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This randomized, open-label, phase 2b study (NCT01565668) evaluated the efficacy and safety of 2 dosing regimens of quizartinib monotherapy in patients with relapsed/refractory (R/R) FLT3-internal tandem duplication (ITD)-mutated acute myeloid leukemia (AML) who previously underwent transplant or 1 second-line salvage therapy. Patients (N = 76) were randomly assigned to 30- or 60-mg/day doses (escalations to 60 or 90 mg/day, respectively, permitted for lack/loss of response) of single-agent oral quizartinib dihydrochloride. Allelic frequency of at least 10% was defined as FLT3-ITD-mutated disease. Coprimary endpoints were composite complete remission (CRc) rates and incidence of QT interval corrected by Fridericia's formula (QTcF) of more than 480 ms (grade 2 or greater). Secondary endpoints included overall survival (OS), duration of CRc, bridge to transplant, and safety. CRc rates were 47% in both groups, similar to earlier reports with higher quizartinib doses. Incidence of QTcF above 480 ms was 11% and 17%, and QTcF above 500 ms was 5% and 3% in the 30- and 60-mg groups, respectively, which is less than earlier reports with higher doses of quizartinib. Median OS (20.9 and 27.3 weeks), duration of CRc (4.2 and 9.1 weeks), and bridge to transplant rates (32% and 42%) were higher in the 60-mg groups than in the 30-mg group. Dose escalation occurred in 61% and 14% of patients in the 30- and 60-mg groups, respectively. This high clinical activity of quizartinib at the evaluated doses is consistent with previous reports with an improved safety profile. Need to dose-escalate more than half of patients who received quizartinib 30 mg also supports further investigation of treatment with quizartinib 60 mg/day.

This randomized, open-label, phase 2b study (NCT01565668) evaluated the efficacy and safety of 2 dosing regimens of quizartinib monotherapy in patients with relapsed/refractory (R/R)

Background. Previous studies have demonstrated that monosomal karyotype (MK) confers a particularly poor prognosis in patients with acute myeloid leukemia (AML) (Breems et al., J Clin Oncol 2008, 26:4791-7). Purpose of the study. To determine the impact of the type of remission-induction chemotherapy and the impact of having a donor on overall survival (OS) in younger ( Methods. In the EORTC/GIMEMA AML-10 trial (Mandelli et al., J Clin Oncol 2009, 27:5397-403), patients were randomized to receive either daunorubicin (DNR), mitoxantrone (MTX), or idarubicin (IDA) in addition to standard-dose cytarabine (SDAC: 10 days of 100 mg/m2 per day as continuous IV infusion) and etoposide for induction chemotherapy. In the EORTC/GIMEMA AML-12 trial, (Willemze et al., J Clin Oncol 2014, 32:219-28) patients were randomized between either SDAC or high dose cytarabine (HiDAC) (3 g/m2 every 12 hours as a 3-hour IV infusion on days 1, 3, 5, and 7) induction, both with DNR, and etoposide. In both trials, a second cycle of the same remission induction chemotherapy was administered in patients who achieved a partial remission. Patients who achieved a complete remission (CR) or a CR with incomplete counts recovery (CRi) after 1 or 2 courses of induction chemotherapy received a consolidation chemotherapy with the same anthacycline as in the induction course plus intermediate dose cytarabine. Patients in both studies were then scheduled to receive an allogeneic hematopoietic stem cell transplantation (allo-HSCT) if they had a donor or an autologous HSCT (auto-HSCT) otherwise, in first CR. For the current analyses, cytogenetics were centrally reviewed and re-classified using the European Leukemia Net (ELN) definition. MK was defined as the presence of 2 or more monosomies, or a single monosomy in the presence of structural abnormalities. Results. In the AML-10 trial, 2157 patients were randomized to receive DNR, MTX or IDA. The current analyses were performed in a subgroup of 910 patients for whom cytogenetic data were available and who did not have t(8;21), inv(16) nor t(15;17). 709 of them were classified in the ELN intermediate cytogenetic risk group and 201 in the adverse group. Out of the 910 patients, 93 had a MK. In comparison to the 817 remaining (MK-) patients, MK+ patients were less likely to achieve a CR, were more likely to have resistant disease (RD), and had worse OS and disease-free survival (DFS) (Table 1). There was no impact of the type of anthracycline on outcomes. Among the MK+ patients who achieved a CR, 5-yr OS from CR was 26% (95% CI, 10-46%) in patients with a donor (n=21, of whom, 13 received an allo-HSCT in first CR), versus 4% (95% CI, 0-18%) in those without a donor (n=24, of whom, 1 received an allo-HSCT in first CR) (P=0.07). Further, among the 10 MK+ who survived more than 1 year, 7 received an allo-HSCT in first CR (n=4) or beyond (n=3). In the AML-12 trial, 1942 patients were randomized between HiDAC or SDAC. The current analyses were performed in a subgroup of 1079 patients for whom cytogenetic data were available and who did not have t(8;21), inv(16) nor t(15;17). 893 of them were classified in the ELN intermediate cytogenetic risk group and 186 in the adverse group. Out of the 1079 patients, 98 had a MK. In comparison to the 1027 MK- patients, MK+ patients were less likely to achieve a CR, were more likely to have RD, and had worse OS and DFS (Table 1). There was a higher probability of achieving a CR and a trend for better OS in MK- patients randomized in the HiDAC arm (Table 2). In contrast, in MK+ patients, HiDAC did not increase either the CR rate as compared to SDAC, nor the 5-yr OS rate: 7% (HiDAC) vs 2% (SDAC) (Table 2). Finally, among the MK+ patients who achieved a CR, 5-yr OS from CR was 14 % (95% CI, 4-32%) in patients with a donor (n=21, of whom, 12 received an allo-HSCT in first CR), versus 4% (95% CI, 0.3-18%) in those without a donor (n=24) (P=0.60). Further, among the 4 MK+ patients who survived more than 1 year, 3 received an allo-HSCT in first CR (n=2) or beyond (n=1). Conclusions. This analysis confirms the poor prognosis of MK in younger AML patients. Shifting DNR to IDA or MTX or administration of HiDAC in the induction course failed to improve outcome of MK+ patients. Finally, there was a suggestion for a longer OS from CR in MK+ patients who had a donor, suggesting that allo-HSCT should be offered in all fit MK+ patients. FB & MSK contributed equally to this work Disclosures Thomas: Pfizer: Consultancy.

*The 2 first authors contributed equally to the work. Background. The best post-remission treatment for younger acute myeloid leukemia (AML) patients has remained controversial (Cornelissen JJ &Blaise D, Blood 2016, 127, 62-70) and there is paucity of studies comparing intensive chemotherapy to autologous (auto) or allogeneic (allo) bone marrow transplantation (BMT). Aim. The main objective of this study was to provide a long-term follow-up evaluation of patients previously enrolled in the pivotal EORTC/GIMEMA AML-8A (Zittoun et al., New England Journal of Medicine 1995, 332, 217-223). Methods. The EORTC/GIMEMA AML-8A prospectively assessed the impact of 3 post-remission treatments on disease-free survival (DFS) and overall survival (OS) in younger ( Results. In the current report, median follow-up was 11.1 (range, 0-28) years. For the whole population (n=422), the 5-, 10 and 15-year OS rates from inclusion were 35%, 32% and 31%, respectively. After the completion of ICT1, 168 patients were allocated to theallo-BMT arm, while 254 patients were randomized to auto-BMT (n=128) or ICT2 (n=126). DFS from CR was longer afterallo-BMT than auto-BMT and DFS from CR was longer after auto-BMT than ICT2, due to a lower relapse incidence (P Conclusions. This long-term follow-up of the EORTC/GIMEMA AML-8A study confirms a better DFS with allo-BMT or auto-BMT when compared to ICT2 for AML patients in first CR. Further, this long-term follow-up study revealed that the vast majority of patients alive in first CR at 5-year remains disease-free survivors 5 years later. Although indications of allogeneic hematopoietic stem cell transplantation (allo-HCT) are nowadays largely driven by cytogenetic/molecular AML profile, long-term results of AML8A study demonstrate that auto-BMT remained superior to ICT2 in younger AML patients not candidate for an allo-HSCT. Disclosures Efficace: TEVA: Consultancy, Research Funding; Seattle Genetics: Consultancy; Bristol Myers Squibb: Consultancy; Lundbeck: Research Funding. Martinelli:Celgene: Consultancy, Speakers Bureau; BMS: Speakers Bureau; Novartis: Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Genentech: Consultancy; Roche: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau; MSD: Consultancy; Pfizer: Consultancy, Speakers Bureau.

