5% of the DNA was mutated. Table 3 Comparison of EGFR status (wild type (WT) or mutant (M)) of exon 19 and exon 21 determined by big dye sequencing or by pyrosequencing

on 58 NSCLC tissues Exon 19   big dye sequencing Exon 21   big dye sequencing     WT M     WT M pyrosequencing WT 47 / pyrosequencing WT 53 /   M 2 9   M 1 4 We then determined the EGFR status of 213 patients with advanced or metastatic lung adenocarcinomas for selection of to anti EGFR therapies (table 4). MDV3100 nmr Seven (3.3%) samples were inconclusive due to poor DNA quality with no DNA amplification. Of the 206 remaining samples, 18 EGFR mutations were detected (8 of exon 19 and 10 of exon 21) (18/206; 8.7%). Among these 206 specimens, 36 had less than 20% of tumor cells and only one with a mutation was detected (1/36; 2.8%). For the 170 specimens containing more than 20% of tumor cells, 17 with mutations were found (17/170; 10%). Table 4 Prospective evaluation of the PP2 clinical trial EGFR status of exons 19 and 21 % of tumoral tumoral samples (n = 206) EGFR mutations (n = 18)   cells number

% exon 19 exon 21 % <20% 36 17.5 0 1 2.8 from 20 to 50% 98 47.6 3 6 9.2 >50% 72 35 5 3 11.1 Samples may contain at least 20% of tumor cells to allow a IACS-10759 purchase correct detection of mutations Discussion Pyrosequencing is sensitive and enables accurate detection of mutations. A previous study has described the capacity of this method to detect small insertions [9] but this study is the first to demonstrate the application of pyrosequencing to exon 19 deletions. Analysis of exon 21 by pyrosequencing had been succinctly described by Takano et al. [10, 11], but without any data about the specificity, the repeatability or the sensitivity. We first investigated the characteristics of EGFR mutations in the lung cancer cell lines NCI-H1650 and NCI-H1975 and used them as positive controls for the deletion in exon19 and the point mutation in exon 21 respectively. Moreover we used the DNA of these cells mixed with DNA isolated from blood samples from healthy volunteers to evaluate the basic properties of our novel method. We didn’t observe strict linearity

because the two cell lines (NCI-H1650 and NCI-H1975) have respectively 4 and 2.8 EGFR gene copies Vasopressin Receptor [12] but we found good sensitivity. In routine daily practice fixed paraffin-embedded specimens, most often of small size, are the only samples available for both diagnosis and molecular analyses. The DNA is frequently fragmented, which could hamper PCR amplification. However, the PCR conditions described in this study allowed analysis of 96.7% of the paraffin-embedded tissues whatever the type of fixative used or the duration of the fixation. When the samples could be amplified and analyzed, results were concordant (97.4%) with those obtained by conventional BigDye terminator sequencing. The difference in sensitivity between the two methods is illustrated by the 3 samples characterized as mutated only by pyrosequencing.