Complex Mutations & Subpopulations of Deletions at Exon 19 of EGFR in NSCLC Revealed by Next Generation Sequencing: Potential Clinical Implications

<div><p>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 <em>EGFR</em> 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.</p> </div

Sanger sequencing (SS) analysis of two mutated cases (#70 and #31) compared with a wild type reference DNA.

<p>Wild type and deleted alleles are superimposed in SS electropherograms. In case #70, carrying a 2236–2250del, the peaks are perfectly aligned and the starting point of the deletion at base 2236 is easily detectable. Next generation sequencing (NGS) confirmed this type of deletion. In case #31, carrying the same mutation, as detected by NGS, peaks in the SS electropherogram are not well aligned and the starting point of the deletion was incorrectly positioned by the operator at base 2237.</p

Immunohistochemical staining with deletion specific 6B6 monoclonal antibody in Non-Small Cell Lung Cancers carrying different deletions at exon 19 of the <i>EGFR</i> gene detected by Next Generation Sequencing.

a<p>The score system is described in detail in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042164#s4" target="_blank">Materials and Methods</a>.</p

Examples of subpopulations of EGFR deletions at exon 19.

<p>The Figure reports 10 selected cases of tumors (#53, #59, #47, #38, #84, #27, #89, #49, #17, #77) showing subpopulations of EGFR deletions at exon 19. In each case the first line corresponds to the wild type sequence. The different bases are highlighted by different colours. Deleted bases are reported as dashes. The black bars under the sequences indicate the consensus for the different bases involved in deletions. On the right of each case is reported the percentage with which the wild type and deleted molecules were present in tumor DNA. The number (N) of sequences obtained in each case were as follow: #53 (N = 6.484), #59 (N = 5.835), #47 (N = 5.629), #38 (N = 4.776), #84 (N = 2.641), #27 (N = 4.389), #89 (N = 3.855), #49 (N = 2.172), #17 (N = 2.856), #77 (N = 3.526). Cases carrying subpopulations in less than 0.1% of the DNA molecules are labeled with an asterisk.</p

Different interpretation of sequence data in case of complex mutations.

<p>A complex in frame deletion of 15 bp (A) can be considered as composed of two non-in frame deletions of 8 bp and 7 bp separated by a consensus sequence of 7 bp, in other words a double deletion. Conventionally, this mutation could have been interpreted as the sum of a non-in frame deletion of 19 bp and non-in frame insertion of 4 bp. Accordingly, in B and C are reported different interpretations for mutations giving rise to complex in frame deletions of 18 bps. Deleted bases are indicated by dashes. Inserted bases are reported a in a frame under the sequence. Correspondence with numbers of tumor samples: A (#59); B (#22); C (#53).</p

Cases in which the accurate sequence of deleted bases was incorrectly determined or not assessable by Sanger sequencing compared with data obtained by Next generation sequencing.

<p>Abbreviations: NA, the exact sequence of deleted bases could not be accurately defined (not assessable).</p>a<p>Novel mutation.</p

Comparison of data obtained by Sanger Sequencing and Next Generation Sequencing analysis on cases carrying complex deletions.

a<p>Novel mutation.</p