Sequence homology (at the protein level) and expression distribution in human tissues among Gq family genes.

<p>Sequence homology (at the protein level) and expression distribution in human tissues among Gq family genes.</p

Number of samples analyzed for each histological type and the number of mutations identified.

<p><b>(AML</b>: acute myeloid leukemia, <b>GIST</b>: Gastrointestinal Stromal Tumors; In addition, p-values of the Fisher's exact test, used to determine the tissue specificity for <i>GNAQ</i>^Q209 mutations in blue naevi, are listed.)</p

PCR primers used for the mutational profiling.

<p>PCR primers used for the mutational profiling.</p

Example of one of the <i>GNAQ</i> mutations identified in blue naevi.

<p>The arrow indicates the location of the missense change p.Q209L (c.A627T).</p

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

Comparison of <i>EGFR</i> mutations in primary tumors and CTC preparations.

<p>Footnotes: CTCs, Circulating Tumor Cells; SS, Sanger sequencing; NGS, next generation sequencing.</p><p>*case with double or multiple mutations.</p><p>**blood-derived material in the Cell Search cartridge containing CTCs or potential neoplastic elements.</p

Different types of events presented by the CellSearch System.

<p>1–2. Two classical examples of CTC fulfilling all the Veridex criteria: A) intact round to oval cells positive for epithelial cell marker (CK-PE) of more than 4 µm in size; B) positivity for the nuclear dye (DAPI) in an area smaller than the cytoplasmic area inside the cytoplasm (at least 50%); C) Negativity for the leucocyte marker (CD45/APC); D) negativity in the blank channel. 3. A suspicious object satisfying only A, C, and D criteria. 4. A large naked nucleus. 5. A cluster of naked nuclei.</p

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