- Email: [email protected]
The future of Marrow transplantation
The Future
of Marrow
Transplantation
E. Donna11 Thomas
INCE the first successful identical twin transplants in the 1950s’ human marrow transplantation has been in a continuous state of evolution. At the present time, as described in detail elsewhere in this issue, marrow transplants are the accepted best therapy for certain diseases and disease states and controversial for others. Problems related to the complications of marrow transplantation and problems related to the patient’s underlying disease prevent a broader clinical application of marrow transplantation. Some of these problems are being addressed in current clinical studies in human patients. Other problems are being studied in the laboratory but are not yet ready for clinical trial. At the same time, marrow transplant technology is being studied for entirely new applications. Studies that point the way to the future of marrow transplantation are summarized here.
S
PROBLEMS
CURRENTLY
IN CLINICAL STUDY
Recurrent Leukemia
The probability that a given patient will suffer a recurrence of leukemia following a bone marrow transplant (BMT) ranges from approximately 15% to 80% depending upon the type and stage of the disease.’ A number of studies are directed toward reducing the probability of recurrence of leukemia, Chemotherapy. Cyclophosphamide (CY) combined with total body irradiation (TBI) has been the standard preparative regimen. Clinical studies now underway are evaluating other chemotherapeutic agents, with or without TBI, including high dose cytosine arabinoside, etoposide, thiotepa, busulfan, or dimethylbusulfan, and combinations of these agents. Early reports with several of these regimens suggest an improvement over the
From the Fred Hutchinson Cancer Research Center, Seattle. Supported by a Research Career Award Al 02425 from the National Institute of Allergy and Infectious Diseases. Address reprint requests to E. Donna11 Thomas, MD, Fred Hutchinson Cancer Research Center, 1124 Columbia St, Seattle, WA 98104. 0 1988
by Grune
& Stratton,
0749-2081/88/0401-0011$05.00/0
74
Inc.
standard CY plus TBI.3-5 However, final evaluation depends upon direct comparison in randomized trials and long-term follow-up of five or more years. Irradiation. The TBl regimen initiated by the Seattle team two decades ago consisted of irradiation of the entire patient with opposing cobalt 60 sources at a dose rate of 6 to 7 cGy/min.‘j Subsequent studies have looked at different regimens of fractionated irradiation and different rates of irradiation exposure. A hyperfractionated regimen (radiation given three times per day for several days) with CY given after irradiation looks particularly promising.’ Regimens involving total lymphoid irradiation (TLI) in addition to TBI are also being explored. Again, however, a randomized comparison of these regimens has not been carried out, and long-term follow-up results are not available. Nevertheless, from these studies of’chemotherapy and irradiation, it is reasonable to expect that regimens with a greater antileukemic effect will be developed. Monoclonal antibodies combined with radioisotopes or toxins. Since the discovery of monoclo-
nal antibodies, one attractive possibility has been the use of these antibodies alone or combined with a radioisotope, a drug, or toxin as a means of a targeted attack against malignant cells. Unfortunately, no antigen (and therefore no monoclonal antibody) has yet been identified that occurs only on a malignant cell. Monoclonal antibodies are available, however, that react with certain malignant cells as well as with normal cells. Examples include malignant melanoma, T-cells and T-cell tumors, B-cells and B-cell tumors, CALLApositive acute lymphoblastic leukemia cells, and certain early myeloid precursor cells.8,9 Antiidiotype monoclonal antibodies (antibodies directed toward the unique surface immunoglobulin of a malignant clone) combined with a radioisotope such as radioactive iodine are uniquely specific in their reaction with the cells of the particular malignant clone.” Clinical studies of this type are underway with encouraging early results. The graft-versus-leukemia reaction. In rodent systems, it has been possible to demonstrate an antileukemic effect of the graft-versus-host reacSeminars
in Oncology
Nursing,
Vol 4, No
1 (February),
1988:
pp 74-78
THE FUTURE
OF MARROW
TRANSPLANTATION
tion. In human studies, it has been shown that leukemia is less likely to recur if patients have graft-versus-host disease (GVHD). ” Some studies involve an effort to create GVHD (for example, by giving additional donor T-cells) in an effort to take advantage of the graft-versus-leukemia effect. Unfortunately, this approach is limited by our current inability to control and predict the course of GVHD. Lymphoblastic lymphoma. Following a marrow transplant, some patients have developed a rapidly proliferating and fatal lymphoblastic lymphoma in cells of donor origin. These lymphomas have been shown to be due to transformation by the Epstein-Barr virus.‘2 Antiviral agents such as acyclovir are currently being evaluated asa means of preventing these somewhat infrequent occurrencesof lymphoma. GVHD
Even when patient and donor are HLA-identical siblings, GVHD is a significant problem occurring in approximately one half of the patients and constituting a significant source of mortality either directly or indirectly through complicating infections. The problem is worse when donor and recipient are lessperfectly matched with regard to histocompatibility. Prevention and treatment of GVHD are under intensive study. Immunosuppressive drugs. The “standard” regimen of immunosuppressiongiven after marrow grafting to prevent GVHD has consisted of methotrexate given intermittently over the first 100 days.6 This regimen amelioratesGVHD but is associated with severe mucositis and delayed marrow engraftment. The new immunosuppressive agent, cyclosporine, avoids theseproblems, but in a randomized comparison with methotrexate it did not prove superior becauseof its own complications of nephrotoxicity, among others.I3 Most recently, a short course of methotrexate combined with cyclosporine has proved superior to either regimen alone. I4 The combined regimen has lead to 80% one-year survivals for recipients of HLAidentical marrow and is now the gold standard against which other regimens must be compared. A variety of other regimensare under investigation including methylprednisolone, antithymocyte globulin, monoclonal antibodies, and even agents
