Comparison between TaqMan and LightCycler technologies for quantification of minimal residual disease by using immunoglobulin and T-cell receptor genes consensus probes.

Quantification of residual leukemic cells at early time points during therapy can reliably predict the outcome in children with acute lymphoblastic leukemia (ALL). Recently, semiquantitative minimal residual disease (MRD) detection assays such as dot-blot hybridization have been replaced by real-time quantitative PCR. We tested the flexibility of the two most used real-time PCR machines: the SDS 7700 or 'TaqMan' (TM) (Applied Biosystems) and the LightCycler (LC) (Roche) instruments. Clonal T-cell receptor and immunoglobulin gene rearrangements were used for MRD detection with germline hydrolyzation probes and clone-specific primers. Sensitivity tests for 65 clonal gene rearrangements and MRD quantification in 90 bone marrow samples during therapy of 49 children with ALL at diagnosis or relapse were performed with both machines. Both real-time PCR systems provided specific results for MRD quantification in all follow-up samples. In conclusion, we were able to demonstrate that TM and LC real-time PCR technologies produce similar MRD quantification results and that the quantification assays can be easily transferred from one detection system to the other. Using the same detection format, both techniques can be applied in combination in multicenter MRD studies.     

Clinico‐genetic aspects of a pediatric non‐neurofibromatosis type 1 malignant triton tumor with loss of chromosome X

Malignant triton tumor (MTT) is an aggressive peripheral nerve sheath tumor with rhabdomyoblastic differentiation. Less than 100 cases have been described, being mostly male children with type 1 neurofibromatosis. We report a 6‐year‐old female with MTT and no diagnostic criteria for neurofibromatosis type 1. Cytogenetic analysis showed a 46,X,‐X[4]/46,XX[16] karyotype. She underwent a transfemoral amputation and chemotherapy and is free of disease 15 months after diagnosis. The few cytogenetic studies of MTT described in the literature have been inconclusive. Further cytogenetic analyses are needed to understand the role of chromosome X monosomy in the pathogenesis of this rare tumor. Pediatr Blood Cancer 2012; 59: 1320–1323. © 2012 Wiley Periodicals, Inc.     

A coordinated approach for the assessment of molecular subgroups in pediatric ependymomas using low-cost methods

Journal of Molecular Medicine - Although ependymoma (EPN) molecular subgroups have been well established by integrated high-throughput platforms, low- and middle-income countries still need...     

Inhibition of Aurora kinases enhances chemosensitivity to temozolomide and causes radiosensitization in glioblastoma cells

Background  

Glioblastoma remains one of the most devastating human malignancies, and despite therapeutic advances, there are no drugs that significantly improve the patient survival. Altered expression of the Aurora kinases was found in different malignancies, and their inhibition has been studied in cancer therapy. In this study, we analyzed the expression of Aurora A and Aurora B in glioblastoma samples and also analyzed whether the effects of Aurora kinase inhibition were associated with temozolomide or not on cell lines and primary cultures of glioblastoma.     

Mowat–Wilson syndrome: the first report of an association with central nervous system tumors

Mowat–Wilson syndrome (MWS) is a rare genetic condition where variable and multiple congenital anomalies including Hirschsprung's disease, intellectual disability, and prominent facial features are present. At molecular level, MWS is characterized by many different described mutations in the zinc finger E-box protein 2 (ZEB2) gene, ultimately leading to loss of gene function. This report is the first to describe the association of MWS with two different asynchronous malignant brain tumors (medulloblastoma and glioblastoma) occurring in a child.     

