CD33-positive Acute Myeloid Leukemia Research Articles

A Prospective Monocentric Study of Fractionated Gemtuzumab Ozogamicin (GO) Combined to Standard-Dose Cytarabine in Older Patients with Acute Myeloid Leukemia (AML) in First Relapse,

Abstract 3603 Aim.Gemtuzumab Ozogamicin (GO) is a potent antibody-directed chemotherapy against CD33 antigen. We have previously conducted a phase 2 study evaluating fractionated infusion of GO 3 mg/m2 on day 1, 4 and 7 combined to standard 3+7 DNR/cytarabine in patients aged 50 to 70 years with relapsed acute myeloid leukemia (AML) (Farhat et al., AJH, accepted). We decided to determine whether the omission of daunorubicin may retain the efficacy of fractionated GO combined to standard-dose cytarabine. Methods.GO was proposed to patients (pts) with CD33 positive AML in relapse according to the French Temporary Authorization (ATU) program. All consecutive pts from our center were eligible if they were in first relapse and aged of 50 years or more. Refractory patients and patients with a first CR of less than 6 months were excluded. Induction consisted of cytarabine 200 mg/m2/d CI on day 1–7 with GO at 3 mg/m2/d on day 1, 4 and 7. Pts achieving CR/CRp received two consolidation courses with cytarabine 1 g/m2/12h on day 1–2 with GO at 3 mg/m2/d on day 1. A maintenance phase with low-dose cytarabine was allowed. Minimal residual disease (MRD) monitoring based on NPM1 mutation or WT1 expression was assessed in informative and evaluable patients. Results.From October 2007 to June 2011, 22 pts (median age, 67 years) were recruited. All pts received an intensive regimen as part of their first line therapy (3+7 schedule or more intensive). Median duration of first CR was 16 months (6 to 33). Cytogenetic analysis at relapse was successfully assessed in all pts and karyotype was classified as favorable in 3 pts (t(8;21); t(16;16); t(15;17)), intermediate in 18 pts and adverse in 1 pt. Molecular profile analysis of intermediate-risk patients revealed the presence of a FLT3 internal tandem duplication (FLT3 -ITD) in 3 pts, a NPM1 mutation in 8 pts (including 2 cases with a concomitant FLT3 -ITD), a single CEBPA mutation in 1 pt, a EVI1 hyperexpression in 2 pts, and a MLL rearrangement in 1 pt. CR+CRp was achieved in 17/22 pts (77.3%) including 2 CRp. The 5 primary resistant pts corresponded to 1 pt with complex karyotype, 1 pt with a MLL rearrangement, 1 pt with EVI1 hyper expression, 1 pt with FLT3 -ITD, and 1 pt without any identified genetic alteration. There were 2 (9%) induction deaths including one patient that experienced veino-occlusive disease (VOD) and one patient with severe sepsis. A second case of reversible grade 2 VOD was documented during induction. Four patients (18.1%) experienced G3-4 infections and only one severe mucositis was observed. Median duration of G3-4 neutropenia and thrombocytopenia was 24 days (16–30) and 30 days (20–60), respectively. Prolonged grade ≥ 3 thrombocytopenia was observed in 2 patients. With a median follow-up of 22 months, median DFS was 17.2 months and estimated at 66.3% at 18 months. To date, the duration of CR2 was superior to that of CR1 in 5 non transplanted patients. Median overall survival was 22.6 months. Four pts who received a reduced intensity allogeneic stem cell transplantation in CR2, and one of them subsequently died. We were able to compare the molecular response obtained after CR2 and after CR1 using the same MRD marker in 6 patients that achieved CR2. In 5 out of 6 paired samples, MRD levels after CR2 were found equal or inferior to MRD levels after CR1 (see fig 1). [Display omitted] Conclusion.Fractionated doses of GO (3 mg/m2/d on day 1, 4, and 7) combined to standard-dose cytarabine in older pts with AML in first relapse is associated with a high CR rate, a prolonged survival and a low rate of toxic deaths. A gain in MRD levels was noted in paired CR1 and CR2 samples. Disclosures:Castaigne:Pfizer/Wyeth: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Combination of CD34/CD38 Immunophenotypes and Side Population (SP) Reveals the Putative Leukemia Stem Cell/Leukemia Initiating Cell In Acute Myeloid Leukemia.

AbstractAbstract 1582Although the originally defined CD34+CD38- leukemia stem cell (LSC)/leukemia initiating cell (LIC) in acute myeloid leukemia (AML) still serves as a lead in studies on the characterization of LSC/LIC, in more immuno-compromised mouse models, other stem cell immunophenotypes, i.e. CD34-CD38- and CD34-CD38+ turn out to have LSC/LIC activity (Taussig et al, Blood 2010; 115: 1976). Two functional LSC phenotypes offer the ability not only to restrict the LSC/LIC compartment to a lower frequency compartment, but also to include such CD34- negative immunophenotypes. These concern high activity of aldehyde dehydrogenase (ALDH) or high efflux of Hoechst 44432 (resulting in side population, SP). The present study deals with the possible overlap between CD34/CD38 and SP defined stem cell compartments. Based on our previous results (van der Pol, Haematologica 2003; 88: 983), we defined truly CD34 negative AML as AML with a very small CD34+ population only (usually≤1%), with particular scatter properties, and proven to be of normal origin. Using expression of cell surface markers present on LSC/LIC, but absent on HSC (van Rhenen et al, Leukemia 2007; 21: 1700; Blood 2007; 110: 2659), the LSC/LIC compartment in this type of leukemia (5 cases studied) was shown to be present in the CD34-CD38+ compartment, while the HSC was found in the CD34+CD38- compartment. When determining aberrant cell surface markers within the SP, the only malignant cells present in the SP population were CD34-CD38+. To assess the primitive character of the putative LSC/LIC sub-populations, cell sorting experiments were performed in which all aberrant marker positive SP cells along with marker positive non-SP cells (n=2) and all aberrant marker negative SP cells and marker negative non-SP cells (n=2), were assessed in the liquid culture stem cell assay. SP marker positive cells had 280- and 725-fold more stem cell activity compared to non-SP cells (14,500 versus 20 and 8,400 versus 30, respectively), while for marker negative cells only the SP fraction contained stem cell activity (8,000 and 8,200 colonies per million input cells). Even though the frequency of SP cells is far below that of non-SP cells, the absolute number of colonies was higher in SP than in non-SP cells. In CD34 positive leukemia, in contrast to CD34 negative AML, the SP fraction was largely filled with cells with all combinations of CD34 and CD38, which all fulfill the criteria (marker positivity) for malignancy. Liquid culture assay results were: for marker positive cells, colonies were formed only in the SP compartment in 2/3 cases studied (6,500 and 4,400 colonies per million input cells). In the remaining case colony formation was 195-fold higher in SP versus non-SP (4,100 versus 21 colonies per million input cells). For marker negative cells, in all three cases only the SP fraction produced colonies (5,300, 1,4500 and 90 per million input cells). Malignant character of marker positive cells was confirmed using FISH analysis, immediately after cell sorting prior to cell culturing, and at the end of the liquid culture on plucked colonies. These results show that both in CD34 negative and CD34 positive leukemia the SP fraction contains most primitive cells and has much higher clonogenic potency compared to non-SP cells. In CD34 negative AML, SP HSC are CD34+CD38- and SP LSC/LIC are CD34-CD38+. In CD34 positive AML, SP HSC are CD34+CD38- and SP LSC/LIC may be CD34+CD38-, but also CD34+CD38+, CD34-CD38+ and CD34-CD38-. The absence of CD34 negative engraftment, seen in earlier studies, likely results from relatively high immune competence of the NOD/SCID mice in those studies. The results suggest that CD34 negative leukemia may contain more immune sensitive LSC/LIC. Above that, CD34 negative AML is characterized by lower multidrug resistance (abstract Schuurhuis, Kelder et al, subm for this meeting). From these characteristics favorable outcome for the particular class of patients, defined as truly CD34 negative, can be predicted. This was indeed the case in our large study (394 patients): median overall survival not reached (>41 months) for CD34 negative patients (n=85) and 19 months for CD34 positive patients (n=309) (p=0.006). Our results warrant to consider CD34 negative patients as a completely different entity of AML patients, in terms of stem cell characteristics, which may evoke different therapeutic strategies compared to CD34 positive patients. Disclosures:No relevant conflicts of interest to declare.