75
such as thalidomide. T-cells contained in the marrow inoculum are thought to be the principal cells responsiblefor the initiation of GVHD. T-cells can be removed from the marrow inoculum by a variety of methodsincluding agglutination techniques. immunoadsorption columns, and treatment with anti-T cell monoclonal antibodies either alone or with complement or anti-T cell monoclonal antibodies bound to an immunotoxin. A number of reports describe the use of these techniquesfor the removal of T cells from the donor marrow, and almost all show a significant reduction in the incidence and severity of GVHD. However. these good resultsare marred by a significant increasein failure of engraftment, which often results in the death of the patient. I5 Graft ,failure. With a chemoradiotherapy preparative regimen and untreated donor marrow, graft failure occurs in lessthan 17~of the marrow graft recipients. Most of these failures are due to graft rejection secondary to immunization of the patient against transplantation antigens contained in blood transfusions.r6 When T-cells are removed from the donor marrow, the incidence of graft failure goes up to approximately 10% to 40%. The mechanismof the graft failure is unknown, but less likely possibilities include damage to stem cells during the separationprocedure and the possibility that T-cells elaborate growth factors necessary to sustain marrow engraftment. More likely, based on the cytogenetic demonstration of residual host T-cells. is the possibility that somehost T-cells are not destroyed by the preparative regimen and are responsible for marrow graft re.jection. Current studies involve efforts to destroy these residual host T-cells by increasedintensity of chemoradiotherapy before grafting and/or the use of antithymocyte globulin or use of anti-T-cell monoclonal antibodies postgrafting. T-cell depletion.
Opportunistic Infection, Cytomegalovirus (CMV)
Especially Infection
Marrow graft recipients are targets for all kinds of infections because of incompetent defense mechanismsassociated with the underlying disease, the period of granulocytopenia in the first few weeks after grafting, and the period of immunodeficiency that extends for several months after
76
grafting. Interstitial pneumonia due to CMV is a major cause of death following a marrow graft. l7 CMV is latent in approximately 50% of normal people as shown by the presence of anti-CMV antibodies. CMV infection in the marrow graft recipient may be a consequence of activation of a latent virus in the patient or in the donor cells or may be transmitted in blood products obtained from apparently normal donors. It has now been shown that patients who are CMV antibody-negative with a negative donor who receive blood products only from CMV-negative blood donors have a very low incidence of CMV infection. l8 Current studies are directed toward the prevention of CMV activation by the use of passive immunization with immunoglobulins and by prophylactic therapy with antiviral agents. Expansion of the Donor Pool
In our country, only about one third of the patients will have an HLA identical sibling to serve as marrow donor. Current efforts are directed toward identifying other donors in an effort to make marrow grafting available to those individuals who do not have an HLA-matched sibling. One such effort involves studies of the extended family where donors who are partially HLA matched can be identified. l9 More than 200 such transplants have been done by the Seattle team with excellent results when donor and recipient differ by only one of the HLA loci. A major national and intemational effort is underway to create a pool of volunteer unrelated donors of known HLA type so that a phenotypically HLA identical donor can be selected. More than 100 such transplants have now been carried out, but it is too early to evaluate long-term results. Lesser Problems
The list of less frequently encountered graftrelated problems is too long to enumerate completely here. Factors predicting the development of veno-occlusive disease (VOD) of the liver are under study.*’ The incidence of leukoencephalopathy can be reduced by elimination of cranial irradiation from the routine treatment of children with leukemia.2’ The incidence of cataracts has been reduced by the use of fractionated irradiation.**
E. D. THOMAS
Fortunately, secondary tumors have been very rare in marrow graft recipients. PROBLEMS
NOW IN PRECLINICAL
STUDY
Separation of Stem Cells
Sedimentation techniques, monoclonal antibodies, and immunadsorption columns are being studied for removal of other cells from bone marrow (negative stem cell separation) or for the selective removal of stem cells from the marrow (positive stem cell separation). These methods are of interest for concentration of stem cells, and for preparation of stem cells for marrow transplantation that are free of leukemic or other malignant cells. Separated stem cells for marrow transplantation are of particular interest in the applications of autologous marrow transplantation. Biologic ResponseModifiers Interferon. Interferon is being studied for its possible antileukemic effect for treatment of patients before or after marrow grafting.23Y24 Preliminary studies suggest some degree of efficacy in patients with acute lymphoblastic leukemia or chronic myelogenous leukemia. Growth factors. A variety of growth factors are becoming available in quantity through the applications of DNA recombinant technology. 25 The hematopoietic growth factors include erythropoietin, G-CSF, and IL- 1. These growth factors offer promise in stimulating a more rapid marrow recovery after marrow grafting and in preventing graft failure. These growth factors may make it possible to grow stem cells in vitro. IL-2 should permit the growth in vitro of donor T-cells that may have an antiviral effect when given along with the marrow graft. Studies in rodents and now some studies in primates illustrate the exciting possibilities for these agents. Bone-SeekingIsotopes
