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Tissue Procurement. All specimens were reviewed by a single reference pathologist (M.Z.) to confirm diagnosis and predominance (>70%) of cancer tissue in tumor specimens. Bronchioloalveolar carcinoma (BAC) was defined as described previously (1). Imaging studies were assessed by a single reference radiologist (R.H.), who graded partial responses according to Response Evaluation Criteria in Solid Tumors (RECIST; i.e., at least a 30% decrease in the sum of the longest diameter of target lesions, taking as reference the baseline sum longest distance) (2). Both observers were blinded to patient outcomes.

Patients on gefitinib received 250 or 500 mg orally once daily. Patients on erlotinib received 150 mg orally once daily. Eight of 10 patients with tumors sensitive to gefitinib had partial responses. The two other patients (G6 and G10) had marked clinical improvement as ascertained by two independent reviewing physicians and manifested by lessened dyspnea and cancer-related pain. However, these individuals had radiographic lesions that did not meet RECIST guidelines. Because erlotinib-treated patients were all on a clinical trial, all had disease measurable using RECIST guidelines. For both drugs, tumors were considered refractory if patients experienced progression of disease as defined by RECIST (i.e., at least a 20% increase in the sum of the longest diameter of target lesions, taking as reference the smallest sum longest diameter recorded since the treatment started or the appearance of one or more new lesions). Patients with stable disease were excluded from this study.

The freshly frozen tumors used in this study were derived from a surgical lung tumor bank comprised of 230 samples resected from untreated patients with stage I, II, and IIIA disease. A total of 96 tumors were randomly selected for use, based upon availability of adequate quantities of extractable DNA. Fifteen tumors from "never smokers" with adenocarcinoma histology were identified. The other 81 NSCLCs analyzed had the following histologies: 33 adenocarcinomas, 24 squamous cell carcinomas, 22 adenocarcinomas with BAC features (AWBF), 1 BAC with focal invasion (BWF1), and 1 pure BAC (PBAC).

For paraffin-embedded tumors, matched peripheral blood from patients was used as a source of nontumor DNA. For frozen tumor specimens, both tumor and matched benign tissue (defined as grossly normal-appearing tumor at least 5 cm away from the primary tumor) were snap frozen in liquid nitrogen at the time of resection. All frozen tissue samples were stored at -80°C until use and were reviewed by a reference pathologist as above. Clinical information was collected prospectively and maintained in a database without patient identifiers. DNeasy tissue kits (Qiagen, Valencia, CA) were used to extract genomic DNA. The majority of paraffin-embedded tissues were analyzed at the Memorial Sloan-Kettering Cancer Center (MSKCC), and the majority of fresh-frozen specimens were analyzed at Washington University (St. Louis) (WUStL).

PCR and Sequencing (MSKCC). Ten 5-m m thick sections from paraffin-embedded tissues were cut and deparaffinized with Citrosolv. PCR was performed on genomic DNA with Amplitaq DNA polymerase (Roche Applied Science) and standard buffers with annealing temperatures of 58°C and 35 cycles of amplification, using nested PCR primer sets. Reference EGFR sequence was obtained from LocusLink number 1956 and GenBank accession number NT_033968 (Table 4).

PCR products were purified with a MultiScreen Resist vacuum manifold and PCR₉₆ Cleanup Plates (Millipore). Sequencing reactions were performed by using Applied Biosystems Version 3.1 Big Dye Terminator chemistry and then analyzed on an Applied Biosystems 3730 Sequencer. All sequence chromatograms were read in both forward (F) and reverse (R) directions, using tracings from at least two independent PCRs. Mutations were also checked against the SNP database (http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?locusId = 1956). The status of tumors was reported only if complete readable sequence was obtained for all exons 18-24.

PCR and Sequencing (WUStL). Primers for EGFR exons 17, and 19-23 were obtained from ref. 3. Primers for amplicons 2-10, 12-16, 18, and 25-28 were designed by using EXONPRIMER (http://ihg.gsf.de/ihg/ExonPrimer.html), a script for designing PCR primers that lie 50-100 bases from the intron/exon boundary, using PRIMER3 (http:frodo.wi.mit.edu/primer3/primer3_code.html; Table 5). The mRNA sequence for EGFR was obtained from GenBank (accession number NM_005228). The exon 24 primer pair was obtained from the ELXR set (ref. 4 and http://mutation.swmed.edu/ex-lax/). Additional sets of primers for exons 17-24 were developed by using EXONPRIMER specifically to address those samples derived from paraffin blocks, which performed reliably for PCR only with amplicons of less than 300 bp (Table 5). Here, EXONPRIMER was given partial exon sequences and a decreased minimal distance between primer and the exon/intron boundary setting to design primers within the exons. All PCR primer pairs had appended M13 forward or reverse primer tails, enabling the use of universal primers for all sequencing reactions.

