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UCLA Conference
Bone Marrow Transplantation in Man
Moderator; MARTIN J. CLINE, M.D., F.A.C.P. Discussants: ROBERT P. GALE, M.D..
E. RICHARD STIEHM, M.D., GERHARD OPELZ, M.D., LOWELL S. YOUNG, M.D., F.A.C.P.
STEPHEN A. FEIG, M.D., and JOHN L. FAHEY, M.D., Los Angeles, California
Bone marrow transplantation is emerging as a viabletherapeutic approach to a number of diseases that areusually or uniformly fatal. We review here recent experiencesin bone marrow transplantation in man at UCLA and invarious other institutions tbrougbout the world. We examinemarrow transplantation in immunodeficiency diseases, acuteleukemia, and aplastic anemia and consider the problemsof infection in the transplant recipients. The applications oftissue typing to marrow transplantation and immunologiemanipulations, which may influence engraftment and graft-versuS'host disease, are also reported.
DK. MARTIN J . CI-INE*: Attempts at bone marrow trans-plantation in man are not new; however, the modern devel-opment of this approach to disease requires understandingof the hematopoietie stem cell, of cell-mediated immuneréactions, and advances in tissue typing. At present, mar-row transplantation has been applied to patients with threeeategories of disease: immunodeficiency, apiastie anemia,and acute leukemia. There is potential for its applieationto other neoplastie diseases and to serious nonmalignantblood disorders sueh as sickle-cell disease. Before this po-tential can be realized, eertain problems must be solved.These problems inelude [1] failure of engraftment in 10%to 20% of reeipients; [2J graft-versus-host disease, whichmay be lethal in 25% of patients receiving allogeneiemarrow; [3] failure of eradication of malignant disease byaggressive chemotherapy or whole body irradiation; and[4] the unknown late sequels of the transplantation pro-cedure, In addition, if marrow transplantation is ever to beapplicable for the great majority of patients who do nothave an immunologically compatible family member as apotential donor, a number of basic and clinical problemsmust be solved. Fundamentally, these involve the elimina-tion, by physical or immunologie means, of eells in thedonor marrow capable of attacking the recipient.
• Division of Hematology-Oncoiogy, Department of Medicine, UCLASchool of Medicine.
•• An edited Iranscription of the Clinical Case Conference arranged bythe DL'parimem of Medicine of üic UCLA School of Medicine, LosAngeles, California.
Tn this conference we shall consider these problems, aswell as review reeent experiences in bone marrow trans-plantation at UCLA and throughout the world. It is worthpointing out that a successful transplantation program isan enormous clinical effort requiring a team approach. AtUCLA, the team consists of clinicians from the Depart-ment of Pediatrics and Internal Medicine, clinical patholo-gists, geneticists, basic scientists from the Departments ofMedical Microbiology and Immunology, and experts intissue typing from the Department of Surgery. Given thislarge array of clinical and scientific talent in a single in-stitution, it is possible to tnount an effort as large and com-plex as a bone marrow transplantation program.
Bone Marrow Transplantation in Aplastic Anemia andLeukemia
APLASTIC ANEMtA
Dr. Robert P. Galet: Aplastic anemia is an uncommondisease with an uncertain cause in a majority of eases (1).Aptasia related to chloramphenicoi or hepatitis carries aparticularly grave prognosis (2, 3). The natural history ofthe disease has changed since the introduetion of héma-tologie supportive methods and new antimicrobial agents.Androgens may have some therapeutic effect, but theirvalue is still questionable (4, 5).
If the basic defeet in aplastic anemia is in the hemato-poietie stem cell rather than the microenvironment, in-fusion of normal bone marrow offers a reasonable thera-peutic approach. The first transplant for aplastic anemiawas reported by Osgood, Riddle, and Mathews (6) in1939. A 19-year-old woman with gold-induced aplasia wasgiven 18 ml of marrow from her ABO compatible husband.Engraftment was not observed, and the patient died 5 dayslater.
Animal models of radiation-induced aplasia treated bymarrow reconstitution were developed during the periodfrom 1945 to 1955. As a result of Ihese studies and thedevelopment of human hisfocompattbility testing, we arenow concerned with several areas in allotransplantation ofmarrow: human leukocyte-locus A (HL-A) and mixedleukocyte culture (MLC) testing in donor selection to
t Division of Hematotogy-Oncotogy, Department of Medicine, UCLASchool of Medicine.
Annals of Internal Medicine 83:691-708.1975 691
facilitate engraftment and circumvent graft-versus-host dis-ease f7), immunosuppressive "conditioning" of the re-cipient for successful engraftment (8-10), and the needfor posttranspiant immunosuppression for control of graft-versus-host disease (9-11 ). The principles of marrow graft-ing have been reviewed recently (10, 12).
The first opportunity to apply these principles to manarose when Thomas and colleagues (13) transplanted 3leukemia patients. Differences in the radiation dose anduncertainty regarding histoeompatibility testing make cri-tical interpretation of the results difficult. Bortin (14) re-viewed transplantation for aplastic anemia in 1970 and re-ported no surviving allogeneic chimera among 73 patients.Graw and Herzig (15) in 1972 reviewed 20 transplantsfrom HL-A compatible donors and reported 70% en-graftment with several immunosuppressive regimens. Theincidence of graft-versus-host disease was 15%. and sur-vival was from 3 to 259+ days.
Transplants for aplastic anemia bave increased drama-tically since 1970. Table 1 is a compilation of data fromseveral centers. A considerable amount of the data is un-published, and individual case reports have been excluded.Workers in Seattle have done three transplants in patientswith identical twins (10. 16). Engraftment was successfulin all, and they remain alive 6 to 12 years posttranspiant.Failure of engraftment in identical twins may relate to acritical clinical status at the time of transplantation or toa microenvironmental malfunction, rather than a stem celldefect. In identical twins, pretransplant and posttranspiantinimimosuppression are not required. Clearly, when thereis an identical twin donor, transplantation should be con-sidered ia the initial therapy of aplastic anemia.
The situation is less clear in allogeneie marrow grafting,but encouraging results have been obtained (Table 1 ). TheSeattle group has reported 37 transplants from HL-A-identical siblings (10, 11, 17). The majority of reeipientshad reeeived androgen therapy, and 35 had been multiplytransfused. Nine patients were conditioned by total body
irradiation (1000 rads), and 28 were conditioned withcyclophosphamide (200 mg/kg body wt) ( 18). Thirty-threeof 35 évaluable patients were engrafted; 6 subsequently re-jected their grafts, 5 died from marrow failure, 4 from in-fection, and 6 of graft-versus-host di.sease. Sixteen patientsare alive with functioning grafts (10). The incidence ofgraft-versus-host disease calculated as a function of en-grafted patients .surviving 50 days is 75%.
The data from Baltimore are less encouraging, and 2of 7 recipients are alive at 123 and 319 days posttrans-piant, respectively*. The National Cancer Institute (NCI)experience has been limited to three reported cases, withno long-term survivors (15. 19. i ) . The Boston group hastransplanted 16 patients from histocompatible donorst.Fourteen cases are évaluable, and engraftment was ob-served in all. Graft-versus-host disease was observed in allpatients and was fatal in several. There are 6 survivors outof 16, 3 with mild and 3 with severe graft-versus-hostdisease. The prognosis of the patients correlated with theirclinical status at the time of transplant (20). The FrenchTransplant Group has reported 24 transplants between1958 and 1973 in apla.stic patients conditioned with anti-lymphocyte globulin (21 ). Donors were not HL-A matched,and sufficient data are not available to permit criticalevaluation. The UCLA transplant experience will be dis-cussed separately and is presented in Table 1.
A number of reports detail treatment of aplastic anemiaby marrow transplantation, ineluding four from the Nether-lands (22, 23), one from London (24), three from Switz-erland (25), and two from Minnesota (26). Analysis ofthese random data is complicated by the selective tendencyto report successful cases.
Statistically, less than 25% of aplastic potential re-eipients will have an HL-A-identical, MLC-compatiblesibling donor. Alternatives have iberefore been considered.
• SANTOS GW: Personal communication.t GRAW RG JR: Personal communicaiion.ÎCAMITTA BM: Personal communLcaiion.
rabie 1. Bone Marrow Transplantation in Aplastfc Anemia. Human Leukocyte-Locus A (HL-A) Identical Siblings
TransplantCenier
1
Seattle
BaltimoreUCLA
BostonNational Cancer
Institute
Conditioning
NonellTotal body irradiationCyclophosphaiiiideCyclophosphymiJeCyclophosphamide
CyclophosphamideAniilymphocyle globulinAntilymphocyte globulin
+ total boJy irradiationCyclophosphamide
Patients(Evaluable)
no.
3(^)9(8)
28(27)7(5)6(6)3(3)tt
16(14)1 (1)1(1)
HI)
Grafts*
no.
37
26363
141I
1
%
100879660
100100100100100
100
G rail-Vers LJS-Host
Disease t
no.
04
20110
1400
0
%
0557533160
TOO00
0
Fatal Grafi-Vers us-Host
Disease J
no.
028100200
0
to0
5040
100Ü0
1400
0
Survivors*
no.
31
15252600
0
/o
1001154**28**S3**663800
0
Referencesg
10,11, 16
18
1915
* lîvaluiible patients only.t Patients wiUi erafcs at risk.Î Patients with graft-vcrsus-host disease only.§ Also, THOMAS ED, SANIOS GW, CAMITTA BM, et aJ: Personal communication.II Identical twins.** Includes one palicnt who was not a chimera.tt Two parent-child transplants and one sibling-sibling mismalcbed trutsplänl (see text).