One of the problems with TBI is that the irradiation is not selective so that all tissues in the body, normal as well as malignant, are irradiated. Boneseeking radioactive isotopes can be used either alone or combined with TBI to increase the irradiation dose to the bone marrow without increasing the irradiation of normal tissues. Studies in the
THE FUTURE
OF MARROW
canine marrow grafting model show promising results that may be applicable to man. Monoclonal
77
TRANSPLANTATION
Antibodies as Transport Agents
Radioactive isotopes bound to monoclonal antibodies react with leukemic cells or with normal myeloid marrow cells can also be used to increase irradiation exposure locally in the marrow. Monoclonal antibodies may be bound to toxins or to chemotherapeutic drugs so that these agents can be specifically directed toward target malignant cells.x.y.26 LABORATORY STUDIES WITH PROMISING FUTURE APPLICATIONS
example, thalassemia major and adenosine deaminase deficiency. The application of gene transfer technology to human patients will almost certainly involve hematopoietic marrow stem cells (reviewed in reference 27). Marrow transplant technology has made these cells readily available for in vitro use and for cryopreservation. Further, marrow transplant technology will be necessary for the reintroduction of these cells into their host and for the destruction of the abnormal cells present in the host. Major problems with gene expression and particularly sustained gene expression indicate that it will be some years before gene transfer technology can be successfully applied to human patients. Understanding Histocompatibility and Tolerctnce
Stem Cell Culture
For several decades it has been recognized that if stem cells could be grown in vitro, then these cells could be used for marrow grafting and for a variety of other studies. Unfortunately, it has not been possible to grow these cells in vitro and, in fact, the identity of the truly pluripotent hematopoietic stem cell (the seed cell that can replicate itself and also develop into all the normal blood cells) in man is unknown. Most of the in vitro assays for colony forming cells are thought to identify cells more differentiated than the true stem cell. With the availability of growth factors cited above, it may become possible to culture stem cells. Perhaps it will be possible for a donor to give 5 or 10 ml of marrow as an outpatient for stem cells to be grown in vitro for a subsequent marrow transplant. Gene Trarwfer
An exciting possibility is that normal genes can be transferred into a patient’s cells to correct a genetically determined disorder. These ceils could then be used for an autologous transplant (since they are of host origin), thus avoiding the risk of GVHD. Several laboratories are intensively studying gene transfer technology by several techniques, the most promising being the use of retroviral vectors. These studies have shown that genes can be transferred in vitro in murine, canine, and primate models. Several genetically determined diseases involve the marrow, and some have already been cured by marrow transplantation, for
Despite numerous studies over several decades, factors governing reactivity between donor and host cells (graft rejection or GVHD) or its absence (tolerance) are still not fully understood. For example, some recipients of HLA-identical marrow grafts do not have GVHD. These patients go onto a long-term state of tolerance between cells of the engrafted marrow and tissues of the host. Other such recipients develop severe and fatal GVHD. Undoubtedly, some of these difficulties are related to histocompatibility differences outside the HLA chromosomal region, so-called minor histocompatibility systems, while some may be due to differences within the HLA system. Molecular biology techniques are making it possible to understand the genetic differences at the DNA level. It may soon be possible to perform tissue typing with probes that detect differences at the DNA level rather than the typing sera now in use that detect differences on the cell surface. Perhaps these studies will shed light on the nature of the histocompatibility differences that determine tolerance on the one hand or GVHD on the other. Recurrent Leukemia
The vast majority, some 95%, of recurrent leukemias in marrow graft recipients have been in cells of host-type, indicating that the chemoradiotherapy preparative regimen did not succeed in destroying all of the patient’s leukemic cells. However, some 5% of recurrent leukemias have been in donor-type cells.28 In most instances, these leukemic donor cells have been recognized by cytoge-
78
E. D. THOMAS
netic studies when donor and recipient are of opposite sex. The mechanisms of malignant transformation of donor cells is unknown. An attractive explanation is that of transfection, a process by which some host DNA could be transferred into a normal donor cell. This host DNA from a leukemic cell might contain an oncogene or some similar
structure that could then be responsible for the malignant transformation of a donor cell. Current studies of the process of malignant transformation, and of oncogenes and their gene products (growth factors), may begin to shed light on the etiology of leukemia.
