Bone marrow transplantation in man.

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 viable therapeutic approach to a number of diseases that are usually or uniformly fatal. We review here recent experiences in bone marrow transplantation in man at UCLA and in various other institutions throughout the world. We examine marrow transplantation in immunodeficiency diseases, acute leukemia, and aplastic anemia and consider the problems of infection in the transplant recipients. The applications of tissue typing to marrow transplantation and immunologic manipulations, which may influence engraftment and graftversus-host disease, are also reported.

D R . MARTIN J . CLINE*: Attempts at bone marrow trans-

plantation in man are not new; however, the modern development of this approach to disease requires understanding of the hematopoietic stem cell, of cell-mediated immune reactions, and advances in tissue typing. At present, marrow transplantation has been applied to patients with three categories of disease: immunodeficiency, aplastic anemia, and acute leukemia. There is potential for its application to other neoplastic diseases and to serious nonmalignant blood disorders such as sickle-cell disease. Before this potential can be realized, certain problems must be solved. These problems include [1] failure of engraftment in 10% to 2 0 % of recipients; [2] graft-versus-host disease, which may be lethal in 2 5 % of patients receiving allogeneic marrow; [3] failure of eradication of malignant disease by aggressive chemotherapy or whole body irradiation; and [4] the unknown late sequels of the transplantation procedure. In addition, if marrow transplantation is ever to be applicable for the great majority of patients who do not have an immunologically compatible family member as a potential donor, a number of basic and clinical problems must be solved. Fundamentally, these involve the elimination, by physical or immunologic means, of cells in the donor marrow capable of attacking the recipient. * Division of Hematology-Oncology, Department of Medicine, UCLA School of Medicine. • An edited transcription of the Clinical Case Conference arranged by the Department of Medicine of the UCLA School of Medicine, Los Angeles, California. Annals of Internal Medicine 83:691-708, 1975

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In this conference we shall consider these problems, as well as review recent experiences in bone marrow transplantation at UCLA and throughout the world. It is worth pointing out that a successful transplantation program is an enormous clinical effort requiring a team approach. At UCLA, the team consists of clinicians from the Department of Pediatrics and Internal Medicine, clinical pathologists, geneticists, basic scientists from the Departments of Medical Microbiology and Immunology, and experts in tissue typing from the Department of Surgery. Given this large array of clinical and scientific talent in a single institution, it is possible to mount an effort as large and complex as a bone marrow transplantation program. Bone Marrow Transplantation in Aplastic Anemia and Leukemia APLASTIC ANEMIA

Dr. Robert P. Galef: Aplastic anemia is an uncommon disease with an uncertain cause in a majority of cases ( 1 ) . Aplasia related to chloramphenicol or hepatitis carries a particularly grave prognosis (2, 3 ) . The natural history of the disease has changed since the introduction of hematologic supportive methods and new antimicrobial agents. Androgens may have some therapeutic effect, but their value is still questionable (4, 5 ) . If the basic defect in aplastic anemia is in the hematopoietic stem cell rather than the microenvironment, infusion of normal bone marrow offers a reasonable therapeutic approach. The first transplant for aplastic anemia was reported by Osgood, Riddle, and Mathews (6) in 1939. A 19-year-old woman with gold-induced aplasia was given 18 ml of marrow from her ABO compatible husband. Engraftment was not observed, and the patient died 5 days later. Animal models of radiation-induced aplasia treated by marrow reconstitution were developed during the period from 1945 to 1955. As a result of these studies and the development of human histocompatibility testing, we are now concerned with several areas in allotransplantation of marrow: human leukocyte-locus A (HL-A) and mixed leukocyte culture (MLC) testing in donor selection to t Division of Hematology-Oncology, Department of Medicine, UCLA School of Medicine.

691

irradiation (1000 rads), and 28 were conditioned with cyclophosphamide (200 mg/ kg body wt) (18). Thirty-three of 35 evaluable patients were engrafted; 6 subsequently rejected their grafts, 5 died from marrow failure, 4 from infection, and 6 of graft-versus-host disease. Sixteen patients are alive with functioning grafts (10). The incidence of graft-versus-host disease calculated as a function of engrafted patients surviving 50 days is 75 %. The data from Baltimore are less encouraging, and 2 of 7 recipients are alive at 123 and 319 days posttransplant, respectively*. The National Cancer Institute (NCI) experience has been limited to three reported cases, with no long-term survivors (15, 19, t ) . The Boston group has transplanted 16 patients from histocompatible donors J. Fourteen cases are evaluable, and engraftment was observed in all. Graft-versus-host disease was observed in all patients and was fatal in several. There are 6 survivors out of 16, 3 with mild and 3 with severe graft-versus-host disease. The prognosis of the patients correlated with their clinical status at the time of transplant (20). The French Transplant Group has reported 24 transplants between 1958 and 1973 in aplastic patients conditioned with antilymphocyte globulin (21). Donors were not HL-A matched, and sufficient data are not available to permit critical evaluation. The UCLA transplant experience will be discussed separately and is presented in Table 1. A number of reports detail treatment of aplastic anemia by marrow transplantation, including four from the Netherlands (22, 23), one from London (24), three from Switzerland (25), and two from Minnesota (26). Analysis of these random data is complicated by the selective tendency to report successful cases. Statistically, less than 25% of aplastic potential recipients will have an HL-A-identical, MLC-compatible sibling donor. Alternatives have therefore been considered,

facilitate engraftment and circumvent graft-versus-host disease (7), immunosuppressive "conditioning" of the recipient for successful engraftment (8-10), and the need for posttransplant immunosuppression for control of graftversus-host disease (9-11). The principles of marrow grafting have been reviewed recently (10, 12). The first opportunity to apply these principles to man arose when Thomas and colleagues (13) transplanted 3 leukemia patients. Differences in the radiation dose and uncertainty regarding histocompatibility testing make critical interpretation of the results difficult. Bortin (14) reviewed transplantation for aplastic anemia in 1970 and reported no surviving allogeneic chimera among 73 patients. Graw and Herzig (15) in 1972 reviewed 20 transplants from HL-A compatible donors and reported 70% engraftment with several immunosuppressive regimens. The incidence of graft-versus-host disease was 15%, and survival was from 3 to 259 + days. Transplants for aplastic anemia have increased dramatically since 1970. Table 1 is a compilation of data from several centers. A considerable amount of the data is unpublished, and individual case reports have been excluded. Workers in Seattle have done three transplants in patients with identical twins (10, 16). Engraftment was successful in all, and they remain alive 6 to 12 years posttransplant. Failure of engraftment in identical twins may relate to a critical clinical status at the time of transplantation or to a microenvironmental malfunction, rather than a stem cell defect. In identical twins, pretransplant and posttransplant immunosuppression are not required. Clearly, when there is an identical twin donor, transplantation should be considered in the initial therapy of aplastic anemia. The situation is less clear in allogeneic marrow grafting, but encouraging results have been obtained (Table 1). The Seattle group has reported 37 transplants from HL-Aidentical siblings (10, 11, 17). The majority of recipients had received androgen therapy, and 35 had been multiply transfused. Nine patients were conditioned by total body

* SANTOS GW: Personal communication. t GRAW RG JR: Personal communication. JCAMITTA BM: Personal communication.

Table 1. Bone Marrow Transplantation in Aplastic Anemia. Human Leukocyte-Locus A (HL-A) Identical Siblings

Transplant Center

Seattle Baltimore UCLA Boston National Cancer Institute

Conditioning

None || Total body irradiation Cyclophosphamide Cyclophosphamide Cyclophosphamide Cyclophosphami de Antilymphocyte globulin Antilymphocyte globulin 4- total body irradiation Cyclophosphamide

Patients (Evaluable)

Grafts*

Graft-Vers usHost Disease f

KD

no. 3 7 26 3 6 3 14 1 1

100 87 96 60 100 100 100 100 100

no. 0 4 20 1 1 0 14 0 0

0 55 75 33 16 0 100 0 0

no. 0 2 8 1 0 0 2 0 0

0 50 40 100 0 0 14 0 0

KD

1

100

0

0

0

0

no. 3(3) 9(8) 28 (27) 7(5) 6(6) 3(3)ft 16(14) 1(1)

%

* Evaluable patients only. t Patients with grafts at risk. t Patients with graft-versus-host disease only. § Also, THOMAS ED, SANTOS GW, CAMITTA BM, et al: Personal communication. || Identical twins. * * Includes one patient who was not a chimera. t t Two parent-child transplants and one sibling-sibling mismatched transplant {see text). 692

Fatal GraftVersus-Host Disease X

November 1975 • Annals of Internal Medicine • Volume 83 • Number 5


%

%

Survivors * References!

