The peripheral benzodiazepine receptor ligand PK11195 overcomes different resistance mechanisms to sensitize AML cells to gemtuzumab ozogamicin

The antibody-targeted therapeutic, gemtuzumab ozogamicin (GO, Mylotarg), is approved for treatment of relapsed acute myeloid leukemia (AML). We previously showed that AML blasts from GO refractory patients frequently express the drug transporters P-glycoprotein (Pgp) and/or multidrug resistance protein (MRP). We also previously reported that inhibition of drug transport by the Pgp modulator, cyclosporine A (CSA), can increase GO sensitivity in Pgp(+) AML cells and that the peripheral benzodiazepine receptor ligand, PK11195, sensitizes AML cells to standard chemotherapeutics both by inhibiting Pgp-mediated efflux and by promoting mitochondrial apoptosis. We now show that PK11195 also can overcome multiple resistance mechanisms to increase GO sensitivity in AML cells, including resistance associated with expression of drug transporters and/or antiapoptotic proteins. PK11195 substantially increases GO cytotoxicity in AML cells from many different cell lines and primary patient samples, often more effectively than CSA. We also show that PK11195 is nontoxic in NOD/SCID mice and can sensitize xenografted human AML cells to GO. Since PK11195 is well tolerated in humans as a single agent, its further study as a multifunctional chemosensitizer for anti-AML therapies, including GO-based therapies, is warranted.     

Safety and efficacy of gemtuzumab ozogamicin in pediatric patients with advanced CD33+ acute myeloid leukemia

This open-label, dose-escalation study evaluated the safety and efficacy of single-agent gemtuzumab ozogamicin, a humanized anti-CD33 antibody-targeted chemotherapeutic agent, for pediatric patients with multiple relapsed or primary refractory acute myeloid leukemia (AML). Twenty-nine children 1 to 16 years of age (relapsed disease, 19; refractory disease, 10) received gemtuzumab ozogamicin ranging from 6 to 9 mg/m2 per dose for 2 doses (separated by 2 weeks) infused over 2 hours. All patients had anticipated myelosuppression. Other toxicities included grade 3/4 hyperbilirubinemia (7%) and elevated hepatic transaminase levels (21%); the incidence of grade 3/4 mucositis (3%) or sepsis (24%) was relatively low. One patient treated at 9 mg/m2 developed veno-occlusive disease (VOD) of the liver and defined the dose-limiting toxicity. Thirteen patients underwent hematopoietic stem-cell transplantation less than 3.5 months after the last dose of gemtuzumab ozogamicin; 6 (40%) developed VOD. Eight of 29 (28%) patients achieved overall remission. Remissions were comparable in patients with refractory (30%) and relapsed (26%) disease. Mean multidrug resistance-protein-mediated drug efflux was significantly lower in the leukemic blasts of patients achieving remission (P < .005). Gemtuzumab ozogamicin was relatively well tolerated at 6 mg/m2 for 2 doses and was equally effective in patients with refractory and relapsed disease. Further studies in combination with standard induction therapy for childhood AML are warranted.     

Hepatic sinusoidal obstruction after gemtuzumab ozogamicin (Mylotarg) therapy

Gemtuzumab ozogamicin (Mylotarg) targets leukemia cells expressing the CD33 receptor by means of a monoclonal antibody conjugated to a cytotoxic agent, calicheamicin. Treatment of acute myeloid leukemia (AML) with gemtuzumab ozogamicin may result in liver injury. We reviewed the course of 23 patients who were given gemtuzumab ozogamicin for AML that had relapsed after hematopoietic cell transplantation. Liver toxicity was assessed through physical examination, serum tests, histologic examination, and hepatic venous pressure measurements. Liver injury developed in 11 patients after gemtuzumab ozogamicin administration; it was manifested as weight gain, ascites, and jaundice in 7 patients. Seven patients died with persistent liver dysfunction and either multiorgan failure or sepsis at a median of 40 days after gemtuzumab ozogamicin infusion. Portal pressure measurements were elevated in 2 patients. Results of liver histologic examination in 5 patients showed sinusoidal injury with extensive sinusoidal fibrosis, centrilobular congestion, and hepatocyte necrosis. Six patients experienced AML remission that was sustained for at least 60 days after gemtuzumab ozogamicin infusion. In summary, hepatic sinusoidal liver injury developed after gemtuzumab ozogamicin infusion. Histology showed striking deposition of sinusoidal collagen, suggesting that gemtuzumab ozogamicin targets CD33(+) cells residing in hepatic sinusoids as the mechanism for its hepatic toxicity.     

