Elena Manara

CBFA2T3-GLIS2 fusion transcript is a novel common feature in pediatric, cytogenetically normal AML, not restricted to FAB M7 subtype

Pediatric cytogenetically normal acute myeloid leukemia (CN-AML) is a heterogeneous subgroup of myeloid clonal disorders that do not harbor known mutations. To investigate the mutation spectrum of pediatric CN-AML, we performed whole-transcriptome massively parallel sequencing on blasts from 7 CN-AML pediatric patients. In 3 patients we identified a recurrent cryptic inversion of chromosome 16, encoding a CBFA2T3-GLIS2 fusion transcript. In a validation cohort of 230 pediatric CN-AML samples we identified 17 new cases. Among a total of 20 patients with CBFA2T3-GLIS2 fusion transcript out of 237 investigated (8.4%), 10 patients (50%) did not belong to the French-American-British (FAB) M7 subgroup. The 5-year event-free survival for these 20 children was worse than that for the other CN-AML patients (27.4% vs 59.6%; P = .01). These data suggest that the presence of CBFA2T3-GLIS2 fusion transcript is a novel common feature of pediatric CN-AML, not restricted to the FAB M7 subtype, predicting poorer outcome.

MicroRNA-34b promoter hypermethylation induces CREB overexpression and contributes to myeloid transformation

MicroRNA-34b down-regulation in acute myeloid leukemia was previously shown to induce CREB overexpression, thereby causing leukemia proliferation in vitro and in vivo. The role of microRNA-34b and CREB in patients with myeloid malignancies has never been evaluated. We examined microRNA-34b expression and the methylation status of its promoter in cells from patients diagnosed with myeloid malignancies. We used gene expression profiling to identify signatures of myeloid transformation. We established that microRNA-34b has suppressor ability and that CREB has oncogenic potential in primary bone marrow cell cultures and in vivo. MicroRNA-34b was found to be up-regulated in pediatric patients with juvenile myelomonocytic leukemia (n=17) and myelodysplastic syndromes (n=28), but was down-regulated in acute myeloid leukemia patients at diagnosis (n=112). Our results showed that hypermethylation of the microRNA-34b promoter occurred in 66% of cases of acute myeloid leukemia explaining the low microRNA-34b levels and CREB overexpression, whereas preleukemic myelodysplastic syndromes and juvenile myelomonocytic leukemia were not associated with hypermethylation or CREB overexpression. In paired samples taken from the same patients when they had myelodysplastic syndrome and again during the subsequent acute myeloid leukemia, we confirmed microRNA-34b promoter hypermethylation at leukemia onset, with 103 CREB target genes differentially expressed between the two disease stages. This subset of CREB targets was confirmed to associate with high-risk myelodysplastic syndromes in a separate cohort of patients (n=20). Seventy-eight of these 103 CREB targets were also differentially expressed between healthy samples (n=11) and de novo acute myeloid leukemia (n=72). Further, low microRNA-34b and high CREB expression levels induced aberrant myelopoiesis through CREB-dependent pathways in vitro and in vivo. In conclusion, we suggest that microRNA-34b controls CREB expression and contributes to myeloid transformation from both healthy bone marrow and myelodysplastic syndromes. We identified a subset of CREB target genes that represents a novel transcriptional network that may control myeloid transformation.

Core-binding factor acute myeloid leukemia in pediatric patients enrolled in the AIEOP AML 2002/01 trial: screening and prognostic impact of c-KIT mutations.

Core-binding factor acute myeloid leukemia in pediatric patients enrolled in the AIEOP AML 2002/01 trial: screening and prognostic impact of c - KIT mutations

Minimal residual disease monitored after induction therapy by RQ-PCR can contribute to tailor treatment of patients with t(8;21) RUNX1-RUNX1T1 rearrangement.

Disease relapse still remains the most important cause of treatment failure in childhood acute myeloid leukemia (AML). Molecular monitoring of response to treatment by minimal residual disease (MRD) provides important information, widely used to tailor treatment in childhood acute lymphoblastic leukemia.1–3 On the contrary, prognostic relevance of MRD in pediatric AML has only been recently proposed and needs to be further investigated and confirmed.4–6 So far, the prognostic impact of the quality of response measured by flow cytometry after induction and consolidation therapy has been shown to provide independent prognostic information in pediatric AML,5 able to permit a refinement of risk stratification and to potentially improve AML patient outcome.