Identification of A Pharmacogenomic Profile Associated with High Sensitivity and Low Toxicity to a Combination of Gemtuzumab Ozogamicin Plus Fludarabine, Cytarabine, Idarubicin Regimen In CD33-Positive Acute Myeloid Leukemia (AML) Patients

AbstractAbstract 967Human genome sequence variation, in the form of single nucleotide polymorphisms (SNPs) as well as more complex structural variation (such as insertions, duplications and deletions) in drug-metabolizing enzyme and transporter genes (DMETs), affects each individual's response to anticancer agents and thus the likelihood of experiencing an adverse drug reaction. Therefore, understanding relationships between genetic variation and differential drug responses is a challenge to improve both the safety and efficacy of drugs. Accordingly, in order to investigate a potential association between SNPs and clinical response and toxicity, we comprehensively assessed the allele frequencies of 1931 genetic variations of 225 absorption, distribution, metabolism and excretion genes in 59 AML patients younger than 65 years, using the new Affymetrix drug-metabolizing enzyme and transport (DMET Plus) genotyping platform (Affymetrix, Inc. Santa Clara, CA, USA). All patients were enrolled in a phase III multicenter clinical trial combining low dose of Gemtuzumab-ozogamicin (GO) with FLAI regimen (Fludarabine, Cytarabine, Idarubicin) as Induction chemotherapy i (eudract: 2007–005248-26; ClinicalTrials.gov NCT00909168). The induction regimen (GO-FLAI) included fludarabine (25 mg/sqm) and Ara-C (2 g/sqm) on days 1–5, idarubicin (10 mg/sqm) on days 1, 3, and 5 and GO (3 mg/sqm) on day 6. Hematopoietic stem cell transplant was planned for all high risk AML patients in first complete remission (CR) after consolidation with intermediate doses of Ara-C and idarubicin (ID-AC and IDA). Cytogenetic, multidrug-resistance phenotype, FLT3 and NPM mutation status, WT1 quantitative expression analyses, were performed at diagnosis in all patients. Furthermore, high-resolution SNP array analysis by GeneChip® Human Mapping 250K NspI and Genome-Wide Human SNP 6.0 (Affymetrix) was also performed. DNA processing and genotype identifications were performed according to the Affymetrix DMET platform's instructions. All statistical analyses were performed using the R package 2.11.1. Allele frequencies estimation and principal component analyses (PCAs) were performed using the adegenet library, whereas an ad hoc R function was developed to compute two-side Fisher's exact test p-values for each SNP in order to test their association with clinical response and toxicity.Results of potential interest were limited to those in which the p-value was <0.01. The median SNP call rate was 99.48% (range, from 98.05 – 100%). Two samples were run in duplicate and results with “passed call rate” were compared across all the 1931 polymorphic sites, showing a repeatability of 99.99%. In an initial screening procedure, we tested the association among SNPs and response to the induction cycle (FLAI + Gemtuzumab-Ozogamicin). We compared the genotyping profiling of 50 patients in complete (81%) and partial (5%) remission versus that of patients (14%) with no response finding out that 2 SNPs in the alcohol dehydrogenase enzyme (ADH1A) were highly associated with response to therapy. Since genetic polymorphisms may influence the toxicity of chemotherapy drugs, we stratified SNPs according to liver toxicity and 2 genes (SLC22A5 and ABCC4) were found to be associated with a grade I/II liver toxicity. Moreover, SNPs in SLC7A8 and SULT2B1 genes were found to be strongly associated (p <0.001) with fever to gemtuzumab-ozogamicin and finally SNPs in UGT2B15, CYP2B6 and PPARD (peroxisome proliferator-activated receptor delta) were found to be associated with a higher level of expression of P-glycoprotein (defined as a mean fluorescence index > 6). In conclusion, we performed an exploratory pharmacogenomic study that related SNPs in multidrug enzymes and transporters genes with the efficacy and toxicity of a combination of Gemtuzumab-ozogamicin (GO) with FLAI regimen used in a clinical trial of AML patients younger than 65 years. We identified for the first time a pharmacogenomic panel made up of 1 gene (ADH1A) associated with clinical outcome and 4 genes associated with toxicity. Findings obtained from this exploratory study are being tested in a larger clinical sample sets to confirm the predictive value of the SNP panel. Supported by European LeukemiaNet, AIL, AIRC, FIRB 2006, PRIN 2008, Ateneo RFO grants, Project of integreted program (PIO), Programma di Ricerca Regione – Università 2007 – 2009. Disclosures:Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy.

Targeted delivery of SiRNA to CD33-positive tumor cells with liposomal carrier systems

SiRNA molecules represent promising therapeutic molecules, e.g. for cancer therapy. However, efficient delivery into tumor cells remains a major obstacle for treatment. Here, we describe a liposomal siRNA carrier system for targeted delivery of siRNA to CD33-positive acute myeloid leukemia cells. The siRNA is directed against the t(8;21) translocation resulting in the AML1/MTG8 fusion protein. The siRNA was encapsulated in free or polyethylene imine (PEI)-complexed form into PEGylated liposomes endowed subsequently with an anti-CD33 single-chain Fv fragment (scFv) for targeted delivery. The resulting siRNA-loaded immunoliposomes (IL) and immunolipoplexes (ILP) showed specific binding and internalization by CD33-expressing myeloid leukemia cell lines (SKNO-1, Kasumi-1). Targeted delivery of AML1/MTG8 siRNA, but not of mismatch control siRNA, reduced AML1/MTG8 mRNA and protein levels and decreased leukemic clonogenicity, a hallmark of leukemic self-renewal. Although this study revealed that further modifications are necessary to increase efficacy of siRNA delivery and silencing, we were able to establish a targeted liposomal siRNA delivery system combining recombinant antibody fragments for targeted delivery with tumor cell-specific siRNA molecules as therapeutic agents.

A Phase I/II Trial of Gemtuzumab Ozogamicin in Combination with Cytarabine and Anthracyclines (Daunorubicin or Idarubicin) for CD33-Positive Refractory or Relapsed AML Patients Younger Than 65 Years. Results of Phase I Parts of the JALSG-AML206 Study.

