Monoclonal antibodies and immunoconjugates in acute myeloid leukemia

Best Practice & Research Clinical Haematology Vol. 19, No. 4, pp. 715e736, 2006 doi:10.1016/j.beha.2006.05.001 available online at http://www.sciencedirect.com

6 Monoclonal antibodies and immunoconjugates in acute myeloid leukemia Sergio Amadori* Director Department of Hematology, Tor Vergata University Hospital, Viale Oxford 81, 00133 Rome, Italy

Roberto Stasi Assistant Member Department of Medical Sciences, Regina Apostolorum Hospital, Albano Laziale, Italy

The use of monoclonal antibodies for patients with acute myeloid leukemia is based on targeting cell-surface antigens preferentially expressed on leukemic blasts while sparing normal cells and tissues. The majority of studies performed to date have used antibodies reactive with the CD33 antigen. Phase II studies have demonstrated antileukemic responses with all agents, although less so with unlabeled antibodies. The most promising results have been obtained in the treatment of minimal residual disease in patients with acute promyelocytc leukemia. Antibody-targeted chemotherapy with gemtuzumab ozogamicin has also shown significant activity in patients with relapsed acute myeloid leukemia. Radioimmunotherapy with b-particle emitters may be most effective for the treatment of bulky disease or as part of a conditioning regimen for hematopoietic stem-cell transplantation, whereas radioimmunotherapy with a-particle emitters may be better suited to the treatment of small-volume or minimal residual leukemia. Whether or not monoclonal antibody therapy will improve disease outcome compared with conventional treatment regimens remains to be demonstrated by well-designed clinical trials. Key words: monoclonal antibodies; immunoconjugates; acute myeloid leukemia; gemtuzumab ozogamicin; radioimmunotherapy.

One of the major goals in medicine today is the design of therapeutic agents that can selectively kill and eliminate certain populations of cells in the human body. The availability of monoclonal antibodies (MoAbs) reactive with antigens expressed only by specific cell types has provided clinical investigators with new tools to achieve this goal.

* Corresponding author. Tel.: þ39 06 20903219; Fax: þ39 06 20903221. E-mail address: [email protected] (S. Amadori). 1521-6926/$ - see front matter ª 2006 Elsevier Ltd. All rights reserved.

716 S. Amadori and R. Stasi

Antibody therapy is ideally suited to the treatment of hematologic malignancies because of the ready accessibility of neoplastic cells in the circulation. Hematopoietic stem cells give origin to a wide variety of cell types that, during the course of differentiation, express several stage-specific markers on their cell surfaces. Most leukemic cells express many of the same differentiation antigens, depending on the stage in which the leukemic transformation occurred. The presence of antigens that are not expressed on normal hematopoietic precursors provides a therapeutic opportunity to eradicate leukemic cells by targeting their surface antigens while preserving the normal stem cell and the subsequent ability to recover trilineage hematopoiesis. MoAbs have been generated against such differentiation antigens, and over the past decade they have been used either unlabeled (‘naked’ antibodies), or conjugated with toxins, radioisotopes, or antitumor drugs. Besides, advances in genetic engineering have permitted the construction of humanized or chimeric antibodies that overcome many of the problems encountered with murine antibodies. These include the inability or limited ability to induce antibody-dependent cytotoxicity (ADCC) or complementmediated cytotoxicity (CDC) in humans, and the development of neutralizing human antimouse antibodies following repeated doses. Conversely, humanized or chimeric antibodies are less immunogenic and can mediate ADCC and CDC. The majority of studies in acute myeloid leukemia (AML) performed to date have used antibodies reactive with the CD33 antigen, and they will be the main focus of this review. CD33 ANTIGEN The CD33 antigen is a 67-kD transmembrane cell-surface glycoprotein expressed on both mature and immature myeloid cells, and on erythroid, megakaryocytic, and multipotent progenitors. However, it is not present on normal CD34þ pluripotent hematopoietic stem cells or non-hematopoietic tissues. CD33 contains two immunoglobulin-like domains: a transmembrane region, and a cytoplasmic tail that has two potential immunoreceptor tyrosine-based inhibitory motif (ITIM) sequences.1,2 Although it has been shown that this molecule is a sialic acid-binding receptor,3 its precise function e as well as the nature of its ligand(s) e remain unknown. In vitro experiments indicate that CD33 may act as an inhibitory receptor.4 Furthermore, the addition of an anti-CD33 antibody can induce apoptosis in AML cell cultures.5 Importantly, it has been shown that the antibodyeCD33 antigen complex rapidly internalizes and translocates into lysosomes after engagement by the antibody.6,7 Downstream steps in CD33 signaling are not well characterized, but some experimental models suggest involvement of Syk, c-Cbl, Vav and ZAP-70.8,9 It is unknown whether CD33 engagement by antibodies activates similar downstream signaling events in vivo, or whether these events might affect internalization and intracellular trafficking of anti-CD33 immunoconjugates. CD33 antigen expression is down-regulated with maturation of the myeloid lineage, resulting in low-level expression on peripheral granulocytes and tissue macrophages. Approximately 90% of AML cases are typically CD33-positive (CD33þ), as defined by the presence of the antigen on more than 20% of the marrow blasts. However, CD33 expression is a continuous variable, and this arbitrary cut-off threshold may not be very useful in investigating the biological mechanisms associated with CD33. The level of surface expression of CD33 among myeloid malignancies has been systematically investigated, and significant variations were found.10 The median

