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Acute Myelogenous Leukemia: Current Treatment and Future Directions DAVIDM. MASTRIANNI,M.D., NADINEM. TUNG, M.D., DANIELG. TENEN, M.D., Boston, Massachusetts

A

Acute myelogenous leukemia (AML), although an uncommon disorder, is a useful prototype for the treatment of malignancies in general. Significant advances have been made in both the understanding and treatment of this disease. In particular, clinically relevant molecular mechanisms of disease in AML are being defined that hold future therapeutic promise. We review the classification and biology of AML and the current treatment controversies in the use of chemotherapy and bone marrow transplantation, and suggest directions for future research.

pproximately 10,000 patients are diagnosed with acute myelogenous leukemia (AML) in the United States every year [l]. AML is a disseminated clonal proliferation of immature cells that resemble precursors of normal hematopoietic elements; myelogenous refers to cells of nonlymphoid origin; acute refers to the short life expectancy of untreated patients. These features and its relative sensitivity to cytotoxic chemotherapy make this uncommon disease a prototype for the understanding and treatment of cancers. Advances in both the understanding of the pathogenesis of AML and treatment have provided exciting links between the laboratory and bedside. It is our intent to provide an overview of the clinical features, biologic principles, and therapy of adult AML and suggest directions for future basic research.

CLASSIFICATIONOF ACUTE LEUKEMIA Successful chemotherapeutic treatment of acute leukemia, which began with Farber and colleagues [2] in 1948, demonstrated the need for reproducible disease classifications. In 1976, the French-American-British (FAB) cooperative group classified acute leukemias based on morphologic examination of bone marrow and blood in Romanowsky-stained samples combined with the cytochemical myeloperoxidase reaction. Agreement among reviewers was achieved in approximately 70% of cases classified by these initial criteria [3-61, which have since been modified to define subtypes more precisely and include limited immunologic identification of leukemic cells [7,8]. Clinicians now have multiple cytochemical stains and surface membrane phenotypes to supplement the initial morphologic analysis of acute leukemia. Chromosomal banding or studies of molecular gene rearrangement may further refine disease classification. For the clinician, the initial classification of acute leukemia as myeloid (AML) or lymphoid (ALL) profoundly affects therapy. The lymphoid leukemic cells of ALL often stain with periodic acid-Schiff in a block-like pattern [9], and express T- or B-lymphocyte antigens or the common lymphoblastic antigen (CALLA-typical of B-cell lineage ALL) and the nuclear enzyme terminal deoxynucleotidyl transferase (TdT). Expression of T-cell receptors or

From the Hematology/Oncology Division, Beth Israel Hospital, and the Department of Medicine, Harvard Medical School, Boston, Massachusetts. This work was supported in part by Grants CA41456. CA34183, and HL07516 from the National Institutes of Health. DMM is supported by a research training fellowship of the American Cancer Society. DGT is a Scholar of the Leukemia Society of America. Requests for reprints should be addressed to Daniel G. Tenen, M.D., Department of Hematology, Research East 219, Beth Israel Hospital, 330 Brookline Avenue, Boston, Massachusetts 02215. Manuscript submitted June 21,1991, and accepted August 12, 1991.

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TABLE I Subtypes

of Acute Myelogenous

French-American-British FAB) Subtype (Morphology)

Leukemia Staining (MPO/ME)

Selected Surface Antigens

Chromosomal Abnormalities

MO Undifferentiated (not FAB classification)

-l-

Granulocytic markers CD13, CD33 (or CD14)

Ml Myeloid w/o maturation:

+I-

CD13, CD33

M2 Myeloid with maturation:

ti-

CD13, CD33

t(8,Zl)

in 10%

M3 Acute promyelocytic:

+I-

CD13, CD33, tCD15

t(15,17)

in ail

M4 Myelomonocytic: M4Eo variant (25%):

tit

Granulocytic and monocytic markers

M5 Monocytic:

+I+

Monocytic markers CD1 lb, My& CD14

M6 Erythroleukemia:

Jr/--

Glycophorin

M7 Megakaryoblastic:

-l-

Factor VII Ag, factor VIII Ag, CD42

Clinical Syndromes

DIC common

Inv 16 (p13,q22)

