Acute GVHD(Grades ll-IV)
Patients Studied’ Alive Present Died Present
Chronic GVHDDead withAcute or
Chronic GVHDResolved Died
Needing
Therapy
Number of patients (%)
Marrow plus buffy coat
(groupl) 36 28(78)t 13(36) 4(11) 18(50)
Marrow only
(group 2) 15 1 1 (73) 3 (20) 1 (7) 3 (20)
7(19) 2(6)
0 2 (13)
9(25)
1 (7)
6(17)
3 (20)
Excluded are two patients with successful second grafts.
MARROW GRAFTS FOR A PLASTIC ANEMIA 241
identical donor who gave the first marrow graft (9
patients) or another HLA-identical sibling (2
patients) after a second conditioning regimen.3
Only 2 of the 1 3 rejecting patients are currently
alive with well functioning second grafts (UPNs 1005
and 1010). Both require therapy for chronic GHVD.
Sustained Engraftment
Engraftment was indicated by increasing marrow
cellularity and rising peripheral granulocyte counts
between days 6 and 28 (median 14) after marrow
transplantation (Table 2), with grafts persisting until
the present time or the day of death. Thirty-six of 42
evaluable patients (86%) in group 1 and I 5 of 22
(68%) in group 2 had sustained engraftment of their
first marrow graft (Tables 3-5). One of the 51
patients with sustained engraftment (UPN 1042) had
a mixture of cells with 46,XY and 45,XY,-7 karyo-
types before transplantation. After transplantation,
until the time of development of acute myeloblastic
leukemia approximately 5 mo after grafting, all
marrow cells examined showed a normal 46,XY (pre-
sumably marrow donor) karyotype. At the time of
florid leukemia, marrow cells showed a 45,XY,-7
karyotype.
Twenty-nine of the 50 remaining patients had
donors of opposite sex. Five with a donor of the same
sex were distinguished from their donors by banding
markers. After grafting, predominantly cells with
donor karyotypes were found in direct and phyto-
hemagglutinin-stimulated marrow preparations.
In 25 of 50, differences for erythrocyte genetic
markers between donor and recipient were found
before transplantation. In 16 of the 25, tested at least
90 days after the last transfusion, the patient’s
erythrocytes were replaced by those of the donor’s
cells. In 9 cases, patients have either as yet not been
retested or they died too early for evaluation. Because
of multiple transfusions, red blood cell genetic mark-
ers were not determined before transplantation in 25
long-term survivors. Twenty-four of these were tested
after transplantation on one or more occasions at least
90 days after the last transfusion. Results showed
identity with the marrow donors in regards to all
erythrocyte antigen and enzyme phenotypes. In one
patient (UPN 704) repeat testing was not carried out;
in this case evidence for allogeneic engraftment rested
on the presence ofacute GVHD.
Surviving patients have peripheral blood counts that
are in the normal range, as shown in Table 2.
GHVD
Of the 51 patients with sustained first graft in
groups 1 and 2, 22 had grade I-IV acute GVHD
(Table 3). Six had grade I disease that resolved under
continued methotrexate (MTX) therapy. Sixteen had
grade II-IV disease (Table 6), and they were treated
with prednisone and/or ATG.45’46 Five of the 1 6 (1 1%)
died of complications associated with acute GVHD
(Tables 3 and 6).
Thirteen patients with grade I-IV acute GVHD
went on to develop mild to severe chronic GVHD, and
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242 STORB ET AL.
eight additional patients developed de novo chronic
GVHD without preceding acute GVHD (Tables 3 and
6). In addition, two patients with successful second
grafts developed chronic GVHD. Four of the 23
patients died with infectious complications associated
with their chronic GVHD. In seven, chronic GVHD
resolved under treatment with prednisone given in
combination with azathioprine, CY, or procarbazine.”#{176}
They are receiving no further treatment. Twelve
patients (including the two with successful second
grafts) are still requiring therapy for chronic GVHD.
Of the 19 surviving patients with chronic GVHD, 8
have Karnofsky performance scores of 1 00%, 7 of
90%, 2 of 80%, 1 of 70%, and 1 of 60% (Tables 3 and
4).
