Detection and characterization of classical and “uncommon” exon 19 Epidermal Growth Factor Receptor mutations in lung cancer by pyrosequencing

Abstract

BackgroundThe management of advanced stage non-small cell lung cancer is increasingly based on diagnostic and predictive analyses performed mostly on limited amounts of tumor tissue. The evaluation of Epidermal Growth Factor Receptor (EGFR) mutations have emerged as the strongest predictor of response to EGFR-tyrosine kinase inhibitors mainly in patients with adenocarcinoma. Several EGFR mutation detection techniques are available, having both sensitivity and specificity issues, being the Sanger sequencing technique the reference standard, with the limitation of a relatively high amount of mutated cells needed for the analysis.MethodsA novel nucleotide dispensation order for pyrosequencing was established allowing the identification and characterization of EGFR mutation not definable with commercially and clinically approved kits, and validated in a consecutive series of 321 lung cancer patients (246 biopsies or cytology samples and 75 surgical specimens).Results61/321 (19%) mutated cases were detected, 17 (27.9%) in exon 21 and 44 (72.1%) in exon 19, these latter corresponding to 32/44 (72.7%) classical and 12/44 (27.3%) uncommon mutations. Furthermore, a novel, never reported, point mutation, was found, which determined a premature stop codon in the aminoacidic sequence that resulted in a truncated protein in the tyrosine kinase domain, thus impairing the inhibitory effect of specific therapy.ConclusionsThe novel dispensation order allows to detect and characterize both classical and uncommon EGFR mutations. Although several phase III studies in genotypically defined groups of patients are already available, further prospective studies assessing the role of uncommon EGFR mutations are warranted.

Increase EGFR Mutations Detection Rate in Lung Adenocarcinoma by Real-Time PCR Screening Followed by Direct Sequencing

“…20,21,[27][28][29][30] The majority of EGFR mutations were in-frame deletions in exon 19. In this study, in concordance with data from others, 7,9 the majority of EGFR mutations were located at exons 19 and 21, comprising 53% of EGFR mutations.…”

Molecular and Histological Changes in Post-Treatment Biopsies of Non-Squamous Non-Small Cell Lung Cancer: A Retrospective Study

“…One of the pathologists (LR) re-assessed each tumour sample for adequacy (set at a minimum of 30% of tumor cells [9]) according to current recommendations [10].…”

“…Genomic DNA from FFPE tissue samples was extracted and purified using QIAmp DNA FFPE Tissue kit (Qiagen, Hilden -Germany), according to the manufacturer's instructions and quantified by spectrophotometry (Eppendorf, Hamburg, Germany), as previously described [9].…”

“…Mutational analyses of EGFR (NM_005228) in exon 18 (codon 719 variants), exon 21 (codons 858 and 861 variants) and exon 19 (from codons 746 to 750) were performed using EGFR TKI response (sensitivity) kit (CE-IVD, Diatech, Jesi -Italy) [9]. Furthermore, 100 ng of genomic DNA were used to amplify target regions containing mutational hotspots of KRAS exon 2 (NM_4985.…”

“…Direct sequencing was performed for mutational analysis of genes investigated using PyroMark Q96 machine (Biotage, Uppsala -Sweden). Pyrosequencing results were further validated by allelic-specific PCR (ARMS) of EGFR exons 18,19 and 21, as indicated by guidelines [10] and previously described [9].…”