no. % 3 100 1 11 15 54** 2 28** 5 83** 2 66 6 38 0 0 0 0 0

0

10, 11, 16 18 19 15

including use of HL-A-identical unrelated donors selected from Tissue Typing Banks. Donor pool size required for support of unrelated marrow transplantation has been reviewed (27). In families, HL-A identity and MLC nonreactivity are concordant due to the close linkage between the HL-A and MLC loci. In unrelated persons this is not the case: only 10% of HL-A-identical unrelated persons can be expected to be MLC nonreactive. It is unknown whether identity at the HL-A or MLC loci, or both, is required for successful transplantation (28). Three recent reports suggest that MLC identity may suffice (26, 29, 30). While the results are preliminary, it also seems possible to engraft successfully MLC-reactive, HL-A-identical related persons. The tempo and severity of graft-versus-host disease did not differ from that in MLC nonreactive transplants. Additional cases are required to evaluate this question. Storb and co-workers (31) have clearly shown the importance of histocompatibility testing in canine marrow transplantation, but differences from clinical transplantation in man complicate interpretation. The effect of prior sensitization on success of engraftment is clinically relevant in patients receiving blood and HL-A-mismatched platelets and granulocytes. Transfusion of buffy coat-poor blood, even from histocompatible donors, can lead to marrow rejection in dogs (32), which may be overcome by a conditioning regimen of procarbazine and antithymocyte globulin (33). The patient should be designated a potential transplant recipient when a suitable donor is identified. Transfusion of erythrocytes and platelets should be minimized, and family members should be assiduously avoided as donors of blood products to prevent sensitization. We use HL-A-identical unrelated donors for platelet support. A major problem is the timing of the transplant, for delay may increase both the risks and the time available for recovery. Indices indicating aggressive disease are granulocyte count < 500/mm 3 , platelet count < 20 000/mm 3 , and > 70% nonmyeloid nucleated cells in the bone marrow (2, 34). Each case needs to be individually evaluated. Presently, we favor early transplantation in patients with a poor prognosis. The value of delay to permit a trial of androgen therapy is debatable ( 1 , 4, 5 ) . In stable patients, we have waited up to 3 months. The importance of ABO compatibility in marrow transplantation is unclear, and attempts to cross this barrier ( A - » 0 ) were initially disappointing. Successful transplantation has been reported in acute leukemia (35), and we have engrafted two such patients without complication. The major problem is elimination of cytotoxic antibody (isohemagglutinin) by combined plasmapheresis, transfusion of donor-type erythrocytes, and neutralization with intravenous A substance. ABO antigens do not seem to be a target of graft-versus-host disease, and we have not observed increased incidence or severity of graft-versus-host disease in this setting. In summary, in patients with moderate to severe aplasia and an identical twin donor, it is reasonable to proceed directly to transplantation. Immunosuppression of the recipient is not required, risk to the donor is negligible, and favorable response can be anticipated. In patients with a

histocompatible sibling donor, we presently favor transplantation if the clinical prognosis is guarded. Pretransplant and posttransplant immunosuppression are required, and a 50% success rate can be anticipated. The procedures should be undertaken in transplantation centers with considerable experience and specialized support facilities. ACUTE LEUKEMIA

The strategy of marrow transplantation in hematologic malignancies involves administration of tumoricidal doses of chemoradiotherapy that would otherwise be lethal. Additionally, transplantation may serve in an immunotherapeutic role, just as tumor-specific antigens occur in human tumors, particularly acute leukemia (36). Studies in mice suggested that graft-versus-host disease can have an antileukemic effect (37). Grafting of patients after supralethal radiotherapy was first attempted by Thomas and associates in 1957 (13). The use of bone marrow transplantation in acute leukemia is complex. It requires therapy protocols capable of disease eradication that are not lethal to vital organs other than the marrow, successful engraftment of the recipient, and prevention of fatal graft-versus-host disease. Four transplant centers are currently involved in marrow grafting, with programs involving a significant number of patients with acute leukemia: Seattle, Baltimore, NCI, and UCLA. We will analyze results emphasizing protocols for pretransplant chemoradiotherapy (Table 2 ) . The Seattle group has reported the largest number of leukemic patients treated by marrow transplantation. Two groups were studied: syngeneic and allogeneic combinations (10, 38, 39). Syngeneic transplant recipients are particularly interesting: engraftment is the rule, and graftversus-host disease and posttransplant immunosuppression are not involved. If the graft-versus-host disease reaction is an important modality of leukemia eradication, then these patients are at high risk of relapse. They offer, therefore, a test of the efficacy of chemoradiotherapeutic conditioning regimens. Fefer and co-workers (38) have reported on 16 transplanted identical-twin patients. In most cases, "immunotherapy" consisting of transfusion of donor lymphocytes and immunization with frozen leukemic cells was given. Complete hematologic remission was induced in 8 8 % , and 6 patients remain in remission at 11 to 44 months. Leukemic recurrence in patients at risk, determined by subtracting those dying of other causes before Day 100, is 3 5 % (4 of 11). These results are encouraging and favor transplantation when an identical-twin donor is available. The role of immunotherapy in these patients is not evaluable, but a controlled trial is underway. The origin of the leukemic recurrence in these patients is unknown; it is not possible to determine if it represents persistence of the tumor or de novo oncogenesis in transplanted cells. Thomas and co-workers (10, 39, 40) have reviewed their experiences in allogeneic marrow transplantation. In initial studies total body irradiation was used for conditioning. Cyclophosphamide was added due to recurrent leukemia in a significant number of patients (41). Seventy patients (36 acute myeloblastic leukemia, 34 acute lymCline 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

Baltimore National Cancer Institute

UCLA

Conditioning *

Patients (Evaluable)

Cyclophosphamide + total body irradiation f f Total body irradiation, cyclophosphamide 4- total body irradiation Cyclophosphamide Cyclophosphamide Total body irradiation Cyclophosphamide -f total body irradiation BACT regimen§§ Cyclophosphamide Cyclophosphamide + total body irradiation || || SCARI regimen***

Grafts!

Graft-Versus-Host DiseaseJ

no. 16(16)

no. 16

no. 0

%

100

70 (68)

63

93

46

73

25 (20) 9(9) 2(2) 5(5)

17 8 1 3

85 88 50 60

11 1 1 3

65 12 100 100

7(5)

KD

5(5)

5 1 5

100 100 100

1 0 1

33 0 25

12(8)

8

100

4

50

%

0

* See text. t Evaluable patients only. X Patients with graft at risk and survival > 50 days. § Patients with graft-versus-host disease at risk. || Patients living > 100 days at risk. ** Also, THOMAS ED, SANTOS GW, GRAW RG JR, et al: Personal communication. ft See text. Identical twins: Seattle, 16; UCLA, 1. XX SANTOS GW: Personal communication. §§ BACT regimen: B.C.N.U. (bis-chlorethyl-nitrosurea), cytosine arabinoside, cyclophosphamide, and 6-thioguanine. Illl Two mixed lymphocyte culture (MLC)-reactive donors included. *** SCARI regimen: 6-thioguanine, cyclophosphamide, cytosine arabinoside, rubidimycin (daunorubricin), and total body irradiation. t t t Three additional patients at Days 10, 15, and 25 alive posttransplant without disease.

phoblastic leukemia) have been transplanted. Graft-versushost disease developed in 7 3 % . A steadily improving survival rate has been observed, with 70% of patients, transplanted between January and June 1974, alive 200 days posttransplant. Leukemic relapse was a problem in 30% (11 acute myeloblastic leukemia, 9 acute lymphoblastic leukemia). If one corrects the recurrence rate for patients at risk, as previously discussed, the actual recurrence rate may be as high as 50% (42). The 120-day survival statistics for 70 acute myeloblastic leukemia and acute lymphoblastic leukemia patients are 20% and 2 5 % , respectively, and the median time to relapse is 45 and 75 days, respectively. This clearly represents a significant achievement in patients refractory to conventional and experimental chemotherapy who are frequently in extremely poor clinical condition. The results of the Baltimore experience are summarized in Table 2, and use of cyclophosphamide in marrow transplantation has been reviewed (9, 18, 43,*). Leukemic recurrence was one of the major problems in this series and is 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, including total body irradiation, cyclophosphamide and total body irradiation, and BACTf (19). These data, comparable to those from Baltimore, underscore the high incidence of leukemic recurrence after cyclophosphamide conditioning. Five evaluable patients receiving BACT showed evidence of engraftment, and two identical twins had recurrent leukemia at 50 and 90 days posttransplant. The

incidence of graft-versus-host disease seemed lower with BACT than with the other regimens, but additional data are required to evaluate this (19, 45). The rate of leukemic recurrence remains unacceptably high. Based on these experiences, UCLA has developed a cytoreductive protocol combining total body irradiation, cyclophosphamide, and combination chemotherapy. The SCARIJ regimen consists of a 5-day course of cytosine arabinoside, 15 mg/kg body wt every 24 h, as a continuous intravenous infusion, and 6-thioguanine, 7.5 mg/kg body wt every 12 h by mouth. This is followed by daunorubricin, 60 mg/m 2 body surface, as a rapid intravenous infusion daily for 3 days. The patient is rested for 3 to 5 days to allow regeneration of the gastrointestinal tract mucosa. This is followed by the cyclophosphamide and total body irradiation regimen and marrow transplantation. The SCARI regimen has been well tolerated in 12 patients, and 8 evaluable patients have no evidence of disease at 75, 80, 80, 96, 100, 150, 180, and 200 days posttransplant. Two are in the immediate posttransplant period without evidence of disease. Additional time and patients are required to evaluate this regimen. Mathe and associates have recently reviewed the marrow transplantation experience in France (12, 21). Results are disappointing, but conditioning used and histocompatibility matching were not optimal. Published and unpublished results from Minnesota, Boston, and San Francisco are discouraging (23, 46-48). The role of graft-versus-host disease in leukemia eradication has been difficult to evaluate and is complicated by several factors: patients with persistent or recurrent dis-

* SANTOS: GW: Personal communication. t BACT: B — B.C.N.U. (bis-chlorethyl-nitrosurea); A = cytosine arabinoside; C rz cyclophosphamide; T = 6-thioguanine.