Monoclonal antibodies in AML 717

number of CD33 molecules per cell was highest in AML samples (mean 10,380, range 709e54,894) followed by myelodysplastic syndromes (mean 6671, range 493e53,791), chronic myeloid leukemia (mean 4410, range 801e16,108), and control samples (mean 2997, range 859e5137), and was lowest in samples from patients with myeloproliferative disorders (mean 2295, range 666e4279). Levels of CD33 intensity did not correlate with leukemia karyotype in patients with AML. On average, CD33 intensity in positive cells was significantly higher in the bone marrow compared with peripheral blood, but this study did not evaluate antigen levels on marrow and blood populations within individual patients. CLINICAL EXPERIENCE WITH ANTIBODIES TO CD33 Unlabeled HuM195 HuM195 (lintuzumab) is a recombinant humanized version of the mouse MoAb M195, directed against CD33.11 The antibody consists of a human immunoglobulin G1 (IgG1) framework that contains human constant regions and murine complementarity-determining regions.12 HuM195 is similar to the parent MoAb with respect to specificity, immunoreactivity, and internalization. HuM195 has a higher avidity than mouse M195 and, because the murine constant regions were replaced with human immunoglobulin sequences, has acquired the ability to mediate ADCC against leukemia targets in vitro.12 An initial phase I study in patients with relapsed or refractory AML investigated the biodistribution, pharmacology, and immunogenicity of HuM195 at doses of 0.5e 10 mg/m2.13 The results demonstrated complete bone marrow targeting without blood pooling of radiolabeled HuM195 following administration of 3 mg/m2 of antibody.13 At this dose, partial saturation of CD33 binding sites on leukemic blasts and re-expression of CD33 antigen at 72 hours following administration occurred despite repeated dosing over a 3-week period. On the other hand, the 10 mg/m2 dose level resulted in complete and prolonged saturation of CD33 binding sites. There was no evidence of a humaneantihuman antibody response at any dose. Of 13 patients enrolled, only one e treated at the highest dose level (10 mg/m2) e had a significant antileukemic response. The same investigators conducted a study using supersaturating doses of HuM195 in a similar advanced leukemia population.14 Ten patients with relapsed or refractory AML received HuM195 at a dose of 12, 24, or 36 mg/m2, infused over 4 hours daily on days 1e4 and 15e18. This study demonstrated that HuM195 was well tolerated at higher doses. Toxicity was primarily related to the infusion period and included fever, rigors, and transient hypotension. Pharmacokinetic data indicated that high serum levels and a prolonged half-life (>1 week) were achieved at all dose levels, and that the CD33 antigen on leukemic cells remained saturated throughout the 4-week trial period. Clinical responses were noted in four of ten patients, including one complete remission (CR) in a patient who received the lowest dose level (12 mg/m2). The evaluation of the antileukemic activity of HuM195 has been the objective of a subsequent phase II trial in patients with relapsed or refractory AML. Patients were treated with two courses of HuM195 at a dose of 12 or 36 mg/m2 daily
4 days, given 2 weeks apart. Of 49 evaluable patients entered into the study, one partial and two complete remissions were achieved, and all responses were seen in patients with low marrow blast infiltration (5e30%). Additional decreases in marrow blast counts were observed in other patients but were transient.15

718 S. Amadori and R. Stasi

In the only phase III trial published to date, HuM195 (12 mg/m2) in combination with induction chemotherapy (mitoxantrone 8 mg/m2, etoposide 80 mg/m2, and cytarabine 1 g/m2, daily for 6 days) was compared with chemotherapy alone in 191 adults with first relapsed or primary refractory AML.16 The response rate with the combination regimen was 36% versus 28% in patients treated with chemotherapy alone (P ¼ 0.28). The overall median survival was 156 days and was not different between the two arms of the study. No differences in chemotherapy-related adverse effects were observed with the addition of the monoclonal antibody to standard chemotherapy. The results of these studies indicate that the most beneficial effects of HuM195 are seen in patients with a low leukemia burden, providing a rationale for the incorporation of the MoAb into treatment strategies that can achieve minimal disease states before its administration. Such an approach has been tested in patients with acute promyelocytic leukemia (APL).17,18 After attaining clinical complete remission with all-trans-retinoic acid (ATRA) and/or chemotherapy, patients received 3 mg/m2 of HuM195 twice weekly for 3 weeks. After the initial six doses of HuM195, appropriate candidates proceeded to hematopoietic stem-cell transplantation (HSCT), and the remaining patients received maintenance therapy with HuM195. Among the 31 patients treated in first remission, 28 had minimal residual disease detectable by reverse transcriptase polymerase chain reaction (RT-PCR) for promyelocytic leukemia and retinoic acid receptor-a (PML/RARa). After six doses of HuM195, 12 of the 24 evaluable patients (50%) converted to RT-PCR negativity before receiving any further therapy. In this experience, 29 of 31 patients with APL remained in CR with a median follow-up of 36 months. Although they were not randomized, these results appeared superior to experiences reported in earlier studies with ATRA or chemotherapy alone.19 Gemtuzumab Ozogamicin Conjugated antibodies are generally less capable of eliciting an immune response and rely on the delivered agent for cell kill. The most clinically successful approach thus far has been with gemtuzumab ozogamicin (GO). GO is a humanized IgG4 anti-CD33 monoclonal antibody (hP67.6) conjugated to a powerful antitumor antibiotic, NAc-g calicheamicin DMH, a hydrazide derivative of calicheamicin (Figure 1). The conjugation is obtained via a bifunctional linker, [4-(4-acetylphenoxy)butanoic acid], which allows the most favorable balance between hydrolytic stability in physiological buffers (pH 7.4) and efficient drug release at the pH of lysosomes (w4).20 The average overall ratio of calicheamicin to antibody is approximately 2:3, with about 50% of the antibody unconjugated.21 Calicheamicin comprises several important functional elements, including the enediyne ring that is the DNA-cleaving portion of the molecule.22 The naturally occurring enediyne antibiotics are a unique class of reactive compounds that, upon aromatization, produce cytotoxic biradicals causing phosphodiester bond breakage in DNA.23,24 The binding of the anti-CD33 antibody portion of GO with the CD33 antigen results in the formation of a complex that is rapidly internalized. Upon internalization, the calicheamicin derivative is released from the antibody by acid hydrolysis, and a reactive intermediate of the calicheamicin derivative is formed through reduction by glutathione.7 This reactive intermediate causes double-stranded breaks in DNA at oligopyrimidineeoligopurine tracts following binding to the minor groove of the DNA duplex.25 Calicheamicin can interfere with biological processes not simply

Monoclonal antibodies in AML 719

Figure 1. Gemtuzumab ozogamicin (GO) is composed of a humanized monoclonal antibody (hP67.6) joined to calicheamicin via a bifunctional linker.

by cleaving free DNA but also by displacing a DNA-binding protein through competition or modulation of DNA structure. Resistance to GO has been correlated with a functional multidrug resistance (MDR) phenotype in leukemic blasts and may be reversed in vitro by cyclosporine A (CSA).26 Experimental models using the HL-60 and AML-193 cell lines suggest that GO is also able to exert a CD33-independent cell killing effect via a non-specific phagocytosis of the immunoconjugate.27 In these studies, receptor-independent endocytosis appeared to account for immunoconjugate internalization and cytotoxicity. Lysis of AML-193 cells required continuous exposure to GO at concentrations of 5 mg/mL for at least 24 hours. Since such conditions in culture exceed achievable plasma levels of GO, including levels after the second dose when blast cell numbers may be quite low, this model does not adequately explain why some patients with CD33e AML might respond to GO. Other hypotheses have been formulated, including the presence of ‘background’ levels of surface CD33 that may be sufficient in some cases for antigen-dependent GO uptake and cytotoxicity, or the presence of a small subpopulation of GO-susceptible CD33þ leukemic progenitors responsible for a cytoreductive effect.28 As a matter of fact, recent in vitro observations using CD33e or minimally expressing cell lines and engineered CD33þ sublines support the concept that surface antigen expression is required for GO cytotoxicity.29 In these experiments, sublines expressing higher levels of CD33 also showed greater cell killing, suggesting that with highly susceptible cell targets, antigen density correlated with drug susceptibility. Recent data indicate that a high CD33-antigen load in peripheral blood may impair GO efficacy, probably due to peripheral consumption of the drug.30 In fact, CD33 saturation in the bone marrow was found to be significantly reduced (40e90% saturation) as compared with CD33 saturation in corresponding peripheral-blood samples (>90%). In vitro, such reduced CD33 saturation levels were strongly related to reduced cell kill. The hypothesis is that high CD33-antigen loads in blood consume