ICER expression inhibits leukemia phenotype and controls tumor progression

The inducible cyclic AMP (cAMP) early repressor (ICER) and cAMP response element-binding protein (CREB) are transcriptional regulators of the cAMP-mediated signaling pathway. CREB has been demonstrated to be upregulated in the majority of childhood leukemias contributing to disease progression, whereas ICER, its endogenous repressor, was found to be downregulated. Our research focus has been the function of restored ICER expression. ICER exogenously expressed in cell lines decreases CREB protein level and induces a lowered clonogenic potential in vitro. It decreases the ability of HL60 to invade the extramedullary sites and to promote bone marrow angiogenesis in nonobese diabetic–severe combined immunodeficient mice, demonstrating its potential effects on tumor progression. ICER represses the majority of 96 target genes upregulated by CREB. It binds CRE promoters and controls gene expression restoring the normal regulation of major cellular pathways. ICER is subjected to degradation through a constitutively active form of the extracellular signal-regulated protein kinase, which drives it to the proteasome. We propose that ICER is downregulated in HL60 to preserve CREB overexpression, which disrupts normal myelopoiesis and promotes blast proliferation. These findings define the function of ICER as a tumor suppressor in leukemia. Unbalanced CREB/ICER expression needs to be considered a pathogenetic feature in leukemogenesis. The molecular characterization of this pathway could be useful for novel therapeutic strategies.

MLL-AF6 fusion oncogene sequesters AF6 into the nucleus to trigger RAS activation in myeloid leukemia

A rare location, t(6;11)(q27;q23) (MLL-AF6), is associated with poor outcome in childhood acute myeloid leukemia (AML). The described mechanism by which MLL-AF6, through constitutive self-association and in cooperation with DOT-1L, activates aberrant gene expression does not explain the biological differences existing between t(6;11)-rearranged and other MLL-positive patients nor their different clinical outcome. Here, we show that AF6 is expressed in the cytoplasm of healthy bone marrow cells and controls rat sarcoma viral oncogene (RAS)-guanosine triphosphate (GTP) levels. By contrast, in MLL-AF6-rearranged cells, AF6 is found localized in the nucleus, leading to aberrant activation of RAS and of its downstream targets. Silencing MLL-AF6, we restored AF6 localization in the cytoplasm, thus mediating significant reduction of RAS-GTP levels and of cell clonogenic potential. The rescue of RAS-GTP levels after MLL-AF6 and AF6 co-silencing confirmed that MLL-AF6 oncoprotein potentiates the activity of the RAS pathway through retention of AF6 within the nucleus. Exposure of MLL-AF6-rearranged AML blasts to tipifarnib, a RAS inhibitor, leads to cell autophagy and apoptosis, thus supporting RAS targeting as a novel potential therapeutic strategy in patients carrying t(6;11). Altogether, these data point to a novel role of the MLL-AF6 chimera and show that its gene partner, AF6, is crucial in AML development.

Presence of high-ERG expression is an independent unfavorable prognostic marker in MLL-rearranged childhood myeloid leukemia.

To the editor: Childhood acute myeloid leukemia (AML) is a heterogeneous disease, in terms of genetic/molecular abnormalities resulting into marked differences in outcome.[1][1] A myriad of proteins have been suggested aberrantly regulated in AML, and Ets-related gene ( ERG , 21q22) expression in

Whole transcriptome sequencing of a paediatric case of de novo acute myeloid leukaemia with del(5q) reveals RUNX1-USP42 and PRDM16-SKI fusion transcripts.