X SCARI: S — 6-thioguanine; C = cyclophosphamide; A — cytosine arabinoside; R =z rubidimycin (daunorubricin); and I — total body irradiation.

594



Table 2. (Continued) Fatal Graft-Versus-Host Disease§

Disease-Free Survivors *

Recurrence || At Risk

Cases

Survival Range

36

no. 6

38

months 2-44

20

50

19

28

3-49

13 5 0 3

9 5 0 0

70 100 0 0

1 0 0 0

4 0 0 0

7

0 0 0

4 1 3

2 1 2

50 100 66

1 0 1

14 0 20

36

0

8

1

12

11

90

no. 0

% 0

no. 11

no. 4

%

23

50

41

1 1 1 0

10 100 100 0

0 0 0 0

%

References**

10, 13, 36-38

18 15, 32, «

4

2-7ftt

* See text. t Evaluable patients only. t Patients with graft at risk and survival > 50 days. § Patients with graft-versus-host disease at risk. || Patients living > 100 days at risk. ** Also, THOMAS ED, SANTOS GW, GRAW RG JR, et al: Personal communication. ft See text. Identical twins: Seattle, 16; UCLA, 1. t t SANTOS GW: Personal communication. §§ BACT regimen: B.C.N.U. (bis-chlorethyl-nitrosurea), cytosine arabinoside, cyclophosphamide, and 6-thioguanine. Illl Two mixed lymphocyte culture (MLC)-reactive doners included. •** SCARI regimen: 6-thioguanine, cyclophosphamide, cytosine arabinoside, rubidimycin (daunorubricin), 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-versushost disease; and patients with severe graft-versus-host disease not uncommonly die before 100 days, which falsely lowers the risk of recurrent leukemia. An accurate definition of the role of graft-versus-host disease awaits further clinical trials and careful comparison with patients not at risk to develop graft-versus-host disease, that is, recipients of syngeneic transplants (49). Marrow transplantation is being used increasingly in patients with acute leukemia. One should consider marrow transplantation relatively early in patients with an identical twin donor. All new patients with leukemia and their immediate families should be HL-A typed. When an HL-Aidentical, MLC-nonreactive donor is identified, the patient should be designated a potential transplant recipient, and transfusion therapy should be carefully monitored. Because of the clear correlation between the patient's clinical status at the time of transplantation and his subsequent survival, one may consider transplantation at a relatively early point in refractory disease. In acute myeloblastic leukemia we currently feel that poor-risk patients should be transplanted either during their first relapse or possibly during remission. In acute lymphoblastic leukemia, where chemotherapy is more effective, one should use all conventional modalities before considering transplantation. We considered patients in their third remission or relapse, those with refractory central nervous system disease, or those with progressively decreasing remission durations as suitable candidates. Inability to induce remission is an obvious indication to consider transplantation. It is important that the physician not postpone transplantation until the pa-

tient is in critical condition. When a suitable related donor is unavailable, a search of the tissue typing bank is appropriate. Marrow transplantation should be undertaken in centers where optimal support facilities are available and where controlled clinical trials can be done. Bone Marrow Transplantation in Immunodeficiency Disorders

Dr. E. Richard Stiehm*: With the exception of the aplastic patient with an identical twin, marrow transplants are most successful in patients with certain primary immunodeficiencies. Transplantation theoretically could be of benefit in all five types of human immunodeficiencies (antibody immunodeficiency, combined antibody and cellular immunodeficiency, cellular immunodeficiency, phagocytic immunodeficiency, and complement immunodeficiency) (50), but with rare exceptions transplants are used only in cellular and combined immunodeficiencies. A few attempts to transplant antibody immunodeficiency and phagocytic immunodeficiency have been made, but the results are either negative or unavailable (14, 51). Patients with immunodeficiency are particularly good candidates for transplantation when [1] their cellular immune defect is complete, so that engraftment is readily achieved; [2] a human leukocyte-locus A (HL-A) and mixed leukocyte culture (MLC)-matched sibling donor is available; and [3] the patient is not terminally ill. These criteria are usually achieved in patients with severe combined immunodeficiency, a heterogeneous but relatively common primary immunodeficiency characterized by the following: clinically, by the early onset of diarrhea, pneu* Division of Pediatric Immunology, Department of Pediatrics, UCLA School of Medicine. Cllne et al. • Bone Marrow Transplantation


695

monia, failure to thrive, and rapid downhill course; immunologically, by profound defects in B- and T-cell function; and, pathologically, by a dysplastic thymus gland lacking Hassall's corpuscles. In most other immunodeficiencies there is sufficient cellular immunity present to reject the transplant unless pretransplant immunosuppression is undertaken. Because of the grave risk from infection of suppressing an already immunodeficient and probably chronically infected patient, few attempts and even fewer successes are recorded. However, one of the early successful transplants was done with immunosuppression in a boy with the Wiskott-Aldrich syndrome (52). The first successful marrow transplant in severe combined immunodeficiency was done in 1968 at the University of Minnesota (53). This patient continues to do well (54). To date, 52 patients have received transplants; 18 have functioning grafts, all but a few from HL-A- and MLC-identical siblings (Table 3 ) . The non-successes include failure of engraftment or death, usually from infection. Fatal graft-versus-host reactions are uncommon when HL-A- and MLC-compatible donors are used. We have done two transplantations at UCLA for combined immunodeficiency. Both patients are doing well, but only one has solid immunologic evidence of engraftment. The patient without proved engraftment is a 34-month-old boy with persistent pneumonia who had partial antibody and cellular immunodeficiency (Table 4). After transplantation with 109 marrow cells (given intraperitoneal^) from an HL-A-, MLC-identical brother, there was no evidence of graft-versushost disease or erythrocyte engraftment, but the in-vitro lymphocyte proliferative response to phytohemagglutinin returned to normal, the IgG levels increased so that gammaglobulin injections could be stopped, and the patient improved significantly. The bone marrow transplant may have resulted in limited reconstitution or provided some transient humoral boost to the patient's own immune system; alternatively, it may have contributed nothing to the patient's spontaneous improvement. The other patient transplanted was a 5-month-old boy who presented with Pneumocystis carinii pneumonia and thrush (55). No tonsillar tissue, adenopathy, or thymus gland on chest X ray was present. Lymphocyte numbers in the peripheral blood were normal, but they were nonreactive to phytohemagglutinin or allogeneic lymphocytes in MLC. Immunoglobulins were extremely low, and delayed hypersensitivity skin tests were negative. HL-A typing disclosed multiple extraneous HL-A antigens that were inconsistent from day to day and delayed donor selection (56). From age 5 to 11 months, despite antibiotics, pentamidine, gammaglobulin, plasma, antifungal agents, and transfer factor, he became progressively worse, developing candidal esophagitis and severe failure to thrive. Finally, a potential donor, an MLC-matched sister, was identified, and on 3 February 1971, he received 2 X 109 bone marrow cells intraperitoneally. Two weeks after transplant, a positive monilia skin test deTable 3. Bone Marrow Transplantation in Human Immunodeficiencies Reported to the NIH/ACS* Organ Transplant Registry as of January 1975f Patients transplanted Transplants done Patients alive Patients alive with surviving grafts