720 S. Amadori and R. Stasi

GO and thereby limit its penetration into bone marrow.30 Consequently, CD33 saturation in bone marrow is reduced, which hampers an efficient cell kill. Therefore, GO would probably be more effective if administered at higher or repeated doses, or, preferably, after reduction of the leukemic cell burden by conventional chemotherapy. The pharmacokinetics of GO is best represented by measurements of the hP67.6 antibody. After administration by intravenous infusion, the drug distributes to a relatively limited space. Slow elimination follows, with a mean half-life of 67 hours. Apparently as a result of reduced leukemic cells, there is a significant increase in plasma concentration after the second infused dose. Although highly variable between individuals, changes in concentrations could not be linked to age, sex, weight, body surface area, or ethnicity.31 Studies in animals have demonstrated that unconjugated calicheamicin derivatives represent less than 4% of total derivatives in plasma, suggesting that calicheamicin remains linked to GO in the circulation. Calicheamicin metabolites are detectable transiently in the serum from patients receiving GO therapy; the clinical significance of such exposure has not been established.32 Single-agent studies Preclinical results led to a phase I dose-escalation study of 40 relapsed or refractory AML patients.33 Three to eight patients were treated at each of eight dose levels of GO: 0.25, 0.5, 1, 2, 4, 5, 6 and 9 mg/m2. Similarly to other monoclonal antibodies, infusional-related toxicity, including fever and chills, was common and occurred within 2e4 hours from the start of therapy. Apart from this, non-hematologic toxicity was confined to the liver, with 20% of patients developing elevations of liver transaminases. As expected, significant myelosuppression was observed. Seven patients died within 30 days of study drug administration; six of these deaths were attributed to disease progression and one to sepsis occurring during neutropenia. More than two doses resulted in prolonged myelosuppression in two patients. Safety data suggested 9 mg/m2 as the maximum tolerated dose (MTD), and CD33 receptor site saturation data further supported the choice of 9 mg/m2 as the appropriate dose for phase II studies. Seven patients had clearance of blasts, and two patients entered CR at doses 9 mg/m2. Two patients developed antibodies to the calicheamicinelinker complex, one with clinical symptoms. The results of this study provided the safety and preliminary efficacy data to initiate phase II trials at a dose of 9 mg/m2. The dosing interval of 14 days was based on the half-life of the antibody. A dose would be expected to be cleared from the body in 4e5 half-lives, or approximately 12e15 days. Although analysis of efficacy was not an objective of this study, data were reviewed to monitor response to treatment. In addition to patients achieving full CR, it was found that some of the responders had clearance of bone marrow blast cells but failed to recover a normal platelet count. The term ‘morphologic remission’ was initially adopted to describe these responses; however, this term was subsequently changed to ‘CR with incomplete platelet recovery’ (CRp). This category of response was included in the phase II studies as a secondary efficacy endpoint. Originally, CRp did not include platelet transfusion independence as a criterion. After discussions with regulatory agencies, a more specific definition of CRp was developed for phase II studies. To be classified as having a CRp, patients had to meet all the criteria for CR except recovery to 100,000 platelets/mL. The CRp patients had to have sufficient bone marrow recovery to be independent of platelet transfusion for at least 1 week. This was supposed to be a clinically meaningful parameter in that patients who are

Monoclonal antibodies in AML 721

independent of platelet transfusion are expected to be at a lower risk for bleeding than those who are not. After the preliminary phase I study, three open-label phase II studies (201, 202, and 203) have been conducted in the United States, Canada, and Europe to further evaluate GO as a therapeutic agent. In these studies, designed to assess efficacy, a more selected patient population was chosen. Patients with prior myelodysplastic syndrome (MDS) or secondary AML were excluded. The first two trials admitted patients 18 years of age with at least a 6-month duration of CR after first-line therapy. The third trial admitted patients of 60 years with an initial CR of at least 3 months. Patients with initial white blood cell counts of >30,000/mL received hydroxyurea first to reduce the count below that level. Overall, 142 patients were entered into the phase II trials, and the pooled results were published in 2001 (Table 1).34 In this experience, 16% of patients achieved a CR using accepted criteria. In addition, 14% of patients achieved a CRp. Significant debate continues on the clinical relevance of achieving a CRp. The authors of the manuscript reporting the phase II results have suggested that the reason that some patients do not recover platelet counts fully is due to effects of the immunoconjugate on normal hematopoiesis and is not a reflection of residual leukemia in the marrow. Review of the data, however, shows that patients who achieved CRp and were not submitted to HSCT relapsed within a median of 2.1 months compared with a median relapse-free survival of 8.9 months for responding patients undergoing transplantation.34 Furthermore, of the patients who subsequently received HSCT, patients in CR survived for an average of 14.5 months, and patients in CRp for at least 5.4 months, compared with 4.2 months for patients who did not achieve remission. This finding would suggest that the level of cytoreduction of leukemic cells in patients with CRp is less than that of patients achieving true CR. The toxicities observed in the larger phase II experience were similar to that seen in the phase I study. Grade 3 or 4 infusion-related events reported with an incidence of at least 4% were chills (11%), fever (7%), and hypotension (4%). Preventive therapy with corticosteroids in addition to acetaminophen and diphenhydramine has been reported to eliminate or greatly reduce infusion-related toxicities.35 Other nonhematologic side-effects were moderate. Grade 3 or 4 hepatic toxicity was manifested primarily by transient elevations of liver transaminases (17%) or hyperbilirubinemia (23%). Evidence of more serious hepatic damage was observed in two patients (one with liver failure and one with persistent ascites and hepatosplenomegaly). A followup report describes results with GO given as a single agent to 101 patients, including 80 patients who were treated on the studies described above, who were aged 60 years (median 69 years, range 60e87 years) with their first recurrence of AML.36 The CR rate was 13%, and the CRp rate 15%. The median overall survival was 5.4 months for all patients, 14.5 months for patients who achieved CR, and 11.8 months for patients who achieved CRp. A 99% incidence of grade 3 or 4 neutropenia or thrombocytopenia was observed. Other grade 3 or 4 toxicities included infections (27%), hyperbilirubinemia (24%), transaminitis (15%), and mucositis (4%). Based on the phase II data, in May 2000 the US Food and Drug Administration (FDA) approved the use of GO for patients with relapsed AML who are >60 years of age and who are considered unfit for conventional cytotoxic therapy.37 However, as data on further studies in AML with GO-based regimens accumulate in the literature, initial enthusiasm has become tempered due to the fact that data on the effectiveness of single-agent GO in unselected patients and in patients with newly diagnosed AML were not so encouraging (Table 1). Roboz et al examined the

