ALBERT

Beschreibung: In acute myeloid leukemia (AML) patients, a variety of clinical and biologic parameters, including phenotype, have been examined for potential value in predicting treatment response and survival. The European Group for the Immunological Classification of Leukaemias (EGIL) has proposed that AML be defined immunologically by the expression of 2 or more of the following myeloid markers: myeloperoxidase, CD13, CD33, CDw65, and CD117. With regard to this classification, the prognostic significance of 21 antigens taken separately and with immunophenotypic subgroups were evaluated and compared with other clinical and biological variables in 177 adult AML patients. None of the antigens tested were associated with treatment outcome. In contrast, patients with blasts disclosing a full expression of panmyeloid phenotype (defined by the expression of all 5 myeloid markers) had a higher complete remission rate (P 

Beschreibung: One of the best-characterized resistance mechanisms in acute myeloid leukemia (AML) is the drug extrusion mediated by P-glycoprotein (Pgp). Recently the results of workshops organized by several groups concluded that accurate measurement of low activity of Pgp is a difficult goal in clinical samples. Therefore, highly sensitive and specific assays were developed to assess the functionality of Pgp using JC-1, a fluorescent molecule with the different emission wavelength (green and red fluorescence) according to its concentration in 129 AML samples. It was shown that JC-1 (green and red bands) may define 3 groups of patients: resistant (R) (29% of patients), intermediate (I) (36%), and sensitive (S) (35%). In contrast, rhodamine 123 assay detected only the R group defined by JC-1. Nevertheless, the I group has an intermediate expression of Pgp (0.39, 0.29, and 0.19 for the R, I, and S groups, respectively, P = .002), an intermediate biologic profile (percentage of CD34, 95%, 67%, and 44%, respectively, P 