52 95 22 18

* NIH = National Institutes of Health; ACS = American College of Surgeons. t Courtesy of Mortimer M. Bortin, M.D. 696

veloped, and oral moniliasis disappeared. Four weeks after transplant, immunoglobulins increased; 6 weeks posttransplant, peripheral lymphocytes were reactive to phytohemagglutinin, showed an XX (donor) karyotype, and had the HL-A type of the donor. Bone marrow cells have remained as XY karyotype. From 18 to 28 days posttransplant, the patient developed a severe graft-versus-host disease reaction with skin rash, heart failure, pneumonitis, conjunctivitis, fever, hepatomegaly, and desquamation. He recovered spontaneously, however, and for the last 3 years has been free of infections and has been growing normally. He has persistently eczematoid skin and clear rhinorrhea, but no allergens have been identified. Peripheral blood studies show eosinophilia, normal phytohemagglutinin response, and both donor HL-A types and patient extraneous HL-A types. The multiple extraneous antigens present before and after transplant that delayed donor selection, and the lack of bone marrow chimerism are of particular interest in this case. The successful transplant in these patients has been duplicated many times in patients with severe combined immunodeficiency. It is now known that severe combined immunodeficiency disease represents a heterogeneous group of disorders. Some of these patients have X-linked inheritance, others have autosomal recessive inheritance, and some have no family histories. The typical patient presents early in infancy with failure to thrive, diarrhea, moniliasis, and pneumonia, but others may present with a skin rash, adenopathy, and hepatosplenomegaly resembling the Letterer-Siwe syndrome (57). Some patients lack an enzyme of purine metabolism, adenosine deaminase, in their tissues (58). Some have peripheral blood B and T cells, while others lack one or both of these cell lines. Despite this variability, all variants seemingly are equally responsive to transplantation. Thus, marrow transplants in severe combined immunodeficiency are so well established that a physician caring for such a child would be seriously remiss if he or she did not explore the possibility of transplantation. New approaches are being sought in the treatment of severe combined immunodeficiency when an HL-A, MLCidentical sibling is unavailable. Perhaps the most complex approach is delivery and maintenance of an affected infant in a germ-free isolator. South and colleagues (59) report one such case, an infant who had a previously affected sibling, so the diagnosis was suspected before birth. The child has been in isolation for 4 years and seemingly is developing normally. Further, B cells are spontaneously appearing, suggesting possible slow acquisition of immunity. Transfer factor has been used in several cases of severe combined immunodeficiency, generally without benefit or with only transient benefit (55, 60). It is likely that most severe, combined immunodeficiency patients have such a profound T-cell defect that transfer factor does not have an effective T-lymphocyte target cell for its action. Although fetal thymus transplants have been effective in restoring immunity in thymic aplasia (DiGeorge's syndrome) (61), their use in severe combined immunodeficiency has, with rare exceptions, not been successful. In thymic aplasia, thymus transplants provide a humoral factor that acts on the patient's own lymphocytes to convert them to functioning T cells. Injections of thymosin, an extract of thymus tissue, has been used to restore cell-



Table 4. Bone Marrow Transplantation at UCLA for Combined Immunodeficiency Patient B

Patient A Age at admission Age at transplant Date of transplant Donor Pretransplant Clinical findings Laboratory studies

Posttransplant Clinical results Laboratory results

34 months 38 months 16 December 1971 Human leukocyte-locus A (HL-A)- and mixed lymphocyte culture (MLC)-identical; Male sibling

5 months 11 months 3 February 1971 MLC-identical; female sibling

Ulcerated vaccination; persistent pneumonia; persistent rhinorrhea IgG, 360; IgM, 60; IgA, 0*; lymphocytes, 1800/mm3; phytohemagglutinin response, X 10 f

Thrush, esophagitis; Pneumocystis carinii pneumonia; failure to thrive IgG, 100; IgM, 50; IgA, 20*; lymphocytes, 3500/mm3; phytohemagglutinin response, X Of; extraneous HL-A antigens

Pneumonia and rhinorrhea cleared; gammaglobulin injections stopped; normal growth began IgG, 880; IgM, 80; IgA, 0*; phytohemagglutinin response, X 80; lymphocytes, 3000/mm3

Thrush and esophagitis cleared; no further infections; normal growth began IgG, 800; IgM, 80; IgA, 70*; phytohemagglutinin response, X 70; XX cells in blood

* Values of immunoglobulins in mg/dl. t Ratio of 3H-thymidine incorporatiort into DNA with and without phytohemagglutinin.

ular immunity temporarily in cellular immunodeficiency disorders (62). The combined use of transfer factor and fetal thymus transplantation has been used successfully in three severe, combined immunodeficiency patients (63, 64). In these patients thymus cells are engrafted, and this results in restoration of T-cell function without B-cell function. Nonfatal graft-versus-host disease reactions have been observed. The transfer factor may allow maturation of thymic tissue, which can then become engrafted. Others have attempted to modify the fatal graft-versushost disease reaction when unmatched marrow is engrafted. Some use a cell suicide method in which the donor's marrow and the patient's leukocytes are cultured together in vitro in the presence of large amounts of 3H-thymidine (65, 66). The donor's immunocompetent cells, which proliferate in vitro in response to the patient's histocompatibility antigens (and which presumably cause the in-vivo graftversus-host reaction), incorporate a lethal dose of thymidine into DNA and self-destruct. The marrow cells not destroyed are transplanted, with the expectation that they can reconstitute the patient without causing a fatal graftversus-host reaction. To date, no successes in humans have been recorded. Another approach is to give enhancing antiserum simultaneously with the unmatched marrow (67, 68). This antiserum is directed against the patient's histocompatibility antigens and inhibits the mixed leukocyte reaction of the donor's lymphocytes toward the patient's cells. It is in the form of plasma and must be given repeatedly. Such a procedure has delayed but not permanently abolished graft-versus-host disease. Recently, a severe combined immunodeficient infant was successfully transplanted with marrow from his HL-Anonidentical uncle, who was MLC identical (69). Other recent attempts have used unrelated donors who are MLC nonreactive with the patient's leukocytes*. These experiences emphasize the crucial role of identity at the MLC * HONG R: Personal communication.

locus rather than at the HL-A locus in graft rejection. Fetal liver transplants have been given to several children with severe combined immunodeficiency t- Fetal liver (8 to 12 weeks of gestation) is an excellent source of stem cells, unencumbered with histocompatibility antigens. Engraftment has been achieved in three patients, but one of these succumbed with immune-complex disease of the kidney J. In summary, severe, combined immunodeficiency was identified as a distinct pathologic and clinical entity in 1957. As shown by the cases presented, a "cure" for some of these patients was found a decade later. A cure for all of these cases is on the horizon, perhaps within a few years. Application of Tissue Typing in Bone Marrow Transplantation

Dr. Gerhard Opelz§: The modern history of bone marrow transplantation is closely associated with developments in the field of histocompatibility testing. With very few exceptions, all successful human bone marrow grafts have been done between siblings who were identical for the two main known histocompatibility antigen systems: the HL-A (human leukocyte-locus A) and MLC (mixed lymphocyte culture) systems. HL-A antigens are serologically defined antigens that are typed for by reacting monospecific antiserums against lymphocytes in the complement-dependent microcytotoxicity test (70). The products of the MLC locus are not serologically detectable and are tested for in the lymphocyte-defined MLC test (71, 72). The HL-A antigen system has been extensively studied for over 10 years; today, 27 antigenic specificities belonging to two series of mutually exclusive allelic antigens can be typed for (73, 74). The antigens responsible for MLC reactivity are less well defined. Only recently, with the use t BUCKLEY RH: Personal communication. % COOPER MD: Personal communication. § Department of Surgery, UCLA School of Medicine. Cline et a/. • Bone Marrow Transplantation


697

of homozygous lymphocytes, has it become possible to type for lymphocyte-defined specificities (75). A collaborative study of 15 international laboratories is currently being carried out to characterize more clearly the nature of lymphocyte-defined antigens. Both serologically defined and lymphocyte-defined antigens are genetically controlled by loci on the same chromosome. Since the tissue type of a person is determined by two serologically defined genes and one lymphocyte-defined gene on each of two chromosomes, the total number of serologically defined antigens in any person is four and that of lymphocyte-defined antigens is two. The principle of inheritance of HL-A and MLC determinants is shown in Figure 1. Because each child receives one chromosome from the mother and one from the father, the chance of two siblings having the same tissue type is 25%. HL-A-identical siblings are, in most instances, also MLC identical. Only in the rare case of a genetic crossover (the frequency in man is an estimated 1%) will HL-Aidentical siblings be MLC incompatible. Kidney transplants between HL-A-identical siblings have been shown to constitute a special category, comparable to those between monozygotic twins. The most recent analyses of 215 transplants from the United States and Canada and 79 transplants from Europe showed 1-year success rates of 90% and 8 8 % , respectively (76, 77). The success rate in bone marrow transplantation is substantially lower; only since tissue typing was introduced for selection of histocompatible sibling donors have success rates improved to the current level. Undoubtedly, the frequent occurrence of graft-versus-host disease and the difficulties in providing immunosuppressive treatment without destroying the graft are the main complicating factors in marrow transplantation as compared with renal transplants. Of interest is the relative importance of HL-A and MLC in bone marrow transplantation. This is critical for the selection of the most suitable sibling donor in a case of genetic crossover, and also when a parent is HL-A identical but MLC incompatible with a child, or vice versa (78). It also is important for the future potential of using unrelated donors for bone marrow transplantation. Data from experiments in dogs suggest that lymphocyte-de-