Reference

Number of patients

Sievers et al33

142

Larson et al36

Disease, status

CR/CRp (%)

Induction deaths (%)

Grade 3e4 liver toxicity (%)

Median overall survival

61 (22e84)

AML, first relapse

16/13

13

23/17

5.9 months (all patients) 12.6 months for CR patients 11.1 months for CRp patients

101

69 (60e87)

AML, first relapse

13/15

14

24/15

5.4 months (all patients) 14.5 months for CR patients 11.8 months for CRp patients

Roboz et al38

43

62a (19e84)

AML, untreated AML, relapsed MDS, untreated

9/5

14

21

4 months for CR and CRp patients

Estey et al39

51

71 (65e89)

AML, untreated MDS, untreated

8e36/NR

37

16

2 months in the no IL-11 group 4 months in the IL-11 group

Amadori et al40

40

76 (61e89)

AML, untreated

10/7

17

10/10

4.3 months (all patients) 11.4 months in patients age 61e75 years 1.0 month in patients age >75 years

Estey et al50

19

APL, untreated

84

16

0

NR 74%  14% at 30 months

51

Median age in years (range)

50

Lo-Coco et al

16

51 (17e77)

APL, relapsed

100/0

0

6

52

15

8.9 (0.7e17.3)

AML, relapsed AML, refractory

20/33

0

13

NR

Arceci et al54

29

12 (1e16)

AML, relapsed AML, refractory

14/14

10

7/21

NR

Zwaan et al

CR, complete remission; CRp, complete remission with incomplete platelet recovery; APL, acute promyelocytic leukemia; MDS, myelodysplastic syndrome; NR, not reported. a Mean age.

722 S. Amadori and R. Stasi

Table 1. Phase II studies of gemtuzumab ozogamicin (GO) monotherapy in acute myeloid leukemia (AML).

Monoclonal antibodies in AML 723

effectiveness of GO in patients with poor-prognosis AML. Forty-three patients, age 19e84 years (mean 62), were treated, including seven patients with untreated AML and age >70 years, two patients with AML evolving from a myelodysplastic syndrome, 14 with AML first salvage (first remission 0e6 months), 15 with AML  second salvage, and 14 with myeloid blast phase of chronic myeloid leukemia (CML). The overall response rate was 14%, with 4/43 (9%) patients achieving CR and 2/43 (5%) achieving CRp.38 Estey et al reported the results of a study involving 51 patients aged 65 years or older (median 71 years, range 65e89 years), 14% of whom were at least partly bedridden, with newly diagnosed AML or high-risk MDS.39 GO was given at 9 mg/m2 on days 1 and 8 or 1 and 15, with or without interleukin-11 (IL-11; 15 mg/kg per day on days 3e28). The CR rate was 8% for the arm without IL-11, and 36% for the arm with IL-11. The authors compared these results with historical controls of similar patients treated with idarubicin and cytosine arabinoside (IA), and concluded that GO was inferior to the IA regimen in this non-randomized retrospective comparison. Finally, Amadori et al have administered GO as initial therapy in AML patients 61 years of age deemed not fit for conventional chemotherapy due to either advanced age (>75 years) or a WHO performance status of grade 2 (age 61e75 years).40 The drug was administered at the dose of 9 mg/m2 as a single 2-hour intravenous infusion on days 1 and 15. Patients who achieved a complete remission (CR/CRp) were to receive a consolidation with two additional injections of the immunoconjugate at the same dose. The overall CR/CRp rate was 17% (95% confidence interval 8e32%). The CR/CRp rate in patients 61e75 years old was 33% (6/18), and 5% (1/22) in patients older than 75 years. Induction death occurred in seven patients (17%), all aged above 75 years. Overall survival was significantly longer in patients aged 61e75 years than in older individuals (P ¼ 0.05), and in CD33þ cases than in CD33e ones (P ¼ 0.05). The authors concluded that the dose/schedule of GO used in this trial is too toxic in the age group over 75 years. Combination regimens Much interest is now focused on the combination of GO with conventional anti-AML chemotherapy. Several pilot trials investigating combination regimens involving GO have been conducted at the M. D. Anderson Cancer Center (Table 2). In one study, GO (9 mg/m2 intravenously over 2 hours on day 1) was combined with ara-C (1 g/m2 over 2 hours intravenously daily on days 1e5) and topotecan (1.25 mg/m2 by continuous intravenous infusion daily on days 1e5).41 Seventeen patients (nine with primary resistant disease and eight with recurrent disease) with AML or advanced MDS received 20 courses of therapy. The median age was 55 years (range 20e70 years). Two patients (12%) achieved CR. The median survival was 8.2 weeks. Five patients (29%) developed grade 3 or 4 transaminitis, including one patient (6%) who died from veno-occlusive disease (VOD) of the liver. In another study, GO was combined with idarubicin and ara-C (MIA) in patients with refractory AML. GO was given at a dose of 6 mg/m2 intravenously over 2 hours on days 1 and 15, idarubicin was given at a dose of 9 mg/m2 intravenously over 30 minutes daily on days 2e4, and ara-C was given at a dose of 1.5 g/m2 by continuous intravenous infusion daily on days 2e5.42 Of 14 patients who were treated on that study, four (29%) had primary refractory AML, and ten (71%) had recurrent disease. Seven patients were aged 60 years or older. MIA induced CR in three patients (21%) and CRp in three patients (21%). The median survival was 8 weeks (range 2e64 weeks), and the median failure-free survival of complete responders was 27 weeks (range 11e64 weeks). All patients developed grade

Reference

Number of patients

Median age in years (range)

Disease, status

Other agents

CR/CRp (%)

Induction deaths (%)

Grade 3e4 liver toxicity (%)

Median overall survival

17

55 (20e70)

AML, relapsed

Topotecan, Ara-C

12

29

18/30

8.2 weeks

Alvarado et al

14

61 (34e74)

AML, relapsed AML, refractory

Idarubicin, Ara-C

21/21

43

NR/29

8 weeks

Apostilodou et al43

11

37 (16e67)

AML, relapsed AML, refractory

Ara-C, DNX, CSA

9/9

18

54/9

3 months

Tsimberidou et al44

59

57 (27e76)

AML, untreated MDS, untreated

Fludarabine, Ara-CCSA

46/2

25

31/7

8 months

Kell et al47

64

46.5 (18e59)

AML, untreated

DNR, Ara-C, 6-TG or Fludarabine, Ara-C, Idarubicin

84

9

47/28

78% at 8 months

Amadori et al48

57

68 (71e73)

AML, untreated

Mitoxantrone, Ara-C, VP-16

35/19

8/5

10.4 months

Cortes et al41 42

14

CR, complete remission; CRp, complete remission with incomplete platelet recovery; APL, acute promyelocytic leukemia; MDS, myelodysplastic syndrome; Ara-C, cytosine arabinoside; DNX, liposome-encapsulated daunorubicin; CSA, cyclosporine A; 6-TG, 6-thioguanine; VP-16, etoposide; NR, not reported.