692 November 1975 • Annals of Internal Medicine • Volume 83 • Number 5
including use of HL-A-identical unrelated donors selectedfrom Tissue Typing Banks, Donor pool size required forsupport of unrelated marrow transplantation has been re-viewed (27). In families, HL-A identity and MLC non-reactivity are concordant due to the close linkage betweenthe HL-A and MLC loci. In unrelated persons this is notthe case: only 10% of HL-A-identical unrelated personsean be expected to be MLC nonreactive. It is unknownwhether identity at the HL-A or MLC loci, or both, is re-quired for successful transplantation f28). Three recentreports suggest that MLC identity may suffice (26, 29, 30).While the results are preliminary, it also seems possible toengraft successfully MLC-reactive, HL-A-identica! relatedpersons. The tempo and severity of graft-versus-host dis-ease did not differ from that in MLC nonreactive trans-plants. Additional cases are required to evaluate this ques-tion. Storb and co-workers (31) have clearly shown theimportance of histocompatibility testing in eanine marrowtransplantation, but differences from elinieal transplanta-tion in man eomplieate interpretation.
The efïeet of prior sensitization on success of engraft-ment is clinically relevant in patients reeeiving blood andHL-A-misniatched platelets and granulocytes. Transfusionof bufïy coat-poor blood, even from histocompatibledonors, can lead to marrow rejection in dogs (32), whiehmay be overcome by a conditioning regimen of procar-bazine and antithymocyte globulin (33).
The patient should be designated a potential transplantrecipient when a suitable donor is identified. Transfusion oferythrocytes and platelets should be minimized, and familymembers should be assiduously avoided as donors of bloodproducts to prevent sensitization. We use HL-A-identicalunrelated donors for platelet support.
A major problem is the timing of the transplant, for de-lay may increase both the risks and the time available forrecovery. Indices indicating aggressive disease are granu-locyte count < 500/mm^, platelet count < 20 000/mm^,and > 70% nonmyeloid nucleated cells in the hone mar-row (2, 34). Each ease needs to be individually evaluated.Presently, we favor early transplantation in patients with apoor prognosis. The value of delay to permit a trial ofandrogen therapy is debatable (1, 4, 5). In stable patients,we have waited up to 3 months.
The importance of ABO compatibility in marrow trans-plantation is unelear, and attempts to cross this barrier(A^O) were initially disappointing. Successful transplan-tation has been reported in acute leukemia (35), and wehave engrafted two such patients without complication. Themajor problem is elimination of cytotoxic antibody (Íso-hemagglutinin) by combined plasmapheresis, transfusionof donor-type erythrocytes, and neutralization with in-travenous A substance. ABO antigens do not seem to be atarget of graft-versus-host disease, and we have not ob-served increased incidence or severity of graft-versus-hostdisease in this setting.
In summary, in patients with moderate to severe aplasiaand an identical twin donor, it is reasonable to proceeddirectly to transplantation. Immunosuppression of the re-cipient is not required, risk to tbe donor is negligible, andfavorable response can be anticipated. In patients with a
histocompatible sibling donor, we presently favor trans-plantation if the clinical prognosis is guarded. Pretransplantand posttransplant immunosuppression are required, and a50% success rate ean be anticipated. The proceduresshould be undertaken in transplantation centers with con-siderable experience and specialized support facilities.
I
ACUTE LEUKEMIA
The Strategy of marrow transplantation in hématologiemalignancies involves administration of tumoricidal dosesof ehemoradiotherapy that would otherwise be lethal. Ad-ditionally, transplantation may serve in an immunothera-peutic role, just as tumor-specific antigens occur in humantumors, particularly acute leukemia (36). Studies in micesuggested that graft-versus-host disease ean have an anti-leukemic efïect (37).
Grafting of patients after supralethal radiotherapy wasfirst attempted by Thomas and associates in 1957 (13).The use of bone marrow transplantation in acute leukemiais complex. It requires therapy protocols capable of dis-ease eradication that are not lethal to vital organs otherthan the marrow, successful engraftnient of the recipient,and prevention of fatal graft-versus-host disease.
Four transplant centers are currently involved in mar-row grafting, with programs involving a significant numberof patients with acute leukemia: Seattle, Baltimore. NCI,and UCLA. We will analyze results emphasizing protocolsfor pretransplant ehemoradiotherapy (Table 2 ),
The Seattle group has reported the largest number ofleukemic patients treated hy marrow transplantation. Twogroups were studied: syngeneic and allogeneic combina-tions (10. 38, 39). Syngeneic transplant recipients are par-ticularly interesting: engraftment is the rule, and graft-versus-host disease and posttransplant immunosuppressionare not involved. If the graft-versus-host disease reactionis an important modality of leukemia eradication, thenthese patients are at high risk of relapse. They offer, there-fore, a test of the effieaey of chemoradiothcrapcutic con-ditioning regimens. Fefer and co-workers (38) have re-ported on 16 transplanted identical-twin patients. In mostcases, "immunotherapy" consisting of transfusion of donorlymphocytes and immunization with frozen leukemic cellswas given. Complete hématologie remission was inducedin 88%, and 6 patients remain in remission at 11 to 44months. Leukemic recurrence in patients at risk, de-termined hy subtracting those dying of other causes beforeDay 100, is 35% (4 of 11). These results are encouragingand favor transplantation when an identical-twin donor isavailable. The role of immunotherapy in these patients isnot évaluable, but a controlled trial is underway. Theorigin of the leukemie recurrenee in these patients is un-known; it is not possible to determine if it represents per-sistence of the tumor or de novo oncogenesis in trans-planted cells.
Thomas and co-workers (10, 39, 40) have reviewedtheir experiences in allogeneic marrow transplantation. Ininitial studies total body irradiation was used for condi-tioning. Cyclophosphamide was added due to recurrentleukemia in a significant number of patients (41). Seventypatients (36 acute myeloblastic leukemia, 34 acute lym-
Cline et al. • Bone Marrow Transplantation 693
Table 2. Bone Marrow Transplantation in Acute Leukemia. Human Leukocyte-Locus A (HL-A) Identical Donors
Transplant Center
Seattle
BaltimoreNational Cancer Institute
UCLA
Cündilioniiig*
Cyclopliosphamide-\- total body irradiation ft
Total body irradiation.cyclophosphamide4- total body irradiation
CyclophosphamideCyclophosphamideTotal body irradiationCyclophosphamide
+ total body irradiationBACT reu;imen§§CyclopliosphamideCyclophosphamide
+ total body irradiationl] HSCARI regimen***
Patients(Evaluable)
no.
16(16)
70 (68)
25 (20)9(9).
7 miw
12(8)
Grafts t
no.
16
63
17813
5I5
8
%
100
93
85885060
100100100
100
Graft-Versus-HostDisease J
no.
0
46
111I3
I01
4
%0
73
6512
100100
330
25
50
• See text,t Evaluahle paiicnis only.Í Patients with graft at risk and survival > 50 days.5 Patients wiih graft-versus-host disease at risk.II Patients living > 100 days at risk.• • Also. THOMAS ED, SANTOS GW, GRAW RG JH. et al: Personal communication.tt See text. Identical twins: Seattle, 16; UCLA. 1.t í SANTOS GW: Personal communication.§S BACT regimen: B.C.N.U. (bis-chlorelhyl-nitrosurea). cytosine arabinoside, cyctophosphannide, and 6-tliioguanine.nil Two mixed lymphocyte culture tMLC)-reactive donors included.• • ' SCARI regimen: fi-thioeuanine, cyclophosphamide, cytosine arabinoside, rubidimycin (daunorubricin). and total body irradiation.t t t Three additional patients at Days 10, IS, and 25 alive poattransplant without disease.
phoblastic leukemia) have been transplanted. Graft-versus-host disease developed in 73%. A steadily improving sur-vival rate has been observed, with 70% of patients, trans-planted between January and June 1974, alive 200 daysposttransplant. Leukemic relapse was a problem in 30%(II acute myeloblastic leukemia, 9 acute îymphoblasticleukemia). If one corrects the recurrence rate for patientsat risk, as previously discussed, the actual recurrence ratemay be as high as 50% (42). The 120-day survival sta-tistics for 70 acute myeloblastic leukemia and acule Iym-phoblastic leukemia patients are 20% and 25%, respec-tively, and the median time to relapse is 45 and 75 days,respectively. This clearly represents a significant achieve-ment in patients refractory to conventional and experi-mental chemotherapy who are frequently in extremely poorclinical condition.
The results of the Baltimore experience are summarizedin Table 2, and use of cyciophosphamide in marrow trans-plantation has been reviewed (9, 18, 43,*). Leukemic re-currence was one of the major problems in this series andis apparent only if one analyzes patients at risk.
