Features, reason for testing, and changes with time of 583 paroxysmal nocturnal hemoglobinuria clones from 529 patients: a multicenter Italian study.

In this study, we aimed at disclosing the main features of paroxysmal nocturnal hemoglobinuria (PNH) clones, their association with presentation syndromes, and their changes during follow-up. A large-scale, cooperative collection (583 clones from 529 patients) of flow cytometric and clinical data was entered into a national repository. Reason for testing guidelines were provided to the 41 participating laboratories, which followed the 2010 technical recommendations for PNH testing by Borowitz. Subsequently, the 30 second-level laboratories adopted the 2012 guidelines for high-resolution PNH testing, both upon order by the local clinicians and as an independent laboratory initiative in selected cases. Type3 and Type2 PNH clones (total and partial absence of glycosyl-phosphatidyl-inositol-anchor, respectively) were simultaneously present in 54 patients. In these patients, Type3 component was sevenfold larger than Type2 (p < 0.001). Frequency distribution analysis of solitary Type3 clone size (N = 442) evidenced two discrete patterns: small (20% of peripheral neutrophils) and large (> 70%) clones. The first pattern was significantly associated with bone marrow failure and myelodysplastic syndromes, the second one with hemolysis, hemoglobinuria, and thrombosis. Pediatric patients (N = 34) showed significant preponderance of small clones and bone marrow failure. The majority of PNH clones involved neutrophils, monocytes, and erythrocytes. Nevertheless, we found clones made exclusively by white cells (N = 13) or erythrocytes (N = 3). Rare cases showed clonal white cells restricted only to monocytes (6 cases) or neutrophils (3 cases). Retesting over 1-year follow-up in 151 cases showed a marked clone size increase in 4 cases and a decrease in 13, demonstrating that early breaking-down of PNH clones is not a rare event (8.6% of cases). This collaborative nationwide study demonstrates a clear-cut difference in size between Type2 and Type3 clones, emphasizes the existence of just two classes of PNH presentations based on Type3 clone size, depicts an asymmetric cellular composition of PNH clones, and documents the possible occurrence of changes in clone size during the follow-up.

14q32/miRNA clusters loss of heterozygosity in acute lymphoblastic leukemia is associated with up-regulation of BCL11a.

This study evaluated the loss and expression level of miRNAs 14q32 clusters in acute lymphoblastic leukemia (ALL) patients with cryptic deletions at 14q32 chromosomal band to investigate their involvement in this disease. We demonstrate that a subset of ALL cases bearing 14q32 LOH showed a down-regulation of miRNA 14q32 clusters, which is directly linked to the submicroscopic chromosomal deletion. As a consequence of miRNAs deregulation we reported an inverse correlation with the expression of their target BCL11a, a transcription factor involved in lymphoid differentiation. These results suggest that 14q32/miRNA clusters LOH may be another mechanism involved in lymphoid B cell transformation and differentiation and therefore, could be used as a diagnostic marker and therapeutic target in subsets of ALL.

Differential expression of specific microRNA and their targets in acute myeloid leukemia.

Acute myeloid leukemia (AML) the most common acute leukemia in adults is characterized by various cytogenetic and molecular abnormalities. However, the genetic etiology of the disease is not yet fully understood. MicroRNAs (miRNA) are small noncoding RNAs which regulate the expression of target mRNAs both at transcriptional and translational level. In recent years, miRNAs have been identified as a novel mechanism in gene regulation, which show variable expression during myeloid differentiation. We studied miRNA expression of leukemic blasts of 29 cases of newly diagnosed and genetically defined AML using quantitative reverse transcription polymerase chain reaction (RT-PCR) for 365 human miRNA. We showed that miRNA expression profiling reveals distinctive miRNA signatures that correlate with cytogenetic and molecular subtypes of AML. Specific miRNAs with consolidated role on cell proliferation and differentiation such as miR-155, miR-221, let-7, miR-126 and miR-196b appear to be associated with particular subtypes. We observed a significant differentially expressed miRNA profile that characterizes two subgroups of AML with different mechanism of leukemogenesis: core binding factor (CBF) and cytogenetically normal AML with mutations in the genes of NPM1 and FLT3-ITD. We demonstrated, for the first time, the inverse correlation of expression levels between miRNA and their targets in specific AML genetic groups. We suggest that miRNA deregulation may act as complementary hit in the multisteps mechanism of leukemogenesis offering new therapeutic strategies.