724 S. Amadori and R. Stasi

Table 2. Phase II studies of gemtuzumab ozogamicin- (GO-)based combination chemotherapy in acute myeloid leukemia (AML).

Monoclonal antibodies in AML 725

3/4 myelosuppression, with severe sepsis occurring in ten patients (71%). Other grade 3/4 non-hematologic toxicities included transaminitis, oral mucositis, and diarrhea. Two patients (14%) developed hepatic VOD. A report from the same group concerned the MDAC regimen (ara-C 1 g/m2 intravenously over 2 hours daily on days 1e5; GO 6 mg/m2 intravenously over 2 hours on day 6; cyclosporine A 6 mg/kg over 2 hours on day 6 as a loading dose followed by 16 mg/kg as a continuous intravenous infusion on days 6e8; and liposome-encapsulated daunorubicin 75 mg/m2 daily as a continuous intravenous infusion on days 6e8) in patients with refractory AML.43 One of 11 patients (9%) achieved CR, and a second patient achieved CRp. Grade 3/4 toxicities included sepsis (63%), hyperbilirubinemia (54%), mucositis (27%), and transaminitis (9%). Cyclosporine A (CSA) has also been combined with GO, fludarabine, and ara-C (MFAC) in the de-novo and recurrent AML settings.44e46 The activity of the MFAC regimen (GO 4.5 mg/m2 intravenously over 2 hours after a loading dose of CSA on day 1; fludarabine 15 mg/m2 intravenously over 30 min every 12 hours for six doses on days 2e4; ara-C 0.5 g/m2 over 2 hours every 12 hours for six doses on days 2e4; CSA 6 mg/kg over 2 hours, followed by 16 mg/kg continuous intravenous infusion on days 1 and 2) as induction therapy has been evaluated in 39 patients with previously untreated AML and 20 patients with refractory anemia with excess blasts (RAEB), or RAEB in transformation (RAEB-t).45 Their median age was 57 years (range 27e76 years). The MFAC regimen induced CR in 27 patients (46%) and CRp in one patient (2%). The median overall survival was 8 months. At 12 months, the survival rate was 38% and the event-free survival rate in patients with CR/CRp was 27%. Infections complicated 38% of the courses of chemotherapy. Grade 3/4 toxicity included hyperbilirubinemia in 31% and transaminitis in 7% of the patients. Four patients (7%) developed hepatic VOD. The MFAC regimen was also evaluated as post-remission therapy in patients with AML after a GO-containing induction regimen.46 Patients in CR commenced idarubicin and ara-C (IA) alternating with MFAC, or vice versa, for 9 months from the date of CR. Idarubicin was administered at 8 mg/m2 on days 1 and 2 and ara-C at 1.5 g/m2 on days 1 and 2. A total of 22 patients received 76 courses of MFAC (35 courses) alternating with IA (41 courses), or vice versa. Failure-free and survival rates at 12 months were 32% and 55%, respectively. Grade 3/4 toxicities e including sepsis, neutropenic fever, and nausea/vomiting e were equivalent with MFAC and IA. These findings indicate that post-remission therapy with MFAC is feasible and well tolerated in patients with AML. Finally, MFAC was tested in 32 patients with primary resistant AML (34%) or recurrent AML (66%).44 Nine patients (28%) achieved CR, and two patients (6%) achieved CRp. The median survival was 5.3 months, and the 12-month survival rate was 19%. Fourteen patients (44%) developed grade 3/4 hyperbilirubinemia, six patients (18%) had grade 3/4 transaminitis, and three patients (9%) had hepatic VOD. Other groups have recently reported their experience with GO-based combination regimens (Table 2). Kell et al evaluated the feasibility of combining GO with intensive chemotherapy as first-line treatment of AML in 72 patients, aged 17e59 years, as a prelude to the United Kingdom Medical Research Council (MRC) AML15 trial.47 Sixtyfour patients received various remission induction regimens (DAT [daunorubicin, ara-C, thioguanine], DA [daunorubicin, ara-C], or FLAG-Ida [fludarabine, ara-C, G-CSF, idarubicin]), all including GO on day 1. It was possible to give GO 3 mg/m2 with course 1, but DAT was associated with liver toxicity. Combining GO 6 mg/m2 with course 1, or GO 3 mg/m2 with two consecutive induction courses was not feasible because of hepatotoxicity and delayed hematopoietic recovery. Thirty-one patients who were treated in consolidation with MACE (amsacrine, ara-C, etoposide) or high-dose ara-C (HiDAC) and GO (3 mg/m2) tolerated the combination well.