The NCI data in 20 patients are reviewed in Table 2(15, 44). Several conditioning regimens were used, includ-ing total body irradiation, cyclophosphamide and totalbody irradiation, and BACTf (19). These data, compar-able to those from Baltimore, underscore the high inci-dence of leukemic recurrence after cyclophosphamide con-ditioning. Five évaluable patients receiving BACT showedevidence of engraftment, and two identical twins had re-current leukemia at 50 and 90 days posttransplant. The
* SANTOS: GW: Personal communication.t BACT: B — B.C.N.U. (bis-chloretbyl-nitrosurea) ; A — cytosine ara-
binoside; C := cyclophosphamide; T = 6-tluoguaninc.
incidence of graft-versus-host disease seemed lower withBACT than with the other regimens, but additional dataare required to evaluate this (19, 45). The rate of leukemicrecurrence remains unacceptably high. Based on these ex-periences, UCLA has developed a cytoreductive protocolcombining total body irradiation, cyclophosphamide, andcombination chemotherapy. The SCARIJ regimen con-sists of a 5-day course of cytosine arabinoside, 15 mg/kgbody wt every 24 h, as a continuous intravenous infusion,and 6-thioguanine, 7.5 mg/kg body wt every 12 h bymouth. This is followed by daunorubricin, 60 mg/m- bodysurface, as a rapid intravenous infusion daily for 3 days.The patient is rested for 3 to 5 days to allow regenerationof the gastrointestinal tract mucosa. This is followed by thecyclophosphamide and total body irradiation regimen andmarrow transplantation. The SCARI regimen has been welltolerated in 12 patients, and 8 évaluable patients have noevidence of disease at 75, 80, 80, 96, 100, 150, 180, and200 days posttransplant- Two are in the immediate post-transplant period without evidence of disease. Additionaltime and patients are required to evaluate this regimen.
Mathé and associates have recently reviewed the marrowtransplantation experience in France (12. 21). Results aredisappointing, but conditioning used and histocompatibilitymatching were not optimal. Published and unpublished re-sults from Minnesota, Boston, and San Francisco are dis-couraging (23, 46-48).
The role of graft-versus-host disease in leukemia eradica-tion has been difficult to evaluate and is complicated byseveral factors: patients witb persistent or recurrent dis-
t SCAR!; S i 6-thioguanine; C := cyclophosphamide; A =. cylosincarabinoside; R = rubidimycin (daunorubricin) ; and I — total body irradi-ation.
694 November 1975 * Annals of Internal Medicine • Volume 83 • Numbers
Table 2. (Continued)
Fatal Graft
no.
0
23
1110
000
0
-Versus-Hostease§
%0
50
10100100
0
000
0
At Risk
no.
n41
13303
413
8
Recurrence 1
Cases
no.
4
20
9500
212
1
36
50
70100
00
5010066
12
Disease-FreeSurvivors*
no.
6
19
1000
I01
11
38
28
4000
140
20
90
SurvivalRange
months
2-44
3-49
7
36
2-7ttt
References**
10, 13, 36-38
' 1815, 32, i t
• See text.t Evaluable patients only.t Patients with graft at riak and survival > SO dayi.§ Patients with graíi-versus-host disease at risk.II Patients living > 100 days at rislc." Also, THOMAS ED, SANTns GW, GRAW RG jR, ct aJ: Personal communication.tt See text. Identical twins: Seattle, 16; UCLA, 1.i t SANTOS GW: Personal communication.§5 BACT regimen: B.C.N.U. (bis-chlorethyl-nitrosurea), cytosine arabinoside, cyciophosphamide, and 6-thioguanine.l!|l Two mixed lymphocyte culture (MLO-reactive doners included,*"* SCARI regimen: 6-thioguanine, cyclophosphamide, cytosine arabinoside. rubidimycin (daunonibricin), and total body irradiation.t t t Three additional patients at Days 10, 15, and 25 alive posttransplant without disease.
ease not infrequently reject or "crowd out" their grafts,which tends falsely to lower the incidence of graft-versus-host disease; and patients with severe graft-versus-host dis-ease not uncommonly die before 100 days, which falselylowers the risk of recurrent leukemia. An accurate defini-tion of the role of graft-versus-host disease awaits furtherelinical trials and careful comparison with patients not atrisk to develop graft-versus-host disease, that is, recipientsof syngeneie transplants (49).
Marrow transplantation is being used increasingly in pa-tients with acute leukemia. One should consider marrowtransplantation relatively early in patients with an identicaltwin donor. AU new patients with leukemia and their im-mediate families should be HL-A typed. When an HL-A-identical, MLC-nonreaetive donor is identified, the patientshould be designated a potential transplant recipient, andtransfusion therapy should be carefully monitored. Be-eause of the clear correlation between the patient's clinicalstatus at the time of transplantation and his subsequentsurvival, one may consider transplantation at a relativelyearly point in refractory disease. In acute myeloblasticleukemia we currently feel that poor-risk patients shouldbe transplanted either during their first relapse or possiblyduring remission. In acute lymphoblastic leukemia, wherechemotherapy is more effective, one should use all con-ventional modalities before considering transplantation. Weconsidered patients in their third remission or relapse, thosewith refractory central nervous system disease, or thosewith progressively decreasing remission durations as suit-able candidates. Inability to induce remission is an obviousindication to consider transplantation. It is important thatthe physician not postpone transplantation until the pa-
tient is in critical eondition. When a suitable related donoris unavailable, a search of the tissue typing bank is ap-propriate. Marrow transplantation should be undertakenin centers where optimal support facilities are available andwhere controlled clinical trials can be done.
Bone Marrow Transplantation in Immunodeficiency Disorders
Dr. E. Richard Stiehm*: With the exception of theaplastie patient with an identical twin, marrow transplantsare most successful in patients with certain primary im-munodeficiencies. Transplantation theoretically could beof benefit in all five types of human immunodeficiencies(antibody immunodeficiency, combined antihody and cellu-lar immunodeficiency, cellular immunodeficiency, phago-cytie immunodeficiency, and complement immunodefici-ency) (50), but with rare exceptions transplants are usedonly in cellular and combined immunodeficiencies. A fewattempts to transplant antibody immunodeficiency andphagocytic immunodeficiency have been made, but the re-sults are either negative or unavailable (14, 51).
Patients with immunodeficiency are particularly goodcandidates for transplantation when [1] their cellular im-mune defect is complete, so that engraftment is readilyachieved; [2] a human leukoeyte-Iocus A (HL-A) andmixed leukoeyte culture (MLC)-matehed sibling donor isavailable; and [3] the patient is not terminally ill. Thesecriteria are usually achieved in patients with severe com-bined immunodeficiency, a heterogeneous but relativelycommon primary immunodeficiency characterized by thefollowing: clinieally, by the early onset of diarrhea, pneu-
• Division of Pédiatrie Immunology, Department oí Pediatrics, UCLASchool of Medicine.
Cllne et al. • Bone Marrow Transplantation 695
monia, failure to thrive, and rapid downhill course; im-munologically, by profound defects in B- and T-eeil func-tion; and, pathologically, by a dysplastic thymus glandlacking Hassall's corpuscles. In most other immunodefi-ciencies there is sufficient cellular immunity present to re-ject the transplant unless pretransplant immunosuppressionis undertaken. Because of the grave risk from infection ofsuppressing an already immunodeficient and probablychronically infected patient, few attempts and even fewersuccesses are recorded. However, one of the early suc-cessful transplants was done with immunosuppression ina boy with the Wiskott-Aid rieh syndrome (52).
The first successful marrow transplant in severe com-bined immunodeficiency was done in 1968 at the Univer-sity of Minnesota (53). This patient continues to do well(54). To date, 52 patients have received transplants; 18have functioning grafts, all but a few from HL-A- andMLC-identical siblings (Table 3). The non-successes in-clude failure of engraftment or death, usually from infec-tion. Fatal graft-versus-host reactions are uncommon whenHL-A- and MLC-compatible donors are used.
We have done two transplantations at UCLA for com-bined immunodeficiency. Both patients are doing well, butonly one has solid immunologie evidence of engraftment.
The patient without proved engraftment is a 34-moQth-oMboy with persistent pneumonia who had partial antibody andcellular immunodeficiency (Table 4). After transplantationwith 10' marrow cells (given iniraperitoneally) from an HL-A-,MLC-identical brother, there was no evidence of graft-versus-host disease or erythrocyte engraftment, but the in-vitrolymphocyte proliferative response to phytohemaggluLinin re-turned to normal, the IgG levels increased so that gamma-globulin injections could be stopped, and the patient improvedsignificantly. The bone marrow transplant may have resultedin limited reconstitution or provided some transient humoralboost to the patient's own immune system; alternatively, it mayhave contributed nothing to the patient's spontaneous improve-ment.
The other patient transplanted was a 5-month-old boy whopresented with Pneumocystis carinii pneumonia and thrush (55).No lonsillar tissue, adenopathy, or thymus gland on chestX ray was present. Lymphocyte numbers in the peripheralblood were normal, but they were nonreactive to phytohemag-glutinin or allogeneie lymphocytes in MLC. Immunoglobulinswere extremely low, and delayed hypersensitivity skin tests werenegative. HL-A typing disclosed multiple extraneous HL-Aantigens that were inconsistent from day to day and delayeddonor selection (56). From age 5 to 11 months, despite anti-biotics, pentamldine, gammaglobulin, plasma, antifungal agents,and transfer factor, he became progressively woi'se, developingcandidal esophagitis and severe failure to thrive. Finally, a po-tential donor, an MLC-matched sister, was identified, and on3 February 1971, he received 2 X 10' bone marrow cells intra-peritoncally.
Two weeks after transplant, a positive monüia skin test de-
Table 3. Bone Marrow Transplantation in Human Immunodefi-
ciencíes Reported to the NIH/ACS* Organ Transplant Registry as
of January 19751
Patients transplantedTransplants donePatients alivePatients alive wilh surviving grafts
52952218
• NIH = Naüonal Institutes of Health; ACS = American College ofSurgeons.
^ Courtesy of Mortimer M, Bortin, M.D.
veloped, and oral moniliasis disappeared. Four weeks aftertransplant, immunoglobulins increased; 6 weeks posttransplant,peripheral lymphocytes were reactive to phytohemagglutinin,showed an XX (donor) karyotype, and had the HL-A type ofthe donor. Bone marrow cells have remained as XY karyotype.
