Search

Summary Aims The transgenic enhanced green fluorescent protein (EGFP) expressing ‘green’ mouse (C57BL/6-TgN(ACTbEGFP)IOsb) is a widely used tool in stem cell research, where the ubiquitous nature of EGFP expression is critical to track the fate of single or small groups of transplanted haematopoietic stem cells (HSC). Our aim was to investigate this assumed ubiquitous expression by performing a detailed histological survey of EGFP expression in these mice. Methods Fluorescent microscopy of frozen tissue sections was used to perform a detailed histological survey of the pattern of EGFP expression in these mice. Flow cytometry was also used to determine the expression pattern in blood and bone marrow. Results Three patterns of EGFP expression were noted. In most tissues there was an apparently stochastic variegation of the transgene, with individual cell types demonstrating highly variable rates of EGFP expression. Certain specific cell types such as pancreatic ductal epithelium, cerebral cortical neurones and glial cells and glomerular mesangial cells consistently lacked EGFP expression, while others, including pancreatic islet cells, expressed EGFP only at extremely low levels, barely distinguishable from background. Lastly, in the colon and stomach the pattern of EGFP expression was suggestive of clonal inactivation. Only cardiac and skeletal muscle showed near ubiquitous expression. Conclusions These findings raise questions regarding the ‘ubiquitous’ expression of EGFP in these transgenic mice and suggest caution in relying overly on EGFP alone as an infallible marker of donor cell origin.

Intratumoral heterogeneity mirrors subclonal diversity and might affect treatment response. To investigate molecular heterogeneity of primary breast cancer specimens, we determined the amplification status of growth regulatory genes (c-erbB2, topoisomerase IIalpha, c-myc, and cyclinD1) in macroscopically and microscopically separate areas of individual tumors (n = 21). Using laser-assisted microdissection and quantitative PCR, we found marked intratumoral heterogeneity with different patterns for each gene. Molecular heterogeneity in amplification pattern could be demonstrated between both macroscopically (0.5 to several centimeters) and microscopically (10 to several hundred micrometers) distant tumor areas. C-erbB2 amplification proved to be the most stable amplification in individual tumors, with heterogeneity occurring in only 36% of amplified cases. By contrast, amplification of c-myc and cyclinD1 revealed varying patterns in the vast majority of amplified cases (100% and 83%). The constancy of c-erbB2 amplification underlines its presumed importance in breast cancer biology. We conclude that the molecular heterogeneity of breast cancer as evidenced in this study requires thorough and representative sampling of different tumor areas when the biologic significance of somatic mutations is considered. Patterns of heterogeneity can be used to trace the clonal evolution within different compartments of an individual tumor.

Ductal carcinoma in situ (DCIS), as an identifiable progenitor lesion of invasive breast cancer, represents a morphologically, biologically, and prognostically heterogeneous disease. It is not clear which molecular mechanisms are involved in progression to infiltrative growth. In this study, 83 DCIS classified according to the Van Nuys grading scheme were examined for amplification of growth regulatory genes that have been found to be amplified in invasive breast cancer (c-erbB2, topoisomerase IIalpha, c-myc, and cyclinD1 genes). Exact quantification of gene amplification was enabled by a combination of laser microdissection of paraffin-embedded tissue with real-time PCR. In DCIS, gene amplifications of all tested genes were found. The most frequently amplified gene was c-erbB2 found in 21 of 83 (25%) cases. Amplification of the other genes under investigation was observed in 4% to 6% of cases, high-grade DCIS being predominantly affected. High-grade DCIS differed significantly from low- and intermediate-grade DCIS in frequency and level of c-erbB2 amplification. In addition, high-grade DCIS revealed an accumulation of genetic aberrations. Amplification status in pure in situ lesions did not differ from intraductal carcinoma with an infiltrative component, indicating that although associated with a higher nuclear grade gene amplification might not represent an independent prognostic marker of disease progression.