Investigational New Drugs18: 57–82, 2000.© 2000Kluwer Academic Publishers. Printed in the Netherlands.

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Invited review

Promising approaches in acute leukemia

Jorge Cortes and Hagop M. KantarjianDepartment of Leukemia, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA

Key words:Acute leukemia, therapy, monoclonal antibodies, immunomodulation

Summary

In the last few decades, there has been a significant improvement in the prognosis of patients with acute leukemias.Still, the majority of patients succumb to these diseases. In recent years there has been a great surge in theunderstanding of the molecular mechanisms of disease which have provided us with new targets for anti-leukemiatherapy. These range from chemotherapeutic agents with novel mechanisms of action, such as topoisomerase Iinhibitors or demethylating agents, to reversal of drug-resistance mechanisms, to monoclonal antibodies directedagainst specific antigens, and targeted therapy that inhibit the function of molecules such as tyrosine kinases or Ras.The research on many of these agents is still in the early phases, but these new approaches offer the promise offinding a cure for the majority of patients with leukemia in the near future. Here we describe some of the promisingapproaches that are currently being investigated in the treatment of acute leukemias.

Introduction

The prognosis of adult patients with acute leukemiahas improved significantly over the past 3 decades.Still, most patients succumb to complications of dis-ease progression and therapy With standard chemo-therapy, 60% to 80% of patients with acute myelo-genous leukemia (AML) and 75% to 85% of thosewith acute lymphocytic leukemia (ALL) achieve acomplete remission (CR). Long-term disease-free sur-vival (DFS) rates are 20% to 30%, and 30% to 40%,respectively, with prognosis depending on patient-and leukemia-associated characteristics including age,performance status, cytogenetics, and immunopheno-type [1–5]. This emphasizes our need to discover newstrategies, and perhaps revisit old ones, to improve thelong-term prognosis.

Current investigations aim at discovering ap-proaches which might improve the prognosis of the60% to 75% of patients in whom the disease ultimatelyprogresses with established modalities. A variety ofsuch avenues exist and are probably better investigatedin pilot and later confirmatory trials. These include: 1)

new agents with different mechanism of actionfrom known active drugs; 2) exploring older drugswith new schedules or improved formulations whichoffer a pharmacologic advantage; 3) host immun-omodulation to enhance specific anti-leukemia effect(e.g interferons alpha or gamma, interleukin-2, li-nomide); 3) targeting leukemia-specific molecular orcellular components; and 4) selective enhancement ofleukemia cell differentiation (Table 1).

In this review, promising approaches for adultacute leukemia will be discussed with focus on 1) newor “rediscovered” chemotherapeutic agents, 2) mono-clonal antibodies, and 3) immune modulation.

New and revisited chemotherapeutic agents

There has been a recent surge in the interest in discov-ering new chemotherapeutic agents for the treatmentof acute leukemia. This followed several success-ful discoveries of agents with significant activity inboth solid tumors and hematologic malignancies (e.gtaxanes, topo I inhibitors, navelbine, gemcitabine).

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Table 1. Promising approaches in acute leukemia therapy

1. Agents with novel antileukemic mechanisms, e.g., topoisomerase I inhibitors

2. New formulations of older drugs, e.g., liposomal agents, IV 6-MP

3. Dose intensive therapy, e.g. allogeneic and autologous stem cell transplantation

4. Leukemic cell sensitization e.g. G-CSF, GM-CSF, IL-3, other cytokines

5. Reversing leukemia drug-resistance e.g. MDR blocking agents

6. Immune modulation e.g. interferon alpha or gamma, interleukin 2, linomide

7. Cellular component therapy e.g. allogeneic or modified autologous lymphocyte re-infusion

8. Cellular targeted therapy e.g. CD33-monoclonal antibodies with or without immunotoxins

9. Selective enhancement of leukemia cell apoptosis e.g, anti-bcl-2, retinoids + cytokines

10. Leukemic cell differentiation e.g. retinoids

11. Gene-directed therapy, e.g. tyrosine kinase inhibitors, Ras inhibitors

I. Nucleoside analogues

The class of nucleoside analogues has been amongthe richest source of agents with active antileukemiceffects (Table 2).

A. Deoxyadenosine analogues

These include fludarabine and chlorodeoxyadenosineboth of which have demonstrated major efficacyin lymphoproliferative disorders including chroniclymphocytic leukemia (CLL), indolent lymphoma,hairy cell leukemia and Waldenström (macroglobu-linemic) lymphoma.

1. FludarabineThe phase I studies of fludarabine in acute leukemiashowed definite anti-AML activity, but at doses of 50to 100 mg/m2 daily for 5 days (i.e., total dose 250 to500 mg/m2 per course), a dose schedule associatedwith significant central and peripheral neurotoxicity(depressed consciousness, coma, blindness, paralysis)[6,7]. However, when used at doses shown to be effect-ive with acceptable toxicities in indolent lymphomaand CLL (25 to 30 mg/m2 daily for 5 days, total dose125 to 150 mg/m2 per course) fludarabine could in-crease phosphorylation of ara-C, increasing 3 to 5 foldthe intracellular levels of its active compound ara-CTPin leukemic cellsin vivo [8,9]. This led to a seriesof investigations of fludarabine and ara-C in differentschedules, either alone (FA), or combined with leuk-emic sensitizing agents such as G-CSF (FLAG), orother cytotoxic agents such as idarubicin (FLAG-IDA)in both AML and ALL [10–14]. In AML salvage, thefludarabine-ara-C combination was associated with atrend for higher CR rates compared with intermedi-

ate or high-doses of ara-C alone, particularly in themore sensitive leukemias, i.e. those associated with along first CR duration [10]. Fludarabine is currentlyused in many frontline and salvage trials in combina-tion with anthracyclines and ara-C [10–14]. However,comparative trials of standard induction-consolidationtherapy in AML with or without fludarabine in patientsubsets (e.g., good versus poor risk AML) are neededbefore fludarabine is established as beneficial in AMLtherapy.

2. 2-chlorodeoxyadenosine (2-CDA)2-CDA was reported to have significant activity inchildhood AML, with 50% of children treated in sal-vage achieving CR [15]. 2-CDA was then used innewly-diagnosed childhood AML before initiation ofstandard therapy (“therapeutic window”), producingCR rates of 27% after 2-CDA alone, and 72% aftermulti-agent chemotherapy [16]. In adult AML, twophase I studies using 2-CDA at doses up to 5 to 21mg/m2 daily for 5 to 7 days (cumulative doses of25 to 105 mg/m2 per course) produced three object-ive responses among 57 patients treated with onlyoccasional toxicity (neurologic, renal, prolonged my-elosuppression) [17,18]. The combination of 2-CDAand ara-C potentiates the antileukemic effect as a res-ult of the incorporation of 2-CDAMP and ara-CTP,inhibition of ribonucleotide reductase, and increasedaccumulation of ara-CTP, resulting in significant in-hibition of DNA synthesis [19]. Salvage therapy withconcomitant administration of 2-CDA and ara-C inAML resulted in 2 CR among 15 patients treated(13%) [18].

New deoxyadenosine analogues (e.g. chlorofluoroara-A) may also be effective and should be pursued[20].

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Table 2. Nucleoside analogues of interest in acute leukemia

Deoxyadenosine analogues Deoxythimidine analogues

Fludarabine 5 fluorouracil

2′-chlorodeoxyadenosine Antiviral agents (e.g. AZT, DDI)

Deoxycoformycin

Chloro-fluoro adenosine arabinoside

Deoxyguanosine analogues Deoxycytidine analogues

6-thioguanine Cytosine arabinoside (ara-C)

6-mercaptopurine Gemcitabine

Guanosine arabinoside (ara-G) Decitabine

Compound 506 (GW506U78, ara-G prodrug) Azacytidine

Tiazofurin Troxacitabine (BCH-4556)

Fazarabine

B. Deoxyguanosine analogues

1. Guanosine arabinoside (ara-G) and compoundGW506U78Preclinical studies indicated the selective antileukemiceffect of ara-G in T cell leukemia [21]. However,ara-G is insoluble preventing its clinical development.Compound GW506U78 is a methoxy derivative ofara-G which upon administration is rapidly demeth-oxylated to ara-G, the active compound [22]. Ini-tial phase I clinical studies of compound 506U78 inchildhood and adult leukemia documented signific-ant responses in T-cell acute lymphoblastic leukemia,T-lymphoid blast phase chronic myeloid leukemia(CML), T-cell lymphomas and B-chronic lymphocyticleukemias (B-CLL) [22–24]. Among 13 evaluable pa-tients with these diseases, 5 (38%) achieved a CR and5 (38%) a partial remission (PR). Patients with B-ALL, B-lymphoma, AML or T-CLL did not respond[22,23]. Response correlated strongly with intracel-lular accumulation of ara-GTP [22]. The maximallytolerated dose was reported to be 40 to 60 mg/kg/dayfor 5 days and was age-dependent, with peripheralneuropathy (numbness, paresthesias, paresis) beingthe dose limiting toxicity [24]. Ongoing studies inboth childhood and adult hematologic malignanciesare investigating different schedules (i.e., day 1, 3,and 5 schedule) and using biochemical modulationwith fludarabine which can significantly increase ara-G phosphorylation and accumulation of ara-GTP [25].These schedules have produced remissions in patientswith refractory CLL [25].