From 18 to 28 days posttransplant, the patient developed asevere graft-versus-host disease reaction with skin rash, heartfailure, pneumonitis, conjunctivitis, fever, hepatomegaly, anddesquamation. He recovered spontaneously, however, and forthe last 3 years has been free of infections and has been grow-ing normally. He has persistently eczematoid skin and clearrhinorrhea, but no allergens have been identified. Peripheralblood studies show eosinophilia, normal phytohemaggiutininresponse, and both donor HL-A types and patient extraneousHL-A types.
The multiple extraneous antigens present before and aftertransplant that delayed donor selection, and the lack of bonemarrow chimerism are of particular interest in (his case.
The successful transplant in these patients has beenduplicated many times in patients with severe combinedimmunodeficiency. It is now known that severe com-bined immunodeficiency disease represents a heterogeneousgroup of disorders. Some of these patients have X-linkedinheritance, others have autosomal recessive inheritance,and some have no family histories. The typieal patientpresents early in infancy with failure to thrive, diarrhea,moniliasis, and pneumonia, but others may present with askin rash, adenopathy, and hepatosplenomegaly resemblingthe Letterer-Siwe syndrome (57). Some patients lack anenzyme of purine metabolism, adenosine deaminase. intheir tissues (58). Some have peripheral blood B and Tcells, while others lack one or both of these cell lines. De-spite this variabihty, all variants seemingly are equally re-sponsive to transplantation. Thus, marrow transplants insevere combined immunodeficiency are so well establishedthat a physician caring for sueh a child would be seriouslyremiss if he or she did not explore the possibility of trans-plantation.
New approaches are being sought in the treatment ofsevere combined immunodeficiency when an HL-A, MLC-identical sibling is unavailable. Perhaps the most complexapproach is delivery and maintenance of an affected infantin a germ-free isolator. South and colleagues (59) reportone such case, an infant who had a previously affectedsibling, so the diagnosis was suspected before birth. Thechild has been in isolation for 4 years and seemingly isdeveloping normally. Further, B cells are spontaneouslyappearing, suggesting possible slow acquisition of immun-ity.
Transfer factor has been used in several cases of severecombined immunodeficiency, generaily without benefit orwith only transient benefit (55, 60). It is likely that mostsevere, combined immunodeficiency patients have such aprofound T-cell defect that transfer factor does not havean effective T-lymphocyte target cell for its action.
Although fetal thymus transplants have been effeetivein restoring immunity in thymie aplasia (DiGeorge'ssyndrome) (61), their use in severe combined immuno-defieiency has, with rare exceptions, not been sueeessful.In thymie aplasia, thymus transplants provide a humoralfactor that acts on the patient's own lymphocytes to con-vert them to functioning T ceils. Injections of thymosin,an extract of thymus tissue, has been used to restore cell-
696 November 1975 • Annals of Internal Medicine • Volume 83 • Numbers
Table 4. Bone Marrow Transplantation at UCLA for Combined immunodeficiency
Patient A Patient B
Age at admissionAge at transplantDate of transplantDonor
PretransplantClinical findings
Laboratory studies
PosttranspiantClinical results
Laboratory results
,14 months38 months16 December 1971Human leuktxiyte-locus A (HL-A)- and mixed
lymphocyte culture {MLC)-identical;Male sibling
Ulcerated vaccination; persistent pneumonia;persistent rhinorrhea
IgG, 360; IgM. 60; igA. 0*; lymphocytes,I800/mm^ phytohemagglutinin response, X lOf
Pneumonia and rhinorrhea cleared ;gammaglobulin injections stopped;normal growth began
IgG, 880; IgM, 80; IgA, 0*;phytohemaggiutinin response, X 80;lymphocytes, 3000/mm'
5 months11 months3 February 1971MLC-identical; female sibling
Thrush, esophagitis; Preiimocystiscurinii pneumonia; failure to thrive
IgG, 100: IgM, 50; IgA. 20*;lymphocytes, 3500 mm';phvtohemaggliifinin response, X Of;extraneous HL-A antigens
Thrush and esophagitis cleared ; nofurther infections; normal growthbegan
IgG, 800; IgM, 80; IgA, 70*;phytohemagglutinin response, X 70;XX cells in blooil
• Values of ¡mmiinoelobulins ¡n mg/dl.t Ratio of 3H-thymidine incorporation into DNA with and without phytohemagglulinin.
tilar immunity temporarily in cellular immunodeficiencydisorders (62). The combined use of transfer factor andfetal thymus transplantation has been used successfully inthree severe, combined immunodeficiency patients (63,64). In these patients thymus cells are engrafted, and thisresults in restoration of T-celi function without B-cell func-tion. Nonfatal graft-versus-host disease reactions have beenobserved. The transfer factor may allow maturation ofthymic tissue, which can then become engrafted.
Others have attempted to modify the fatal graft-versus-bost disease reaction when unmatched marrow is engrafted.Some use a cell suicide method in which the donor'smarrow and the patient's leukocytes are cultured togetherin vitro in the presence of large amounts of 3H-thymidine(65, 66). The donor's immunocompetcnt cells, which pro-liferate in vitro in response to the patient's histoeompatibil-ity antigens (and which presumably cause the in-vivo graft-versus-host reaction), incorporate a lethal dose of thymidineinto DNA and self-destruct. The marrow cells not de-stroyed are transplanted, with the expectation that theycan reconstitute the patient without causing a fatal graft-versus-host reaction. To date, no successes in humans havebeen recorded. Another approach is to give enhancinganliserum simultaneously with the unmatched marrow(67, 68). This antiserum is directed against the patient'shistoeompatibility antigens and inhibits the mixed leukocytereaction of the donor's lymphocytes toward the patient'scells. It is in the form of plasma and must be given repeat-edly. Such a procedure has delayed but not permanentlyabolished graft-versus-host disease.
Recently, a severe combined immunodeficient infantwas successfully transplanted with marrow from his HL-A-nonidentical uncle, who was MLC identical (69). Otherrecent attempts have used unrelated donors who are MLCnonreactive with the patient's leukocytes*. These expe-riences emphasize the crucial role of identity at the MLC
locus rather than at the HL-A locus in graft rejection.Fetal liver transplants have been given to several chil-
dren with severe combined immunodeficiencyt- Fetalliver (8 to 12 weeks of gestation) is an excellent source ofstem celts, unencumbered with histoeompatibility antigens.Engraftment has been achieved in three patients, but oneof these succumbed with immune-complex disease of thekidneyt.
In summary, severe, combined immunodeficiency wasidentified as a distinct pathologic and clinical entity in1957. As shown by the cases presented, a "cure" for someof these patients was found a decade later. A cure for allof these cases is on the horizon, perhaps within a fewyears.
Application of Tissue Typing in Bone Marrow Transplantation
Dr. Gerhard Opelz§: The modern history of bone mar-row transplantation is closely associated with developmentsin the field of histocompatibiliiy testing. With very fewexceptions, all successful human bone marrow grafts havebeen done between siblings who were identical for the twomain known histoeompatibility antigen systems: the HL-A(human leukocyte-locus A) and MLC (mixed lymphocyteculture) systems. HL-A antigens are serologically definedantigens that are typed for by reacting monospecificantiserums against lymphocytes in the complement-de-pendent microcytotoxicity test (70). The products of theMLC locus are not serologically detectable and are testedfor in the lymphocyte-defined MLC test (71, 72). TheHL-A antigen system has been extensively studied forover 10 years; today, 27 antigenic specificities belongingto two scries of mutually exclusive allelic antigens can betyped for (73, 74). The antigens responsible for MLCreactivity are less well defined. Only recently, with the use
' HnNO R: Personal comm uni catiotï.
t BUCKLEY R H : Personal communication,t COOPER MD: Personal communication.§ Department of Surgery, UCLA School of Medicine.
C/tne et a\. • Bore Marrow Transplantation 697
of homozygous lymphocytes, has it become possible totype for lymphocyte-defined specificities (75). A collab-orative study of 15 international laboratories is currentlybeing carried out to characterize more clearly the natureof lymphocyte-defined antigens.
Both serologically defined and lymphocyte-defined anti-gens are genetically contralled by loci on the same chromo-some. Since the tissue type of a person is determined bytwo serologically defined genes and one lymphocyte-de-fined gene on each of two chromosomes, the total num-ber of serologically defined antigens in any person isfour and that of lymphocyte-defined antigens is two. Theprinciple of inheritance of HL-A and MLC determinantsis shown in Figure 1. Because each child receives onechromosome from the mother and one from the father,the chance of two siblings having the same tissue type is25%. HL-A-identical siblings are, in most instances, alsoMLC identical. Only in the rare case of a genetic crossover(the frequency in man is an estimated 1%) will HL-A-identical siblings be MLC incompatible.
KJdncy transplants between HL-A-identical siblings havebeen shown to constitute a special category, comparable tothose between monozygotic twins. The most recent analysesof 215 transplants from the United States and Canadaand 79 transplants from Europe showed 1-year successrates of 90% and 88%, respectively {76, 77). The successrate in bone marrow transplantation is substantially lower;only since tissue typing was introduced for selection ofhistocompatible sibling donors have success rates im-proved to the current level. Undoubtedly, the frequent oc-currence of graft-versus-host disease and the difficulties inproviding immunosuppressive treatment without destroyingthe graft arc the main complicating factors in marrowtransplantation as compared with renal transplants.
