Document bBYO7EyqY3xJa4nZoNGvQgmQ3

PLAINTIFF'S Differentiation-Inducing Agents in the Treatment of Myelodysplastic Syndromes Masahiro Kizaki and H. Phillip Koeffler THE MYELODYSPLASTIC syndromes (MDS) are a group of hematopoietic stem cell disorders, characterized by bone marrow dysplasia and chronic cytopenias leading to frequent hemorrhage, infections, and need for red cell transfusions; these individuals have an increased risk of transformation to acute myelogenous leukemia (AML).'.' The defective maturation, which involve one or more of the hepatopoietic lineages, is the central pathophysiological feature of MDS.'" No specificor effective therapies for MDS exist at this time. Supportive treatment with blood products (red cells and platelets) combined with antibiotics in case of infection has been the basic therapy for MDS. Patients with MDS who receive conventional treatment with combination chemotherapy often have prolonged pancytopenia and the therapy can be fatal in elderly individuals."." Moreover, MDS patients are often too old for bone marrow transplantation. Because cells of the MDS clone phenotypically have a block in differentiation,agents that stimulate differentiation of leukemic cells in vitro may have a potential therapeutic Laboratory studies using leukemic cell lines have provided examples of a variety of agents that are capable of reversing the block in differentiation. These observations have provided the basis for many recent clinical trials aimed at improvingmarrow function in MDS by stimulating differentiation. This approach is particularly attractive in elderly MDS patients who are unable to tolerate aggressive forms of therapie~.'~ TOOLS TO STUDY DIFFERENTIATION OF LEUKEMIC CELLS Acute myeloid leukemia arises from neoplastic transformation at the level of the pluripotent hematopoietic stem cells. These stem cells can either replicate or differentiate to committed stem cells that mature into functional blood cells. Induction of differentiation to mature macrophages and granulocytes by physiologic and nonphysiologic agents was demonstrated in vitro in murine" and subsequentlyhuman leuke- mic cell lines.16The first studiesused the murine myeloid leukemic cell line, M-1, established from spontaneous myeloid leukemia in a SL strain of These cells can be induced to differentiate both in vitro and in vivo into macrophages, and granulocytes by treatment with various compounds including differentia- tion inducing factor (D-factor), colony stimulat- ing factors (CSF), interleukin-1 (ILl), IL6, lipopolysaccharide (LPS), glucocorticoids, 1,25- dihydroxyvitamin D,, dimethyl sulfoxide (DMSO) and 1ectins.l' Another intensively studied cell line is the murine erythroleukemia cell lines (MEL), derived from spleens infected with the Friend virus complex.2oThese cells differentiate to red cells after culture with a variety of chemicals, including planar-polar compounds such as hexamethylene bisacetamide (HMBA), purines and their derivatives, hemin, short- chain fatty acids, as well as inhibitorsof DNA or RNA synthesis, such as ultraviolet irradiation and X irradiation.20 Several human myeloid leukemia cell lines, such as HL-60, 362, KG-1, ML-1, and ML-3 cells, can be induced by various compounds to differentiateinto granulocytes, macrophages, or erythroid cells. These leukemic cell lines are blocked at different stages of maturation: KG-1 cells are myeloblasts; HL-60 cells are promyelo- cytes; ML-1 and ML-3 cells are myelomono- blasts; U937 and THP-1 cells are monocytoid lines; K562 cells have characteristics of very immature cells being able to differentiate down the red cells, megakaryocyte, and macrophage ~~ From the Division ofHematologylOncology, UCLA School of Medicine, Los Angeles, CA,and Division of Hematology1 Oncobgy, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, CA. Supported in part by grants by US Public Health Senrice Grants No. DK4I936-02, CA26038-II, CA43277-0.5, CA33936-08,R86LA022, IRT370, the We& Family Foundation, 4E Leukemia Fund in memory of Marilyn Levine and the Realtors of Real Estate Industry Division to H.P.K. Address