Copy-number analysis identified new prognostic marker in acute myeloid leukemia.

Recent advances in genomic technologies have revolutionized acute myeloid leukemia (AML) understanding by identifying potential novel actionable genomic alterations. Consequently, current risk stratification at diagnosis not only relies on cytogenetics, but also on the inclusion of several of these abnormalities. Despite this progress, AML remains a heterogeneous and complex malignancy with variable response to current therapy. Although copy-number alterations (CNAs) are accepted prognostic markers in cancers, large-scale genomic studies aiming at identifying specific prognostic CNA-based markers in AML are still lacking. Using 367 AML, we identified four recurrent CNA on chromosomes 11 and 21 that predicted outcome even after adjusting for standard prognostic risk factors and potentially delineated two new subclasses of AML with poor prognosis. ERG amplification, the most frequent CNA, was related to cytarabine resistance, a cornerstone drug of AML therapy. These findings were further validated in The Cancer Genome Atlas data. Our results demonstrate that specific CNA are of independent prognostic relevance, and provide new molecular information into the genomic basis of AML and cytarabine response. Finally, these CNA identified two potential novel risk groups of AML, which when confirmed prospectively, may improve the clinical risk stratification and potentially the AML outcome.

Evaluation of allogeneic hematopoietic SCT in younger adults with adverse karyotype AML.

To illustrate methodological issues, we compared donor vs no-donor to transplant vs no-transplant comparisons in a cohort of 107 patients aged 50 years with adverse karyotype AML in first CR. Adverse karyotypes were defined as -7, del(7q), -5, del(5q), t(9;22), 11q23, 3q26 or complex abnormalities. Mantel-Byar estimations and hematopoietic SCT (HSCT) as a time-dependent variable were used to compare transplant vs no-transplant cumulative incidence of relapse (CIR), relapse-free survival (RFS) and OS. In all, 52 patients had a sibling donor, but only 35 of them were transplanted in first CR, whereas 9 patients received HSCT from alternative stem cell sources. Donor-based analysis showed lower CIR in the donor group, not translating in prolonged RFS or OS. Conversely, transplant-based analysis showed that HSCT in the first CR improved the three CIR (multivariate hazard ratio (HR), 0.31; P<0.001), RFS (multivariate HR, 0.57; P=0.047) and OS (multivariate HR, 0.54; P=0.03) endpoints. At 5 years, OS was estimated at 33% in transplanted vs 18% in non-transplanted patients. The positive effect of HSCT was more pronounced in patients aged 35 years and/or in those transplanted in the more recent years. These results confirm that HSCT is likely the best curative option in younger patients with adverse karyotype AML.

Prognostic significance of DNA methyltransferase 3A mutations in cytogenetically normal acute myeloid leukemia: a study by the Acute Leukemia French Association.

Recently, DNA methyltransferase 3A (DNMT3A) mutations have been identified in acute myeloid leukemia (AML), the highest frequency being found within cytogenetically normal (CN) AML. In this study, diagnostic samples from 123 adults younger than 60 years with primary CN-AML homogeneously treated in the Acute Leukemia French Association-9801 and -9802 trials were screened for mutations in DNMT3A-conserved domains by direct sequencing. Patients were also assessed for the presence of FLT3 (fms-like tyrosine kinase receptor-3), NPM1 (nucleophosmin), CEBPA, WT1 (Wilms tumor 1), IDH1 (isocitrate dehydrogenase 1) and IDH2 mutations. Thirty-eight mutations were detected in 36 patients (29%): 36 nucleotide substitutions, mostly affecting amino-acid residue R882 and two frameshift deletions. DNMT3A mutations were significantly associated with the French-American-British subtypes M4/M5 and the presence of NPM1 mutations. In the whole cohort, DNMT3A mutated patients had a shorter event-free survival (5-year EFS: 13% vs 32%, P = 0.02) and overall survival (5-year OS: 23% vs 45%, P = 0.02) compared with DNMT3A wild-type patients. In multivariate analysis including age, white blood cell count, NPM1/FLT3-internal tandem duplication/CEBPA risk group and DNMT3A mutational status, the presence of a DNMT3A mutation remained an independent adverse prognostic factor for EFS and OS, suggesting that testing for DNMT3A mutations could help further improve risk stratification in CN-AML.