Of interest is the relative importance of HL-A and MLCin bone marrow transplantation. This is critical for theselection of the most suitable sibling donor in a case ofgenetic crossover, and also when a parent is HL-A iden-tical but MLC incompatible with a child, or vice versa(78). It also is important for the future potential of usingunrelated donors for bone marrow transplantation. Datafrom experiments in dogs suggest that lymphocyte-de-
Inheritance of Histocompatibility Antigens
Fathera Q
I 7 b
Mother
2 Ig c
Ti 5 d"
ChildrenBo 3 7 b 7 b
12 It 5 d 12 c 5 d
Figure 1. Inberitance of histocompatibility chromosomes results infour different possible tissue types in children. Tbe numbersrepresent tbe buman leukocyte-locus A (HL-A, serologically defined)specificities of tbe two segregant series of antigens, wbile tbe let-ters represent hypotbetical mixed lymphocyte culture (MLC,lymphocyte-defined) specificities.
fined antigens are slightly more important than serolog-ically defined antigens; however, matching for both antigensystems is of benefit and their effect is additive*. In man,successful transplants have been reported from MLCidentical but HL-A-different donors (79, 80). In our ex-perience, temporary engraftment was achieved in a patientwith aplaslic anemia who was transplanted from his MLC-identical, HL-A-incompatible mother (78). Although thegraft was subsequently lost, the patient is alive and clin-ically well. In three transplants from HL-A-compatible,MLC-incompatible related donors, temporary engraftmentwas achieved in each instance, but all three patients diedof either recurrent leukemia or graft-versus-host disease.Although the limited experience in a few patients doesnot positively establish that MLC cottipatibility is necessaryfor successful bone marrow transplantation, the absenceof a documented successful transplant across the MLCbarrier should be reason for extreme caution.
With the improving clinical success of bone marrowtransplantation, the possibility of using unrelated marrowdonors for patients with no histocompatible family mem-bers is becoming an issue of increasing importance. Largenumbers of volunteer blood donors have already beenHL-A typed, mainly for the purpose of providing histo-compatible platelets and granulocytes for supportive ther-apy (27, 81). Since it is estimated that about 10% ofunrelated persons who have the same HL-A type are alsoMLC identical, selection of unrelated donors for bone mar-row transplantation does not seem impossible. Aside fromethical considerations, the practicality of this approachremains to be tested, however. The still moderate successrate between histocompatibie siblings indicates that othertransplantation loci besides HL-A and MLC do exist.These undefined histocompatibility loci are more likelyto match in siblings than in unrelated persons. It is there-fore essential that the search for new antigen systems thatcould be of importance in bone marrow transplantation beintensified. For practical purposes, it will also be importantto determine the individual strength of each antigen sys-tem.
Infectious Complications of Marrow Transplantation
Dr. Lowell S. Youngt : Infection is the most commonfatal complication of acute leukemia, aplastic anemia, andcongenital immunodeficiency states. In addition to pre-existing defects in host defense, the therapy given beforetransplantation results in extreme vulnerability to infection.Thus, it is not surprising that the infectious complicationsof marrow transplantation are similar to, but perhaps moresevere than, those of acute leukemia or congential im-munodeficiencies (82-85).
Table 5 summarizes the common pathogens that we andothers have encountered, their reservoir, and their likelymode of transmission. Opportunistic pathogens fall into twobroad categories: endogenous pathogens from the host'snormal microbial flora that become invasive after impair-ment of host resistance, and exogenous infecting organisms
• VBIESENDOW H M ; Personal commun ¡c a don,t Diviüíun of Infectious Diseases, Department of Medicine, UCLA
School of Medicine.
698 November 1975 • Annals of Internal Medicine • Volume 83 • Number 5
Table 5. Infections Complicating Bone Marrow Transpiantation:Epidemio1<^ic Patterns
Origin Examples of Pathogens
Endogenous infection reservoirGastrointestinal tract
Upper
LowerSkinFemale genital tractCells (nerve, macrophages, and
so forth)Exogenous infection; mode of
transmissionContact
DirectAerosol droplets
AirborneTrue droplet nucleiDust
Oral (ingestion)
Stapbylococci, Gram-negativebacilli
Gram-negative bacilliStaphylococci, Candida speciesAnaerobic organismsHerpes viruses, Toxoplasma
StaphylococciRespiratory viruses
Varicella? AsperfiillusPseudomonas
acquired frotn the environment. Aspergillus pneumoniais probably an exogenous infection, with transmission oc-curring by the airborne route, whereas Toxoplasma andvaricella are acquired exogenously, remain latent in hosttissue for long periods, and cause recrudescent infectionafter immunosuppression. With several pathogens, theepidemiologic pattern is either unclear or both mechanismstnay be involved. For instance, Pseudomonas aeruginosais not part of the normal fecal flora of healthy humans(86), but the prevalence of stool colonization by Pseu-domonas rises strikingly in patients with underlying disease(87), on special diets (88), or in those who receive broad-spectrum antimicrobials (89, 90). Gastrointestinal coloni-zation precedes pseudomonas bacteremia in many debil-itated patients (83, 87, 91).
There are. other microbes, such as the presumed parasitePneumocystis carinii, whose epidemiology and mechanismof disease production are poorly understood. Experimentalexamples and the bulk of epidemiologic evidence supportthe view that the endogenous route is the most commonpathway for opportunistic infection, but there are alsomany clearcut examples of horizontal transmission ofdisease within the hospital.
After the initial course of cytotoxic therapy, total gran-ulocyte counts of less than lOO/mm^ are usually observed.During this phase, staphylococci and antihiotic-sensitiveGram-negative bacilli predominate as causes of systemicinfection. We have observed a direct correlation betweenneutropenia and low or negligible titers of heat-stableopsonizing of antibodies against autologous infectingstrains (92). Thus, the defects in host defense can beboth cellular and humoral and may not be immediatelycorrected by successful marrow engraftment. Infectionscaused by more antibiotic-resistant Gram-negative bacillisuch as Fseudomonas are usually encountered after aninitial course of systemic antibiotics. The deep mycosesand P. carinii pneumonia are most commonly "late" in-fections, developing after engraftment has been unsuccess-ful or has been complicated by graft-versus-host disease.
A common experience is that infection can developwith dramatic suddenness during periods of marked leuko-penia. The clinical course is well illustrated in Figure 2,which shows the pulmonary changes associated with bac-teremic pseudomonas pneumonia during an interval ofonly 36 hours. If patients have preexisting infection, suc-cessful marrow engraftment may be curative, whereas un-successful engraftment is associated with persistent andoften lethal infection. Figure 3 {top) shows a patient witha dense left upper lobe consolidation who was found tohave an Aspergillus species in his sputum before trans-plantation. This lesion persisted for several weeks duringan abortive transplantation attempt, and it was present atnecropsy despite treatment with granulocyte transfusionsand amphotericin B. Terminal spread of infection to alllobes of the lung resulted in a diffuse aspergillus pneu-monitis (Figure 3, bottom).
We have found that mixed or dual infections arecommon, particularly the association of Gram-negativebacillary infection with fungal penumonia. This emphasizesthat documentation of one infectious process hardly ex-cludes another. As a corollary, fever and signs of infectionthat develop after one treatment course should not be at-tributed to a recrudescence of an antecedent infection.
Figure 2. Top. A patient with posthepatic apiastic anemia developedhigh fever and right-sided chest pain but had a normal chestX ray. Bottom. Thirty-six hours later, shock and extensive consolida-tion in both lung fieids developed, which at autopsy proved to bepseudomonas pneumonia with vasculitis.
Cli'ne et a\. • Bone Marrow Transplantation 699
Figure 3. Top. A 12-year-old boy with aplastic anemia had a denseleft upper lobe infiltrate and AspergiJ/us fumigatus in his sputum.Bottom. Despite amphotericin B and granulocyte transfusions, thislesion persisted during an unsuccessful transplant attempt, and thepatient died with a diffuse aspergillus pneumonia.
and the patient must be thoroughly reevaluated. Further-more, there is still significant infection risk after marrowengraftment. Serial laboratory studies in several patientssuccessfully transplanted continue to show impaired cell-mediated immunity for long periods, and we have observedinterstitial pneumonia due to Pneumocystis organisms andpneumococcal bacteremia months after successful trans-plantation.
Our approach to the management of these patients isderived from experience with hématologie malignanciesand is based on the principle of prevention, aggressivediagnostic measures, and early presumptive treatment (82,83). Many of the methods are experimental, require com-plex equipment, and have not been critically evaluated inlarge-scale clinical trials. They are, however, based on anincreasing understanding of the epidemiology of nosoco-mial infection and reflect our ability to compensate forhost factors that are known to be impaired.
Patients are managed in protective isolation and receiveoral nonabsorbable antimicrobials that inhibit most aerobicbacteria and Candida species. Because of high rates ofcontamination of fresh vegetables with certain bacteriastich as Pseudomonas, patients receive only cooked ma-
terials (93). The skin is bathed frequently, and "surveil-lance" cultures of the nares, oropharynx, axilla, and stoolare taken twice weekly. These often give clues to thenature of an incipient septic process.
It is unclear at this point whether the beneficial resultsof laminar flow units in reducing infection rates are dueto the isolation alone, laminar flow per se, or the oralantimicrobials (94, 95). Oral antibiotic regimens by them-selves may be useful in reducing infection (95).
The rapidity with which systemic bacterial infectionscan develop has led us to endorse the principle of "pre-sumptive antimicrobial therapy" for fever associated withclinical signs of infection. Broad spectrum antimicrobials,particularly the combination of an aminoglycoside andcarbenicillin, are preferred because of the high mortalityrate associated with proteus, pseudomonas, and serratiainfections. This formula may be modified if surveillancecultures show Klebsiella or staphylococcal organisms, inwhich case we substitute a cephalosporin for carbenicillin.