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

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

In adult acute myeloid leukemia (AML), the weight of the contribution of the combined activity of Pgp and MRP1 to drug resistance is not known. To address this question, we compared the activity of these proteins to the in vitro resistance to daunorubicin (DNR), etoposide, and cytosine arabinoside (Ara-C), using the calcein-AM uptake and the 3-[4, 5-di-methyl-thiazol-2, 5-diphenyl] tetrazolium bromide (MTT) assay in 80 adult AML patients. We found no correlation or only a weak correlation between the in vitro drug resistance to DNR and etoposide and MRP1 or Pgp expression or function when tested separately. However, a strong correlation was observed between the simultaneous activity of MRP1 and Pgp (quantified as the modulation of calcein-AM uptake by cyclosporin A and probenecid) and the LC50 of DNR (r = .77, P < .0001). This emphasized the role of these two proteins, not separately, but together in the resistance to DNR. In contrast, Mvp/LRP expression did not correlate with the LC50 of DNR. A high level of simultaneous activity of Pgp and MRP1 was predictive of a poor treatment outcome (for achievement of CR [P = .008], duration of relapse-free survival [RFS; P = .01], and duration of overall survival [OS; P = .02]). In addition, high LC50 of DNR and high LC50 of etoposide together were also predictive of a poor treatment outcome (for duration of RFS [P= .02] and duration of OS [P = .02]). The unfavorable cytogenetic category was more closely associated with the combined activity of both MRP1 and Pgp (P = .002) than with the activity of Pgp or MRP1 separately. This could explain the poor prognosis and the in vitro resistance to daunorubicin in this group of patients. These data suggest that treatment outcome may be improved when cellular DNR and etoposide resistance can be circumvented or modulated. Modulation of not only Pgp but also MRP1 could be essential to attain this aim in adult AML.

Inhibitors of differentiation (Id) are a group of dominant inhibitors of basic helix-loop-helix transcriptional factors, which promote excessive proliferation, and also protect cells against drug-induced apoptosis in mammalians. Recently, Id1 has been identified as a common downstream target of several constitutively activated oncogenic tyrosine kinase, such as FLT3 internal tandem duplication, in leukemia cells. We analyzed Id1 expression as possible prognostic factor in 237 acute myeloid leukemia (AML) patients. High Id1 expression was associated with older age (P = .009) and with FLT3 internal tandem duplication (P = .003). However, 61% of the patients in the group of FLT3− AML were Id1+, suggesting that other tyrosine kinases are involved. In whole population, high Id1 expression independently predicted shorter disease-free survival (P = .05) and overall survival (P = .003). In young patients (age ≤ 60 years) with normal cytogenetics, Id1+ was, in multivariate analysis, associated with lower complete remission rates (P = .02), shorter disease-free survival (P = .02), and overall survival (P = .006). In conclusion, our data provide a new molecular marker for refining the risk classification of AML, especially in young patients with normal cytogenetic. Id1− patients with normal cytogenetic should be classified as favorable-risk leukemia. Id1, as a downstream target of constitutively activated tyrosine kinase, could be a suitable candidate for targeted therapy.

Resistance to multiple chemotherapeutic agents has been related to the production of P-glycoprotein, a trans-membrane drug efflux pump that is encoded by the multidrug resistance (MDR) gene mdr1. To investigate whether mdr1 could be involved in clinical resistance to chemotherapy in acute leukemias, we have analyzed retrospectively the RNA from adult acute leukemia cells by slot-blot hybridization with a human mdr1 probe. Units of mdr1 expression were defined by reference to drug- sensitive human sarcoma and K562 leukemia cell lines (1 U) and the highly resistant doxorubicin selected leukemia cells K562/R7 (50 U). We studied 41 adult patients with acute leukemias: 5 acute lymphoblastic leukemias, 23 acute myeloid leukemias, and 13 secondary leukemias or blast crisis of chronic myelogenous leukemia. Expression of 10 U or more of mdr1 was found in 6 of 31 (19%) leukemias at diagnosis, versus 5 of 10 (50%) after relapse from therapy, P = .06. The complete remission rate and in vitro sensitivity to daunorubicin were both correlated with low expression (1 U, v 2 U or more) of mdr1. Among 36 evaluable attempts to induce remission, the complete remission rate was 67% (8 of 12) for patients with undetectable or minimal mdr1 expression (1 U), versus 29% (7 of 24) in patients with 2 U or more of expression, P = .03. In vitro resistance to daunorubicin or other MDR-related drugs was associated with expression of 2 U or more of mdr1 in 11 of 11 cases, while specimens that were sensitive to these agents were negative for mdr1 expression in 5 of 11 cases, P = .03. These data suggest that mdr1 expression contributes to chemoresistance in acute leukemia. Determination of mdr1 gene expression may be useful in designing therapy for patients with leukemia.

RW and FB are co-senior authors. Background: The prognosis of younger patients with intermediate/bad risk AML or high-risk MDS remains unsatisfactory. Although with current remission induction chemotherapy, 60-85% of patients achieves complete remission (CR), only 30-50% of them remains alive for more than 5 years. Clofarabine, a second-generation purine analog, is highly active as a single agent in AML. Willemze et al (Ann Hematol, 2014) recently reported the results of phase I of the AML-14A study and identified clofarabine at 10 mg/m2/day for 5 days as the maximum tolerated dose (given either in a 1-h infusion or as push injection) in combination with cytosine arabinoside (Ara-C) and idarubicin. We herein report the final results of the combined phase I and II parts of the AML-14A study that explored the antitumor activity of clofarabine containing induction combination regimens at the aforementioned phase I selected dosage schedules. Methods: Patients aged 18-60 years with intermediate/bad-risk AML or high-risk MDS (≥10% bone marrow blasts), adequate renal and hepatic function, and WBC count 65% or not. Using a Fleming design, the regimen was considered active if ≥ 23 out of 30 patients per arm achieved CR/CRi. Secondary endpoints included safety, CR/CRi rate after consolidation, hematopoietic recovery, ability of CD34 harvesting after consolidation, disease-free survival (DFS) and survival from CR/CRi, and overall survival (OS). Randomization was stratified by institution and by presence of poor prognostic features (WBC at diagnosis >=100 x 109/L or very high-risk cytogenetics/FLT3-ITD). Results: A total of 64 patients was randomized: 12 in the phase I part and 52 in the phase II part of the study. Two patients did not meet the inclusion criteria and were excluded. Among the remaining 62 patients, 5 had high-risk MDS. Median age was 50 yrs (range 20-60). Baseline characteristics were well balanced between the two arms. The CR/CRi rate after induction was 84% (26 of 31 patients) in each arm (95% CI: 66-95%) (Table 1). In Arm A vs Arm B, the most frequent grade >2 non-hematological and non-infectious adverse events over the induction-consolidation period were anorexia (29% vs 32%), and diarrhea (26% vs 32%). Finally, during treatment period there were 2 toxic deaths in Arm-A and 1 in Arm-B. Table 1: Patient outcomes. Median follow-up was 1.8 (range, 1 – 5.25) yrs. Arm-A (n=31) Arm-B (n=31) CR/CRi after 1-2 courses of induction, # pts (%) 23 (74) / 3 (10) 25 (81) / 1 (3) CR/CRi after 1 course of induction, # pts (%) 23 (74) / 3 (10) 24 (77) / 1 (3) OS median (95%CI), yrs 2.5 (1-NR) NR OS at 1-yr (95%CI), % 74 (55-86) 74 (55-86) # of infectious episodes with G3-4 neutropenia / # of patients with infection episodes 47 / 30 59 / 31 In patients who achieved CR/CRi Time to recovery from start of course 1 # of days with neutrophils NR= not reached. Conclusions: The 2 tested clofarabine (5x10 mg/m2) containing regimens yielded an impressive (84%) CR/CRi rate among patients with intermediate/bad-risk AML and high-risk MDS patients. Toxicity profiles in the two arms appeared relatively comparable. Disclosures Off Label Use: Clofarabine was used off label..

