Gene amplification of CCNE1, CCND1 and CDK6 in gastric cancers detected by multiplex ligation-dependent probe amplification and fluorescence in situ hybridization.

New and effective treatments for advanced gastric cancer are urgently needed. Cyclins E and D1 form a complex with cyclin-dependent kinase 2 (CDK2), 4 or 6 to regulate G1-S transition. The G1-S regulatory genes encoding cyclin E (CCNE1), cyclin D1 (CCND1) and CDK6 (CDK6) are frequently amplified in gastric cancer and may therefore influence molecularly targeted therapies against ERBB2 or EGFR when co-amplified. A total of 179 formalin-fixed and paraffin-embedded gastric cancer specimens were examined for these gene amplifications by multiplex ligation-dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH). Amplification of at least one G1-S regulatory gene was found in 35 tumors (CCNE1 amplification, 15% of samples; CCND1, 6%; CDK6, 1%). In 13 of the 35 tumors, dual-color FISH identified co-amplification of the G1-S regulatory genes with ERBB2, EGFR and/or KRAS in single cancer nuclei. The observation that cells with G1-S regulatory gene amplification contained clonal subpopulations with co-amplification of ERBB2, EGFR or KRAS in five early and three advanced cancers suggests that amplification of the G1-S regulatory genes represents an early event which precedes ERBB2, EGFR or KRAS amplification. Amplified CCNE1, CCND1 and CDK6 in advanced gastric cancer may be potentially useful as direct targets for molecular therapy, or for combination therapy with ERBB2 or EGFR inhibitors. MLPA could be a useful tool for identification of patients who would benefit from such therapies.