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HER2 amplification and/or overexpression in breast cancer are adverse prognostic factors and can predict the response to trastuzumab therapy. As assessment of HER2 status in breast cancer is of great importance for clinical decision-making, it is crucial to have reliable methods to quantify HER2. In the present project, we have developed a modification of the direct-double-differential PCR method (dddPCR) for gene dosage quantification of HER2 in breast cancer samples. The study was also undertaken to establish the potential application of the newly developed dddPCR method for HER2 testing in a diagnostic laboratory. The dddPCR validity for determining HER2 amplification in comparison to IHC-based HercepTest for the detection of protein levels was performed. Modified dddPCR was proven to be highly reproducible and reliable. A statistically significant correlation between protein expression and gene dosage of HER2 was found in the examined material. The obtained results indicate that dddPCR provides a fast and reliable diagnostic tool for HER2 quantification and may be considered as a supplementary method to verify equivocal results obtained with IHC-based HercepTest. Notably, dddPCR can be performed in every basic molecular biology laboratory, and is inexpensive and time-saving.

ErbB-1, -2, -3 and -4 proteins are growth factor receptors, encoded by the family of respective erbB protooncogenes. These receptor-encoding proto-oncogenes frequently undergo amplification, and less frequently, a deletion, in several human neoplasms. The role of the ErbB family in human endocrine neoplasms, including pheochromocytoma (PHEO), was not extensively tested and not previously established. The expression/overexpression of erbB oncogenes in pheochromocytoma tissue was determined only in a few cases, and to the best of our knowledge, their mutations (amplification or deletion) were not examined in any series of PHEO cases. We, therefore, used a double differential polymerase chain reaction (ddPCR) for determination of the amplification/deletion profiles of erbB-1, -2, -3 and -4 genes in formalin-fixed, paraffin embedded (FFPE) specimens of human PHEOs. We examined the average gene copy number (AGCN) of the genes in 36 samples of pheochromocytomas (2 extra-adrenal and 34 adrenal tumors). We found the mean AGCNs of the oncogenes equal 1.18 for erbB-1 [amplification was found in 11/35 cases (31%) and deletion in 6/35 cases (17%)], 2.00 for erbB-2 [amplification was found in 8/34 cases (24%), no deletion was found], 1.36 for erbB-3 [amplification was found in 4/36 cases (11%) and deletion in 1/36 cases (3%)], and 1.22 for erbB-4 [amplification was found in 5/30 cases (17%) and deletion in 1/30 cases (3%)]. A mutation(s) of any erbB oncogene was found in 25/36 (69%) samples tested. Some abnormalities of the erbB oncogenes showed interesting correlations with one another and with clinical features of the tumors. The frequent occurrence of amplifications and deletions of the erbB oncogenes in human pheochromocytoma implies the importance of the gene family in the development of these tumors.