Background: FLT3-ITD mutational status is a well known prognostic factor in AML patients. However, its prognostic value in case of low mutational burden is currently under debate, especially in patients with intermediate karyotype and NPM1 mutations. Indeed some studies suggested that patients with an allelic ratio (i.e FLT3-ITD/FLT3 WT) lower than 50 % might have the same prognosis as unmutated patients. Patients and methods: Four hundred and sixty nine (469) patients diagnosed with AML in a single center from 1995 to 2014 and who received intensive chemotherapy based on anthracycline and cytarabine were included. Patients with t(15;17)(q24;q21) or t(9;22)(q34;q11) translocations were excluded. 134 patients underwent allogeneic stem cell transplantation. FLT3-ITD allelic burden was determined using fresh or frozen cells drawn at time of diagnosis in all patients. The mutational status for FLT3-ITD was determined using high-resolution sizing of fluorescent dye-labeled PCR amplification of exons 14-15. Beyond its contribution to mutation detection, high-resolution sizing also allows to estimate the allelic burden by measuring peak height ratios (mutant/wild type) on a fluorescence scale. Low allelic burden was defined by an allelic ratio lower than 50%. NPM1 and CEBPA mutational status were determined in most patients (n=409 and n=349 respectively). Median follow-up time for patients who were still alive was 37 months. Results: Median age was 56 years (range 16-81). Karyotype was unfavorable in 110 (24%) patients, favorable in 45 (10%), intermediate in 298 (63%) patients and normal in 229 (48 %). Overall, FLT3-ITD was detected in 83 (17%) patients. Twenty three patients (5%) had a low allelic ratio and 60 (13%) had a high allelic ratio. NPM1 mutation was found in 100 (24%) patients and bi-allelic CEBPA mutations in 21 (6%). In the overall population, patients with FLT3-ITD had poorer outcome compared to other patients (OS at 3 years: 33+/-5% vs. 52 +/-3%, p =0.02). There was no difference between patients with low allelic burden or patients with high allelic burden for leukemia-free survival (7+/-7% vs. 38+/-8%, p=0.12), overall survival (25+/-5% vs. 37+/-6%, p=0.67) or CR reaching (70% in the two groups). In multivariate analysis, factors independently associated with OS were cytogenetic risk group, age, leukocyte count at diagnosis, NPM1 status, CEBPA status and FLT3-ITD status. There were no difference between patients with high vs. low allelic burden (HR=1.20 [0.59-2.42]). Results were the same when focusing on patients with intermediate karyotype. FLT3-ITD was associated with lower survival (p=0.003), but the mutational burden was not (OS at 3 years 30+/-12% vs. 36+/-7%, for low or high burden respectively, p=0.86). In multivariate analysis, factors independently associated with OS were age, leukocyte count, NPM1 status, CEBPA status and FLT3-ITD status. Again, there were no difference between patients with high versus low allelic burden (HR=0.98[0.45-2.08]. Conclusions: In this series, low FLT3-ITD allelic burden had a similar prognostic significance compared to a higher allelic burden. Therefore, patients with low FLT3-ITD allelic burden should likely be considered as having the same prognosis as patients with high burden, and FLT3-ITD detection, but not FLT3-ITD allelic burden, should be the major factor to be considered for therapeutic decisions. Disclosures No relevant conflicts of interest to declare.

In myelodysplastic syndromes (MDS), anemia responds to recombinant human erythropoietin (rHuEPO) alone and in combination with recombinant human granulocyte-colony-stimulating factor (rHuGCSF) in 10% to 20% and in 35% to 40% of patients, respectively. We randomly divided 60 patients with low-grade anemic MDS and serum EPO levels lower than 500 IU/L (500 mU/mL) into 2 groups: rHuEPO + rHuG-CSF (arm A) and supportive care (arm B). After 12 weeks, those who had erythroid responses were given rHuEPO alone for 40 additional weeks. They were also given rHuG-CSF if they had relapses. A response was considered major if the hemoglobin (Hb) level was 115 g/L (11.5 g/dL) or higher and minor Hb increase was 15 g/L (1.5 g/dL) or more or if it remained stable without transfusion. Ten of 24 patients responded in arm A, and 0 of 26 responded in arm B (P =.01). Eight patients in arm A continued rHuEPO therapy alone, and 6 had relapses. Responses were always restored when rHuG-CSF was reintroduced. Mean direct costs per patient were 26,723 euros (arm A) and 8,746 euros (arm B). Quality of life was assessed with a Functional Assessment of Cancer Therapy-Anemia (FACT-An) scale. Similar percentages of patients from both arms showed significant clinical improvement. rHuEPO plus rHuG-CSF led to responses in 41.7% of MDS patients. This treatment was expensive. No effect on quality of life was demonstrated.

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 (p2 m² (who underwent chemotherapy capping) was not different from other patients: BSA > 2 m² did not impact OS (p=0.65;HR=0.84;95%CI [0.39-1.8]), or LFS (p=0.6;HR=0.82;95%CI [0.38-1.75]), or CR1 (p=0.5;HR=0.63;CI [0.162-2.432]). Conclusion BMI doesn’t seem to be a prognostic factor in intensively treated AML patients. Chemotherapy dose calculation capping at 2m² of BSA is not associated with a poorer outcome in this population. Disclosures: No relevant conflicts of interest to declare.

