RESUMEN
BACKGROUND: The epidermal growth factor receptor (EGFR) mutation status assessment has become increasingly important given the significant impact of tyrosine kinase inhibitors in lung cancer management. Our aim was to compare real life operational characteristics for three EGFR mutation assays - two targeted approaches and a next generation sequencing (NGS) technique. METHODS: EGFR mutation status was assessed for lung adenocarcinoma samples (formalin fixed- paraffin embedded samples) using qPCR, SNaPshot and NGS (Ion Torrent™) techniques. RESULTS: A total of 15 high clinical significance mutations were identified by at least one technique from the total of 64 samples. All mutations were identified by the TaqMan qPCR technique while SNaPshot in conjunction with fragment analysis identified 11 EGFR mutations. The NGS approach followed by an automatic analysis using the default calling parameters identified 10 mutations from the SNaPshot/qPCR panel and other three insertions, five point mutations and 58 silent variants; manual data review identified all 15 high significance mutations. CONCLUSIONS: Performance was similar for high tumor content samples but careful data analysis and post hoc variant calling filter alterations were necessary in order to obtain robust results from low tumor content samples by NGS. NGS is able to generate a comprehensive mutational profile albeit at a higher cost and workload. Result interpretation should take into account not only general run parameters such as mean read depth but also relative coverage and read distribution; currently there is an acute need to define firm recommendations/standards concerning NGS data interpretation and quality control.
Asunto(s)
Adenocarcinoma/genética , Análisis Mutacional de ADN/métodos , Receptores ErbB/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Neoplasias Pulmonares/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Adenocarcinoma del Pulmón , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , MutaciónRESUMEN
INTRODUCTION: Lung cancer's dismal prognosis led to new therapeutic approaches among which TKIs being among most promising; MATERIAL AND METHOD: Retrospective study at the Regional Institute of Oncology Iasi of non small cell lung cancer patients which underwent molecular investigations between November 2013 - September 2014. EGFR mutation status (positive, negative, undetermined) was assessed with an Entrogen EGFR kit using DNA extracted from paraffin embedded samples (surgical or endobronchial biopsies) with the Macherey-Nagel "NucleoSpin FFPE DNAkit" and then amplified on a Applied Biosystem 7500 Real Time PCR System. RESULTS: There were 63 adenocarcinoma samples (17 females, mean age 60,9 +/- 9 years): 49 primary lung tumors and 14 secondary lesions (brain, lymph nodes, pleural). There was insufficient bioptic material for three cases. TTF1 status was determined for 46 patients--six were negative. There were twelve mutations identified (7 female subjects, 5 male)--six L858R, five Del 19 and one G719X; ten were TTF1 positive for the remaining two TTF1 status was unknown. Female sex predominance was statistically significant (p = 0.02, chi squared). Mean age for mutation positive patients was 64 +/- 10 years; there were three never smokers, three active smokers and no data on smoking status was available for six subjects. CONCLUSION: Although small dimension of the study group precludes statistical significance EGFR mutations seem to correlate with TTF1 status.