reprint requests to H. Phillip Koefler, MD, CedarsSinai Medical Center, Division ofHematologyIOncology, 8700 Beverly Blvd, LosAngeles, CA 90048. Copyright 0 1992 by W.B.Saunders Company 0093- 7754192 I 1 901-0007$05.0010 Seminars in Oncology, Vol 19, No 1 (Februaw), 1992: pp 95-105 95 96 KlZAKl AND KOEFFLER lineages.'6 A variety of compounds induce the differentiation of HL-60 cells towards granulocytes includingDMSO, HMBA, retinoids, interferon alfa (IFNa), and granulocyte-colonystimulating factor." Phorbor esters, 1,25(OH),B, and IFN-I, predominantly induce monocytic differentiation of HL-60, KG-1, ML-1, ML-3, THP-1 and U937 cells.'6.22uK562 cells differentiate towards erythroblasts with synthesis of fetal hemoglobin when cultured with either hemin or butyrate." The use of glucose-6-phosphatasedehydrogenase (G6PD) isoenzymes, karyotypic analysis, and more recently analysis of DNA restriction fragment-lengthpolymorphisms (RFLP) showed that leukemic and preleukemic blast cells can differentiate in vivo to form mature blood cell^.^-*^ Theoretically, therefore, agents that foster differentiationin vitromay have therapeutic potential. The remaining chapter reviews the use of specific agents. COMPOUNDS.USED TO INDUCE DIFFERENTIATION Retinoids Vitamin A is an essential micronutrient and deficiencies of this molecule can cause night blindness and x e r ~ p h t h a l m i aV. ~ita~m~in A and its analogs (retinoids) have been known since the 1920s to influence growth and differentiation of normal and malignant cells. Retinoids are potent anticarcinogenic agents in many experimental models and they inhibit growth and induce differentiation of transformed neoplastic cells.MRetinoids initially were demonstrated to inhibit growth of murine lymphoma and myeloma cell lines.31Subsequently, retinoids were found to have profound effects on leukemia cells in vitro. Retinoic acids (RA) inhibit proliferation and induce differentiation of cells from several human AML cell lines.32All-trans retinoic acid (mns-RA) induces differentiation and inhibits proliferation of HL-60cells.21More than 90% of the cells differentiate towards granulocytes after 72 to 96 hours of exposure to 10-6MtransRA. Growth of these cells is also inhibited by RA33t;rans and 13-ci$forms of RA are equally effective in inhibiting proliferation. U937 cells can differentiate towards mature monocytes by treatment with RA;differentiation of the cells is associated with decreased proliferation without loss of viability." The ML-3 cells show partial monocytic differentiationafter exposureto RA3'; KG-1 cells do not differentiate upon exposure to retinoids, but these cells are very sensitive to the growth-inhibitingeffects of retinoids, showing a 50% inhibition of colony formation at 2.4 x M RA.33In contrast, retinoids stimulate the clonal growth of normal human myeloid and erythroid precursors in vitr0.3~The reason for stimulation of proliferation in vitro of normal hematopoietic progenitor cells and for inhibition of clonal proliferation of leukemic cells is unclear. Mechanism of action of RA on hematopoietic cells is unknown, but is more than a nonspecific toxic effects. Cytoplasmic retinoic acid binding protein (CRABP) was hypothesized as the biological mediator of RA.36However, we were unable to detect CRABP in HL-60, KG-1, or leukemicblasts from patients.32Recently,cDNA clones coding for several retinoic acid receptors (RAR) have been isolated and identified as members of the steroid/thyroid hormone receptor family.37.3T*hese receptors are intracellular proteins that mediate complex effects on development, growth, and physiological homeostasis by selective modulation of gene tran~cription.