FMS-like tyrosine kinase 3 internal tandem duplications (FLT3-ITD) in acute myeloid leukemia (AML) are associated with early relapse after standard chemotherapy and poor survival. Quizartinib (AC220) is an oral FLT3 receptor tyrosine kinase inhibitor that has shown the highest level of single agent activity seen with a FLT3 targeted agent in FLT3+ relapsed AML to date. This Phase 2 study was conducted to assess the efficacy and safety of two lower doses of quizartinib monotherapy in FLT3-ITD positive (+) pts aged 18 yrs or older with AML relapsed or refractory to 2nd-line, salvage chemotherapy or relapsed after hematopoietic stem cell transplantation (HSCT) to identify the optimal dose for future studies. The co-primary endpoints of the study were the composite complete remission (CRc) rate, which included complete remission (CR), complete remission with incomplete platelet recovery (CRp), and complete remission with incomplete hematologic recovery (CRi) and the rate of grade ³2 QTc prolongation. The Phase 2 study enrolled a total of 76 pts who were randomized to either 30 mg or 60 mg daily. Preliminary data from the entire study of 76 pts are included here; updated information will be provided for presentation. Thirty eight patients each were randomized to each arm and treated continuously during 28-day cycles. 58% of the randomized patients were male. The pts had a median age of 53 yrs (range 19 to 77). For 30 mg pts the derived CRc rate was 50% (5% CR, and 45% CRi). For 60 mg pts the CRc rate was 50 % (3% CR, 3% CRp, and 45% CRi). 29% of pts at 30mg and 37% of pts at 60mg were successfully bridged to HSCT. Efficacy Results in AML Pts Relapsed/Refractory to 2nd-line Therapy or HSCT30 mg Arm N = 3860 mg Arm N = 38*Cumulative CRc, n (%)19 (50)19 (50)*CRc + PR, n (%)22 (63)26 (68)*Subjects receiving HSCT after discontinuing quizartinib, n (%)11 (29)14 (37)*Median overall survival, days146197*Two subjects randomized did not receive drug due to ineligibility but are included in the ITT analysis.CRc = composite complete remission; HSCT = hematopoietic stem cell transplantation; PR = partial remission. The rate of grade 2 or higher QTc prolongation assessed by central reading was 11% and 17% in 30 mg and 60 mg arms respectively, grade 3 QT prolongation was 3% in both arms and there was no grade 4. The mean maximum prolongation from baseline was 31.5 ms and 39.7 ms in 30 mg and 60 mg arms respectively. A majority of patients (78%) had a treatment related adverse event. The most common (10%) treatment-related adverse events (AEs) were anemia (18%), nausea (15%), fatigue (12%) and diarrhea (11%). Sixteen patients (22%) had a serious adverse event (SAE) possibly related to study drug. The most common (5%) treatment-related SAE was febrile neutropenia (5%). A total of 4 pts (5%) experienced a treatment-related AE resulting in discontinuation of quizartinib. The data from this Phase 2 study confirm the high degree of activity of quizartinib monotherapy in FLT3-ITD(+) AML pts relapsed/refractory to 2nd-line treatment or HSCT, previously shown in the larger Phase 2 ACE study of 333 patients treated at higher doses (90mg, 135mg or 200mg). Importantly, 25 of 76 pts (33%) were successfully bridged to a potentially curative HSCT, an almost identical rate to that seen with higher doses. The safety profile is improved specifically decreasing QT prolongation rate at lower doses. Other safety findings were manageable, and were primarily gastrointestinal adverse events and myelosuppression. The data from this Phase 2 study confirms an improved risk:benefit profile with lower doses of quizartinib and multiple randomized Phase 3 studies with quizartinib in both relapsed and newly diagnosed AML patients are planned. Disclosures: Cortes: Ambit: Research Funding. Schiller:Astellas: Consultancy. Trone:Ambit: Employment. Gammon:Ambit: Employment. Goldberg:Astellas: Research Funding. Marie:Astellas: Travel expenses and medical writing services provided by Astellas Other. Martinelli:Pfizer: Consultancy; BMS: Consultancy, Speakers Bureau; Ariad: Consultancy; Novartis: Consultancy, Speakers Bureau. Levis:Ambit Biosciences: Consultancy, Research Funding.

Background Secondary acute myeloid leukemia (sAML) describes patients (pts) with a history of malignant or non-malignant disease or AML secondary to environmental, occupational or therapeutic exposures. They are generally associated with poor outcome despite the use of intensive treatments. The impact of clinical features and type of treatment on pts' outcome is still not well established. In the current analysis we evaluated sAML pts who were treated in 13 EORTC collaborative trials conducted between May 1986 and January 2008. sAML pts in the database were pooled to characterize clinical features of the disease and evaluate changes in survival over these years (yrs). Method Main selection criteria were AML with bone marrows blasts ≥20% and documented history of prior malignancy, non-malignant disease and/or toxic exposure. AML-M3 and MDS without confirmed diagnosis ≥2 months before AML were excluded. All pts were eligible for standard treatment. Induction regimens were anthracycline and AraC based: 7+3, including etoposide, intensified with high dose (HD)-AraC randomized to standard doses (SD) in younger (AML12) or gemtuzumab ozogamicin in elderly pts. Consolidation regimens were age adapted. In mid-1980s, autologous transplant was tested vs a 2nd consolidation cycle (AML8A) in pts ≤45 yrs and thereafter used systematically in pts ≤60 yrs without available donor. Allogeneic transplant (Allo-SCT) was offered to pts ≤46 yrs with HLA-compatible sibling since mid-1980s and expanded in the last decade to pts up to 59 yrs. Selected pts were divided into 3 sAML cohorts, cohort A after MDS, cohort B after other malignant diseases and cohort C after non-malignant conditions and/or toxic exposure. Results Of 8858 pts enrolled in the 13 evaluated studies, 962 were sAML. Median age was 63 yrs (range 16-85), 413 were young (≤60 yrs) and 549 were elderly (≥61 yrs); 54% were males. Cohort A consisted of 509 pts (median age 64 yrs), cohort B of 362 pts (median age 59 yrs) and cohort C of 91 pts (median age 61 yrs). In cohort B, breast cancer (24%) and lymphoma (14%) were the most frequent primary tumors. Autoimmune diseases represented 22% of non-malignant conditions. In young pts, complete remissions (CR/CRi) rate was 59%; 55% in SD-AraC vs 89% in HD-AraC treated pts. Allo-SCT in CR1 was performed in 21% of all pts. The Allo-SCT rate increased from 5% before 1990, 20% in 1990-1999 to 25% from 2000 (20% in SD-AraC vs 31% of HD-AraC treated pts). CR/CRi was achieved in 45% of elderly pts. Median follow-up was 6 yrs. Median overall-survival (OS) was 14.5 months in young and 9 months in elderly pts. The 5-yr OS was 28% and 7% respectively. Five-yr OS was 11% in cohort A and 22% in both cohort B and C. Treatment outcome of younger pts according to disease features and treatment type over time in cohort A and B are detailed in table 1 & 2. Using Cox model stratified by cohort age, gender, WBC, risk group, year of treatment and HD-AraC were independent prognostic factors for OS. In the AML12 study, compared to denovo pts, sAML pts ≤45 yrs had worse outcome if treated with SD-AraC whereas a better OS was seen if treated with HD-AraC. In elderly pts only the good/intermediate risk group of cohort B had a relatively better 5-yr OS (15%). Conclusions The outcome of sAML in younger pts has improved over the yrs in parallel with HD-AraC introduction in induction of remission. HD-AraC should be considered for younger pts with sAML. Disclosures: Ramadan: Alwaleed Bin Talal Foundation : A research funding is under advanced negotiation with the foundation Other. Suciu:Alwaleed Bin Talal Foundation : A research funding is under advanced negotiation with the foundation Other. Meert:Alwaleed Bin Talal Foundation : A research funding is under advanced negotiation with the foundation Other. de Schaetzen:Alwaleed Bin Talal Foundation : A research funding is under advanced negotiation with the foundation Other Other.

Background Patients with acute myeloid leukemia (AML) frequently harbor mutations in genes involved in the DNA (hydroxy)methylation pathway (DNMT3A, TET2, IDH1, and IDH2). In addition, changes in DNA methylation have been implicated in the pathogenesis of AML. Recently it was discovered that TET proteins are able to convert 5-methylcytosine into 5-hydroxymethylcytosine (5hmC), which is an important intermediate in the demethylation pathway. In this study, we measured 5-hydroxymethylcytosine levels in AML patients, and correlated these with mutational status and overall survival (OS). Patients and methods Samples from 206 clinically and molecularly well-characterized younger adult AML patients (≤60 years), included in the EORTC/GIMEMA AML-12 06991 clinical trial, were analyzed for mutations in DNMT3A, TET2, IDH1 and IDH2. 5-hydroxymethylcytosine levels were measured using HPLC-MS/MS. Results In healthy control cells, 5hmC levels were confined to a narrow range (1.5 fold difference), whereas in AML cells, a much wider range was detected (15 fold difference). In remission, 5hmC values were normalized to levels comparable to healthy bone marrow and peripheral blood, indicating that the aberrant 5hmC levels at diagnosis are intrinsic to the leukemic cells. Patients with mutations in TET2 and patients with mutations in IDH1/2 had significantly lower levels of 5hmC compared to patients without mutated TET2 and IDH1/2 (both P Conclusion Both low and very high levels of 5hmC are markers of poor prognosis in AML, lending further support for testing therapies targeting the DNA hydroxymethylation pathway. Disclosures: No relevant conflicts of interest to declare.