Tiazofurin has also demonstrated activity in acuteleukemia and CML in blastic phase. The dose sched-

ule targeted the biologic endpoint of inhibiting inosine5′-phosphate dehydrogenase which, when combinedwith inhibition of the guanine-salvaging activity ofhypoxantine-guanine phosphoribosyltransferase, res-ults in suppression of guanosine triphosphate (GTP)pools [26]. However, this dose schedule was associ-ated with significant toxicity including neurotoxicity,pericarditis, renal dysfunction and others. Improvingthe therapeutic toxic ratio of Tiazofurin through dose-schedule modulations may render it a more attractiveinvestigational agent.

2. Intravenous 6-mercaptopurine (6-MP) and6-thioguanine (6-TG)

Both 6-TG and 6-MP have shown anti-leukemic ef-ficacy and have been used as part of induction (e.g.,6-thioguanine, daunorubicin, ara-C [TAD]) and main-tenance therapy in AML and ALL. Both drugs havebeen used in oral formulations because of ease of ad-ministration. However, oral 6-MP has erratic absorp-tion with bioavailability of 20% to 60%, with first-passrelated hepatic toxicity [27]. In childhood ALL, serumconcentrations of 6-MP are variable, and a directrelationship exists between 6-MP concentration-timecurve and relapse-free survival [28]. The intracellularlevels of the active metabolite of 6-MP, thioguan-ine nucleotides (6-TGN), are also variable, with lowlevels associated with a poor prognosis [29,30]. Anintravenous 6-MP formulation has been available forover 40 years, but has only recently been incorporatedmore extensively into the therapy of acute leukemias[27]. It offers 100% bioavailability, higher drug deliv-ery without dose-limiting hepatotoxicity, avoidance of

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patient non-compliance with oral therapy, and cyto-toxic levels can be achieved in the CSF [27]. Asimilar dose-response relationship exists for metho-trexate (MTX) [31], and regimens using intravenous6-MP and MTX have reported improved results inchildhood [32–34] and adult ALL [35], but studiesdirectly comparing oral versus intravenous 6-MP areneeded. 6-MP has also demonstrated synergistic activ-ity with ara-C, increasing the intracellular levels ofara-CTP [36]. The combination of high-dose intraven-ous 6-MP and high-dose ara-C resulted in CR in 6 of13 children (46%) with refractory or relapsed AMLand in 1 of 10 (10%) with ALL [37]. This combin-ation was also used in post-induction chemotherapyfor children with newly diagnosed AML, with 7 of the14 children treated remaining in complete remission[38]. Late intensification with a combination of mer-captopurine, vincristine, methotrexate, and prednisone(POMP) prolonged survival in adult AML [39]. Theseresults indicate the need to revisit the role of intraven-ous 6-MP combinations in both childhood and adultAML and ALL.

6-TG has been used mostly in AML in the TADregimen and in maintenance (6-TG + ara-C). 6-TGis better tolerated than 6-MP, induces 4-fold higherintracellular levels of 6-TGN [40], and is probably amore potent cytotoxic drug than 6-MP [41]. A phaseI study in children showed that doses of 20 mg/m2/hadministered intravenously in a continuous infusionover 36 hours result in plasma concentrations wellabove cytotoxic concentrations with myelosupressionas the dose-limiting toxicity [42]. Further investigationof intravenous 6-TG in acute leukemias is warranted.

C. Deoxycytidine analogues

Based on the major activity of ara-C in acute leuk-emias and other hematologic disorders, other deoxy-citidine analogues have been explored. Interestingly, arelated structural analogue, gemcitabine, with signific-ant activity in solid tumors has shown little activity inleukemia. Despite the structural similarities of ara-Cand fazarabine, azacytidine and decitabine, fazara-bine was inactive while the latter two, through novelmechanisms of action, might prove complementary orsynergistic with ara-C in leukemia.

1. Azacytidine, decitabineHypermethylation is a general property of tumor pro-gression and resistance: it has been reported in 10to 50% of patients with newly diagnosed hematolo-

gic malignancies, and in 100% of recurrent ALL andCML in transformation [43]. Both azacytidine anddeoxyazacytidine are potent hypomethylating agents.Azacytidine has established efficacy in AML and isoffered on a compassionate basis for patients with re-fractory AML [44–46]. In myelodysplastic syndrome(MDS), azacytidine in a low-dose schedule has pro-duced CR in 10% to 15% of patients and objectiveresponses in 35% to 50% [47]. It is currently under-going phase III comparative trials with the standard ofcare in MDS.

Decitabine has been investigated mostly in Europe,with encouraging activity observed in AML, MDS andchronic myelomonocytic leukemia (CMML) (Table3). In AML salvage, decitabine has induced CR in5% to 30% of patients as a single agent, and in 25%to 50% in combination with anthracyclines [48–52].Schwartzman et al. reported 6 CR in 8 patients (75%)with newly-diagnosed AML treated with daunorubicin50 mg/m2 daily for 3 days and decitabine 90 mg/m2

daily for 5 days [52]. Lower dose schedules of de-citabine (15 mg/m2 over 4 hours every 8 hours×3 days; 135 mg/m2 per courses) have also producedobjective responses in 40% to 50% of patients withCMML and MDS. Wijermans et al. [53] treated 29elderly patients with high-risk MDS with decitabine40 to 50 mg/m2 daily for 3 days by continuous infu-sion. Eight achieved a CR (28%), 5 had a PR (17%),and 2 improved, for an overall response rate of 54%.Five patients (17%) died during induction. The me-dian remission duration was 8 months and the mediansurvival 11 months. Similar results were reported byPinto et al. [51] treated 46 patients (36 evaluable, me-dian age 69 years) with decitabine 405 mg/m2 percourse. In MDS, 8 of 35 patients achieved a CR and5 had PR. In CMML 3 of 11 patients achieved CRand 5 had PR. The CR duration was 10 months. Re-sponses required a median of 3 to 4 courses of therapyto develop fully.

Decitabine is associated with an unusual pattern ofdelayed and prolonged myelosuppression, indicatingits potential value as part of dose-intensive regimensfollowed by stem cell support to improve antitumorefficacy, or as salvage therapy with stem cell support[54]. In CML transformation, decitabine has producedobjective responses in 25% of patients in blastic phase,and in 40% of patients with accelerated phase [55].Hypomethylating agents should be investigated fur-ther in hematologic malignancies and perhaps lymph-oproliferative disorders, and as part of regimens usingstem cell support. Correlating the degree of tumor-

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Table 3. Decitabine in hematologic malignancies

Disease (reference) No. CR (%) Overall

response∗ (%)

AML salvage

Momparler [48] 6 2 (33)

Debusscher [49] 20 1 (5)

Willemze [50]

DAC+AMSA 30 8 (27)

DAC+Ida 33 15 (45)

Overall 63 23 (37)

Kantarjian [55] 8 1 (12)

Previously untreated AML

Schwartsmann [52] 8 6 (75)

MDS and CMML

Wijermans [53] 29 8 (28) 15 (54)

Pinto [51]

MDS 35 8 (23) 13 (37)

CMML 11 3 (27) 8 (73)

CML

Kantarjian [55]

Accelerated 17 5 (29) 8 (47)

Blastic 20 2 (10) 5 (25)

∗Overall response = Complete or partial remission, or hematologicimprovement (MDS, CMML), or complete or partial remission, orback top chronic phase (transformed CML).

specific DNA methylation patterns with response willhelp selecting patients who may benefit from decit-abine therapy. In AML, the value of decitabine couldbe evaluated in all patients as part of combinationchemotherapy or in high-risk patients who have com-pleted induction-consolidation, and who could then berandomized to no further therapy or to maintenancetherapy with decitabine. Based on thein vitro syn-ergism between decitabine and topo I reactive agents[56], such combinations should also be investigated inleukemia.

