RESUMO
The increase of mixed chimerism (MC) after allogeneic hematopoietic stem cell transplantation has been associated with a high risk of relapse. A variety of techniques that use polymorphic markers have been established to survey hematopoietic chimerism status. The highest sensitivity is achieved using real-time quantitative polymerase chain reaction (RQ-PCR) analysis of insertion/deletion polymorphism, which allows the detection of disease recurrence and subsequently the earlier initiation of therapeutic intervention. The purpose of this study is the evaluation of multiplex RQ-PCR for MC assessment (six biallelic genetic systems and Y-specific locus), allowing the amplification and detection of target gene of interest and glyceraldehyde-3-phosphate dehydrogenase reference housekeeping gene in a single microtube. With optimized amounts of primers and probe, the quantification of target DNA was shown to be linear throughout the tested range (100%-0.05%). The efficiencies of multiplex RQ-PCR were in a range of 0.89 to 1.07. The sensitivity of individual systems ranged 0.02% to 0.04% with an average of 0.034%. A high degree of linear correlation between the chimerism results obtained by multiplex RQ-PCR vs singleplex RQ-PCR was observed (P < 0.0001, Spearman's coefficient = 0.9927), while correlation between multiplex RQ-PCR vs short tandem repeat analysis was also statistically significant (P < 0.0001, Spearman's coefficient = 0.9769). This new multiplex RQ-PCR assay is a quick, sensitive, reproducible, and cost-effective method for accurate MC assessment.
Assuntos
Quimerismo , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Reação em Cadeia da Polimerase Multiplex/métodos , Polimorfismo Genético , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Quimeras de Transplante/genética , Alelos , Primers do DNA , Humanos , Leucemia/terapia , Recidiva Local de Neoplasia/diagnóstico , Recidiva Local de Neoplasia/etiologia , Recidiva Local de Neoplasia/prevenção & controle , Quimeras de Transplante/sangueAssuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteínas WT1/metabolismo , Processamento Alternativo , Perfilação da Expressão Gênica , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mieloide Aguda/genética , Isoformas de Proteínas/análise , Reação em Cadeia da Polimerase em Tempo Real , Proteínas WT1/genéticaRESUMO
We monitored DLI treatment of 13 post-SCT relapses using quantitative competitive (QC) RT-PCR for BCR-ABL (sensitivity 10(-5)) and compared responses to DLI alone and DLI in combination with interferon-alpha (IFN). Ten relapses (one blast crisis, five cytogenetic and four molecular) were treated with DLI+IFN, three relapses (one cytogenetic, two molecular) were treated with DLI alone. Except the patient treated in blast crisis, who died, all the patients treated with DLI+IFN achieved complete molecular remission, with the median time interval of 3.9 months (range 0.25-10.5 months). None of the three patients treated with DLI alone have achieved complete molecular remission up to now, i.e. 32, 45, and 50 months after DLI. However, in all of them some decrease of BCR-ABL transcript level was detected. Although the retrospective analyses did not confirm that IFN improved the response to DLI, our results based on sensitive molecular monitoring suggest that DLI effect, at least in some patients, is supported by IFN administration.