Overall, patients in group 1 receiving buffy coat in
addition to the marrow inoculum developed acute and,
in particular, chronic (p < 0.05, chi-square test)
GVHD more frequently than those in group 2 given
marrow alone (Table 6). However, the percentages of
patients dying with acute or chronic GVHD in the two
groups of patients were similar.
Influence ofDonor Sex on Survival
All patients. Forty-three recipients had male
donors. Twenty-five (58%) are alive and 18 (42%)
died. Twenty-two recipients had female donors.
Sixteen (73%) are alive and 6 (27%) died. Results
were identical for male and female recipients. When
data were analyzed separately for patients in groups 1
and 2, results were as follows: Group 1-70% ofrecipients given male marrow are surviving compared
to 70% of recipients of female marrow; group 2-38%
of recipients given male marrow are surviving
compared to 67% of recipients of female marrow.
Patients with sustainedfirst graft (n = 51). Thir-
ty-one recipients had male donors. Twenty-three
(75%) are alive and 8 (25%) died. Twenty recipients
had female donors. Fifteen (75%) are alive and 5
(25%) died. Results were identical for male and
female recipients.
Miscellaneous Problems, Interstitial Pneumonia, and
Toxicities
Patient UPN 697 died on day 6 after grafting of
progressive heart failure; this was interpreted to be the
result of CY-cardiotoxicity.2 Patient UPN 716 was
admitted for transplantation with an aspergillus si-
nusitis and died of hemorrhage into his infected
sinuses.
Knowledge before transplantation of the diagnosis
“preleukemia” in patients UPN 691 and 1042 would
have dictated a different approach at transplantation,
probably using a protocol involving TB! and perhaps
changing the outcome.
� Fourteen patients developed interstitial pneumonia
between 13 and 186 days (median 59) after the first
graft. Biopsies in 12 showed idiopathic pneumonia in
five, cytomegalovirus in three, pneumocystis carinii in
two, pneumocystis carinii and cytomegalovirus in one,
and herpes simplex virus in one. Pneumonia was seen
in 8 of 5 1 (1 6%) patients with sustained first grafts,
and 3 (6%) died. Six of 1 3 (46%) patients with graft
rejection developed pneumonia, and 4 (3 1%) died.
DISCUSSION
Allogeneic marrow engraftment in patients with
aplastic anemia depends on the efficacy with which
the conditioning regimen suppresses the immune
response of the recipient to non-HLA antigens of the
marrow donor. CY, first tested in rodents,47 dogs,48
monkeys,49 and three patients with leukemia5#{176} was
capable of abrogating the primary immune response to
donor antigens in patients who were untransfused
before transplantation. More than 90% of these
patients showed sustained engraftment of the HLA-
identical marrow and more than 80% are surviving”
(and unpublished).
CY was less effective for conditioning patients who
had transfusions before transplantation. Graft rejec-
tion was a common cause of failure, with an incidence
of 30%�60%.lb0 Presumably, patients were sensitized
to non-HLA antigens of the marrow donor by transfu-
sion of blood products. CY, like most immunosuppres-
sive agents, is less effective in suppressing a secondary
than a primary immune response. The non-HLA anti-
gens involved in rejection are unknown. Studies in
dogs showed that at least two polymorphic antigen
systems outside of the major histocompatibility region
play a role in sensitization to marrow grafts.5’
The best way to avoid graft rejection is to avoid
transfusions before transplantation. If transfusions are
necessary, siblings, parents, and immediate family
members should not be used as donors since they are
most likely to sensitize the patient against those non-
HLA antigens for which he/she and the donor differ.
Single donors are less likely to sensitize than multiple
donors.
Studies in dogs suggested that the antigens involved
in sensitization are expressed on B lymphocytes and
monocytes but not on T lymphocytes, granulocytes,
erythrocytes, or platelets.51’52 Serologic data in human
marrow graft recipients also provide circumstantial
evidence of an involvement of monocytes in sensitiza-
tion.53 Perhaps removal of mononuclear cells will
result in human blood products that carry a lesser risk
of sensitization.
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MARROW GRAFTS FOR A PLASTIC ANEMIA 243
At present, most patients with aplastic anemia have
had multiple transfusions and, thus, are at risk of graft
rejection. We demonstrated a correlation between two
in vitro tests of sensitization of recipient against donor
and marrow graft rejection in patients conditioned
with CY.3336 Between 22% and 26% of “nonsensi-
tized” patients rejected their grafts compared to 62%-
73% of “sensitized” patients. This correlation was
statistically significant even when other factors possi-
bly predictive of rejection were considered.2’ It
provided additional circumstantial evidence that rejec-
tion was immune-mediated.
