Detection of EGFR mutations in samples with few cancer cells in non-small cell lung cancer (NSCLC) patients (p) and distribution of exon 20 mutations

Abstract

8073 Background: Detection of EGFR mutations in advanced NSCLC p is very often limited by the small amount of tumor tissue. We developed a protocol to determine EGFR mutations in tumor samples containing less than 150 cancer cells. We also studied the distribution of EGFR mutations throughout tumor samples in p progressing after erlotinib treatment. Methods: Cancer cells were microdissected directly into PCR buffer and amplified by a nested PCR. This preliminary microdissection ensures that only cancer cells are collected. Exon 19 del were subsequently detected by sequencing and Genescan, and exon 20 (T790M) and exon 21 (L858R) mutations by sequencing and TaqMan assay. Results: We examined 268 samples of primary NSCLC having less than 150 cancer cells. EGFR gene status was assessable in 213 samples (79%); 45/45 fresh cytological specimens - pleural effusions, fine-needle aspirates, broncoalveolar lavages, sputum - and 168/223 paraffin-embedded samples. Samples having as little as 8 cancer cells were successfully amplified. Overall, 11.7% of p had exon 19 del and 5.6% exon 21 mutations. The presence of either mutation was predictive of the response to erlotinib. In p progressing to erlotinib, exon 20 mutation was detected in 6/15 p (40%) and was found more frequently in p harboring exon 19 del (5/9) than those with the L858R mutation (1/6). In the 6 p harboring the exon 20 mutation, the original mutation was homogenously present. However, in two cases, the exon 20 mutation could not be detected in some groups of cells or areas within the tumor. Conclusions: The protocol presented allows for detection of EGFR mutations in samples containing few cancer cells, thus widening the range of NSCLC p that can be analyzed. In addition, the distribution of the exon 20 mutation is frequently heterogeneous in mutated tumors from p progressing to erlotinib, suggesting that microdissection of several groups of cancer cells might be the most appropriate way to test for this mutation. E20 wt E20 T790M Primary mutation Del Exon 19 4 5 L858R 5 1 No significant financial relationships to disclose.