Marrow eosinophilia; CNS disease

1 lq23 region often involved

Gum/skin infiltrates; increased lysozyme

Myelofibrosis common

PO = myeloperoxidase; ME = monocyte &erase; DIC = disseminatedintravascular coagulation;CNS = central nervoussystem.

immunoglobulin gene rearrangement may also be useful in identifying cells of lymphocytic lineage [lo]. Acute myeloid leukemias (with the exception of megakaryoblastic leukemia) generally stain for myeloperoxidase and express myeloid surface markers. Five to 10 percent of leukemias have no evident lineage features or have hybrid or biphenotypic characteristics in one or many clones [ll]. Once the diagnosis of AML is made, subtyping requires defining the differentiation of the dominant leukemic blasts (granulocytic, monocytic, erythrocytic, or megakaryocytic) as summarized in Table I [12-E]. In general, both the morphology and surface membrane antigens reflect cell lineage and maturity. Most granulocytic and monocytic leukemias express CD13 or CD33 or both. Leukemias with nondiagnostic morphology that express these antigens (or CD14) have been defined as AML-MO [16]. Clinically, the diagnosis of acute promyelocytic leukemia (AML-M3 or APML), in which the blast cells have abnormal promyelocytelike primary granules visible by light microscopy (typical M3) or by electron microscopy (microgranular variant M3V) [17], now has implications for treatment with all-trans-retinoic acid as discussed below. Other subtypes may form unique clinical syndromes, but currently have less relevance in planning treatment. Ultimately, understanding the molecular basis for these heterogeneous phenotypes (both morphologic and immunologic) will be critical to understanding the pathogenesis of AML and planning future treatments.

CLINICAL PRESENTATION Ninety percent of acute leukemias in adults are myelogenous and incidence increases with age [lS]. Ionizing radiation is clearly leukemogenic, as illustrated by the increased risk of leukemia among patients treated with radiotherapy for ankylosing spondylitis in Britain [19] and the survivors of the atomic bombs in Hiroshima and Nagasaki [20]. Chemotherapeutic agents, particularly the alkylating agents [21], procarbazine [22], and nitrosoureas [23], are well-documented leukemogens. Treatment-related leukemias often evolve from a myelodysplastic syndrome, are usually the M2 or M4 subtypes, and may contain deletions in chromosome 5 or 7 (see below) [24]. Benzene, in addition to its known relationship to aplastic anemia, may predispose to AML, particularly erythroleukemia (M6)

D51.

Classically, acute leukemia presents with cytopenias and peripheral blasts. Neutropenic fevers, anemia, or thrombocytopenia that requires blood products, and metabolic derangements of hyperuricemia, hyperkalemia, and hyperphosphatemia are well known. In approximately 20% of patients, peripheral blast counts are over 50,000/mm3 [26]. An increase in blood viscosity [27], blast cell rigidity [28], agglutination [29], and local metabolic effects place these patients at risk for leukostatic complications, particularly hemorrhage in the central nervous system and pulmonary and renal failure during the first week of diagnosis. The risk markedly increases when the blast count is greater than March

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100,000; in such cases, leukapheresis supplemented with the antimetabolite hydroxyurea is indicated until the blast count is definitively reduced [30]. Distinct clinical features are associated with FAB subtypes. In nearly all patients with acute promyelocytic leukemia (AML-M3 or APML), release of the granular contents of promyelocytes results in laboratory evidence of disseminated intravascular coagulation and is often accompanied by clinical hemorrhage. Heparin may be administered to interrupt the coagulopathy before chemotherapy-induced cell lysis occurs, and factor replacement may be required [31]. The need for anticoagulation has been questioned by the demonstration of heparinresistant thrombin generation and the clinical efficacy of blood product support with rapid-induction chemotherapy [32,33]. The myelomonocytic AML variant, designated M4Eo, is characterized by atypical eosinophilia in the bone marrow and may be associated with myeloblastomas of the central nervous system and leptomeningeal disease [34]. Monocytic (AML-M5) and less commonly myelomonocytic (AML-M4) leukemias are classically accompanied by gum and skin infiltration. Erythroleukemia (AML-M6) often evolves from myelodysplasia and occasionally from polycythemia vera or chronic myelogenous leukemia (CML), and may transform into an AML-Ml or AML-MB subtype [ 181. Hepatosplenomegaly and demonstration of visceral organ involvement at autopsy are not unusual [35]. Megakaryoblastic leukemias (AML-M7) often exhibit striking marrow fibrosis that may preclude a marrow aspiration [36].