Loss of heterozygosity in acute leukemia: evidence of frequent submicroscopic deletions.

Although chromosomal abnormalities are detected by conventional cytogenetic analysis (CCA) in 40-60% of patients with acute myeloid leukemia (AML), cryptic chromosomal deletions may only be detected by molecular analysis. To determine their frequency, we studied 74 cases of AML by microsatellite allelotype assay using 35 microsatellites spanning eight chromosomal regions known to be frequently involved in AML. In 42 (57%) we found DNA imbalance at the screened loci. This was detected by CCA only in 4 cases. Our data show that cryptic deletions are a common event in AML.

miR-155 regulative network in FLT3 mutated acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) represents a heterogeneous disorder with recurrent chromosomal alterations and molecular abnormalities. Among AML with normal karyotype (NK-AML) FLT3 activating mutation, internal tandem duplication (FLT3-ITD), is present in about 30% of patients, conferring unfavorable outcome. Our previous data demonstrated specific up-regulation of miR-155 in FLT3-ITD+ AML. miR-155 is known to be directly implicated in normal hematopoiesis and in some pathologies such as myeloid hyperplasia and acute lymphoblastic leukemia. METHODS AND RESULTS: To investigate about the potential influence of miR-155 de-regulation in FLT3-mutated AML we generated a transcription factors regulatory network and combined this with data from multiple sources that predict miR-155 interactions. From these analyses, we derived a sub-network, called "miR-155 module" that describes functional relationship among miR-155 and transcription factors in FLT3-mutated AML. We found that "miR-155 module" is characterized by the presence of six transcription factors as central hubs: four miR-155 regulators (JUN, RUNX1, FOSb, JUNB) and two targets of miR-155 (SPI1, CEBPB) all known to be "master" genes of myelopoiesis. We found, in FLT3-mutated AML, a significant down-regulation of miR-155 target genes CEBPB and SPI1 and up-regulation of miR-155 regulator genes JUN and RUNX1. We also showed that PKC412-related FLT3 inhibition, in MV4-11 cell line, causes down-regulation of miR-155 and increased level of mRNA and protein of miR-155 target SPI1. We showed in experiments of miR-155 mimic in K562 cell line, a high increase of miR-155 and an inverse correlation with the mRNA levels of its targets SPI1 and CEBPB. Moreover silencing of miR-155 in primary AMLs causes mRNA up-regulation of its target SPI1 and CEBPB. CONCLUSION: Our results suggest that activating mutation of FLT3 in AML can lead, through the induction of JUN, to an increased expression of miR-155, which then causes down-regulation of SPI1 and CEBPB and consequently may causes block of myeloid differentiation.

Carboxyamidotriazole inhibits cell growth of imatinib-resistant chronic myeloid leukaemia cells including T315I Bcr-Abl mutant by a redox-mediated mechanism.

Mutation of the Bcr-Abl oncoprotein is one of most frequent mechanisms by which chronic myelogenous leukemia (CML) cells become resistant to imatinib. Here, we show that treatment of cell lines harbouring wild type or mutant BCR-ABL with carboxyamidotriazole (CAI), a calcium influx and signal transduction inhibitor, inhibits cell growth, the expression of Bcr-Abl and its downstream signalling, and induces apoptosis. Moreover, we show that CAI acts by increasing intracellular ROS. Clinically significant, CAI has also inhibitory effects on T315I Bcr-Abl mutant, a mutation that causes CML cells to become insensitive to imatinib and second generation abl kinase inhibitors.