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.

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of resting lymphocytes. The identification of p27kip1, a cyclin-dependent kinase inhibitor that contributes to cell cycle arrest and represents a link between extracellular signals and cell cycle, prompted us to study p27 protein in the lymphocytes from 88 patients with B-CLL and 32 patients with other chronic B-lymphoproliferative disorders. The expression of p27 protein was higher in B-CLL samples with variations among them. In B-CLL, p27 levels were independent of absolute number of circulating lymphocytes, but strongly correlated with both lymphocyte and total tumor mass (TTM) doubling time. High p27 expression was associated with a poorer overall prognosis. In vitro, there was an increased spontaneous survival of B-CLL cells expressing high p27 levels. Interleukin-4 (IL-4) upregulated p27 levels in B-CLL cells, while fludarabine decreased p27 levels. Thus, our results indicate that p27 may be a valuable kinetic marker in B-CLL by providing instantaneous estimation of the disease doubling time. In addition, these results suggest that there is a link between p27 expression and the ability of CLL cells to undergo apoptosis.

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.

Abstract 2804 Background: The “monosomal karyotype” cytogenetic subgroup (MK+), as defined by Breems et al. (1) encompasses mostly complex karyotypes (CK, >3 abnormalities) and is associated with very poor outcome in AML (1, 2) and MDS (3). Discussions are ongoing regarding treatment response prediction, and a prognosis-modifying effect within the CK+ AML subgroup (1, 4). In a study of 227 AML pts >60 years, 38 of whom were MK+, we found that particularly pts with at least 2 monosomal autosomes (MK2+, by definition also CK+) appeared to benefit from DAC (5). We now applied this question to higher-risk MDS pts randomized to either DAC or Best supportive care (BSC) in the 06011 trial (6) which explicitly enrolled pts with IPSS poor-risk cytogenetics. Methods: Of 233 randomized pts, 206 had cytogenetics informative for MK status: 63 had normal cytogenetics (CN), 143 had cytogenetic abnormalities (CA) without MK (MK-, n=73) or with MK (MK+, n=70). This last group was further subdivided into pts with 1, 2 or at least 3 monosomal autosomes (MK1, MK2, MK3+, n=17, 22, 31, resp.). Endpoints were overall survival (OS), progression-free survival (PFS) and response rate (RR, complete and partial remissions, hematologic improvement). Analysis was based on the intent-to-treat principle. Results: As recently published (6), in the overall 233 pts, PFS was significantly improved in the DAC arm as compared to BSC (median 0.55 vs 0.25 years, hazard ratio [HR] 0.68, p=0.004), but not OS (HR 0.88), probably due to post-progression treatments, suboptimal DAC schedule and treatment duration as possible confounders. In the 206 pts with informative cytogenetics, significant improvement in outcome with DAC vs. BSC was also seen for PFS (p=0.022, HR 0.72, Table 1) but not OS (HR 0.93). The improvement of PFS with DAC vs BSC was quite pronounced in the 63 pts with CN (HR 0.55, p=0.03), but less impressive and not significant in the 143 pts with CA (HR 0.76, p=0.11). When subdividing the latter by MK categories, pts with either 2 (MK2) or more than 2 (MK3+) monosomal autosomes also had significantly prolonged PFS with DAC, reflected in RR of 67% in MK2 and 33% in MK3+ DAC-treated pts (Table 1). This effect was not obvious in the MK1 subgroup, where the PFS outcome was favorable in both treatment groups, and in the MK− subgroups, either without (“MK−/CK−”) or with complex karyotype (“MK−/CK+”), the latter having a very poor outcome in both treatment groups. With caution (considering the limited size of some subgroups), these results support our observation that AML pts with a complex karyotype harboring 2+ monosomies can gain benefit from this hypomethylating agent. Conclusions: This first randomized trial addressing the predictive value of the MK genotype in the presence of 2+ monosomies embedded in a complex karyotype demonstrates a very rapid deterioration of MDS pts receiving BSC (within Disclosures: No relevant conflicts of interest to declare.

Abstract 2610 Introduction: Remission induction treatment in previously untreated intermediate/bad risk AML or high risk MDS needs continuous improvement. Clofarabine is a nucleoside analog with proven activity in acute leukemias. The aim of the study was to determine the recommended/maximum tolerated dose (MTD) of Clofarabine when administered with standard remission induction regimen (Ara-C and Idarubicin) in patients suffering from AML/MDS. Methods: This open label, 2-arm, multi-center, phase I trial included adult patients (pts) with previously untreated intermediate/bad risk AML or high risk MDS. All FAB subtypes except M3 and the cytogenetic groups, t(8;21) or inv(16) with WBC 0.5 × 109/l and platelets >50 × 109/l) later than 8 weeks after start of remission induction. If 1 out of the first 3 pts experienced a DLT, 3 additional pts were added (a maximum of 6 pts per dose level) in order to ensure the safety profile of this dose level. If a second patient experienced a DLT, no further pts were entered at that particular dose level. Dose escalation stopped and additional pts included at the previous lower dose to have a minimum of 6 pts treated at the recommended dose. Results: 25 pts have been entered into this phase I part in 3 participating sites. WHO PS status was 0 (N=16), 1 (N=8) or 2 (N=1). Age range was 25–60 years (median 55 years). 20 pts had de novo AML, 3 pts had secondary AML, 2 pts had de novo MDS (WHO RAEB-2). 7 pts had high cytogenetic risk features. In arm A and in arm B 10 mg/m2/d Clofarabine, and in arm A 15 mg/m2/d Clofarabine 6 pts each have been included. In arm B 15 mg/m2/d Clofarabine 7 pts were included, as one patient was substituted since she received 60% of total dose of Clofarabine and refused further treatment with Clofarabine due to related adverse event. All 25 pts were evaluable for DLT analysis. Overall, 5 pts with DLTs were observed: 1 in Arm A 10mg/m2/d Clofarabine, 3 in Arm A 15 mg/m2/d Clofarabine, 1 in Arm B 15 mg/m2/d Clofarabine. One patient with prolonged hematological toxicity as DLT reported in Arm A 10mg/m2/d Clofarabine. Two deaths in cytopenia/aplasia and one toxic death following sepsis/multiorgan failure have been reported as DLTs in 3 patients in both Arm A and B 15 mg/m2/d Clofarabine. The 5th patient had grade 4 anorexia, diarrhea, infection, prolonged hypoplasia and grade 3 confusion which classified as DLT in Arm A 15 mg/m2/d Clofarabine. In addition, three pts in the 15 mg/m2/d Clofarabine arms needed to be withdrawn from Clofarabine due to adverse events not classified as DLT. Main toxicity was bone marrow toxicity resulting in prolonged aplasia. Out of 25 pts, complete remission (CR) following induction 1/2 has been achieved in 19 pts: in 10/12 pts using 10 mg/m2/d Clofarabine (5 pts each in Arm A and Arm B) and in 9/13 pts using 15 mg/m2/d Clofarabine (4 pts in Arm A and 5 pts in Arm B). In addition CR with incomplete blood count recovery (CRi) following induction 1/2 was observed in 2 pts: 1 in Arm A 10 mg/m2/d Clofarabine and 1 in Arm B 15 mg/m2/d Clofarabine. Conclusion: Infusion of 15 mg/m2/d Clofarabine resulted in a high rate of DLTs (4/13 pts) and early treatment withdrawals (3/13 pts). The infusion of 10 mg/m2/d Clofarabine had an acceptable rate of DLTs (1/12 pts), no early treatment withdrawals occurred, and a high CR/CRi rate (11/12 pts) was observed. Therefore the MTD of Clofarabine in combination with Ara-C and Idarubicin is defined at 10 mg/m2/d and will be the recommended dose for the phase II part of this study. Disclosures: Willemze: Genzyme: member advisory committee clofarabine. Muus:Amgen: Membership on an entity's Board of Directors or advisory committees. de Witte:Novartis: Consultancy, Honoraria, Speakers Bureau.

