Oncogene (2007) 26, 3679–3690 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc REVIEW The role of Gemtuzumab Ozogamicin in the treatment of acute myeloid patients

L Pagano, L Fianchi, M Caira, S Rutella and G Leone

Istituto di Ematologia, Universita` Cattolica del Sacro Cuore, Roma, Italia

Gemtuzumab Ozogamicin (GO) is an antibody-targeted (HiDARAC) (NCCN, 2002).Unfortunately, a high agent consisting of the humanized murine percentage of patients relapses or is resistant to the CD33 antibody (clone P67.6) to which the - induction treatment: about 10–50% of patients with g1 derivative is attached via a hydrolysable bifunctional newly diagnosed AML may not achieve complete linker. GO is able to induce apoptosis in vitro in CD33- remission (CR).The AML relapse may be attributable expressing cells and it has been approved in USA and in to the resistance of leukemic cells to currently available Europe as monotherapy for the treatment of elderly chemotherapy (Montillo et al., 1998). The treatment of patients (older than 60 years) with relapsed acute myeloid refractory/relapsed AML is a relevant problem, parti- leukemia (AML). GO administered as a single agent cularly in elderly patients, for which treatment-related has resulted in overall response rates of about 30% in morbidity and mortality contraindicate a high-dose previously relapsed adults AML patients (including also cytotoxic chemotherapy.As well as age, duration of with incomplete platelet recovery). Preliminary data first CR is another important outcome predictive factor indicate a potential role for GO also as a component of in relapsed/refractory AML (Estey, 2000).Of note CR induction or consolidation regimens in adults and children. rate is 40–60% in patients with a 1 year or longer first As for adverse events, veno-occlusive syndrome charac- remission duration, but it is only 10–15% in those with a terizes its tolerability profile, but GO is comparatively shorter first CR (Thalhammer et al., 1996). At present, well tolerated by most patients. there are no standard treatment regimens for this Oncogene (2007) 26, 3679–3690. doi:10.1038/sj.onc.1210364 category of patients and a wide variety of empirical approaches have been used.Different drug combina- Keywords: ; elderly; mylotarg tions have been experienced (i.e. ara-c plus mitoxan- trone or HiDARAC), obtaining a further CR in about 40% of patients in first relapse (range 5–80%) (Vogler et al., 1994). It is clear that results of conventional treatments, Introduction particularly in refractory/relapsed patients, are com- pletely unsatisfactory.Consequently, new therapies are Acute myeloid leukemia (AML) is the most common necessary to improve survival. type of acute leukemia occurring in adults.Approxi- Opportunities for major therapeutic advances have mately 11 000 individuals are diagnosed annually in the come from the identification of novel targets to which United States as having AML.The median age at specific therapy can be directed, theoretically sparing presentation is approximately 65 years.The incidence of normal tissues.The development of AML and myelodysplasia appears to be rising, particu- with monoclonal antibodies, specifically Gemtuzumab larly in patients older than 60 years (O’Donnell, 2004). Ozogamicin (GO; Mylotarg, Wyeth-Ayerst, St Davids, The prognosis of AML adults patients is generally poor: PA, USA), has provided the first such agent in the only 15–25% of them is alive at 5 years from diagnosis armamentarium of antileukemic drugs. and the median survival among relapsed patients is about 3–12 months (Keating et al., 1989). More dramatic is the situation of elderly AML that presented a percentage of patients alive after 5 years lower than 5– Monoclonal antibodies: GO 10%.Current standard therapy consists of induction with cytotoxic chemotherapy including an anthracycline GO is a new -targeted antineo- and cytarabine (ara-c) and consolidation with cyclic plastic agent consisting of a recombinant humanized intensive chemotherapy, usually high doses of ara-c antibody to CD33, which is linked to calicheamicin, a potent antitumor antibiotic that cleaves double-stranded DNA at specific sequences (Zein et al., 1989). Approxi- Correspondence: Professor L Pagano, Istituto di Ematologia, Universita` Cattolica del Sacro Cuore, Largo Francesco Vito, 1, I-00168 Roma, mately 90% of AML patients have myeloid blast cells Italia. expressing the CD33 surface antigen (Legrand et al., E-mail: [email protected] 2000).Therefore, GO was designed to deliver cytotoxic Gemtuzumab Ozogamicin and acute myeloid leukemia L Pagano et al 3680 chemotherapy selectively to AML cells while limiting which is released inside the lysosomes because of their non-hematologic toxicity (Sievers et al., 2000). The acidic pH, binds to DNA in the minor groove in a conventional schedule of GO administration is a 2-h relatively sequence-specific manner and causes DNA infusion (9 mg/m2) on days 1 and 15 of treatment.GO is double-strand breaks and cell death (Ikemoto et al., licensed for the treatment of elderly AML patients in 1995).Several mechanisms responsible for sensitivity of first relapse as single agent.However, in the recent years AML cells to GO have been described, including surface various studies have tested the GO combined to other levels of CD33, binding of GO to cell surface CD33, antileukemic agents also in younger or pediatric patients antibody-receptor internalization, re-expression of either at relapse or at diagnosis. CD33, P-glycoprotein-mediated efflux of GO (Figure 1). Recently, the existence of a quantitative relationship between CD33 expression levels and in vitro response to Biological characteristics and therapeutical opportunities GO has been demonstrated by use of lentivirus- mediated gene transfer to manipulate CD33 expression The CD33 antigen is a 67-kDa type I transmembrane in myeloid cell lines that normally lack CD33 or have sialoglycoprotein, a founding member of a rapidly very low levels of CD33 (Walter et al., 2004). evolving immunoglobulin superfamily subset of sialic Furthermore, disruption of the CD33 cytoplasmic acid binding immunoglobulin-related lectins (siglecs; ITIMs, which are crucial for the internalization of siglec-3) (Crocker, 2002).Functionally, CD33 is in- antibody bound to CD33, by introduction of point volved in cell–cell interactions and signalling in the mutations not only prevents internalization of Ab- hematopoietic and the immune system.CD33 is bound CD33, but also reduces GO-induced cytotoxicity. characterized by the presence of two conserved immu- It must be also mentioned that efficient non-CD33- noreceptor tyrosine-based inhibitory motifs (ITIMs) in mediated GO uptake can occur via endocytosis in the cytoplasmic region, a property that is shared with all human CD33neg acute lymphoblastic (Jedema CD33-related siglecs discovered so far.If phosphory- et al., 2004), providing a potential rationale for the use lated upon treatment with pervanadate or anti-CD33 of GO in the treatment of CD33neg malignancies with antibody plus a cross-linking secondary antibody, these endocytic capacity. tyrosine motifs recruit and activate Src homology-2 Manipulation of the expression level of CD33 on (SH2) domain-containing tyrosine phosphatases (SHP-1 AML blasts has been advocated as a novel therapeutic and SHP-2).Also, cross-linking of CD33 with mono- approach to improve the clinical efficacy of GO. clonal antibodies on normal CD34 þ cells cultured with Unfortunately, scarce information is currently available stem cell factor (SCF) and granulocyte macrophage on the regulation of CD33 expression on both normal colony-stimulating factor (GM-CSF) has been asso- and malignant cells.Preliminary in vitro studies suggest ciated with inhibition of cell growth, suggesting a role that G-CSF, GM-CSF, interferon-a, interleukin (IL)-3 for CD33 in the negative regulation of cell proliferation and IL-6 may modestly increase CD33 expression on the (Mingari et al., 2001). It has been shown that expression surface of CD34 þ CD33 þ blast cells (Sivaraman et al., of the protein kinase Syk in AML cells correlates with 2002).Recently, we showed that in vivo priming with G- responsiveness to CD33 ligation (Balailan et al., 2003). CSF in elderly patients with AML might be associated In the responding AML cases, CD33 ligation induces with an increase in the percentage of AML blasts phosphorylation of Syk as well as the CD33 molecule, expressing the CD33 antigen (Leone et al., 2004), raising and the formation of a Syk/CD33 complex.None- the possibility that augmented CD33 expression in vivo theless, a proportion of Sykneg AML cases might might translate into better clinical responses.Theoretically, respond to CD33 ligation by substitution of Syk with the functionally overlapping ZAP-70 protein kinase. Approximately 90% of AML patients have myeloid blast cells that express the CD33 surface antigen 6 (Dinndorf et al., 1986; Legrand et al., 2000). The CD33 2 1. CD33 expression level CD33 antigen is virtually absent on steady-state, bone 2. CD33 saturation marrow resident hematopoietic stem cells (HSC) and on 5 3. Internalization 4. Activation of calicheamicin non-hematopoietic tissues but is highly expressed on CD33 3 5. New expression of CD33 granulocyte colony-stimulating factor (G-CSF-mobi- 4 6. Binding of GO lized HSCs) (Rutella et al., 2000). 1 7. Efflux of calicheamicin 8. Induction of DNA breaks GO is composed of the humanized immunoglobulin G4 (IgG4) monoclonal antibody anti-CD33 (mAb) hP67.6, linked to N-acetyl g calicheamicin dimethyl 7 hydrazide.Unlike the HuM195 anti-CD33 antibody, 8 which contains a human IgG1 framework, the IgG4 Drug pump component of GO (hP67.6) is not cytotoxic in vitro nor does it induce complement-mediated or antibody- Figure 1 The cellular parameters that affect AML cell sensitivity to GO have been listed.The response of AML blast cells to GO dependent cellular cytotoxicity. might depend on GO binding, internalization of GO–CD33 On binding of GO to CD33, the compound is rapidly complexes, re-expression of surface membrane CD33 and P-gp- internalized (van der Velden et al., 2001) and calicheamicin, mediated efflux of calicheamicin.