Figure 1. Inheritance of histocompatibility chromosomes results in four different possible tissue types in children. The numbers represent the human leukocyte-locus A (HL-A, serologically defined) specificities of the two segregant series of antigens, while the letters represent hypothetical mixed lymphocyte culture (MLC, lymphocyte-defined) specificities. 698

fined antigens are slightly more important than serologically defined antigens; however, matching for both antigen systems is of benefit and their effect is additive*. In man, successful transplants have been reported from MLC identical but HL-A-different donors (79, 80). In our experience, temporary engraftment was achieved in a patient with aplastic anemia who was transplanted from his MLCidentical, HL-A-incompatible mother (78). Although the graft was subsequently lost, the patient is alive and clinically well. In three transplants from HL-A-compatible, MLC-incompatible related donors, temporary engraftment was achieved in each instance, but all three patients died of either recurrent leukemia or graft-versus-host disease. Although the limited experience in a few patients does not positively establish that MLC compatibility is necessary for successful bone marrow transplantation, the absence of a documented successful transplant across the MLC barrier should be reason for extreme caution. With the improving clinical success of bone marrow transplantation, the possibility of using unrelated marrow donors for patients with no histocompatible family members is becoming an issue of increasing importance. Large numbers of volunteer blood donors have already been HL-A typed, mainly for the purpose of providing histocompatible platelets and granulocytes for supportive therapy (27, 81). Since it is estimated that about 10% of unrelated persons who have the same HL-A type are also MLC identical, selection of unrelated donors for bone marrow transplantation does not seem impossible. Aside from ethical considerations, the practicality of this approach remains to be tested, however. The still moderate success rate between histocompatible siblings indicates that other transplantation loci besides HL-A and MLC do exist. These undefined histocompatibility loci are more likely to match in siblings than in unrelated persons. It is therefore essential that the search for new antigen systems that could be of importance in bone marrow transplantation be intensified. For practical purposes, it will also be important to determine the individual strength of each antigen system. Infectious Complications of Marrow Transplantation

Dr. Lowell S. Youngf: Infection is the most common fatal complication of acute leukemia, aplastic anemia, and congenital immunodeficiency states. In addition to preexisting defects in host defense, the therapy given before transplantation results in extreme vulnerability to infection. Thus, it is not surprising that the infectious complications of marrow transplantation are similar to, but perhaps more severe than, those of acute leukemia or congential immunodeficiencies (82-85). Table 5 summarizes the common pathogens that we and others have encountered, their reservoir, and their likely mode of transmission. Opportunistic pathogens fall into two broad categories: endogenous pathogens from the host's normal microbial flora that become invasive after impairment of host resistance, and exogenous infecting organisms * VRIESENDORP HM: Personal communication. t Division of Infectious Diseases, Department of Medicine, School of Medicine.



UCLA

Table 5. Infections Complicating Bone Marrow Transplantation: Epidemiologic Patterns Origin

Examples of Pathogens

Endogenous infection reservoir Gastrointestinal tract Upper

Staphylococci, Gram-negative bacilli Lower Gram-negative bacilli Skin Staphylococci, Candida species Female genital tract Anaerobic organisms Cells (nerve, macrophages, and Herpes viruses, Toxoplasma so forth) Exogenous infection: mode of transmission Contact Direct Staphylococci Aerosol droplets Respiratory viruses Airborne True droplet nuclei Varicella Dust ? Aspergillus Oral (ingestion) Pseudomonas

acquired from the environment. Aspergillus pneumonia is probably an exogenous infection, with transmission occurring by the airborne route, whereas Toxoplasma and varicella are acquired exogenously, remain latent in host tissue for long periods, and cause recrudescent infection after immunosuppression. With several pathogens, the epidemiologic pattern is either unclear or both mechanisms may be involved. For instance, Pseudomonas aeruginosa is not part of the normal fecal flora of healthy humans (86), but the prevalence of stool colonization by Pseudomonas rises strikingly in patients with underlying disease (87), on special diets (88), or in those who receive broadspectrum antimicrobials (89, 90). Gastrointestinal colonization precedes pseudomonas bacteremia in many debilitated patients (83, 87, 91). There are, other microbes, such as the presumed parasite Pneumocystis carinii, whose epidemiology and mechanism of disease production are poorly understood. Experimental examples and the bulk of epidemiologic evidence support the view that the endogenous route is the most common pathway for opportunistic infection, but there are also many clearcut examples of horizontal transmission of disease within the hospital. After the initial course of cytotoxic therapy, total granulocyte counts of less than 100/mm 3 are usually observed. During this phase, staphylococci and antibiotic-sensitive Gram-negative bacilli predominate as causes of systemic infection. We have observed a direct correlation between neutropenia and low or negligible titers of heat-stable opsonizing of antibodies against autologous infecting strains (92). Thus, the defects in host defense can be both cellular and humoral and may not be immediately corrected by successful marrow engraftment. Infections caused by more antibiotic-resistant Gram-negative bacilli such as Pseudomonas are usually encountered after an initial course of systemic antibiotics. The deep mycoses and P. carinii pneumonia are most commonly "late" infections, developing after engraftment has been unsuccessful or has been complicated by graft-versus-host disease.

A common experience is that infection can develop with dramatic suddenness during periods of marked leukopenia. The clinical course is well illustrated in Figure 2, which shows the pulmonary changes associated with bacteremic pseudomonas pneumonia during an interval of only 36 hours. If patients have preexisting infection, successful marrow engraftment may be curative, whereas unsuccessful engraftment is associated with persistent and often lethal infection. Figure 3 {top) shows a patient with a dense left upper lobe consolidation who was found to have an Aspergillus species in his sputum before transplantation. This lesion persisted for several weeks during an abortive transplantation attempt, and it was present at necropsy despite treatment with granulocyte transfusions and amphotericin B. Terminal spread of infection to all lobes of the lung resulted in a diffuse aspergillus pneumonitis (Figure 3, bottom). We have found that mixed or dual infections are common, particularly the association of Gram-negative bacillary infection with fungal penumonia. This emphasizes that documentation of one infectious process hardly excludes another. As a corollary, fever and signs of infection that develop after one treatment course should not be attributed to a recrudescence of an antecedent infection,

Figure 2. Top. A patient with posthepatic aplastic anemia developed high fever and right-sided chest pain but had a normal chest X ray. Bottom. Thirty-six hours later, shock and extensive consolidation in both lung fields developed, which at autopsy proved to be pseudomonas pneumonia with vasculitis. C//ne et a/. • Bone Marrow Transplantation


699

Figure 3. Top. A 12-year-old boy with aplastic anemia had a dense left upper lobe infiltrate and Aspergillus fumigatus in his sputum. Bottom. Despite amphotericin B and granulocyte transfusions, this lesion persisted during an unsuccessful transplant attempt and the patient died with a diffuse aspergillus pneumonia.

and the patient must be thoroughly reevaluated. Furthermore, there is still significant infection risk after marrow engraftment. Serial laboratory studies in several patients successfully transplanted continue to show impaired cellmediated immunity for long periods, and we have observed interstitial pneumonia due to Pneumocystis organisms and pneumococcal bacteremia months after successful transplantation. Our approach to the management of these patients is derived from experience with hematologic malignancies and is based on the principle of prevention, aggressive diagnostic measures, and early presumptive treatment (82, 83). Many of the methods are experimental, require complex equipment, and have not been critically evaluated in large-scale clinical trials. They are, however, based on an increasing understanding of the epidemiology of nosocomial infection and reflect our ability to compensate for host factors that are known to be impaired. Patients are managed in protective isolation and receive oral nonabsorbable antimicrobials that inhibit most aerobic bacteria and Candida species. Because of high rates of contamination of fresh vegetables with certain bacteria such as Pseudomonas, patients receive only cooked ma700