2. Other deoxycitidine analogues

Fazarabine combines properties of both ara-C and aza-cytidine. Despite encouraging preclinical data, thephase I-II study of fazarabine in acute leukemiashowed little activity. It also required about 15 dose es-calations from the phase II solid tumor dose limited bymyelosuppression (from 2 mg/m2 by continuous infu-sion daily× 5 up to 450 mg/m2 daily× 5). Among 72

patients entered on study, only one objective responsewas noted [57].

Gemcitabine (2′,2′-difluorodeoxycytidine; dFdC)is active in several solid tumors, andin vitro stud-ies showing activity against leukemia cell lines [58–60] stimulated interest in gemcitabine in acute leuk-emias. Pilot studies suggested that a dose rate of 10mg/m2/minute produce plasma concentrations greaterthan 20µmol/L and maximized the rate of dFdCTPaccumulation [61]. Hence phase I studies used a fixedincremental design of dose-per-unit time, where doseintensity was increased by prolonging the duration ofthe infusion at a fixed dose rate. A phase I study ofgemcitabine given weekly defined the MTD at 4800mg/m2 over 480 minutes, and was dose limited bymucositis and skin rashes [62]. No complete responseswere seen in this heavily pretreated group of patients[62]. A second phase I study of gemcitabine givenevery 3–4 weeks is ongoing with the current doseschedule at 12,500 mg/m2 over 1250 minutes (Es-tey E. – unpublished data). Another potential rolefor gemcitabine in acute leukemias is to increase theactivity of deoxycytidine kinase, which is the rate-limiting enzyme in the phosphorylation of ara-C intoits active metabolite [63]. This results in a 3-fold in-crease in intracellular accumulation of ara-CTP, andincreased cytotoxicity in leukemic cell lines [63,64].Gemcitabine could thus be used for modulation ofara-C activity in acute leukemia in a similar way asfludarabine. The higher intrinsic cytotoxic effect ofgemcitabine compared with fludarabine [64] makesthis modulation an interesting approach.

II. Topoisomerase I inhibitors

Uncoiling of DNA is through an excisional processcarried by two essential cellular enzymes, topoi-somerase II (topo II) for double-stranded DNA, andtopo I for single stranded DNA. Topo II inhibitors haveestablished anti-tumor activity and are part of currenttreatment schedules in several solid and hematologicmalignancies. Camptothecin (CPT) was investigatedin the 70’s and abandoned as being too toxic. The re-cent discovery of the topo I inhibitory effect of CPTrenewed interest in the drug and its analogues. TopoI levels were found to be significantly higher in tu-mor cells compared to their normal counterparts [65].Hence, inhibition of topo I enzyme may provide aselective tumor versus normal cell kill [65-67].

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1. Topotecan

Topotecan is a semisynthetic analogue of the alkal-oid camptothecin which acts as a specific inhibitor oftopoisomerase I by stabilization of the topoisomeraseI-DNA complex leading to cell death. Two phase Istudies of topotecan as a continuous infusion scheduleover 5 days in acute leukemia defined the MTD at 2mg/m2 daily for 5 days, with mucositis as the dose-limiting toxicity. Objective response were observedin these trials with reduction in the number of circu-lating blasts in all patients included in one trial, andCR in 11% in the other [68,69]. A phase II studyof topotecan as a single agent in our institution didnot result in objective CRs among 14 patients withrefractory or relapsed AML (unpublished data). Thisfinding, together with the results obtained in MDS andCMML (discussed later), emphasize the risk of false-negative results even when using active agents, aspatients are treated with more effective frontline ther-apy and relapsing with more refractory disease. Usingan upfront “window” therapy approach has been sug-gested to explore activity of potentially effective drugsin a better-risk population but this is still controversial.

Topotecan was first investigated in MDS andCMML based on: 1) similarities between MDS andAML in terms of disease biology and treatment re-sponse; 2) less exposure of MDS than AML tochemotherapy thus providing a “therapeutic window”of opportunity to investigate topotecan in relativelychemotherapy-naive patients; and 3) the poor resultswith standard therapy. Sixty patients with high-riskMDS (refractory anemia with excess blasts [RAEB],or in transformation [RAEBT]) or CMML received to-potecan 2 mg/m2 as a continuous infusion for 5 days,every 3–4 weeks. The CR rate was 30% and was sim-ilar for MDS and CMML [70]. Patients with mutationsin the RAS-gene had a lower response rate, as didthose with monocytosis or thrombocytopenia.

Because of the positive results, combination ther-apy of topotecan with other agents, including ara-C, etoposide, anthracyclines, and cyclophosphamidewere initiated in different dose schedules in MDS andAML [71–73]. The combination of topotecan 1.25mg/m2 continuous infusion daily for 5 days and ara-C 1 g/m2 daily for 5 days was used to treat 86 patientswith MDS (n=59) or CMML (n=27), of which 57were previously untreated. CR was achieved in 48 pa-tients (56%), and it was higher in MDS (61%) thanCMML (44%) [73]. Preclinical studies indicated thattopoisomerase I inhibitory agents may potentiate DNA

damage induced by exposure of tumor cells to radi-ation or alkylating agents (e.g. cyclophosphamide) in aschedule-dependent way by preventing repair of DNAdamage [74,75]. Designed on the basis of this syn-ergism, the combination of cyclophosphamide, ara-Cand topotecan has been investigated in patients withrefractory or relapsed AML with a reported responserate of 23% [76], and is currently being evaluated infront-line therapy.

Topotecan is also available in an oral formulationwhich may offer practical, and perhaps more effect-ive longer-exposure schedules (21-day oral topotecanschedules) in indolent hematologic disorders (MDS,CMML, lymphoma, myeloma) [77].

2. Other topoisomerase I inhibitors

Camptothecin-11 (CPT-11) showed activity in the ini-tial phase I studies in acute leukemia [78] whichhas been confirmed in early phase II trials [79]. 9-aminocamptothecin (9-AC) has promising preclinicalactivity, and phase I studies of 3- to 21-day intra-venous and oral schedules are under investigation.9-nitrocamptothecin (9-NC) also offers the advantageof oral route delivery, and has produced objective re-sponses in solid tumors, and in MDS, CMML andPh-negative CML [80]. Correlative studies of topo I-DNA complex formation and response may help selectpatient who benefit specifically from therapy with topoI inhibitors.

III. Homoharringtonine (HHT)

HHT is a plant alkaloid derived from the bark ofCe-falotaxus fortuneii. Its antineoplastic mechanism ofaction is not clear, but it inhibits protein synthesis [81]resulting in cytotoxicity. HHT also induces differenti-ation of leukemic cell-line HL-60 into cells resemblingmonocytesin vitro [82], and of fresh leukemia cellswhen combined with cytarabine [83]. HHT has beenused in China for the treatment of acute and chronicleukemias, alone [84,85] and in combination withother agents [86,87]. Phase I studies in the UnitedStates with HHT bolus schedules, showed hypoten-sion, arrhythmias and myelosuppression to be dose-limiting [88,84], but hypotension and arrhythmiaswere reduced significantly when HHT was given ina continuous infusion schedule [89–91]. Several in-vestigators have used HHT alone or in combinationsfor the treatment of patients with acute leukemias,

