Ricardo Defavery

Childhood B lineage acute lymphoblastic leukemia clonality study by the polymerase chain reaction

Purpose B cell precursors acute lymphoblastic leukemia (ALL) present rearrangements in the heavy chain immunoglobulin and T cell receptor genes, especially in the complementarity determining region 3 (CDR-3) and T cell receptor δ (TCRδ) (Vδ2Dδ3) regions. These rearrangements may be amplified by the polymerase chain reaction (PCR) and used as clonal markers of B lineage ALL. Our purpose was to study clonality at the DNA level by PCR in B lineage ALL. Patients and Methods Fifty-three pediatric patients (36 with B lineage ALL, 7 with ALL-T, and 10 with nonlymphocytic disease) were investigated using consensus primers for the CDR-3 regions of IgH and TCRδ. Results Clonality was detected in 86.1% of the patients with B lineage ALL when the primers for the CDR-3 regions were used, in 41.6% when the primers for TCRδ were used, and in 91.6% when the two primers were used together. Biclonality was found in 22.5% and 6.6% of patients that have shown clonality for CDR-3 and TCRδ, respectively. Clonality was not detected in any other samples using these primers. Conclusions PCR using CDR-3 and TCRδ primers can be used as an aid for B lineage ALL diagnosis and clonal evolution of theses disease.

B lineage acute lymphoblastic leukemia transformation in a child with juvenile myelomonocytic leukemia, type 1 neurofibromatosis and monosomy of chromosome 7 - Possible implications in the leukemogenesis

This report describes the case of an 8-month-old infant with a diagnosis of juvenile myelomonocytic leukemia (JMML) and type 1 neurofibromatosis that presented progression to B lineage acute lymphoid leukemia (ALL). The same rearrangement of gene T-cell receptor gamma (TCR gamma) was detected upon diagnosis of JMML and ALL, suggesting that both neoplasias may have evolved from the same clone. Our results support the theory that JMML may derive from pluripotential cells and that the occurrence of monosomy of chromosome 7 within a clone of cells having an aberrant neurofibromatosis type 1 (NF1) gene may be the cause of JMML and acute leukemia.

Analysis of p16 gene mutations and deletions in childhood acute lymphoblastic leukemias

CONTEXT The p16 tumor suppressor gene encodes a cyclin-dependent kinase 4 inhibitor that blocks cell division during the G1 phase of the cell cycle. Alterations in this gene have been reported for various neoplasia types, including acute lymphoblastic leukemias (ALL), especially T-cell acute lymphoblastic leukemias (ALL). OBJECTIVE To determine probable alterations in the p16 gene in children with acute lymphoblastic leukemias using the polymerase chain reaction (PCR) and direct DNA sequencing and also to analyze event-free survival (EFS). DESIGN Retrospective study. SETTING Department of Child Care and Pediatrics, Faculty of Medicine of Ribeirão Preto, Universidade Federal de São Paulo. PARTICIPANTS Fifty-six children with ALL (mean age 4 years). Forty (71.43%) had B-cell and 12 (21.43%) had T-cell ALL; 4 (7.1%) were biphenotypic. SAMPLE DNA samples were extracted from bone marrow upon diagnosis and/or relapse. In 2 T-cell cases, DNA from cerebrospinal fluid (CSF) was analyzed. MAIN MEASUREMENTS Deletions or nucleotide substitutions in exons 1, 2 and 3 of the p16 gene were determined by PCR and nucleotide sequencing. Event-free survival was determined by the Kaplan-Meyer and log-rank test for patients carrying normal and altered p16. RESULTS Deletions in exon 3 were observed in five cases. Abnormal migration in PCR was observed in seven cases for exon 1, six for exon 2, and five for exon 3. Mutations in exon 1 were confirmed by direct DNA sequencing in four cases and in exon 2 in two cases. The Kaplan-Meyer survival curves and the log-rank test showed no significant differences in 5-year EFS between children with normal or altered p16, or between patients with B-ALL carrying normal or altered p16 gene. Patients with T-ALL could not be evaluated via Kaplan-Meier due to the small number of cases. CONCLUSIONS Our results, particularly regarding deletion frequency, agree with others suggesting that deletions in the p16 are initial events in leukemia genesis. The small number of samples did not allow establishment of correlation between childhood ALL and the p16 point mutations found in our study. Kaplan-Meier analysis revealed no significant correlation between EFS and alterations in ALL. The p16 alterations frequency observed for B and T-ALL agreed with reports from other centers.

Translocation (1;5)(q32;q35) in CD30+ anaplastic large cell non-Hodgkin lymphoma of childhood: A case report

The authors studied cytogenetically a case of CD30+ anaplastic large cell non-Hodgkin lymphoma previously diagnosed as malignant histiocytosis and detected a translocation involving chromosomes 1 and 5, t(1;5)(q32:q35). After comparing their findings with those from reports in the literature, they comment about the importance of breakpoint q35 on chromosome 5 and point out the importance of associating morphologic, immunoperoxidase, and cytogenetic findings to confirm the diagnosis of this tumor.

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 (TCR&ggr;) 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 TCR&ggr; gene rearrangements was not a rare event among children in Brazil and, when present, interfered with the detection of minimal residual disease.