Abstract 4145 INTRODUCTION:Gemtuzumab ozogamicin (GO) is an antibody-targeted chemotherapeutic agent consisted of a humanized anti-CD33 monoclonal antibody conjugated to a calicheamicin. Several trials evaluated the efficacy of GO in combination with cytotoxic chemotherapy for treating refractory/ relapsed acute myeloid leukemia (AML) or as front-line therapy. However, there is a still open question what is optimal dose or timing of GO as well as optimal partner of antileukemic drugs. In order to investigate better molecular-target therapy for AML, we initiated phase I/II trial named JALSG AML206 study. PATIENTS and METHODS:The primary goal of this study is to evaluate the safety and the antileukemic activity of combination of GO, cytarabine (Ara-C) and either daunorubicin (DNR)[DAG regimen] or idarubicin (IDR)[IAG regimen] in patients with relapsed/refractory CD33-positive AML. Phase I part was dose-finding study for GO, IDR and DNR. Eligibility criteria included age (20-64yrs), AML(except APL) in first relapse more than 6 months from induction therapy or refractory to the first induction therapy, ECOG performance status 0-2, CD33 expression >20%. All registered patients were received standard dose of Ara-C(100 mg/m2/CIV) on days 1-7 combining GO with either DNR or IDR. GO was planned to administer one day after last administration of DNR or IDR. In the DAG regimen, DNR (50 mg/m2/IV) on days 1-3 + GO(3 mg/m2/DIV) on day 4 as level-1, DNR(50 mg/m2/IV on days 1-4 + GO(3mg/m2/DIV) on day 5 as level-2, and DNR(50 mg/m2/IV) on days 1-5 + GO(3mg/m2/DIV) on day 6 as level-3 were administered. In the IAG regimen, IDR(10 mg/m2/IV) on days 1-3 + GO(3mg/m2/DIV) on day 4 as level-1, IDR(12 mg/m2/IV) on days 1-3 + GO(3 mg/m2/DIV) as level-2, and IDR(12 mg/m2/IV) on days 1-3 + GO (5 mg/m2/DIV) on day 4 as level-3 were administered. Dose and schedule level indicated above was increased using a standard 3+3 design. RESULTS:Nineteen patients (10 patients in the DAG, 9 patients in the IAG) have enrolled: median age was 59 yrs (range 33-64) and 53% were male. Eight patients were refractory and 11 patients were relapsed AML. Cytogenetic risk (according to CALGB): poor 19%, intermediate 63%, and good 19%. The median value of blasts in the bone marrow before treatment was 93%(range 39-100%). No DLT was observed at levels 1 and 2 in both the DAG and the IAG regimens. At level 3 in the DAG, no DLT was observed although one patient had Grade 4 neutropenia for 4 weeks due to progression of AML. In the IAG regimen, 3 of 3 patients had a DLT at level 3; these were Grade 3/4 infection due to severe neutropenia and prolongation of Grade 4 neutropenia independent to progression of leukemia. Two patients had Grade 3 of increase of AST/ALT, but none had VOD/SOS. Among 15 patients assessable for response, 6 patients (4 in DAG, 2 for IAG) (40%) attained CR, and 6 patients had hematologic improvement. One patient died of CNS bleeding within 30-days of therapy due to progression of AML. CONCLUSION:The MTD of GO just after administration of anthracycline combining with standard Ara-C was 5 mg/m2. Addition to standard Ara-C, GO (3 mg/m2) on day 6 with dose-dense DNR(50 mg/m2 × 5 days) and GO(3 mg/m2) on day 4 with IDR(12 mg/m2 × 3days) can be safely administered. Antileukemic activity of these level-3s of DAG and IAG regimens will be evaluated in phase 2 part of this study. Disclosures:No relevant conflicts of interest to declare.

Immunological Intervention Triggered by Decreasing CD34 + Lineage-Specific Donor Cell Chimerism to Prevent Relapse of Acute Myeloid Leukemia (AML) or Myelodysplastic Syndrome (MDS) After Allogeneic Hematopoietic Stem Cell Transplantation (HSCT).

Abstract 3317Poster Board III-205Relapse of the underlying malignant disease remains the most frequent cause of treatment failure in patients with AML or MDS after allogeneic HSCT. Analysis of CD34+ lineage-specific donor cell chimerism (DCC) is a sensitive method for monitoring of minimal residual disease (MRD) in patients with AML or MDS with CD34 expression. Early detection of increasing MRD in this group of transplanted patients may provide an indication for clinical interventions as it has been shown for patients with chronic myeloid leukemia. Therefore, we retrospectively addressed the question whether immunological interventions in case of decreasing lineage-specific DCC may prevent overt hematological relapse. Between May 1999 and December 2008, 221 patients with AML (206) or MDS (15) received an allogeneic HSCT at our center. Inclusion criteria for this study were >100 days of follow up after HSCT and ≥3 informative chimerism analyses from bone marrow samples. Eighty-eight patients matched those criteria. At initial diagnosis, CD34 expressing leukemic cells were found in 72/88 patients. Unsorted and lineage-specific DCC (cell subset purification was performed by flow sorting) were measured on days +30, +60, +100, +180, +270 and +360 after transplantation and every 3 months in the second year. Impending relapse was defined as CD34+ donor cell chimerism <90%, bone marrow blasts <5% and no evidence of extramedullary relapse. Isolated decrease of CD34+ DCC was found in 26 patients (30%; 25/26 patients with CD34 positive AML at initial diagnosis). Twenty-one of the 26 patients received treatment for impending relapse consisting of rapid tapering of immunosuppressive medications (14) and/or infusion of donor lymphocytes (7). Twelve of these patients remained in hematological remission, 9 of the 21 patients relapsed of whom 5 achieved sustained complete remission after salvage treatment. Four of the 5 patients without intervention after detection of decreasing CD34+ DCC relapsed within 14-69 days. The remaining patient without relapse developed severe chronic graft-versus-host disease without intervention shortly after detection of impending relapse and converted to full DCC. Twelve of the 88 patients (14%) relapsed without evidence of isolated decreasing CD34+ DCC prior to hematological relapse. Mixed chimerism of the whole bone marrow in combination with mixed lineage-specific DCC was found in 4 patients and 9 patients showed stable DCC until hematological relapse was diagnosed. Six of these patients achieved sustained remission after salvage treatment. Median follow-up of surviving patients with impending relapse was 806 days after transplantation and 716 days after first detection of decreasing CD34+ DCC. Relapse free survival at 2 years of all 88 patients and of the 26 patients with impending relapse was 60% (95% C.I., 48-72%) and 56% (95% C.I., 36-76%), respectively. Overall survival rates were 76% (95% C.I., 66-86%) and 65% (95% C.I., 45-85%), respectively. These preliminary results with 46% of patients with impending relapse successfully treated only with immunological intervention suggest that analysis of CD34+ DCC may be an important tool in the management of AML and MDS patients after allogeneic HSCT. Furthermore, isolated decrease of CD34+ DCC with complete unsorted DCC indicated that CD34+ DCC is an important and highly sensitive marker for MRD in patients with AML or MDS expressing CD34 on their malignant cells. DisclosuresNo relevant conflicts of interest to declare.

Decitabine-Based Salvage Therapy in Adults with Acute Myeloid Leukemia.