Multidrug resistance protein attenuates gemtuzumab ozogamicin-induced cytotoxicity in acute myeloid leukemia cells

Gemtuzumab ozogamicin (GO) is a novel immunoconjugate therapy for acute myeloid leukemia (AML). P-glycoprotein (Pgp) confers resistance to GO and is associated with a worse clinical response. To address whether multidrug resistance protein (MRP) affects GO susceptibility, we characterized Pgp, MRP1, and MRP2 expression in CD33+ cell lines and CD33+ AML samples and analyzed the effect of the Pgp inhibitor cyclosporine (CSA) and the MRP inhibitor MK-571 on GO-induced cytotoxicity. MRP1, but not MRP2, expression correlated with MRP activity. MK-571 enhanced GO-induced cytotoxicity in Pgp-negative/MRP-positive NB4 and HL-60 cells. CSA, but not MK-571 alone, restored GO susceptibility in Pgp-positive/MRP-positive TF1 cells; however, MK-571 enhanced cytotoxicity in the presence of CSA. All patient samples exhibited MRP activity, and 17 of 23 exhibited Pgp activity. CSA increased GO-induced cytotoxicity in 12 Pgp-positive samples, whereas MK-571 alone was effective in only one sample with minimal Pgp activity. In 3 Pgp-positive/MRP-positive samples, MK-571 enhanced GO-induced cytotoxicity in the presence of CSA. Thus, MRP1 may attenuate susceptibility to GO. This effect was comparatively less than that for Pgp and required the inhibition of Pgp for detection in cells that coexpressed both transporters. Because MK-571 and CSA failed to affect cytotoxicity in a portion of Pgp-positive/MRP-positive AML samples, additional resistance mechanisms are likely important.     

Detection of activity of P-glycoprotein in human tumour samples using rhodamine 123

Based on the fluorescent properties of the dye rhodamine 123 (Rh123), which is transported by the membrane efflux pump P-glycoprotein (P-gp), we developed a functional flow cytometric assay for the detection of multidrug-resistant (MDR) cells. Using drug sensitive cell lines (KB-3-1) and MDR mutants (KB-8-5, KB-C1) experimental conditions were established that enabled demonstration of significant differences in Rh123 efflux and accumulation. Subsequently we investigated the applicability of this functional assay for the prediction of MDR in human peripheral blood and bone marrow samples. Using two-colour flow cytometry, the leukaemic blast cells of six patients suffering from acute myeloid leukaemia (AML) were analysed. In three cases the blast cells showed a rapid and marked Rh123 efflux. In the presence of MDR inhibitors these cells retained Rh123. To determine whether the efflux of Rh123 was associated with P-gp expression, the leukaemic cells were stained with the monoclonal antibody MRK-16. In addition extracted RNA was analysed by polymerase chain reaction to evaluate the expression of mdr 1 mRNA. In all three Rh123+ cases mdr 1 mRNA was detectable whereas only one AML case expressed P-gp. In comparing Rh123 with daunorubicin, which also allows the detection of MDR cells, accumulation studies proved Rh123 to be the more sensitive drug for flow cytometric MDR screening. Additionally, two-colour flow cytometry was much easier to perform with Rh123 than with daunorubicin. Our results indicate that flow cytometric measurement of Rh123 accumulation/efflux proves applicable to detect MDR cells in heterogenous clinical samples.     

