RESUMO
The hotspot c.7541_7542delCT NOTCH1 mutation has been proven to have a negative clinical impact in chronic lymphocytic leukemia (CLL). However, an optimal method for its detection has not yet been specified. The aim of our study was to examine the presence of the NOTCH1 mutation in CLL using three commonly used molecular methods. Sanger sequencing, fragment analysis and allele-specific PCR were compared in the detection of the c.7541_7542delCT NOTCH1 mutation in 201 CLL patients. In 7 patients with inconclusive mutational analysis results, the presence of the NOTCH1 mutation was also confirmed using ultra-deep next generation sequencing. The NOTCH1 mutation was detected in 15% (30/201) of examined patients. Only fragment analysis was able to identify all 30 NOTCH1-mutated patients. Sanger sequencing and allele-specific PCR showed a lower detection efficiency, determining 93% (28/30) and 80% (24/30) of the present NOTCH1 mutations, respectively. Considering these three most commonly used methodologies for c.7541_7542delCT NOTCH1 mutation screening in CLL, we defined fragment analysis as the most suitable approach for detecting the hotspot NOTCH1 mutation.
Assuntos
Análise Mutacional de DNA/métodos , Leucemia Linfocítica Crônica de Células B/genética , Mutação , Fragmentos de Peptídeos/análise , Receptor Notch1/genética , Sequenciamento de Nucleotídeos em Larga Escala , HumanosAssuntos
Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/genética , Receptor Notch1/genética , Proteína Supressora de Tumor p53/genética , Alelos , Evolução Clonal/genética , Análise Mutacional de DNA/métodos , Genótipo , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Análise de Célula Única/métodosRESUMO
The canonical Wnt pathway, dependent on ß-catenin-controlled transcription, is the most explored Wnt pathway, known to drive the malignant transformation of multiple cell types. Several reports have suggested that this pathway also participates in chronic lymphocytic leukaemia (CLL) pathogenesis. To get a better insight into the role of the Wnt/ß-catenin pathway in CLL we analysed in detail the expression of the most overexpressed Wnt ligand, encoded by the WNT3 gene, in a well-defined cohort of 137 CLL patients. Our analysis demonstrated that (i) untreated patients with more aggressive disease (with a notable exception of patients with 11q deletion) express less WNT3, (ii) WNT3 declines with disease progression in a significant proportion of patients and (iii) low WNT3 was identified as a strong independent marker indicating shorter treatment-free survival in CLL patients with IGHV mutation. Interestingly, CLL-related lymphoid cell lines, but not stromal cells, failed to respond to the ligand-induced activation of the Wnt/ß-catenin pathway. This opens the possibility that CLL cells use Wnt-3 to communicate with the cells in the microenvironment. We thus propose that the Wnt/ß-catenin pathway plays a more complex role in CLL pathogenesis than previously anticipated.
Assuntos
Regulação Neoplásica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/diagnóstico , Proteína Wnt3/genética , Comunicação Celular , Linhagem Celular , Estudos de Coortes , Progressão da Doença , Feminino , Genes de Cadeia Pesada de Imunoglobulina/genética , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Masculino , Mutação , Prognóstico , Via de Sinalização WntRESUMO
TP53 gene defects represent a strong adverse prognostic factor for patient survival and treatment resistance in chronic lymphocytic leukemia (CLL). Although various methods for TP53 mutation analysis have been reported, none of them allow the identification of all occurring sequence variants, and the most suitable methodology is still being discussed. The aim of this study was to determine the limitations of commonly used methods for TP53 mutation examination in CLL and propose an optimal approach for their detection. We examined 182 CLL patients enriched for high-risk cases using denaturing high-performance liquid chromatography (DHPLC), functional analysis of separated alleles in yeast (FASAY), and the AmpliChip p53 Research Test in parallel. The presence of T53 gene mutations was also evaluated using ultra-deep next generation sequencing (NGS) in 69 patients. In total, 79 TP53 mutations in 57 (31 %) patients were found; among them, missense substitutions predominated (68 % of detected mutations). Comparing the efficacy of the methods used, DHPLC and FASAY both combined with direct Sanger sequencing achieved the best results, identifying 95 % and 93 % of TP53-mutated patients. Nevertheless, we showed that in CLL patients carrying low-proportion TP53 mutation, the more sensitive approach, e.g., ultra-deep NGS, might be more appropriate. TP53 gene analysis using DHPLC or FASAY is a suitable approach for mutation detection. Ultra-deep NGS has the potential to overcome shortcomings of methods currently used, allows the detection of minor proportion mutations, and represents thus a promising methodology for near future.