REFERENCES 1. Thomas ED, Lochte HL Jr, Cannon JH, et al: Supralethal whole body irradiation and isologous marrow transplantation in man. J Clin Invest 38:1709-1716, 1959 2. Thomas ED: Marrow transplantation for malignant diseases (Kamofsky Memorial Lecture). J Clin Oncol 1:5 17-53 1, 1983 3. Coccia PF, Strandjord SE, Gordon EM, et al: High dose cytosine arabinoside (Ara-C) and fractionated total body irradiation (F-TBI) as preparation for bone marrow transplantation (BMT) for childhood acute leukemia in remission-A preliminary report. Proceedings, American Society of Clinical Oncology, 19th Annual Meeting, May 22-24, 1983, San Diego, p 175, (abstr C-680) 4. Blume KG, Forman SJ, O’Donnell MR, et al: Total body irradiation and high-dose etoposide: A new preparatory regimen for bone marrow transplantation in patients with advanced hematologic malignancies. Blood 69: 1015-1020, 1987 5. Santos GW, Tutschka PJ, Brookmeyer R, et al: Marrow transplantation for acute nonlymphocytic leukemia after treatment with busulfan and cyclophosphamide. N Engl J Med 309:1347-1353, 1983 6. Thomas ED, Storb R, Clift RA, et al: Bone-marrow transplantation. N Engl J Med 292:832-843, 895-902, 1975 7. Dinsmore R, Kirkpatrick D, Flomenberg N, et al: Allogeneic bone marrow transplantation for patients with acute nonlymphocytic leukemia. Blood 63:649-656, 1984 8. Vallera DA, Kersey JH, Quinones RR, et al: Antibodyricin conjugates: Purgative reagents for murine and human allogeneic bone marrow transplantation, in Gale RP (ed): Recent Advances in Bone Marrow Transplantation, vol 7, New York, Liss, 1983, pp 209-222 9. Raso V, Ritz J, Basala M, et al: Monoclonal antibodyricin A chain conjugate selectively cytotoxic for cells bearing the common acute lymphoblastic leukemia antigen. Cancer Res 42:457-464, 1982 10. Miller RA, Maloney DC, Wamke R, et al: Treatment B-cell lymphoma with monoclonal anti-idiotype antibody. Engl J Med 306:517-522, 1982
of N
11. Weiden PL, Floumoy N, Thomas ED, et al: Antileukemic effect of graft-versus-host disease in human recipients of allogeneic-marrow grafts. N Engl J Med 300:1068-1073, 1979 12. Schubach WH, Hackman R, Neiman PE: A monoclonal immunoblastic sarcoma in donor cells bearing Epstein-Barr virus genomes following allogeneic marrow grafting for acute lymphoblastic leukemia. Blood 60:180-187, 1982 13. Storb R, Deeg HJ, Thomas ED, et al: Marrow transplantation for chronic myelocytic leukemia: A controlled trial of cyclosporine versus methotrexate for prophylaxis of graftversus-host disease. Blood 66:698-702, 1985
cyclosporine compared with cyclosporine alone for prophylaxis of acute graft versus host disease after marrow transplantation for leukemia. N Engl J Med 314:729-735, 1986 15. Martin PJ, Hansen JA, Buckner CD, et al: Effects of in vitro depeletion of T cells in HLA-identical allogeneic marrow grafts. Blood 66:664-672, 1985 16. Storb R, Weiden PL, Deeg HJ, et al: Rejection of marrow from DLA-identical canine littermates given transfusions before grafting: Antigens involved are expressed on leukocytes and skin epithelial cells but not on platelets and red blood cells. Blood 54~477.484, 1979 17. Meyers JD, Flournoy N, Thomas ED: Nonbacterial pneumonia after allogeneic marrow transplantation: A review of ten years’ experience. Rev Infect Dis 4:1119-l 132, 1982 18. Bowden RA, Sayers M, Floumoy N, et al: Cytomegalovirus immune globulin and seronegative blood products to prevent primary cytomegalovirus infection after marrow transplant. N Engl J Med 314:1006-1010, 1986 19. Beatty PC, Clift RA, Mickelson EM, et al: Marrow transplantation from related donors other than HLA-identical siblings. N Engl J Med 313:765-771, 1985 20. McDonald GB, Sharma P, Matthews DE, et al: Venoocclusive disease of the liver after bone marrow transplantation: Diagnosis, incidence, and predisposing factors. Hepatology 4:116-122, 1984 21. Thompson CB, Sanders JE, Floumoy N, et al: The risks of central nervous system relapse and leukoencephalopathy in patients receiving marrow transplants for acute leukemia. Blood 67:195-199, 1986 22. Deeg HJ, Floumoy N, Sullivan KM, et al: Cataracts after total body irradiation and marrow transplantation: A sparing effect of dose fractionation. Int J Radiat Oncol Biol Phys 10:957-964, 1984 23. Meyers JD, Floumoy N, Sanders JE, et al: Prophylactic human leukocyte interferon after allogeneic marrow transplantation. Ann Intern Med (in press) 24. Talpaz M, Kantarjian HM, McCredie KB, et al: Clinical investigation of human alpha interferon in chronic myelogenous leukemia. Blood 69:1280-1288, 1987 25. Metcalf D: The molecular biology and functions of the granulocyte-macrophage colony-stimulating factors. Blood 67~257-267, 1986 26. Kronke M, Depper JM, Leonard WJ, et al: Adult T cell leukemia: A potential target for ricin A chain immunotoxins. Blood 65:1416-1421, 1985 27. Thomas ED: Marrow transplantation and gene transfer as therapy for hematopoietic diseases, in Molecular Biology of Homo Sapiens, Cold Springs Harbor Symposia on Quantitative Biology, vol 51(2). 1986, pp 1009-1012 28. Boyd CN, Ramberg RC, Thomas ED: The incidence of recurrence of leukemia in donor cells after allogeneic bone
of Marrow
Transplantation
E. Donna11 Thomas
INCE the first successful identical twin transplants in the 1950s’ human marrow transplantation has been in a continuous state of evolution. At the present time, as described in detail elsewhere in this issue, marrow transplants are the accepted best therapy for certain diseases and disease states and controversial for others. Problems related to the complications of marrow transplantation and problems related to the patient’s underlying disease prevent a broader clinical application of marrow transplantation. Some of these problems are being addressed in current clinical studies in human patients. Other problems are being studied in the laboratory but are not yet ready for clinical trial. At the same time, marrow transplant technology is being studied for entirely new applications. Studies that point the way to the future of marrow transplantation are summarized here.
