Direct resequencing of the complete ERBB2 coding sequence reveals an absence of activating mutations in ERBB2 amplified breast cancer.
Gene amplification is among the most common genetic abnormalities that cause cancer. One of the most clinically important gene amplifications in human cancer causes extensive reduplication of ERBB2. A variety of cancers also occasionally harbor somatic mutations in ERBB2. Gene amplification and activating mutations both have predictive value for clinical response to targeted inhibitors. Since the number of gene copies in an amplicon may exceed 100, and since amplicons may encompass multiple genes, high-resolution analysis of gene amplifications poses considerable technical challenges. We have overcome this obstacle by using emulsion-based resequencing to determine the sequence of many independently-amplified individual DNA molecules in parallel. We used this high throughput sequencing technology to analyze ERBB2 mutational status in five ERBB2 amplified cell lines (four breast, one ovarian) and two breast tumors. Genomic DNA was isolated and the 28 exons of ERBB2 were independently amplified. Amplicons were then pooled at equimolar ratios, subjected to emulsion PCR (emPCR) and finally to picotiter plate pyrosequencing. High-quality sequence data were obtained for all amplicons analyzed and no activating mutations within ERBB2 were identified. Although we did not find activating mutations within the multiple copies of ERBB2 in these samples, the results establish the utility of this technology as a feasible and cost-effective approach for high resolution analysis of amplified genes.
Zito
Christina I
CI
Department of Pathology, Yale University School of Medicine, New Haven, CT 06520, USA. christina.zito@yale.edu
Riches
David
D
Kolmakova
Julia
J
Simons
Jan
J
Egholm
Michael
M
Stern
David F
DF
D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism; D004252:DNA Mutational Analysis; D004273:DNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D005260:Female; D005784:Gene Amplification; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D047468:Immunoprecipitation; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014162:Transfection; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; D018719:Erb-b2 Receptor Tyrosine Kinases