726 S. Amadori and R. Stasi

Complete remission with course 1 was seen in 86% of patients. GO given both during induction (DA or FLAG-Ida) and consolidation (MACE or HidAC) was well tolerated. These schedules are now being compared in the MRC AML15 trial in patients younger than 60 years of age. The European Organization for Research and Treatment of Cancer Leukemia Group (EORTC-LG), in collaboration with the Italian Cooperative Group GIMEMA, conducted a phase II study of GO and conventional chemotherapy with mitoxantrone, ara-C and etoposide (MICE) for induction of remission in previously untreated AML patients aged 61e75 years.48 Eligible patients received frontline treatment with GO 9 mg/m2 infused intravenously on days 1 and 15. Following response assessment to GO, patients were started on conventional chemotherapy with the MICE regimen. No further treatment was planned for complete responders. Among the 57 evaluable patients, 38 (67%) completed the whole sequential treatment as planned. The overall response rate to the entire induction sequence was 54.4% (31/57), with CR in 35.1% and CRp in 19.3%. Rates of failure due to treatment-related mortality or resistant disease were 14.1% (three toxic deaths during the GO segment, five during MICE) and 29.9%, respectively. An initial response to GO was documented in 20 patients (35.1%), with CR in 22.8% and CRp in 12.3%; six additional patients entered a partial remission. Reversible myelosuppression and liver toxicity were the main adverse events during both segments of induction. Frontline GO was associated with modest mucosal and gastrointestinal toxicity, but grade 3/4 myelosuppression was universal and prolonged. Hepatic VOD developed in three patients after GO and two after MICE, resulting in four deaths from liver failure. One-year survival at follow-up was 34%. Twelve patients continue in CR/CRp after a median of 226 days. This novel regimen is now being compared in a randomized phase III trial (AML-17). Trials in acute promyelocytic leukemia APL is an ideal model to test the efficacy of GO. In fact, APL is characterized by a high and homogeneous expression of the CD33 antigen in virtually 100% of cases and in 100% of individual patient blasts, as well as by the lack of, or very low expression of, the MDR glycoprotein.49 Two recent reports highlight the efficacy of GO in patients with APL (Table 2). Estey et al administered GO 9 mg/m2 with ATRA to 19 patients with previously untreated APL.50 Once they achieved CR, patients were to receive 8 courses of GO (9 mg/m2 every 4e5 weeks) and ATRA; idarubicin was added only for persistent or recurrent RT-PCR positivity. Sixteen patients (84%) achieved CR. All 12 patients who were tested at the time of the report had negative PCR results at 4e6 months from CR; none of seven patients who were evaluated subsequently reverted to positive PCR results (median follow-up in CR, 5 months; maximum 14 months). GO was not associated with clinically significant hepatotoxicity. Lo-Coco et al administered GO to 16 patients with APL who had relapsed at the molecular level. Of these patients, eight were experiencing a first, five a second, two a third, and one a fourth relapse.51 GO was administered at 6 mg/m2 for two doses, and patients achieving a new molecular remission (i.e., negativity of the RT-PCR test for PML/RARa) received a third dose. A molecular remission was obtained in nine (91%) of 11 patients tested after two doses and in 13 (100%) of 13 patients tested after the third dose. Of the remaining three patients, one achieved molecular remission after one GO administration and received no further therapy owing to hepatic toxicity, and two showed disease progression during treatment. Quantitative RT-PCR studies

Monoclonal antibodies in AML 727

showed that responding patients experienced a remarkable decline of the PML/RARa transcript after the first GO dose. Of 14 responders, seven remained in sustained molecular remission for a median of 15 months (range 7e31 months) while seven experienced relapse at 3e15 months. GO was administered again in two patients with relapse, and both obtained a new molecular remission. Trials in children and adolescents Limited data exist regarding the use of GO for the treatment of refractory or relapsed AML in children. In two European trials, GO was used to treat children with refractory/relapsed AML on a compassionate-use basis (Table 2).52,53 As reported by Zwaan et al, 15 children (four de novo, 11 relapsed/refractory) were administered GO at doses ranging from 4 to 9 mg/m2 per course.52 Clinical response included three patients achieving CR, five patients achieving CRp, and three patients with no change in bone marrow blast count. Although survival rates were low, many of these patients had relapsed at least once, if not multiple times, and were resistant to multiple chemotherapeutic agents and combinations. Results reported by Reinhardt et al in 12 relapsed/refractory patients receiving GO at 1.8e9 mg/m2 per course showed no evidence of antileukemic response.53 One patient in each compassionate-use report experienced hepatic VOD. Recently, Arceci et al reported the results of an open-label dose-escalation study in patients younger than 18 years of age with refractory and relapsed AML.54 Twentynine children and adolescents 1e16 years of age received GO ranging from 6 to 9 mg/m2 for two doses (separated by 2 weeks) infused over 2 hours. All patients had anticipated myelosuppression. Other toxicities included grade 3/4 hyperbilirubinemia (7%) and elevated hepatic transaminases (21%); the incidence of grade 3/4 mucositis (3%) or sepsis (24%) was relatively low. One patient treated at 9 mg/m2 developed hepatic VOD and defined the dose-limiting toxicity. The MTD was determined to be 6 mg/m2. Thirteen patients received HSCT <3.5 months after the last GO dose; six (40%) developed hepatic VOD. Eight of 29 patients achieved CR (28%). Remissions were comparable in refractory (30%) and relapsed (26%) patients. Flow cytometry studies revealed that the mean MDR-mediated drug efflux was significantly lower in the leukemic blasts of patients achieving remission (P < 0.005). Studies on liver toxicity A summary of the main topics associated with GO-related liver toxicity is reported in Table 3. In the pivotal phase II studies, the overall incidence of hepatic VOD was 3% when GO was administered to 277 patients with AML in first relapse.55 However, the incidence rate increased up to 15% and 20% in patients treated with GO in the pre- or post-HSCT setting, respectively. In a retrospective study, the incidence of VOD was 64% (9/14) when GO was administered to patients with AML prior to undergoing myeloablative allogeneic HSCT.56 A report by Giles et al indicates that GO is associated with a high risk of developing a potentially fatal VOD even outside the HSCT setting.57 In this study, a total of 119 patients with AML (n ¼ 92), advanced MDS (n ¼ 25), or CML in blast phase (n ¼ 2) were reviewed. VOD developed in 14 patients (12%). There was no evident relationship between the risk of developing VOD and age, performance status, baseline peripheral blood counts, presence of an antecedent hematological disorder, leukemia karyotype, FAB diagnosis, GO regimen and total dose, or baseline bilirubin and

728 S. Amadori and R. Stasi

Table 3. Gemtuzumab ozogamicin (GO) and liver toxicity.  Reversible increases in levels of serum bilirubin and aminotransferases in more than 50% of patients  Hyperbilirubinemia of grade 3/4 severity in 13e24% of patients  VOD occurs in <5% of patients when GO is used at the approved, single-agent dose schedule for AML in first recurrence; the incidence varies from 5% to 12% when GO is combined with other cytotoxic agents  The incidence of VOD increases to 20% in patients treated with GO in the post-HSCT setting, and to 15% in those who have received GO prior to undergoing HSCT  GO-related VOD is unpredictable in timing and may be fatal; apart from a prior HSCT, no significant risk factors have been identified  Hepatic injury is characterized by damage to sinusoidal endothelial cells, activation of stellate cells, sinusoidal vasoconstriction, and ischemic hepatocyte necrosis  Accordingly, GO-related VOD may be better defined by the term sinusoidal obstruction syndrome (SOS)  Preliminary reports support the prophylactic use of defibrotide for patients who receive HSCT after GO exposure AML, acute myeloid leukemia; VOD, veno-occlusive disease; HSCT, hematopoietic stem-cell transplantation.