S
PROBLEMS
CURRENTLY
IN CLINICAL STUDY
Recurrent Leukemia
The probability that a given patient will suffer a recurrence of leukemia following a bone marrow transplant (BMT) ranges from approximately 15% to 80% depending upon the type and stage of the disease.’ A number of studies are directed toward reducing the probability of recurrence of leukemia, Chemotherapy. Cyclophosphamide (CY) combined with total body irradiation (TBI) has been the standard preparative regimen. Clinical studies now underway are evaluating other chemotherapeutic agents, with or without TBI, including high dose cytosine arabinoside, etoposide, thiotepa, busulfan, or dimethylbusulfan, and combinations of these agents. Early reports with several of these regimens suggest an improvement over the
From the Fred Hutchinson Cancer Research Center, Seattle. Supported by a Research Career Award Al 02425 from the National Institute of Allergy and Infectious Diseases. Address reprint requests to E. Donna11 Thomas, MD, Fred Hutchinson Cancer Research Center, 1124 Columbia St, Seattle, WA 98104. 0 1988
by Grune
& Stratton,
0749-2081/88/0401-0011$05.00/0
74
Inc.
standard CY plus TBI.3-5 However, final evaluation depends upon direct comparison in randomized trials and long-term follow-up of five or more years. Irradiation. The TBl regimen initiated by the Seattle team two decades ago consisted of irradiation of the entire patient with opposing cobalt 60 sources at a dose rate of 6 to 7 cGy/min.‘j Subsequent studies have looked at different regimens of fractionated irradiation and different rates of irradiation exposure. A hyperfractionated regimen (radiation given three times per day for several days) with CY given after irradiation looks particularly promising.’ Regimens involving total lymphoid irradiation (TLI) in addition to TBI are also being explored. Again, however, a randomized comparison of these regimens has not been carried out, and long-term follow-up results are not available. Nevertheless, from these studies of’chemotherapy and irradiation, it is reasonable to expect that regimens with a greater antileukemic effect will be developed. Monoclonal antibodies combined with radioisotopes or toxins. Since the discovery of monoclo-
nal antibodies, one attractive possibility has been the use of these antibodies alone or combined with a radioisotope, a drug, or toxin as a means of a targeted attack against malignant cells. Unfortunately, no antigen (and therefore no monoclonal antibody) has yet been identified that occurs only on a malignant cell. Monoclonal antibodies are available, however, that react with certain malignant cells as well as with normal cells. Examples include malignant melanoma, T-cells and T-cell tumors, B-cells and B-cell tumors, CALLApositive acute lymphoblastic leukemia cells, and certain early myeloid precursor cells.8,9 Antiidiotype monoclonal antibodies (antibodies directed toward the unique surface immunoglobulin of a malignant clone) combined with a radioisotope such as radioactive iodine are uniquely specific in their reaction with the cells of the particular malignant clone.” Clinical studies of this type are underway with encouraging early results. The graft-versus-leukemia reaction. In rodent systems, it has been possible to demonstrate an antileukemic effect of the graft-versus-host reacSeminars
in Oncology
Nursing,
Vol 4, No
1 (February),
1988:
pp 74-78
THE FUTURE
OF MARROW
TRANSPLANTATION
tion. In human studies, it has been shown that leukemia is less likely to recur if patients have graft-versus-host disease (GVHD). ” Some studies involve an effort to create GVHD (for example, by giving additional donor T-cells) in an effort to take advantage of the graft-versus-leukemia effect. Unfortunately, this approach is limited by our current inability to control and predict the course of GVHD. Lymphoblastic lymphoma. Following a marrow transplant, some patients have developed a rapidly proliferating and fatal lymphoblastic lymphoma in cells of donor origin. These lymphomas have been shown to be due to transformation by the Epstein-Barr virus.‘2 Antiviral agents such as acyclovir are currently being evaluated asa means of preventing these somewhat infrequent occurrencesof lymphoma. GVHD
Even when patient and donor are HLA-identical siblings, GVHD is a significant problem occurring in approximately one half of the patients and constituting a significant source of mortality either directly or indirectly through complicating infections. The problem is worse when donor and recipient are lessperfectly matched with regard to histocompatibility. Prevention and treatment of GVHD are under intensive study. Immunosuppressive drugs. The “standard” regimen of immunosuppressiongiven after marrow grafting to prevent GVHD has consisted of methotrexate given intermittently over the first 100 days.6 This regimen amelioratesGVHD but is associated with severe mucositis and delayed marrow engraftment. The new immunosuppressive agent, cyclosporine, avoids theseproblems, but in a randomized comparison with methotrexate it did not prove superior becauseof its own complications of nephrotoxicity, among others.I3 Most recently, a short course of methotrexate combined with cyclosporine has proved superior to either regimen alone. I4 The combined regimen has lead to 80% one-year survivals for recipients of HLAidentical marrow and is now the gold standard against which other regimens must be compared. A variety of other regimensare under investigation including methylprednisolone, antithymocyte globulin, monoclonal antibodies, and even agents
75