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.

Abstract 3124 Poster Board III-61 Aim of the study Tissue factor is normally absent from monocytes in circulating blood. It can be induced by inflammatory mediators leading to the formation of monocyte-associated prothrombinase activity, which participates to thrombin generation. This activity appears to be important in both thrombosis (venous and arterial) and in chronic inflammation by inducing the release of inflammatory cytokines. This could be attributed to the activation of PAR-1 and PAR-2 (protease-activated receptors) by factor Xa or by thrombin. In this study, we compared the action of Rivaroxaban and Fondaparinux, two specific factor Xa inhibitors, on the activation of coagulation and on the secretion of inflammatory cytokines in both activated monocytes and activated human monocyte/macrophage cell line THP1. Methods 1-Monocytes were isolated from healthy volunteers and THP-1 cells were used as macrophages. 2- Activation of cells was performed by adding 1 μg/ml LPS for 2 hours at 37°C, in the presence of defibrinated human plasma which provides plasma coagulation factors, in the absence (control) or presence of Fondaparinux (500, 1000 and 1500 ng/ml) or Rivaroxaban (150, 250, 350 ng/ml, final concentrations). After incubation, the cells were isolated and their supernatants collected. 3- The procoagulant activity of cells was tested by measuring their effect on the clotting time of normal plasma in presence of calcium. 4- The release of cytokines was tested by antibody-cytokines array in the supernatants (RayBio®). 5-To test the neutralization of factor Xa bound to monocytes, activated monocytes were incubated with human purified factor Xa (100 ng/ml) for 10 min at room temperature. After isolation of the cells, 250 ng/ml Rivaroxaban or the appropriate solvent was added for 1, 10 and 30 min. The Xa activity associated to monocytes was measured by its amidolytic activity. Results 1- The procoagulant activity of monocytes and macrophages was reduced by Rivaroxaban and not by Fondaparinux. Results of prothrombinase activity in the presence of Rivaroxaban at 150, 250, 350 ng/ml, expressed as percentage of the control value was: 30 ± 3, 16 ± 4 and 12 ± 2 % for monocytes and: 43 ± 2, 24 ±3, 15 ± 1 % for THP-1 cells. In contrast, Fondaparinux did not modify the prothrombinase activity of monocytes (105 ± 12%). It was also shown that in the conditions used, Rivaroxaban (250 ng/ml) inhibited completely the amidolytic activity of factor Xa bound to activated monocytes. 2- Both Rivaroxaban and Fondaparinux modify the profile of chemokines secretion by activated monocytes and THP-1. In monocytes and THP-1 cells, LPS induced an important increase in Il-8 and angiogenin and a moderate increase in MIP-1d (Macrophage Inflammatory Protein-1) and RANTES, a member of the Il-8 superfamily. Both Rivaroxaban and Fondaparinux decreased the secretion of these chemokines to the basal level of secretion by non activated monocytes or THP-1. The secretion of leptin was only induced by LPS- treated THP-1 and was strongly decreased by both FXa inhibitors. In contrast, secretion of EGF (epithelial growth factor) was only induced by activated monocytes and strongly decreased by Rivaroxaban and moderately by Fondaparinux. Discussion and conclusion The results show that 1- Rivaroxaban induced a concentration-dependent inhibition of the procoagulant activity of activated monocytes and macrophages, whereas fondaparinux was devoid of this effect. This difference is attributed to a better access of Rivaroxaban to FXa bound to monocytes, as compared to the Fondaparinux-antithrombin complex. This inhibitory effect of Rivaroxaban could contribute to its antithrombotic activity. 2- Rivaroxaban inhibited the secretion of inflammatory chemokines by activated monocytes and THP-1. This decreased secretion was also observed with Fondaparinux, suggesting that it could be due to the decrease in thrombin generation in plasma, affecting the PAR-1 cell signaling system. As it was reported that that elevated IL-8 is associated with recurrent venous thrombosis and that these inflammatory cytokines are involved in plaque progression and rupture by recruitment of subpopulations of leukocytes and by potent angiogenic activity, this decrease in cytokines could also contribute to the antithrombotic efficacy of Rivaroxaban. This study received a support from Bayer Schering Pharma, France. Equal contribution of Marc Laurent and Rémi Varin Disclosures No relevant conflicts of interest to declare.

Abstract 791 The AML-12 randomized phase III trial of the EORTC and GIMEMA Leukemia Groups assessed the efficacy and toxicity of HD-AraC (3 g/m2/12 hrs for 4 days) combined with daunorubicin (50 mg/sqm for 3 days) and etoposide (50 mg/sqm for 5 days) vs SD-AraC (100 mg/sqm for 10 days) combined with the same drugs. Patients (pts) in complete remission (CR) received consolidation consisting of AraC (500 mg/sqm/12 hrs for 6 days) and daunorubicin (50 mg/sqm for 3 days). Subsequently an allogeneic (allo-SCT) or autologous stem cell transplantation (auto-SCT) was planned according to donor availability, cytogenetics and age. A 2nd randomization was performed after consolidation in pts without a donor: auto-SCT followed or not by low dose IL-2 (4-8 × 106 IU s.c. for 5 days per month) during one year. A total of 577 patients were required to be randomized for the 2nd question in order to reach 255 events (relapses or deaths) which would allow to detect a 11.5% increase in the 3-year disease-free survival (DFS) from 50% to 61.5% corresponding to hazard ratio (HR) = 0.70 (2-sided alpha=5%, statistical power=80%). Randomization was performed centrally; the 2nd randomization was stratified for induction treatment, cytogenetic/molecular genetic group, number of courses to reach CR, auto-SCT planned (No/Yes) and center. Intent-to-treat analysis was done. From 9/1999 till 1/2008, 2005 previously untreated AML pts (APL excluded), age Disclosures: Muus: Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract 4687 Inhibitors of differentiation (Id) or DNA binding helix-loop-helix (HLH) proteins are a group of dominant inhibitors of basic HLH transcriptional factors, which act as positive regulators of cell growth or promote excessive proliferation, and also protect cells against drug-induced apoptosis in mammalians. Recently, Id1 has been identified as a common downstream target of several constitutively activated oncogenic tyrosine kinase(TK), such as FLT3/ITD, in leukemia cells. We analyzed Id1 expression as possible prognostic factor in 237 AML patients. High Id1 expression was associated with older age(p=0.009); and with Ftl3/ITD(p=0.003). However, 61% of the patients in the group of FLT3- AML were Id1+, suggesting that other TK are involved. In whole population, high Id1 expression independently predicted shorter disease free survival (DFS)(p=0.05), and overall survival (OS)(p=0.003). In the subgroup of young patients (age'60 years) with normal cytogenetic, Id1+ was, in multivariate analysis, associated with lower CR rates(p=0.02), shorter DFS(p=0.02) and OS(p=0.006). In conclusion our data provides a new molecular marker for refining the risk classification of AML especially in young patients with normal cytogenetic. Id1- patients with normal cytogenetic should be classified as favourable-risk leukemia. Id1, as a downstream target of constitutively activated TK, could be a suitable candidate for targeted therapy. Disclosures: No relevant conflicts of interest to declare.