T cell receptor gamma (TCRG) gene rearrangements in Brazilian children with acute lymphoblastic leukemia: analysis and implications for the study of minimal residual disease

The high frequency of T cell receptor gamma (TCRG) gene rearrangements in both B-lineage and T cell acute lymphoblastic leukemia (ALL), its easy detection and the lower incidence of oligoclonality make this gene one of the main target for the detection of minimal residual disease by PCR in childhood ALL. We analyzed the frequency and type of TCRG rearrangements in DNA samples obtained from the bone marrow of 102 Brazilian children at diagnosis using PCR and automatic sequencing. TCRG rearrangements were found in 69% of patients with B-lineage ALL and in 94% of patients with T cell ALL. In contrast to other studies, rearrangements involving the Vgamma9 segment reported to be uncommon were the most frequent both in B-lineage and T cell ALL and involved 49/109 (45%) of the rearranged alleles. This fact should be considered when standardizing consensus primers for the study of minimal residual disease in different populations.     

Clonal evolution as the limiting factor in the detection of minimal residual disease by polymerase chain reaction in children in Brazil with acute lymphoid leukemia

BACKGROUND: The purpose of this investigation was to analyze the incidence of clonal evolution in children in Brazil children with acute lymphoblastic leukemia and its interference with the detection of minimal residual disease by polymerase chain reaction using clone-specific primers. PATIENTS AND METHODS: The authors analyzed DNA samples from 12 children with acute lymphoblastic leukemia at diagnosis and after relapse using polymerase chain reaction and automatic sequencing to determine the presence of T-cell receptor gamma (TCRgamma) gene rearrangements. A clone-specific primer was synthesized based on the sequence obtained at diagnosis for each patient and at relapse for those with clonal evolution for the study of minimal residual disease. RESULTS: A change of the original clone was detected in 3 of 12 patients (25%), involving the same rearrangement detected at diagnosis, suggesting the development of subclones. Minimal residual disease was detected at the end of treatment or before the relapse in all patients who had maintained the same rearrangements detected at diagnosis. Minimal residual disease was investigated at the end of treatment in two of the three patients with clonal evolution and was not detected with the use of clone-specific primers. CONCLUSIONS: These data suggest that clonal evolution for TCRgamma gene rearrangements was not a rare event among children in Brazil and, when present, interfered with the detection of minimal residual disease.     

Pediatric glioblastoma cell line shows different patterns of expression of transmembrane ABC transporters after in vitro exposure to vinblastine

Background  Resistance to drug is a major cause of treatment failure in pediatric brain cancer. The multidrug resistance (MDR) phenotype can be mediated by the superfamily of adenosine triphosphate-binding cassette (ABC) transporters. The dynamics of expression of the MDR genes after exposure to chemotherapy, especially the comparison between pediatric brain tumors of different histology, is poorly described. Objective  To compare the expression profiles of the multidrug resistance genes ABCB1, ABCC1, and ABCG2 in different neuroepithelial pediatric brain tumor cell lines prior and following short-term culture with vinblastine. Methods  Immortalized lineages from pilocytic astrocytoma (R286), anaplasic astrocytoma (UW467), glioblastoma (SF188), and medulloblastoma (UW3) were exposed to vinblastine sulphate at different schedules (10 and 60 nM for 24 and 72 h). Relative amounts of mRNA expression were analyzed by real-time quantitative polymerase chain reaction. Protein expression was assessed by immunohistochemistry for ABCB1, ABCC1, and ABCG2. Results  mRNA expression of ABCB1 increased together with augmenting concentration and time of exposure to vinblastine for R286, UW467, and UW3 cell lines. Interestingly, ABCB1 levels of expression diminished in SF188. Following chemotherapy, mRNA expression of ABCC1 decreased in all cell lines other than glioblastoma. ABCG2 expression was influenced by vinblastine only for UW3. The mRNA levels showed consistent association to protein expression in the selected sets of cell lines analyzed. Conclusions  The pediatric glioblastoma cell line SF188 shows different pattern of expression of multidrug resistance genes when exposed to vinblastine. These preliminary findings may be useful in determining novel strategies of treatment for neuroepithelial pediatric brain tumors. Financial support: FAPESP (grant 2007/04065-9).