CD33 expression and P-glycoprotein-mediated drug efflux inversely correlate and predict clinical outcome in patients with acute myeloid leukemia treated with gemtuzumab ozogamicin monotherapy

Gemtuzumab ozogamicin (GO) contains an anti-CD33 antibody to facilitate uptake of a toxic calicheamicin-gamma(1) derivative. While recent in vitro data demonstrated a quantitative relationship between CD33 expression and GO cytotoxicity, previous correlative studies failed to identify a significant association between CD33 expression and clinical outcome. Studying patients undergoing GO monotherapy for relapsed acute myeloid leukemia (AML), we now find that AML blasts of responders have a significantly higher mean CD33 level and lower P-glycoprotein (Pgp) activity compared with nonresponders. CD33 expression and Pgp activity are inversely correlated. While both variables are associated with outcome, Pgp remains significantly associated with outcome even after adjusting for CD33, whereas CD33 does not show such an association after adjusting for Pgp. The inverse relationship between CD33 and Pgp suggests a maturation-stage-dependent expression of both proteins, and offers the rationale for using cell differentiation-promoting agents to enhance GO-induced cytotoxicity.     

Multiparametric analysis of apoptotic and multi-drug resistance phenotypes according to the blast cell maturation stage in elderly patients with acute myeloid leukemia.

BACKGROUND AND OBJECTIVES: Acute myeloid leukemia (AML) is a heterogeneous group of malignant diseases, often characterized by coexistence of more than one subpopulation of blast cells. Multiparametric flow cytometry immunophenotyping has proven to be a reliable and sensitive approach for the discrimination of myeloid blast cells from residual normal cells present in bone marrow samples from AML patients and, at the same time, allows the identification of different maturation compartments among myeloid blasts. Therefore, it provides a unique tool for assessing apoptotic and multidrug resistance (MDR)-associated phenotypes in individual subsets of leukemic cells. DESIGN AND METHODS: The aim of the present study was to explore the simultaneous expression of proteins related to both apoptosis (APO2.7, bcl-2, bax) and multidrug resistance (MDR1, MRP, LRP) in the different blast cell subpopulations detected at diagnosis in a group of 72 elderly patients with AML. In addition, we included 5 bone marrow samples from healthy adult donors in the analysis. RESULTS: Immature blast cells (CD34+: subset I) showed a significantly higher level of bcl-2 expression (p <0.0001) together with a lower reactivity for APO 2.7 (p=0.02) as compared to the other more mature CD34- cell subsets. The expression of Bax parallelled that of APO 2.7, although the difference between immature CD34+ blast cells and the mature blast cell subsets did not reach statistical significance (p=0.18). These results translated into a significantly (p<0.0001) higher bcl-2/bax ratio for the CD34+ blast cells as compared to that of the two CD34- blast cell subpopulations. Regarding the expression of the multidrug resistance-associated proteins Pgp and MRP, CD34+ blast cells displayed a greater expression of both proteins as compared to the more mature CD34- AML blast cells, but differences according to maturation stage of AML blast cells did not reach statistical significance. In contrast, LRP expression was significantly lower in the more immature CD34+ blast cell subset than in the more mature ones (p=0.01). INTERPRETATIONS AND CONCLUSIONS: As far as normal bone marrow is concerned our results suggest that all blast cell subpopulations are more protected from apoptosis than their normal counterparts. We conclude that in elderly patients with AML the more immature blast cells are more resistant to apoptotic processes, which could explain why, when AML relapses, the blast cells frequently display a more immature phenotype than that observed at diagnosis. Contradictory results in multidrug resistance profile support the hypothesis that failure to respond to chemotherapeutic drugs in AML is a multifactorial phenomenon.     

Molecular remission of Philadelphia/bcr-abl-positive acute myeloid leukaemia after treatment with anti-CD33 calicheamicin conjugate (gemtuzumab ozogamicin, CMA-676)

The anti-CD33 monoclonal antibody linked to NAc-gamma calicheamicin gemtuzumab ozogamicin (CMA-676) was used to treat a patient with Philadelphia/bcr-abl-positive acute myeloid leukaemia. We report a morphological and cytogenetic complete remission after treatment with two doses of gemtuzumab ozogamicin as a single agent. Using real-time polymerase chain reaction (PCR), gemtuzumab ozogamicin treatment resulted in a 3-log tumour mass reduction in bone marrow.     