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mostly myeloid (Table 4). These trials used variabledose schedules and resulted in response rates of 0% to25% in patients with refractory or relapsed AML [92–96]. Warrel et al. reported 7 CRs among 28 patients(25%) treated with a total dose of 45 mg/m2 or higher,given as a seven-day continuous infusion of 7 mg/m2

daily or a nine-day continuous infusion of 5 mg/m2

daily [92]. The higher dose was associated with hy-potension in 63% of patients. Feldman et al. used asimilar schedule and reported CR in 16% of patients,with hypotension in 44% of patients [93]. Hypotensionwas also a significant problem in 40% of patients in anECOG study using a total dose of 52.5 mg/m2 over8 days, and no CR were achieved [94]. In contrast, aschedule of 2.5–3.0 mg/m2 daily continuous infusionover 15 to 21 days (total dose 37.5 to 52.5 mg/m2)resulted in a response rate of only 3%, but cardiovas-cular complications were minimal [95]. Feldman etal. used HHT 5 mg/m2 daily continuous infusion for9 days in patients with newly diagnosed myelodys-plastic syndrome (MDS, n=16) or AML evolving afteran MDS (n=12). Seven patients (25%) achieved a CR,and hypotension was reported only in 3 patients [96].HHT has also been used in combination with ara-Cin 22 patients with relapsed or refractory AML whoreceived ara-C 100 mg/m2 continuous infusion dailyfor 7 days and HHT 1.5 to 5 mg/m2 CI daily for 7days [97]. Five achieved CR (23%), and four of theresponders received doses of 4 mg/m2 which was thedose recommended for further studies. Interestingly,4 of the 5 responders in this study had acute promy-elocytic leukemia (APL). Responses in patients withAPL have been reported in other small series [98–100] ranging from 46% to 100%. In one study of 10patients with APL (nine were previously untreated),CR was achieved in 7 (70%), and treatment producedbone marrow aplasia in only one, while 3 had mildhypoplasia, and six maintained a hypercellular bonemarrow [100]. Feldman et al. reported a 60% remis-sion rate in patients with relapsed APL [98]. Theseresults, together with the differentiating properties ofHHT [82,83], and the sensitivity of the HL-60 cellline to HHT compared to other leukemia or lymph-oma derived cell-lines, suggest that HHT could be aninteresting agent in patients with relapsed APL [112].Only a few patients with ALL have been treated withHHT, and no responses were observed [92,93].

Therefore, there is clear evidence that HHT hasantileukemic effect, and trials of combinations includ-ing HHT are warranted. Particularly intriguing is itspotential role in patients with APL.

IV. Alkylating agents

Alkylating agents have been used for a long time inthe treatment of hematologic malignancies and solidtumors. The availability of new alkylating agents, andnew formulations and schedules for old alkylators,have renewed the interest in this group of drugs.

1. Tallimustine

Tallimustine, a distamycin A derivative, is a novel al-kylating benzoyl mustard which alkylates N3 adeninein the minor groove of DNA with very stringent se-quence specificity [101]. This mechanism of actionis different from other alkylating agents and thereforeresults in different cell cycle perturbation compared tothe more conventional alkylators [102], and differentmechanism of resistance [103]. Although significantactivity was found in preclinical studies against avariety of malignancies, initial trials in solid tumorshave yielded modest results [104,105]. In phase I andII trials the dose limiting toxicity was neutropenia,which prompted the interest of tallimustine in leuk-emia [104–105]. In a human leukemia model usingSCID mice, tallimustine at 0.86 to 3.0 mg/kg dailyfor 3 days resulted in the cure of most mice [107].Based on this model, a phase I trial was conductedin patients with refractory or relapsed acute leukemia.The MTD was 900 mcg/m2 daily for 3 days, every 3to 4 weeks, which is 3 times higher than the MTDin solid tumors, with the dose limiting toxicity be-ing mucositis. Magnesium and potasium wasting werealso observed. Two of nineteen patients (11%) withAML or AUL achieved CR, and two others had a mar-row CR but with persistent thrombocytopenia [107].Based on the SCID mouse model demonstrating thesafety and efficacy of combining tallimustine with ara-C, studies with combinations including tallimustineare needed, as well as additional trials of tallimustinein other hematologic malignancies (lymphomas, my-eloma, Waldenström’s macroglobulinemia).

2. Platinum analogues

Despite the low degree of activity of cisplatin inhematologic malignancies, a synergistic effect of cis-platin and ara-C was demonstrated bothin vitro andin clinical trials in lymphoma. Combination studiesof cisplatin plus amsacrine, or cisplatin with VP16,ara-C, and steroids (ESHAP) have resulted in highCR rates (Kantarjian H. – unpublished data). Phar-macologic modulation of cisplatin with fludarabine

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Table 4. Results with homoharringtonine (HHT) in AML

Author (reference) No. CR (%) Comments

Warrell [92] 31 7 (23) – 7/28 CR when cumulative dose≥ 45 mg/m2

– Hypotension in 63% using 7 mg/m2 ×7 days

Feldman [93] 57 7 (12) – All CR among patients with relapse AML; no CR among 14 patients withprimary refractory AML

Stewart [94] 20 0 (0)

Kantarjian [95] 31 1 (3) – Low-dose, continuous infusion for 15 to 21 days

Feldman [96] 22 5 (23) – HHT + ara-C

– No CR among 8 patients with primary refractory disease

–4/5 CR among patients with APL

results in increased cytotoxicity and inhibition of re-pair of cisplatin-induced cross links [108]. Other plat-inum analogues have also been investigated. The mostpromising results were achieved with carboplatin,which was associated with CR rates of 5% to 20%in patients with refractory or relapsed AML or ALL[109,110], and of 40% to 60% in newly-diagnosedhigh-risk AML. Salvage therapy with carboplatin-based combination regimens have resulted in CR ratesof 8% to 50% [110–116].

CI-973 is a new platinum analogue with signific-ant anti-leukemic effect against a variety of leukemiacell lines at concentrations 10-fold lower than the peakplasma levels achieved in phase I trials for solid tu-mors [117]. In a phase I study of CI-973 in acute leuk-emia, no responses were achieved among 22 patientswith refractory or relapsed acute leukemia (18 AML,4 ALL) [118]. There might be a role for platinumanalogues, particularly carboplatin, in combination forsalvage therapy, or as frontline/consolidation therapyin AML, but further studies are required.

3. Other alkylating agents

Alkylating agents have been used for the treatment ofacute leukemias mostly in the context of bone marrowtransplantation, but may have a role in the setting ofnon-myeloablative therapy. The self-renewal capacityof AML progenitors, rather than terminal divisionsmight be differentially affected by alkylating agents[119,120]. Some alkylating agents may be more ef-fective inhibitors of self-renewal capacity than ara-C[120]. Therefore, drugs such as melphalan, busulfanand cyclophosphamide should be re-evaluated, par-ticularly considering that intravenous formulations ofmelphalan and busulfan could make drug deliverymore reliable, accurate, and adjustable to effective

blood levels. Alkylating agents may be used in syner-gistic combinations. Topo I inhibitors and nucleosideanalogues inhibit DNA repair thus enhancing DNAdamage by alkylating agents [74,75]. These combin-ations are under investigation.

V. Liposomal agents

Liposomal encapsulation of chemotherapeutic agentsand other drugs changes the pharmacologic propertiesof the active compound and has been advocated to re-duce toxicity, therefore potentially allowing for high-dose therapy and potentially improve the therapeuticbenefit of these drugs [121]. Dose-intensive anthra-cycline therapy is associated with improved prognosisin acute promyelocytic leukemia, AML, and ALL butdose escalation is limited by mucositis and, in thelong term, dose-dependent cardiac toxicity. Liposomaldaunorubicin (DNX) has been used in phase I trialsin solid tumors with no extramedullary toxicity, andlimited only by myelosuppression at doses of between100 mg/m2 and 120 mg/m2 repeated every three weeks[122] DNX has demonstrated significant activity inAIDS-associated Kaposi’s sarcoma, and no cardiactoxicity has been reported among patients receivingcumulative daunorubicin doses of>600 mg/m2 (range600 to 3159 mg/m2) [123]. In a phase I study inpatients with refractory or relapsed acute leukemia,mucositis was the dose-limiting toxicity at a doseof 150 mg/m2 [124]. No significant cardiac toxicitywas reported and 2 patients (1 with CML in blasticphase and 1 with acute promyelocytic leukemia insecond relapse) achieved a complete remission [124].Preliminary results of a study using DNX in combina-tion with high-dose ara-C (1 g/m2 continuous infusiondaily for 4 days) reported a CR rate of 21% with an

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additional 11% with hematologic improvement (i.e.,met all criteria for CR except platelet count improvedbut<100× 109/L) [125]. The MTD for DNX was 125mg/m2. Preliminary results of a combination chemo-therapy regimen for salvage ALL have produced aCR rate of 33% [126]. Dose escalation of liposomaldoxorubicin has been limited by hand-foot syndrome[127], although this could be possibly ameliorated bypyridoxine [128]. Whether these anthracyclines willbe safer in the long term and therefore valuable inhigh-risk populations (e.g., elderly) and the role ofhigh-dose anthracyclines using these formulations iscurrently being investigated.