~~ Cloning of genes coding for human glucocorticoid and estrogen receptors has allowed a detailed biochemical characterization of this family of molecules including identification of discrete DNA- and ligand-binding domains.39 RAR is represented by at least three species, RAR-a,37*38RAR-p,40 and RAR-y.4',42The RAR-CYgene is located on human chromosome 17q22encoding a 462 amino acid protein with a DNA binding domain (C region) and a ligandbinding domain (E region)." The RAR-P gene is expressed in epithelial cells and expression of RAR-y may be restricted to skin." More recently, Mangelsdorf et al# discovered a novel receptor (RXR-a), which is a member of the steroid superfamily and which binds and responds specifically to retinoids.MThese data suggest that different receptors may mediate different functions of retinoic acid. In addition, the temporal and spatial patterns of expression of RAR transcripts suggest that important yet separate roles exist for each of the RAR subtypes during limb morphogene~is.4W~e, as well DIFFERENTIATIONAGENTS IN MDS 97 as others, have examined the expression of subanalysis of 39 nonsideroblastic refractory RAR mRNA in hematopoietic cellsw9 and we anemia patients showed an increase in survival have found that all hematopoietic cell lines in the group who received the retinoic acid. We expressed RAR-y mRNA including KG-1 (my- reported a multicenter trial in which 68 patients eloblasts), HL-60 (promyelocytes), ML-3, were randomly assigned to either 134s retinoic THP-1, U937 (myelomonoblasts and mono- acid or placebo; no significant difference was blasts), W62 (erythroblasts), and S-LB1 (T- noted between the two groups.'* Another study lymphocytes). Steady-state levels of RAR-a suggested that long-term administration of the mRNA were not affected by induction of termi- drug was important and responses can require a nal differentiation of HL-60 cells to either number of months of therapy.@' granulocytes or macrophages. Furthermore, both Hepatotoxicity, as manifested by hyperbiliru- actively proliferating and resting lymphocytes binemia and serum glutamic pyruvic transami- from the same individuals expressed equal con- nase (GOT) elevations, are dose-limiting fac- centrations of RAR-a mRNA. This data sug- tors. Other reported side effects include gest that level of expression of RAR-cx mRNA is cheliosis, mucositis, erythema of the skin, con- not related to cellular proliferation or stage of junctivitis, nausea, joint pains, lethargy, and differentiation. Further studies showed that elevation of serum triglyceride levels?'75A8lpha induction of differentiation of HL-60 by RA is tocopherol taken orally and applied to dry skin mediated through RARa?' Taken together, the can reverse some of the skin toxicity of retinoic molecular and cellular biological data suggest acid. To avoid these side effects, fenretinide that expression of RARa is necessary, but not [N-(Chydroxyphenyl) retinamide], a synthetic sufficient to induce differentiation and inhibit amide derivative of retionic acid has been stud- proliferation of leukemic cells. ied; however, no responses occurred in 14 I At least 150 reported MDS patients have evaluable MDS patients:' Moreover, one pa- been treated with 13-cisretinoic acid in hopes of tient experienced evolution into acute my- improving hematopoiesis and decreasing their elomonocyticleukemia. I incidence of evolution to AML (Table 1). Dose All-trans and 134s retinoic acid are naturally of drug and duration of therapy varied between occurring isomers of retinoic acid. Hung et a16* studies. Response rates were approximately found that all-trans retinoic acid induced 23 20%. Gold et al" reported the first study in 1983 complete remissions in 24 patients with acute showing that 5 out of 15 evaluable patients had promyelocytic leukemia (APL, M3).62Recently, improvement in hematologic parameters; re- Castaigne et a163reported 14 complete remis- sponses were not generally observed until at sions of 22 APL patients using all-trans retinoic least 3 weeks of therapy?' Clark et alS7randomly acid and Warrell et a1- showed that 9 of the 11 assigned 98 MDS patients to either 13-cis reti- APL patients entered complete remission while noic acid or observational control group and receiving all-trans RA. In APL, the RAR-a is found no significant difference in survival be- translocated from chromosome 17 to a gene tween treated and control groups. However, known as my1 on chromosome 15.6'The t(15;17) Table 1. Trials of 1 3 4 s Retinoic Acid Therapy in