RESUMO
Molecular testing is extremely important in cancer care, starting as early as at diagnosis. In order to address the challenge of providing reliable results within the timeframe adapted to patient management and suitable to guide clinical decisions, a capturebased nextgeneration sequencing (NGS) panel focusing on ten genes known to harbor genetic variations which may be targeted by approved drugs in patients with cancer was designed and validated. Very favorable analytical performances were obtained for both solid and liquid biopsies. For solid biopsies, a low read depth (80X per nucleotide) led to the genotype detection accuracy of 100%. The read of raw data for liquid biopsies resulted in the 91.19% result concordance between paired solid and liquid samples. The present method met all the requirements for the ISO15189 certification. During our threeyear experience of routinely using this panel, almost 2,300 samples from lung and colorectal cancers, melanomas and gastrointestinal stromal tumors have been analyzed. It was found that our panel detected slightly more gainoffunction variants than described in the literature. Surprisingly, lossoffunction variants were also detected in certain of the analyzed genes. Finally, liquid biopsy data revealed statistically different mutated allele frequencies between tumor types, but also between mutated genes and variants themselves. In conclusion, the use of our capturebased NGS panel is perfectly adapted to perform relevant molecular diagnosis in a time frame compatible with patient care.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Neoplasias , Biópsia , Frequência do Gene , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Mutação/genética , Neoplasias/diagnóstico , Neoplasias/genéticaRESUMO
Since January 16(th) 2010, the French legislation requires that the medical laboratories must be accredited according to ISO 15189 standards. This concerned all the biological medical technics, including molecular biology technics. In this work, we described the validation steps by real time quantitative PCR of L858R mutation in EGFR gene, frequently detected in non-small lung cancers (NSCLC). Epidermal growth factor - tyrosine kinase inhibitors (EGFR-TKIs) are authorized in Europe for the treatment of metastatic NSCLC after failure of, at least one, prior chemotherapy. Thus, in view of accreditation of this analysis, we have used the recommendation of the COFRAC (Comité français d'accréditation) and INCa (Institut national du cancer). Several parameters have been tested, such as the primers, the limit of detection, and the sensitivity and specificity of the method. In addition, a risk study has been evaluated. Although long and fastidious, the method of validation is required to perform analysis in optimal conditions to guaranty optimal results for the patients.