A therapeutic dilemma is the patient who ¡s persistentlyleukopenic and febrile without objective evidence of infec-tion. Our experience has been that the abrupt withdrawalof antimicrobials is often associated with clinical deteriora-tion and the eventual documentation of a septic process.It may be that all markedly leukopenic patients havelocalized infection due to usually saprophytic or stoolcolonizing organisms whose invasiveness is limited by sys-temic antimicrobials. If fever or documented infectionpersist despite antimicrobials, we give daily granulocytetransfusions; experimental evidence of their efficacy ispersuasive (96-98).
The most challenging infections are those caused byfungi and the cytomegaloviruses; safe, effective therapy isnot currently available for these organisms. The develop-ment of new pulmonary infiltrates and sustained feverafter a response to antibiotics for Gram-negative bacterialinfection often heralds fungal superinfection (99). Am-photericin B, the most consistently effective agent for deepmycoses, is fungistatic raiher than fungicidal, and thechance for successful therapy without marrow engraftmentis remote. Pneumonia caused by Pneumocystis has beentreated successfully with several agents, including pentami-dine isethionate, but co-trim ox azoic now seems to be aneffective and safe oral agent.
We have little doubt that the quality of supportive careand frequent clinical observations are vital elements in thesuccessful management of these patients. Such factors, aswell as scrupulous measures to minimize exogenous in-fection, may be far more important than antimicrobialprophylaxis or the use of laminar air flow. Factors affect-ing successful outcome include the presence of preexistinginfection (negative), the quality of the match, and thedevelopment of graft-versus-host disease. There is evi-dence that the use of antithymocyte globulin is associatedwith an interstitial pneumonia caused by the cytomegalo-virus, but the association of this interstitial pneumonitiswith antilymphocyte globulin is so strong that it raises thepossibility of a hypersensitivity reaction (100). Thus, apoor match increases the likelihood of graft rejection orgrafl-versus-host disease and the latter necessitates the
700 November J975 • Annals of Internal Medicine • Vo/ume 83 • Numbers
use of measures, such as antilymphocyte globulin admin-istration, which in turn increase infection risk.
It is not unusual to observe defervescence and clinicalimprovement as a harbinger of successful engraftment.While much attention has been focused on the absoluteperipheral granulocyte count as the niajor index of thelikelihood of infeetion (101), comparably depressed pe-ripheral neutrophil counts in a patient with a regeneratingmarrow and a patient without engraftment have entirelydifferent prognostie implications. The important perspec-tive is that all the measures involved to prevent and treatinfection are, in effeet, a form of temporizing, intended toprotect the host before an adequate mass of donor marrowbecomes functional. Ultimately, successful treatment ofsupervening infection coincides with successful engraft-ment of transplanted marrow.
The Bone Marrow Transplant Experience at UCLA
Dr. Stephen A. Feig*: A program of bone marrowtransplantation has been in operation at UCLA since 1973.Tbe purposes of (he program are twofold: the applicationof a new therapeutic modality to the management ofmalignant disorders with poor prognosis; and researchdirected toward improving the efficacy, broadening the ap-plicability, and understanding the long-term effeets ofmarrow transplantation. To date, 30 transplants have beendone at UCLA for severe aplastie anemia and acuteleukemia. Sixteen patients are presented in this report.Fo'"'teen addiiioii.:' patients have been transplanted soreeently that the results cannot be evaluated yet. Prelim-inary data were presented in Tables 1 and 2. While thenumbers are still small, several tentative conclusions aresuggested by this experience.
Patients were prepared for transplantation with oneof several regimens outlined in Figure 4. During the trans-plant period, all patients were kept in strict isolation withaccess limited to immediate family, the responsible phy-sicians, and special nurses. The patients were bathed dailyin betadine and reeeived gastrointestinal antibiotic pro-phylaxis.
Under general anesthesia, marrow was obtained fromthe donor by multiple aspirations and processed by themethod of Thomas and Storb (102). Posttransplant, as aprophylaxis against graft-versus-host disease, the patient
• Division of Hematology-Oncology, Department of Pediatrics, UCLASchool of McditLne.
received methotrexate (103). All posttransplant bloodproducts were irradiated with 1500 rads to inhibit engraft-ment with transfused lymphoeytes (103).
Graft-versus-host disease was graded by the stagingcriteria of Storb and colleagues (104) and treated accord-ing to their recommendations.
APLASTIC ANEMIA '
Six patients have been transplanted for severe aplastieanemia (Table 6). Two patients (1-2 and 1-3) died ofsepsis in the immediate posttransplant period, despite in-tensive antibiotic therapy and granuloeyte transfusions.Neither had evidenee of engraftnient at postmortem ex-amination. Both patients had long-standing aplasia, hadbeen multiply transfused with erythroeytes and platelets,and were severely ill and malnourished. There may nothave been sufficient time for engraftment before theirdeaths in the face of these adverse faetors.
Patient I-l is the only long-lerm chimera in this series. Hiscourse was compliealed by a eytoniegaiovirus and pneumococ-cal infection at 3 and 8 months after transplantation. In eachinstance, recovery was nomial. The patient continues to havesignificant impairment of cell-mediated immune function 18months after transplantation. He has reduced tritiated ihymidineCHTdR) incorporation by his lymphocytes after stimulationwith phytohemagglutinin, quantitatively decreased circulating Tcells, and impaired mixed lymphocyte reactivity against un-related lymphoeytes. Humoral immune function, assessed byquantilation of circulating B cells and by quantitative imniuno-globulins, has been normal since 3 months after transplant;ition.
Patient 1-4 developed severe aplastic anemia after repeatedtreatment with chloramphenicol. Marrow biopsy showed aretieulin myelofibrosis. He underwent marrow transplantationfrom his HL-A-mismatched, MLC-unreactive mother. No evi-dence of engraftment was ever observed, yet this patient isalive with partial hématologie recovery of his own hema-topoietic tissues at 250 days. He continues to require eryth-rocyte transfusions, but he maintains a platelet count of 50 000and a granulocyte count of 1000/mm^
Patients 1-5 and 1-6 are still too early in their courses to befully analyzed. Patient 1-5, with aplaslic anemia after hepatitis,has clear evidenee of engraftment and shows early hématologieimprovement. Patient f-6 had a moderately cellular bone mar-row at 14 days posttransplant. with only donor male karyo-types. Her peripheral blood counts rose then fell again withthe reappearance of aplasia at 28 days. She has just receiveda second transplant from another sibling.
ACUTE LYMPHOBLASTIC LEUKEMIA
Four patients have undergone bone marrow transplan-
Figure 4, Regimens for preparationof bone marrow transplantation.AG: 1 unit of donor whole blood orleukocyte concentrate: CYCLO (A):cyclophosphamide, 45 mg/kg bodywt day, intravenously; CYCLO (B):cyclophosphamide, 60 mg/kg bodywt • day, intravenously; TBI: 1000rads total body irradiation at ^ 8rads/min; ARA C: cytosine arabino-side. 10 to 15 mg/kg body wt •day, continuous infusion; 6-TG: 6-thioguanine, 5 to 7.5 mg/kg bodywt by mouth every 12 h; DM:daunomycin, 60 mg/m^ • day, in-travenously for 1 to 3 days; BM;~- 3 X l C filtered nucleated mar-row cel ls/kg intravenously.
PREPARATION FOR TRANSPLANTATION
I -16 I -15 I -14 1 -13 I -12 I -11 I -10 I -9 I -8 I -7 I -6 I -5 i -4 i -3 i -2 i -1 i 0 i
1. APtASTIC ANEMIA
2. ACUTE LfUKEMlA
A.
AG CYCLO (A)
ARA-C
6-TGDM
crine et al. • Bone Marrow Transplantation 701
Table 6. Patients Transplanted for Severe Aplastic Anemia
Recipienl
Patient
M
1-2
1-3
1-4
1-5
1-6
Age
yrs
12
26
12
27
18
20
Sex
M
M
M
M
M
F
Donor
Relation-ship*
Sibling
Sibling
Molher
Mother
Sibling
Sibling
Sibling
Sex
F
M
F
F
F
M
F
Preparatory
(Regimen)
1
1
I
1
1 (Cyclophos-phamidecystitis)
1
i
Histocompatibility
HumanLetikocyle-
Locus A(HL-A)
Identical
Identical
Nonidenlical
Nonidentical
Identical
Identical
Identical
MixedLymphocyte
Culture(MLC)
Nonreactive
Nonreactive
R^Di
Nonreactive
•
NonreM;tive
Nonreactive
Nonreactive
Graft (Evidence)
+ (Chromosomes,ABO bloodgroup marker)
None (Autopsy)
None (Autopsy)
None (Chromo-
Graft-
vers US-Host
Disease(Therapy)
None
> . •
. . •
somes, ABO bloodgroup marker,erythrocyte phos-phoglucomutaseisoenzyme marker,erythrocyteadenosine deami-nase isoenzymemarker)
-^ (Chromosomes)
Transient(Chromosomes)
None
Stage i I(None)
None
None
Survival (Status, or
Cause ol Ueatnj
days
> 500 (Completehématologierecovery)
19 (Pseudomonassepsis)
21 (Aspergillussepsis)
> 250 (Hématologieimprovement)
> 80 (Hématologieimprovement)
> 32
> 35 (Aptastic)
" Kclajiunship of donor to recipient.Î R ^ D: Recipient reactive to donor; donor nontcactive to recipient.
tation for drug-resistant acute lyraphoblastic leukemia(Table 7).