Minimal residual disease in Brazilian children with acute lymphoid leukemia: comparison of three detection methods by PCR

The minimal residual disease (MRD) detection by the polymerase chain reaction (PCR) in children with acute lymphoblastic leukemia has been pointed to be an adverse prognostic factor. Detection methods based on this technique using clone-specific primers are cumbersome and time consuming. The detection of monoclonal gene rearrangements of gamma T-cell receptors (TCRgamma) is a simpler although less sensitive method. In the present study, we analyzed the presence of MRD during four different phases of treatment (week 4; 3-6, 12-24 months, and end of treatment) in 34 Brazilian children with lymphoid leukemia by three detection methods based on the PCR technique: (1) using consensus primers for the detection of a clonal population for TCRgamma; (2) clone-specific primers for the junctional region of TCRgamma; and (3) a semi-nested reaction with an initial cycle with consensus primers followed by a second cycle with clone-specific primers. MRD presence was associated with a shorter event-free survival and was the major independent prognostic factor in most of the phases analyzed. The use of consensus primers for the detection of TCRgamma clonality, although less sensitive, proved to be a simpler, faster and less costly method whose positivity was associated with more than 90% relapse rates during all phases analyzed.

Prognostic significance of bi/oligoclonality in childhood acute lymphoblastic leukemia as determined by polymerase chain reaction.

CONTEXT The CDR-3 region of heavy-chain immunoglobulin has been used as a clonal marker in the study of minimal residual disease in children with acute lymphoblastic leukemia. Southern blot and polymerase chain reaction studies have demonstrated the occurrence of bi/oligoclonality in a variable number of cases of B-lineage acute lymphoblastic leukemia, a fact that may strongly interfere with the detection of minimal residual disease. Oligoclonality has also been associated with a poorer prognosis and a higher chance of relapse. OBJECTIVES To correlate bi/oligoclonality, detected by polymerase chain reaction in Brazilian children with B-lineage acute lymphoblastic leukemia with a chance of relapse, with immunophenotype, risk group, and disease-free survival. DESIGN Prospective study of patients outcome. SETTING Pediatric Oncology Unit of the University Hospital, Faculty of Medicine of Ribeirão Preto, University of São Paulo. PARTICIPANTS 47 children with acute lymphoblastic leukemia DIAGNOSTIC TEST Polymerase chain reaction using consensus primers for the CDR-3 region of heavy chain immunoglobulin (FR3A, LJH and VLJH) for the detection of clonality. RESULTS Bi/oligoclonality was detected in 15 patients (31.9%). There was no significant difference between the groups with monoclonality and biclonality in terms of the occurrence of a relapse (28.1% versus 26.1%), presence of CALLA+ (81.2% versus 80%) or risk group (62.5% versus 60%). Disease-free survival was similar in both groups, with no significant difference (p: 0.7695). CONCLUSIONS We conclude that bi/oligoclonality was not associated with the factors investigated in the present study and that its detection in 31.9% of the patients may be important for the study and monitoring of minimal residual disease.

Lack of evidence for mutations or deletions in the CDKN2A/p16 and CDKN2B/p15 genes of Brazilian neuroblastoma patients

Neuroblastoma, the most common extracranial tumor in childhood, has a wide spectrum of clinical and biological features. The loss of heterozygosity within the 9p21 region has been reported as a prognostic factor. Two tumor suppressor genes located in this region, the CDKN2B/p15 and CDKN2A/p16 (cyclin-dependent kinase inhibitors 2B and 2A, respectively) genes, play a critical role in cell cycle progression and are considered to be targets for tumor inactivation. We analyzed CDKN2B/p15 and CDKN2A/p16 gene alterations in 11 patients, who ranged in age from 4 months to 13 years (male/female ratio was 1.2:1). The most frequent stage of the tumor was stage IV (50%), followed by stages II and III (20%) and stage I (10%). The samples were submitted to the multiplex PCR technique for homozygous deletion analysis and to single-strand conformation polymorphism and nucleotide sequencing for mutation analysis. All exons of both genes were analyzed, but no deletion was detected. One sample exhibited shift mobility specific for exon 2 in the CDKN2B/p15 gene, not confirmed by DNA sequencing. Homozygous deletions and mutations are not involved in the inactivation mechanism of the CDKN2B/p15 and CDKN2A/p16 genes in neuroblastoma; however, these two abnormalities do not exclude other inactivation pathways. Recent evidence has shown that the expression of these genes is altered in this disease. Therefore, other mechanisms of inactivation, such as methylation of promoter region and unproperly function of proteins, may be considered in order to estimate the real contribution of these genes to neuroblastoma genesis or disease progression.

Inactivation of the p15 gene in children with acute lymphoblastic leukemia

CONTEXT Tumor suppressor genes act on the control of cell cycle progression. In pediatric neoplasias, some of these genes may be considered to be markers for diagnosis or relapse, thus probably representing prognostic indicators. OBJECTIVE To study the inactivation of the p15 gene in children with acute lymphoblastic leukemia. TYPE OF STUDY Retrospective study. SETTING Laboratory of Molecular Biology, Department of Pediatrics, Faculdade de Medicina de Ribeiráo Preto, Universidade de São Paulo. PARTICIPANTS Eighty-three children and adolescents with acute lymphoblastic leukemia were studied, with the examination of 83 bone marrow samples obtained at diagnosis, four obtained also during relapse, and two cerebrospinal fluid samples obtained from two cases of isolated relapse in the central nervous system. MAIN MEASUREMENTS Homologous deletion of the p15 gene by multiplex polymerase chain reaction, and screening for point mutations by polymerase chain reaction/single-strand conformational polymorphism. RESULTS Deletion of exon 2 of the p15 gene was observed in 15 children, including one case in which deletion was only verified during isolated central nervous system relapse. No case of exon 1 deletion, or that was suggestive of point mutations, was observed and no association between p15 gene inactivation and classic risk factors was established. CONCLUSION According to the literature, inactivation of the p15 gene by deletion of exon 2 in acute lymphoblastic leukemia found in the population studied would be considered to be a molecular marker for diagnosis or relapse. However, no correlation between p15 gene deletion and clinical prognostic indicators was observed.