such as thalidomide. T-cells contained in the marrow inoculum are thought to be the principal cells responsiblefor the initiation of GVHD. T-cells can be removed from the marrow inoculum by a variety of methodsincluding agglutination techniques. immunoadsorption columns, and treatment with anti-T cell monoclonal antibodies either alone or with complement or anti-T cell monoclonal antibodies bound to an immunotoxin. A number of reports describe the use of these techniquesfor the removal of T cells from the donor marrow, and almost all show a significant reduction in the incidence and severity of GVHD. However. these good resultsare marred by a significant increasein failure of engraftment, which often results in the death of the patient. I5 Graft ,failure. With a chemoradiotherapy preparative regimen and untreated donor marrow, graft failure occurs in lessthan 17~of the marrow graft recipients. Most of these failures are due to graft rejection secondary to immunization of the patient against transplantation antigens contained in blood transfusions.r6 When T-cells are removed from the donor marrow, the incidence of graft failure goes up to approximately 10% to 40%. The mechanismof the graft failure is unknown, but less likely possibilities include damage to stem cells during the separationprocedure and the possibility that T-cells elaborate growth factors necessary to sustain marrow engraftment. More likely, based on the cytogenetic demonstration of residual host T-cells. is the possibility that somehost T-cells are not destroyed by the preparative regimen and are responsible for marrow graft re.jection. Current studies involve efforts to destroy these residual host T-cells by increasedintensity of chemoradiotherapy before grafting and/or the use of antithymocyte globulin or use of anti-T-cell monoclonal antibodies postgrafting. T-cell depletion.
Opportunistic Infection, Cytomegalovirus (CMV)
Especially Infection
Marrow graft recipients are targets for all kinds of infections because of incompetent defense mechanismsassociated with the underlying disease, the period of granulocytopenia in the first few weeks after grafting, and the period of immunodeficiency that extends for several months after
76
grafting. Interstitial pneumonia due to CMV is a major cause of death following a marrow graft. l7 CMV is latent in approximately 50% of normal people as shown by the presence of anti-CMV antibodies. CMV infection in the marrow graft recipient may be a consequence of activation of a latent virus in the patient or in the donor cells or may be transmitted in blood products obtained from apparently normal donors. It has now been shown that patients who are CMV antibody-negative with a negative donor who receive blood products only from CMV-negative blood donors have a very low incidence of CMV infection. l8 Current studies are directed toward the prevention of CMV activation by the use of passive immunization with immunoglobulins and by prophylactic therapy with antiviral agents. Expansion of the Donor Pool
In our country, only about one third of the patients will have an HLA identical sibling to serve as marrow donor. Current efforts are directed toward identifying other donors in an effort to make marrow grafting available to those individuals who do not have an HLA-matched sibling. One such effort involves studies of the extended family where donors who are partially HLA matched can be identified. l9 More than 200 such transplants have been done by the Seattle team with excellent results when donor and recipient differ by only one of the HLA loci. A major national and intemational effort is underway to create a pool of volunteer unrelated donors of known HLA type so that a phenotypically HLA identical donor can be selected. More than 100 such transplants have now been carried out, but it is too early to evaluate long-term results. Lesser Problems
The list of less frequently encountered graftrelated problems is too long to enumerate completely here. Factors predicting the development of veno-occlusive disease (VOD) of the liver are under study.*’ The incidence of leukoencephalopathy can be reduced by elimination of cranial irradiation from the routine treatment of children with leukemia.2’ The incidence of cataracts has been reduced by the use of fractionated irradiation.**
E. D. THOMAS
Fortunately, secondary tumors have been very rare in marrow graft recipients. PROBLEMS
NOW IN PRECLINICAL
STUDY
Separation of Stem Cells
Sedimentation techniques, monoclonal antibodies, and immunadsorption columns are being studied for removal of other cells from bone marrow (negative stem cell separation) or for the selective removal of stem cells from the marrow (positive stem cell separation). These methods are of interest for concentration of stem cells, and for preparation of stem cells for marrow transplantation that are free of leukemic or other malignant cells. Separated stem cells for marrow transplantation are of particular interest in the applications of autologous marrow transplantation. Biologic ResponseModifiers Interferon. Interferon is being studied for its possible antileukemic effect for treatment of patients before or after marrow grafting.23Y24 Preliminary studies suggest some degree of efficacy in patients with acute lymphoblastic leukemia or chronic myelogenous leukemia. Growth factors. A variety of growth factors are becoming available in quantity through the applications of DNA recombinant technology. 25 The hematopoietic growth factors include erythropoietin, G-CSF, and IL- 1. These growth factors offer promise in stimulating a more rapid marrow recovery after marrow grafting and in preventing graft failure. These growth factors may make it possible to grow stem cells in vitro. IL-2 should permit the growth in vitro of donor T-cells that may have an antiviral effect when given along with the marrow graft. Studies in rodents and now some studies in primates illustrate the exciting possibilities for these agents. Bone-SeekingIsotopes