Mydodysplastic Syndromes Investigators Gold et als1 Greenberg et al" Swanson et aI' Picozziet aly Abraham et a P Kerndrupet al" Clark et al" Koeffler et alY Leoniet al" Total No. of Patients 15 18 10 15 1 6 33 35 20 153 Dose 20-125 mgJm2/d 1-2 mglkgld 2.5-4 mg1kgJd 2.5-4 mglkgld 100 mg/m2/d 20-100 mglmzld 20 mglmzld 100 mg/m2/d 50-100 mg/mz/d Duration of Therapy 7-30 W >8W 8W 8W 16 W 6W 8W 6W >4w Abbreviations: GR, good response; PR. partial response; W, weeks. GR + PR (%I 5 (33) 3 (17) 3 (30) 5 (33) 1 (100) 0 (0) 3 (9) 3 (9) 12 (60) 35 (23) 98 KlZAKl AND KOEFFLER translocation creates a fusion gene between the amino-termillus of my1 and C-terminus of RAR-a. Further studies must determine if the responsiveness of APL to all-tmns RA is either related to unique properties of the fusion protein or reflects the maturity of APL cells allowing them to be more responsive to differentia- tion agents. In summary, the role of retinoic acid for treatment of MDS syndromes is still not fully defined. 1 3 4 retinoic acid may have moderate effects on 20% to 30% of patients, but studies have not shown an increased survival in these patients. Most of these studies had small numbers of patients and were not designed to look at survival. If long-term therapy is required for responsiveness to retinoic acid, only MDS patients with relatively indolent disease would be suitable candidates for this therapy. Future studieswill determine if all-trans retinoic acid is more effective than 1 3 4 s retinoic acid for therapy of MDS. and induction of differentiation of HL-60 cells along either the monocytic or granulocytic pathways does not change their levels of steady-state VDR RNA.= Furthermore, almost all neoplastic hematopoietic cells express VDR. Further studies should elucidate the physiological role of l,Z(OH),D, receptors in hematopoietic cells. The VDR appear to be identical in both normal and neoplastic proliferating hematopoieticcells and their expression, similar to RAR, is probably necessary, but not sufficient for induction of differentiation of normal and leukemic hematopoietic cells. The 1,25(OH),D3induce murine myeloid leukemia cells (Ml) to differentiate to monocyte/ macrophage-like cells as assessedby morphological and functional criteria."' Upon treatment with 1,25(OH),D,, M1 cells become adherent, acquiremorphological characteristicsof mature macrophages, express Fc and C3 receptors, and develop lysozyme and phagocytic activity. Also, the proliferation of M1 cells is inhibited by 1,25(OH),D,. Similar findings were made for 1,25 DlHYDROXYVlTAMlN D, AND ITS the human HL-60 and U937 leukemic cells.n- ANALOGS 75.79-82 A large proportion of HL60cells (30% to The biologically active metabolite of vitamin 50%) became multinucleated and gained the I D,, 1,Z-dihydroxyvitaminD, [1,25(OH),D,], is ability to bind and degrade bone matrix.', This I ' recognized as a major hormonal regulator of hormone also reduced the clonal growth of calcium metabolism in individuals.66Recently, freshly harvested leukemic blast cells from pa- evidence has been obtained for a wider biologi- tients with myeloid leukemia.= In contrast, cal role of 1,25(OH),D3 in tissues not primarily similar concentrations of 1,25(OH),D, slightly related to mineral metabolism. Studies suggest stimulated the clonal growth of normal myeloid that this active metabolite may play an irnpor- committed stem cells; this is similar to the tant role as an immunohematopoietic regula- paradoxical action of re ti no id^.%*^^ The reason tory hormone?'-@' that 1,25(OH),D3 stimulates clonal growth of The 1,25(OH),D, is a sew-steroid that is normal and inhibits clonal growth of leukemic taken up by the target cell and binds to specific hematopoietic cells is unclear. vitamin D receptors (VDR) located in the The ability of 1,25(OH),D3to reduce prolifer- nucleus. The hormone-receptor complex binds ation and induce differentiation of myeloid to DNA and modulates transcriptional and leukemic cells in vitro formed the basis for translationalevents. The putative DNA-binding several small therapeutic trials of the seco- regions of VDRs have strong homologies to the steroid for MDS. Table 2 shows these results. structures of other steroid hormone recep- tor~.