The first patient (II-l) had moderate graft-versus-host dis-ease after engraftmenl and was treated with antithymocyte glob-ulin. Three months after transplantation, the patient developedan acute interstitial pneumonitis and succumbed afier a rapidlyprogressive 48-hour illness. Pneumocystis organisms, but noleukemia, were found at postmortem examination.
The transplant in Patient 11-2 was unusual in that her bloodgroup was O, while the donor's blood group was A. The daybefore transplant, the recipient underwent an 18-Utre plasmaexchange with fresh-frozen type A plasma, using the Amincocell separator (American Instrument Co,. Division of TravenolLaboratory, Inc., Silver Spring, Maryland). Isoheniagglutinintiters were further reduced by the administration of Witebskysubstance. The patient's subsequent course has been quitebenign, and her isohemagglutinin titer and Coombs' test remainnegative.
Patient 11-4 has had a remarkably stormy course. He de-veloped hemorrhagic cystitis, presumably secondary to cyclo-phosphamide. This became so severe that ure tero vesical ob-struction occurred. The patient required bilateral cutaneousureterostomies 21 days after transplantation. At that timethere was negligible karyotypic and morphologic evidence ofengraftment. After surgery his condition improved, and he hashad evidence of engraftment and good wound healing,
ACUTE MYELOBLASTIC LEUKEMIA
Six patients received marrow transplants for acutemyeloblastic leukemia (Table 8). Patients III-l and III-2were transplanted during a rapidly proHferative phase oftheir disease. Despite intense preparation, only transientevidence of engraftment was observed. Leukemia recurred
early, and the patients died as a result of their primarydisease.
Because of this experience. Patients 111-3, 111-4, andIÏI-6 were prepared with a more intense regimen ofchemotherapy and total body irradiation (Figure 4). Pa-tient III-3 developed severe gastrointestinal toxicity, be-came septic, and died before engraftment could be shown.Intense cytoreductive chemotherapy has resulted in sur-vival without recurrent leukemia thus far in Patients 111-4and 111-6. These patients have been successfully engraftedand are living at home without disease or the need ofhématologie support.
Patient III-5 received a transplant from his HL-A-identical,MLC-reactive mother. Successful engraftment was shown with-in 2 weeks. Weekly chromosome analysis of bone marrow cellsshowed only female cells. The patient developed moderatelysevere graft-versus-host disease and hemorrhagic cystitis. Hepa-tic and gastrointestinal symptoms resulted in electrolyte im-balance. The patient required peripheral hyperalimentation.Terminally, he developed sudden anuria and respiratory dis-tress. Postmortem examination showed total obstruction of theurinary tract, with a clot filling the entire collecting system. Healso had hemorrhagic pneumonitis that grew Pseudomonasorganisms. His gastrointestinal tract and bladder were entirelydenuded of endothelium but were coated with an abundant,acute inflammatory response. Although pre-terminal peripheralblood counts showed pancytopenia. at postmortem examinationhis marrow was hypercellular, with normal maturation of allcell lines.
DISCUSSION
A rapidly growing program of bone marrow transplanta-
702 November 1975 • Annals of Internal Medicine • Volum« S3 • Numbers
Table 7. Patients Transplanted for Refractory Lymphoblastic Leukemia
Recipient
Patient
II-1
II-2
n-3
11-4
Age
yrs
10
15
48
13
Sex
F
F
M
M
Donor
Relation-ship*
Sibling
Sibling
Idenlicaltwin
Sibling
Sex
F
M
M
F
Preparatory
(Regimen)
2A
2A
2B
2A (Cyclophos-phamidecystitis)
Histocompatibility
HumanLeukocyte-
Locus A(HL-A)
Idenlical
Identical
Identical
Identical
MixedLymphocyte
Culture(MLC)
Nonreactive
Nonreactive
Nonreactive
Nonreactive
Graft (Evidence)
+ (Erythrocyteacid phos-phatase iso-enzyme marker.erythrocyte glu-tamic pyruvatetransaminaseisoenzymemarker, erythro-cyte antigen)
+ (Chromosomes,ABO bloodgroup marker
+ (Not applicable)
+ (Chromosomes)
Graft-
Verstis-Host
Disease(Therapy)
Stage in(Antithy-mocyteglobulin)
None
None
None
Survival (Status, or
Cause ol Ueatn)
days
100 (No evidenceof disease)(InterstitialpneumonitiS;
> 120 (No evidenceof disease)
> 100 (No evidenceof disease)
> 75 (No evidenceof disease)
• Relationship of donor to recipient.
tion has been established at UCLA. The results of trans-plantation in patients witb severe aplastic anemia havebeen shown to be clearly superior to alternate forms oftherapy (105). We concur that, if a suitably matcheddonor is available, transplantation is the treatment ofchoice for that condition.
Paradoxically, Patient 1-4 achieved long-term survivalwith partial hématologie recovery in the presence of clearevidence of graft rejection. This observation suggeststhat aplastic anemia is a heterogeneous group of diseases.Some forms may be the result of damage to hematopoietiestem cells, while others may be due to an unfavorablemicroenvironmenl. In theory, the former category shouldlend itself ideally to therapy by marrow transplantation,whereas the latter might possibly have an increased risk oftransplant rejection. One might speculate, however, thatrecovery of this patient's own marrow after the intenseimmunosuppression of transplant preparation is consistentwith tbe presence of an unfavorable environment in hismarrow and with a possible immunologie cause for bisaplastic process.
Our early experiences in transplanting patients witbacute myeloblastic leukemia suggest that cyclophosphamidealone, or in combination with total body irradiation, maybe inadequate to eradicate this disease when it is presentin a rapidly proliferative phase. This observation is furtherstrengthened by tbe accumulating experience in severalcenters of late relapses in patients transplanted for bothacute myeloblastic leukemia and acute lympboblastic leu-kemia.
Two potential solutions are possible. Either tbe cytore-ductive tberapy presently recommended should be doneat a time when there is less tumor bulk (that is, patientsshould be transplanted earlier in their course), or the
patients sbould be prepared for transplantation with moreintense cytoreductive therapy. These alternatives are cur-rently under study at UCLA.
Four patients in the current series were transplantedagainst histocompatibility barriers. The only survivingpatient of this group is bematologically improved butsbows no evidence of engraftment (Patient 1-4). In twootber patients, potential success was thwarted by earlyfungal sepsis (Patient 1-3) or recurrent acute myeloblasticleukemia (Patient III-2). In one patient, it was possibleto eradicate acute myeloblastic leukemia and acbieve per-sistent engraftment of maternal marrow (Patient 111-5);tbis patient died because of the combined effects of severeenteritis and cystitis. It was not possible to document tbepresence of graft-versus-host disease by skin or rectalbiopsy, or at autopsy, although tbe clinical features werequite typical. Tbis experience does not lead to optimismthat persistent engraftment against histocompatibility bar-riers can be accomplished by current methods. If moreeffective means to control graft-versus-host disease aredeveloped, such transplants might be feasible for selectedpatients witb severe marrow failure or terminal leukemia.
Finally, severe and prolonged impairment of cell-medi-ated immune function was shown in one long-term sur-vivor (Patient I-l). Although the patient bas tolerated in-fections moderately well, the ultimate implications of bisimmunologie incotnpetence cannot be evaluated at thistime.
Immunologie Problems and Opportunities in Bone MarrowTransplantation
Dr. John L. Fahey* : Immunology has often been en-
• Department of Microbiology and Immunology, UCLA School ofMedicine.
Cline et al. • Bone Marrow Transplantation 703
Table 8. Patients Transplanted for Refractory Myeloblastic Leukemia
Recipient
Patient Age Sex Relaiion- Sexship*
Donor Preparatory HIstocompatibiiity(Regimen)
Graft (Evidence)
Human MixedLeukocyte- Lymphocyte
Locus A Culture(HL-A) (MLO
Graft-Versus-Host
Disease(Therapy)
Survival (Status, orCause of Death)
yrs
III-l a 43 M Sibling M
Ill-Ib
Identical Nonreactive Transient(Cliromosome)
None
III-2 13 M Sihling F
Cyclophos-phamide,cytosinearabinoside,6-thio-guanine,daunomycin,vincristine
2A Identical
Transient None(Chromosome)
III-3
I [1-4
III-5
29 M Sibling F Procarbazine, Identical Nonreactivecytosinearabinoside,6-mercapto-purine, anti-thymocyteglobulin,tolal bodyirradiation
2B
Transient(Chromosome)
Not applicable
None
Not appli-cable
daysRetransplanied at 39
days (recurrentacute myelo-blastic leukemia)
76 (Recurrentacute myelo-blaslicleukemia)
39 (Recurrentacute myelo-blasticleukemia)
8 (Severeintestinaltoxicity)
26 M Sibling M Identical Nonreactive
10 M Mother F 2A (Cyclo- Identicalphosphamidecysiitis)
D
43 M Sibling M 2B Identical Nonreactive
+ (ABO bloodgroup marker,erythrocyte phos-phoglucomutaseisoenzymemarker)
-|- (Chromosome)
+ (Erythrocyte acidphosphatase iso-enzyme marker,erythrocyteglutamic pyruvatetransaminaseisoenzymemarker)
Stage III(steroids)
Stage III(Anti-thymocyteglobulin,steroids)None
> 150 (No evidenceof disease)
47 CNo evidenceof disease)(renal andpulmonaryfailure)
> 100 (No evidenceof disease)
• Relationship of donor to recipient.t D ^ R ; Donor and recipient reactive to each oiher.
riched by careful observation of the differences betweenclinical findings and inbred animal systems. The findingthat kidney transplantation could be successfully donebetween non-hiinian-leukocyte-locus-A (HL-A)-matcheddonors and recipients indicated that the immune systemcould be successfully modified by nonspecific immune sup-pressive agents, and that genetic dlíTerences per se neednot be decisive in the transplantation of certain organs inman.
