Complex mutations & subpopulations of deletions at exon 19 of EGFR in NSCLC revealed by next generation sequencing

Potential clinical implications

Antonio Marchetti, Maela Del Grammastro, Giampaolo Filice, Lara Felicioni, Giulio Rossi, Paolo Graziano, Giuliana Sartori, Alvaro Leone, Sara Malatesta, Michele Iacono, Luigi Guetti, Patrizia Viola, Felice Mucilli, Franco Cuccurullo, Fiamma Buttitta

Research output: Contribution to journalArticle

41 Citations (Scopus)

Abstract

Microdeletions at exon 19 are the most frequent genetic alterations affecting the Epidermal Growth Factor Receptor (EGFR) gene in non-small cell lung cancer (NSCLC) and they are strongly associated with response to treatment with tyrosine kinase inhibitors. A series of 116 NSCLC DNA samples investigated by Sanger Sequencing (SS), including 106 samples carrying exon 19 EGFR deletions and 10 without deletions (control samples), were subjected to deep next generation sequencing (NGS). All samples with deletions at SS showed deletions with NGS. No deletions were seen in control cases. In 93 (88%) cases, deletions detected by NGS were exactly corresponding to those identified by SS. In 13 cases (12%) NGS resolved deletions not accurately characterized by SS. In 21 (20%) cases the NGS showed presence of complex (double/multiple) frameshift deletions producing a net in-frame change. In 5 of these cases the SS could not define the exact sequence of mutant alleles, in the other 16 cases the results obtained by SS were conventionally considered as deletions plus insertions. Different interpretative hypotheses for complex mutations are discussed. In 46 (43%) tumors deep NGS showed, for the first time to our knowledge, subpopulations of DNA molecules carrying EGFR deletions different from the main one. Each of these subpopulations accounted for 0.1% to 17% of the genomic DNA in the different tumors investigated. Our findings suggest that a region in exon 19 is highly unstable in a large proportion of patients carrying EGFR deletions. As a corollary to this study, NGS data were compared with those obtained by immunohistochemistry using the 6B6 anti-mutant EGFR antibody. The immunoreaction was E746-A750del specific. In conclusion, NGS analysis of EGFR exon 19 in NSCLCs allowed us to formulate a new interpretative hypothesis for complex mutations and revealed the presence of subpopulations of deletions with potential pathogenetic and clinical impact.

Original languageEnglish
Article numbere42164
JournalPLoS One
Volume7
Issue number7
DOIs
Publication statusPublished - Jul 27 2012

Fingerprint

lung neoplasms
Epidermal Growth Factor Receptor
Non-Small Cell Lung Carcinoma
exons
Exons
mutation
Mutation
cells
DNA
Tumors
erbB-1 Genes
sampling
mutants
neoplasms
Protein-Tyrosine Kinases
Neoplasms
Immunohistochemistry
Alleles
immunohistochemistry
tyrosine

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Complex mutations & subpopulations of deletions at exon 19 of EGFR in NSCLC revealed by next generation sequencing : Potential clinical implications. / Marchetti, Antonio; Del Grammastro, Maela; Filice, Giampaolo; Felicioni, Lara; Rossi, Giulio; Graziano, Paolo; Sartori, Giuliana; Leone, Alvaro; Malatesta, Sara; Iacono, Michele; Guetti, Luigi; Viola, Patrizia; Mucilli, Felice; Cuccurullo, Franco; Buttitta, Fiamma.

In: PLoS One, Vol. 7, No. 7, e42164, 27.07.2012.

Research output: Contribution to journalArticle

Marchetti, A, Del Grammastro, M, Filice, G, Felicioni, L, Rossi, G, Graziano, P, Sartori, G, Leone, A, Malatesta, S, Iacono, M, Guetti, L, Viola, P, Mucilli, F, Cuccurullo, F & Buttitta, F 2012, 'Complex mutations & subpopulations of deletions at exon 19 of EGFR in NSCLC revealed by next generation sequencing: Potential clinical implications', PLoS One, vol. 7, no. 7, e42164. https://doi.org/10.1371/journal.pone.0042164
Marchetti, Antonio ; Del Grammastro, Maela ; Filice, Giampaolo ; Felicioni, Lara ; Rossi, Giulio ; Graziano, Paolo ; Sartori, Giuliana ; Leone, Alvaro ; Malatesta, Sara ; Iacono, Michele ; Guetti, Luigi ; Viola, Patrizia ; Mucilli, Felice ; Cuccurullo, Franco ; Buttitta, Fiamma. / Complex mutations & subpopulations of deletions at exon 19 of EGFR in NSCLC revealed by next generation sequencing : Potential clinical implications. In: PLoS One. 2012 ; Vol. 7, No. 7.
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abstract = "Microdeletions at exon 19 are the most frequent genetic alterations affecting the Epidermal Growth Factor Receptor (EGFR) gene in non-small cell lung cancer (NSCLC) and they are strongly associated with response to treatment with tyrosine kinase inhibitors. A series of 116 NSCLC DNA samples investigated by Sanger Sequencing (SS), including 106 samples carrying exon 19 EGFR deletions and 10 without deletions (control samples), were subjected to deep next generation sequencing (NGS). All samples with deletions at SS showed deletions with NGS. No deletions were seen in control cases. In 93 (88{\%}) cases, deletions detected by NGS were exactly corresponding to those identified by SS. In 13 cases (12{\%}) NGS resolved deletions not accurately characterized by SS. In 21 (20{\%}) cases the NGS showed presence of complex (double/multiple) frameshift deletions producing a net in-frame change. In 5 of these cases the SS could not define the exact sequence of mutant alleles, in the other 16 cases the results obtained by SS were conventionally considered as deletions plus insertions. Different interpretative hypotheses for complex mutations are discussed. In 46 (43{\%}) tumors deep NGS showed, for the first time to our knowledge, subpopulations of DNA molecules carrying EGFR deletions different from the main one. Each of these subpopulations accounted for 0.1{\%} to 17{\%} of the genomic DNA in the different tumors investigated. Our findings suggest that a region in exon 19 is highly unstable in a large proportion of patients carrying EGFR deletions. As a corollary to this study, NGS data were compared with those obtained by immunohistochemistry using the 6B6 anti-mutant EGFR antibody. The immunoreaction was E746-A750del specific. In conclusion, NGS analysis of EGFR exon 19 in NSCLCs allowed us to formulate a new interpretative hypothesis for complex mutations and revealed the presence of subpopulations of deletions with potential pathogenetic and clinical impact.",
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