growth factors, which have been produced recombinantly, include the glycoprotein granulocyte colony-stimulating factor (G-CSF), which induces granulocytic differentiation; macrophage colonystimulating factor (M-CSF), which induces macrophage differentiation; and granulocyte-macrophage colony-stimulating factor (GM-CSF), which induces differentiation of both cell types (and eosinophils). Interleukin-3 (IL-3) induces a broader spectrum of cells [43]. The response of leukemic cells to these agents varies, but in general IL-3 and GM-CSF increase blast proliferation and are enhanced by G-CSF [44]. Erythropoietin increases proliferation of many erythroleukemias [45]. These proliferative effects (without complete differentiation) suggest roles for these factors in maintaining growth of the leukemic clone. Yet the number of growth factor receptors on leukemic blasts appears to be normal [46]. Constitutive expression of GMCSF by leukemic blasts has been reported in select cases [47], but is of uncertain importance in uiuo [48]. Abnormal secretion of colony-stimulating factors by subclones (particularly monocytic cells) [49], by stromal elements [50], or by internal autostimulation that bypasses membrane receptors is under investigation [51]. A growth factor that acts on early hematopoietic stem cells, termed stem cell factor (SCF), has recently been identified [52]. SCF acts as the ligand for the tyrosine kinase-type receptor c-kit-a receptor known to mediate stem cell proliferation [53]. Analysis of the role of this ligand and receptor in normal hematopoiesis and leukemia is underway.

BIOLOGYOF AML

CHROMOSOMALAND MOLECULAR ABNORMALITIESIN AML

Normal hematopoietic cells arise from a pluripotential stem cell that can self-renew. With early maturation, this ability to self-renew is lost, although the ability to produce more mature progeny remains. Subsequent cell divisions result in the acquisition of lineage traits (“differentiation”) with the eventual loss of proliferative ability (“terminal division”). Leukemias may arise at various points in the hematopoietic hierarchy. CML, for example, arises in an early stem cell and involves multiple cell lines. AML typically arises in a cell committed to the myeloid lineage but may involve multiple cell lines as demonstrated using glucose-g-phosphate dehydrogenase (GGPD) isoenzymes [37] or cytogenetic markers [38]. In culture, most leukemic blasts are hypoproliferative-the progeny of rapidly dividing subgroups (colony-forming units) [39,40] that undergo only limited differentiation [41]. In vitro proliferation of these colony-forming units usually depends on the addition of exogenous growth factors [42]. These 288

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Leukemia probably results from multiple alterations of critical genes. In particular, derangements of highly conserved proto-oncogenes (c-one) have been implicated in the development of AML and of other malignancies. Experimentally, insertion of altered forms of these critical genes into susceptible hosts results in tumorigenesis. Various mechanisms, including point mutations, translocations, or deletions, may result in gene amplification, loss of suppressor genes and their products, or production of abnormal proteins. In AML, mutations of the Nras gene on chromosome 1 can be demonstrated in 25% of patients [54]. The C-MS family encodes a signal-transducing protein that, when mutated, is thought to cause signal overexpression. Translocation of an active gene near a second gene may result in a fusion protein (as in the Philadelphia translocation of CML [55]) or deregulation of a proto-oncogene (c-myc in Burkitt’s lymphoma [56]). Deletions may involve critical protective antioncogenes (as in 92