ADDITIONAL HISTOCOMPATlBn.rrY SYSTEMS IN MAN
Kidneys can be successfully transplanted at times de-spite histoincanipatibility. Bone marrow transplantation,
however, can produce damaging immunologie reactions(for instance, graft-versus-host disease) even when donorand recipient are histocompatible. Indeed, the frequencyof graft-versus-host disease may be as high as 50% to 70%with matched donors and recipients. Clearly, such reac-tions derive from histocompatibility systems in additionto the known HL-A, mixed lymphocyte culture (MLC),and ABO systems. These undefined histocompatibility sys-tems await identification and assessment of their sigoifi-cance in man. Studies in mice currently indicate the pres-ence of 16 histocompatibilily systems and the probabilitythat 25 or more occur. There seems no reason to believethat man is greatly diïïerent from mouse in this respect.
704 November 1975 • Annais of Internal Medicine • Volume S3 • Number 5
Studies in animals hy Graff and associates (106) clearlyindicate that a summation of minor histocompatibilityditïerences may restilt in severe graft rejection reaction,which approximates that seen when there are differencesat a single major locus. Accordingly, definition of theminor or tissue-speeific histocompatibility systems in manmay well be important, so that appropriate remedial orpreventive measures can be taken to avert unnecessarygraft-versus-host reaction.
Successful treatment of graft-versus-host reaetion in-volving minor histocompatihility regions by antilymphucyteglobulin or other approaches that cause a general suppres-sion of the immune system are important at this stage ofdevelopment of clinical marrow transplantation. They do,however, contribute to the general immune suppression,and subnormal function of the immune system presents asevere and often fatal hazard to the successful transplantrecipient. This further emphasizes the need for speeifiemeans of averting the minor, as well as the major, histoin-compatihilities that may produce graft-versus-host reae-tions.
PRKVENTION OR CONTROL OF GRAFT-VERSUS-HOSTRFLACTIONS
Bone marrow transplantation is restricted to a smallpercentage of potential recipients because of histocom-patibility barriers than cannot be overcome. In the Amer-ican society of today, it has been calculated that the pa-tient needing marrow transplantation has about a 40%chance of finding an HL-A matched donor within thefamily (107). The actual chances of finding a suitabledonor are considerahly less (perhaps 1 in 4) when ABOtyping and such faetors as geographic location and healthof the donor are taken into account. Clearly, the preven-tion or control of graft-versus-host disease are majorchallenges to extension of bone marrow transplantation.
Two general approaches have been explored: removalof reacting cells, especially in the period when marrow hasbeen obtained from the donor and before if is transfusedinto the new host; and suppression or impairment ofundesired immunologie reactions. These two approachesand some of the means of achieving them are outlined inTable 9.
Physical methods based on differenees in density orsize have been used to separate marrow cells into thosemost effeetive in restoring marrow versus those mostlikely to cause undesirable reactions. Beneficial effects havebeen claimed by Gelfand and co-workers (68), using thisgeneral approach. Possibly, physical methods could hecombined with immunologically speeific reactions (perhapsadding additional mass or charges to cells recognizinghistocompatibility antigens) and thus facilitate ready sep-aration of undesirable eellular components from the trans-planted bone marrow.
Specificity of immunologie recognition may make itpossible to remove all of the cells capable of recognizinghost histocompatibiiity factors from the mixture of cellsin the donor bone marrow. Bonavida and Kedar (108)have removed donor lymphoid cells capable of producinggraft-versus-host disease by a selective adherence method
Table 9. Approaches to Prevention and Reduction of Graft-Versus-Host Reaction
Removal from donor bone marrow of cells reading wiili histo-compatibility factorsPhysicalImmunologie- specificPhysical binding of cells with histocompatibility receptors"BLirnout" methods
Specific stimulation plus radioisotope incorporationHistocompaiibility factors labeled with isotope or toxin
Blocking antibodyImmunologie—nonspecificAntliymphocyte globulin"Chalones"—siipprcssive tissue factors
Suppression of cells involved in graft-versus-host reactionSoluble antigen or Ag:Ah complexesStimulation of suppressor cellsAdvantageous biilance of humoral and cellular immune factors
before reconstitution of the recipient. In other animalstudies (109). there was an inability to retain anti-tumorimmunity while selectively removing graft-versus-host dis-ease producing (antihistocompatibility) cells. Extension ofthis line of work is under way. With isolation and puri-fication of histocompatibility factors, these eouid be boundas molecular entities rather than as whole cells to insoluhlematrices, so that marrow eells with histocompatibilityrecognition capacity could be removed, and the hema-topoietic ceils of the donor could be successfully clearedfor nontoxic transfer to a new host.
Removal of potentially reactive cells may be more diffi-cult than removing cells that already arc capable of rec-ognizing and reacting with histoeompatibility faetors. Pre-sumably, stem cells do not have a recognition unit forhistocompatibility factors on their surface, and the pre-cursors of graft-versus-host active eells cannot be selec-tively removed. Gross removal of all stem cells, on theother hand, would be extremely disadvantageous. It ispossible, however, that in-vitro stimulation could lead toselective receptor expression and thus allow specific cellremoval. Removal of reacting eells hy a combination ofmaneuvers ultimately may prove to be most effective.
"Burnout" methods have heen proposed by which radio-isotopes are used to kill the cells capable of reacting withspecific histocompatibility antigens. Salmon and associates(65) have induced 3H-thymidine incorporation into graft-versus-host disease-producing cells by exposing them invitro to the histocompatibility antigens of the new host.These cells were stimulated to blastogenie transformation(DNA synthesis) and suffered irreparable damage fromincorporation of highly radioactive DNA precursor (thy-midine). Several infants have been treated with bonemarrow modified in this way (66).
Purified histocompatibility factors theoretically could belabeled with isotope or toxins (such as diphtheria) andused to kill graft-versus-host disease-producing cells. His-tocompatibility antigens remain to be purified sufiiL'icntlyfor a trial of this approach. An additional difficulty is thevariety of histoeompatibility faetors (HL-A antigens andso forth) that would be needed to make this effective.
Blocking antibody offers the opportunity to interfere
C/ino et al. • Bone Marrow Transplantation 705
with graft-versus-host disease, as it can help prolong graftsurvival. Preliminary experiments with this approach intnan have been reported by Buckley and colleagues (67).Here, also, the multiplicity of histocompatibility antigens,as well as the paucity of potent antiserum, impairs thefeasibility of this approach.
Immunologie reagents, as well as drugs, can nonspe-cifically reduce immune responses. Antilymphocyte glob-ulin, for example, can effectively modify graft-versus-hostreactions. The exact antigens involved and the optimalconditions for producing most effective antiserums forclinical use are still being investigated.
Extracts of iymphoid tissue (chalones) have been ex-plored as nonspecific immunosuppressive agents (110,111). These seem to be effective in averting graft-versus-host disease in animal systems.
Soluble antigeniantibody complexes can impair cellularimmunity (112). Presumably, such agents act on specifi-cally sensitized lymphocytes. It is possible that some of theeffects attributable to blocking antibody (in protectingagainst graft rejection) may, in fact, involve antigen:antibody complexes.
Administration of soluble antigen tends to induce agreater amount of serum blocking activity and less directcell-mediated immunity than do whole cells (113). Pre-treatment of recipients vi-ith soluble histocompatibility an-tigens have enabled grafts to survive longer in some ani-mal systems (113, 114). Whether such a procedure canbe modified for clinical application to bone marrow trans-plantation remains to be determined.
Suppressor cells that tend to reduce or minimize thedimensions of immune response have been identified in anumber of antigen systems. These have been defined most-ly in terms of B-cell antibody-producing systems, butthere is reason to believe that similar suppressor cells mayact in the immune response to histocompatibility factors.Suppressor systems in man have been identified in patientswith immune deficiency syndromes (115). Apparently,these systems are acting broadly on large populations ofIymphoid cells. It remains to be seen whether these canbe used more selectively to eradicate graft-versus-hostdisease.
The immune response to complex systems, such ashistocompatibility antigens, involves multiple factors. Notonly do many humoral and cellular components participate,but amplification systems involving other cells and serumcomponents may participate. Furthermore, not ail aspectsof an immune response tend toward attack against antigen-bearing target cells. Regulatory and controlling factors inthe immune system tend to limit or diminish the immuneresponse. Clearly, with greater appreciation of the mech-anisms that reduce immune response and with bettermethods for removal of particular cell types, efforts tocontrol and prevent graft-versus-host reactions will beextended, and barriers that currently prevent extensiveuse of bone marrow transplantation will be breached.
ACKNOWLEDGMENTS: Grant support: by grants from theGwynne Hii/en Cherry Memorial Laboratory; from the Celia HagenMemorial Fund; and from the U.S. Public Health Service, grantsRK-5354 and CA 1. 68«.
Received 27 June 1975; accepted 15 July 1975.
•• Requests for reprints should be addressed to Martin J. Cline,M.D., Division of Hematology-Oncology. UCLA School of Medicine,Los Angeles. CA 90024.
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708 November 1975 • Annals of Internal Medicine • Volume 83 • Number 5