@'~T~he receptors for 1,25(OH),D, are by no means limited to the classical target tissues of intestine, bone and kidney, but they have a wide distribution in a number of tissues including hematopoietic cells.66972A-c7tsivated, but not quiescent lymphocytes express large numbers of VDR.'6-n In contrast, nondividing monocytes do express VDR and do respond to 1,25(OH),D, Table 2. Trials of 1,25(OH),D, Therapy in Myelodysplastic Syndromes Investigators No. of Patients Dose Durationof PR + MR Therapy (%) Koeffleretal" 18 2.0rngld 4-20 W 8(44) Metha et ala' 6 l.Omg/d 12 W O(0) RichardetaIm 7 2.5ma/d >8 W O(0) ~~ Abbreviations: PR, partial response; MR, minor response: W, weeks. DIFFERENTIATION AGENTS IN MDS 99 We have treated 18 MDS patients with 2 pg/ tion of higher concentrations of the compound day of 1,25(OH),D, for at least 12 weeks.% than 1,25(OH),D, in our murine model. During the course of therapy, most developed Other znalogs of 1,25(OH),D3 have been an increase in their peripheral blood granulo- synthesized that also have little calcemicactivity cytes, macrophages, and/or platelets. However, but retain the ability to differentiate myeloid no significant change occurred in median levels leukemia cells. 24-homo-1,25(OH),D3 can in- of these cellsbetween the initiation and comple- duce differentiation of HL-60 cells, but do not tion of treatment. Only one patient showed a increase serum calcium when administered to partial response with a 50% increase in plate- vitamin D-deficient rats.% Similar differential lets, granulocytes, and monocytes for more than activity occurs with MC903 [1,24(OH),-22ene- 5 weeks. Seven patients revealed a minor re- 24cyclopropyl-D,], a 1,25(OH),D, analog with a sponse. Six patients had progressive disease cyclopropyl group at the end of the side chain.95 with evolution to AML by the end of the study. 22-oxa-1,25(OH),D3 ( O m ) also has very low Serum calcium rose from a median of 8.9 bone calcium mobilizing activity in vitro.%OCT mg/dL at pretreatment to a peak of 10.8mg/dL exhibited an activity 10 times more potent than during treatment. Five patients developedmildly 1,25(OH),D, in suppressing growth of WEHI-3 symptomatichypercalcemia with anorexia, nau- cells and in inducing differentiation of both sea, polydypsia, polyuria, and/or lethargy. Con- HL-60 and WEHI-3 leukemic cells. The binding centrations of the drug that were required in affinity of O(X for the 1,25(OH),D, receptors vitro to induce differentiation produced hyper- in HL-60 cells was weaker than that of calcemia in vivo. l,Z(OH),D, for its receptors.% This suggests Attempts are currently being directed toward that factors other than the binding affinity for the identification of chemically modified vita- the receptor such as stability,cellular uptake, or min D, analogs that induce hematopoietic cell intracellular metabolism of vitamin D com- differentiation without inducing hypercalce- pounds, probably are also important in separat- mia.@ We identified over 15 new analogs of ing the differentiation-inducing activity from 1,25(OH),D, that were either equivalent or the hypercalcemic action of these new analogs. more potent than 1,25(OH),D, as assessed by In summary, these novel vitamin D analogs (1) inhibition of clonal proliferation of HL-60, are more potent than 1,25(OH),D, in inducing U937, and patients' myeloid leukemic cells; and differentiation and inhibiting proliferation of (2) induction of differentiationof HL-60 ~ells.8~-~' leukemic cells. Moreover, these compounds Also these analogs stimulated clonal growth of appear to have the potential to be markedly less normal human myeloid stem cells. We assessed toxic as measured by the development of hyper- the effects on calcium metabolism of these calcemia. For example, about 10-fold more novel analogs in vivo by intestinal calcium ab- 1,25(OH),-16ene-23yne-D3is required to pro- sorption (ICA) and bone calcium mobilization duce hypercalcemia as compared to 1,25(OH),D3. (BCM). Each of the analogs mediated markedly These novel vitamin D, analogs may prove less (10- to 200-fold) ICA and BCM as com- superior to l,Z(OH),D, in a number of clinical pared with 1,25(0H)2D3.89-9A1mong them, situations including MDS and acute leukemia. 