terials ( 9 3 ) . The skin is bathed frequently, and "surveillance" cultures of the nares, oropharynx, axilla, and stool are taken twice weekly. These often give clues to the nature of an incipient septic process. It is unclear at this point whether the beneficial results of laminar flow units in reducing infection rates are due to the isolation alone, laminar flow per se, or the oral antimicrobials (94, 95). Oral antibiotic regimens by themselves may be useful in reducing infection (95). The rapidity with which systemic bacterial infections can develop has led us to endorse the principle of "presumptive antimicrobial therapy" for fever associated with clinical signs of infection. Broad spectrum antimicrobials, particularly the combination of an aminoglycoside and carbenicillin, are preferred because of the high mortality rate associated with proteus, pseudomonas, and serratia infections. This formula may be modified if surveillance cultures show Klebsiella or staphylococcal organisms, in which case we substitute a cephalosporin for carbenicillin. A therapeutic dilemma is the patient who is persistently leukopenic and febrile without objective evidence of infection. Our experience has been that the abrupt withdrawal of antimicrobials is often associated with clinical deterioration and the eventual documentation of a septic process. It may be that all markedly leukopenic patients have localized infection due to usually saprophytic or stool colonizing organisms whose invasiveness is limited by systemic antimicrobials. If fever or documented infection persist despite antimicrobials, we give daily granulocyte transfusions; experimental evidence of their efficacy is persuasive (96-98). The most challenging infections are those caused by fungi and the cytomegaloviruses; safe, effective therapy is not currently available for these organisms. The development of new pulmonary infiltrates and sustained fever after a response to antibiotics for Gram-negative bacterial infection often heralds fungal superinfection (99). Amphotericin B, the most consistently effective agent for deep mycoses, is fungistatic rather than fungicidal, and the chance for successful therapy without marrow engraftment is remote. Pneumonia caused by Pneumocystis has been treated successfully with several agents, including pentamidine isethionate, but co-trimoxazole now seems to be an effective and safe oral agent. We have little doubt that the quality of supportive care and frequent clinical observations are vital elements in the successful management of these patients. Such factors, as well as scrupulous measures to minimize exogenous infection, may be far more important than antimicrobial prophylaxis or the use of laminar air flow. Factors affecting successful outcome include the presence of preexisting infection (negative), the quality of the match, and the development of graft-versus-host disease. There is evidence that the use of antithymocyte globulin is associated with an interstitial pneumonia caused by the cytomegalovirus, but the association of this interstitial pneumonitis with antilymphocyte globulin is so strong that it raises the possibility of a hypersensitivity reaction (100). Thus, a poor match increases the likelihood of graft rejection or graft-versus-host disease and the latter necessitates the

November 1975 • Annals of Internal Medicine • Volume 83 • Number


5

use of measures, such as antilymphocyte globulin administration, which in turn increase infection risk. It is not unusual to observe defervescence and clinical improvement as a harbinger of successful engraftment. While much attention has been focused on the absolute peripheral granulocyte count as the major index of the likelihood of infection (101), comparably depressed peripheral neutrophil counts in a patient with a regenerating marrow and a patient without engraftment have entirely different prognostic implications. The important perspective is that all the measures involved to prevent and treat infection are, in effect, a form of temporizing, intended to protect the host before an adequate mass of donor marrow becomes functional. Ultimately, successful treatment of supervening infection coincides with successful engraftment of transplanted marrow. The Bone Marrow Transplant Experience at UCLA

Dr. Stephen A. Feig*: A program of bone marrow transplantation has been in operation at UCLA since 1973. The purposes of the program are twofold: the application of a new therapeutic modality to the management of malignant disorders with poor prognosis; and research directed toward improving the efficacy, broadening the applicability, and understanding the long-term effects of marrow transplantation. To date, 30 transplants have been done at UCLA for severe aplastic anemia and acute leukemia. Sixteen patients are presented in this report. Fourteen additional patients have been transplanted so recently that the results cannot be evaluated yet. Preliminary data were presented in Tables 1 and 2. While the numbers are still small, several tentative conclusions are suggested by this experience. Patients were prepared for transplantation with one of several regimens outlined in Figure 4. During the transplant period, all patients were kept in strict isolation with access limited to immediate family, the responsible physicians, and special nurses. The patients were bathed daily in betadine and received gastrointestinal antibiotic prophylaxis. Under general anesthesia, marrow was obtained from the donor by multiple aspirations and processed by the method of Thomas and Storb (102). Posttransplant, as a prophylaxis against graft-versus-host disease, the patient

received methotrexate (103). All posttransplant blood products were irradiated with 1500 rads to inhibit engraftment with transfused lymphocytes (103). Graft-versus-host disease was graded by the staging criteria of Storb and colleagues (104) and treated according to their recommendations. APLASTIC ANEMIA

Six patients have been transplanted for severe aplastic anemia (Table 6 ) . Two patients (1-2 and 1-3) died of sepsis in the immediate posttransplant period, despite intensive antibiotic therapy and granulocyte transfusions. Neither had evidence of engraftment at postmortem examination. Both patients had long-standing aplasia, had been multiply transfused with erythrocytes and platelets, and were severely ill and malnourished. There may not have been sufficient time for engraftment before their deaths in the face of these adverse factors. Patient 1-1 is the only long-term chimera in this series. His course was complicated by a cytomegalovirus and pneumococcal infection at 3 and 8 months after transplantation. In each instance, recovery was normal. The patient continues to have significant impairment of cell-mediated immune function 18 months after transplantation. He has reduced tritiated thymidine (3HTdR) incorporation by his lymphocytes after stimulation with phytohemagglutinin, quantitatively decreased circulating T cells, and impaired mixed lymphocyte reactivity against unrelated lymphocytes. Humoral immune function, assessed by quantitation of circulating B cells and by quantitative immunoglobulins, has been normal since 3 months after transplantation. Patient 1-4 developed severe aplastic anemia after repeated treatment with chloramphenicol. Marrow biopsy showed a reticulin myelofibrosis. He underwent marrow transplantation from his HL-A-mismatched, MLC-unreactive mother. No evidence of engraftment was ever observed, yet this patient is alive with partial hematologic recovery of his own hematopoietic tissues at 250 days. He continues to require erythrocyte transfusions, but he maintains a platelet count of 50 000 and a granulocyte count of 1000/mm3. Patients 1-5 and 1-6 are stil] too early in their courses to be fully analyzed. Patient 1-5, with aplastic anemia after hepatitis, has clear evidence of engraftment and shows early hematologic improvement. Patient 1-6 had a moderately cellular bone marrow at 14 days posttransplant, with only donor male karyotypes. Her peripheral blood counts rose then fell again with the reappearance of aplasia at 28 days. She has just received a second transplant from another sibling. ACUTE LYMPHOBLASTIC LEUKEMIA

* Division of Hematology-Oncology, Department of Pediatrics, UCLA School of Medicine.

Four patients have undergone bone marrow transplan-

Figure 4. Regimens for preparation of bone marrow transplantation. AG: 1 unit of donor whole blood or leukocyte concentrate; CYCLO (A): cyclophosphamide, 45 mg/kg body wt • day, intravenously; CYCLO (B): cyclophosphamide, 60 mg/kg body wt • day, intravenously; TBI: 1000 rads total body irradiation at ~ 8 rads/min; ARA-C: cytosine arabinoside, 10 to 15 mg/kg body wt • day, continuous infusion; 6-TG: 6thioguanine, 5 to 7.5 mg/kg body wt by mouth every 12 h; DM: daunomycin, 60 mg/m 2 • day, intravenously for 1 to 3 days; BM: ~ 3 X 108 filtered nucleated marrow cells/kg intravenously. Cline et al. • Bone Marrow Transplantation


701

Table 6. Patients Transplanted for Severe Aplastic Anemia Recipient

Donor

Patient Age Sex Relation- Sex ship*

Preparatory (Regimen)

Histocompatibility Human LeukocyteLocus A (HL-A)

Mixed Lymphocyte Culture (MLC)

1-1

yrs 12

M

Sibling

F

1

Identical

Nonreactive

1-2

26

M

Sibling

M

1

Identical

Nonreactive

1-3

12

M

Mother

F

1

Nonidentical

1-4

27

M

Mother

F

1

Nonidentical Nonreactive

1-5

18

M

Sibling

F

1-6

20

F

Sibling

M

Sibling

F

1 (Cyclophos- Identical phamide cystitis) 1 Identical 1

Identical

Graft (Evidence)

R^Df

Nonreactive

GraftversusHost Disease (Therapy)

+ (Chromosomes, None ABO blood group marker) None (Autopsy) None (Autopsy) None (Chromo... somes, ABO blood group marker, erythrocyte phosphoglucomutase isoenzyme marker, erythrocyte adenosine deaminase isoenzyme marker) + (Chromosomes) Stage II (None)

Nonreactive Transient (Chromosomes) Nonreactive None

Survival (Status, or Cause of Death)

days > 500 (Complete hematologic recovery) 19 (Pseudomonas sepsis) 21 (Aspergillus sepsis) > 250 (Hematologic improvement)

> 80 (Hematologic improvement)

None

> 32

None

> 35 (Aplastic)

* Relationship of donor to recipient. t R ^ r D : Recipient reactive to donor; donor nonreactive to recipient.

tation for drug-resistant acute lymphoblastic (Table 7 ) .