Oncogene Gemtuzumab Ozogamicin and acute myeloid leukemia L Pagano et al 3681 the combination of myeloid growth factors and GO patients, as it might have implications for the treatment might also enhance drug sensitivity through mechan- of AMLs. isms that are independent of a cytokine effect on surface CD33 expression, for example, stimulation of cell proliferation or reversal of GO resistance at the molecular level. Clinical experiences with GO The in vitro response of AML cell lines to GO has been shown to fall into three groups (Amico As already reported, GO has been approved as et al., 2003): cells that become blocked in the G2 phase monotherapy in USA and in Europe for the treatment of cell cycle but do not go into apoptosis (THP-1); of elderly patients with relapse AML; however, better those that are blocked in G2 but rapidly go into results seem to be obtained when it is administered in apoptosis (HL-60 and NB-4), and, finally, cells combination with other drugs. resistant to even high doses of GO (KG-1).Further- more, it has been shown that apoptosis involves caspase GO as monotherapy 3 activation and that G2 arrest correlates with phos- In a phase I dose-escalation study, Sievers et al.(1999) phorylation of ataxia-telangiectasia mutated/ataxia- treated 40 patients with refractory or relapsed AML telangiectasia-related (ATM/ATR)–Chk1/Chk2 kinases, with GO as a single-agent with doses ranging from 0.25 but not with induction of the other major players to 9 mg/m2.Inclusion criteria were: a good performance in both G1 and G2 arrest following DNA damage, status, a white blood cell count o30 Â 109/l, and absence namely, p53 and p21. of renal or liver dysfunction.Eighteen patients had We recently investigated whether the in vitro exposure received prior stem cell transplant (SCT).After 1–3 to G-CSF sensitizes AML cell lines and primary AML doses of GO, eight patients (20%) achieved a reduction blast cells to the killing effect of GO (Rutella et al., in marrow blast count to less than 5% at morphological 2006).The GO-sensitive HL-60 and NB-4 AML cell exam.Five of these eight patients recovered neutrophil lines but not GO-insensitive K562, TF-1 and KG-1a count, but only three of them achieved a normal platelet cells, displayed enhanced cell death in response to GO level.Those patients who achieved a CR but without after in vitro priming with G-CSF.Pretreatment with recovering a platelet count greater than 100 Â 109/l, were G-CSF did not affect cell cycle distribution either in considered to have achieved CR with thrombocytopenia GO-insensitive or in GO-sensitive AML cell lines.Also, (CRp).Toxicities were primarily infusion-related in vitro priming with G-CSF exerted negligible effects on and chills, and myelosuppression.Based on these CD33 expression levels, suggesting that mechanisms results, a three open-label, multicenter phase II trials other than activation of cell division or increase of with GO at 9 mg/m2 dose were started (Sievers et al., CD33-binding sites on the cell membrane might 2001).All included patients were in first relapse, with the be involved in the G-CSF-induced enhancement of evidence of CD33 þ AML blasts.One hundred and GO-specific cell death.The in vitro effects of G-CSF on forty-two patients with a median age of 61 years were GO-induced cytotoxicity were also evaluated in 10 enrolled in these three studies.The overall response rate consecutive elderly patients with AML.Response to (including both CR and CRp) was 30% with a median GO was heterogeneous, with half of the cases showing time to remission of 60 days.There were not differences induction of cell death and half of the cases being among patients when we stratified them for age or refractory to the cytocidal effect of GO.As already cytogenetic risk groups.The median relapse-free survi- shown with AML lines, G-CSF exerted no significant val (RFS) for all patients achieving a remission was 5.3 effects either on cell cycle distribution or on CD33 months.Myelosuppression was the first side effect, but expression level in primary AML blast cells when about 30% of patients developed treatment-related compared with cultures maintained with SCF and IL-3 infections and/or increase of bilirubinemia and tran- alone.It has been shown that AML blasts from GO- saminases.Hepatic veno-occlusive disease (VOD) refractory patients might express the ATP-binding occurred in seven patients (3%) and was fatal in two cassette (ABC) membrane transporter P-gp/ABCB of them. (Pallis and Russell, 2004), the product of the MDR Similarly to Sivers, the Mylotarg Study Group gene, and possess drug efflux capacity.In our study, (Larson et al., 2002) treated 101 patients aging 60 years G-CSF exerted a dose-dependent inhibition of P-gp/ or older at first relapse of AML.The CR rate was 13%, ABCB1 function in efflux-competent AML cells lines.P- with a CRp rate of 15%.The median overall survival gp/ABCB1 expression levels were unchanged in AML (OS) was 5.4 months for all patients, 14.5 months for cells lines exposed to G-CSF when compared with those patients who achieved a CR and 11.8 months for not treated with the cytokine, suggesting that the patients who achieved a CRp.Grade 3 or 4 toxicities modulation of P-gp/ABCB1 function occurred at the included neutropenia, thrombocytopenia, infections, functional level.Collectively, these studies demonstrate hyperbilirubinemia, increase of transaminases and that G-CSF might sensitize AML blast cells to the mucositis. killing effect of GO, and the observation that in vitro GO was also used in the treatment of high-risk pretreatment with G-CSF might sensitize GO-resistant myelodysplastic syndrome (MDS) patients, but results primary AML blast cells to the cytocydal effects of GO were very poor.Among 26 MDS patients, none may deserve further investigation in a wider cohort of achieved a CR and toxicities included grade 3/4

