RESUMO
Real-time quantitative PCR (qPCR) is routinely used to detect minimal residual disease in chronic myeloid leukemia patients. The absolute quantification with droplet digital PCR (ddPCR) could reduce the inherent variability of qPCR. We established a duplex ddPCR assay using the Europe against Cancer (EAC) primer/probe system for breakpoint cluster region protein-tyrosine-protein kinase ABL1 (BCR-ABL1) and ABL1 and compared the results with qPCR. cDNA samples (n = 230) from patients with chronic myeloid leukemia were analyzed using both procedures. A second, commercially developed ddPCR assay for BCR-ABL1 was also evaluated. ABL1 and BCR-ABL1 transcript levels were similar with all assays, but the proportion of deep molecular responses was lower with ddPCR than with qPCR. The EAC ddPCR assay had a false-positive rate of 4% using a cutoff of three BCR-ABL1 copies per duplicate, compared with 2% without cutoff for the commercial ddPCR. The detection rate for molecular response 4.5 was 100, and a shift toward more minimal residual disease was seen in patient samples. In conclusion, using the EAC protocol for BCR-ABL1 quantification with ddPCR is feasible and shows low intra-assay and interassay variation but requires a cutoff that reduces sensitivity. The commercial ddPCR assay is highly sensitive and specific for BCR-ABL1. The use of either ddPCR assay resulted in a shift to lower molecular response classes compared with qPCR aligned to international scale.
Assuntos
Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Proteínas Proto-Oncogênicas c-abl/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Estudos de Coortes , Estudos de Viabilidade , Células HeLa , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Monitorização Fisiológica/métodos , Neoplasia Residual/genética , Inibidores de Proteínas Quinases/uso terapêutico , Sensibilidade e EspecificidadeRESUMO
Quantitative real-time PCR methods are commonly used to monitor BCR-ABL transcript levels in patients with chronic myelogenous leukemia. However, standard techniques involve separate measurements of target and reference DNAs, require standard curves, and are susceptible to PCR inhibition. An optimized duplex droplet digital PCR (ddPCR) should provide absolute quantification without the need for standard curves. The combination of high sensitivity and low background is particularly important for reliable monitoring of minimal residual disease. In this report, we describe primer probe set testing and step by step optimization of a duplex ddPCR for BCR-ABL/ABL. The optimization of ddPCR parameters increased ABL and BCR-ABL fluorescence signals by two- and fivefold, respectively, and enhanced the resolution between positive and negative drops. The optimized procedure generates a background false-positive rate of 5% of samples and reliably detects BCR-ABL/ABL down to 1/100,000 (CV <10%), with a single BCR-ABL copy being detected in 54% of reactions performed in duplicate from a molecular remission (MR) 5 sample (limit of detection = 1). Assay of duplicates resulted in a detection rate of 100% and 92% for MR4 and MR4.5, respectively. Detection of MR4.5 was increased to 100% by analyzing quadruplicates. Selection of an optimal primer and probe combination and stepwise optimization of the ddPCR conditions has yielded a robust low-background duplex ddPCR procedure for BCR-ABL/ABL.
Assuntos
Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Reação em Cadeia da Polimerase/métodos , Linhagem Celular Tumoral , Primers do DNA/genética , Sondas de DNA/genética , Dosagem de Genes , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/diagnóstico , Neoplasia Residual/diagnóstico , Neoplasia Residual/genéticaRESUMO
In acute myeloid leukemia (AML), leukemia-initiating cells exist within the CD34+/CD38- cell compartment. They are assumed to be more resistant to chemotherapy, enriched in minimal residual disease cell populations, and responsible for relapse. Here we evaluated clinical and biological associations and the prognostic impact of a high diagnostic CD34+/CD38- cell burden in 169 AML patients receiving an allogeneic stem cell transplantation in complete remission. Here, the therapeutic approach is mainly based on immunological graft-versus-leukemia effects. Percentage of bone marrow CD34+/CD38- cell burden at diagnosis was measured using flow cytometry and was highly variable (median 0.5%, range 0%-89% of all mononuclear cells). A high CD34+/CD38- cell burden at diagnosis associated with worse genetic risk and secondary AML. Patients with a high CD34+/CD38- cell burden had shorter relapse-free and overall survival which may be mediated by residual leukemia-initiating cells in the CD34+/CD38- cell population, escaping the graft-versus-leukemia effect after allogeneic transplantation. Evaluating the CD34+/CD38- cell burden at diagnosis may help to identify patients at high risk of relapse after allogeneic transplantation. Further studies to understand leukemia-initiating cell biology and develop targeting therapies to improve outcomes of AML patients are needed.