Abstract 2063Poster Board II-40 Introduction:Adults with primary refractory or relapsed acute myeloid leukemia (AML) have a poor prognosis with complete responses to salvage therapy from 13-15% and a median survival of 1.5-6 months[1, 2]. From 2006-2009, 79 patients with relapsed or refractory acute myeloid leukemia were given salvage chemotherapy with decitabine 20mg/m2 daily for 10 days or decitabine 20mg/m2 × 5 days with gemtuzumab ozogamicin (GO) 3mg/m2 on day 5 at Weill Cornell Medical Center. Methods:Medical records of 79 patients who received decitabine-based salvage therapy were reviewed from September 2006 through July 2009 at Weill Cornell Medical College. Survival was calculated by the Kaplan Meir method and differences in survival calculated by the log-rank test using STATA software. Results:Twenty-five patients received decitabine-based therapy as first salvage, 32 patients as second salvage, and 22 patients as third or greater salvage. Fifty-one patients were treated with decitabine/GO and 29 patients received decitabine alone. Median age of patients was 65.5 years with a range of 24 -89 years (first salvage 75 years, second salvage 62 years, and third or greater salvage 64 years). Median survival of all patients was 205 days, range 7-732 days. Overall 34% patients responded: 16% CR (<5% blasts in bone marrow, recovery ANC >1000 and Plts > 100,000) with median survival not yet reached; 5% CRp (< 5% blasts in bone marrow, ANC >1000 and plts < 100,000) with median survival 223 days; 13% PR (blasts 6-11%) with median survival 205 days; and 66% no response with median survival 118 days. Patients receiving first salvage had median survival 181 days with CR 13%, CRp 6%, PR 17%. Patients receiving second salvage had median survival 207 days with CR 9%, CRp 4%, PR 9%. Patients with third or greater salvage had median survival 209 days with CR 23%, CRp 0%, PR 14%. Patients receiving decitabine alone had a median survival of 209 days and those receiving decitabine/GO had a median survival of 177 days but the difference was not significant. Median survival for normal, favorable, intermediate and unfavorable cytogenetics was 282, 224, 157 and 176 days respectively (p=0.06). Conclusions: Decitabine-based treatment for relapsed and refractory AML is a low intensity alternative that has activity rivaling more intensive regimens. This retrospective study suggests that further investigation of decitabine-based salvage is warranted.1. Sievers, E.L., et al., Efficacy and safety of gemtuzumab ozogamicin in patients with CD33-positive acute myeloid leukemia in first relapse. J Clin Oncol, 2001. 19(13): p. 3244-54.2. Giles, F., et al., Outcome of patients with acute myelogenous leukemia after second salvage therapy. Cancer, 2005. 104(3): p. 547-54. Disclosures:Off Label Use: Phase I trial of decitabine in AML is off-label .

Phase I/II study of humanized anti-CD33 antibody conjugated with calicheamicin, gemtuzumab ozogamicin, in relapsed or refractory acute myeloid leukemia: final results of Japanese multicenter cooperative study

The primary objective of this study was to investigate the tolerability, efficacy and pharmacokinetic profile of gemtuzumab ozogamicin (GO) in patients with relapsed and/or refractory CD33-positive acute myeloid leukemia (AML). Patients received 2-h infusions of GO twice with an interval of approximately 14 days. Tolerability was assessed using the National Cancer Institute Common Toxicity Criteria Version 2.0. Samples for pharmacokinetics were taken on day 1 and day 8 of the first treatment cycle. The dose was increased stepwise and, in each cohort, patients were treated at the same dose. Forty patients, median age 58 years (range 28-68) were treated; 20 and 20 patients were enrolled to the phase I and II parts, respectively. In the phase I part, dose-limiting toxicities (DLTs) were hepatotoxicities, and the recommended dose was established as 9 mg/m2 given as two intravenous infusions separated by approximately 14 days. The pharmacokinetic study revealed that Cmax and AUC were equivalent to those of non-Japanese patients. In the phase II part, complete remission was observed in 5 patients, and one patient had complete remission without platelet recovery. Four of these 6 in remission and one in the phase I are long-term survivors (alive for at least 44 months). GO is safe and effective as a single agent among Japanese CD33-positive AML patients. Remission lasted longer in a subset of patients than in non-Japanese patients in earlier studies. Further studies of this agent are warranted to establish standard therapy.

Gemtuzumab Ozogamicin (GO) and Continuous Infusion Cytarabine (ARA-C) for Relapsed/Refractory Acute Myeloid Leukemia (AML) Prior to Allogeneic Stem Cell Transplantation (SCT).

The question whether to re-induce AML patients (pts) in first or subsequent relapse to reduce leukemia disease burden prior to SCT is not well studied. Several groups reported a 28–30% disease-free survival rate at five years for matched related donor SCT in first relapse without re-induction. Finally, re-induction regimen choices prior to SCT are relatively limited. Specifically, GO-containing regimens have been suspected to be associated with increased incidence of liver sinusoidal occlusive syndrome (SOS) when used prior to SCT. We evaluated our institutional experience of using GO and ARA-C for relapsed/refractory CD33-positive AML pts prior to allogeneic SCT. A total of 18 pts (median age 45.4 years; range 28–66) with relapsed/refractory AML were treated with GO/ARA-C. The median initial complete remission duration was 4.4 (range 0.5–14.5) months. The median time from relapse to GO/ ARA-C was 57 (range 6–142) days. Pts received GO (6 mg/m2 on day 1, 4 mg/m2 on day 8) and continuous infusion ARA-C (100 mg/m2) daily for 7 days. Bone marrow aspiration and biopsy were performed at a median of 15 (range 11–39) days post induction (one pt had >20% peripheral blasts on day 14 and therefore did not undergo bone marrow aspiration and biopsy). Three (17%) pts developed infectious complications following re-induction and one pt had disease progression that prevented them from undergoing bone marrow aspiration and biopsy and continuing on to SCT. Marrow aplasia (defined as <5% blasts and <20% cellularity) was reached in six of 14 (43%) evaluable pts. Three pts (all without achieving marrow aplasia) developed infectious complications that precluded SCT. A total of 11 pts proceeded onto SCT at a median of 30 (range 21–73) days after re-induction, including six pts who achieved marrow aplasia and five who did not. Six pts underwent matched unrelated donor SCT (two were mismatched at one allele) and five received matched sibling SCT. Reduced intensity preparatory regimens (fludarabine/melphalan) were used in all but one pt who received a fully myeloablative regimen (busulfan/cyclophosphamide). Engraftment to absolute neutrophil count ≥0.5x109/L for three days was achieved in all 11 pts at a median of 15 (range 9–21) days. During SCT, all pts were treated with prophylactic enoxaparin 40 mg subcutaneously once daily to prevent SOS of the liver. The two pts with mismatched unrelated donor SCT died three and nine days after achieving count recovery from pulmonary infiltrates and severe graft versus host disease (GVHD), respectively. Acute GVHD grade II-IV developed in six of the pts with matched donors (three related and three unrelated donors). Chronic GVHD developed in four (one related and three unrelated donors) of the pts. None developed liver SOS. Four pts are alive from starting GO/ARA-C at a median of 7.2 (range 1.2–52.7) months; two of them with and two of them without GVHD. Marrow aplasia prior to SCT was achieved in two of them. In summary, the question of whether to re-induce pts with relapsed/refractory AML to reduce leukemia burden prior to allogeneic SCT remains controversial. However, if one chooses to proceed with re-induction, then the combination of GO/ARA-C represents a feasible re-induction strategy with acceptable toxicity, achievement of marrow aplasia in approximately half of evaluable pts, and no increased risk of liver SOS in the setting of enoxaparin prophylaxis and low intensity conditioning regimens.