P-glycoprotein and multidrug resistance-associated protein, but not lung resistance protein, lower the intracellular daunorubicin accumulation in acute myeloid leukaemic cells

The in vitro intracellular daunorubicin accumulation (IDA) of blast cells from 69 patients with newly diagnosed acute myeloid leukaemia (AML) was correlated with the expression and functional activity of the multidrug resistance (MDR) proteins, P-glycoprotein (Pgp), multidrug resistance-associated protein (MRP) and lung-resistance protein (LRP). An inverse and significant association was found between IDA and Pgp-related efflux activity (r = -0.31, P = 0.01) and also MRP (r = -0.25, P = 0.04) but not with LRP (r = -0.13, P = 0.28). Coexpression of the MDR proteins had an additive effect in further lowering of IDA levels, suggesting that the clinical MDR phenotype is dependent on the sum of multiple MDR factors available to the leukaemic cell. Thus, the median IDA of leukaemic cells without any MDR proteins was significantly higher than that of blasts carrying two MDR proteins (0.466 vs. 0.296, P = 0.046). Seven patients with no expression of Pgp, MRP and LRP still had low IDA levels, suggesting the presence of efflux MDR mechanisms other than those studied. The relation of IDA to clinical parameters known to be associated with poor prognosis, such as age, secondary AML, karyotype, peripheral blood blast and CD34 counts, was also studied, but no significance was found on multifactorial analysis. There was a non-significant trend for earlier relapse in patients with low IDA levels (leukaemia-free survival of 16.3 months compared with 21.1 months in patients with high IDA levels). Our data suggest that, while the IDA assay is a quick and relatively easy test for the combined efflux MDR phenotype, it is unable to detect other MDR mechanisms, such as LRP, which may be important to the clinical outcome of patients with AML.     

Multidrug resistance 1 gene expression and AgNOR in childhood acute leukemias

The multidrug resistance 1 (MDR1) gene product, P-glycoprotein (Pgp/p170) is a membrane protein, which acts as an ATP dependant efflux pump that expels a wide variety of organic compounds including chemotherapeutic agents from the cell. Pgp over expression has been demonstrated to be linked with poor treatment outcome and poor prognosis in a number of malignant tumors. AgNORs is a simple, reliable and inexpensive method of evaluating the proliferative activity of a tumor. We have studied MDR1 expression and AgNORS in 41 cases of acute leukemia in children. In this study, AgNOR counts in patients with acute lymphoblastic leukemia (ALL) L2 subtype (FAB classification) were significantly higher as compared to the ALL L1 subtype. Similarly, mean AgNOR count in the acute myeloid Leukemia (AML) M2 subtype was significantly higher as compared to the ALL L1 subtype. However, there was no correlation between AgNOR and treatment outcome or between AgNOR counts and MDR1 expression in any of the subtypes of acute leukemia included in this series. In AML, MDR1 gene expression was found to be related to reduced remission induction rates and hence poorer prognosis. In ALL, our study has shown no difference in remission induction between MDR1 positive and MDR1 negative cases. This would suggest that factors other than MDR1 may be of relevance in Pediatric ALL.     

Molecular response in two children with relapsed acute myeloid leukemia treated with a combination of gemtuzumab ozogamicin and cytarabine

Phase I/II studies of gemtuzumab ozogamicin (GO) in pediatric refractory/relapsed acute myeloid leukemia (AML) have been reported. We present the cases of two children with relapsed AML who were treated with GO plus cytarabine, leading to a decrease of minimal residual disease down to levels not previously obtained. The toxicity profile of this treatment was relatively mild, except for severe but manageable myelosuppression.     

Fractionated gemtuzumab ozogamicin combined with intermediate-dose cytarabine and daunorubicin as salvage therapy in very high-risk AML patients: a bridge to reduced intensity conditioning transplant?

Outcome of patients with primary refractory/relapsed (R/R) acute myeloid leukemia (AML) remains dismal. Herein, we present a retrospective monocentric study of 24 very high-risk AML patients who received a combination of fractionated gemtuzumab ozogamicin (GO) with intermediate-dose cytarabine and daunorubicin as salvage therapy. Median age was 55.3 years. Diagnostic was secondary AML for 33% of them. Seven patients had favorable risk, 8 had intermediate-1 or intermediate-2, and 6 had unfavorable risk of AML according to the European LeukemiaNet prognostic index. Complete remission was achieved in 50% of cases (46% in refractory and 55% in relapsed AML) without excessive toxicity. Thirteen patients could be referred for transplant. Only allogeneic hematopoietic stem cell transplantation provided a benefit in this patient cohort with a 1-year overall survival of 50.7 versus 18.1% in the absence of transplantation. Patients treated with reduced intensity conditioning (RIC) showed a longer survival as compared to those undergoing myeloablative conditioning regimen mainly because of decreased toxicity.Our data suggest that salvage therapy with fractionated GO combined with intermediate-dose cytarabine and daunorubicin in very high-risk patients may serve as a potential bridge therapy to RIC transplant.     