transaminase levels. The authors hypothesized that in phase II studies the incidence of VOD was underreported because, prior to the use of GO, the occurrence of VOD outside the HSCT setting had been very uncommon.58 They also noted that in the APL trials or in the M.D. Anderson studies on the MFAC regimen as post-remission therapy, no patient developed hepatic VOD. To explain these findings, they speculated on a correlation between the circulating tumor load, tumor load in the liver, or circulating soluble CD33 levels and ensuing GO-associated VOD and/or hepatotoxicity.59 Rajvanshi et al reviewed the course of 23 patients who were given GO for AML that had relapsed after HSCT.60 Liver toxicity was assessed through physical examination, serum tests, histologic examination, and hepatic venous pressure measurements. Liver injury developed in 11 patients after GO administration; it was manifested as weight gain, ascites, and jaundice in seven patients. Seven patients died with persistent liver dysfunction and either multiorgan failure or sepsis at a median of 40 days after GO infusion. Portal pressure measurements were elevated in two patients. Results of liver histology in five patients showed sinusoidal injury with extensive sinusoidal fibrosis, centrilobular congestion, and hepatocyte necrosis. These findings suggest that GO targets CD33þ cells residing in hepatic sinusoids as a likely mechanism for its hepatic toxicity. McDonald has proposed using the term sinusoidal obstruction syndrome (SOS) for GO-related VOD, because it is more descriptive of the histological pattern seen in patients.61 In addition to the direct injury of Kupffer cells, which are known to be CD33þ, he also advanced an alternative pathophysiological hypothesis. In this model, defective secretion of gluthionyl calicheamicin by hepatocytes into bile leads to accumulation of calicheamicin in hepatocytes and sinusoids, which leads to activation of stellate cells and apoptosis of sinusoidal endothelial cells.61 A few trials have addressed the issue of the pharmacologic prevention of GO-related SOS/VOD. The M. D. Anderson group tested the efficacy of ursodiol in reducing the incidence and/or severity of liver toxicity in patients with refractory AML or MDS undergoing GO therapy.62 Ursodiol is a hydrophilic, non-hepatotoxic bile salt variably reported to decrease

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the incidence of HSCT-associated VOD.58 A total of 85 consecutive patients who were receiving treatment with GO-based regimens received ursodiol orally at a dose of 600 mg daily for 21 days, beginning 1 day prior to therapy. VOD developed in ten of 85 patients (12%). This incidence is superimposable on that reported in a prior cohort of 119 patients who received the same GO-based regimens without ursodiol. Recently, the prophylactic use of defibrotide has been shown to prevent SOS/VOD in children who had undergone stem-cell transplantation after GO exposure.63 Radiolabeled anti-CD33 antibodies The development of radiolabeled anti-CD33 MoAbs has been based primarily on the characteristics (physical half-life, emission range, labeling efficiency) of the radioisotope used. Most studies have employed b-particle-emitting isotopes. Because b particles have a relatively long path length (0.8e5.0 mm), they have the potential to destroy the targeted and surrounding cells, including normal cells. Therefore, their use has generally been confined to patients undergoing marrow transplantation, in which marrow ablation has been the goal. Initial studies used anti-CD33 antibodies labeled with iodine-131 (131I). This radioisotope has a half-life of 8 days, and has both a b decay with a path length of 0.7 mm and a g component that allows the quantitative determination of antibody localization in patients by g camera imaging, but which necessitates that patients be treated in radiation isolation. Early studies examined the biodistribution of 131I-labeled M195 and p67 murine antibodies in patients with AML beyond first remission.64,65 For each anti-CD33 antibody, the antibody dose resulting in optimal biodistribution was low (<5 mg/m2) because the relative numbers of CD33 antigen sites were rapidly saturated and the circulation of unbound antibody at higher antibody doses led to increased radiation delivery to extramedullary tissues. A therapeutic dose-escalation study of 131I-M195 was conducted in 24 patients with relapsed or refractory AML, including seven who had failed prior HSCT.65 Divided doses ranging from 50 mCi/m2 to 210 mCi/m2 were administered. Twenty-three patients (96%) demonstrated declines in peripheral blood cell counts, with decreased percentages of bone marrow blasts seen in 83% of cases. For 131I-M195 doses of 135 mCi/m2 or greater, pancytopenia was profound and lasted for at least 12 days. Eight patients had sufficient marrow cytoreduction to proceed to HSCT. Of assessable patients, 37% developed human anti-mouse antibody (HAMA). Significant hepatic toxicity was seen in one patient, and the maximum tolerated dose (MTD) was not reached. 131 I-M195 has been evaluated in patients with relapsed APL to treat minimal residual disease.66 After attaining a second remission with all-trans retinoic acid, seven patients received either 50 or 70 mCi/m2 of 131I-M195. No immediate toxicity was seen, and late toxicity was limited to myelosuppression. The maximum tolerated dose of 131 I-M195 that resulted in neutropenia lasting fewer than 2 weeks was 50 mCi/m2. Of six patients who were RT-PCR-positive before 131I-M195, two transiently converted to negative. The median disease-free survival of the seven patients was 8 months (range 3e14 months). Neutralizing HAMA developed in five of the seven patients, allowing only one dose of 131I-M195 to be administered. More recently, the same group investigated whether 131I-labeled M195 and HuM195 could be combined safely with busulfan and cyclophosphamide (BuCy) as preparative regimen for allogeneic HSCT.67 A total of 31 patients with relapsed/refractory AML (n ¼ 16), blast phase of CML (n ¼ 14), or