Specific Detection of Aberrant and Normal Stem Cells in Acute Myeloid Leukemia Patients Opens the Way for Defining Highly Specific Targets for Stem Cell Therapy.

In acute myeloid leukemia (AML), a small fraction of blast cells contains the tumor initiating cells, further referred to as leukemic stem cells (LSCs). LSC resemble hematopoietic stem cells (HSCs) with respect to self renewal capacity and quiescence. Therefore, LSCs are proposed to be therapy resistant. In order to optimally target LSCs and sparing HSC and to monitor therapy, discrimination between LSC is HSC is required. We showed that within the CD34+CD38− stem cell compartment, LSCs can be discriminated from HSC by aberrant expression of markers, including lineage markers like CD7, CD19 and CD56 and the novel LSC marker CLL-1 (van Rhenen et al., Leukemia 2007 and Blood 2007). Too low aberrant marker expression, however, hampers discrimination in part of the cases. Therefore, we investigated additional parameters that would allow to distinguish LSCs from HSCs in CD34 positive AML patients. In 14 out of 48 cases studied, flow cytometry revealed a double population within the CD34+CD38− compartment, characterized by a small but clear difference in forward scatter (FSC, reflecting cell size) and sideward scatter (SSC, reflecting granularity). In 7/14 cases with high marker expression, FSChighSSChigh population coincided completely with the population with aberrant marker expression. In the other cases, marker expression was too moderate to show a complete overlap. The FSClowSSClow population within the CD34+CD38− stem cell compartment is the minor population at diagnosis (median 16%, range 0.2%–92%; n=14), had no expression of aberrant markers and, moreover, closely resembled the FSC/SSC characteristics of normal BM HSCs. In addition, in these patients, the normality of the FSClowSSClow population was also supported by the fact that the CD34 and CD45 antigen density was similar to that of normal BM HSCs. Altogether, this enabled to use FSC/SSC characteristics together with aberrant CD34 and CD45 expression to discriminate between LSC and HSC in cases with low or absent aberrant marker expression (8/48). In addition, the malignant character of the FSChighSSChigh population and the normal character of the FSClowSSClow population could be proven in three AML patients with cytogenetic aberrancies. Patient 1 had a t(8;21) translocation and presented with a CD34+CD38−- population that was CD19 positive (81% of the stem cell compartment) and had FSChighSSChigh properties. FACSsorted cells contained the translocation in 90% of the cells. The CD19 negative population (19% of the stem cell compartment) had FSClowSSClow characteristics and contained 0% t(8;21) cells. In two other AML cases with a cytogenetic aberrancy (t(8;21) and t(15;17), respectively), FSC/SSC characteristics, CD34/CD45 antigen density and aberrant marker expression (CD56 in one case and CLL-1 in the other) were partly overlapping (estimated LSC contribution to the CD34+CD38− compartment was 85% in both cases). Cell sorting on the highest FSC/SSC and marker expression nevertheless resulted in enrichment of cytogenetically aberrant cells (63% and 73%, respectively), while the corresponding FSClowSSClow cells, which missed CD56 and CLL-1 expression, were enriched for cytogenetically normal HSCs (87% and 67%, respectively). The marker and scatter parameters discussed above have generated the possibility to discriminate between LSCs and HSCs and now allows specific detection of LSC in >75 % of the patients. Discrimination between LSCs and HSCs in AML might not only facilitate to establish the therapeutic window of current therapies in terms of LSC specificity, but also allow the identification of new highly AML stem cells specific therapeutic targets. This should ultimately result in more selective therapies, which would be highly effective for AML stem cells, while leaving the normal HSC intact. This work was supported by Netherlands Cancer Foundation KWF.

Gemtuzumab-ozogamicin in combination with fludarabine, cytarabine, idarubicin (FLAI-GO) as induction therapy in CD33-positive AML patients younger than 65 years

Introduction The addition of gemtuzumab-ozogamicin (GO) to an induction regimen including synergistic drugs, such as intermediate dose of cytarabine (Ara-C), idarubicin and fludarabine (FLAI), could reduce treatment failure in acute myeloid leukemia (AML) patients. Nevertheless, the role and safety of this antibody target-therapy in first-line chemotherapy in patients younger than 65 years has not yet been defined. Patients and methods The primary goal of this prospective phase II pilot study was to evaluate the efficacy and the safety profile of FLAI plus GO as induction regimen. Thirty consecutive AML patients were included. All patients were younger than 65 with a median age of 53 years and CD33 expression exceeded 20% in all cases. The M/F ratio was 16/14 and 21/30 (70%) of patients were poor-risk at diagnosis. The induction regimen (FLAI-GO) included fludarabine (30 mg/m 2) and Ara-C (2 g/m 2) on days 1–5, idarubicin (10 mg/m 2) on days 1, 3, and 5 and GO (3 mg/m 2) on day 6. Hematopoietic stem cell transplant (HSCT) was planned for all high risk AML patients in first complete remission (CR) after consolidation with intermediate doses of Ara-C and idarubicin (IDAC-IDA). Cytogenetic, multidrug-resistance phenotype, FLT3 mutation status, and WT1 quantitative expression analyses were performed at diagnosis in all patients. WT1 expression and cytogenetic (in positive cases) analyses were performed after induction to detect and follow minimal residual disease. Results Patients were evaluated for response rate, treatment-related adverse events, overall survival and relapse free survival. After induction with FLAI-GO, CR rate was 90% (26 of 29 evaluable pts); one patient achieved partial remission and two were resistant. There was only one case of death during induction (DDI). After FLAI-GO, the mean value of WT1 dropped from 4200 ± 2777 copies/10 4ABL to 192 ± 399 copies/10 4ABL. The toxicity of FLAI-GO was acceptable; 57% of patients experienced transient and reversible GO infusion-related adverse events (especially fever and chills), but no cases of veno-occlusive disease occurred during CHT or after HSCT. After a median follow-up of 16 months (range 2–25), 24/30 (80%) patients are alive (24/24 in CR). The probability of 1-year OS and RFS was 90 and 85%, respectively. Allogeneic and autologus HSCT was performed in 19 (63%) and 4 (13%) patients, respectively. Conclusions These preliminary results suggest that FLAI-GO is an effective and well tolerated induction regimen for CD33 positive AML patients younger than 65 years, with a high complete response rate, favourable safety profile, low DDI. These results encourage the testing of this regimen in a multicenter prospective trial.

Expression of myeloperoxidase enhances the chemosensitivity of leukemia cells through the generation of reactive oxygen species and the nitration of protein

Myeloperoxidase (MPO), a pivotal lineage marker for acute myeloid leukemia (AML), has been also shown to have a prognostic value: a high percentage of MPO-positive blasts correlates to favorable prognosis. To understand the relationship between the expression of MPO in leukemia cells and the response to chemotherapeutic agents, we established MPO-expressing K562 leukemia cell lines and then treated them with cytosine arabinocide (AraC). Cells expressing wild-type MPO, but not mutant MPO that could not mature, died earlier of apoptosis than control K562 cells. Reactive oxygen species (ROS) were generated more in leukemia cells expressing MPO, and the generation was abrogated by MPO inhibitors or antioxidants. Tyrosine nitration of cellular protein also increased more in MPO-expressing K562 cells than control cells after treatment with AraC. In clinical samples, CD34-positive AML cells from high-MPO cases showed a tendency to be sensitive to AraC in the colony-formation assay, and the generation of ROS and the nitration of protein were observed only when the percentage of MPO-expressing cells was high. These data suggest that MPO enhances the chemosensitivity of AML through the generation of ROS and the nitration of proteins.

