title: Next-generation sequencing of lung cancer EGFR exons 18-21 allows effective molecular diagnosis of small routine samples (cytology and biopsy).
abstract: Selection of lung cancer patients for therapy with tyrosine kinase inhibitors directed at EGFR requires the identification of specific EGFR mutations. In most patients with advanced, inoperable lung carcinoma limited tumor samples often represent the only material available for both histologic typing and molecular analysis. We defined a next generation sequencing protocol targeted to EGFR exons 18-21 suitable for the routine diagnosis of such clinical samples. The protocol was validated in an unselected series of 80 small biopsies (n=14) and cytology (n=66) specimens representative of the material ordinarily submitted for diagnostic evaluation to three referral medical centers in Italy. Specimens were systematically evaluated for tumor cell number and proportion relative to non-neoplastic cells. They were analyzed in batches of 100-150 amplicons per run, reaching an analytical sensitivity of 1% and obtaining an adequate number of reads, to cover all exons on all samples analyzed. Next generation sequencing was compared with Sanger sequencing. The latter identified 15 EGFR mutations in 14/80 cases (17.5%) but did not detected mutations when the proportion of neoplastic cells was below 40%. Next generation sequencing identified 31 EGFR mutations in 24/80 cases (30.0%). Mutations were detected with a proportion of neoplastic cells as low as 5%. All mutations identified by the Sanger method were confirmed. In 6 cases next generation sequencing identified exon 19 deletions or the L858R mutation not seen after Sanger sequencing, allowing the patient to be treated with tyrosine kinase inhibitors. In one additional case the R831H mutation associated with treatment resistance was identified in an EGFR wild type tumor after Sanger sequencing. Next generation sequencing is robust, cost-effective and greatly improves the detection of EGFR mutations. Its use should be promoted for the clinical diagnosis of mutations in specimens with unfavorable tumor cell content.
authors.affiliation: Department of Medicine (DIMES) - Anatomic Pathology Unit, Bellaria Hospital, University of Bologna, Bologna, Italy.
authors.forename: Dario
authors.initials: D
authors.lastname: de Biase
authors.affiliation: Department of Pharmacology and Biotechnology (FaBiT), University of Bologna, Bologna, Italy.
authors.forename: Michela
authors.initials: M
authors.lastname: Visani
authors.affiliation: Department of Public Health, University of Naples Federico II, Naples, Italy.
authors.forename: Umberto
authors.initials: U
authors.lastname: Malapelle
authors.affiliation: Department of Medicine (DIMED) - Surgical Pathology & Cytopathology Unit, University of Padua, Padua, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Simonato
authors.affiliation: Department of Medicine (DIMES) - Anatomic Pathology Unit, Bellaria Hospital, University of Bologna, Bologna, Italy ; Department of Pharmacology and Biotechnology (FaBiT), University of Bologna, Bologna, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Cesari
authors.affiliation: Department of Public Health, University of Naples Federico II, Naples, Italy.
authors.forename: Claudio
authors.initials: C
authors.lastname: Bellevicine
authors.affiliation: Department of Pharmacology and Biotechnology (FaBiT), University of Bologna, Bologna, Italy.
authors.forename: Annalisa
authors.initials: A
authors.lastname: Pession
authors.affiliation: Department of Public Health, University of Naples Federico II, Naples, Italy.
authors.forename: Giancarlo
authors.initials: G
authors.lastname: Troncone
authors.affiliation: Department of Medicine (DIMED) - Surgical Pathology & Cytopathology Unit, University of Padua, Padua, Italy.
authors.forename: Ambrogio
authors.initials: A
authors.lastname: Fassina
authors.affiliation: Department of Medicine (DIMES) - Anatomic Pathology Unit, Bellaria Hospital, University of Bologna, Bologna, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Tallini
mesh_terms: D000368:Aged; D002289:Carcinoma, Non-Small-Cell Lung / Q000175:diagnosis* / Q000235:genetics / Q000473:pathology; D004252:DNA Mutational Analysis / Q000379:methods*; D066246:ErbB Receptors / Q000235:genetics*; D005091:Exons / Q000235:genetics*; D005260:Female; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008175:Lung Neoplasms / Q000175:diagnosis* / Q000235:genetics / Q000473:pathology; D008297:Male; D025202:Molecular Diagnostic Techniques / Q000379:methods*
chemical_list: D066246:ErbB Receptors