Oncogene Gemtuzumab Ozogamicin and acute myeloid leukemia L Pagano et al 3682 infections in 11 patients and grade 3/4 hepatotoxicity in CRp.No major liver toxicities were observed and the eight patients (Raza et al., 2002). authors concluded that GO administered in fractioned GO as a single agent at conventional doses was doses showed an excellent efficacy/safety profile (Taksin compared with HiDARAC in the treatment of AML et al., 2007). patients at first relapse.One hundred and twenty-eight In the last years, GO as monotherapy was used also in patients were enrolled in each arm.Patients treated with induction of first remission particularly in high-risk GO achieved a CR in the 38% of cases (increasing to elderly AML. 41% considering also CRp).GO treatment was charac- A study by GIMEMA-EORTC evaluated the efficacy terized by a higher remission duration if the first of GO as single-agent treatment for frail elderly AML remission duration was of 3–10 months, whereas patients (AML-15B trial).Forty untreated patients HiDARAC resulted the most efficacious when the received GO 9 mg/m2 in two doses separated by 2 duration of first CR was longer than 19 months.No weeks.More than 50% of them aged over 75 years.A differences were observed if the duration of first total of seven patients (four CR and three CRp) were remission was between 10 and 19 months (Leopold considered responsive (17%), whereas 24 patients were et al., 2003). resistant (60%) and other seven patients (17%) died in The efficacy of GO as monotherapy at different doses induction (two patients were not valuable).The median was tested in adult, elderly and pediatric patients.In a OS was 4.3 months with a 1- and 2-year survival rates of study including eight relapsed/refractory elderly AML, 34 and 7%.Myelosuppression occurred in the majority GO (9 mg/m2) was administered after a priming with of patients, and the most frequently related complica- recombinant human granulocyte colony-stimulating tions were microbiologically documented infections factor (rhG-CSF) before treatment both for increasing (40%).Severe liver toxicity was observed in four cases proportion of CD34 þ /CD33 þ positive blast cells and (10%), but only one of them developed a fatal VOD. for sensitizing blast cells to calicheamicin.A CR was The authors stated that GO at this schedule resulted too observed in four patients (50%) with a median event- toxic in patients aging over 75 years (Amadori et al., free survival of 4.7 months. It is noteworthy that VOD 2005).Conversely, in another small pilot study by represented the most relevant side effect, observed in Nabhan et al.(2004) 12 patients with AML aging over three patients (37.5%) (Leone et al., 2004). 65 years were treated with GO as first induction; among Similarly, Piccaluga et al.(2004a) treated 24 relapsed/ 11 valuable cases, a response rate was observed in three refractory elderly AML patients (median age 63 years) patients (27%) with an RFS of 7.6 months. Adverse with standard doses (9 mg/m2) of GO, administered for events were considered acceptable by authors: grade 3 2–3 doses with a 2-week interval.The CR and CRp rate and 4 cardiac toxicity in five patients (41%) (Nabhan was 13 and 8%, respectively, with a median RFS of 6 et al., 2005). In 2005, Thomas et al.(2005) reported their months.The most common non-hematological toxicities experience with GO in two different schedules (6 mg/m2 were infections and liver dysfunction, but VOD on day 1; 15 or 3 mg/m2 on days 1, 4 and 7).A total of occurred in one patient only. 30 patients were treated and an overall response of 30% Recently, the Mylotarg Study Group, including USA, was obtained (nine patients).Better results were France, The Netherlands and Germany centres (Larson observed in younger patients (lower than 60 years).As et al., 2005), presented the results of three open-label, for side effects, a high percentage of febrile neutropenia single arm, phase II studies.They included 277 first and infections was observed (63%).However, the results relapsed AML patients (median age 61 years) that of this trial were very poor because of a median RFS of received monotherapy with GO at 9 mg/m2 in two doses 3 months. separated by 2 weeks.A total of 71 patients achieved More recently the Leiden University Group reported remission (26%).In particular, 35 patients (13%) its experience in 38 AML patients, both at diagnosis and achieved a CR and other 36 (13%) a CRp.The median at relapse, treated with GO at 6 and 9 mg/m2.A CR was RFS was 6.4 months for the first category and 4.5 observed in 12 patients, including five patients with months for the second one.As for treatment-related CRp, whereas one patient achieved a PR.The best complications, nine patients developed an intracranial results were observed in AML at diagnosis (47% CR in hemorrhage (3%) and eight of them died for this newly diagnosed vs 22% in relapsed group) and in those condition, whereas other 13 patients died for infection. with de novo AML (60% CR vs 22% in secondary Liver function abnormalities with grade 3 or 4 AML; P ¼ 0.015) (Van der Heiden et al., 2006). hyperbilirubinemia were reported in 80 patients (29%); The results of the main studies about GO used in alanine aminotransferase (ALT) or ALT abnormalities monotherapy are reported in Table 1. were less frequently detected (18 and 9%, respectively). Considering pediatric AML, some studies investi- A total of 16 episodes of VOD were documented (5%): gated the efficacy of GO in this subgroup patients.In the it was fatal in eight patients. first report, about 15 patients with relapsed/refractory The Alfa Group reported the results of a pivotal AML, eight patients showed an improvement with a phase II study in which 57 first relapsed AML patients reduction of blasts count, but only one were treated with GO at a dose of 3 mg/m2 on days 1, 4, CRp was documented (Zwaan et al., 2003). More 7.Fifteen patients (26%) achieved CR and other recently, 12 multiple relapsed or refractory AML four patients CRp (7%).The median RFS was children received GO as compassionate use.Five 11 months in both the group of patients, CR and children presented a hematological improvement with