1,25(OH),-16ene-23yne-Dw3 as the most potent In addition, they will provide a tool to dissect modulator of growth and differentiationof leu- the mechanism of action of vitamin D, com- kemic cells.8939T2he therapeutic potential of pounds in promoting cellular differentiation. 1,25(OH),-16ene-23yne-D3prompted us to de- velop in vivo models of AML.wWe found that INTERFERONS WEHI 3BD+,a murine myeloid leukemia line, can be induced by either 1,25(OH),D, or Interferons are a family of proteins sharing the capacity to exert distinctive effects on cell 1,25(OH),-16ene-23yne-D,to lose clonal proliferative capacity as the cells terminally differen- functions. IFNs are secreted by many cell types in response to various stimuli. Interferon gamma tiate. Importantly, 1,25(OH),-16ene-23yne-D3 is produced by immunocompetent T lympho- was 10 to 25 times less active than 1,25(OH),D3 cytes, in response to a sensitizing antigen.97 in causing hypercalcemia, allowing administra- Fibroblasts usually produce only IFN-p.98Leu- 100 KlZAKl AND KOEFFLER kocytes predominantly produce IFN-a and a small percentage of IFN-p.99Interferons render cells resistant to viral growth.@' ' They also play a physiological role in the regulation of immune responses. Depending on the target cell, IFNs can function as differentiation factors, as cellu- lar growth inhibitors, and as modulators of gene expression in differentiated cells. They have also been reported to contribute to control of autocrine growth. Human IFN-a, -p, and -y enhance the differentiation of the myeloblastic leukemic HL-60 cells into macrophages.10'~1I0n4 vitro IFN-y also induces monocytic differentia- tion of blasts from AML and MDS patients.'" Interferon alfa has not been found to be clini- cally effective in AML'06; but it is effective for treatment of patients with chronic myeloid leu- kemia.Im Partial remissions with IFN-a were reported in MDS'OB.'OP;however, confirmatory data from clinicaltrials are not available. In one patient the complete disappearance of the cyto- genetic anomalyof trisomy 8was observed after 34 days of IFN-a treatment, indicative of eradi- cation of the malignant hematopoietic clone.''o Recently, a study examined the effects of long- term application of IFN-a at higher dosages in patients with "low-risk" MDS."' Ten patients were included in the study; 8 were treated for 6 to 36 months. IFN-a was administered at a median dosage of 9, 6, and 4 million units per week during the first, second, and third years, respectively. Responses occurred in 3 of 10 (30%) of the patients; disease progression was thought to have been retarded during the third year of therapy in an additional case. Only a few case studies have been published on treatment of MDS patients with IFN-y in Only minimal benefit was observed. Flu-like symp- toms, which can be debilitating at times, are examples of the side-effects of therapy with interferons. HEXAMETHYLENE BISACETAMIDE AND 5-AZACMIDINE A number of nonphysicological agents induce in vitro differentiation of leukemic cells and some of them have been tried in patients with AML and MDS. Hexamethylene bisacetamide, a low-molecular weight polar-planar compound, was introduced into clinical trials be- cause of its potent tumor-differentiatingcapabilities in v i t r ~ . " ~ -H"e~xamethylene bisacetamide induces differentiation of Friend MEL and human myeloblasticHL-60 cell^."^^^'^ Hexameth- ylene bisacetamide concentrations that induced differentiation in vitro (2-5 mM) inhibited the clonal growth of myeloid and erythroid committed hematopoietic stem cells in 15patients with MDS and these concentrations of HMBA are achievable in clinical trials (1-2 mM).IZOHowever, these same concentrations also inhibited in vitro clonal growth of committed hematopoietic stem cells of normal individuals suggesting lack of a selective cytotoxicity to neoplastic cells. Phase I trials have demonstrated that HMBA produces some hematologic toxicity.'21,1uTherapeutic efficacy of HMBA in MDS is currently being e ~ a l u a t e d . ' ~T'h~es~e~trials are examiningthe effectsof maximally tolerated HMBA concentrations infused for 5 and 10 consecutivedayson peripheral blood cell counts and blast cell fractions. 