False positive galactomannan Platelia due to piperacillin-tazobactam.

INTRODUCTION: Invasive aspergillosis is a serious disease, the lethality of which is important among hematology patients. Early diagnosis is crucial for treatment options and the prognosis. Detection of the antigen galactomannan is the most frequently used microbiological tools. But galactomannan detection may be falsely positive, and this false positivity has been associated with piperacillin-tazobactam treatment, the main antibiotic combination used in clinical hematology. OBJECTIVE: The purpose of our study, carried out from January 2009 to December 2010 at the Versailles hospital on in-patients with hematological disorders, was to evaluate the association between false galactomannan positivity and administration of piperacillin-tazobactam, and to study a possible variability of products issued by three manufacturers. PATIENTS AND METHOD: We noted that 207 patients were included (n=207), accounting for 69 false positive and 138 true negative results. The intrinsic galactomannan values in the study were sensitivity 100%, specificity 68%, positive and negative predictive values respectively 16%, 100%, and a likelihood positive and negative test at respectively 3.12, and 0. RESULTS: The statistical analysis did not determine any association between false positivity in galactomannan and piperacillin-tazobactam issued by two manufacturers (P=0.87 and P=0.94). But, there was a significant association between false galactomannan positivity and piperacillin-tazobactam issued by the third manufacturer (P=0.02). Four of the 25 batches issued by this manufacturer were tested and negative "in vitro" for galactomannan. DISCUSSION: This study results suggest that the association between false galactomannan positivity and piperacillin-tazobactam is not longer systematic, but can still prevail depending on the manufacturers. It also confirmed the positive contribution of testing piperacillin-tazobactam batches "in vitro" before using the antibiotic.

Effect of priming with granulocyte-macrophage colony-stimulating factor in younger adults with newly diagnosed acute myeloid leukemia: a trial by the Acute Leukemia French Association (ALFA) Group.

In a multicenter trial, 259 young adults (15-49 years) with newly diagnosed acute myeloid leukemia (AML) were first randomized to receive a timed-sequential induction regimen given either alone (135 patients) or concomitantly with granulocyte-macrophage colony-stimulating factor (GM-CSF) (124 patients). Patients reaching complete remission (CR) were then randomized to compare a timed-sequential consolidation to a postremission chemotherapy including four cycles of high-dose cytarabine followed by maintenance courses. In the appropriate arm, GM-CSF was given concurrently with chemotherapy during all cycles of consolidation. CR rates were significantly better in the GM-CSF arm (88 vs 78%, P<0.04), but did not differ after salvage. Patients receiving GM-CSF had a higher 3-year event-free survival (EFS) estimate (42 vs 34%), but GM-CSF did not impact on overall survival. Patients with intermediate-risk cytogenetics benefited more from GM-CSF therapy (P=0.05) in terms of EFS than patients with other cytogenetics. This was also confirmed when considering only patients following the second randomization, or subgroups defined by a prognostic index based on cytogenetics and the number of courses required for achieving CR. Priming of leukemic cells with hematopoietic growth factors is a means of enhancing the efficacy of chemotherapy in younger adults with AML.

High efficacy and safety profile of fractionated doses of Mylotarg as induction therapy in patients with relapsed acute myeloblastic leukemia: a prospective study of the alfa group.

Pivotal phase II studies in acute myeloblastic leukemia (AML) patients in first relapse have used gemtuzumab ozogamicin (GO) (Mylotarg) at a dose of 9 mg/m(2) on days 1 and 14. These studies showed a 26% response rate (13% complete remission (CR) and 13% CRp (complete remission with incomplete platelet recovery)) but with high degree of hematological and liver toxicities. Based on in vitro studies showing a re-expression of CD33 antigenic sites on the cell surface of blasts cells after exposure to GO, we hypothesized that fractionated doses of GO may be efficient and better tolerated. Fifty-seven patients with AML in first relapse received GO at a dose of 3 mg/m(2) on days 1, 4 and 7 for one course. Fifteen patients (26%) achieved CR and four (7%) CRp. Remission rate correlated strongly with P-glycoprotein and MRP1 activities. The median relapse-free survival was 11 months, similar for CR or CRp patients. Median duration of neutropenia < 500/microl and thrombocytopenia < 50,000/microl were, respectively, 23 and 21 days. No grade 3 or 4 liver toxicity was observed. No veno-occlusive disease occurred after GO or after hematopoietic stem cell transplantation given after GO in seven patients. Mylotarg administered in fractionated doses demonstrated an excellent efficacy/safety profile.