Oncogene Gemtuzumab Ozogamicin and acute myeloid leukemia L Pagano et al 3683 Table 1 Results of the main trials with GO used in monotherapy in adult AML patients Authors No. of Median age State of disease Treatment CR % ORa % RFS OS patients (years) (months) (months)

Sievers et al. (2001) 142 61 (22–84) 1st relapse GO 16 30 6.8 5.9 (9 mg/m2) 11,141 g Larson et al. (2002) 101 69 (60–87) 1st relapse GO 13 28 3.3 5.4 (9 mg/m2) 11,141 g Piccaluga et al. (2004a) 24 63 (20–75) Relapsed GO 13 21 7 2 (6–9 mg/m2) 11,141 g Larson et al. (2005) 277 61 (20–87) 1st relapse GO 13 26 5.2 4.9 (9 mg/m2) 11,141 g Amadori et al. (2005) 41 76 (61–89) At diagnosis GO 10 17 NR 4 (9 mg/m2) 11,141 g Nabhan et al. (2005) 12 75 (33–79) At diagnosis GO 27 27 7.6 NR (9 mg/m2) 11,141 g Thomas et al. (2005) 30 63 (18–74) At diagnosis/relapsed GO 30 30 3 6.8 (6 mg/m2) 11,141 g OR (3 mg/m2) 11,41,71 g Van der Heiden et al. (2006) 38 58 (27–77) At diagnosis/relapsed GO 18 32 NR NR (6–9 mg/m2) Taksin et al. (2007) 57 64 (22–80) 1st relapse GO 26 33 11 8.4 (3 mg/m2) 11,41,71 g