5-azacytidineis a ring analog of cytidine that is capable of inducing remissions in approximately 20% of patients with relapsed AML.lz 5-azacytidineis incorporated into newly synthesized DNA.'25Incorporation leads to a decrease in the activity of DNA methyl transferase thus reducingmethylationof newly synthesizedDNA; this hypomethylation can modify gene expression.'%Differentiation of HL-60 cells along the myeloid lineage occurs after exposure to 5-azacytidine.'27Therefore, a study was conducted of low-dose 5-azacytidine (75 mg/m' per day) administered as a continuous infusion for 7 days in 11 patients with AML;no patient had a re- sponse as defined by bone marrow remission.1zB However, in 44 evaluable patients with MDS, administration of 5-azacytidine in the same dose and schedule resulted in normalization of peripheral blood and bone marrow in 5 patients (11%)and improvement in an additional 16,for an overall response rate of 48%.129Further clinicaltrials are necessary,however, before this approach can be recommended for therapy of MDS. COMBINATIONS OF DIFFERENTIATION INDUCING AGENTS Numerous reports on low-dose cytarabine treatment in MDS and AML have been pub- DIFFERENTIATION AGENTS IN MDS 101 lished since the initial successful case report in 1979." Recently,combination of differentiationinducing agents with DNA synthesis inhibitors, such as cytarabine, resulted in vitro in a 10- to 1,000-fold greater differentiation of leukemic cells in vitro than either agent a10ne.I~'A phase I1 clinical trial with low-dose cytarabine (5 mg/MZper 12h subcutaneously[SC]) and 134s retinoic acid (60 mg/M2 per day orally) in 14 patients with MDS resulted in one partial and one minor response.13A2 nother innovative study combined IFN-a (3 M U/day, SC), 13-cis retinoic acid (1mg/kg per day) and 1,25(OH),D, (1 p+g/d)for treatment of MDS.'33Four complete and 9 partial remissionswere obtained out of 47 patients. This result was comparable to the response rate of treatment with low-dose cytarabine; in addition, this treatment had a high rate of side effects, perhaps due to IFN-y. This therapy did not appear to prolong survivals of the patients. In conclusion, combinations of cytarabine and inducers of differentiation does not seem to offer a therapeutic benefit to patients with MDS as compared to single agents alone. CONCLUSIONS Myelodysplastic syndromes are clonal hematologic malignancies with an increased risk of transformationto AML. Natural history of MDS ranges from a relatively benign slowly progressive to a very aggressive disease resulting in either life-threatening cytopenias or evolution to AML. Therapies for MDS have included hormones, chemotherapy, bone marrow transplantation, and differentiatingagents. Recently, hematopoietic growth factors have been tested in clinical trials of MDS because they increase the numberof neutrophils, platelets, and erythrocytesof these patients.'"'39However, hematopoietic growth factors, especially granulocytemacrophage colony stimulating factors, have a risk for increasing the number of blast cells in patients with refractory anemia with excess blasts in transition and also chronicmyelomonocytic leukemia.'%139Inducers of differentiation are often well tolerated by elderly patients as compared to cytotoxic agents. However, results with these agents have not been satisfactory. Further studies should continue to try to identify new compounds that can induce differentiation of the most immature hematopoietic stem cells of the MDS clone. In addition, major effort should be directed towards understanding the molecular biology of MDS; such an understanding may permit us to taylor our therapy to correct chemically the molecular defect. REFERENCES 1. 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