Imatinib and methylprednisolone alternated with chemotherapy improve the outcome of elderly patients with Philadelphia-positive acute lymphoblastic leukemia: results of the GRAALL AFR09 study.

Acute lymphoblastic leukemia (ALL) in the elderly is characterized by its ominous prognosis. On the other hand, imatinib has demonstrated remarkable, although transient, activity in relapsed and refractory Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL), which prompted us to assess the use of imatinib in previously untreated elderly patients. ALL patients aged 55 years or older were given steroids during 1 week. Ph+ve cases were then offered a chemotherapy-based induction followed by a consolidation phase with imatinib and steroids during 2 months. Patients in complete response (CR) after consolidation were given 10 maintenance blocks of alternating chemotherapy, including two additional 2-month blocks of imatinib. Thirty patients were included in this study and are compared with 21 historical controls. Out of 29 assessable patients, 21 (72%, confidence interval (CI): 53-87%) were in CR after induction chemotherapy vs 6/21 (29%, CI: 11-52%) in controls (P=0.003). Five additional CRs were obtained after salvage with imatinib and four after salvage with additional chemotherapy in the control group. Overall survival (OS) is 66% at 1 year vs 43% in the control group (P=0.005). The 1-year relapse-free survival is 58 vs 11% (P=0.0003). The use of imatinib in elderly patients with Ph+ ALL is very likely to improve outcome, including OS.

Report of 34 patients with clonal chromosomal abnormalities in Philadelphia-negative cells during imatinib treatment of Philadelphia-positive chronic myeloid leukemia.

Imatinib mesylate (Gleevec), an inhibitor of the BCR-ABL tyrosine kinase, was introduced recently into the therapy of chronic myeloid leukemia (CML). Several cases of emergence of clonal chromosomal abnormalities after therapy with imatinib have been reported, but their incidence, etiology and prognosis remain to be clarified. We report here a large series of 34 CML patients treated with imatinib who developed Philadelphia (Ph)-negative clones. Among 1001 patients with Ph-positive CML treated with imatinib, 34 (3.4%) developed clonal chromosomal abnormalities in Ph-negative cells. Three patients were treated with imatinib up-front. The most common cytogenetic abnormalities were trisomy 8 and monosomy 7 in twelve and seven patients, respectively. In 15 patients, fluorescent in situ hybridization with specific probes was performed in materials archived before the initiation of imatinib. The Ph-negative clone was related to previous therapy in three patients, and represented a minor pre-existing clone that expanded after the eradication of Ph-positive cells with imatinib in two others. However, in 11 patients, the new clonal chromosomal abnormalities were not detected and imatinib may have had a direct effect. No myelodysplasia was found in our cohort. With a median follow-up of 24 months, one patient showed CML acceleration and two relapsed.

Secondary antifungal prophylaxis with voriconazole to adhere to scheduled treatment in leukemic patients and stem cell transplant recipients.

Although the efficacy and safety of voriconazole to treat invasive fungal infections have been demonstrated in prospective trials, its use for secondary prophylaxis to prevent reactivation of these infections remains unknown. Delaying the scheduled treatment of leukemia until complete resolution of fungal infection may have major implications for prognosis. We report 11 leukemic patients with previous aspergillus (n=10) and candida (n=1) infection who received voriconazole 400 mg/day intravenously or orally for between 44 and 245 days. Nine patients were scheduled for allogeneic stem cell transplant, and two for consolidation therapy for acute leukemia. None of the patients had a relapse of fungal infection, and scheduled treatment was delayed only once. Voriconazole was well tolerated, except in one patient who had abnormal liver tests secondary to hepatic graft-versus-host disease, and one who had visual disturbances. This small but homogeneous series indicates that voriconazole may be useful to prevent fungal relapse during at-risk periods in leukemic patients. Prospective trials are warranted to confirm these encouraging results.