A liposomal formulation of vincristine has demon-strated increased efficacy against leukemia cell lines[129,130]. The MTD in a phase I study in solid tu-mors was defined as 2.4 mg/m2 [131] and studiesin hematologic malignancies are currently underway.Liposomal formulations of topoisomerase I inhibit-ors, including topotecan [132] and its analogue GL147211C [133] are available with increased activity inpreclinical models.

Monoclonal antibodies

Monoclonal antibodies (MoAb) may target cytotoxictherapy (e.g., radioisotopes, toxins) specifically to ma-lignant cells. MoAb may also induce on their ownimmune-mediated killing of leukemic cells. Severalconditions need to be met for MoAb to become ef-fective therapy, including: 1) identification of tumor-specific antigens, 2) development of humanized MoAbto avoid antibodies against murine MoAb (HAMA),and 3) use of toxins with tight binding to MoAb andproper targeting to tumor cells to avoid normal tis-sue toxicities, or else, antibodies that are capable ofinducing immune destruction of tumor cells withoutthe need for toxin complexes. Some MoAb have beendeveloped and used with promising results in acuteleukemia.

I. Anti-CD33

CD33 is a surface glycoprotein expressed in commit-ted normal myelomonocytic and erythroid progenitorcells but not on stem cells [134]. It is also found onmost myeloid leukemic blasts and leukemic progen-itors [135]. Monoclonal anti-CD33 antibodies wereinitially IgG2 derived from mice, and were able to

bind avidly their target and kill cells in the presenceof rabbit but not human complement [136]. In phaseI trials, there was saturation of binding sites at doses≥5 mg/m2, with uptake in the bone marrow within 1hour after administration [137]. Murine anti-CD33 an-tibodies have no intrinsic cytotoxic activity, but whenlabeled with131I-significant selective irradiation wasdelivered to the bone marrow [137]. Using this131I-labeled anti-CD33, Schwartz et al. [138] treated 24patients with myeloid malignancies, including 16 withrefractory or relapsed AML, 5 with “blastic” MDS,2 with secondary leukemias, and 1 with CML inblastic phase. Ninety six percent of patients developedpancytopenia, and 89% had significant decreases inthe percentage of marrow blasts. Eight patients hadsufficient cytoreduction to proceed to bone marrowtransplantation (BMT). However, 37% of patients de-veloped human anti-mouse antibodies which resultedin loss of activity of the antibody when patients werere-treated [138].

Recently, a humanized anti-CD33 antibody hasbeen constructed, which retained its specificity, hada higher binding avidity than the murine antibody and,unlike the murine counterpart, mediated antibody-dependent cell-mediated cytotoxicity [139]. In a phaseI study, 13 patients with refractory or relapsed AMLwere treated at dose levels from 0.5 to 10 mg/m2/dosefor 6 doses over 18 days [140]. Total doses as high as216 mg could be safely administered, with the optimalbiodistribution and saturation of binding sites occur-ring at 3 mg/m2. The antibody was rapidly internal-ized, and no patient developed antihuman antibodies[140]. In a recent trial, 7 patients with acute promy-elocytic leukemia received ATRA followed by131I-labeled anti-CD33. Two of six patients with detectablePML/RARα mRNA after treatment with ATRA hadnegative determinations after receiving the antibody[141]. The treatment was well tolerated.

These antibodies have also been used in the settingof BMT. When used in conjunction with conventionalconditioning for allogeneic bone marrow transplant-ation, 131I-labeled anti-CD33 antibodies was admin-istered safely, delivered significantly higher doses ofradiotherapy to the bone marrow and spleen, but thehalf-life of the radioactive component in the bone mar-row was relatively short (9 to 41 hours) which maylimit its use in this setting [142]. However, these anti-bodies may be of value for bone marrowin vitro pur-ging. The sequential treatment of bone marrow withVP16 plus ara-C followed by complement-mediatedlysis using an anti-CD33 antibody synergistically

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killed CFU-L, sparing early normal hematopoieticprogenitor cells [143].

MoAb have recently been conjugated with othermolecules in an attempt to increase specific cytotox-icity. An anti-CD33 MoAb conjugated to the potentcytotoxic agent colicheamicin was used to treat 23patients with AML in first relapse; three patientsachieved a CR and 7 others met all criteria for CRexcept for platelets< 100×109/L (but> 50×109/L)[143a]. Other combinations include anti-CD33 anti-body conjugated to gelonin, a toxin which inhibitsribosomal protein synthesis. This conjugate resultedin 4500 increased activity in inhibiting myeloid celllines while conserving the specificity of the antibody[144] and significantly suppressed leukemia growthin a mouse model [145]. Anti-CD33 antibodies havebeen conjugated to another toxin, ricin, to increase thespecific cytotoxicity [146]. Incubation of normal bonemarrow with this conjugate resulted in enrichment oflong-term culture initiating cells [146]. Bispecific an-tibodies to CD33 and CD 16 have been effective inaugmenting the killing of resistant leukemic cellsinvitro by peripheral blood lymphocytes and NK cells[147].

Further studies are underway using these and newantibodies in the treatment of patients with AML, butthe early results offer the potential of a useful approachfor the therapy of AML.

II. Anti-CD52 (CAMPATH-1)

CD52 is a 21 to 28 kDa phosphatidylinositolglycan(PIG)-anchoredglycoprotein expressed at high densityon normal lymphocytes, monocytes and macrophages[148]. Although its function is unknown, it may parti-cipate in the activation of T-lymphocytes [149]. CD52is expressed in the majority of lymphoid malignan-cies including non-Hodgkin’s lymphomas, CLL, hairycell leukemia, prolymphocytic leukemia, Sezary’ssyndrome, and ALL, although only a few myeloidmalignancies express CD52 [150]. CAMPATH-1 is amonoclonal antibody which binds to most circulatinglymphocytes and has been used successfully to purgebone marrow from T-lymphocytes prior to bone mar-row transplantation [151]. Several subtypes are avail-able with distinctive properties. The IgM rat antibodyfixes human complement and has been very effectivefor bone marrow purging [151,152]. The IgG vari-ety can be cytolytic with human complement, and theIgG2b isotype can mediate antibody-dependent cell-

mediated cytotoxicity [153]. A humanized version ofCAMPATH-1 (CAMPATH-1H) was engineered to re-duce the immunogenecity of the antibody [154]. Haleet al. first treated two patients with non-Hodgkin’slymphoma with CAMPATH-1H and noted clearing oftumor in the blood and bone marrow and resolutionof splenomegaly [154]. Interestingly, administrationof the IgM and IgG2a isotypes have resulted in tran-sient depletion of peripheral blood lymphocytes andconsumption of complement with little effect on tu-mor masses or bone marrow. In contrast, the IgG2bisotype has produced long-lasting lymphocyte deple-tion from peripheral blood and bone marrow, as wellas improvement in splenomegaly [155].

Information is starting to accumulate on the ef-fect of CAMPATH-1H in leukemias. A response rateof 89% was observed in a small series of patientswith CLL treated with CAMPATH-1H for front-linetherapy [156]. Among 29 patients with refractory orrelapsed CLL 11 (38%) achieved a PR and 1 (4%)a CR after treatment with CAMPATH-1H [157]. Al-though most ALL and a large proportion of AMLexpress CD52 [150] only a handful of patients withacute leukemia treated with this antibody have beenreported. Dyer et al. [155] included 5 patients withALL in their trial with CAMPATH-1H: 2 with B-cellALL, 2 with common ALL, and one with Philadelphiachromosome-positive ALL. The first four patients hadfailed conventional chemotherapy, whereas the latterreceived CAMPATH-1H as primary therapy. In allcases the peripheral blood cleared from leukemic cellsand the bone marrow showed a reduction in leukemiccells, complete in 3 patients and partial in 2 (includingthe patient treated with CAMPATH-1H as front-linetherapy, in whom the percentage of blasts in the bonemarrow decreased from 97% to 21%) [155]. There-fore, CAMPATH-1H deserves further investigation assalvage therapy in patients with CLL and ALL, or astherapy for minimal residual disease for patients infirst CR.

