Epidermal Growth Factor Receptor (EGFR) Mutations, KRAS Mutations, and EGFR Gene Amplification by FISH on Cytology Specimens
ME Leon, MA Villalona-Calero, GA Otterson, W Zhao. The Ohio State Univ., Columbus, OH
Background: In patients with non-small cell carcinoma (NSCLC), the presence of a somatic EGFR mutation is significantly associated with response to gefitinib and erlotinib. NSCLC with KRAS mutation do not respond as well to anti-EGFR therapy. Patients with tumors having EGFR gene amplification (EGFR-GA) and/or high polysomy have shown favorable outcomes when treated with tyrosine kinase inhibitors. The experience of these tests in cytology specimens is limited.
Design: 34 consecutive patients with NSCLC that underwent EGFR mutation, KRAS mutation, and EGFR-GA by Fluorescent In Situ Hybridization (FISH) analysis on cytology specimens were studied. Genomic deoxyribonucleic acid (DNA) was extracted from fresh fluid, or formalin fixed paraffin embedded cell blocks (FFPE-CB). Polymerase chain reaction (PCR)-based Fluorescence Fragment Analysis Assays were performed for designated mutations in the Exons 19 and 21; and they were confirmed by direct cycle sequencing methods. The Exon 2 of the KRAS gene was amplified by PCR, and followed by direct nucleotide sequencing to evaluate point mutations. EGFR-GA FISH was studied with a standard method. Amplification was defined as EGFR gene to chromosome 7 ratio > 2. High polysomy was defined as 4 or more copies in > 40% of the cells.
Results: The mean age of the patients was 62.7 years-old.17 males and 17 females were studied. 30 lung adenocarcinomas, 3 NSCLC, favor adenocarcinoma, and one squamous cell carcinoma were studied, including at total of 36 specimens; the specimen was a fine needle aspiration (FNA) in 21 cases (12 lung, 5 neck, 2 mediastinal,1 chest wall, and 1 abdominal wall), a pleural fluid in 12 cases; and a pericardial fluid, a bronchial wash, and a fresh cerebrospinal fluid (CSF), one each. FFPE-CB were studied in 35 specimens; and one fresh CSF was used. Respectively, 5 (13.8%) cases showed a somatic EGFR mutation, 4 in Exon 19 (15 base pair deletion) and one in Exon 21 (L858R). Four of these latter cases showed also EGFR-GA. 11 cases showed Exon 2 KRAS mutations. 1 case with G12A; 4, G12C; 1, G12R; 2, G12S; 2, G12V; and 1, G13C. Two of these cases showed EGFR-GA. 7 of the cases negative for EGFR and KRAS mutations showed EGFR-GA.
Conclusions: In our experience, routine clinical cytology specimens including fluids and cell blocks can be reliably used to perform EGFR-GA by FISH, and assay for KRAS, and EGFR mutations. Therefore, this testing may be useful in the patient's management.
Tuesday, March 23, 2010 2:00 PM
Platform Session: Section D, Tuesday Afternoon