Amplification was performed by using the specified primer pairs in a 10-m l reaction volume containing 1 m l of 5 ng/m l genomic DNA, 5 m l of 2x Amplitaq Gold PCR master mix (Applied Biosystems; 0.05 unit/m l AmpliTaq Gold DNA polymerase/30 mM Tris·HCl, pH 8.05/100 mM KCl/400 m M each dNTP/5 mM MgCl₂), 1 m l of 0.6 m M of either forward or reverse primer, and 1.6 m l of 50% glycerol. The PCR cycling program was: 95°C for 5 min; then 94°C for 30 s, 60°C for 45 s, 72°C for 45 s for 41 cycles, with a final extension at 72°C for 10 min. Aliquots of all PCR products were examined by electrophoresis on a 1.5% agarose gel containing ethidium bromide. The PCR products were treated with 0.037 unit/m l of exonuclease I and 0.18 unit/m l of shrimp alkaline phosphatase (both from Amersham Pharmacia) by incubation at 37°C for 30 min, followed by inactivation at 80°C for 15 min. A 6-m l sequencing reaction containing 2 m l of the treated PCR product, 4 m l of BigDye terminator version 3.1 premix (Applied Biosystems), and 10 m M M13 forward or reverse sequencing primer (as appropriate) was cycled as follows: 96°C for 1 min, then 96°C for 10 s, 50°C for 5 s, 60°C for 4 min, and repeated for a total of 25 cycles. Sequencing electrophoresis was performed on Applied Biosystems 3730 DNA sequencers.

PHRED (5) was used as the basecaller. Traces were assembled using PHRAP (www.phrap.org/phrap.docs/phrap.html) against the EGFR genomic reference sequence obtained from GenBank through the LocusLink site (www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l = 1956, accession number NT_033968.5). POLYPHRED version 4.05 (6) was used to tag potential sequence variants. High-quality variants in one or both sequencing directions were tagged. The final POLYPHRED tag report was piped through Prettybase (Variation Discovery Resource http://pga.gs.washington.edu/) and Report_prettybase.pl (www.genome.ou.edu/informatics.html), which generates a polymorphism report by variant position for each patient sample. In the final report, sequence was analyzed successfully from approximately 90% of target amplicons (exons 2-28). All candidate mutations were verified by using sequence from at least two independent PCRs from the same tumor aliquot.

Functional Analyses of Mutant EGFRs. The del L747-S752 mutant was introduced into the full-length EGFR coding sequence with the following primers: L747-S752 F 5'-AAAATTCCCGTCGCTATCAAGGAACCGAAAGCCAACAAGGAAATCCTC-3' and L747-S752 R

5'-GAGGATTTCCTTGTTGGCTTTCGGTTCCTTGATAGCGACGGGAATTTTAAC-3', with pBluescriptSKII-EGFR as template. The L858R mutant was constructed using the primers: L858R F 5'-ATTTTGGGCGGGCCAAACTG-3' and L858R R

5'- CAGTTTGGCCCGCCCAAAAT-3'. The XbaI-KpnI fragments of both wild-type and mutant EGFRs were subcloned into the multiple cloning site of the pcDNA3.1(-) expression vector (Invitrogen) after digestion with XbaI and KpnI.

Immunoblotting. Cells were lysed in 50 mM Tris·HCl, pH 8.0/150 mM sodium chloride/5 mM magnesium chloride/1% Triton X-100/0.5% sodium deoxycholate/0.1% SDS/40 mM sodium fluoride/1 mM sodium orthovanadate and Complete protease inhibitors (Roche Diagnostics). After quantitation by Bio-Rad protein assays, ~10 m g of each sample was separated by gel electrophoresis on various Tris-Glycine precast gels (Invitrogen) and transferred to nitrocellulose (Schleicher and Scheull) by using a semidry transfer apparatus (Bio-Rad).

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