III. Anti CD19 monoclonal antibodies

The CD19 antigen is expressed in most normal andneoplastic B cells, and can be detected early in theirontogeny [158]. Although CD19 MoAb can recognizemalignant cells of many B-cell malignancies, it hasvery weak direct antitumor effect [159,160]. Whenconjugated to the plant toxin ricin, with potent ribo-some inactivating activity, CD19 has shown specific

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cytotoxicity in vitro against B-cells [161]. Anti-CD19antibodies conjugated with blocked ricin (to preventnonspecific binding of ricin, anti-B4-bR) have shownantitumor activity in SCID mouse models of sev-eral CD19-positive human neoplasms, including ALL[162]. Phase I/II trials have been conducted in pa-tients with B-cell neoplasms, documenting the safetyof these antibodies, with the dose limiting toxicity be-ing elevation of liver enzymes [163,164]. Thirty five to70% of patients treated in phase I trials developed an-timouse antibodies and/or anti-ricin antibodies whichcould potentially limit the long-term treatment effic-acy [163,164]. The effect of anti-B4-bR on minimalresidual disease in patients with B-lineage ALL wasrecently reported [165]. Forty six adults with CD19-positive ALL who were in CR after one courseof consolidation were treated with anti-B4-bR at 30µg/kg/day as IV continuous infusion on days 1 to 7and 15 to 21. Of 5 patients with BCR-ABL positivedisease at diagnosis, 3 were positive prior to receivingthe immunotoxin and all remained positive, whereas1 of 4 who had become negative after chemotherapyconverted to positive. Of 7 patients with T-cell receptoror Ig heavy chain rearrangement at diagnosis 4 re-mained positive, 1 converted from positive to negative,and 2 converted from negative to positive after anti-B4-bR [165]. Although these results are not encour-aging, other antibodies and different strategies mayprove more useful in the setting of ALL. One possibil-ity is to combine the immunotoxin with chemotherapy.In vitro studies have shown synergistic activity of anti-B4-bR with chemotherapy in lymphoma cell lines,including some multidrug-resistant [166]. This effectmay be related to downregulation of P-glycoproteinexpression in resistant cells after exposure to theimmunotoxin [166]. This approach has resulted inimproved survival of mice treated with the combina-tion of anti-B4-bR and doxorubicin or etoposide [166]or with combination chemotherapy [167]. Anti-B4-bR has also been used effectively for purging bonemarrow grafts of leukemia or lymphoma cells [168].A reduction of more than 3 logs of malignant cellshas been reported with minimal effect against normalhematopoietic cells [168].

IV. Anti CD20 monoclonal antibodies

CD20, a 32 kD non-glycosylated phosphorylated pro-tein, is expressed on the surface of the majority ofnormal B cells but not early pre-B cells [169]. A vari-

ety of lymphoid malignancies express CD20, includ-ing non-Hodgkin’s lymphoma, ALL and CLL [170].Radiolabeled anti-CD20 antibodies were investigatedin the treatment for non-Hodgkin’s lymphomas withpromising results, but responses were observed withonly trace doses of the labeled antibody, suggest-ing activity of the unconjugated antibody [171,172].A humanized, unconjugated version of anti-CD20MoAb showed significant anti-B cell activityin vitroand in vivo in animal models through ill antibody-dependent cell-mediated cytotoxicity [173]. Severaltrials have been performed in B-cell lymphomas [174–176]. A 48% response rate was reported among 166patients with relapsed low-grade or follicular lymph-oma treated with anti-CD20 as a single agent [176].Toxicity was minimal and no immune response to theinfused antibody was reported [174–176]. In combin-ation with CHOP chemotherapy, 95% of 40 patientstreated responded (55% CR, 40% PR) [177]. Re-sponses have also been observed in CLL [178] and ag-gressive lymphomas [179]. The activity of anti-CD20MoAb therapy alone or in combination with chemo-therapy is currently under investigation in patientswith ALL who express CD20 on leukemic blasts.

Multidrug resistance modulation

The effectiveness of chemotherapeutic agents in acuteleukemia is limited by mechanisms of leukemic cellsresistance. Resistance can be induced by exposure ofleukemic cells to these agents or may be intrinsic to theneoplastic cells. Several mechanisms leading to multi-drug resistance (MDR) have been identified in acuteleukemia and their pharmacologic manipulation is anactive area of research [180]. Probably the best knownis the MDR1 gene, which translates into a membraneglycoprotein, P-glycoprotein (PgP) that can functionas a drug efflux pump [181]. The overexpression ofPgP has been linked to resistance to anthracyclines,vinca alkaloids, taxanes, epipodophyllotoxins, andsome alkylating agents. Overexpression of MDR1 andPgP have been reported with variable frequencies inAML, and has been associated with a poor response tochemotherapy and shortened survival [180,182,183].Measures of PgP function such as retention of rhodam-ine [184] and intracellular daunorubicin accumulation[185] also correlate with outcome in some reports.

Several agents have been used to inhibit PgP func-tion as a means to prevent development of resistanceor to restore leukemic cell sensitivity. These include

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quinine, verapamil and dexverapamil, tamoxifen, cyc-losporin A and its non-immunosuppressive derivativePSC 833. These agents have shown efficacyin vitro,and clinical trials have produced some interestingresults.

The clinical applications of verapamil in MDRmodulation are limited by cardiovascular toxicitywhen used at doses that produce plasma levels withPgP inhibitory activity [186]. R-verapamil has demon-strated similar potency as verapamil in reversing MDRbut has a better toxicity profile [187,188] and is cur-rently being evaluated in acute leukemias [189].

Quinine therapy was well tolerated and plasmalevels within vitro inhibitory activity against PgP wereachieved in patients [190]. A large randomized trialincluding 315 patients with a variety of acute leuk-emias, most of them refractory or relapsed, found asimilar response rate for patients treated with (53%)and without (46%) quinine together with chemother-apy [191]. There was a trend for better CR rates amongMDR-positive patients who received quinine (60% vs35%; p=0.06) [191].

Cyclosporine-A (CsA) is a potent inhibitor of PgPat concentrations which can be achievedin vivo (500to 2,000 ng/ml). When used with chemotherapy forAML, CsA prolonged the clearance of anthracyclines[192]. Initial trials in AML have reported CR rates in82% of patients with relapsed AML and in 25% ofthose with refractory disease [193]. The response ratedid not correlate with MDR phenotype but correlatedwith the development of hyperbilirubinemia, whichoccurred in over 60% of patients [193]. In a recentmulticenter trial, patients with refractory or relapsedAML, secondary AML or RAEB-T were randomizedto receive daunorubicin and ara-C alone or in combin-ation with CsA. There was no difference in the CRrate between the two groups, but patients treated withCsA had a superior relapse-free survival and overallsurvival [194]. PSC 833 is a non immunosuppress-ive analog of CsA with a 10- to 30-fold more potentinhibiting activity against PgP [195,196]. PSC 833also caused hyperbilirrubinemia in most patients, andat doses of 12 mg/kg/d ataxia has been observed inphase I trials [197]. Studies in acute leukemia arecurrently underway [180], but preliminary results aredisappointing.

Some caution notes should be considered regard-ing approaches aimed at MDR modulation Thesedrugs may increase the toxicity of chemotherapy byaltering the clearance of the antineoplastic agents. Pre-clinical data with PSC 833 suggest that both acute

and delayed toxicity can be potentiated, and increasedconcentrations of doxorubicin can be found in theheart and other organs [198,199]. Increased acutetoxicity has been reported in some trials in acuteleukemia [191], and the effect on long-term toxicityremains to be determined (i.e., cardiotoxicity, second-ary malignancies). Another concern is the appearanceof alternative mechanisms of resistance. List et al.[193] reported 5 patients who achieved CR after ther-apy with cytarabine and daunorubicine plus CsA whoeventually relapsed. In 4 of these patients the diseasewas MDR-negative at relapse suggesting that a clonewith alternative mechanisms of resistance had beenselected [193]. Thus, even when MDR expression iseffectively inhibited in MDR-positive leukemia, othermechanisms of resistance may persist or evolve andmake cells unresponsive to therapyin vitro or in vivoin animals [200,201]. At least two other mechanismsof resistance mediated by non-PgP transport proteinshave been described. MDR-related protein (MRP) isexpressed in normal hematopoietic cells as well as inacute leukemias particularly resistant AML [202,203].The MRP gene is located in chromosome 16p13.1[204] and, interestingly, in patients with AML andinv(16)(p13q22), deletion of the MRP gene is associ-ated with prolonged failure-free survival [205]. Lungresistance protein (LRP) has been demonstrated in avariety of cell lines and has been detected in 37% ofpatients with AML, including patients with de novo(33%) and relapsed (38%) disease [206]. In contrastto MDR, LRP expression had an independent adverseprognostic effect for response [206]. The LRP is alsolocalized on the short arm of chromosome 16, closeto the MRP gene [207]. Unfortunately, these mechan-isms seem to be only weakly sensitive to known PgPinhibitors [208,209]. Additional mechanisms of drugresistance exist, for which no drug manipulation hasbeen designed and which are not related to transportproteins. Therefore, MDR manipulation as an area ofresearch remains in its early phase of development.