One of the problems with TBI is that the irradiation is not selective so that all tissues in the body, normal as well as malignant, are irradiated. Boneseeking radioactive isotopes can be used either alone or combined with TBI to increase the irradiation dose to the bone marrow without increasing the irradiation of normal tissues. Studies in the
THE FUTURE
OF MARROW
canine marrow grafting model show promising results that may be applicable to man. Monoclonal
77
TRANSPLANTATION
Antibodies as Transport Agents
Radioactive isotopes bound to monoclonal antibodies react with leukemic cells or with normal myeloid marrow cells can also be used to increase irradiation exposure locally in the marrow. Monoclonal antibodies may be bound to toxins or to chemotherapeutic drugs so that these agents can be specifically directed toward target malignant cells.x.y.26 LABORATORY STUDIES WITH PROMISING FUTURE APPLICATIONS
example, thalassemia major and adenosine deaminase deficiency. The application of gene transfer technology to human patients will almost certainly involve hematopoietic marrow stem cells (reviewed in reference 27). Marrow transplant technology has made these cells readily available for in vitro use and for cryopreservation. Further, marrow transplant technology will be necessary for the reintroduction of these cells into their host and for the destruction of the abnormal cells present in the host. Major problems with gene expression and particularly sustained gene expression indicate that it will be some years before gene transfer technology can be successfully applied to human patients. Understanding Histocompatibility and Tolerctnce
Stem Cell Culture
For several decades it has been recognized that if stem cells could be grown in vitro, then these cells could be used for marrow grafting and for a variety of other studies. Unfortunately, it has not been possible to grow these cells in vitro and, in fact, the identity of the truly pluripotent hematopoietic stem cell (the seed cell that can replicate itself and also develop into all the normal blood cells) in man is unknown. Most of the in vitro assays for colony forming cells are thought to identify cells more differentiated than the true stem cell. With the availability of growth factors cited above, it may become possible to culture stem cells. Perhaps it will be possible for a donor to give 5 or 10 ml of marrow as an outpatient for stem cells to be grown in vitro for a subsequent marrow transplant. Gene Trarwfer
An exciting possibility is that normal genes can be transferred into a patient’s cells to correct a genetically determined disorder. These ceils could then be used for an autologous transplant (since they are of host origin), thus avoiding the risk of GVHD. Several laboratories are intensively studying gene transfer technology by several techniques, the most promising being the use of retroviral vectors. These studies have shown that genes can be transferred in vitro in murine, canine, and primate models. Several genetically determined diseases involve the marrow, and some have already been cured by marrow transplantation, for
Despite numerous studies over several decades, factors governing reactivity between donor and host cells (graft rejection or GVHD) or its absence (tolerance) are still not fully understood. For example, some recipients of HLA-identical marrow grafts do not have GVHD. These patients go onto a long-term state of tolerance between cells of the engrafted marrow and tissues of the host. Other such recipients develop severe and fatal GVHD. Undoubtedly, some of these difficulties are related to histocompatibility differences outside the HLA chromosomal region, so-called minor histocompatibility systems, while some may be due to differences within the HLA system. Molecular biology techniques are making it possible to understand the genetic differences at the DNA level. It may soon be possible to perform tissue typing with probes that detect differences at the DNA level rather than the typing sera now in use that detect differences on the cell surface. Perhaps these studies will shed light on the nature of the histocompatibility differences that determine tolerance on the one hand or GVHD on the other. Recurrent Leukemia
The vast majority, some 95%, of recurrent leukemias in marrow graft recipients have been in cells of host-type, indicating that the chemoradiotherapy preparative regimen did not succeed in destroying all of the patient’s leukemic cells. However, some 5% of recurrent leukemias have been in donor-type cells.28 In most instances, these leukemic donor cells have been recognized by cytoge-
78
E. D. THOMAS
netic studies when donor and recipient are of opposite sex. The mechanisms of malignant transformation of donor cells is unknown. An attractive explanation is that of transfection, a process by which some host DNA could be transferred into a normal donor cell. This host DNA from a leukemic cell might contain an oncogene or some similar
structure that could then be responsible for the malignant transformation of a donor cell. Current studies of the process of malignant transformation, and of oncogenes and their gene products (growth factors), may begin to shed light on the etiology of leukemia.