leukemia

The first patient (II-l) had moderate graft-versus-host disease after engraftment and was treated with antithymocyte globulin. Three months after transplantation, the patient developed an acute interstitial pneumonitis and succumbed after a rapidly progressive 48-hour illness. Pneumocystis organisms, but no leukemia, were found at postmortem examination. The transplant in Patient II-2 was unusual in that her blood group was O, while the donor's blood group was A. The day before transplant, the recipient underwent an 18-litre plasma exchange with fresh-frozen type A plasma, using the Aminco cell separator (American Instrument Co., Division of Travenol Laboratory, Inc., Silver Spring, Maryland). Isohemagglutinin titers were further reduced by the administration of Witebsky substance. The patient's subsequent course has been quite benign, and her isohemagglutinin titer and Coombs' test remain negative. Patient II-4 has had a remarkably stormy course. He developed hemorrhagic cystitis, presumably secondary to cyclophosphamide. This became so severe that ureterovesical obstruction occurred. The patient required bilateral cutaneous ureterostomies 21 days after transplantation. At that time there was negligible karyotypic and morphologic evidence of engraftment. After surgery his condition improved, and he has had evidence of engraftment and good wound healing. ACUTE MYELOBLASTIC LEUKEMIA

Six patients received marrow transplants for acute myeloblastic leukemia (Table 8). Patients III-l and III-2 were transplanted during a rapidly proliferative phase of their disease. Despite intense preparation, only transient evidence of engraftment was observed. Leukemia recurred 702

early, and the patients died as a result of their primary disease. Because of this experience, Patients III-3, III-4, and III-6 were prepared with a more intense regimen of chemotherapy and total body irradiation (Figure 4 ) . Patient III-3 developed severe gastrointestinal toxicity, became septic, and died before engraftment could be shown. Intense cytoreductive chemotherapy has resulted in survival without recurrent leukemia thus far in Patients III-4 and III-6. These patients have been successfully engrafted and are living at home without disease or the need of hematologic support. Patient III-5 received a transplant from his HL-A-identical, MLC-reactive mother. Successful engraftment was shown within 2 weeks. Weekly chromosome analysis of bone marrow cells showed only female cells. The patient developed moderately severe graft-versus-host disease and hemorrhagic cystitis. Hepatic and gastrointestinal symptoms resulted in electrolyte imbalance. The patient required peripheral hyperalimentation. Terminally, he developed sudden anuria and respiratory distress. Postmortem examination showed total obstruction of the urinary tract, with a clot filling the entire collecting system. He also had hemorrhagic pneumonitis that grew Pseudomonas organisms. His gastrointestinal tract and bladder were entirely denuded of endothelium but were coated with an abundant, acute inflammatory response. Although pre-terminal peripheral blood counts showed pancytopenia, at postmortem examination his marrow was hypercellular, with normal maturation of all cell lines. DISCUSSION

A rapidly growing program of bone marrow transplanta-



Table 7. Patierits

Tr;ansplanted

Recipient

f 120 (No evidence + (Chromosomes, None of disease) ABO blood group marker > 100 (No evidence + (Not applicable) None of disease) > 75 (No evidence + (Chromosomes) None of disease)

* Relationship• of dc>nor to recipic;nt.

tion has been established at UCLA. The results of transplantation in patients with severe aplastic anemia have been shown to be clearly superior to alternate forms of therapy (105). We concur that, if a suitably matched donor is available, transplantation is the treatment of choice for that condition. Paradoxically, Patient 1-4 achieved long-term survival with partial hematologic recovery in the presence of clear evidence of graft rejection. This observation suggests that aplastic anemia is a heterogeneous group of diseases. Some forms may be the result of damage to hematopoietic stem cells, while others may be due to an unfavorable microenvironment. In theory, the former category should lend itself ideally to therapy by marrow transplantation, whereas the latter might possibly have an increased risk of transplant rejection. One might speculate, however, that recovery of this patient's own marrow after the intense immunosuppression of transplant preparation is consistent with the presence of an unfavorable environment in his marrow and with a possible immunologic cause for his aplastic process. Our early experiences in transplanting patients with acute myeloblastic leukemia suggest that cyclophosphamide alone, or in combination with total body irradiation, may be inadequate to eradicate this disease when it is present in a rapidly proliferative phase. This observation is further strengthened by the accumulating experience in several centers of late relapses in patients transplanted for both acute myeloblastic leukemia and acute lymphoblastic leukemia. Two potential solutions are possible. Either the cytoreductive therapy presently recommended should be done at a time when there is less tumor bulk (that is, patients should be transplanted earlier in their course), or the

patients should be prepared for transplantation with more intense cytoreductive therapy. These alternatives are currently under study at UCLA. Four patients in the current series were transplanted against histocompatibility barriers. The only surviving patient of this group is hematologically improved but shows no evidence of engraftment (Patient 1-4). In two other patients, potential success was thwarted by early fungal sepsis (Patient 1-3) or recurrent acute myeloblastic leukemia (Patient III-2). In one patient, it was possible to eradicate acute myeloblastic leukemia and achieve persistent engraftment of maternal marrow (Patient III-5); this patient died because of the combined effects of severe enteritis and cystitis. It was not possible to document the presence of graft-versus-host disease by skin or rectal biopsy, or at autopsy, although the clinical features were quite typical. This experience does not lead to optimism that persistent engraftment against histocompatibility barriers can be accomplished by current methods. If more effective means to control graft-versus-host disease are developed, such transplants might be feasible for selected patients with severe marrow failure or terminal leukemia. Finally, severe and prolonged impairment of cell-mediated immune function was shown in one long-term survivor (Patient 1-1). Although the patient has tolerated infections moderately well, the ultimate implications of his immunologic incompetence cannot be evaluated at this time. Immunologic Problems and Opportunities in Bone Marrow Transplantation

Dr. John L. Fahey*: Immunology has often been en* Department of Microbiology and Immunology, UCLA School of Medicine. Cline et al. • Bone Marrow Transplantation


703

Table 8. Patients Transplanted for Refractory Myeloblasts Leukemia Recipient

Donor

Patient Age Sex Relation- Sex ship*

Ill-la

yrs 43

M

Sibling

M

IIMb

III-2

13

M

Sibling

F

III-3

29

M

Sibling

F

III-4

26

M

Sibling

M

III-5

10

M

Mother

F

III-6

43

M

Sibling

M

Preparatory (Regimen)

1

Cyclophosphamide, cytosine arabinoside, 6-thioguanine, daunomycin, vincristine 2A

Procarbazine, cytosine arabinoside, 6-mercaptopurine, antithymocyte globulin, total body irradiation 2B

2A (Cyclophosphamide cystitis) 2B

Histocompatibility Human LeukocyteLocus A (HL-A)

Mixed Lymphocyte Culture (MLC)

Identical

Nonreactive

Graft (Evidence)

GraftVersusHost Disease (Therapy)

Transient (Chromosome)

None


None


None

Not applicable

_i-

Survival (Status, or Cause of Death)

days Retransplanted at 39 days (recurrent acute myeloblastic leukemia) 76 (Recurrent acute myeloblasts leukemia)

39 (Recurrent acute myeloblast^ leukemia) 8 (Severe intestinal toxicity)

Identical

D^Rf

Identical

Nonreactive

Not applicable

Identical

Nonreactive

Identical

D^Rf

Identical

Nonreactive

> 150 (No evidence + (ABO blood Stage III of disease) group marker, (steroids) erythrocyte phosphoglucomutase isoenzyme marker) 47 (No evidence + (Chromosome) Stage III (Antiof disease) thymocyte (renal and globulin, pulmonary steroids) failure) + (Erythrocyte acid None > 100 (No evidence phosphatase isoof disease) enzyme marker, erythrocyte glutamic pyruvate transaminase isoenzyme marker)

* Relationship of donor to recipient. R: Donor and recipient reactive to each other.

tD^

riched by careful observation of the differences between clinical findings and inbred animal systems. The finding that kidney transplantation could be successfully done between non-human-leukocyte-locus-A (HL-A)-matched donors and recipients indicated that the immune system could be successfully modified by nonspecific immune suppressive agents, and that genetic differences per se need not be decisive in the transplantation of certain organs in man. ADDITIONAL HISTOCOMPATIBILITY SYSTEMS IN MAN

Kidneys can be successfully transplanted at times despite histoincompatibility. Bone marrow transplantation, 704

November

1975

however, can produce damaging immunologic reactions (for instance, graft-versus-host disease) even when donor and recipient are histocompatible. Indeed, the frequency of graft-versus-host disease may be as high as 50% to 70% with matched donors and recipients. Clearly, such reactions derive from histocompatibility systems in addition to the known HL-A, mixed lymphocyte culture (MLC), and ABO systems. These undefined histocompatibility systems await identification and assessment of their significance in man. Studies in mice currently indicate the presence of 16 histocompatibility systems and the probability that 25 or more occur. There seems no reason to believe that man is greatly different from mouse in this respect.