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retinoblastoma [57] or ~53 [58]) or genes required for normal differentiation (postulated in the 5qsyndrome discussed below [59]). Cytogenetic abnormalities may be detected in 60% to 90% of patients with AML and provide important links between molecular defects and clinical syndromes [60]. The most dramatic evidence of this is the translocation t(15,17)(q22,q12)-the cytogenetic hallmark of both variants of promyelocytic leukemia (AML-M3). This translocation splits the retinoic acid receptor a! on chromosome 17 [61,62]. Normally, retinoic acid plays a significant role in cell differentiation via specific receptors, including RAR-a, and is involved in the regulation of the rate of transcription (of DNA into RNA) of target genes [63]. In the t(15,17) translocation, critical elements of the retinoic acid receptor CYare replaced by DNA sequences from chromosome 15 (termed the PML gene). This translocation creates a fused PMLIRAR-a gene, which produces a novel protein [64]. The fusion protein may act to suppress the normal RAR-a! allele-referred to as a dominant negative mutant-and thus block expression of retinoic acid-controlled genes required for differentiation [65]. Treatment of acute promyelocytic leukemia with all-trans-retinoic acid has been uniquely effective and complete morphologic responses have been obtained, although the duration of effect is uncertain [66-681. Elegant parallel studies of blast cells from patients treated with trunsretinoic acid have demonstrated in vitro differentiation that correlates with in uiuo response [69]. The mechanism of action by which trans-retinoic acid corrects the block in differentiation is under intensive study and may suggest future therapeutic strategies. The most common translocation in AML, t(8,21)(q22,q22), is seen in 10% of patients with M2 leukemias and is often associated with a loss of the sex chromosome [70]. These patients are typically younger and are reported to respond well to initial chemotherapy, particularly when they present with granulocytopenia [71]. The proto-oncogene Hu-ets2, the human counterpart of the transforming gene of the avian erythroblastosis virus, is moved from chromosome 21 to chromosome 8 by this translocation; however, the importance of this event is not yet clear [72]. Another common cytogenetic abnormality in AML is the inversion of fragments of chromosome 16 (invl6[p13,q22]) seen in the myelomonocytic subtype with marrow eosinophilia (M4Eo) [73]. Patients with this variant appear to have a better survival and have been reported to have an increased incidence of disease in the central nervous system [74]. The inversion results in two breakpoints on chromosome 16. On the long arm,

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the metallothionein gene complex was initially reported to be split by the inversion [75], a finding now disputed [76]. Also common are the deletions of the long arm of chromosome 5 (5q-) or 7 (7q-) seen in about 8% of patients with AML; disease in such patients is often associated with chemotherapy or exposure to mutagens or ionizing radiation, and prognosis is often poor. The region of the 5q deletion contains genes encoding such proteins as MCSF, GM-CSF, IL-3 (and other interleukins), and their receptors, including c-fms (the receptor for MCSF). As with the t(15,17) translocation, defining these cytogenetic events on a molecular level will be critical to understanding both the cellular and the clinical pathophysiology of the disease.

CURRENTTHERAPYOF AML Chemotherapy AML is not clinically evident until approximately lOlo tumor cells exist; a lethal tumor burden is 1012 to 1013 cells [18]. Chemotherapy for AML begins with intensive induction treatment designed to cause marrow aplasia and render the leukemic clone undetectable (a complete remission). Induction with cytosine arabinoside and an anthracycline (usually daunorubicin) achieves a complete remission in 60% to 80% of patients [77]. Despite a vast cell kill, lo7 leukemic cells may remain-including lo4 leukemic stem cells [18]. Further intense treatment (variously referred to as “consolidation” or “intensification” therapy) attempts to eradicate the remaining leukemic stem cells. Long-term disease-free survival is reported in 15% to 20% of patients, although late relapses do occur [78]. The most aggressive consolidation therapies, which involve multiple cycles of high-intensity chemotherapy and often include high doses of cytosine arabinoside, result in S-year disease-free rates of 40% to 50% in patients less than 45 years old who achieve a complete remission with induction treatment [79-811. The major obstacle to the cure of AML is leukemic relapse in the bone marrow. Clinically relevant adverse prognostic features include increasing age (which is possibly associated with a more primitive defective stem cell [37]) and history of a treatment-induced myelodysplastic syndrome. Other possible prognostic factors, such as FAB classification or chromosomal abnormalities, may be useful for comparisons of therapeutic trials, but have limited clinical utility at present. The median duration of complete remissions is 1 to 2 years; the response to further chemotherapy after relapse is usually poor. Multiple agents have been tested and the best complete remission rates in selected patients after relapse approximate 40%-responses are rarely longer than 1 year [82,83]. March