REFERENCES 1. Thomas ED, Lochte HL Jr, Cannon JH, et al: Supralethal whole body irradiation and isologous marrow transplantation in man. J Clin Invest 38:1709-1716, 1959 2. Thomas ED: Marrow transplantation for malignant diseases (Kamofsky Memorial Lecture). J Clin Oncol 1:5 17-53 1, 1983 3. Coccia PF, Strandjord SE, Gordon EM, et al: High dose cytosine arabinoside (Ara-C) and fractionated total body irradiation (F-TBI) as preparation for bone marrow transplantation (BMT) for childhood acute leukemia in remission-A preliminary report. Proceedings, American Society of Clinical Oncology, 19th Annual Meeting, May 22-24, 1983, San Diego, p 175, (abstr C-680) 4. Blume KG, Forman SJ, O’Donnell MR, et al: Total body irradiation and high-dose etoposide: A new preparatory regimen for bone marrow transplantation in patients with advanced hematologic malignancies. Blood 69: 1015-1020, 1987 5. Santos GW, Tutschka PJ, Brookmeyer R, et al: Marrow transplantation for acute nonlymphocytic leukemia after treatment with busulfan and cyclophosphamide. N Engl J Med 309:1347-1353, 1983 6. Thomas ED, Storb R, Clift RA, et al: Bone-marrow transplantation. N Engl J Med 292:832-843, 895-902, 1975 7. Dinsmore R, Kirkpatrick D, Flomenberg N, et al: Allogeneic bone marrow transplantation for patients with acute nonlymphocytic leukemia. Blood 63:649-656, 1984 8. Vallera DA, Kersey JH, Quinones RR, et al: Antibodyricin conjugates: Purgative reagents for murine and human allogeneic bone marrow transplantation, in Gale RP (ed): Recent Advances in Bone Marrow Transplantation, vol 7, New York, Liss, 1983, pp 209-222 9. Raso V, Ritz J, Basala M, et al: Monoclonal antibodyricin A chain conjugate selectively cytotoxic for cells bearing the common acute lymphoblastic leukemia antigen. Cancer Res 42:457-464, 1982 10. Miller RA, Maloney DC, Wamke R, et al: Treatment B-cell lymphoma with monoclonal anti-idiotype antibody. Engl J Med 306:517-522, 1982
of N
11. Weiden PL, Floumoy N, Thomas ED, et al: Antileukemic effect of graft-versus-host disease in human recipients of allogeneic-marrow grafts. N Engl J Med 300:1068-1073, 1979 12. Schubach WH, Hackman R, Neiman PE: A monoclonal immunoblastic sarcoma in donor cells bearing Epstein-Barr virus genomes following allogeneic marrow grafting for acute lymphoblastic leukemia. Blood 60:180-187, 1982 13. Storb R, Deeg HJ, Thomas ED, et al: Marrow transplantation for chronic myelocytic leukemia: A controlled trial of cyclosporine versus methotrexate for prophylaxis of graftversus-host disease. Blood 66:698-702, 1985
cyclosporine compared with cyclosporine alone for prophylaxis of acute graft versus host disease after marrow transplantation for leukemia. N Engl J Med 314:729-735, 1986 15. Martin PJ, Hansen JA, Buckner CD, et al: Effects of in vitro depeletion of T cells in HLA-identical allogeneic marrow grafts. Blood 66:664-672, 1985 16. Storb R, Weiden PL, Deeg HJ, et al: Rejection of marrow from DLA-identical canine littermates given transfusions before grafting: Antigens involved are expressed on leukocytes and skin epithelial cells but not on platelets and red blood cells. Blood 54~477.484, 1979 17. Meyers JD, Flournoy N, Thomas ED: Nonbacterial pneumonia after allogeneic marrow transplantation: A review of ten years’ experience. Rev Infect Dis 4:1119-l 132, 1982 18. Bowden RA, Sayers M, Floumoy N, et al: Cytomegalovirus immune globulin and seronegative blood products to prevent primary cytomegalovirus infection after marrow transplant. N Engl J Med 314:1006-1010, 1986 19. Beatty PC, Clift RA, Mickelson EM, et al: Marrow transplantation from related donors other than HLA-identical siblings. N Engl J Med 313:765-771, 1985 20. McDonald GB, Sharma P, Matthews DE, et al: Venoocclusive disease of the liver after bone marrow transplantation: Diagnosis, incidence, and predisposing factors. Hepatology 4:116-122, 1984 21. Thompson CB, Sanders JE, Floumoy N, et al: The risks of central nervous system relapse and leukoencephalopathy in patients receiving marrow transplants for acute leukemia. Blood 67:195-199, 1986 22. Deeg HJ, Floumoy N, Sullivan KM, et al: Cataracts after total body irradiation and marrow transplantation: A sparing effect of dose fractionation. Int J Radiat Oncol Biol Phys 10:957-964, 1984 23. Meyers JD, Floumoy N, Sanders JE, et al: Prophylactic human leukocyte interferon after allogeneic marrow transplantation. Ann Intern Med (in press) 24. Talpaz M, Kantarjian HM, McCredie KB, et al: Clinical investigation of human alpha interferon in chronic myelogenous leukemia. Blood 69:1280-1288, 1987 25. Metcalf D: The molecular biology and functions of the granulocyte-macrophage colony-stimulating factors. Blood 67~257-267, 1986 26. Kronke M, Depper JM, Leonard WJ, et al: Adult T cell leukemia: A potential target for ricin A chain immunotoxins. Blood 65:1416-1421, 1985 27. Thomas ED: Marrow transplantation and gene transfer as therapy for hematopoietic diseases, in Molecular Biology of Homo Sapiens, Cold Springs Harbor Symposia on Quantitative Biology, vol 51(2). 1986, pp 1009-1012 28. Boyd CN, Ramberg RC, Thomas ED: The incidence of recurrence of leukemia in donor cells after allogeneic bone