The Role of Gemtuzumab Ozogamicin in Elderly AML Patients in Complete Remission.

The majority of patients (pts) with acute myeloid leukemia (AML) are diagnosed in their 6th and 7th decade of life. AML in elderly pts is associated with poor response to conventional chemotherapy and limited long-term survival, reflecting a higher incidence multidrug resistance mechanisms, a low bone marrow reserve which may prevent/delay the recovery of hematopoiesis after treatment, and the occurrence of co-morbidities. Gemtuzumab ozogamicin (GO) is an immunoconjugate with a humanized anti-CD33 that after internalization, releases a cytotoxic drug, calicheamicin; ≥80% of AML pts have myeloid blast cells that express the CD33 surface antigen. GO as a single agent has low antileukemic activity (Sievers et al, J Clin Oncol 2001; 19:3244–54). However, GO is being used at lower doses in combined chemotherapy regimens as induction or postremission therapy.Results of limited studies to date appear encouraging and phase III trials are ongoing. We report our experience with 14 consecutive AML pts with a median age of 72 years (Table) treated between May 2004 and May 2007 with standard induction protocols. These included 1 or 2 courses of Fluda (FLAG, 10 pts) or non-Fluda induction regimens (daunorubicin+Ara-C, 1 pt, and idarubicin+Ara-C+etoposide, 4 pts). followed by 2 postremission courses of MFAC, consisting of fludarabine 15 mg/m2/bid IV on days 2–4, Ara-C 0.5 g/m2/bid IV on days 2–4, GO 4.5 mg/m2/d IV on day 1 with the addition of cyclosporin A 6 mg/kg bw IV followed by 16 mg/kg bw on days 1–2. Among 9 pts with available baseline cytogenetic data, 6 were in the poor-risk group and 3 were in the favorable-risk group. Pts were determined to have CD33-positive AML by analysis of bone marrow aspirates and by immunophenotyping. The rate of clinical remission (CR), characterized by ≤5% blast in the marrow, recovery of neutrophils to ≥1500 μ/L and RBC and platelet transfusion-independent, was 64.2% (9/14). Seven pts received 2 M-FAC courses, 2 pts did not receive the recommended second M-FAC because of disease progression (1 pt) or infection (1 pt). Subsequently, M-FAC was given to 2 pts twice every 4 weeks for maintenance therapy. Seven pts (50%) obtained a durable CR and the median response duration was 12 months. The most common non-hematological toxicities were infections and elevation of bilirubin and hepatic enzymes. Interestingly, no veno-occlusive disease was noted in these pts in CR and with low tumor load. This positive outcome compares favorably with that of the few published studies in which a similar approach was used, and further supports the use of GO in combined regimens for AML. MFAC was feasible and well tolerated, and the CR rates obtained in pts receiving MFAC after standard induction regimens were particularly encouraging. We conclude that combination with other cytotoxic agents may increase GO efficacy in elderly pts with AML.Patient characteristics and outcomeN=14Median (range)Age, years72 (56–83)CD33+ blasts, %91.1 (84–95)WHO Performance StatusPts (%)0–19(64%)21 (7.1)34 (28.6)DiagnosisAML de novo7( 50)Secondary AML1 (7.1)AML developed from MDS5 (35.8)AML in first relapse1 (7.1)Induction regimenFluda (FLAG)10 (71.4)Non-Fluda (3+7, ICE)4 (28.6)Time from MDS to AML (yrs)< 12 (14.3)< 31 (7.1)unknown2 (14.3)Cytogenetic grouppoor risk6 (42.9)favorable risk3 (21.4)not tested5 (35.7)MFAC1 course9 (64.2)2 courses7 (50)Induction death rate5 (35.7)MFAC death rate1 (7.1)CR rate7 (50)Median (range)Disease-free survival (months)12 (2–24)

An Open Label, Dose Escalation Study of AVE9633 Administered as a Single Agent by Intravenous (IV) Infusion Weekly for 2 Weeks in 4-Week Cycle to Patients with Relapsed or Refractory CD33-Positive Acute Myeloid Leukemia (AML).

AVE9633/huMy9-6-DM4 is an immunoconjugate composed of a humanized monoclonal IgG1 antibody, huMy9-6, which specifically targets the CD33 antigen, conjugated through a disulfide link to the maytansine derivative DM4, a potent tubulin inhibitor. The CD33 antigen is expressed on the surface of myeloid cells. After the conjugate is bound to the CD33 antigen it is internalized and the cytotoxic is released within the target cell. We report preliminary results of the ongoing phase I dose escalation study of AVE9633 in patients with refractory/relapsed CD33+ AML. The study regimen consists of AVE9633 IV infusion on Day 1 and Day 8 of a 28-day cycle. To date, dose levels of 30 (n=1), 50 (n=3), 75 (n=3), 105 (n=3) and 130 (n=3) mg/m2 on Day 1 and Day 8 have been investigated. Patients received 1 (n=2), 2 (n=5), 3 (n=2) and 4 (n=4) cycles of AVE9633. No dose-limiting toxicity was noted so far. Nine patients experienced mild to moderate infusion reactions, mostly on Day1 of Cycle1. One patient at 130 mg/m2 presented grade 3 ALT elevation of 3 days duration. Free DM4, measured by LC/MS/MS was detectable at 105 and 130 mg/m2 in the range of 5 to 10 ng/mL. AVE9633/huMy9-6-DM4 exposure (measuring, by ELISA method, all antibodies containing at least one molecule of DM4) increased proportionally with the administered dose. Using Flow Cytometry Assay, saturation and down regulation of CD33 on peripheral and marrow blasts were observed from the dose of 50 mg/m2. There was one CRp in a 68-year old woman with refractory AML, achieved after 4 cycles of AVE9633 given at 105 mg/m2 × 2, with a 4-month continuing CRp as of 7/07 (she is receiving AVE9633 105 mg/m2 once monthly). One PR occurred in a 81-year old woman with refractory AML after one cycle at 130 mg/m2 × 2, persistent after the second cycle (third cycle is ongoing). Two patients had >50% decrease in bone marrow blasts, at 105 mg/m2 × 2 (from 25% to 8% after two cycles) and at 130 mg/m2 × 2 (from 35% to 9% after one cycle). One additional patient at 75 mg/m2 × 2 presented clearance of peripheral blasts by Day 10 of Cycle1. Mean DM4/IgG on the surface of peripheral blasts (available for one patient at 130 mg/m2) progressively decreased after the infusion, from 3.09 on day 1 to 0.28 on Day 8, and then increased to 2.64 after the second infusion of AVE9633 on Day 8. These preliminary results show that AVE9633 is well tolerated, with manageable safety profile, allowing outpatient treatment. Evidence of anti-leukemia activity was observed in 5 patients. The study is continuing with the evaluation of 150 mg/m2 × 2. Evaluation of closer drug administrations which might enhance efficacy appears warranted.