Beschreibung: Abstract 1698 Background: Many clinical and biological features are commonly used in order to predict the probability of response to standard treatment in adult acute myeloid leukaemia (AML). Although cytogenetic analysis provides the major information for prognosis, many molecular alterations like FLT3/ITD, mutations of NPM1, mutations of CEBPα, or BAALC over expression have been described these last years, in order to guide therapeutic choices, especially in patients with normal karyotype. In the other hand, prognostic implications of ABC proteins' expression and functionality, like ABCB1 (Pgp) and others, have been known for years but relationships between ABC proteins and those molecular markers haven't been fully studied. Material and methods: In this retrospective study, we explore the relationships between ABC proteins and these molecular markers, and evaluate whether ABC proteins' activity is an independent prognosis factor in 206 AML, homogenously treated in EORTC protocols. ABC proteins' activity has been assessed with JC1 functional test performed at the time of diagnosis, and all patients were screened from frozen bone marrow or peripheral blood samples for FLT3/ITD, NPM1 mutations, CEBPα mutations, and expression of BAALC. 206 patients aged from 16 to 81 years and treated either in EORTC AML 10, AML 12 or AML 13 from 1996 to 2008 have been included, with a median follow up time for alive patients of five years. Results: In the whole population, 42 patients (20%) showed a high ABC functional assay (JC1+). High ABC activity was associated with NPM1 wild type (p=0.03) and higher BAALC expression (p=0.007). FLT3-ITD was detected in only 2 patients with high ABC functional assay. There was no relationship between high ABC functionality and CEBPα mutational status. In multivariate analysis including age, leukocyte count, cytogenetic, existence of a pre leukemic phase, NPM1, FLT3/ITD, CEBPα and BAALC status and JC1 assay, high ABC proteins' activity was an independent prognosis factor for OS in the whole population (p=0.03). Others independent prognosis factors for OS were age (p=0.0004), cytogenetic (p=0,045), FLT3/ITD (p=0.0096), and NPM1 mutation (p= 0.07). When interesting to normal cytogenetic population (112 patients), high ABC proteins' activity was an independent prognosis factor for DFS (p=0.0107) and OS (p=0.0038). Age (p=0.0012), and FLT3/ITD (p=0.0173) were the only other prognosis factors for OS. In patients with normal cytogenetic, and both NPM1 WT and no FLT3/ITD, only age (p=0.0008) and high ABC proteins' activity (p=0.004) were independent prognosis factors for OS. Conclusion: ABC proteins' activity remains an independent prognosis factor in adult AML, when compared to these molecular factors. Because of its relatively high frequency (around 20 % of patients), and the easiness to perform this test, we think JC1 probe should be used in every AML patients in order to refine the treatment strategy at the time of diagnosis. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Thirteen cell lines with different levels of Pgp and MRP expression were used to assess the ability of calcein acetoxymethyl ester (calcein-AM) uptake and calcein efflux to measure Pgp and MRP functions, respectively. There was a good correlation between MRP expression and the modulatory effect of probenecid (a specific modulator of MRP) on the calcein efflux (r = .91, P= .0003) and between Pgp expression and the modulatory effect of CsA on calcein-AM uptake (r = .96, P 〈 .0001). In light of the high correlations for both proteins, we tested calcein-AM uptake and efflux in fresh myeloid leukemic cells. In 53 acute myeloid leukemia (AML) patients, there was also a good correlation between MRP expression (measured by reverse transcription-polymerase chain reaction and by MRPm6 expression by flow cytometry) and the modulatory effect of probenecid on the calcein fluorescence (r = .92, P 〈 .0001) and between Pgp expression as measured by UIC2 antibody binding on flow cytometry and the modulatory effect of cyclosporin A on calcein-AM uptake (r = .83,P 〈 .0001). Pgp activity was higher in CD34+leukemia than in CD34− leukemia (2.26 ± 1.50 v1.46 ± 1.21, respectively; P = .003), and MRP activity was higher in CD34− leukemia than in CD34+leukemia (1.77 ± 0.40 v 1.4 ± 0.29, respectively; P= .004). Pgp expression and activity (P = .004 andP = .01, respectively) and MRP activity (P = .03) but not MRP expression were prognostic factors for achievement of complete remission. These results suggest that functional testing (with calcein-AM ± modulators) for the presence of both MRP and Pgp activities is of prognostic value and that MRP contributes to drug resistance in AML.

Beschreibung: Background Obesity is an increasing matter of concern worldwide. A few studies suggested that BMI might be a risk factor for outcome in different malignancies including AML. However, not all studies could confirm the prognostic value of BMI in AML. Also, capping chemotherapy dose calculation at 2m² of Body Surface Area (BSA) is widely used, but there is no strong evidence-based rationale for this. With this background, this single centre report aimed to assess whether BMI and chemotherapy dose capping could be correlated with outcome in AML patients receiving intensive induction and consolidation chemotherapy. Patients and Methods Between 2003 and 2012, all consecutive AML patients referred to our centre were prospectively included in this analysis. Eligible patients were those treated with intensive induction chemotherapy (consisting in cytosine arabinoside and anthracyclines). Acute promyelocytic leukemia cases were excluded. Anthropomorphic, patients, cytogenetic and molecular AML characteristics were collected and analyzed to assess their impact on Overall Survival (OS), Leukemia-Free Survival (LFS) and Complete Remission Rate (CR1). Analyses were performed using the logistic regression method. For every patient, BSA was capped at 2m² for chemotherapy dose calculation. Results In all, data were available for 233 patients: median age was 56 (range, 16-80) years; 60% were men; median weight was 69 kg (range, 40-158); median height 169 cm (range, 146-207); BSA 〉 2m² was observed in 15% of cases; underweight was seen in 7% of cases, while overweight represented 33%, and truly obese patients 10%. 64% had a Performance Status (PS) of 0 or 1. In terms of AML characteristics, secondary AML was diagnosed in 16% of cases. The good, intermediate, and poor cytogenetic risk groups represented 9%, 67%, and 24%, respectively. FLT3-ITD was observed in 17% of cases; (bi allelic) CEBPα mutation in 6% and NPM1 mutation in 23%. 27% of patients could proceed to allogeneic stem cell transplantation. With a median follow-up of 40.2 (range, 0.69-107.7) months, CR1 was achieved in 71% of cases. Overall median OS was 51±4 months, and median LFS was 45±4 months. In multivariate analysis including all relevant factors from the univariate analysis, and after adjustment for gender, BMI did not prove to be significantly associated with OS (p=0.98; HR=1.0;95%CI [0.951-1.052]), nor with LFS (p=0.88;HR=1.0;95%CI [0.957-1.05]). Also, obesity was not associated with CR1 achievement (p=0.70;HR=1.3;95%CI [0.360-4.56]). Factors significantly associated with OS were age under 60 years (p

