DNA structural features on borders of ERBB2 amplicons in breast cancer

In 25–30% of cases of breast cancer tumors, the amplification of the chromosome fragment around ERBB2 underlies the increased expression of genes adjacent to ERBB2. The increased expression of genes within ERBB2-containing amplicons may impact not only the growth and development of the tumor, but also the sensitivity of the tumor to different types of anti-cancer therapies. The initial cause of the amplification and the exact borders of ERBB2-amplified chromosome fragment are still not completely characterized. No specific DNA sequences were found on the junction regions during intrachromosomal DNA amplification. We hypothesized that amplification borders can be specified by the structural peculiarities of DNA, rather than the particular DNA sequence. This study focused on the mapping of ERBB2 amplification borders in breast cancer and the search for unusual structural features of DNA at the borders of the identified amplicons. The copy number of ten genes adjacent to ERBB2 was evaluated by real time PCR in 162 breast cancer samples. Several ERBB2-containing amplicons of various lengths were revealed. In the majority of the analyzed samples, the borders of these amplicons were located within ZNFN1A3 and RARA genes. A bioinformatics analysis of the nucleotide sequence peculiarities around ERBB2 gene revealed the presence of AT-rich DNA regions with a high degree of flexibility. These regions were able to form stable secondary structures. Positions of these sites strongly coincide with the positions of the ERBB2-containing amplicon borders found in real time PCR experiments. Based on the obtained results, one can suppose that the structural features of DNA are involved in the formation of ERBB2-containing amplicon borders in breast cancer cells and the data are of importance for understanding the mechanisms of oncogene amplification.     

Her-2 gene dosage analysis in human breast cancer by PCR with internal standard

PCR assay with internal control was developed to quantify the her-2 gene dosage in human breast cancer tumor samples. Recombinant plasmid with a fragment of the her-2 gene containing the fragment of T7 phage DNA was designed especially to be used as an internal control in the PCR her-2 gene dosage assay. PCR conditions were optimized for the simultaneous usage of two templates--human genomic DNA and DNA of recombinant plasmid (internal control). The ability of the technology to discriminate a twofold increase of the her-2 gene dosage was demonstrated. Increased level of her-2 gene amplification was observed in 6 of 38 samples investigated. This new simple rapid assay can be an alternative to fluorescence in situ hybridization (FISH) and immunochemistry (ICH) tests for the detection of her-2 amplification in human tumors. This technique may be a useful tool for large randomized, prospective cooperative group trials and may support selection of optimal therapy for breast cancer patients in the future.