Assuntos
ADP-Ribosil Ciclase 1/análise , Antígenos CD34/análise , Leucemia Mieloide Aguda/patologia , Células-Tronco Neoplásicas/patologia , Adulto , Idoso , Aloenxertos , Intervalo Livre de Doença , Feminino , Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/terapia , Masculino , Pessoa de Meia-Idade , Prognóstico , Recidiva , Taxa de Sobrevida , Adulto JovemRESUMO
A broad range of malignant diseases, such as mantle cell lymphoma (MCL), is associated with complex genomic alterations, demanding multimodal functional testing of candidate genes. To assess such candidate disease genes, we have developed a bidirectional targeted transgenesis tool, which allows well-controlled modulation of individual gene activities within a cellular MCL system. The engineered versatile transgenesis system permits functional analysis of virtually any candidate gene: for tumor suppressor genes by complementation via integration of respective genomic DNA or for oncogenes by inactivation via integrated shRNA coding plasmids. Complementation by genomic DNA ensures wild-type (WT) regulated gene expression, whereas genomic integration of shRNA coding inserts by an advanced RNAi-strategy mediates specific knock-down of gene expression. Site-specific genomic integration of an unmodified BAC, which contains the CDKN2A/B genes absent in the MCL model system, restored CDKN2A/B expression resulting in the inhibition of cell proliferation. CCND1, strongly overexpressed in the model system, was down-regulated via shRNA expression, again inhibiting proliferation. Notably, the presented site-specific shRNA-strategy circumvents interference by IFN-response induced when using other RNAi gene knock-down methods. In conclusion, we here demonstrate that adequate restoration of a range of different gene activities yields in a desired antiproliferative effect in MCL-derived cells. By antagonizing inactivated tumor suppressor genes or activated oncogenes, the presented approach can be readily used for the functional analysis of a broad range of disease-related genetic defects.
Assuntos
Marcação de Genes/métodos , Genes Supressores de Tumor , Linfoma de Célula do Manto/genética , Oncogenes , Transgenes , Linhagem Celular Tumoral , Proliferação de Células , Cromossomos Artificiais Bacterianos , Regulação da Expressão Gênica , Genoma Humano , Humanos , Interferons/metabolismo , Linfoma de Célula do Manto/metabolismo , Interferência de RNARESUMO
Deletion of chromosome region 11q22-q23 defines a subgroup of patients with B-cell chronic lymphocytic leukemia (B-CLL) characterized by poor survival. Although the tumor-suppressor gene ATM in the consensus deletion region was found to be biallelically inactivated in about one third of B-CLL cases, in the majority of those who have this deletion, inactivation of the remaining ATM allele was not observed. To identify a second disease-associated gene, we investigated two B-CLL cases with translocation breakpoints in the critical 11q23 deletion region. In one case, a t(X;11)(q13;q23) was cloned and two novel genes were isolated. The breakpoint on 11q23 affected the ARHGAP20 gene, which encodes a protein predicted to be involved in the regulation of Rho family GTPases. The breakpoint on Xq13 occurred in BRWD3, which codes for a putative novel transcription factor. The rearrangement of ARHGAP20 and BRWD3 did not result in fusion transcripts, but it disrupted both genes. Mutation analysis of 28 B-CLL samples with monoallelic deletions and two B-CLL samples with 11q23 translocations detected no deleterious mutation in the remaining copy of ARHGAP20. Quantitative expression analysis in 22 B-CLLs revealed significant up-regulation of ARHGAP20 in CLL B cells, whereas BRWD3 was slightly down-regulated. Thus, deregulation of ARHGAP20 by altered gene expression or by gene disruption (but not point mutation) might be a general molecular mechanism of B-CLL leukemogenesis.