We attempted to prevent it by giving more intensive
conditioning therapy with procarbazine, ATG, and
1000 rad TBI, or CY and TBI.’6 Eleven “sensitized”
patients were so treated. Although all but one
engrafted, survival was poor because of complicating
GVHD and interstitial pneumonia.
Since March 1 976, “nonsensitized” patients were
given the customary CY regimen followed by marrow
infusion, while “sensitized” patients (and, between
November 1978 and September 1980, all transfused
patients) were given CY, bone marrow, and additional
viable donor buffy coat. The rejection rate of 32% in
“nonsensitized” patients was slightly but not signifi-
cantly higher than the 22%-26% observed in the
past21’3336 and points out the difficulties of establishing
reliable in vitro tests of sensitization to non-HLA
antigens. Perhaps assays involving inhibition of
erythroid colonies in vitro are more consistent.54’55
The rejection rate of 14% in transfused patients
given additional donor buffy coat was lower than the
38% observed in the past.2’3 This difference becomes
even greater when current results are compared with
previous ones in “sensitized” patients where rejection
rates of 62�Yo-73% were observed.21’3336 When a multi-
factorial analysis using a binary logistic regression
model was carried out involving the first 1 58 patients
conditioned with CY in Seattle, the use of buffy coat
was significantly associated with a low rejection rate
(unpublished). As a result of the decreased rejection
rate, survival has increased from 43% to 70%. The
mechanism by which buffy coat enhances engraftment
is unclear. It could be related to an increase in the
number of pluripotent stem cells infused, as suggested
by studies in experimental animals,2227 to a facilita-
tion of marrow engraftment by lymphoid cells, as
suggested by in vivo studies in mice and dogs283#{176}and
in vitro studies in dogs and humans,3”32 or a combina-
tion of the two mechanisms. “Facilitation” of engraft-
ment could also be the result of a graft-versus-host
reaction.
One concern has been that the addition of viable
buffy coat would increase the incidence and severity of
GVHD. Analysis of the results in patients with
sustained grafts supports that concern. Both acute and
chronic GVHD were more frequent in patients given
buffy coat compared to those given marrow only.
However, the mortality from acute and chronic
GVHD was comparable in the two groups of patients
( 1 7% versus 20%), and the major difference was in the
percentage of patients requiring continued therapy for
chronic GVHD (25% versus 7%). It can be concluded
that the addition of buffy coat has reduced the risk of
rejection and increased long-term survival of trans-
fused patients despite an increased risk of chronic
GVHD(Fig. 1).
Other transplant teams have used other approaches
to reduce the graft rejection rate. Procarbazine and
ATG,56 administered with the standard CY regimen,
did not alter the incidence of graft rejection in Seattle3
but was thought to be effective by the transplant team
in Boston, although without improvement in survival.57
The team at UCLA has used CY followed by 300 rad
TBI.’9 The rejection rate declined, and 12 of 23
patients are surviving between 6 mo and 2.5 yr. Gluck-
man et al.2#{176}have used CY followed by 800 rad TBI
with shielding of the lung to 400 rad in an attempt to
reduce interstitial pneumonia. The rejection rate was
low, but survival was not improved. Santos et al.
(personal communication) have used CY and 800 rad
TB! wherein the lungs are shielded to get only 600 rad.
A comprehensive review of recent data from 24 world-
wide teams (not including Seattle) by the Interna-
tional Bone Marrow Transplant Registry involving
144 patients indicated a I-yr survival of 44%; more
than half of these patients were conditioned by regi-
mens including TBI.’7 A separate review by 13 Euro-
pean transplant teams involving I 59 transplants
showed an overall 1-yr survival of 41.2%.18 The
Minneapolis team has used CY followed by exposure
to 750 rad of total lymphoid irradiation in I 9
patients.58 Only one patient suffered graft rejection,
and 1 5 were alive between 1 and 27 (median 4) mo.