Semiquantitative RT-PCR evaluation of the MDR1 gene expression in patients with acute myeloid leukemia

Resistance to chemotherapy is one of the major obstacles to effective treatment in acute myeloid leukemia (AML). The most extensively studied protein involved in multidrug resistance (MDR) is the transmembrane glycoprotein P (P-gp), the product of the multidrug resistance gene 1 (MDR1). MDR1/P-gp overexpression is frequently observed in hematological malignancies, especially in acute leukemia, and has been reported to correlate with poor prognosis in acute myeloid leukemia (AML). The aim of this study was to evaluate the level of MDR1 gene expression in bone marrow and/or peripheral blood samples in 92 AML patients in relation to their prognosis. The analyzed group was stratified according to presence or absence of prognostically favorable aberrations (PFAs), such as t(15;17) with PML/RARalpha fusion gene, t(8;21) with AML1/ETO fusion gene or inv(16)/ t(16;16) with CBFbeta/MYH11 fusion gene. These prognostically favorable aberrations were detected by RT-PCR and/or standard cytogenetic techniques. MDR1 expression was detected by semiquantitative comparative RT-PCR using software-based evaluation. The levels of MDR1 expression in the bone marrow predicted induction of complete remission in the whole group of analyzed patients (P = 0.032). They were significantly lower in PFA negative patients who achieved complete remission compared to those who failed to achieve complete remission (P = 0.008). In PFA negative patients, MDR1 expression was higher when compared to PFA positive patients (P = 0.055). No such difference was found when analyzing peripheral blood samples. Our experiments showed no impact of MDR1 expression in bone marrow or peripheral blood cells on overall survival (P = 1.000 and P = 0.903 respectively). In summary, the present study shows the prognostic impact of MDR1 expression on induction of complete remission in AML patients. We confirmed that MDR1 overexpression is an unfavorable prognostic factor in AML, which may help to stratify the risk rate of PFA negative patients. In future studies, quantitative detection of MDR1 expression might be a valuable tool to predict prognosis in this patient subset.     

Altered multidrug resistance phenotype caused by anthracycline analogues and cytosine arabinoside in myeloid leukemia.

The expression of P-glycoprotein (Pgp) is often increased in acute myeloid leukemia (AML). However, little is known of the regulation of Pgp expression by cytotoxics in AML. We examined whether Pgp expression and function in leukemic blasts was altered after a short exposure to cytotoxics. Blasts were isolated from 19 patients with AML (15 patients) or chronic myeloid leukemia in blastic transformation (BT-CML, 4 patients). Pgp expression and function were analyzed by flow cytometric analysis of MRK 16 binding and Rhodamine 123 retention, respectively. At equitoxic concentrations, ex vivo exposure for 16 hours to the anthracyclines epirubicin (EPI), daunomycin (DAU), idarubicin (IDA), or MX2 or the nucleoside analogue cytosine arabinoside (AraC) differentially upregulated MDR1/Pgp expression in Pgp-negative and Pgp-positive blast cells. In Pgp-negative blasts, all four anthracyclines and AraC significantly increased Pgp expression (P =.01) and Pgp function (P =.03). In contrast, MX2, DAU, and AraC were the most potent in inducing Pgp expression and function in Pgp positive blasts (P <.05). A good correlation between increased Pgp expression and function was observed in Pgp-negative (r =.90, P =.0001) and Pgp-positive blasts (r =.77, P =.0002). This increase in Pgp expression and function was inhibited by the addition of 1 micromol/L PSC 833 to blast cells at the time of their exposure to these cytotoxics. In 1 patient with AML, an increase in Pgp levels was observed in vivo at 4 and 16 hours after the administration of standard chemotherapy with DAU/AraC. Upregulation of Pgp expression was also demonstrated ex vivo in blasts harvested from this patient before the commencement of treatment. In 3 other cases (1 patient with AML and 2 with BT-CML) in which blasts were Pgp negative at the time of initial clinical presentation, serial samples at 1 to 5 months after chemotherapy showed the presence of Pgp-positive blasts. All 3 patients had refractory disease. Interestingly, in all 3 cases, upregulation of Pgp by cytotoxics was demonstrated ex vivo in blasts harvested at the time of presentation. These data suggest that upregulation of the MDR1 gene may represent a normal response of leukemic cells to cytotoxic stress and may contribute to clinical drug resistance.     

