Comparison between qualitative and real-time polymerase chain reaction to evaluate minimal residual disease in children with acute lymphoblastic leukemia

Introduction: Minimal residual disease is an important independent prognostic factor that can identify poor responders among patients with acute lymphoblastic leukemia. Objective: The aim of this study was to analyze minimal residual disease using immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements by conventional polymerase chain reaction followed by homo-heteroduplex analysis and to compare this with real-time polymerase chain reaction at the end of the induction period in children with acute lymphoblastic leukemia. Methods: Seventy-four patients diagnosed with acute lymphoblastic leukemia were enrolled. Minimal residual disease was evaluated by qualitative polymerase chain reaction in 57 and by both tests in 44. The Kaplan-Meier and multivariate Cox methods and the log-rank test were used for statistical analysis. Results: Nine patients (15.8%) were positive for minimal residual disease by qualitative polymerase chain reaction and 11 (25%) by real-time polymerase chain reaction considering a cut-off point of 1 × 10−3 for precursor B-cell acute lymphoblastic leukemia and 1 × 10−2 for T-cell acute lymphoblastic leukemia. Using the qualitative method, the 3.5-year leukemia- free survival was significantly higher in children negative for minimal residual disease compared to those with positive results (84.1% ± 5.6% versus 41.7% ± 17.3%, respectively; p-value = 0.004). There was no significant association between leukemia-free survival and minimal residual disease by real-time polymerase chain reaction. Minimal residual disease by qualitative polymerase chain reaction was the only variable significantly correlated to leukemia-free survival. Conclusion: Given the difficulties in the implementation of minimal residual disease monitoring by real-time polymerase chain reaction in most treatment centers in Brazil, the qualitative polymerase chain reaction strategy may be a cost-effective alternative.

Gene rearrangement study for minimal residual disease monitoring in children with acute lymphocytic leukemia

OBJECTIVE To detect markers for minimal residual disease monitoring based on conventional polymerase chain reaction for immunoglobulin, T-cell receptor rearrangements and the Sil-Tal1 deletion in patients with acute lymphocytic leukemia. METHODS Fifty-nine children with acute lymphocytic leukemia from three institutions in Minas Gerais, Brazil, were prospectively studied. Clonal rearrangements were detected by polymerase chain reaction followed by homo/heteroduplex clonality analysis in DNA samples from diagnostic bone marrow. Follow-up samples were collected on Days 14 and 28-35 of the induction phase. The Kaplan-Meier and multivariate Cox methods were used for survival analysis. RESULTS Immunoglobulin/T-cell receptor rearrangements were not detected in 5/55 children screened (9.0%). For precursor-B acute lymphocytic leukemia, the most frequent rearrangement was IgH (72.7%), then TCRG (61.4%), and TCRD and IgK (47.7%); for T-acute lymphocytic leukemia, TCRG (80.0%), and TCRD and Sil-Tal deletion (20.0%) were the most common. Minimal residual disease was detected in 35% of the cases on Day 14 and in 22.5% on Day 28-35. Minimal residual disease on Day 28-35, T-acute lymphocytic leukemia, and leukocyte count above 50 x 109/L at diagnosis were bad prognostic factors for leukemia-free survival in univariate analysis. Relapse risk for minimal residual disease positive relative to minimal residual disease negative children was 8.5 times higher (95% confidence interval: 1.02-70.7). CONCLUSION Immunoglobulin/T-cell receptor rearrangement frequencies were similar to those reported before. Minimal residual disease is an independent prognostic factor for leukemia-free survival, even when based on a non-quantitative technique, but longer follow-ups are needed.

Molecular detection of HPV 16 and 18 in cervical samples of patients from Belo Horizonte, Minas Gerais, Brazil

PURPOSE: The aim of this study was to investigate the frequency of HPV infection and the types 16 and 18 in cervical samples from patients attended at two public health services of the city of Belo Horizonte, MG. METHODS: Cervical samples from 174 patients were collected for cytopathological and molecular tests. HPV infection was searched by PCR utilizing MY09 and MY11 primers or HPV 16 and HPV 18 specific primers. RESULTS: Amongst the 174 samples analyzed, 20.7 percent presented squamous intraepithelial and/or invasive lesions detected on cytopathological analysis and of those, 94.4 percent were infected by HPV. HPV 16 was found in 20 percent of the cases of low-grade squamous intraepithelial lesions and in 40 percent and 50 percent of high-grade squamous intraepithelial lesion and squamous invasive carcinoma, respectively. HPV 18 was detected in 6.7 percent of the low-grade lesion samples and in two HPV16 co-infected samples. In 50 percent of the cases of high-grade lesion, the HPV type was not determined. CONCLUSION: The HPV 16 was the virus type more frequently detected. However, more than 50 percent of the positive samples at the cytopathological analysis were negative for HPV 16 and 18, indicating that possibly other virus types are present in relative high frequencies in the studied population.

OBJETIVOS: O objetivo deste estudo foi investigar a freqüência da infecção por HPV e dos tipos 16 e 18 em amostras cervicais de pacientes atendidas em dois serviços públicos da cidade de Belo Horizonte-MG. MÉTODOS: Amostras cervicais de 174 pacientes foram coletadas para estudo citopatológico e molecular. A pesquisa da infecção por HPV foi feita através da PCR utilizando os oligonucleotídeos MY09/MY11. Os tipos virais 16 e 18 foram pesquisados através da utilização de oligonucleotídeos específicos. RESULTADOS: Dentre as 174 amostras analisadas, 20,7 por cento apresentaram lesões escamosas intra-epiteliais e/ou invasoras detectadas na análise citopatológica, das quais 94,4 por cento mostraram infecção por HPV. O HPV 16 foi encontrado em torno de 20 por cento dos casos de lesão escamosa intra-epitelial de baixo grau e em 40 por cento e 50 por cento dos casos de lesão escamosa intra-epitelial de alto grau e carcinoma escamoso invasor, respectivamente. O HPV 18 foi encontrado em 6,7 por cento das amostras com lesão de baixo grau e em dois casos de co-infecção com HPV 16. Em 50 por cento dos casos de lesão de alto grau, o tipo de HPV não foi determinado. CONCLUSÕES: O HPV 16 foi o tipo viral mais freqüentemente detectado. No entanto, mais de 50 por cento das amostras positivas no exame citopatológico não apresentaram HPV 16 e 18, indicando que possivelmente outros tipos virais estejam presentes em freqüências relativamente altas na população estudada.