Beschreibung: Thirteen cell lines with different levels of Pgp and MRP expression were used to assess the ability of calcein acetoxymethyl ester (calcein-AM) uptake and calcein efflux to measure Pgp and MRP functions, respectively. There was a good correlation between MRP expression and the modulatory effect of probenecid (a specific modulator of MRP) on the calcein efflux (r = .91, P= .0003) and between Pgp expression and the modulatory effect of CsA on calcein-AM uptake (r = .96, P 〈 .0001). In light of the high correlations for both proteins, we tested calcein-AM uptake and efflux in fresh myeloid leukemic cells. In 53 acute myeloid leukemia (AML) patients, there was also a good correlation between MRP expression (measured by reverse transcription-polymerase chain reaction and by MRPm6 expression by flow cytometry) and the modulatory effect of probenecid on the calcein fluorescence (r = .92, P 〈 .0001) and between Pgp expression as measured by UIC2 antibody binding on flow cytometry and the modulatory effect of cyclosporin A on calcein-AM uptake (r = .83,P 〈 .0001). Pgp activity was higher in CD34+leukemia than in CD34− leukemia (2.26 ± 1.50 v1.46 ± 1.21, respectively; P = .003), and MRP activity was higher in CD34− leukemia than in CD34+leukemia (1.77 ± 0.40 v 1.4 ± 0.29, respectively; P= .004). Pgp expression and activity (P = .004 andP = .01, respectively) and MRP activity (P = .03) but not MRP expression were prognostic factors for achievement of complete remission. These results suggest that functional testing (with calcein-AM ± modulators) for the presence of both MRP and Pgp activities is of prognostic value and that MRP contributes to drug resistance in AML.

Beschreibung: Adult acute myeloid leukemia (AML) with intermediate cytogenetic remains a very heterogeneous group of patients with highly variable individual prognosis. Emerging data suggest that some molecular markers could help to refine cytogenetic stratification. Here we focused on ABCB1 (MDR1/Pgp) ATP-binding cassette transporter activity and fms-like tyrosine kinase mutations (Flt3/ITD) because their individual prognosis value is well demonstrated and because they may lead to targeted therapy. Therefore we assessed Pgp activity using JC-1 probe and Flt3 exon 14 mutational status by standard PCR and realised a multivariate analysis in 171 adult AML patients treated in the EORTC protocols. Flt3/ITD (ITD+) and high Pgp activity (Pgp+) were found in 26/171 (15%) and 55/171 (32%) of the patients, respectively. The repartition was remarkable in that, as defined with our criteria, both were negative in 94/171 (55%, Pgp-ITD- group) or mutually exclusive in 73/171 (43%, Pgp-ITD+ and Pgp+ITD- groups) and only 4/171 (2%, Pgp+ITD+ group) were positive for both. In univariate analysis complete remission (CR) rates for ITD+ were 38% vs 61% for ITD- patients (p=0.034) and 47% vs 62% (p=0.06) for Pgp+ vs Pgp- patients. Individually, both parameters were strong predicators for overall survival (OS) (p=0,02) but not for disease free survival. In multivariate analysis Pgp+ITD+ (p