Long-term outcome with pentostatin treatment in hairy cell leukemia patients. A French retrospective study of 238 patients.

With the introduction of new drugs such as alpha-interferon (IFN) and purine analogs, the management of hairy cell leukemia (HCL) patients has changed. However, deoxycoformycin (DCF) produced higher complete remission rates than IFN. The current study was undertaken to provide long-term data on duration of overall survival (OS) and disease-free survival (DFS) and incidence of subsequent malignancies. We retrospectively analyzed the data of patients treated with DCF (4 mg/m2/day, every 2 weeks) from 39 French centers. In 84 of 238 included patients, DCF was the first-line therapy. Pretreatment variables influencing the achievement of complete remission, DFS, and OS were identified by multivariate analysis. Two hundred and thirty-eight patients received a median of nine cycles (range, 1-19 cycles). A complete remission was obtained in 182 of 230 evaluable patients (79%) and a partial response was obtained in 38 patients, for an overall response rate of 95.6%. In the multivariate analysis hemoglobin level less than 100 g/l and leukocytes less than 2 x 10(9)/l were parameters adversely influencing complete remission achievement. With a median follow-up of 63.5 months (range, 0.39-138.4 months), disease recurrence was observed in 34 of 220 responding patients (15%). The estimated 5-years and 10-years DFS was 88.1% and 68.8%, respectively. Hemoglobin level less than 100 g/l and leukocytes less than 2 x 10(9)/l were the pre-treatment variables associated with a shorter DFS. The estimated 5-year and 10-year OS were 89.4% and 88.7%, respectively. Hemoglobin level less than 100 g/l, leukocytes less than 2 x 10(9)/l, and adenopathy were significant factors of reduced survival. Hematologic toxicity was the main side-effect, followed by infection and emesis. During the period of follow-up, 18 patients developed second cancer, but the standardized incidence ratio was 0.95. Pentostatin is a highly effective regimen for hairy cell leukemia that produces durable complete responses. Toxicity of DCF is acceptable. Subsequent malignancies do not appear to be increased with pentostatin treatment.

Prognostic significance of FLT3 internal tandem repeat in patients with de novo acute myeloid leukemia treated with reinforced courses of chemotherapy.

FLT3 internal tandem duplications (FLT3-ITDs) are present in nearly 25% of patients with AML and have been associated with poor response to conventional therapy and poor outcome. We retrospectively evaluated the effect of reinforced courses of chemotherapy on the prognostic value of FLT3-ITDs in 159 AML patients prospectively enrolled in the ALFA-9000 trial, which randomly compared three reinforced induction regimens (standard 3+7 including high-dose daunorubicin, double induction, and timed-sequential therapy). FLT3-ITD was present in 40/159 (25%) blast samples and associated with high WBC (P = 0.002) and cytogenetics (P < 0.001) with a higher incidence (35%) in patients with a normal karyotype. There was no difference in CR rate between FLT3-wt and FLT3-ITD patients (80% vs 78%). Relapse-free survival (RFS) was similar in both groups (5-year RFS, 33% vs 32%; P = 0.41), even after adjustment for age, sex, WBC, cytogenetics, and treatment arm. A trend to a worse survival was observed in the FLT3-ITD group (estimated 5-year OS, 23% vs 37%; P = 0.09), mainly in patients with a normal karyotype. This was associated with a dramatic outcome in relapsing FLT3-ITD patients (estimated 3-year post-relapse survival, 0% vs 27%; P = 0.04). These results suggest that the bad prognosis associated with FLT3-ITDs in AML might be partly overcome using reinforced chemotherapy. Early detection of FLT3 mutations might thus be useful to intensify induction as well as post-remission therapy in FLT3-ITD patients.

BCR/ABL fusion gene detected on 9q34 by fluorescence in situ hybridization in an acute leukemia with two BCR/ABL positive clones, one Ph-negative and one Ph-positive.

We report cytogenetic, fluorescence in situ hybridization (FISH), and molecular analyses in the first reported case of an acute leukemia with two BCR-positive clones: one cell Ph-positive and all others Ph-negative. A BCR/ABL fusion gene on 9q34 was detected only with a BCR/ABL dual color translocation probe. These FISH interphase signals must be confirmed on a metaphase to avoid an erroneous interpretation. This observation appears to indicate a 2-step mechanism for this aberrant fusion gene localization: first, a classical t(9;22), and then the transfer of the fusion gene formed on chromosome 22 to chromosome 9 by a second translocation between the long arms of the derivative chromosomes 9q+ and 22q-, masking the first chromosome exchange.

