Highly Sensitive Real-Time PCR for the Detection of EGFR Mutations in Lung Adenocarcinoma. Is It Worth It?
Natalia Rodon, Ruth Roman, Montse Verdu, Beatriz Garcia, Merce Pujol, Xavier Puig. BIOPAT.Biopatologia Molecular, SL, Grup Assistencia, Barcelona, Spain; Hospital de Barcelona, SCIAS, Grup Assistencia, Barcelona, Spain; Histopat Laboratoris, Barcelona, Spain
Background: Mutations involving the epidermal growth factor receptor (EGFR) correlate with responsiveness to tyrosine kinase inhibitors (TKIs), which significantly improves patient survival in lung adenocarcinoma (AC). These mutations are described to be more frequent in non-mucinous lepidic adenocarcinomas (nmBAC). The unquestionable importance of determining the presence of these mutations, even in samples with a small tumor representation, has boosted the appearance of highly sensitive methods which would allow the detection of 1-5% mutated DNA. The aim of this study was to test the ability of a real-time PCR kit to detect EGFR mutations in a series of lung AC considered wild-type by the Sanger's sequencing method.
Design: 52 primary lung ACs were revised by two independent pathologists to establish histological type and immunoprofile (CK7, CK5/6, 34βE12, CK20, p63 and TTF1) of every case. DNA was extracted from formalin-fixed paraffin embedded sections of every tumor sample. EGFR mutational status analysis of exons 19, 20 and 21 was performed by Sanger's direct sequencing and by the real-time PCR kit Therascreen EGFR PCR (Qiagen). Fluorescent in-situ hybridization (FISH) was performed to assess the copy number status of EGFR gene using Vysis LSI EGFR SpectrumOrange/CEP7 SpectrumGreen Probe (Abbot Molecular).
Results: Sanger's direct sequencing method allowed the determination of 7 cases with EGFR mutations (13.5%) and 45 EGFR wild-type tumor samples. Therascreen EGFR PCR kit detected 4 new cases with EGFR mutations, three of which showed 100% infiltrating nmBAC pattern and one acinar with 50% nmBAC component, increasing the global number of mutant cases to 11 (21.2 %). The most prevalent histological subtype in the mutated group corresponds to nmBAC pattern, being present in 8 of 11 (72.7%) cases. FISH analysis reveals 9 of 11 (81.8%) EGFR mutated cases being FISH positive, 2 by EGFR amplification and 7 with high polisomy.
Conclusions: The use of real-time PCR in the global series increases the percentage of EGFR mutated samples detected by 7.7%. Considering that all 4 recovered cases have nmBAC component, the use of highly sensitive techniques in this subtype of AC should be recommended despite their substantial cost.
Tuesday, March 20, 2012 1:00 PM
Poster Session IV # 297, Tuesday Afternoon