Abstract 1064 Poster Board I-86 Introduction: Decreased fibrinolysis has been reported in venous thrombosis. Thrombus degradation depends on its structure: thicker fibrin fibers are permeable to blood flow and highly susceptible to fibrinolytic enzymes, while thinner fibers are poorly permeable to flow and are resistant to fibrinolysis. Thrombin concentration present at the time of gelation profoundly influences fibrin clot structure: decrease in thrombin generation leads to the formation of thick fibrin fibers and to a decrease in activation of thrombin-activated fibrinolysis inhibitor (TAFI). Rivaroxaban, an oral direct factor Xa inhibitor, is in late stage clinical development for the prevention and treatment of venous and arterial thrombosis. The objective of this study was to evaluate the effect of Rivaroxaban on whole blood (WB) clot structure and degradability by t-PA. Compared to plasma clots, WB clots might better represent the in vivo formed thrombi. Methods: 1- Clots were formed by adding to WB or to corresponding plasma, low concentration of tissue factor and calcium in the presence or absence of Rivaroxaban at therapeutic concentrations (0.15 and 0.25 μg/ml). 2- Clot permeability was calculated by measuring the flow rate of liquid through the clot. It was expressed as Darcy constant. 3- Clot degradability was evaluated by D dimers generation during clot perfusion with plasminogen and tissue-type plasminogen activator (t-PA). Results: 1- In the absence of Rivaroxaban, WB clots had a lower porosity than that of corresponding plasma clots: Darcy constant of WB clots was 3.1 –fold lower than that of plasma clots. This decreased porosity of WB clots leads to thrombolysis resistance by preventing access of fibrinolytic enzymes to fibrin network: D dimers generation in t-PA-perfused clots for 60 min was 38 -fold lower in WB clots compared to plasma clots. 2- Rivaroxaban increased the permeation rate of WB clots and thrombolysis by t-PA: the addition of Rivaroxaban at 0.15 μg/ml in WB (corresponding in fact to plasma concentration of 0.25 μg/ml), increased the Darcy constant by 5.5 –fold and the clot degradability in 60 min by 108 -fold. These effects of Rivaroxaban were higher in WB clots than in corresponding plasma clots, as Rivaroxaban at 0.25 μg/ml in plasma clots increased the Darcy constant by 2.5-fold and clot degradation by 9.6-fold. In the presence of Rivaroxaban, the Darcy constant and the degradability of WB clots and of plasma clots were nearly identical. 3- To explain the greater efficacy of Rivaroxaban on WB permeation constants and thrombolysis in comparison to plasma clots a) we tested the possibility for Rivaroxaban to reduce the entrapment of red blood cells (RBC) into the network of fibrin as RBC can be responsible for fibrin pore occlusion. This possibility was excluded since Rivaroxaban had no effect on clot permeation rate in clots formed by clotting purified fibrinogen with thrombin in the presence or in the absence of RBC (condition in which there is no generation of thrombin): RBC induced a 2.5 times decrease in permeation rate due to entrapment of RBC into fibrin network, regardless of presence or absence of Rivaroxaban. b) we analyzed the effect of RBC on thrombin generation and its modification by Rivaroxaban: the addition of 0.1 ml RBC diluted ½ to 0,2 ml plasma increased the thrombin generation (540 % of control without RBC). This is probably due to exposure of phosphatidyl serine at surface of RBC during thrombin generation. The increase in thrombin generation by RBC was reduced to 140 % in presence of Rivaroxaban at 0.15 μg/ml. This is explained by Rivaroxaban's inhibition of factor Xa bound to cells. Conclusion: Thrombin generation was greater in WB than in plasma, leading to a lower porosity and degradability of WB clots as compared to plasma clots. Rivaroxaban, by decreasing thrombin generation, increased clot permeability and degradability to the same level in WB clots and plasma clots. This property of Rivaroxaban may contribute to its antithrombotic effect. This study received a support from Bayer-Schering-Pharma France. Disclosures: No relevant conflicts of interest to declare.

The AML-12 randomized phase III trial of EORTC-LG and GIMEMA assessed the efficacy and toxicity of HD-AraC (3 g/m2/12 hrs for 4 days) combined with daunorubicin (50 mg/sqm for 3 days) and etoposide (50 mg/sqm for 5 days) vs SD-AraC (100 mg/sqm for 10 days) with the same drugs. Patients (pts) in complete remission (CR) received consolidation consisting of AraC (500 mg/sqm/12 hrs for 6 days) and daunorubicin. Subsequently an allogeneic (allo-SCT) or autologous stem cell transplantation (auto-SCT) was planned according to donor availability and age. A 2nd randomization was performed after CR in pts without a donor: auto-SCT followed or not by low dose IL-2. The trial was powered to detect an 8% difference in the 5-yr survival rate; secondary endpoints were response to induction, DFS, toxicity. Randomization was performed centrally; the 1st randomization was stratified for age, performance status, WBC and center. Intent-to-treat analysis was done. From 9/1999 till 1/2008, 2005 previously untreated AML pts (APL excluded), age

Treatment of high-risk leukemia with transplantation of allogenic bone marrow or stem cells from peripheral blood is limited by the scarcity of HLA-matched related donors; only 30% of patients were eligible. Unrelated cord blood transplantation (UCBT) have become emerging therapies for adult patients without matched donors, We previously demonstrated that NK cells generated after haplo-mismatched hematopoietic stem cells transplantation are blocked at an immature state characterized by specific phenotypic features and impaired functioning, having negative impact for clinical relapse. In the present work, we studied NK cells after UCBT. This study included 27 adults’ patients (median age: 42 years), with high-risk hematologic malignancies, following a non-myeloablatif regimen. Fourthy one percent (11/27) of patients experienced initial engraftment, whereas 16 subjects (59%) experienced either primary or secondary graft failure. Neutrophil engraftment occurred at a median of 20.5 days. The incidence of the relapse was 26% (7/27) and was a major cause of death in 5 subjects. The cumulative incidence of grades II, and III acute GVHD was 33.3%. Infection occurred only in 2 patients (7.4%). The survival rate was 73% at 6 months. Analysis of DNA polymorphisms in the peripheral blood revealed full (median 99.7) donor-type chimerism. Peripheral blood samples were collected during the first year following UCBT in order to realize an extensive phenotypic study of NK cells. This analysis was realized on the cytotoxic NK CD56dim cell subset, which reaches a count number at one-month post graft (M1) close to healthy volunteer. Our results shown that NK cells phenotype post-UCBT was closed to adult controls, contrasting with NK cells generated after haplo-mismatch allograft. Indeed, excepted for the receptors NKG2A and KIR2DL1/DS1, which are respectively increased (M1: p

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.

In organ transplant, alloreactivity is supposed to induce modifications on the vascular endothelium. Complications occurring in the course of allogeneic haematopoietic stem cell (HSC) transplantation, such as acute or chronic graft-versus-host disease (GVHD), have been related to endothelial cell alterations. Moreover, the incidence of GVHD is significantly different following allogeneic bone marrow, peripheral blood stem cell or umbilical cord blood transplantation (BMT, PBSCT and UCBT respectively). Considering these different incidences of acute and chronic GVHD and taking into account the organ transplantation-related vasculopathy, we aimed to further clarify the vascular impact of the three aforementioned sources of HSC. We studied 21 patients (pts), 13 male and 8 female. Mean age was 42 years old (21–64). The underlying diseases were: acute leukaemia (myeloblastic 15pts; lymphoblastic 3pts), multiple myeloma (1pt), aplastic anemia (1pt) and chronic idiopathic myelofibrosis (1pt). Seven pts underwent BMT after myeloablative conditioning regimen (cyclophosphamide and busulphan or 12Gy total body irradiation-TBI), 6 pts PBSCT following non-myeloablative preparative regimen (fludarabine and 2Gy TBI) and 8 pts UCBT after reduced intensity conditioning regimen (cyclophosphamide, fludarabine and 2Gy TBI). We assessed the kinetics of plasma levels of 4 endothelial markers: soluble thrombomodulin (sTM), vWF-Ag, FVIII and the vascular endothelial growth factor (VEGF). Measurements were performed before the conditioning regimen and on days 0, 15, 30, 60, 90. Student paired t-test was used for statistical comparison. Differences were significant if p0.05). Similarly, vWF-Ag was significantly increased on day 0 in BMT and PBSCT (217±66% and 214±93% respectively, p