• Annals of Internal Medicine • Volume 83 • Number


5

Studies in animals by Graff and associates (106) clearly indicate that a summation of minor histocompatibility differences may result in severe graft rejection reaction, which approximates that seen when there are differences at a single major locus. Accordingly, definition of the minor or tissue-specific histocompatibility systems in man may well be important, so that appropriate remedial or preventive measures can be taken to avert unnecessary graft-versus-host reaction. Successful treatment of graft-versus-host reaction involving minor histocompatibility regions by antilymphocyte globulin or other approaches that cause a general suppression of the immune system are important at this stage of development of clinical marrow transplantation. They do, however, contribute to the general immune suppression, and subnormal function of the immune system presents a severe and often fatal hazard to the successful transplant recipient. This further emphasizes the need for specific means of averting the minor, as well as the major, histoincompatibilities that may produce graft-versus-host reactions. PREVENTION OR CONTROL OF GRAFT-VERSUS-HOST REACTIONS

Bone marrow transplantation is restricted to a small percentage of potential recipients because of histocompatibility barriers than cannot be overcome. In the American society of today, it has been calculated that the patient needing marrow transplantation has about a 40% chance of finding an HL-A matched donor within the family (107). The actual chances of finding a suitable donor are considerably less (perhaps 1 in 4) when ABO typing and such factors as geographic location and health of the donor are taken into account. Clearly, the prevention or control of graft-versus-host disease are major challenges to extension of bone marrow transplantation. Two general approaches have been explored: removal of reacting cells, especially in the period when marrow has been obtained from the donor and before it is transfused into the new host; and suppression or impairment of undesired immunologic reactions. These two approaches and some of the means of achieving them are outlined in Table 9. Physical methods based on differences in density or size have been used to separate marrow cells into those most effective in restoring marrow versus those most likely to cause undesirable reactions. Beneficial effects have been claimed by Gelfand and co-workers (68), using this general approach. Possibly, physical methods could be combined with immunologically specific reactions (perhaps adding additional mass or charges to cells recognizing histocompatibility antigens) and thus facilitate ready separation of undesirable cellular components from the transplanted bone marrow. Specificity of immunologic recognition may make it possible to remove all of the cells capable of recognizing host histocompatibility factors from the mixture of cells in the donor bone marrow. Bonavida and Kedar (108) have removed donor lymphoid cells capable of producing graft-versus-host disease by a selective adherence method

Table 9. Approaches to Prevention and Reduction of Graft-VersusHost Reaction Removal from donor bone marrow of cells reacting with histocompatibility factors Physical Immunologic—specific Physical binding of cells with histocompatibility receptors "Burnout" methods Specific stimulation plus radioisotope incorporation Histocompatibility factors labeled with isotope or toxin Blocking antibody Immunologic—nonspecific Antilymphocyte globulin "Chalones"—suppressive tissue factors Suppression of cells involved in graft-versus-host reaction Soluble antigen or Ag:Ab complexes Stimulation of suppressor cells Advantageous balance of humoral and cellular immune factors

before reconstitution of the recipient. In other animal studies (109), there was an inability to retain anti-tumor immunity while selectively removing graft-versus-host disease producing (antihistocompatibility) cells. Extension of this line of work is under way. With isolation and purification of histocompatibility factors, these could be bound as molecular entities rather than as whole cells to insoluble matrices, so that marrow cells with histocompatibility recognition capacity could be removed, and the hematopoietic cells of the donor could be successfully cleared for nontoxic transfer to a new host. Removal of potentially reactive cells may be more difficult than removing cells that already are capable of recognizing and reacting with histocompatibility factors. Presumably, stem cells do not have a recognition unit for histocompatibility factors on their surface, and the precursors of graft-versus-host active cells cannot be selectively removed. Gross removal of all stem cells, on the other hand, would be extremely disadvantageous. It is possible, however, that in-vitro stimulation could lead to selective receptor expression and thus allow specific cell removal. Removal of reacting cells by a combination of maneuvers ultimately may prove to be most effective. "Burnout" methods have been proposed by which radioisotopes are used to kill the cells capable of reacting with specific histocompatibility antigens. Salmon and associates (65) have induced 3H-thymidine incorporation into graftversus-host disease-producing cells by exposing them in vitro to the histocompatibility antigens of the new host. These cells were stimulated to blastogenic transformation (DNA synthesis) and suffered irreparable damage from incorporation of highly radioactive DNA precursor (thymidine). Several infants have been treated with bone marrow modified in this way (66). Purified histocompatibility factors theoretically could be labeled with isotope or toxins (such as diphtheria) and used to kill graft-versus-host disease-producing cells. Histocompatibility antigens remain to be purified sufficiently for a trial of this approach. An additional difficulty is the variety of histocompatibility factors (HL-A antigens and so forth) that would be needed to make this effective. Blocking antibody offers the opportunity to interfere Cline et a/. • Bone Marrow Transplantation


705

with graft-versus-host disease, as it can help prolong graft survival. Preliminary experiments with this approach in man have been reported by Buckley and colleagues ( 6 7 ) . Here, also, the multiplicity of histocompatibility antigens, as well as the paucity of potent antiserum, impairs the feasibility of this approach. Immunologic reagents, as well as drugs, can nonspecifically reduce immune responses. Antilymphocyte globulin, for example, can effectively modify graft-versus-host reactions. The exact antigens involved and the optimal conditions for producing most effective antiserums for clinical use are still being investigated. Extracts of lymphoid tissue (chalones) have been explored as nonspecific immunosuppressive agents (110, 111). These seem to be effective in averting graft-versushost disease in animal systems. Soluble antigen: antibody complexes can impair cellular immunity (112). Presumably, such agents act on specifically sensitized lymphocytes. It is possible that some of the effects attributable to blocking antibody (in protecting against graft rejection) may, in fact, involve antigen: antibody complexes. Administration of soluble antigen tends to induce a greater amount of serum blocking activity and less direct cell-mediated immunity than do whole cells (113). Pretreatment of recipients with soluble histocompatibility antigens have enabled grafts to survive longer in some animal systems (113, 114). Whether such a procedure can be modified for clinical application to bone marrow transplantation remains to be determined. Suppressor cells that tend to reduce or minimize the dimensions of immune response have been identified in a number of antigen systems. These have been defined mostly in terms of B-cell antibody-producing systems, but there is reason to believe that similar suppressor cells may act in the immune response to histocompatibility factors. Suppressor systems in man have been identified in patients with immune deficiency syndromes (115). Apparently, these systems are acting broadly on large populations of lymphoid cells. It remains to be seen whether these can be used more selectively to eradicate graft-versus-host disease. The immune response to complex systems, such as histocompatibility antigens, involves multiple factors. Not only do many humoral and cellular components participate, but amplification systems involving other cells and serum components may participate. Furthermore, not all aspects of an immune response tend toward attack against antigenbearing target cells. Regulatory and controlling factors in the immune system tend to limit or diminish the immune response. Clearly, with greater appreciation of the mechanisms that reduce immune response and with better methods for removal of particular cell types, efforts to control and prevent graft-versus-host reactions will be extended, and barriers that currently prevent extensive use of bone marrow transplantation will be breached. ACKNOWLEDGMENTS: Grant support: by grants from the Gwynne Hazen Cherry Memorial Laboratory; from the Celia Hagen Memorial Fund; and from the U.S. Public Health Service, grants RR-5354 and CA 15688. Received 27 June 1975; accepted 15 July 1975. 706

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Human bone marrow transplantation.

Bone Marrow Transplantation.

Bone marrow transplantation. Preface.

Editorial: Bone marrow transplantation.

Experimental bone marrow transplantation.

Bone-marrow transplantation in osteopetrosis.

Bone marrow transplantation.

Marrow repopulation after bone marrow transplantation.

Allogeneic bone marrow transplantation in multiple myeloma. European Group for Bone Marrow Transplantation.

Technique of bone marrow transplantation.

Psoriasis and bone marrow transplantation.

Immunoglobulin synthesis following allogeneic bone marrow transplantation in man. Conversion to donor allotype.

Bone marrow transplantation: a survey.

HLA-mismatched bone marrow transplantation.

Migraine after bone-marrow transplantation.

Bone-marrow transplantation in acute leukaemia.

Autologous bone marrow transplantation in solid tumors.

Bone marrow transplantation in patients with gold-induced marrow aplasia.

Cytogenetics in autologous bone marrow transplantation.

Bone-marrow transplantation in acute leukaemia.

Legal issues in bone marrow transplantation.

Bone marrow transplantation in patients with thalassemia.

Allogeneic bone marrow transplantation in childhood leukemia.

Second bone marrow transplantation in eight children.