Three cases of typical aplastic anaemia associated with a Philadelphia chromosome.

We report three cases of typical aplastic anaemia (AA) associated with a Philadelphia chromosome. This translocation was detected at the time of diagnosis of AA (one patient) and when overt leukaemia was diagnosed (two patients: one chronic myeloid leukaemia and one acute lymphoblastic leukaemia) after AA therapy and recovery of blood counts. We discuss the literature arguments about considering some cases of AA as preleukaemic disorders and suggest that our cases illustrate the association of AA with a clonal malignant disorder. We conclude that cytogenetic analysis is necessary at diagnosis of AA or after recovery of blood counts.

Additional chromosomal abnormalities in patients with acute promyelocytic leukaemia (APL) do not confer poor prognosis: results of APL 93 trial.

In spite of the recent improvement in the outcome of acute promyelocytic leukaemia (APL) with treatment combining all trans retinoic acid (ATRA) and chemotherapy (CT), some patients with this disease still have a poor outcome. The prognostic significance of chromosomal abnormalities in addition to t(15;17) in APL is uncertain. We examined the prognostic significance of secondary chromosomal changes in 292 patients included in a European trial who were treated with ATRA and CT. The incidence of chromosomal abnormalities in addition to t(15;17) was 26% and trisomy 8 was the most frequent secondary change (46% of the cases with secondary changes). No significant differences were seen with regard to age, sex, initial white blood cell count, % of circulating blasts, platelet count, fibrinogen level and incidence of microgranular variants between patients with or without additional rearrangements. Outcome was also similar between patients with t(15;17) alone and patients with t(15;17) and other clonal abnormalities for complete remission (92% vs. 93% respectively), event-free survival at 2 years (76.1% vs. 78.1% respectively), relapse at 2 years (16.7% vs. 11.6% respectively) and overall survival at 2 years (79.9% vs. 79.5% respectively). Analysis according to the type of induction treatment (ATRA followed by CT or ATRA plus CT) or the type of maintenance treatment (with ATRA, low-dose CT or both) also failed to show any difference between the two groups. Thus, in a large cohort of APL patients treated with ATRA and CT, additional chromosomal abnormalities had no impact on prognosis.

Oral 9-cis retinoic acid (Alitretinoin) in the treatment of myelodysplastic syndromes: results from a pilot study.

A multicenter phase II study was initiated to investigate the efficacy, toxicity and tolerability of an oral regimen of 9-cis retinoic acid (9CRA) as a differentiation-inducing agent stimulating both retinoic acid receptor (RAR) and retinoic X receptor (RXR). Thirty patients with myelodysplastic syndromes (MDS) were enrolled into the study. The MDS subtypes were distributed as follows: 14 refractory anaemia (RA), four refractory anaemia with ringed sideroblasts (RARS), and 12 refractory anaemia with excess blasts (RAEB). The age ranged from 40 to 81 years (median 70). None of these had previously received treatment for MDS other than supportive therapy. 9CRA (Alitretinoin capsules, kindly provided by Allergan-Ligand Retinoid Therapeutics) was given daily at 60 mg/m2 p.o. for 1 week, followed by an intra-patient escalation to 100 mg/m2 during the second week, up to a maximum of 140 mg/m2. The planned treatment duration was 48 weeks. Twenty-five were available for assessment. One patient (4%) with RA achieved complete hematological remission. Four (16%), two with RA, two with RAEB, had minor responses resulting in decreased transfusion requirements or increased neutrophils. Thus, the overall response rate was 20% in evaluable patients with MDS and 17% in the study group on an intention-to-treat basis. The most frequent side-effects included headache (77%), dry skin (57%), arthralgias (30%), and rash (23%). In conclusion, although modest responses were noted in this study, the treatment tolerability was suboptimal. It is conceivable that a lower dosage schedule may be efficacious and better tolerated so enabling prolonged exposure which may be required to induce a differentiation effect.