Adult ALL still have poor outcome compared with childhood ALL, with an expected OS of less than 40%. Recent retrospective studies have shown that adolescents and young adults ( Methods: 28 consecutive adults Philadelphia negative ALL aged from 16 to 57 years, with a median follow-up of alive patients of 35 months, received treatment courses according to FRALLE 2000 from 2001 to 2007. After a prednisone prephase and a four-drugs induction (prednisone, daunorubicin, vincristine and 9 infusions of L-asparaginase), patients in CR received a consolidation course, two delayed intensifications with L-asparaginase separated by an interphase, and a maintenance chemotherapy during two years. Results were compared with the outcome from 20 consecutive patients treated in our institution with the historic EORTC ALL-4 adults protocol from 1998 to 2001. Results: All the clinical (age, WHO performance status, gender) and biological (WBC, phenotype, cytogenetic) parameters of patients treated in FRALLE protocol were statistically similar with those of patients treated in ALL-4 adults protocol. CR rate was achieved in 82% of patients after FRALLE induction, and 100% after a salvage therapy with high doses cytarabine. The good early response was evaluated by cortico-sensitivity and chemo-sensitivity (after 14 days of chemotherapy). All patients who achieved cortico and chemo-sensitivity were alive in persistant CR, with a better survival than other patients (p=0.008) (fig1). When we compared patients treated by FRALLE or ALL-4 protocol, the 4-years DFS and OS are 90% +/−6% vs 47%+/−12%, (p= 0,01) and 83%+/−9% vs 35%+/−16%, (p=0,05) respectively (figures 2 and 3). This better outcome is not explained by significant differences in patients characteristics nor by a better CR rate but rather by a lower relapse rate in the pediatric treatment group. This indicates a major role of the dose intensity, especially for L-asparaginase, corticosteroid, methotrexate, and purinethol. No treatment related mortality and no severe side effect, except one pulmonary embolism, were observed during treatment with supportive cares including parenteral nutrition, granular growth factor, infectious prophylaxis, and antithrombine III infusions. This pilot study shows that adults up to 57 years with Ph negative-ALL have a dramatically better outcome when there are treated with childhood ALL protocol without any major side effect. This therapeutic strategy has to be confirmed by the current prospective study performed by the EORTC/HOVON group. Figure Figure

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 Figure Figure

The treatment of elderly patients with acute myeloid leukemia (AML) is very disappointing. Studies have established that if half of the intensivity treated patients achieve complete remission (CR), the high rate of relapse within the first year jeopardize the long term survival. No clearly effective postremission therapy had been established. Therefore we retrospective analyzed 141 elderly patients (>60 yo) in first CR, to evaluate the effectiveness of postremission therapy, consolidation (3+7or 2+5) and/or maintenance (low dose AraC). All patiens received a 3+7 induction therapy. In these 141 patients DFS and OS at 4 years are respectively 14%, and 19%. According to the clinical status of the patient after induction, 20 (14%) patients did not receive therapy after induction, 30 (21%) patients received only maintenance therapy, 53 (38%) patients received only consolidation therapy, and 38 patients (27%) received both consolidation and maintenance therapy. These 4 groups were stratified according to age (< >70 yo) and WBC (< or > 30 x 109/l) (See Tables). The outcome of these patients receiving or not post induction therapy is shown in these Tables. Patients DFS at 4 years OS at 4 years Consolidation No consolidation P value consolidation No consolidation P value Outcome of patients receiving or not consolidation therapy in 4 groups stratified according to age and WBC count < 70yo and WBC< 30x109/l 19% 13% p=0.01 27% 13% p=0.0074 30x109/l 26% 0% p=0.08 38% 0% p=0.05 >70 yo and WBC < 30x109/l 0% 30% p=0.001 6% 30% p=0.01 >70 yo and WBC> 30x109/l 2% 0% NS 4% 0% NS In patients > 70 yo and WBC < 30 x 109/l patients who received consolidation have a poorer prognosis than patients who did not because the mortality in first complete remission (mortality during consolidation) was high (25% versus 0% respectively, P=0.01). Patients DFS at 4 years OS at 4 years Maintenance No Maintenance p value Maintenance No Maintenance P value Outcome of patients receiving or not maintenance therapy in 4 groups stratified according to age and WBC count 30x109/l 23% 14% p=0.05 32% 23% NS >70 yo and WBC70 yo and WBC >30x109/l 2% 0% NS 4% 0% NS In conclusion, in patients < 70 yo consolidation ± maintenance therapy (for DFS in patients with > 30 x109/l) improves the outcome of these patients (DFS and OS). In patients > 70 yo and with WBC < 30 x109/l maintenance therapy without consolidation, improves outcome. In these later patients (> 70 yo and with WBC < 30 x 109/l) consolidation therapy decrease outcome. In patients > 70yo and with WBC > 30 x 109/l, both consolidation and/or maintenance therapy does not improve outcome.

Purpose: Prospective evaluation of a PCR-ELISA method for the early diagnosis of invasive aspergillosis (IA) in hematological patients. Patients and Methods: The PCR-ELISA assay was evaluated using 1,494 sera samples from 201 hematological adult patients. Monitoring was performed twice weekly during 256 consecutive high-risk treatment periods. Results: Underlying diseases were acute leukemia 52.7%, lymphoma 20.9%, multiple myeloma 12.4%. Eighty four patients (41.8%) were stem cell transplant recipients. Fifty-five patients were diagnosed with IA. PCR-ELISA positivity (i) preceded the empirical antifungal therapy in 20/31 patients (median 10.5 days), (ii) preceded or was simultaneous to the radiological diagnosis by chest CT in 21/31 patients (median 8 days), (iii) preceded the mycological/histological diagnosis in 7/12 patients (median 70 days) and, (iv) anticipated or was simultaneous to the galactomannan (GM) antigen positivity in 50% (median 16.5 days) and in 89 % of the patients (median 33 days) using a 0.5 and a 1.5 optical density index (ODI) cut-off for the GM assay, respectively. For at least two positive samples the sensitivity, specificity, positive and negative predictive values of the PCR-ELISA assay, were 63.6%, 82.2%, 57.4% and 85.7%, respectively. When the PCR-ELISA and GM detections were combined, values of the combined test for at least two positive samples with either assay were 80%, 47.3%, 36.4%, 86.2% using the 0.5 GM ODI cutoff, and 67.3%, 82.2%, 58.7% and 87% using the 1.5 GM ODI cutoff. Conclusion: In addition to serial screening with GM antigenemia and chest CT surveillance, the PCR-ELISA assay may improve the early diagnosis of IA.

Detection of membrane markers and ultrastructural peroxidase activity was carried out on the blasts of 16 apparently nonmyeloid adult acute leukemias. These patients were selected from 73 adult leukemic patients by the negativity of their routine cytochemical myeloid markers: i.e., myeloperoxidase, chloresterase activity, and Sudan Black B staining. B and T acute lymphoid leukemias (ALL) were excluded from the study. After concurrent testing from human T lymphocyte antigen (HuTLA), common ALL antigen (cALL), la-like antigens, and peroxidase activity at the electron microscopic level (POEM), only two patients remained undifferentiated (cALL-, POEM-). The other cases were classified as following : 6 common ALL (cALL+, POEM-), 1 pre-T-ALL (cALL+, HuTLA+, POEM-), 5 very poorly differentiated acute myelogenous leukemia (AML) (cALL-, POEM+), and 2 mixed leukemias (cALL+, POEM+). Terminal deoxynucleotidyl transferase activity (TdT) was measured in 7 cases and was found to be present at high levels in 4 cases of cALL and in the 2 cases of acute undifferentiated leukemias (AUL): it was absent in two cases of AML. Cytogenetic analysis had showed that 2 of the cALLs, 3 of the AMLs, and the 2 mixed leukemias were PH1+. We conclude that POEM detection is useful in apparently nonmyeloid leukemias with negative immunologic lymphoid markers, and that the existence of a Ph1 chromosome should be investigated, particularly in the unusual case of mixed (lymphoid-myeloid) acute leukemia.