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aplasia that requires transfusion support and is associated with infections is well described. Graft failure is infrequent, but is more likely with T-cell depletion. Invariable mucositis may be accompanied by major organ toxicity, including hepatic venoocclusive disease or interstitial pneumonitis (often secondary to cytomegalovirus). Moderate to severe acute GVHD, clinically manifest as rash, hepatic dysfunction, diarrhea, and fever, is reported in nearly half of the patients receiving HLA-identical marrow transplants [87]. Chronic GVHD resembles a collagen-vascular condition with skin, gastrointestinal, hepatic, and joint involvement accompanied by an immunosuppressed state that is exacerbated by treatment. Mild cases of GVHD appear to be associated with better survival-possibly because of an increase in the effect of the graft-versusleukemia interaction. Severe cases may result in fatal multiorgan system disease, are increased in older patients, and can be reduced by immunosuppressive drugs such as methotrexate and cyclosporine. Alternatively, GVHD may be decreased by depleting the transfused marrow of T cells by monoclonal antibodies linked to a variety of toxins (such as ricin, diphtheria toxin, and various chemotherapeutic agents), density separation, or E-rosette agglutination. Unfortunately, T-cell depletion has been associated with increased rates of graft rejection or failure and a higher risk of relapse, which is attributed to a partial loss of the graftversus-leukemia interaction. Selective depletion of cells responsible for GVHD with preservation of cells with graft-versus-leukemia interaction is an important goal of future therapy [87,92,93]. In sum, bone marrow transplant is an effective, but highly toxic, antileukemia therapy. For young patients in first remission with sibling donors who are HLA-identical, the net survival consistently ranges from 40% to 60% [87,94]. Several comparative nonrandomized studies demonstrate a survival advantage for patients who undergo bone marrow transplant; however, comparisons with conventional chemotherapy are highly biased by patient selection [95,96]. Other analyses suggest that given the cure rate with conventional treatment, net survival in the young patients who would be transplant candidates would be similar if transplantation were delayed until the first relapse [97,98]. Bone marrow transplantation may be less effective after relapse, but patients cured by chemotherapy would be spared the potential morbidity of the transplant procedure. Patients in their first remission with a single antigen-mismatched sibling appear to have the same prognosis after bone marrow transplant as patients with complete HLA matches, and a similar analysis of treatment options may apply [99,100].

Failure to eradicate disease reflects an inability of the drugs to differentiate sufficiently between leukemic and normal stem cells. Drug insensitivity of leukemic cells may be worsened by a mutation or an increase in expression of the multidrug resistance gene 1841, alterations in drug transport, changes in the activation or detoxification of the drug inuiuo, or changes in drug targets. Synchronization of leukemic cell cycles, use of drugs with differing mechanisms of resistance, or specific pharmacologic agents to block drug resistance are among the methods proposed for increasing the effectiveness of chemotherapy 185,861. Current practical approaches to drug resistance include dose escalation as seen in intense induction therapy, use of high-dose cytosine arabinoside, and bone marrow transplantation. Allogeneic Bone Marrow Transplantation The fact that the efficacy of many antileukemic agents is dose-related provided the rationale for using hematologically lethal doses of therapy followed by hematologic rescue through marrow transplantation. The success rate of allogeneic bone marrow transplantation (marrow harvested from another person) has been difficult to assess because of the necessity of relying on retrospective or, at best, nonrandomized prospective studies that were undertaken while transplant technology was changing and conventional chemotherapy was becoming more effective. Most transplant data refer to patients treated in remission (initial or subsequent) with regimens such as cyclophosphamide and totalbody irradiation or busulfan and cyclophosphamide and rescued with marrow provided by an HLA-identical sibling [87,88]. The procedure appears to have the greatest efficacy in first remission-when residual disease can be eradicated in most patients. The efficacy of bone marrow transplant depends upon both the treatment regimen and a complex graft-versus-leukemia interaction probably mediated by cytotoxic T cells. That this interaction is antileukemic is supported by the higher rate of relapse in patients with syngeneic transplants (marrow harvested from an identical twin) and in patients given T-cell-depleted marrow in an attempt to decrease graft-versus-host disease (GVHD) (see below) [89]. Relapses after bone marrow transplant are usually due to a failure to control extant disease; occasional relapses are related to the donor marrow [go]. One third of patients under age 45 who receive allogeneic bone marrow transplants die during the immediate peritransplant period, and mortality increases with age; thus, the procedure is used only for patients under 45 years old [91]. Prolonged marrow 290