Beschreibung: In acute myeloid leukemia (AML) patients, a variety of clinical and biologic parameters, including phenotype, have been examined for potential value in predicting treatment response and survival. The European Group for the Immunological Classification of Leukaemias (EGIL) has proposed that AML be defined immunologically by the expression of 2 or more of the following myeloid markers: myeloperoxidase, CD13, CD33, CDw65, and CD117. With regard to this classification, the prognostic significance of 21 antigens taken separately and with immunophenotypic subgroups were evaluated and compared with other clinical and biological variables in 177 adult AML patients. None of the antigens tested were associated with treatment outcome. In contrast, patients with blasts disclosing a full expression of panmyeloid phenotype (defined by the expression of all 5 myeloid markers) had a higher complete remission rate (P 

Beschreibung: Abstract 4909 Background: A major issue in the treatment of acute myeloid leukemia (AML) is resistance to chemotherapeutic drugs. The role of ABC proteins, and specially ABCB1 (PgP/mdr1), in this resistance has been well established, and higher ABC proteins' activity, assessed with functional tests, has been associated with poorer complete remission rates and poorer overall prognosis (Marzac et al, Haematologica, 2011). Furthermore, the evaluation of molecular minimal residual disease (MRD), using mutated nucleophosmin (NPM1)expression quantification has been related to patients' global prognosis (Krönke et al, J. Clin. Oncol., 2011), and to response to treatments. In this study, we evaluate the impact of ABC proteins' activity on MRD after one course of induction chemotherapy, in 26 patients with NPM1 mutated AML. Material and methods: We retrospectively identified 26 AML patients with NPM1 mutation treated in our center and with MRD data. MRD was evaluated as the ratio of NPM1 mutated allele and total NPM1, using PCR DNA quantification and the delta delta Ct method. MRD was measured at the time of diagnosis and after one course of anthracycline-based induction chemotherapy. ABC proteins' activity was evaluated at the time of diagnosis using JC1 +/− cyclosporine A assay (Legrand et al, Blood, 2001). Correlations between ABC proteins' activity and the level of post induction MRD were evaluated with the Mann-Whitney test. Survival was evaluated using the Cox model. For all analyses, P values were considered significant when lower than 0. 05. Results: Median age at diagnosis was 53 years old. Twenty-two patients had normal cytogenetic analysis at diagnosis, and the other 4 patients had intermediate prognosis cytogenetic analysis. Nine patients harboured FLT3-ITD mutation. Median ABC proteins' activity was 0. 11 (0 – 0. 77). After one course of induction chemotherapy, 3 patients did not reach cytological complete remission. In 17 patients MRD level after induction therapy was inferior to 1 %, in 11 patients MRD was inferior to 0. 1 % and in 7 patients MRD was inferior to 0. 01 %. Overall, higher MRD level after induction (defined by MRD level higher than 0. 1 %) was associated with poorer prognosis for disease free survival (HR= 4. 25 [95% CI 1. 049–17. 27]; p=0. 04), and for overall survival HR=11. 25 [95% CI 1. 22–103. 23]; p=0. 03). Higher ABC proteins' activity was associated with higher MRD levels post induction, and patients who did not reach MRD level lower than 0. 1 % had significantly higher ABC proteins' activity than other patients (p=0. 008). ABC proteins' activity was also associated with overall survival in our patients (p=0. 04). Conclusion: Higher ABC proteins' activity is associated with higher MRD levels after one course of induction chemotherapy in 26 NPM1 mutated AML patients, and is also associated with poorer overall survival. The poorer prognosis associated with high ABC proteins' activity in AML seems to be in part related to direct resistance to chemotherapy. These data should be confirmed in larger studies. Disclosures: No relevant conflicts of interest to declare.