Immune modulation and other biologic therapies

Several biologic agents and manipulations are cur-rently being investigated in the management of acuteleukemias. In many cases, these biologic agents in-duce immune mediated killing of the leukemic cells,whereas in others the mechanism may be more com-plex.

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I. Interferon alpha (IFN-A)

IFN-A has potent inhibitory activity against AMLcells and cell lines [210–212]. Inhibition may bethrough blocking IL-1 and IL-6 mediated growth[212]. IFN-A can also increase the lysing activity ofheterologous natural killer (NK) cells against AMLcells [213]. In somein vitro conditions, IFN-A hassynergistic cytotoxic activity against AML cell lines[214]. IFN-A has been used as maintenance therapyin adult AML after induction of CR with TAD: noimprovement in CR duration or survival was seencompared to no maintenance or maintenance withthioguanine and cytarabine [214]. Maintenance withIFN-A in patients with Ph-positive ALL in CR alsodid not result in improved CR duration (KantarjianH; unpublished data). In contrast, an induction re-gimen combining IFN-A and ara-C showed remark-able activity in patients with refractory or relapsedAML (27/35 CR, 77%) andde novoAML (15/15CR, 100%) [214]. Similar activity has been reportedin patients with refractory or relapsed non-Hodgkin’slymphoma [214,215]. A combination of IFN-A, ara-C, and thymopentin also had significant activity inpatients with myelodysplastic syndromes (overall re-sponse rate 75%) [216]. Further studies are neededto determine the role of IFN-A and IFN-A-basedcombinations in acute leukemias.

II. Interleukin 2 (IL-2)

Interleukin-2 is a pleomorphic cytokine capable ofstimulating the activity of cytotoxic T-cells and NKcells, and can induce the generation of lymphokine-activated killer (LAK) cells [217]. IL-2 with orwithout LAK cells can inhibit the growth of murineleukemia cellsin vivo, and NK-resistant myeloid orlymphoid leukemic cells can be lysedin vitro by LAKcells [218–221]. This effect seems to be mediatedby the Fas/APO-1 pathway [222]. IL-2 was extens-ively used after BMT, mostly autologous [223]. IL-2has also been used in treating acute leukemias aloneor in combination with chemotherapy or LAK cells(Table 5). Several trials have documented CRs in upto 57% of patients with AML and low percentage ofbone marrow blasts at relapse (e.g., less than 20% to25%); little effect was seen in patients with higherpercentages [224–226]. IL-2 may have prolonged CRduration when used as maintenance [227] or consolid-ation [228] therapy in patients in second CR. A pilotstudy using a 12 week continuous infusion of IL-2with weekly bolus in patients in first CR suggested

an improved CR duration compared to historical con-trols [229]. IL-2 has been used as maintenance therapyin combination with histamine with promising results[230]. IL-2 may also have a role in the management ofmyelodysplastic syndromes [231], and anecdotal re-sponses have been reported using low-dose IL-2 [232].There is little if any information on the use of IL-2in ALL, but ALL blasts may be less susceptible toLAK cell-mediated lysis than AML cells [233]. Trans-duction of IL-2 into acute leukemia cells reduces theoncogenic potential of these cells in animal modelsand may be a model for manipulation of this suscept-ibility [234]. The exact role of IL-2 in the managementof acute leukemia needs to be better defined, includingthe optimal dose, schedule and timing. However, IL-2 may be an valuable addition to the therapy of acuteleukemia [235].

III. Linomide

Linomide (roquinimex) is a quinoline derivative withtumor suppressing activity in some melanoma animalmodels [236]. Linomide has a variety of immun-omodulatory activities including stimulation of NKcell [237] and lymphokine-activated killer (LAK) cell[238] activity, enhancement of delayed-type hyper-sensitivity (DTH) reactions [239] and proliferativeT-cell responses [240], and increased IL-6 production[241]. Low concentrations of linomide are synergisticwith IL-2 in stimulating lymphocyte and NK-cell pro-liferation, but at higher concentrations the prolifera-tion decreases [242]. A pilot study in patients withsolid tumors used linomide at doses of 0.05 to 0.6mg/kg orally once a week. An increase number ofCD16+ NK cells and CD14+ monocytes was docu-mented, with increase NK activity at a dose of 0.2mg/kg, but not at 0.6 mg/kg [243]. In hematologicmalignancies, linomide has been considered for themanagement of AML and CML after autologous bonemarrow transplantation in an effort to produce a graft-versus-leukemia effect after this procedure [241,244].In several cases, an acute graft-versus-host reactionhas been induced after autologous bone marrow trans-plantation followed by linomide therapy in patientswith acute or chronic myeloid leukemias [244,245].Five patients with AML were treated with linomide0.3 mg/kg/week orally in cycles of three weeks fol-lowed by three weeks of rest for up to six months afteran autologous BMT [246]. During treatment periodsthere was an increase in the number and cytotoxicactivity of circulating NK cells, as well as increased

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Table 5. Interleukin 2 in AML

Author No Disease status (No.) CR (%) Outcome Comments

(reference)

Foa [224] 12 Refractory (2) 4 (33) 1 CCR (30+ months) All patients who responded

Relapse (10) 3 relapsed (20 days, 4 mo, 9 had<20% BM blasts

mo)

Meloni [226] 14 Refractory (3) 8 (57) 5 CCR (14+ to 68+ months) All pts had<25% BM blasts

Relapse (11) 3 relapsed (2,5,9 months) No patient with refractory

AML responded

Lim [225] 13 Refractory (1) 1 (17) 1 relapsed (6 months)

Relapse (5)

1st CR (3) 1st CR in CCR (13+,13+,22+

2nd CR (4) months)

2nd CR: 2 relapsed (5,6

months), 2 CCR (7+,12+

months)

Bergmann [228] 21 2nd CR 7 CCR at 7+ to 49+ months

5 pts CR2 duration> CR1

duration

Wiernick [227] 7 2nd CR CR duration 4–49 months PEG-IL-2

2 CCR (43+,49+ months)

3 pts CR2 duration> CR1

duration

Brune [230] 28 1st CR (17) Median CR1 duration 12+ Ara-C + 6-TG between IL-2

2nd CR (8) months (range 7–51 months) courses

3rd CR (2) (12/17 remain in CR)

4th CR (1) Median CR> 1 duration 13

months (range 6–49) (5/11

remain in CR)

8/10 evaluable CR> 1 longer

than previous CR

Cortes [229] 18 1st CR 6 CCR after 4 years

TNFα, IFNγ , and IL-1 secretion [246]. All patientsengrafted, but one died of intracerebral hemorrhage 13weeks after the transplant, and one relapsed 4 monthsafter transplant [246]. Linomide has also been usedafter autologous BMT for patients with CML [244].Six patients (one in second chronic phase, one in ac-celerated phase, four in first chronic phase) receivedLinomide after engraftment and continued for twoyears. The preliminary analysis had little informationon cytogenetic responses, but interestingly 3 patientsdeveloped graft-versus-host disease [244].