A major problem with irradiation regimens in
patients with nonmalignant diseases is an increased
cancer risk. The risk of developing a malignancy in
canine irradiation chimeras is 6.9 times that of control
dogs.59 That study also reported on the development ofnonhematologic malignancies in two human radiation
chimeras transplanted for leukemia. An association
between radiation and malignancy in man is estab-
lished beyond doubt.60’6’ Even relatively low exposure
to irradiation has been reported to lead to cancer. A
study involving I 1,000 children receiving approxi-
mately 140 rad to the head for ringworm infection
showed a sixfold risk for head and neck tumors
compared to their nonirradiated siblings.62 The chil-
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244 STORB ET AL.
dren also showed a significant increase in thyroid
tumors, although thyroid glands had only been
exposed to 9 rad.63 Evidence for radiation-induced
breast cancer has been obtained in A-bomb survivors
exposed to less than 100 rad.64
Interstitial pneumonia, either associated with a
pathogen or idiopathic, was seen among 22% of the
present patients. The highest incidence was in patients
with graft rejection (46%), usually after the second
conditioning regimen and presumably related to the
prolonged and severe therapy-associated depression of
the immune system, absence of a functioning graft,
and more intensive transfusion requirements. Patients
without rejection had an incidence of 16% and a
mortality of 6%. This incidence, although still of
concern, is lower than that reported by transplant
teams using TBI conditioning regimens. UCLA
reported 1 1 cases of interstitial pneumonia among 21
evaluable cases (53%) with a mortality of 19%.’� The
Paris team reported 6 cases among 1 5 patients with
sufficient follow-up (40%) and a mortality of 20%.20 A
similarly high incidence was seen in patients with
leukemia conditioned by CY and TB!.65 The reasons
for the higher incidence of and mortality from intersti-
tial pneumonia in patients conditioned with TBI are
not clear but are presumably related to the pulmonary
toxicity of TB!.
There were limited short- and long-term toxicities
of CY. The most serious toxicity was cardiac, with an
incidence of less than 1 %. One patient of the current
study and one of a previously reported study2 died of
this complication. Hemorrhagic cystitis, usually last-
ing no longer than 7 days, occurred in less than
one-third of the patients and resolved in all without
specific therapy.3 Cystitis can be modified by 2-
mercaptoethane sulphonate sodium, already in use in
Europe.66 Among 1 00 Seattle patients who have
survived between 1 and 1 0 yr after grafting, there have
been no serious late CY effects, in particular no
malignant tumors. Sterility may not be a problem.
Two patients of the current study (UPNs 626 and
758) and one previously reported67 had normal chil-
dren.
The suspected etiologies of the patients’ aplastic
anemia did not appear to influence the outcome of the
marrow transplants. Both patients with Fanconi’s
anemia are surviving with successful grafts.
The International Marrow Transplant Registry
Report stressed that aplastic anemia patients given
marrow from male donors had a better survival than
those given marrow from female donors.’7 The report
encouraged transplant teams to prefer male over
female donors when given the choice. The present
results and those in previously reported transfused and
untransfused patients from 2168 do not
support that recommendation. Overall, recipients of
marrow from female donors survived slightly better
than those given marrow from male donors (73%
versus 58%). This survival advantage was largely
explained by a lesser rate of rejection of female
marrow. When only patients with sustained first grafts
were analyzed, recipients of both male and female
marrow showed a survival rate of 75%. The differences
between the Registry results and ours remain unex-
plained but may be related to differences in condi-
tioning regimens. The present results also do not
confirm our previous suggestion68 that recipients of
marrow from donors of opposite sex have a worse
survival than those from sex-matched donors.
ACKNOWLEDGMENT
The authors thank the physicians, nurses, ward clerks, secre-
taries, technologists, and data analysts of the Adult Leukemia
Center and the patients’ referring physicians for their cooperation in
this study, and Dr. E. Giblett, Puget Sound Blood Center, for
carrying out erythrocyte marker studies.
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1982 59: 236-246
Hackman, JA Hansen, J Sanders, K Sullivan, PL Weiden and RP WitherspoonR Storb, KC Doney, ED Thomas, F Appelbaum, CD Buckner, RA Clift, HJ Deeg, BW Goodell, R transfused aplastic anemia patientsMarrow transplantation with or without donor buffy coat cells for 65
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