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Relapse of AML is almost invariably fatal when treated with conventional chemotherapy, whereas bone marrow transplant may offer cure in limited cases [101,102]. In any analysis, it must be recognized that only 10% of patients with AML will be candidates for allogeneic bone marrow transplantation either because of age (median age in AML is 60), lack of a donor, or failure to survive induction therapy [103]. Unrelated HLA-compatible transplantation is now available through donor registries for some patients without compatible siblings, although the logistical and financial obstacles are formidable. Autologous Bone Marrow Transplantation Autologous bone marrow transplantation allows high doses of chemotherapy and radiotherapy to be administered to many patients who are not candidates for allogeneic transplant. Bone marrow, and often peripheral stem cells, are removed from the patient, stored during the administration of highdose chemotherapy, and later reinfused. Because the risk of GVHD is eliminated, autologous transplantation is less toxic than allogeneic transplantation, but its effectiveness is lessened by the absence of the graft-versus-leukemia interaction, and the possible reinfusion of leukemic stem cells. Most centers harvest marrow from patients in a complete remission and administer treatment similar to that used in allogeneic transplants. Given the risk of reinfusing occult leukemic stem cells, “purging” the stored marrow of occult leukemic cells may be attempted in vitro. Successful purging of leukemic cells in rodent marrows using 4-hydroperoxy-cyclophosphamide encouraged human trials using this and related compounds [104]. Leukemia-associated monoclonal antibodies have also been used for the same purpose [105], as has long-term marrow culture to select normal cells for reinfusion [106]. The benefit of these procedures is uncertain. The published results of autologous bone marrow transplant encompass a variety of purging and conditioning techniques. The often significant delay from achievement of complete remission to transplantation (selecting patients who may be already cured) and the young median ages in most series further restrict interpretation. Reports of selected patients undergoing autologous transplantation demonstrate a l- to S-year disease-free survival rate of 30% to 50% of patients in first remission and roughly 20% for patients in second remission. Longer follow-up is clearly needed for significant longterm survival to be demonstrated [107-1091. Because of the potentially wide applicability and relatively low toxicity of autologous bone marrow transplantation, an increase in the antileukemic ef-

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ficacy by improving treatment regimens and purging techniques would have considerable implications for the treatment of AML. Differentiating Agents Inducing terminal differentiation of leukemic blasts is a conceptually attractive therapy for AML because the more mature leukemic cells would not replicate and might be functional. As mentioned previously, all-trans-retinoic acid has been uniquely successful in inducing differentiation in patients with acute promyelocytic leukemia (AML-M3). Complete morphologic responses may be obtained without the risk of hemorrhage induced by cell lysis [66-681. The persistence of a genetically abnormal and presumably unstable leukemic clone suggests that additional ablative therapy will be required for cure. Multiple other agents will induce human myeloid cell lines to differentiate in culture, including polar compounds such as dimethylsulfoxide and hexamethylene bisacetamide, vitamin D3, and such cytotoxic agents as cytosine arabinoside and &azacytidine [llO]. However, many agents that cause established cell cultures to differentiate may be ineffective on heterogeneous fresh leukemic cells or toxic in vitro. Small clinical trials or case reports in elderly patients or those with refractory disease describe transient responses to low-dose cytosine arabinoside [ill].

FUTUREDIRECTIONS:BIOLOGIC UNDERSTANDINGSOF AML THAT HOLD PROMISEFORFUTURETHERAPIES Chemotherapy and bone marrow transplantation have clearly advanced the treatment of AML. Future research in the use of other drug combinations, attempts to synchronize leukemic cell cycles, and methods of overcoming drug resistance are likely to improve the efficacy of chemotherapy. The separation of the graft-versus-leukemia effect from GVHD will hopefully reduce the toxicity of allogeneic bone marrow transplantation and may provide new treatment strategies. Improved techniques of “purging” bone marrow of leukemic cells prior to reinfusion in autologous marrow transplantation may improve the efficacy of this procedure. Identification of more precise prognostic factors, particularly chromosomal abnormalities, will help select treatment. Ultimately, continued advances in understanding the biology of AML may also lead to treatment alternatives that are less toxic and more effective. The demonstration that promyelocytic leukemia (AML-M3), defined by the t(15,17) translocation, is characterized by a disruption in the retinoic acid March

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expression or function of this gene will be studied to determine if it has any role in AML [57]. Similarly, a role has been postulated for the antioncogene ~53 in AML [122,123]. If it can be demonstrated that patients with AML lack these protective genes and their products, appropriate therapeutic replacement strategies could be explored.

receptor (Yand that the blast cells of these patients (in uivo and in. vitro) will differentiate in response to all-trans-retinoic acid provides an exciting link between the hematologist, cytogeneticist, and molecular biologist. Treatments tailored to the molecular defect in other forms of AML may eventually be designed to curb self-renewal of the malignant stem cell, to promote differentiation of the malignant blasts, or both.

