Mariadomenica Divona

Retinoic acid and arsenic trioxide for acute promyelocytic leukemia.

BACKGROUNDnAll-trans retinoic acid (ATRA) with chemotherapy is the standard of care for acute promyelocytic leukemia (APL), resulting in cure rates exceeding 80%. Pilot studies of treatment with arsenic trioxide with or without ATRA have shown high efficacy and reduced hematologic toxicity.nnnMETHODSnWe conducted a phase 3, multicenter trial comparing ATRA plus chemotherapy with ATRA plus arsenic trioxide in patients with APL classified as low-to-intermediate risk (white-cell count, ≤10×10(9) per liter). Patients were randomly assigned to receive either ATRA plus arsenic trioxide for induction and consolidation therapy or standard ATRA-idarubicin induction therapy followed by three cycles of consolidation therapy with ATRA plus chemotherapy and maintenance therapy with low-dose chemotherapy and ATRA. The study was designed as a noninferiority trial to show that the difference between the rates of event-free survival at 2 years in the two groups was not greater than 5%.nnnRESULTSnComplete remission was achieved in all 77 patients in the ATRA-arsenic trioxide group who could be evaluated (100%) and in 75 of 79 patients in the ATRA-chemotherapy group (95%) (P=0.12). The median follow-up was 34.4 months. Two-year event-free survival rates were 97% in the ATRA-arsenic trioxide group and 86% in the ATRA-chemotherapy group (95% confidence interval for the difference, 2 to 22 percentage points; P<0.001 for noninferiority and P=0.02 for superiority of ATRA-arsenic trioxide). Overall survival was also better with ATRA-arsenic trioxide (P=0.02). As compared with ATRA-chemotherapy, ATRA-arsenic trioxide was associated with less hematologic toxicity and fewer infections but with more hepatic toxicity.nnnCONCLUSIONSnATRA plus arsenic trioxide is at least not inferior and may be superior to ATRA plus chemotherapy in the treatment of patients with low-to-intermediate-risk APL. (Funded by Associazione Italiana contro le Leucemie and others; ClinicalTrials.gov number, NCT00482833.).

Alterations of the FLT3 gene in acute promyelocytic leukemia: association with diagnostic characteristics and analysis of clinical outcome in patients treated with the Italian AIDA protocol.

Alterations in the FLT3 gene, including internal tandem duplications (ITDs) and D835 mutations occur frequently in acute myelogenous leukemia. We investigated the prevalence and clinico-biological correlations of FLT3 ITDs and D835 mutations in 90 patients with acute promyelocytic leukemia (APL) receiving the AIDA protocol. Twenty patients in which both presentation and relapse material was available were analyzed sequentially. Thirty-three patients (37%) harbored the ITD, and seven (7.7%) the D835 mutation in blasts obtained at diagnosis. Presence of ITDs was strongly associated with high WBC count (P = 0.0001), M3 variant (P = 0.0004), and the short (BCR3) PML/RARα isoform (P = 0.003). There was no difference in response to induction in the two ITD+ve and ITD−ve groups, while a trend towards inferior outcome was observed for ITD+ve cases when analyzing disease-free survival (DFS) and relapse risk (RR). These differences, however, did not reach statistical significance. Sequential studies showed variable patterns in diagnostic and relapse material, ie ITD (−ve/−ve, +ve/+ve, +ve/−ve, −ve/+ve) and D835 (−ve/−ve, +ve/−ve, −ve/+ve). Our results indicate that FLT3 alterations are associated in APL with more aggressive clinical features and suggest that these lesions may not play a major role in leukemia progression.

Simultaneous detection of NPM1 and FLT3-ITD mutations by capillary electrophoresis in acute myeloid leukemia

Mutations in the Nucleophosmin (NPM1) gene have been recently described to occur in about one-third of acute myeloid leukemias (AML) and represent the most frequent genetic alteration currently known in this subset. These mutations generate an elongated NPM1 protein that localizes aberrantly in the cytoplasm. In analogy with Flt3 alterations, NPM1 mutations are mostly detectable in AML with normal karyotype and their recognition may be relevant to identify distinct response to treatment. Hence, in addition to conventional karyotyping and RT-PCR of fusion genes, combined analysis of both Flt3 and NPM1 mutations will be increasingly relevant in the genetic diagnosis work-up of AML. We developed a multiplex RT-PCR assay followed by capillary electrophoresis to simultaneously analyze NPM1 and Flt3 gene alterations (NFmPCR assay). The assay was validated in leukemic cell RNAs extracted from 38 AML patients, which had been previously characterized for Flt3 status by conventional RT-PCR. Direct sequencing of NPM1 RT-PCR products was carried out in 15 cases to verify results obtained by capillary electrophoresis. Both NPM1 sequencing and conventional RT-PCR Flt3 results showed 100% concordance with the results of the NFmPCR assay. We suggest that this assay may be introduced in routine analysis of genetic alterations in AML.