730 S. Amadori and R. Stasi

advanced MDS (n ¼ 1) received 131I-M195 or 131I-HuM195 (total doses ranging from 122 to 437 mCi) plus BuCy. Hyperbilirubinemia was the most common extramedullary toxicity, occurring in 69% of patients during the first 28 days after transplant. The median survival was 4.9 months (range 0.3e90þ months). Investigators at Memorial Sloan Kettering Cancer Center have developed a radioimmunotherapy with a-particle emitters to treat AML. In contrast to b particles, a particles have high energy but a short path length, such as 50e80 mm for bismuth-213 (213Bi). Most available a particles have a very short half-life, and the 46-minute half-life for 213Bi necessitates that the antibody be administered immediately after radiolabeling.68 It is also critical that the antibody has rapid access to target cells before radioisotope decay. In a phase I non-transplant trial in 18 patients with relapsed AML, doses of 10.36e37 MBq/kg 213Bi-HuM195 were administered over 2e4 days in 3e7 fractions. Early scans demonstrated localization of isotope in marrow, spleen, and liver within 10 minutes of infusion, with better localization to marrow with later as opposed to earlier infusions. The maximum tolerated dose in this study was not reached because escalation beyond 37 MBq/kg was not performed. Median time of marrow recovery was 22 days, but myelosuppression lasted up to 34 days in patients treated at the highest dose levels. No complete responses were observed, but there was a decrease in percentage of marrow blasts in 14 patients.69 Because of the risk of delivering toxic radiation doses to extramedullary tissues when large doses of a-emitting radioisotopes are used, these investigators are now focusing on treating patients with less advanced disease. These considerations led to a trial in which 213Bi-HuM195 is given for the elimination of minimal disease after partial cytoreduction with cytosine arabinoside in patients with advanced AML. CLINICAL EXPERIENCE WITH ANTIBODIES TO OTHER ANTIGENS Radiolabeled antibodies have been developed against other cell-surface antigens on myeloid cells, including CD45 and CD66. In contrast to the relatively narrow expression and low copy numbers for CD33, the CD45 antigen is broadly expressed by all leukocytes and their precursors at an average copy number of w200,000 per cell.70 It is expressed by most AML and the majority of ALL samples, and is not appreciably internalized after antibody binding.71 Since it is expressed by both normal and malignant cells, radiolabeled anti-CD45 antibodies can be used to deliver radiation to marrow, spleen, and lymph nodes for patients with acute leukemia, whether in remission or relapse. For patients in remission, even blasts not expressing CD45 may be killed if surrounded predominantly by non-malignant hematopoietic cells, because of the bystander effect. The largest experience with murine 131I-labeled anti-CD45 (BC8) monoclonal antibody has been reported by the Fred Hutchinson Cancer Research Center.72 In a phase I clinical trial, 131I-BC8 was administered to 25 patients in remission and in relapse, to ascertain factors influencing antibody biodistribution, and to estimate the MTD of radiation delivered by antibody that could be combined with the conventional transplant preparative regimen of high-dose cyclophosphamide and 12 Gy TBI. Eligible patients were those at high risk of relapsing post-transplant, including those with advanced (e.g. primary refractory or beyond first remission) acute myeloid or lymphoid leukemia or myelodysplastic syndrome, with HLA-matched related donors or autologous HSCs available. Thirty-four patients received a therapy dose of antibody labeled with 76e613 mCi 131I, estimated to deliver 3.5e12.25 Gy to the normal bone

Monoclonal antibodies in AML 731

marrow. Dose-limiting regimen-related toxicity was seen in one of six patients (grade 3 VOD of the liver) treated at 10.5 Gy, and in both of two patients treated at 12.25 Gy (grade 3e4 mucositis), and thus the MTD was estimated to be 10.5 Gy. Although this phase I study was not designed to determine efficacy of the combined preparative regimen, 30% of the patients with advanced leukemia (seven of 25 with AML or MDS and three of nine with ALL) survive disease-free 38e112 months after transplantation. This study demonstrated that appreciable doses of supplemental targeted radiation to hematopoietic tissues (average estimated doses of 24 Gy to marrow and 50 Gy to spleen with an MTD of 10.5 Gy to liver) can be delivered when combined with a conventional preparatory regimen. Phase II studies are now ongoing. CD66, also known as non-specific cross-reacting antigen (NCA), is a glycoprotein expressed on myeloid cells but not on leukemia cells. A phase I dosimetry trial showed that administration of rhenium-188 (188Re)-labeled anti-CD66 resulted in a favorable biodistribution in 11 of 12 patients, with significant amounts of radiation delivered to the marrow.73,74 Bunjes et al reported encouraging results of a phase I/II study on the ability of 188Re-anti-CD66 MoAb to intensify the conditioning regimen prior to HSCT for patients with high-risk AML or MDS.75 Thirty-two patients were allografted after a conditioning with the radiolabeled MoAb in combination with TBI or busulfan-based regimens. Thirty patients received T-cell-depleted peripheral blood or bone marrow stem cells. The authors observed a transplantationrelated mortality of 24%. Only three patients developed a clinically relevant acute graft-versus-host disease grade II. Klein et al further reported their results on 19 patients receiving allografts after conditioning regimens intensified by 188Re-labeled anti-CD66 MoAbs.76 In contrast to Bunjes et al, they reported a very high rate of severe acute graft-versus-host disease and transplantation-related mortality, although it has to be underlined that a different immunosuppressive regimen was employed. CONCLUSION Passive immunotherapy with monoclonal antibodies is a promising strategy for the treatment of AML. However, because of the difficulty in targeting large disease burdens, the elimination of minimal residual disease remains its most promising application. As a matter of fact, the most encouraging results have been obtained in patients with APL who had evidence of disease only at the molecular level. The role of GO in elderly patients clearly needs to be better defined with randomized clinical studies. It is still to be determined whether this agent, when incorporated into AML treatment regimens, will produce a clinically significant benefit. Several studies are under way that incorporate GO as post-remission therapy or combine it with standard induction chemotherapy to evaluate this question. An issue in some of these studies is that the addition of GO requires dose reductions of the established agents. A situation where the introduction of a novel agent requires such dose reductions is always fraught with controversy, and benefits of the novel regimen need to be very convincing indeed to justify a change in therapy. Another critical issue is the validity of the concept of CRp. In fact, patients who are unable to undergo further cytotoxic therapy owing to long-lasting thrombocytopenia are likely to relapse and die rapidly. Therefore, for patients with AML in relapse, achievement of a CRp represents an antileukemic response that appears to be of benefit only to those patients for whom a transplant strategy is available.

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Radioimmunotherapy with b-particle emitters may be most effective for the treatment of bulky disease or as part of a preparative regimen for hematopoietic stem-cell transplantation, whereas radioimmunotherapy with a-particle emitters may be better suited for the treatment of small-volume or minimal residual leukemia. Whether the use of these monoclonal antibodies in combination with, or as a substitute for, currently available therapy will lead to improved outcomes for patients with AML remains to be demonstrated by carefully designed clinical trials.

Practice points  both labeled and unlabeled monoclonal antibodies have been shown to be useful for the control of minimal residual disease in acute promyelocytic leukemia  single-agent gemtuzumab ozogamicin has significant activity in patients aged 61e75 years with relapsed or refractory AML who are not considered suitable for conventional chemotherapy  gemtuzumab ozogamicin can be safely incorporated in combination regimens with standard chemotherapy agents  veno-occlusive disease of the liver is a potentially serious complication of gemtuzumab ozogamicin treatment, and may occur even outside of the hematopoietic stem-cell transplantation setting

Research agenda  randomized phase III trials to assess the efficacy of monoclonal antibodies and immunoconjugates in improving the outcome of patients with acute myeloid leukemia  further evaluation of gemtuzumab ozogamicin in very old individuals (75 years)  development of strategies to prevent liver toxicity associated with gemtuzumab ozogamicin-based regimens  studies that incorporate multidrug resistance-reverting agents to enhance the antileukemic activity of gemtuzumab ozogamicin  explore the activity of monoclonal antibodies and immunoconjugates directed against other cell-surface antigens

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