Lucília Norton

Molecular characterization of the MLL-SEPT6 fusion gene in acute myeloid leukemia: identification of novel fusion transcripts and cloning of genomic breakpoint junctions

This report describes a detailed RNA and DNA analysis in three new patients with acute myeloid leukemia carrying the MLL-SEPT6 rearrangement. One of the MLL fusion partners in leukemia is the SEPT6 gene, which belongs to the evolutionarily conserved family of genes of septins. In this work we aimed to characterize at both the RNA and DNA levels three acute myeloid leukemias with cytogenetic evidence of a rearrangement between 11q23 and Xq24. Molecular analysis led to the identification of several MLL-SEPT6 fusion transcripts in all cases, including a novel MLL-SEPT6 rearrangement (MLL exon 6 fused with SEPT6 exon 2). Genomic DNA breakpoints were found inside or near Alu or LINE repeats in the MLL breakpoint cluster region, whereas the breakpoint junctions in the SEPT6 intron 1 mapped to the vicinity of GC-rich low-complexity repeats, Alu repeats, and a topoisomerase II consensus cleavage site. These data suggest that a non-homologous end-joining repair mechanism may be involved in the generation of MLL-SEPT6 rearrangements in acute myeloid leukemia.

Expression pattern of the septin gene family in acute myeloid leukemias with and without MLL-SEPT fusion genes

Septins are proteins associated with crucial steps in cell division and cellular integrity. In humans, 14 septin genes have been identified, of which five (SEPT2, SEPT5, SEPT6, SEPT9, and SEPT11) are known to participate in reciprocal translocations with the MLL gene in myeloid neoplasias. We have recently shown a significant down-regulation of both SEPT2 and MLL in myeloid neoplasias with the MLL-SEPT2 fusion gene. In this study, we examined the expression pattern of the other 13 known septin genes in altogether 67 cases of myeloid neoplasia, including three patients with the MLL-SEPT2 fusion gene, four with MLL-SEPT6 fusion, and three patients with the MLL-SEPT9 fusion gene. When compared with normal controls, a statistically significant down-regulation was observed for the expression of both MLL (6.4-fold; p=0.008) and SEPT6 (1.7-fold; p=0.002) in MLL-SEPT6 leukemia. Significant down-regulation of MLL was also found in MLL-MLLT3 leukemias. In addition, there was a trend for SEPT9 down-regulation in MLL-SEPT9 leukemias (4.6-fold; p=0.077). Using hierarchical clustering analysis to compare acute myeloid leukemia genetic subgroups based on their similarity of septin expression changes, we found that MLL-SEPT2 and MLL-SEPT6 neoplasias cluster together apart from the remaining subgroups and that PML-RARA leukemia presents under-expression of most septin family genes.

ANAPHYLACTOID REACTION TO HIGH-DOSE METHOTREXATE AND RE-ADMINISTRATION AFTER A SUCCESSFUL DESENSITIZATION

Methotrexate (MTX) is an antimetabolite with a major role in the treatment of acute lymphoblastic leukaemia (ALL). The authors report the management of a 9-year-old boy who developed an anaphylactoid reaction to high-dose MTX infusion, after a first challenge with intrathecal administration, during induction therapy for ALL. A desensitization protocol was followed and MTX tolerance has been successfully achieved, allowing this patient to complete the needed chemotherapy.

Both SEPT2 and MLL are down-regulated in MLL-SEPT2 therapy-related myeloid neoplasia