Improved Outcomes With Retinoic Acid and Arsenic Trioxide Compared With Retinoic Acid and Chemotherapy in Non–High-Risk Acute Promyelocytic Leukemia: Final Results of the Randomized Italian-German APL0406 Trial

Purpose The initial results of the APL0406 trial showed that the combination of all- trans-retinoic acid (ATRA) and arsenic trioxide (ATO) is at least not inferior to standard ATRA and chemotherapy (CHT) in first-line therapy of low- or intermediate-risk acute promyelocytic leukemia (APL). We herein report the final analysis on the complete series of patients enrolled onto this trial. Patients and Methods The APL0406 study was a prospective, randomized, multicenter, open-label, phase III noninferiority trial. Eligible patients were adults between 18 and 71 years of age with newly diagnosed, low- or intermediate-risk APL (WBC at diagnosis ≤ 10 × 109/L). Overall, 276 patients were randomly assigned to receive ATRA-ATO or ATRA-CHT between October 2007 and January 2013. Results Of 263 patients evaluable for response to induction, 127 (100%) of 127 patients and 132 (97%) of 136 patients achieved complete remission (CR) in the ATRA-ATO and ATRA-CHT arms, respectively ( P = .12). After a median follow-up of 40.6 months, the event-free survival, cumulative incidence of relapse, and overall survival at 50 months for patients in the ATRA-ATO versus ATRA-CHT arms were 97.3% v 80%, 1.9% v 13.9%, and 99.2% v 92.6%, respectively ( P < .001, P = .0013, and P = .0073, respectively). Postinduction events included two relapses and one death in CR in the ATRA-ATO arm and two instances of molecular resistance after third consolidation, 15 relapses, and five deaths in CR in the ATRA-CHT arm. Two patients in the ATRA-CHT arm developed a therapy-related myeloid neoplasm. Conclusion These results show that the advantages of ATRA-ATO over ATRA-CHT increase over time and that there is significantly greater and more sustained antileukemic efficacy of ATO-ATRA compared with ATRA-CHT in low- and intermediate-risk APL.

Nucleophosmin/B26 regulates PTEN through interaction with HAUSP in acute myeloid leukemia.

PTEN (phosphatase and tensin homolog deleted in chromosome 10) is a bona fide dual lipid and protein phosphatase with cytoplasmic (Cy) and nuclear localization. PTEN nuclear exclusion has been associated with tumorigenesis. Nucleophosmin (NPM1) is frequently mutated in acute myeloid leukemia (AML) and displays Cy localization in mutated nucleophosmin (NPMc+) AML. Here we show that NPM1 directly interacts with herpes virus-associated ubiquitin specific protease (HAUSP), which is known as a PTEN deubiquitinating enzyme. Strikingly, PTEN is aberrantly localized in AML carrying NPMc+. Mechanistically, NPM1 in the nucleus opposes HAUSP-mediated deubiquitination and this promotes the shuttle of PTEN to the cytoplasm. In the cytoplasm, NPMc+ prevents HAUSP from deubiquitinating PTEN, causing the latter to stay in the cytoplasm where it is polyubiquitinated and degraded. Our findings delineate a new NPM1–HAUSP molecular interaction controlling PTEN deubiquitination and trafficking.

A monoclonal antibody against mutated nucleophosmin 1 for the molecular diagnosis of acute myeloid leukemias.

Mutations in the nucleophosmin 1 (NPM1) gene are the most frequent genetic aberrations of acute myeloid leukemia (AML) and define a clinically distinct subset of AML. A monoclonal antibody (T26) was raised against a 19-amino acid polypeptide containing the unique C-terminus of the type A NPM1 mutant protein. T26 recognized 10 of the 21 known NPM1 mutants, including the A, B, and D types, which cover approximately 95% of all cases, and did not cross-react with wild-type NPM1 or unrelated cellular proteins. It performed efficiently with different detection technologies, including immunofluorescence, immunohistochemistry, and flow cytometry. Within a series of consecutive de novo AML patients, 44 of 110 (40%) and 15 of 39 (38%) cases scored positive using the T26 antibody in immunofluorescence and flow cytometry assays, respectively. T26-positive cases were found to be all carrying mutations of NPM1 exclusively, as determined by molecular analysis. T26 is the first antibody that specifically recognizes a leukemia-associated mutant protein. Immunofluorescence or flow cytometry using T26 may thus become a new tool for a rapid, simple, and cost-effective molecular diagnosis of AMLs.

Argonaute 2 sustains the gene expression program driving human monocytic differentiation of acute myeloid leukemia cells

MicroRNAs are key regulators of many biological processes, including cell differentiation. These small RNAs exert their function assembled in the RNA-induced silencing complexes (RISCs), where members of Argonaute (Ago) family of proteins provide a unique platform for target recognition and gene silencing. Here, by using myeloid cell lines and primary blasts, we show that Ago2 has a key role in human monocytic cell fate determination and in LPS-induced inflammatory response of 1,25-dihydroxyvitamin D3 (D3)-treated myeloid cells. The silencing of Ago2 impairs the D3-dependent miR-17-5p/20a/106a, miR-125b and miR-155 downregulation, the accumulation of their translational targets AML1, VDR and C/EBPβ and monocytic cell differentiation. Moreover, we show that Ago2 is recruited on miR-155 host gene promoter and on the upstream region of an overlapping antisense lncRNA, determining their epigenetic silencing, and miR-155 downregulation. These findings highlight Ago2 as a new factor in myeloid cell fate determination in acute myeloid leukemia cells.

