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The anti-CD33 antibody calicheamicinconjugate gemtuzumab ozogamicin (GO) was used to treat 16 patients with acute promyelocytic leukemia (APL) who had relapsed at the molecular level. Of these patients, 8 were experiencing a first, 5 a second, 2 a third, and 1 a fourth relapse. GO was administered at 6 mg/m 2 for 2 doses, and patients achieving a new molecular remission (MR) (ie, negativity of the reverse transcriptase-polymerase chain reaction [RT-PCR] test for PML/ RAR␣) received a third dose. MR was obtained in 9 (91%) of 11 patients tested after 2 doses and in 13 (100%) of 13 patients tested after the third dose. Of the 3 remaining patients, 1 achieved MR after one GO administration and received no further therapy owing to hepatic toxicity, and 2 showed disease progression during treatment. Quantitative RT-PCR studies showed that responding patients experienced a dramatic decline (at least 2 logs) of the PML/RAR␣ transcript after the first GO dose. Of 14 responders, 7 remained in sustained MR for a median of 15 months (range, 7-31 months) while 7 experienced relapse at 3 to 15 months. GO was administered again in 2 patients with relapse, and both obtained a new MR. These data indicate that GO is highly effective as a single treatment for patients with molecularly relapsed APL including those with very advanced

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.

This review highlights new findings that have deepened our understanding of the mechanisms of leukemogenesis, therapy and resistance in acute promyelocytic leukemia (APL). Promyelocytic leukemia-retinoic acid receptor α (PML-RARa) sets the cellular landscape of acute promyelocytic leukemia (APL) by repressing the transcription of RARa target genes and disrupting PML-NBs. The RAR receptors control the homeostasis of tissue growth, modeling and regeneration, and PML-NBs are involved in self-renewal of normal and cancer stem cells, DNA damage response, senescence and stress response. The additional somatic mutations in APL mainly involve FLT3, WT1, NRAS, KRAS, ARID1B and ARID1A genes. The treatment outcomes in patients with newly diagnosed APL improved dramatically since the advent of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). ATRA activates the transcription of blocked genes and degrades PML-RARα, while ATO degrades PML-RARa by promoting apoptosis and has a pro-oxidant effect. The resistance to ATRA and ATO may derive from the mutations in the RARa ligand binding domain (LBD) and in the PML-B2 domain of PML-RARa, but such mutations cannot explain the majority of resistances experienced in the clinic, globally accounting for 5–10% of cases. Several studies are ongoing to unravel clonal evolution and resistance, suggesting the therapeutic potential of new retinoid molecules and combinatorial treatments of ATRA or ATO with different drugs acting through alternative mechanisms of action, which may lead to synergistic effects on growth control or the induction of apoptosis in APL cells.

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 þ cell fraction, we identified a discrete population expressing high levels of the IL3 receptor a-chain (CD123) and MIC-2 (CD99) in combination with the IL2 receptor a-chain (CD25). The presence of this population positively correlated with the internal tandem duplications (ITD) mutation in the FLT3 gene (r ¼ 0.71). Receiver operating characteristics showed that, within the CD34 þ cell fraction a percentage of CD123/CD99/CD25 þ cells !11.7% predicted FLT3-ITD mutations with a specificity and sensitivity of >90%. CD34/CD123/ CD99/CD25 þ clones were also detectable at presentation in 3 patients with FLT3 wild-type/NPM1 þ AML who relapsed withQuantitative 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/CD99þ LAIP is strictly associated with FLT3-ITD-positive cells.

The use of high-dose ascorbate (ASC) for the treatment of human cancer has been attempted several decades ago and has been recently revived by several in vitro and in vivo studies in solid tumors. We tested the cytotoxic effects of ASC, alone or in combination with arsenic trioxide (ATO) in acute myeloid leukemia (AML) and acute promyelocytic leukemia (APL). Leukemic cell lines and primary blasts from AML and APL patients were treated with graded concentrations of ASC, alone or in association with standard concentration (1 μM) of ATO. The ASC/ATO combination killed myeloid blasts, including leukemic CD34+ cells, while sparing CD34+ progenitors obtained from normal cord blood and bone marrow. Actually, approximately one-third (11/36) of primary AML cases were highly sensitive to the ASC/ATO combination. The mechanism of cell killing appeared to be related to increased oxidative stress and overproduction of ROS in a non-quantitative fashion, which resulted in induction of apoptosis. These effects were reverted by the addition of the antioxidant N-Acetyl-Cysteine (NAC). In the APL NB4 model, ASC induced direct degradation of the PML and PML/RARA proteins via caspase activation, while the transcriptional repressor DAXX was recruited in re-constituted PML nuclear bodies. Our findings encourage the design of pilot studies to explore the potential clinical benefit of ASC alone or in combination with ATO in advanced AML and APL.

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.

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