Journal: PLoS ONE

Article Title: The Use of a Two-Tiered Testing Strategy for the Simultaneous Detection of Small EGFR Mutations and EGFR Amplification in Lung Cancer

doi: 10.1371/journal.pone.0117983

Figure Lengend Snippet: A) Map of the EGFR gene with the positions of the RT-PCR amplicons (EGFR-RT52) and MLPA probes (EGFRmut+ assay) indicated (vertical lines under the map). The mutation-sensitive EGFRmut+ probes are indicated in red. The positions of oncogenic EGFR mutations are indicated over the map. B) The RT-PCR results representing (from the top) (i) the reference sample (a sample with no mutations or amplification), (ii) a sample with the most common in-frame deletion in exon 19 (c.2235_2249del15), (iii) a sample with both L858R in exon 21 and T790M in exon 20, and (iv) a sample with an in-frame deletion in exon 19 (c.2235_2249del15) and EGFR amplification. In each graph, the overlapping results of the 8 RT-PCR reactions covering 29 EGFR mutations are shown. The red lines indicate the positive amplification-curves of specific EGFR mutations (pointed on graph), and the green base line represents the non-amplified signal due to the lack of the evaluated mutation in the analyzed sample. C) MLPA electropherograms of samples analyzed by RT-PCR (panel B). The probe IDs are indicated under the electropherograms. The asterisks indicate the control probes; the pink arrowheads indicate reduced signal of MS- probes and increased signal of MS+ probes, respectively; and the black arrowheads indicate amplified signals of EGFR -specific probes. D) Bar plots corresponding to the electropherograms shown in panel C and representing the normalized copy number value (y-axis) of each probe (x-axis). The pink arrowheads indicate a reduced copy number value of the MS- probes and an increased copy number value of the MS+ probes, respectively.

Article Snippet: All 239 samples were analyzed as blind samples by two methods: (i) a commercial RT-PCR assay (EGFR-RT52; Entrogen Inc.) that covers 29 of the most common oncogenic mutations in exons 18, 19, 20 and 21 of EGFR (for details, see the manufacturers webpage; http://www.entrogen.com/web2/egfr-mutation-analysis-kit ) and (ii) a custom-designed MLPA assay (EGFRmut+) that simultaneously allows the detection of amplifications and small mutations in EGFR (for details see [ ]) ( ).

Techniques: Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Amplification

Journal: PLoS ONE

Article Title: The Use of a Two-Tiered Testing Strategy for the Simultaneous Detection of Small EGFR Mutations and EGFR Amplification in Lung Cancer

doi: 10.1371/journal.pone.0117983

Figure Lengend Snippet: A) Examples of EGFR (samples v-vi), MET (samples vii-viii) and ERBB2 (samples ix-x) amplification detected by the EGFRmut+ assay. B) Characteristics of samples with EGFR , MET and ERBB2 gains/amplifications. The samples shown in (ii-iii) and (vi-x) are indicated in the first column. The sex and EGFR mutation status of each sample are indicated in the second and the third columns, respectively. Columns 4–6 show the copy number values of EGFR , MET and ERBB2 , respectively. The dark blue cells indicate amplifications (copy number ≥4), and the light blue cells indicate gains (copy number 3–4). C) Frequency of EGFR mutations in samples with EGFR amplification, EGFR gain and normal EGFR copy number.

Article Snippet: All 239 samples were analyzed as blind samples by two methods: (i) a commercial RT-PCR assay (EGFR-RT52; Entrogen Inc.) that covers 29 of the most common oncogenic mutations in exons 18, 19, 20 and 21 of EGFR (for details, see the manufacturers webpage; http://www.entrogen.com/web2/egfr-mutation-analysis-kit ) and (ii) a custom-designed MLPA assay (EGFRmut+) that simultaneously allows the detection of amplifications and small mutations in EGFR (for details see [ ]) ( ).

Techniques: Amplification, Mutagenesis