PML–RARα kinetics and impact of FLT3 –ITD mutations in newly diagnosed acute promyelocytic leukaemia treated with ATRA and ATO or ATRA and chemotherapy

The APL0406 study showed that arsenic trioxide (ATO) and all-trans retinoic acid (ATRA) are not inferior to standard ATRA and chemotherapy (CHT) in newly diagnosed, low–intermediaterisk acute promyelocytic leukaemia (APL). We analysed the kinetics of promyelocytic leukaemia–retinoic acid receptor-α (PML–RARα) transcripts by real-time quantitative PCR (RQ-PCR) in bone marrow samples from 184 patients and assessed the prognostic impact of fms-related tyrosine kinase 3–internal tandem duplication (FLT3–ITD) in 159 patients enrolled in this trial in Italy. After induction therapy, the reduction of PML–RARα transcripts was significantly greater in patients receiving ATRA-CHT as compared with those treated with ATRA–ATO (3.4 vs 2.9 logs; P=0.0182). Conversely, at the end of consolidation, a greater log reduction of PML–RARα transcripts was detected in the ATRA–ATO as compared with the ATRA–CHT group (6.3 vs 5.3 logs; P=0.0024). FLT3–ITD mutations had no significant impact on either event-free survival (EFS) or cumulative incidence of relapse in patients receiving ATRA–ATO, whereas a trend for inferior EFS was observed in FLT3–ITD-positive patients receiving ATRA-CHT. Our study shows at the molecular level that ATRA–ATO exerts at least equal and probably superior antileukaemic efficacy compared with ATRA–CHT in low–intermediaterisk APL. The data also suggest that ATRA–ATO may abrogate the negative prognostic impact of FLT3–ITD.

Identification of a potential “hotspot” DNA region in the RUNX1 gene targeted by mitoxantrone in therapy‐related acute myeloid leukemia with t(16;21) translocation

The translocation t(16;21) involving RUNX1 (AML1) and resulting in the RUNX1‐CBFA2T3 fusion is a rare but recurrent abnormality mostly found in therapy‐related acute myeloid leukemia (t‐AML) associated with agents targeting topoisomerase II (topo II). We characterized, at the genomic level, the t(16;21) translocation in a patient who developed t‐AML after treatment of multiple sclerosis with mitoxantrone (MTZ). Long template nested PCR of genomic DNA followed by direct sequencing enabled the localization of RUNX1 and CBFA2T3 (ETO2) breakpoints in introns 5 and 3, respectively. Sequencing of the cDNA with specific primers showed the presence of the expected RUNX1‐CBFA2T3 fusion transcript in leukemic cells. The RUNX1 intron 5 breakpoint was located at nucleotide position 24,785. This region contained an ATGCCCCAG nucleotide sequence showing ∼90% homology to a “hotspot” DNA region ATGCCCTAG present in intron 6 of PML previously identified in therapy‐related acute promyelocytic leukemia cases arising following treatment with MTZ. This study suggests a wider distribution in the human genome, and particularly at genes involved in chromosome translocations observed in t‐AML, of DNA regions (hotspot) targeted by specific topo II drugs.

A Leukemia-Associated CD34/CD123/CD25/CD99+ Immunophenotype Identifies FLT3-Mutated Clones in Acute Myeloid Leukemia

Purpose: We evaluated leukemia-associated immunophenotypes (LAIP) and their correlation with fms-like tyrosine kinase 3 (FLT3) and nucleophosmin (NPM1) gene mutational status in order to contribute a better identification of patients at highest risk of relapse in acute myeloid leukemia (AML). Experimental Design: Bone marrow samples from 132 patients with AML were analyzed by nine-color multiparametric flow cytometry. We confirmed the presence of the mutation in diagnostic samples and in sorted cells by conventional RT-PCR and by patient-specific RQ-PCR. Results: Within the CD34+ cell fraction, we identified a discrete population expressing high levels of the IL3 receptor α-chain (CD123) and MIC-2 (CD99) in combination with the IL2 receptor α-chain (CD25). The presence of this population positively correlated with the internal tandem duplications (ITD) mutation in the FLT3 gene (r = 0.71). Receiver operating characteristics showed that, within the CD34+ cell fraction a percentage of CD123/CD99/CD25+ cells ≥11.7% predicted FLT3–ITD mutations with a specificity and sensitivity of >90%. CD34/CD123/CD99/CD25+ clones were also detectable at presentation in 3 patients with FLT3 wild-type/NPM1+ AML who relapsed with FLT3-ITD/NPM1+ AML. Quantitative real-time PCR designed at relapse for each FLT3-ITD in these three cases confirmed the presence of low copy numbers of the mutation in diagnostic samples. Conclusions: Our results suggest that the CD34/CD25/CD123/CD99+ LAIP is strictly associated with FLT3-ITD–positive cells. Clin Cancer Res; 21(17); 3977–85. ©2015 AACR.