Failure of three novel regimens to improve outcome for patients with relapsed or refractory acute myeloid leukaemia: a report from the Eastern Cooperative Oncology Group

The treatment of relapsed acute myeloid leukaemia (AML) remains unsatisfactory. We conducted a phase II randomized trial where patients received intermediate-dose cytarabine for 4 d followed by gemtuzumab ozogamicin on day 5 (Arm A), or combined with liposomal daunorubicin for 3 d (Arm B), or cytarabine given for 5 d combined with cyclophosphamide for 3 d and topotecan by continuous infusion for 5 d (Arm C). Eligible patients had primary refractory AML, a first relapse after a remission of <1 year, or a second or greater relapse. The primary objective of this trial was attainment of a conventional complete remission (CR) or a CR without platelet recovery (CRp) in at least 40% of patients. The CR/CRp rates for the 82 eligible patients were 3/26 (12%) in Arm A, 2/29 (7%) in Arm B, and 1/27 (4%) in Arm C. No patients who had relapsed within 6 months of initial CR or who had suffered multiple relapses responded. More than 95% of patients subsequently died of AML. No unexpected toxicities were encountered. We conclude that none of these three regimens were effective enough in the treatment of high-risk relapsed or refractory AML to warrant further study. This trial was registered at http://www.clinicaltrials.gov as #NCT00005962.     

Modulation of multidrug resistance by BIBW22BS in blasts of de novo or relapsed or persistent acute myeloid leukemia ex vivo

The phenylpteridine derivative BIBW22BS (BIBW22) is a potent modulator of multidrug resistance (MDR). We investigated BIBW22 in comparison to dexniguldipine and verapamil as modifier of MDR in blasts of de novo, relapsed or persistent acute myeloid leukemia (AML) in vitro. All patients with relapsed or persistent AML had been pretreated with idarubicin and cytosine arabinoside. The degree of MDR was determined by efflux kinetics of rhodamine 123 (R123), daunorubicin, and idarubicin measured by flow cytometry (FACS). A total of 51 patients with AML, 25 de novo and 26 relapsed or persistent, were investigated. While only 6 out of 25 de novo AML blast populations showed moderate efflux of R123 and daunorubicin, 17 out of 26 blast populations of relapsed or persistent AML had an efflux between 20% and 44% within 15 min ex vivo. This efflux could be significantly inhibited by 1 M BIBW22, 1 M dexniguldipine, or 10 M verapamil. For idarubicin we found an effusion of 40±9% within 15 min in all blast populations that could not be inhibited by the modulators. Clinically achievable drug concentrations causing only moderate side-effects are in the range of 0.5 M dexniguldipine and 3 M verapamil. Up to now, BIBW22 has not been investigated clinically. Thus, the potential toxicity of concentrations of 0.5–1 M BIBW22, sufficient for an optimal efflux inhibition ex vivo, is not known yet. We conclude from our ex vivo investigations in blast populations of de novo, relapsed or persistent AML that BIBW22 is a potent modulator of MDR.Abbreviations AML acute myeloid leukemia - MDR multidrug resistance     

Modulation of drug resistance transporters as a strategy for treating myelodysplastic syndrome

