Intratumoral heterogeneous amplification of ERBB2 and subclonal genetic diversity in gastric cancers revealed by multiple ligation-dependent probe amplification and fluorescence in situ hybridization

SummaryA humanized monoclonal antibody against ERBB2 is used in neoadjuvant therapy for patients with gastric cancer. A critical factor in determining patient eligibility and predicting outcomes of this therapy is the intratumoral heterogeneity of ERBB2 amplification in gastric adenocarcinomas. The aims of this study are to assess the underlying mechanisms of intratumoral heterogeneity of ERBB2 amplification; to characterize the diversity of coamplified oncogenes such as EGFR, FGFR2, MET, MYC, CCND1, and MDM2; and to examine the usefulness of multiple ligation-dependent probe amplification (MLPA) in the semicomprehensive detection of these gene amplifications. A combined analysis of immunohistochemistry and fluorescence in situ hybridization revealed ERBB2-amplified cancer cells in 51 of 475 formalin-fixed, paraffin-embedded gastric adenocarcinomas. The fraction of amplification-positive cells in each tumor ranged from less than 10% to almost 100%. Intratumoral heterogeneity of ERBB2 amplification, defined as less than 50% of cancer cells positive for ERBB2 amplification, was found in 41% (21/51) of ERBB2-amplified tumors. The combined analysis of MLPA and fluorescence in situ hybridization revealed that ERBB2 was coamplified with EGFR in 7 tumors, FGFR2 in 1 tumor, and FGFR2 and MET in 1 tumor; however, the respective genes were amplified in mutually exclusive cells. Coamplified ERBB2 and MYC coexisted within single nuclei in 4 tumors, and one of these cases had suspected coamplification in the same amplicon of ERBB2 with MYC. In conclusion, the amplification status of ERBB2 and other genes can be obtained semicomprehensively by MLPA and could be useful to plan individualized molecularly targeted therapy against gastric cancers.