Monoclonal antibodies as targeted therapy in hematologic malignancies in older adults

Background: Biological agents are proving to be increasingly useful and exciting additions to the antineoplastic armamentarium, but many clinicians are unfamiliar with the properties of these types of agents. Objectives: This review focuses on monoclonal antibodies (MAbs) that are used in the treatment of hematologic malignancies. Our goal was to provide the reader with information on trials that led to US Food and Drug Administration (FDA) approval of commonly used MAbs in hematologic malignancies, including their mechanisms of action and pharmacokinetics, with specific emphasis on use in elderly patients; we also present data on toxicities and precautions to be aware of when administering these drugs. Methods: Materials for this review were gathered based on a computerized literature search (English-language articles only) using the PubMed database covering the period January 1998 to December 2005. Search terms used included the following: elderly, monoclonal antibodies, and neoplasms. Results: Alemtuzumab is a recombinant DNA-derived, humanized MAb directed against the CD52 B-cell antigen. It is indicated for the treatment of B-cell chronic lymphocytic leukemia (CLL) in patients who have been treated with alkylating agents and who have failed to respond to fludarabine therapy. Gemtuzumab ozogamicin is an MAb conjugated with a cytotoxic antitumor antibiotic, calicheamicin. It has been approved for use in patients with CD33-positive acute myeloid leukemia (AML) in first relapse who are aged ≥60 years and who are not considered candidates for other cytotoxic chemotherapy. Rituximab, one of the first MAbs approved by the FDA for use in human cancers, is an antibody directed against the CD20 antigen found on the surface of normal and malignant B lymphocytes. It is extensively used in the treatment of B-cell malignancies, such as CLL, and non-Hodgkin's lymphomas (NHLs), such as follicular lymphoma and diffuse large B-cell lymphoma. Conclusions: It is noteworthy that while there have been a number of studies using these agents in the younger population, there continues to be a paucity of clinical trials targeting the elderly patient in particular; this continues to be an area of research interest. More clinical studies of these agents—conducted specifically in elderly patients with CLL, NHL, AML, and other hematologic malignancies—are needed.

Aberrant marker expression patterns on the CD34+CD38− stem cell compartment in acute myeloid leukemia allows to distinguish the malignant from the normal stem cell compartment both at diagnosis and in remission

Acute myeloid leukemia (AML) is generally regarded as a stem cell disease. In CD34-positive AML, the leukemic stem cell has been recognized as CD38 negative. This CD34+CD38- population survives chemotherapy and is most probable the cause of minimal residual disease (MRD). The outgrowth of MRD causes relapse and MRD can therefore serve as a prognostic marker. The key role of leukemogenic CD34+CD38- cells led us to investigate whether they can be detected under MRD conditions. Various markers were identified to be aberrantly expressed on the CD34+CD38- population in AML and high-risk MDS samples at diagnosis, including C-type lectin-like molecule-1 and several lineage markers/marker-combinations. Fluorescent in situ hybridization analysis revealed that marker-positive cells were indeed of malignant origin. The markers were neither expressed on normal CD34+CD38- cells in steady-state bone marrow (BM) nor in BM after chemotherapy. We found that these markers were indeed expressed in part of the patients on malignant CD34+CD38- cells in complete remission, indicating the presence of malignant CD34+CD38- cells. Thus, by identifying residual malignant CD34+CD38- cells after chemotherapy, MRD detection at the stem cell level turned out to be possible. This might facilitate characterization of these chemotherapy-resistant leukemogenic cells, thereby being of help to identify new targets for therapy.

Proteasome Inhibitor Bortezomib Has Antitumour Activity Against Both CD34 Negative and CD34 Positive Acute Myeloid Leukemia Cells.

Proteasome inhibition represents a promising novel anticancer therapy, and Bortezomib is a highly selective reversible inhibitor of the 26S subunit of the proteasome complex. Acute Myeloid Leukemia (AML) is a heterogeneous group of diseases in which coexistence of leukemic cells at different stages of differentiation is frequently found, and CD34, a marker of immaturity, has been associated with drug resistance and poorer outcome. In order to explore the activity of Bortezomib in AML, we have evaluated its effectiveness against freshly isolated cells from 27 newly diagnosed AML patients (excluding APL). Mononuclear cells (MNC) were isolated from fresh bone marrow samples by Ficoll-Hipaque density sedimentation. The percentage of blasts after purification was 88±9% (Mean±SD). In order to assess the effective dose of Bortezomib in AML, four cell lines (HL-60, KG-1, HEL y MV4-11) were treated for 24h with increasing doses of Bortezomib. IC50 was 50nM (Range 10nM to 100nM). Based on these results, patients' samples were treated with Bortezomib for 18 hours. Doxorubicin 1μM was also used for treatment in order to compare with Bortezomib activity in 23 paired samples. Using quadruple staining (Annexin V/CD33/CD34/CD45), the number of apoptotic cells was measured by multiparametric flow cytometry. At least 50.000 events were acquired in a FACScalibur cytometer (BDB). Annexin V+ (AV+) events were quantified over total cellularity, as well as in CD34+, CD34− blast cell fractions and normal residual lymphocytes. The percentage of apoptotic events was corrected according to the proportion of apoptotic cells in the control tube (absence of drug). The proportion of AV+ cells in the total blast cell population was 55±27%. CD34+ and CD34− blast cells populations were separately analyzed (only one leukemic cell population CD34+, or CD34− were detected in 7 and 13 cases, respectively; while in the remaining 6 cases both populations existed). Interestingly, Bortezomib showed similar activity on CD34+ (52±26%, n=13) and CD34− (55±26%, n=19) blast cell populations (p=0.68). Moreover, in the 6 cases in which CD34+ and CD34− leukemic cells coexisted, no difference was detected on the proportion of AV+ events after Bortezomib treatment in both cellular subsets (42±26% and 48±16% respectively, p=NS). In addition, the proportion of AV+ cells within the normal residual lymphocytes with bortezomid was significantly lower (20±12%) when compared with blast cells (p< 0.01). Bortezomib was more effective for total blast cells compared to doxorubicin (56±32% vs 40±26%, respectively; p=0.009). Interestingly, when CD34+ and CD34− blast cell populations were separately analyzed, differences were only significant for the CD34+ populations (n=13) (52±26% vs 32±28% AV + cells for Bortezomib and doxorubicin, respectively; p=0.009), with no differences in either CD34− populations or normal residual lymphocytes. In summary, our results show a high antitumour activity of Bortezomib in AML blast cells, with similar efficacy against the more immature CD34+ and more mature CD34− leukemic cell subsets, while the toxicity against residual lymphocytes is significantly lower. These results support a therapeutic role for Bortezomib in adult Acute Myeloid Leukemia.

Phase I Study of AVE9633, an AntiCD33-Maytansinoid Immunoconjugate, Administered as an Intravenous Infusion in Patients with Refractory/Relapsed CD33-Positive Acute Myeloid Leukemia (AML).