Resistance to chemotherapy is an obstacle to the successful treatment of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). The failure of therapeutic treatment may be due to the development of multidrug resistance (MDR), mechanisms of which include upregulation of membrane-resident transporters which efflux chemotherapeutic drugs from tumor cells, and failure of the cancer cell to undergo apoptosis in response to chemotherapy. Membrane transporter-based drug efflux transporters have been extensively studied, and agents that block drug efflux have been found and investigated. Presence of P-glycoprotein (Pgp, MDR1, ABCB1), a member of the ATP-binding cassette (ABC) transporter family, has been reported to correlate with poor prognosis in AML and MDS. In MDS, Pgp expression increases as the disease progresses. Overexpression of other transporters, such as the multidrug resistance protein (MRP1, ABCC1), and the vault-associated transporter lung resistance protein have been shown as well in both MDS and AML, but their prognostic relevance is not clear. Recently, a novel ABC half-transporter, the breast cancer resistance protein (ABCG2) has been found in approximately 30% of AML cases, and may play a role in resistance to chemotherapy. In clinical trials in MDS, first-generation Pgp blockers, such as cyclosporin-A and verapamil, were minimally effective, non-specific, and toxic. However, another first-generation blocker, quinine, was used in MDS and may specifically benefit MDS patients overexpressing Pgp. A second-generation drug, the non-immunosuppressive cyclosporine analog valspodar (PSC833), was studied in AML and MDS, and was highly toxic, resulting in the need to reduce the dosage of the chemotherapeutic drugs as a result of valspodar reducing the clearance of the chemotherapeutic agents. Third-generation drugs, which are highly specific for Pgp and which seem to have only modest effects on drug clearance, include tariquidar, zosuquidar, laniquidar, and ONT-093. These are all in phase I/II trials and show promise for future treatment.     

Granulocyte colony-stimulating factor enhances the in vitro cytotoxicity of gemtuzumab ozogamicin against acute myeloid leukemia cell lines and primary blast cells

OBJECTIVE: To evaluate the effects of granulocyte colony-stimulating factor (G-CSF) on the in vitro sensitivity of acute myeloid leukemia (AML) cell lines and primary AML blast cells to gemtuzumab ozogamicin (GO). MATERIALS AND METHODS: AML cell lines and primary blasts from 10 patients with AML were first incubated for 72 hours in the presence of G-CSF (5 or 100 ng/mL) and then exposed to increasing concentrations of GO (1-1,000 ng/mL) for an additional 72 hours. RESULTS: Pretreatment with G-CSF translated into significant enhancement of GO-induced cytotoxicity in the GO-sensitive HL-60 and NB-4 cells. Conversely, the response of GO-insensitive KG-1a, TF-1, and K562 cells was unaffected by in vitro priming with G-CSF. In vitro exposure to G-CSF augmented GO-induced apoptosis in 7 of 10 primary AML samples and rendered blast cells from three refractory patients sensitive to killing effect of GO. The G-CSF-induced increase of the cytocidal activity of GO was independent of effects on the cell cycle and on the expression levels of CD33 antigen. Of potential interest, G-CSF induced dose-dependent inhibition of P-glycoprotein (P-gp/ABCB1) function in the GO-sensitive HL-60 and NB-4 cells and in blasts from three patients with AML that we tested. CONCLUSION: Collectively, our findings point to G-CSF as a potential sensitizing agent that can be exploited therapeutically to improve the clinical efficacy of GO.     

Successful repeated treatment of acute myeloid leukemia in early relapse with gemtuzumab ozogamicin alone

A 68-year-old female was diagnosed with acute myeloid leukemia (AML-M2 without 8/21 translocation) in December 2006. Although a complete remission (CR) was obtained after induction chemotherapy, the first post-remission therapy was discontinued because of severe cardiovascular complications. She had a relapse of AML with CD33-positive myeloblasts which comprised 38.4 % of the bone marrow cells in November 2007. She received two courses of low-dose chemotherapy because of the previous complications. The amount of Wilm’s tumor 1 (WT1) mRNA in the peripheral blood was 13,000 copies/μg RNA after the first course of the chemotherapy, and 4.8 % myeloblasts remained in the bone marrow after the second course. She was treated with a single course of gemtuzumab ozogamicin (GO), with a subsequent CR with 0.9 % marrow myeloblasts and fewer than 50 copies of WT-1 mRNA (normal level). Thereafter, she received five courses of GO monotherapy at each occasion of early AML relapse. Hematological remission has been sustained over a period of about 24 months with the GO monotherapy alone. This case suggests that GO monotherapy is a useful salvage therapy for early relapse of CD33-positive AML in situations in which standard chemotherapy is not indicated.