Fusion genes are frequently detected in childhood acute myeloid leukaemia (AML) and other haematopoietic cancers. Recent decades have witnessed the identification, through chromosomal analysis techniques or reverse transcription polymerase chain reaction (RT-PCR), of several fusion genes influencing either proliferation/apoptosis or differentiation ability of AML cells (Gianfelici et al, 2012). The advent of next generation sequencing (NGS) has dramatically improved our ability to investigate the mutational profile of cancer cells, and several studies used NGS to identify novel mutations and chromosomal alterations in haematological neoplasms (Masetti et al, 2013). Along this line of research, we performed whole-transcriptome sequencing (WTS) of blast cells from a paediatric AML patient with del(5q) to elucidate the mutational profile of this rare type of childhood leukaemia associated with resistance to chemotherapy and grim survival (Van den Berghe et al, 1985). An 8-year-old child was diagnosed with AML FrenchAmerican-British (FAB) type M5b. Clinical presentation was characterized by hyperleucocytosis (96 18 x 10/l), anaemia (7 6 g/l) and thrombocytopenia (105 x 10/l). Bone marrow (BM) smears showed 60% blasts; conventional cytogenetics on BM cells revealed an abnormal karyotype, 46, XY, del(5) (q14q34), in 20 metaphases. No recurrent genetic abnormality involving KMT2A (also termed MLL), CBFB, NPM1 and FLT3 genes was found. The patient was enrolled in the Associazione Italiana di Ematologia e Oncologia Pediatrica (AIEOP) AML 2002/01 Protocol. After first induction course, BM evaluation on day +21 documented induction failure (88% blasts). Two cycles of salvage treatment allowed the patient to achieve complete remission. Thereafter, he received allogeneic haematopoietic transplantation from a human leucocyte antigen (HLA)-identical unrelated donor. 5 years after the allograft, the patient is alive and disease-free. The massively parallel sequencing of the transcriptome allowed us to map 50 3 million reads at a mean depth of 20X. Giving value to only concordant results emerging from the use of three distinct algorithms, namely Chimerascan, deFuse and FusionMap, two fusion transcripts, RUNX1USP42 and PRDM16-SKI, were identified in the patient. Perusal of the reads mapping these chimeric transcripts showed that the first was an in-frame fusion, while PRDM16SKI was a novel out-of-frame fusion (Fig 1A). RT-PCR analysis and Sanger sequencing confirmed the presence of both chimeric transcripts (Fig 1B). To date, the cryptic t (7;21) leading to the fusion between RUNX1 and USP42 has been reported only once in children (Paulsson et al, 2006), while seven adult cases of myeloid neoplasms harbouring the RUNX1-USP42 transcript have been published (Table I), suggesting that this genetic abnormality is a rare, but non-random, feature of myeloid malignancies in adults. To assess the recurrence of RUNX1-USP42 fusion transcript in childhood AML, we examined 132 children with cytogenetically normal (CN) de novo AML enrolled in the AIEOP AML 2002/01 Protocol. No additional patient was found to carry this chimeric transcript. To the best of our knowledge, this is the second paediatric case in which the RUNX1-USP42 fusion transcript has been detected and, unprecedentedly, it was observed at diagnosis together with del(5q), whereas in the case described by Paulsson et al (2006) the 5qoccurred later in the disease course. In our case, the fusion occurred between exon 6 of RUNX1 and exon 3 of USP42 (Fig 1A-B). However, as previously reported, owing to different breakpoints and alternative splicing in RUNX1, several splice-variants of RUNX1-USP42 chimeric transcript may be generated by the cryptic t(7;21) (Foster et al, 2010) (Table I). This notwithstanding, all RUNX1-USP42 isoforms harbour both the catalytic domain (UCH) of USP42 and the highly conserved Runt homology domain (RHD) of RUNX1, which mediates DNA binding and heterodimerization of RUNX1 with CBFb. Several mechanisms through which this chimeric transcript could contribute to the leukaemogenesis have been proposed: (i) dominant negative inhibition of wild-type RUNX1 transcription activation activity (Paulsson et al, 2006; Panagopoulos et al, 2013), (ii) USP42-mediated stabilization of RUNX1 from ubiquitin-proteasome degradation (Paulsson et al, 2006), and iii) ectopic over-expression of USP42 and deregulation of TP53-dependent cell-cycle arrest (Gigu ere & H ebert, 2011). In accordance with the last hypothesis, through RTqPCR analysis, we demonstrated an over-expression of USP42 in t(7;21)-positive cells when compared with other CN-AML cells lacking this translocation, and CD34+ haematopoietic progenitors (P = 0 02) (Fig 1C). This finding was further validated through gene expression analysis performed on the WTS data (Fig 1D). Taken together, these correspondence

Screening of novel genetic aberrations in pediatric acute myeloid leukemia: a report from the AIEOP AML-2002 study group

To the editor: Acute myeloid leukemia (AML) is a heterogeneous disease with known specific recurrent genetic aberrations. The continuous and increasing identification of new genetic lesions has permitted the identification of new subgroups of patients with different prognosis.[1][1] In the present

Characterization of children with FLT3-ITD acute myeloid leukemia: a report from the AIEOP AML-2002 study group.

Recurrent molecular markers have been routinely used in acute myeloid leukemia (AML) for risk assessment at diagnosis, whereas their post-induction monitoring still represents a debated issue. We evaluated the prognostic value and biological impact of minimal residual disease (MRD) and of the allelic ratio (AR) of FLT3-internal-tandem duplication (ITD) in childhood AML. We retrospectively screened 494 children with de novo AML for FLT3-ITD mutation, identifying 54 harboring the mutation; 51% of them presented high ITD-AR at diagnosis and had worse event-free survival (EFS, 19.2 versus 63.5% for low ITD-AR, <0.05). Forty-one percent of children with high levels of MRD after the 1st induction course, measured by a patient-specific real-time-PCR, had worse EFS (22.2 versus 59.4% in low-MRD patients, P<0.05). Next, we correlated these parameters with gene expression, showing that patients with high ITD-AR or persistent MRD had characteristic expression profiles with deregulated genes involved in methylation and acetylation. Moreover, patients with high CyclinA1 expression presented an unfavorable EFS (20.3 versus 51.2% in low CyclinA1 group, P<0.01). Our results suggest that ITD-AR levels and molecular MRD should be considered in planning clinical management of FLT3-ITD patients. Different transcriptional activation of epigenetic and oncogenic profiles may explain variability in outcome among these patients, for whom novel therapeutic approaches are desirable.