AVE9633 is an immunoconjugate created by conjugation of the cytotoxic maytansinoid, DM4, to the monoclonal IgG1 antibody, huMy9-6 (average of 3.5 molecules of DM4 per antibody). The huMy9-6 antibody is a humanized version of a murine monoclonal antibody, My9-6, which is specific for the CD33 antigen expressed on the surface of myeloid cells, including the majority of cases of AML. Because CD33 has little expression outside the hematopoietic system, it represents an attractive target for antibody-based therapy in patients with AML. The humanized antibody, huMy9-6, binds to the CD33 antigen with an apparent KD in the range of 10−10 M. Maytansinoids are anti-mitotics that inhibit tubulin polymerization and microtubule assembly, inhibiting cells during the G2/M phase of the mitotic cycle. In order to link maytansinoids to antibodies via disulfide bonds, a new thiol-containing maytansinoid (DM4) was synthesized. Attachment of potent maytansinoids to an antibody via disulfide bonds provides a satisfactory stability in the bloodstream. After the conjugate is bound at the specific tumor site it is internalized and the cytotoxic agent is released within the target cell. A phase I study of AVE9633 is being conducted in patients with refractory/relapsed CD33+ AML. The study regimen consists of AVE9633 IV infusion on Day 1 of a 3 weeks cycle. To date dose levels of 15 (N=3), 30 (N=5), 50 (N=4), 75 (N=4), 105 (N=2), 200 (N=3) and 260 (N=1) mg/m2 have been investigated. Hypersensitivity reactions during perfusion were noted, requiring prophylaxis with steroids. No other AVE9633- attributable extramedullary Grade 3 AE has been observed to date. Free DM4, measured by LC/MS/MS was detectable from the 75 mg/m2 dose level; its Cmax (at the end of infusion) increased from 10 ng/mL at the 75 mg/m2 dose level to 70 ng/mL at 200 mg/m2. Neither AVE9633-associated myelosuppression nor responses have been noted. Using Flow Cytometry Assay on peripheral blasts and monocytes, total saturation and down regulation of CD33 were observed following administration of doses ≥ 30 mg/m2. AVE9633 exposure (measuring, by ELISA method, all antibodies containing at lease one molecule of DM4) increased proportionally with the administered dose in the dose range 15 to 200 mg/m2. Updated PK results and potential explanations for the lack of efficacy using this treatment schedule will be presented.

In Acute Myeloid Leukemia Both Malignant and Normal Stem Cells Can Be Detected in Remission Bone Marrow.

In CD34-positive acute myeloid leukemia (AML), the leukemia-initiating event is thought to occur in the CD34+CD38− stem cell compartment. Survival of these cells after chemotherapy hypothetically results in minimal residual disease (MRD) and leads to relapse.C-type lectin-like molecule 1 (CLL-1) could serve as a possible target for therapy, as we previously showed that it is expressed on malignant CD34+CD38− cells at diagnosis and not on normal CD34+CD38− cells (van Rhenen et al., Blood 106: 6a, 2005). CLL-1 expression is also present on malignant CD34+CD38− cells after chemotherapy.In the present study we investigated whether other antigens are also aberrantly expressed on CD34+CD38− cells in AML patients at diagnosis which would allow detection of AML CD34+CD38− cells in remission bone marrow. Such would offer opportunities for patient risk stratification and guidance of therapeutic intervention.Flowcytometry was performed on bone marrow CD34+CD38− cells in AML at diagnosis, after chemotherapy and in normal (NBM) and regenerating (RBM) bone marrow. Antibodies were anti-CD34, anti-CD45 and anti-CD38 together with antibodies against CLL-1, CD2, CD5, CD7, CD11b, CD19 and CD56, apart from CLL-1 all used as leukemia-associated phenotypes (LAPs) in immunophenotypic MRD detection (Feller et al., Leukemia 8:1380, 2004; further referred to as “whole blast” MRD). In diagnosis AML marker expression on CD34+CD38- cells was scored as <50% or >50%, because at least 50% expression is needed for accurate measurements of malignant CD34+CD38- cells after chemotherapy.At diagnosis, 60/77 AML samples were CD34-positive. A reliable number of CD34+CD38- events (>20) could be measured in 56/60 cases. CLL-1 expression >50% was observed in 15/60 cases, LAP expression in 9/60 cases and both CLL-1 and LAP in 8/60 cases. Altogether, in 32/60 CD34-positive cases, detection of malignant CD34+CD38- cells was possible. In NBM (n=4) as well as in RBM, the CD34+CD38- cells did show low CLL-1 (n=6) and low LAP (n=2, for all antigens) expression (all <3%). For three AML samples in which a leukemia-specific translocation was present, FISH analysis showed the translocation in CD34+CD38-LAP+ cells.Therefore, CLL-1 and/or LAP staining might enable to accurately discriminate between residual normal and malignant CD34+CD38- cells after chemotherapy. Firstly, we found that irrespective of the aberrant phenotype or the time point of analysis the frequency of residual malignant CD34+CD38- cells significantly correlated with “whole blast” MRD (r=0.42, p=0.008, n=38). Secondly the ratio between malignant and normal CD34+CD38- cells (stem cell ratio) was found to parallel both CD34+CD38- cell frequency and “whole blast” MRD. Both parameters, similar to “whole blast” MRD, parallelled clinical outcome with decreases when entering remission, no changes in non-responders and increases with forthcoming relapses.In 3/60 cases in which “whole blast” MRD could not be used, stem cell MRD could be performed. Since the populations are so well defined [CD34+/CD38−/CD45dim/aberrant marker] stem cell MRD will require less extensive experience than currently used MRD frequency assessment. These advantages are expected to increase as the use of 5 or more fluorescence channels will improve the success of stem cell MRD.In conclusion it is possible to detect malignant stem cells in AML patients at diagnosis and after chemotherapy, which would offer opportunities for future patient risk stratification and guidance of therapeutic intervention.

CD7-restricted activation of Fas-mediated apoptosis: a novel therapeutic approach for acute T-cell leukemia

AbstractAgonistic anti-Fas antibodies and multimeric recombinant Fas ligand (FasL) preparations show high tumoricidal activity against leukemic cells, but are unsuitable for clinical application due to unacceptable systemic toxicity. Consequently, new antileukemia strategies based on Fas activation have to meet the criterion of strictly localized action at the tumor-cell surface. Recent insight into the FasL/Fas system has revealed that soluble homotrimeric FasL (sFasL) is in fact nontoxic to normal cells, but also lacks tumoricidal activity. We report on a novel fusion protein, designated scFvCD7:sFasL, that is designed to have leukemia-restricted activity. ScFvCD7:sFasL consists of sFasL genetically linked to a high-affinity single-chain fragment of variable regions (scFv) antibody fragment specific for the T-cell leukemia-associated antigen CD7. Soluble homotrimeric scFvCD7:sFasL is inactive and acquires tumoricidal activity only after specific binding to tumor cell-surface-expressed CD7. Treatment of T-cell acute lymphoblastic leukemia (T-ALL) cell lines and patient-derived T-ALL, peripheral T-cell lymphoma (PTCL), and CD7-positive acute myeloid leukemia (AML) cells with homotrimeric scFvCD7:sFasL revealed potent CD7-restricted induction of apoptosis that was augmented by conventional drugs, farnesyl transferase inhibitor L-744832, and the proteasome inhibitor bortezomib (Velcade; Millenium, Cambridge, MA). Importantly, identical treatment did not affect normal human peripheral-blood lymphocytes (PBLs) and endothelial cells, with only moderate apoptosis in interleukin-2 (IL-2)/CD3-activated T cells. CD7-restricted activation of Fas in T-cell leukemic cells by scFvCD7:sFasL revitalizes interest in the applicability of Fas signaling in leukemia therapy.