BackgroundA relevant role of septins in leukemogenesis has been uncovered by their involvement as fusion partners in MLL-related leukemia. Recently, we have established the MLL-SEPT2 gene fusion as the molecular abnormality subjacent to the translocation t(2;11)(q37;q23) in therapy-related acute myeloid leukemia. In this work we quantified MLL and SEPT2 gene expression in 58 acute myeloid leukemia patients selected to represent the major AML genetic subgroups, as well as in all three cases of MLL-SEPT2-associated myeloid neoplasms so far described in the literature.MethodsCytogenetics, fluorescence in situ hybridization (FISH) and molecular studies (RT-PCR, qRT-PCR and qMSP) were used to characterize 58 acute myeloid leukemia patients (AML) at diagnosis selected to represent the major AML genetic subgroups: CBFB-MYH11 (n = 13), PML-RARA (n = 12); RUNX1-RUNX1T1 (n = 12), normal karyotype (n = 11), and MLL gene fusions other than MLL-SEPT2 (n = 10). We also studied all three MLL-SEPT2 myeloid neoplasia cases reported in the literature, namely two AML patients and a t-MDS patient.ResultsWhen compared with normal controls, we found a 12.8-fold reduction of wild-type SEPT2 and MLL-SEPT2 combined expression in cases with the MLL-SEPT2 gene fusion (p = 0.007), which is accompanied by a 12.4-fold down-regulation of wild-type MLL and MLL-SEPT2 combined expression (p = 0.028). The down-regulation of SEPT2 in MLL-SEPT2 myeloid neoplasias was statistically significant when compared with all other leukemia genetic subgroups (including those with other MLL gene fusions). In addition, MLL expression was also down-regulated in the group of MLL fusions other than MLL-SEPT2, when compared with the normal control group (p = 0.023)ConclusionWe found a significant down-regulation of both SEPT2 and MLL in MLL-SEPT2 myeloid neoplasias. In addition, we also found that MLL is under-expressed in AML patients with MLL fusions other than MLL-SEPT2.

Genetic diagnosis of alveolar rhabdomyosarcoma in the bone marrow of a patient without evidence of primary tumor

Alveolar rhabdomyosarcoma (ARMS) is characterized by two pathognomonic translocations, both involving the FOXO1 gene. We describe a case of a 10‐year‐old child with multiple lytic lesions involving all the vertebral bodies, sternum and femur and a bone marrow biopsy compatible with a small round cell neoplasia, but no evidence of a primary tumor. Interphase FISH analysis with specific probes evidenced a rearrangement involving the FOXO1 gene and RT‐PCR identified the PAX7‐FOXO1 fusion transcript. These data show a case of ARMS with no evidence of primary tumor presenting the PAX7‐FOXO1 fusion gene. Pediatr Blood Cancer 2008;51:554–557.

Successful treatment with daclizumab of refractory anaplastic lymphoma

Anaplastic large cell lymphoma (ALCL) is a relatively rare and highly malignant form of non‐Hodgkin lymphoma (NHL) which accounts for 10–15% of these childhood lymphomas. Current treatment protocols for ALCL in children consist of a short course of high intensity polychemotherapy. Here we describe an 8‐year‐old female with relapsed ALCL who achieved good response with anti‐CD25 monoclonal antibody daclizumab. Daclizumab appears to offer a safe treatment option, but further research needs to be conducted in order to define its role in children with ALCL who do not respond to intensive chemotherapy. Pediatr Blood Cancer 2009;53:1130–1131.

The EORTC Children's Leukemia Group: Preclinical and clinical research and resulting achievements

Abstract The EORTC Childrens Leukemia Group (CLG) is a spin-off from the EORTC Hemopathies Working Party (adults and children). After a decade of collaboration in the adult-pediatric group it became clear that there was not only a large difference in cure rates between children and adults, but many chemotherapeutic-toxic borders were substantially different. During the following decade the CLG was not only a witness of a very exciting battle against childhood leukemia, but it also contributed substantially to better cure rates for these diseases. The main activity of the CLG was concentrated on the field of acute lymphoblastic leukemia (ALL). Fine tuning of treatment elements using the BFM design as a backbone has been done very successfully over the past decades. The CLG has many achievements, and the major ones include: the 58831 trial showing the superfluity of the prophylactic Central Nervous System (CNS) radiotherapy in ALL patients when a adequate systemic and CNS directed chemotherapy is ascertained. The 58881 trial demonstrated that the assessment of minimal residual disease (MRD) at completion of induction in ALL is a key step in the process to categorizing and allocating patients into different risk groups, and that MRD is a powerful and independent prognostic factor. This same 58881 trial showed the clear difference in efficacy of different asparaginases resulting in an optimization of the use these drugs and a revival of interest for the asparaginases in the treatment of ALL. In the near future the CLG will focus mainly on translational research projects and innovative biologically targeted treatment approaches, on collaborations with other childrens leukemia groups in intergroup studies, and on the evaluation of the long-term outcome of childhood cancer survivors.