Abbreviations: AML, acute myeloid leukemia; CR, complete remission; CRp, complete remission without platelet count recovery; GO, Gemtuzumab Ozogamicin; NR, not reported; OS, overall survival; RFS, relapse free survival. aCR+CRp.

a bone marrow blastic count lower than 5% without GO in refractory/relapsed AML achieving a CR.Only one patient presented a VOD that In one of the first studies, GO was employed at 9 mg/m2 recovered after defibrotide treatment (Reinhardt et al., combined with ara-c and topotecan (Cortes et al., 2002). 2004).In another series, 29 refractory or relapsed Seventeen patients with AML or advanced MDS (nine children received two doses of GO (separated by 2 resistant and eight relapsed disease) were treated.Two weeks) at 6, 7.5 or 9 mg/m2.In 13 of them, GO patients (12%) achieved a CR.The median OS was 8.2 administration was followed by hematopoietic stem cell weeks.Liver toxicity was observed in five patients (29%) transplantation (HSCT).A total of eight patients (28%) and one of them died for VOD.GO was tested in achieved overall remission without differences between combination with ara-c, mitoxantrone and amifostine in resistant or relapsed disease.All of them developed a 19 patients with either relapsed or post-MDS AML high myelosuppression, complicated by sepsis in 24% of (Venugopal et al., 2002). The results were dramatic. cases.Almost all patients had transient and reversible Eight patients had treatment-related liver toxicity and alterations in liver function test results (aspartate three of them died for progression of this complication. aminotransferase, ALT and total bilirubin).Grade 3 Eleven of the 19 enrolled patients died for sepsis, and 4 elevations were observed in eight (28%) patients bleeding, multiorgan failure or progressive disease. and VOD occurred in seven patients (Arceci et al., During the following years, GO was combined with 2005). many other different drugs.It was tested in association with idarubicin and ara-c (MIA) in 14 patients with AML (four refractory and 10 relapsed).GO was administered at a dose of 6 mg/m2 (Alvarado et al., G-O combined with other 2003).Six patients were responsive: three of them (21%) achieved a CR, whereas remaining three (21%) achieved Other trials evaluated the efficacy of GO in combination a CRp.Six patients (43%) died early during the aplasia, with cytotoxic chemotherapy for treating refractory/ and two patients were refractory to therapy.The median relapsed AML or as front-line therapy.GO was OS was 8 weeks (range 2–64 weeks).The median RFS of associated with different drugs in order to achieve a patients who achieved a CR or a CRp was 27 weeks higher CR rate and to prolong RFS. (range 11–64 weeks).The treatment was associated with

Oncogene Gemtuzumab Ozogamicin and acute myeloid leukemia L Pagano et al 3684 myelosuppression.Grade 3 or 4 sepsis occurred in 10 for the treatment of secondary or relapsed AML patients (71%).Eight patients (57%) showed an increase (Langston et al., 2004). Nineteen patients were treated of transaminases, and two of them (14%) developed and nine of them (47%) achieved a CR.None hepatic VOD. experienced VOD or other unexpected toxicities. In a pilot study, GO was associated to ciclosporin Chevallier et al.(2005) combined GO (9 mg/m 2)with (CSA), ara-c and liposomal daunorubicin (MDAC) intermediate doses of ara-c and mitoxantrone (MID- (Apostolidou et al., 2003) to treat 11 patients with AM) in 17 AML patients (13 refractory and four refractory AML.Only one patient (9%) achieved a CR, relapsed).A total of 13 patients (76%) resulted and another one achieved a CRp.The most relevant responsive: 12 patients (70%) achieved a CR and toxicities observed were: sepsis (63%), hyperbilirubine- another one patient achieved a CRp.Four patients mia, (54%), mucositis (27%) and increased transami- presented a grade 3/4 liver toxicity, including a patient nases levels (9%).The association of GO–CSA was who died for VOD.Seven of 13 responder patients employed in a larger study including also ara-c and relapsed and the median OS and RFS were 11 and 11 fludarabine (MFAC) (Tsimberidou et al., 2003a). GO months, respectively. was administered at a dose of 4.5 mg/m2 in 32 patients In a more recent study, GO was associated to Bcl-2 with refractory (34%) or relapsed AML (66%).Nine antisense (oblimersen) in AML patients in first relapse. patients (28%) achieved a CR, and two patients (6%) Oblimersen was administered at 7 mg/kg/day on days achieved a CRp.The median OS was 5.3months, and 1–7 and 15–21, whereas GO at the dose of 9 mg/m2 on the 12-month survival rate was 19%.Fourteen patients days 4 and 18.Among 48 treated patients, 12 showed (44%) developed an important hyperbilirubinemia, and a response to the treatment (five CR and seven CRp) three patients (9%) had a VOD. with a survival among these patients over 6 months.The CALGB Group (Stone et al., 2004) treated patients addition of oblimersen to GO did not cause an increase with relapsed or refractory AML with HiDARAC of serious adverse events (Moore et al., 2006). associated to GO at different doses (9 or 4.5 mg/m2). The results of the main studies on GO in relapsed/ The HiDARAC plus GO at 9 mg/m2 was characterized refractory patients are reported in Table 2. by a high toxicity; in fact eight of nine treated patients died during induction.Among the 44 following patients GO in untreated AML treated with HiDARAC plus GO at 4.5 mg/m2, seven In a report by Estey et al.(2002a), 51 untreated AML or patients (19%) achieved CR and other two CRp.About high-risk MDS patients aged older than 65 years were 20% of patients presented a liver toxicity, but none randomized to receive induction therapy with GO at a presented a VOD.HiDARAC was also combined to dose of 9 mg/m2 with or without IL-11 at a dose of topotecan and GO at different doses (6, 4.5 and 2 mg/m2) 15 mg/kg per day on days 3–28.CR was seen in 2/26

