RESUMO
INTRODUCTION: Non-invasive mutation testing using circulating tumour DNA (ctDNA) is an attractive premise. This could enable patients without available tumour sample to access more treatment options. MATERIALS & METHODS: Peripheral blood and matched tumours were analysed from 45 NSCLC patients. We investigated the impact of pre-analytical variables on DNA yield and/or KRAS mutation detection: sample collection tube type, incubation time, centrifugation steps, plasma input volume and DNA extraction kits. RESULTS: 2 hr incubation time and double plasma centrifugation (2000 x g) reduced overall DNA yield resulting in lowered levels of contaminating genomic DNA (gDNA). Reduced "contamination" and increased KRAS mutation detection was observed using cell-free DNA Blood Collection Tubes (cfDNA BCT) (Streck), after 72 hrs following blood draw compared to EDTA tubes. Plasma input volume and use of different DNA extraction kits impacted DNA yield. CONCLUSION: This study demonstrated that successful ctDNA recovery for mutation detection in NSCLC is dependent on pre-analytical steps. Development of standardised methods for the detection of KRAS mutations from ctDNA specimens is recommended to minimise the impact of pre-analytical steps on mutation detection rates. Where rapid sample processing is not possible the use of cfDNA BCT tubes would be advantageous.
Assuntos
Adenocarcinoma/genética , Coleta de Amostras Sanguíneas/métodos , Carcinoma Pulmonar de Células não Pequenas/genética , Análise Mutacional de DNA/métodos , DNA de Neoplasias/genética , Genes ras , Neoplasias Pulmonares/genética , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Adenocarcinoma/sangue , Adenocarcinoma/química , Automação , Coleta de Amostras Sanguíneas/instrumentação , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/química , Centrifugação/métodos , DNA de Neoplasias/sangue , DNA de Neoplasias/isolamento & purificação , Humanos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/química , Kit de Reagentes para Diagnóstico , Soluções , Manejo de Espécimes/métodos , Temperatura , Fatores de TempoRESUMO
The DNA methyl-transferase 3A gene (DNMT3A) is the third most frequently mutated gene in cytogenetically normal acute myeloid leukemia (CN-AML) patients (20-30 %), who belong to a group of patients with intermediate risk. About 60 % of mutations in this gene have been identified in the arginine codon R882. To date, there is no consensus on whether these mutations can be used as biomarkers for monitoring of minimal residual disease and management of preemptive AML therapy. We studied the occurrence of mutations in the DNMT3A gene in our cohort of patients and their persistence during AML treatment. Using next-generation sequencing, we identified four mutations in 11/25 of our analyzed patients--frequent R882C and R882H mutations, rare Y735S mutation, and a novel L347P mutation. Mutation R882C was detected in 5/11, R882H in 4/11 patients, and Y735S and L347P in one patient each. In 4/7 patients initially carrying mutations in the R882 codon, we found the persistence of mutations also during complete remission with, however, no correlation to AML kinetics. Our findings suggest that mutations in the DNMT3A gene can only be used as a biomarker for those AML patients in whom DNMT3A mutation is lost after therapy.