RESUMO
AIMS: Patients with colorectal cancer harbouring KRAS mutations do not respond to antiepidermal growth factor receptor (anti-EGFR) therapy. Community screening for KRAS mutation selects patients for treatment. When a KRAS mutation is identified by direct sequencing, mutant and wild type alleles are seen on the sequencing electropherograms. KRAS mutant allele-specific imbalance (MASI) occurs when the mutant allele peak is higher than the wild type one. The aims of this study were to verify the rate and tissue distribution of KRAS MASI as well as its clinical relevance. METHODS: A total of 437 sequencing electropherograms showing KRAS exon 2 mutation was reviewed and in 30 cases next generation sequencing (NGS) was also carried out. Five primary tumours were extensively laser capture microdissected to investigated KRAS MASI tissue spatial distribution. KRAS MASI influence on the overall survival was evaluated in 58 patients. In vitro response to anti-EGFR therapy in relation to different G13D KRAS MASI status was also evaluated. RESULTS: On the overall, KRAS MASI occurred in 58/436 cases (12.8%), being more frequently associated with G13D mutation (p=0.05) and having a heterogeneous tissue distribution. KRAS MASI detection by Sanger Sequencing and NGS showed 94% (28/30) concordance. The longer overall survival of KRAS MASI negative patients did not reach statistical significance (p=0.08). In cell line model G13D KRAS MASI conferred resistance to cetuximab treatment. CONCLUSIONS: KRAS MASI is a significant event in colorectal cancer, specifically associated with G13D mutation, and featuring a heterogeneous spatial distribution, that may have a role to predict the response to EGFR inhibitors. The foreseen implementation of NGS in community KRAS testing may help to define KRAS MASI prognostic and predictive significance.
Assuntos
Desequilíbrio Alélico , Neoplasias Colorretais/genética , Análise Mutacional de DNA , Mutação , Proteínas Proto-Oncogênicas/genética , Proteínas ras/genética , Anticorpos Monoclonais Humanizados/uso terapêutico , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Cetuximab , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Éxons , Predisposição Genética para Doença , Humanos , Itália , Estimativa de Kaplan-Meier , Microdissecção e Captura a Laser , Metástase Neoplásica , Seleção de Pacientes , Fenótipo , Medicina de Precisão , Valor Preditivo dos Testes , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas p21(ras) , Fatores de Tempo , Transfecção , Resultado do Tratamento , Proteínas ras/metabolismoRESUMO
AIMS: To evaluate the accuracy, consumable cost and time around testing (TAT) of a next-generation sequencing (NGS) assay, the Ion Torrent AmpliSeq Colon and Lung Cancer Panel, as an alternative to Sanger sequencing to genotype KRAS, NRAS and BRAF in colorectal cancer patients. METHODS: The Ion Torrent panel was first verified on cell lines and on control samples and then prospectively applied to routine specimens (n=114), with Sanger sequencing as reference. RESULTS: The Ion Torrent panel detected mutant alleles at the 5% level on cell lines and correctly classified all control tissues. The Ion Torrent assay was successfully carried out on most (95.6%) routine diagnostic samples. Of these, 12 (11%) harboured mutations in the BRAF gene and 47 (43%) in either of the two RAS genes, in two cases with a low abundance of RAS mutant allele which was missed by Sanger sequencing. The mean TAT, from sample receipt to reporting, was 10.4 (Sanger) and 13.0 (Ion Torrent) working days. The consumable cost for genotyping KRAS, NRAS and BRAF was 196 (Sanger) and 187 (Ion Torrent). CONCLUSIONS: Ion Torrent AmpliSeq Colon and Lung Cancer Panel sequencing is as robust as Sanger sequencing in routine diagnostics to select patients for anti-epidermal growth factor receptor (EGFR) therapy for metastatic colorectal cancer.
Assuntos
Neoplasias Colorretais/genética , Análise Mutacional de DNA/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Biomarcadores Tumorais/genética , Análise Mutacional de DNA/economia , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala/economia , HumanosRESUMO
Advanced non-small cell lung cancer samples are tested for epidermal growth factor receptor (EGFR) gene mutations. Their detection by direct sequencing is time-consuming. Conversely, the length analysis of fluorescently labelled PCR products is easier. To avoid labelled primers and the automated capillary electrophoresis apparatus, we validated a fast and sensitive chip-based microfluidic technology. The limit of detection of fragment length assay on microfluidic device was 5%, more sensitive than direct sequencing (12.5%). The novel methodology showed high accuracy in the analysis of samples whose mutational status was known. The accuracy in quantifying mutated alleles (mA) was evaluated by PCR products subcloning; the mA% provided by direct sequencing of subcloned PCR products showed a close correlation with the mA% provided by the microfluidic technology for both exon 19 (R(2)=0.9) and 21 (R(2)=0.9). Microfluidic-based on-chip electrophoresis makes EGFR testing more rapid, sensitive and cost-effective.
