Dear Sir, Gefitinib (Iressa) is a small-molecule inhibitor of the epidermal growth factor receptor (EGFR) that produces markedly favorable responses in a subset of patients with nonsmall cell lung cancer (NSCLC). Activating somatic mutations in the EGFR tyrosine-kinase domain (TKD) are strongly predictive of both clinical and in vitro sensitivity to gefitinib. To date, gefitinib-sensitizing EGFR mutations have been detected almost exclusively in lung adenocarcinomas, with only 1 missense TKD mutation identified in a colorectal carcinoma. The paucity of gefitinib-sensitizing mutations in cancers exclusive of the lung is consistent with the modest therapeutic responses gefitinib has generally shown in early clinical studies of nonlung cancers. However, not all cancers have been clinically evaluated for gefitinib responsiveness, and screening for gefitinib-sensitizing mutations may be an efficient means of detecting additional responsive cancers. Patients with mesothelioma have a notoriously poor prognosis, with few therapeutic options beyond palliative surgery. Although mesotheliomas have been shown to overexpress EGFR and have been considered as targets for EGFR inhibitors, a screen for EGFR TKD mutations in this cancer has not been reported. In this letter, we describe our screening of human malignant mesothelioma for the most common EGFR TKD mutations identified in gefitinib-responsive NSCLC. A point mutation at EGFR nucleotide 2573 (TfiG) in exon 21, conferring an arginine for leucine substitution at position 858 (L858R), and a series of in-frame deletions in exon 19 have together comprised 70–100% of the TKD mutations detected in gefitinib-responsive NSCLC in the 4 relevant studies published to date. Three additional point mutations, at nucleotide 2582 (TfiA) in exon 21 for L861Q and 2 exon 19 substitutions at nucleotide 2155 (GfiT or A) for G719S and G719C, respectively, have been identified with a 0–20% combined frequency. We developed PCR-based assays to rapidly screen tumor DNA for these 5 somatic mutations One primer in each PCR was labeled with a cyanine-based fluorescent dye (D3-PA or D4-PA) for subsequent fragment analysis using the CEQ 8000 Genetic Analysis System (Beckman Coulter, Fullerton, CA). For detection of deletions in the EGFR exon 19, primers 50-D3-PA-GCTGGTAACATCCACCCAGA-30 and 50-GAGAAAAGGTGGGCCTGAG-30 were designed to amplify a 247 bp region encompassing the entire EGFR exon 19, which was directly sized by fragment analysis. The EGFR point mutations were identified by endonuclease digestion and restriction fragment analysis of their respective amplicons. Primers utilized for genotyping the 858 and 861 codons were 50-D3-PA-GCAGAGCTTCTTCCCATGAT-30 and 50-CTGACCTAAAGCCACCTCCTT-30 and for the 760 codon, 50-D4-PA-GCTGAGGTGACCCTTGTCTC-30 and 50-CCTGTGCCAGGGACCTTA-30. For codon 858, Fau I specifically cleaves the TfiG mutation, such that the 235 bp 858/ 861 amplicon yields a 154 bp labeled restriction fragment. For codon 861, the mutant TfiA is cut by Pvu II, reducing the 858/861 amplicon to a 170 bp labeled fragment. The 182 bp codon 760 amplicon was digested with BsiHKA1, which cleaves at 135 bp both GfiT and GfiA mutants, can be subsequently discriminated by Sac I and cuts exclusively the GfiA. PCRs were performed in 5 ll or 10 ll reactions using PCR Master Mix (Promega, Madison, WI) and 5 ng of tumor DNA purified with the DNeasy 96 Tissue Kit (Qiagen, Valencia, CA), both according to manufacturer’s recommendations. Cycling parameters for all PCRs were as follows: primary denaturation at 95 C for 5 min, then 25 to 30 cycles of 95 C denaturation for 30 sec, 62 C annealing for 30 sec and 72 C extension for 30 sec, followed by a final extension for 5 min. All endonucleases were purchased from New England Biolabs (Beverly, MA) and used with appropriate controls according to manufacturer’s recommendations. One microliter of unpurified PCR was digested for 4–6 hours in a 10 ll reaction volume, 5 ll of which was subsequently analyzed in the CEQ8000 using the standard fragment analysis parameters. Sixty-six patients underwent resection for mesothelioma at the Karmanos Cancer Institute at Wayne State University in Detroit, Michigan, from 2001–2004. The case series is an expansion of that described elsewhere and includes 56 males and 10 females, all Caucasian and with a mean age of 66 years (range 41–87). Mesothelioma DNA from each subject was successfully extracted and screened for EGFR mutations. No size polymorphisms in EGFR exon 19 were detected, nor were point mutations found in any of the evaluated codons: 858, 861 and 719. In a concurrent screen of 99 lung adenocarcinoma specimens, 19 (19%) were found to harbor either a heterozygous inframe deletion in exon 19 (9 samples) or a heterozygous L858R (10 samples). Direct DNA sequencing of the amplicons derived from the lung cancer mutants and an equal number of wild-type specimens confirmed the results of our screening assays.