Table 2 GO combined to conventional chemotherapy for the treatment of relapsed/resistant AML Authors No. of Median age State of disease CTX plus GO CR % ORa % RFS Median survival patients (years) (doses of GO) (months) (months)

Cortes et al. (2002) 17 55 (20–70) Resistant or MTA (9 mg/m2)1212NR8.2 relapsed (plus MDS) Venugopal et al. (2002) 19 55 (22–68) Resistant or MMACA (9 mg/m2) 32 32 NR 10.8 relapsed (plus MDS) Alvarado et al. (2003) 14 61 (34–74) Resistant or MIA (6 mg/m2)21436.32 relapsed Apostolidou et al. (2003) 11 37 (16–67) Resistant or MDAC (6 mg/m2) 9 18 9.5 3 relapsed Tsimberidou et al. (2003a) 32 53 (18–78) Resistant or MFAC (4.5 mg/m2) 28 34 5 5.3 relapsed Stone et al. (2004) 24 64 (55–69) Resistant or HiDARAC (9 mg/m2)1924NRNR relapsed Langston et al.(2004) 19 64 (37–77) Relapsed HiDARAC+Topotecan 47 NR 10.4 NR (plus sAML (3–6 mg/m2) and MDS) Chevallier et al. (2005) 17 54 (21–68) Resistant or MIDAM (9 mg/m2)70761111 relapsed Moore et al. (2006) 48 67 (NR) 1st relapse Oblimersen+GO 10 25 3.7 2.3 (9 mg/m2)

Abbreviations: AML, acute myeloid leukemia; CR, complete remission; CRp, complete remission without platelet count recovery; CTX, chemotherapy trial; GO, Gemtuzumab Ozogamicin; NR, not reported; OS, overall survival; RFS, relapse-free survival.Schedules: MTA: Cytarabine+topotecan; MMACA: Cytarabine+Mitoxantrone+Amifostine; MIA: Cytarabine+Idarubicine; MDAC: Cytarabine+Cyclospor- ine+Daunoribicin; MFAC: Cytarabine+Cyclosporine+Fludarabine; HiDARAC: high doses of Cytarabine; MIDAM: Cytarabine+Mitoxan- trone. aCR+CRp.

Oncogene Gemtuzumab Ozogamicin and acute myeloid leukemia L Pagano et al 3685 (8%) of patients without IL-11 and in 9/25 (36%) with et al., 2005) that reported 15 untreated elderly AML IL-11.Survival was not significantly different in the two treated with HiDARAC plus GO (9 mg/m2).With this groups, 8 and 15 weeks, respectively.However, when schedule the authors obtained an overall response rate compared with historical control treated with idarubicin of about 67% and an RFS of 6 months. plus HiDARAC, a high CR rate (48%) and a more More recently, on the basis of the positive results of a prolonged survival was obtained in the control arm. previous study with GO plus G-CSF priming (Leone In a following study performed by the same group, 59 et al., 2004), preliminary data were presented on a series de novo AML/MDS patients were treated with the of 23 secondary AML (12 relapsed and 11 newly schedule previously used for relapsed/refractory patients diagnosed cases): they were treated with G-AraMy (MFAC).This regimen induced a CR in 27 patients combination including rhG-CSF (5 mg/kg, on days 1–8), (46%) and a CRp in another patient (2%).The median ara-c as continuous perfusion (100 mg/m2 on days 4–8) survival period was of 8 months.At 12 months, the and GO (6 mg/m2 on day 9).Fourteen patients (61%) survival rate was 38%.The major toxicities were achieved a CR, whereas other two patients obtained a infectious complications, hyperbilirubinemia and raised PR.The most common adverse event was myelosup- levels of transaminases.Four patients (7%) developed a pression, as expected.VOD episodes were not recorded. VOD (Tsimberidou et al., 2003b). Seven patients (30%) developed a documented infection The Medical Research Council (MRC) reported the (including pulmonary aspergillosis in two cases).The feasibility of combining GO with induction chemothe- median RFS and OS were 6.3 and 7.6 months, rapy in patients younger than 60 years (AML 15 trial). respectively (Fianchi et al., 2006). Seventy-two patients were treated with different chemo- Results obtained using GO in combination with other therapies (H-DAT schedule or FLAG-Ida schedule) and drugs in the induction of first remission for de novo with escalating doses of GO (from 3 to 9 mg/m2) AML are summarized in Table 3. administered on day 1 of each course (both in induction and consolidation).Patients were stratified into six different cohorts considering induction regimen they received; 91% of those treated with FLAG-Ida or DA GO and acute promyelocytic leukemia (without thioguanine) achieved a CR and 78% of them were RFS at 8 months.Overall 29 patients suffered for 3 Many reasons make GO an interesting drug in acute and 4 liver toxicity; seven patients suffered for a VOD, promyelocytic leukemia (APL), even in relapsed phase. also named sinusoidal obstructive syndrome (SOS), It resulted highly active because of the large expression that was fatal in three of them; everyone of them of CD33 in APL cells and because of the low levels of had received thioguanine.Other 12 patients died P-gp function in leukemic promyelocytes (Guglielmi for infectious complication (five aspergillosis and seven et al., 1998; Paietta, 2003). In addition Takeshita et al. bacterial infection) (Kell et al., 2003). (2005) demonstrated that resistance mechanism to GO The interesting results of MRC trials were confirmed differ from those reported to explain resistance to all- in another study by De Angelo et al.(2003).They trans retinoic acid (ATRA) or arsenic trioxide.The treated 53 untreated AML patients with daunorubicin efficacy of GO in APL patients was based also on and ara-c, followed by GO (6 mg/m2), obtaining 44 CR positive results of previous treatment with un-conju- (83%).Other 21 patients received a combination of ara-c gated anti-CD33 monoclonal antibody (HuM195).In and GO (6 mg/m2) with nine CR (43%). fact, in a study on 31 patients with newly diagnosed The EORTC-GIMEMA group recently presented the APL treated with HuM195 twice-weekly for 3 weeks, it results of a trial using GO on days 1 and 15 followed by was demonstrated that among 22 valuable patients, 12 the combination of mitoxantrone, ara-c and etoposide in achieved molecular remission (MR) without additional a cohort of AML elderly patients with either de novo or therapy (Jurcic et al., 2000). secondary AML.A total of 57 patients were enrolled. GO has been successfully used either as a single agent Initial treatment with GO resulted in an overall response or in combination.The first report by Petti et al.(2001) rate (13 CR plus seven CRp) of 35% that increased to about a pretreated patient in third hematological relapse 54.4% (31/57 patients) with remission induction with who achieved a prolonged CR, prompted investigators combination chemotherapy.However, five patients to consider GO as a valid alternative in APL.Further developed a VOD and four of them died for toxicity. single case report showed the efficacy of this approach At 226th day, only 12 patients were in continuous CR/ (Schwarz et al., 2004; Tsimberidou et al., 2004). CRp (21%) (Amadori et al., 2004). In a study by GIMEMA group (Lo-Coco et al., 2004), Another study reported the experience in nine elderly 16 patients relapsed at molecular level were treated with AML patients (five untreated AML and four with GO at 6 mg/m2 for two doses.MR was obtained in 14 relapsed/refractory disease) with GO (6 mg/m2) com- patients (87%).Of 14 responders, seven remained in bined with ara-c in continuous infusion.Among only sustained MR for a median time of 15 months.A total untreated patients, three achieved CR (60%) and two of six patients showed a liver toxicity but none were resistant.The median CR duration was 12 months. developed a VOD. The most common adverse event was bleeding; no cases GO has been combined with ATRA in previously of VOD were observed (Piccaluga et al., 2004b). Little untreated patients in a study by Estey et al.(2002b). more wide is the experience of Duke University (Rao Nineteen patients were treated with ATRA until CR