The experience on linomide outside the transplantsetting is limited. One study reported on 17 patientswith myelodysplastic syndrome (6 refractory anemia,1 refractory anemia with ringed sideroblasts, 10 re-fractory anemia with excess blasts) treated with Li-

nomide [247]. There was an increase in CD8+ andCD56+/CD3-cells, but no increase in NK or LAKcell activity [247]. After therapy, neutrophil countsimproved in 4 patients, platelet counts in two, andhemoglobin in one patient who became transfusionindependent [247]. Linomide, and other immunomod-ulatory drugs (e.g., histamine) are therefore attractivealternatives in the management of acute leukemiaswhich deserve further investigation.

IV. Specific immunotherapy

Although there is little clinical data for specific im-munotherapy, preclinical results make this an excitingopportunity to explore. One approach has been theuse of AML cells transducted with B7.1, a criticalco-stimulatory molecule in the generation of an ef-

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fective immune response [248]. Mice treated withirradiated B7.1-transfected cells 2 weeks prior to in-oculation of untreated leukemia cells are protectedfrom developing leukemia, and the immunity is long-lasting (up to 5 to 6 months) upon rechallenge [249].Furthermore, mice vaccinated early (i.e., one week)after inoculation of untreated leukemia cells reject theleukemia, although later vaccination (i.e., 2 weeksafter AML inoculation) was ineffective [249]. How-ever, the combination of early chemotherapy and latevaccination cured 100% of the mice, suggesting thatsuch an approach could be used in patients withAML [250]. Using a similar strategy, the same au-thors used AML cells transfected with B7.2 or oneof several cytokines with dendritic cell-stimulatingproperties (GM-CSF, IL-4, or TNF-α) [251]. Micevaccinated with GM-CSF-transfected AML cells de-veloped anti-leukemia immunity that protected themwhen challenged with untreated AML cells. Further-more, GM-CSF-transfected AML cells rescued 100%of mice when vaccinated one week prior to injec-tion with untreated AML and up to 80% when givenlate (i.e., 2 weeks after AML inoculation) [251].Therefore, GM-CSF gene immunotherapy might beeven more effective than B7.1-based cell vaccines andconstitute a promising approach to take to the clinic.

Another approach is to develop “vaccines” againstleukemia-specific antigens. One approach has beento use peptides derived from chromosomal transloca-tions resulting in ocogenic fusion proteins. Althoughmany of these proteins are intracellular, their abilityto bind HLA class I molecule and therefore their po-tential to stimulate a specific immunologic responsehas been reported [252]. This approach would belimited to leukemias where a specific fusion proteinis known (e.g., BCR/ABL, PML/RARα, etc.) but itmight prove beneficial in such cases. An alternativeto this strategy is the use of proteins expressed in amore uniform fashion. Proteinase 3 is overexpressedin a variety of myeloid leukemias [253] and it mightbe involved in the process of leukemotrenesis or main-tenance of the leukemic phemotype [254]. PR-1, apeptide contained in proteinase 3, can bind HLA-A2.1and stimulate the generation of cytotoxic T lympho-cytes which preferentially lyse myeloid leukemia cells[255–256]. Clinical studies using these “vaccines” arecurrently underway.

Dendritic cells are potent antigen-presenting cellsresponsible for eliciting T-cell responses and are cur-rently being investigated to generate effective can-cer immunotherapy [257]. Dendritic cells generated

from leukemic cells could present the necessary an-tigens to elicit a specific immune response. Dendriticcells have been generated ex vive from myelomono-cytic precursors from chronic mylogenous leukemia[258] and acute myelogenous leukemia [259]. Auto-logous lymphocytes co-cultured with these dendriticcells were able to lyse autologous leukemia cells, butnot normal cells, raising the possibility of specificimmunotherapy.

Other approaches

Multiple other approaches are currently under devel-opment and might represent the future of the treatmentof acute leukemia. Many of them are in early phases ofdevelopment and the data available is still preliminary.Although not all inclusive, some of the most promisingand challenging new drugs are:

I. Retinoids. The activity of all-trans retinoic acid(ATRA) in acute promyelocytic leukemia has beenwell established and is beyond the scope of this re-view. However, retinoids are being investigated inother acute leukemias with intriguing results. Retin-oids increase the sensitivity of leukemia cells to ara-C[260] and anthracyclines [261]. This effect might bemediated by downregulation of bcl2 [261,262] and ad-dition of G-CSF might augment this phenomenon in asynergistic way [263].

Based on these data, a recent trial investigated theeffect of adding ATRA to chemotherapy on a ran-domized trial of fludarabine, ara-C and idarrubicin±G-CSF± ATRA in patients with poor-prognosis AML(excluding APL) [264]. Patients who received ATRAhad an early survival advantage but it was lost withmore follow-up [264]. ATRA alone has been reportedto induce a complete remission in a patient with AML[265]. In a small series of 13 patients with refractoryor resistant AML, ATRA in combination with low-dose ara-C (20 mg/m2 subcutaneous, twice daily for10 days every month) induced remission in 8 patients.Therefore, the role of retinoids in acute leukemiasneeds to be further explored, looking at schedules thatmight be more appropriate [260,261], new formula-tions such as liposomal ATRA, and other retinoidssuch as 9-cis retinoic acid which have a more potentanti-leukemia activityin vitro against AML [267].

II. Arsenic Trioxide. Arsenic trioxide (As2O3) wasinitially reported to have significant activity in APL,

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with 9 of 10 patients who had relapsed after treatmentwith ATRA achieved a remission when treated withAs2O3 alone [268]. No myelosuppression was seenon these patients. These results have been expanded,including the achievement of molecular remissions in8 of 11 patients who achieved a CR [269]. As2O3 in-duces differentiation [269] and apoptosis [269–271] ofthe leukemic cells, including downregulation of bcl-2[270] and increased caspase expression and activation[269]. Induction of apoptosis may occur without in-duction of differentiation and is associated with theloss of the PML/RAR alpha fusion protein [271]. In-terestingly, As2O3 has been reported to inhibit growthand induce apoptosis independent of PML/RAR ex-pression in a variety of myeloid leukemia cell lines[272], and significant cytotoxicity against other tumorcell-lines, including breats and prostate has been re-ported [273–274]. The activity of As2O3 in patientswith other leukemias is currently being investigated.

II. Targeted Therapy. CGP 57148 (STI 571) is anovel agent that inhibits the tyrosine kinase activity ofABL and the platelet-derived growth factor receptorand other activated ABL tyrosine kinases [275–277],and suppresses the growth of cells expressing theseproteins [275–278]. Early clinical results suggest avery potent anti-leukemia activity with minimal tox-icity in patients with Interferon-resistant Ph-positiveCML [279] and studies in ALL and transformed CMLare ongoing. Another approach is the use of Ras in-hibitors. Ras mutations occur frequently in leukemias,the highest frequency being reported in chronic my-elomonocytic leukemia where as many as 65% ofpatients have been shown to have mutations in Ras.Mutations in AML have been reported in 25% to 44%of patients. Ras may be activated by other mechan-isms in the absence of mutations (e.g., BCR-ABL)[280,281]. Recently, a new class of drugs has beendescribed which targets the posttranslational prenyla-tion of Ras critical for its membrane localization andactivity. These drugs target the enzyme FTase and arecalled farnesyl transferase inhibitors [282].

Conclusions

Several promising strategies exist which will hope-fully find a role and lead to improved prognosis inacute leukemia. Some of them (new chemotherapyagents, MoAb, MDR modulation) have been dis-cussed in this review. One important lesson from

some of these trials is the importance of molecularpharmacology in the rational design of chemother-apy combinations to obtain the most benefit of certaindrugs, and investigate potential synergism (and avoidantagonisms). Appropriate schedules of combinationchemotherapy and even single agents should be de-signed based on their mechanisms of action and phar-macologic properties. Other approaches, such as doseintensive chemotherapy and stem cell transplantationand leukemia sensitization (i.e., using growth factorssuch as G-CSF, GM-CSF, IL-3, and others) have beencovered by excellent recent reviews [283,284]. Thenewer approaches directed to specific targets, such asleukemic cells (e.g., monoclonal antibodies), specificgenes (e.g., tyrosine kinase inhibitors, Ras inhibit-ors), immune modulators (e.g., n-2, dendritic cells),or mechanisms of resistance (e.g., MDR blockers, in-hibitors of DNA repair) offer a refreshing promiseto the treatment of leukemias and might change ourmanagement of these diseases in the coming years.

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Address for offprints:Jorge Cortes, Leukemia Department, Box 61,M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX77030; Fax: 215 728 3639