José Eduardo Bernardes

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.

Polymerase chain reaction on cerebrospinal fluid cells in suspected leptomeningeal involvement in childhood acute lymphoblastic leukemia: comparison to cytomorphological analysis.

The leptomeningeal involvement of central nervous system is defined in the most centers by the presence of blast cells in the CSF or the presence of cranial-nerve palsies. Sometimes, cytology does not allow clear distinction between lymphoblasts and normal cells, and auxiliary methods to the precise identification of leukemic cells in cerebrospinal fluid is necessary. We analyzed CSF from 11 consecutive patients, in whom a differential diagnosis of leptomeningeal involvement was made, including 4 patients at diagnosis and 7 patients during the treatment by cytomorphological analysis and PCR and automatic sequencing. Six patients were considered with leptomeningeal involvement by conventional analysis: unequivocal cytomorphological involvement was considered in 5 patients, and in one it was assumed to be due to cranial-nerve palsy, with no blast cells detected in cerebrospinal fluid. In 2 it was considered suspicious and in 3 negative. PCR and sequencing analysis showed involvement in 6 patients; 5 of the 6 patients were considered to have leptomeningeal involvement based on clinical and cytomorphological criteria, and, in one of the patients, it was suspicious. Our data suggest that the use of PCR and sequencing can be useful in confirming CNS leukemia and eliminating other conditions when used together with the cytomorphological analysis.

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.

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.

Transient pancytopenia induced by parvovirus B19 in a child with hereditary spherocytosis

OBJECTIVE: To describe the occurrence of transient pancytopenia induced by parvovirus B19 infection in a patient with hereditary hemolytic anemia and to discuss the importance of the diagnosis of this pathology.METHODS: Case report of a child whose diagnosis was made by polymerase chain reaction (PCR) and serology, and review of the literature.CLINICAL REPORT: A twelve year-old male patient with hereditary spherocytosis, presenting non-specific symptoms of an infectious syndrome followed by severe and transient pancytopenia, whose diagnosis was a parvovirus B19 infection.CONCLUSION: The diagnosis of parvovirus infection has a particular importance in hematology, especially on some morbid conditions, among them the hereditary hemolytic anemias. PCR is useful because of its rapidness and sensitivity on the specific diagnosis of this disease.

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.

Is p190 bcr-abl rearrangement necessary for acute transformation in some p210 CML of childhood?

Chronic myeloid leukemia (CML) is a rare disease in childhood which is almost exclusively associated with bcr-abl p210 (M-bcr) rearrangements. It has been suggested that co-expression of p190 and p210 may be a pathway of CML progression in adult patients. We report two cases of pediatric patients with a diagnosis of CML who presented co-expression of the p210 and p190 transcripts during progression to the blastic phase. The present data suggest that p190 may be a secondary event in at least some cases of childhood CML, suggesting an association with progression to a blastic crisis in these patients.