Oncogene Gemtuzumab Ozogamicin and acute myeloid leukemia L Pagano et al 3686 Table 3 GO combined to conventional chemotherapy for induction treatment of untreated AML Authors No. of Median age State of disease CTX plus GO CR % ORa % RFS Median survival patients (years) (doses of GO) (months) (months)

Estey et al. (2002a) 51 71 (65–89) At diagnosis GO vs GO+IL11 (9 mg/m2)22b 22 NR 3 (plus MDS) Tsimberidou et al. (2003b) 59 57 (27–64) At diagnosis MFAC (6 mg/m2)464768 (plus MSD)

Kell et al. (2003) 39 (19–56) All at diagnosis 1st group 22 H-DAT+GO (3 mg/m2)91NRNRNR 2ndgroup 9 H-DAT+GO (6 mg/m2)89 3rd group 15 S-DAT+GO (3 mg/m2) Â NR 2 cycles 4th group 10 S-DAT+GO (3 mg/m2)60 5th group 8 S-DA+GO (3 mg/m2) 100 6th group 15 FLAG-IDA+GO 93 (3 mg/m2) De Angelo et al. (2003) 58 (18–78) All at diagnosis 1st group 53 DA+GO (6 mg/m2)83NRNRNR 2nd group 21 AraC+GO (6 mg/m2)43 Amadori et al. (2004) 57 68 (61–73) At diagnosis GO +MICE (9 mg/m2) 35 54 6 NR 11,151 Piccaluga et al. (2004b) 5 65 (63–71) At diagnosis GO+Ara-C (6 mg/m211; 60 60 12 13 4 mg/m2 81) Rao et al. (2005) 15 68 (NR) At diagnosis HiDARAC (9 mg/m2)NR676 NR

Abbreviations: AML, acute myeloid leukemia; CR, complete remission; CRp, complete remission without platelet count recovery; CTX, chemotherapy trial; GO, Gemtuzumab Ozogamicin; NR, not reported; OS, overall survival; RFS, relapse free survival.Schedules: MTA: Cytarabine+topotecan; DAT: Daunorubicin+Cytarabine+Thioguanine; FLAG-IDA: Fludarabine+Cytarabine+Idarubicin; DA: Daunorubicin+ Cytarabine; MICE: Mitoxantrone+Idarubicin+Cytarabine+Etoposide; HiDARAC: high doses of Cytarabine. aCR+CRp. bEleven patients: 2/26 in the GO arm, 9/25 in the GO+IL-11 arm.