Transcription Factors and Myeloid Differentiation The critical role of transcription factors (factors that interact with the complex elements generating RNA from DNA) in inducing normal cell differentiation has become increasingly evident. It is interesting to speculate that a decrease in or lack of a transcription factor required for differentiation of myeloid precursors could be pathogenic in AML. Indeed, transcription factors have been implicated in the pathogenesis of human leukemias through ectopic expression of a “normal” nonmyeloid transcription factor (evi-1) [124] or expression of a hybrid transcription factor formed at the breakpoint of a chromosomal translocation (as in the t[l:19] translocation seen in some cases of ALL) [125]. These findings suggest that isolation of other transcription factors whose abnormal structure or expression (or both) have a role in the pathogenesis of AML may be accomplished by understanding breakpoints in other specific chromosome alterations, such as the common AML translocation t&21) or the inversion of chromosome 16 discussed above. Alternatively, transcription factors could be isolated by analysis of elements that induce the upregulation of myeloid-specific genes expressed in mature monocytes and granulocytes, such as CDllb [126], or, conversely, of factors that induce the down-regulation of genes that are expressed in myeloid precursors and must be turned off, such as CD34 [127]. Whether isolated by these or other genetic techniques, demonstration that specific transcription factors are deficient in AML blasts would suggest that reintroduction of functional genes for these factors could induce blast cell differentiation. The potential for such reintroduction is illustrated by the demonstration that introduction of a single complementary DNA for the myoD gene induces myogenic (muscle) differentiation of fibroblasts [128].

Colony-Stimulating Factors and Blast Differentiation Initially, the use of colony-stimulating factors as differentiating agents in AML was proposed. Yet the relative ineffectiveness of these factors in inducing differentiation of AML blasts [112] and their ability to promote growth of AML blasts have led to concern that administration of these factors may accelerate the leukemic condition [113]. Since some AML blast cells proliferate spontaneously in culture, production of colony-stimulating factors has been demonstrated in some patients [47], and experimental leukemia can be induced by retroviral activation of expression of colony-stimulating factors in mice [114,115], one could consider a therapy designed to block colony-stimulating factors. This may be accomplished either with specific monoclonal antibodies or by introducing genes that produce modified competitive analogues of colonystimulating factors in select patients [116]. Oncogenes, Suppressor Genes, and AML As summarized above, several lines of evidence point toward a role for oncogenes in the pathogenesis of AML [ 1171. Experimental studies suggest that it may be possible to induce differentiation of AML blasts by blocking the expression of specific oncogene products. Blocking expression of the myc or myb oncogene may induce the differentiation of human leukemic cell lines [113-1201. The high frequency of ras-activating mutations in AML suggests a role for this oncogene in AML, perhaps in eliciting production of colony-stimulating factors [121]. Although myc, myb, and n-ras are expressed in a wide variety of cells, success in introducing foreign genes into the stem cells of murine and human bone marrow suggests that it may be possible to target antioncogene therapy specifically to hematopoietic precursors. The identification of the SCF growth factor and its receptor, c-kit (discussed earlier), may aid in this targeting of stem cells for future gene transfer experiments. Antioncogene or “suppressor” genes may be involved in differentiation of human leukemic cell lines. Since the phosphorylation state of the retinoblastoma gene (a prototypical suppressor gene) changes during myeloid differentiation, defects in 292

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CONCLUSION Advances in chemotherapy and bone marrow transplantation make cure possible in select patients with AML. While the heterogeneity of this disease has long been recognized morphologically and clinically, the heterogeneous molecular defects in AML are now being defined. Identification of responsible genetic defects in AML will hopefully 92

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allow the development of specific therapies signed to correct these defects and augment current therapies for this disease.

dethe

ACKNOWLEDGMENT We would like to thank Drs. Stephen Robinson, Paul Eder, and James Griffin for their helpful comments.

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