blood-1982-storb-236-46

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




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




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.


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 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-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




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-




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.


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




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.





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-




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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246 STORB ET AL.

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I980

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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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