with the addition of GO (9 mg/m2) on day 5.Main- other monoclonal antibodies, appear in about 30% of tenance ATRA and GO were used after achievement of patients at the first infusion of GO, but only in the 10% CR.Sixteen patients (84%) achieved a CR, with 14 in a of them during subsequent administrations (Giles et al., MR, suggesting a benefit of the addition of GO to 2001; Larson et al., 2005). Premedication with acetami- ATRA.At present, this is the only available study about nophen and antihistaminics or corticosteroids is effec- the GO as front-line therapy in APL; consequently, tive in reducing these events. further studies are needed to prove the major efficacy of VOD/SOS is the most frequently reported extrahe- the scheme respect to more established associations and matological adverse event.It is usually defined accord- anthracycline-based protocols. ing to the Seattle and Baltimore criteria, as a coexistence At present, no cases of VOD have been reported in of hyperbilirubinaemia, painful hepatomegaly and fluid APL; it may relate to the GO binding to the high level of retention in the absence of other causes (Bearman, CD33 in circulating promyelocytes, which avoids 2000).Many mechanisms have been proposed to explain calicheamicin-induced damage of hepatic cells. the high incidence of hepatotoxicity after GO adminis- tration: (1) the damage to the CD33-bearing Kuppfer and sinusoidal cells; (2) the infiltration of the liver by blast cells; (3) the calicheamicin-related damage Hematological and extrahematological adverse events to sinusoidal cells upon its separation from the anti- body.However, even CD33-negative human hepato- The most important GO associated toxicities are cytes appeared to metabolize GO, as observed by myelosuppression and, consequently, febrile neutrope- Cohen et al.(2002).It is worth noticing that a prior nia: infections incidence ranges from 13 to 50% in therapy with GO represents an independent risk factor different series (Amadori et al., 2005; Larson et al., for VOD in patients undergoing HSCT (Wadleigh et al., 2005).Although pluripotent progenitor cells do not 2003). express CD33, myelosuppression may be due to the Investigators associated with the Research on Adverse CD33-positivity of more differentiated multipotent Drug Events and Reports (RADAR) project reviewed hematopoietic cells, targeted by GO.Indeed, throm- safety reports for GO included in reports of clinical bocytopenia and neutropenia have been constantly trials and observational studies, interim reports from an observed after GO-based regimens and they may lead FDA mandated Prospective Observational Registry, to modify the therapeutic schedule (Sievers et al., 2001). and the Food and Drug Administration’s Adverse Infusion-related adverse reactions (, chills, cuta- Event Reporting System.Medline searches provided neous rash, hypotension, hypertension, hyperglycemia, incidence estimates of GO-associated VOD/SOS and dyspnea, nausea, emesis and headache), similarly to comparative rates of VOD without GO.VOD is

Oncogene Gemtuzumab Ozogamicin and acute myeloid leukemia L Pagano et al 3687 characterized by hyperbilirubinemia, painful hepatome- Council (AML15 trial), patients with AML are rando- galy, ascites and sudden weight gain developing at a mized to receive induction courses with ara-c, dauno- median of 10 days following GO administration for rubicin and etoposide; daunorubicin and ara-c or patients who did not undergo an allogeneic SCT with fludarabine, ara-c, idarubicin and G-CSF, with procedure and 13 days following an allogeneic SCT or without GO on day 1 of course 1.The GIMEMA- for patients who had previously received GO.Among EORTC group is conducting a randomized trial (AML- adult AML patients who received GO in clinical trials, 17) employing standard intensive chemotherapy, with or VOD incidence was 3% for GO doses p6 mg/m2 without GO, as induction and consolidation therapy administered as monotherapy or in combination with in patients aged 64–75 years with untreated AML. non-hepatotoxic agents; conversely it was 28% if GO A second trial (AML-19) includes GO monotherapy was administered at 9 mg/m2 or with thioguanine versus standard supportive care in patients with (McKoy et al., 2006). untreated AML older than 75 years who are not candidates for intensive chemotherapy. The role of GO before SCT has been investigated in Conclusions several phase II studies too.In general, prior exposure to GO significantly increases the risk of hepatic VOD in The role of targeted therapy for the treatment of patients undergoing myeloablative allogeneic SCT, but hematological malignancy is continuing to expand as this risk decreases if at least 3 months have elapsed since new targets are identified and advances in the conjuga- the last dose of GO (Wadleigh et al., 2003). tion of radioactive molecules and toxins occur.GO Current data indicate potential role for GO as a activity in relapsed patients has been demonstrated, with component of induction or consolidation regimens in approximately 30% of patients achieving a remission: adults and, based on an early study, in the treatment of drug allowed equivalent survival and fewer total days of children with AML.The hepatotoxicity does not appear hospitalization than conventional chemotherapy in the to be significantly different when GO is used in same category of patients.Although GO is approved for conjunction with cytotoxic chemotherapy, but the risk older patients with relapsed AML who are intolerant of of SOS does appear greater when standard dosing cytotoxic chemotherapy, its potential roles in other schedules are employed in the peritransplant setting. settings are actively being studied.At present, coopera- However, GO is well tolerated by most patients. tive group studies combining GO with standard-dose In conclusion, GO provides an effective and relatively chemotherapy are ongoing.The Southwest Oncology well-tolerated treatment option for patients with CD33- Group (SWOG) is investigating ara-c and daunorubicin positive AML.It is useful for cytoreduction of the with or without GO followed by HiDARAC followed leukemia tumor burden, allowing recovery of normal by either GO or no additional therapy in patients aging hematopoiesis.At present, the better dosage seems 6 mg/ 18–55 years with untreated de novo AML (SWOG trial m2 that is characterized by the absence of side effects, S0106).The Eastern Cooperative Oncology Group particularly VOD.The combination with other antileu- (ECOG) is studying daunorubicin dose intensification kemic agents does not increase the toxicity, whereas and GO consolidation therapy before autologous SCT improves the percentage of response, particularly when in AML patients (16–60 years).In the Medical Research it is combined with cytosine arabinoside.

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