Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
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In order to investigate the expression of ERC4, ERD3 and ERD5 mRNA expression relative to WT-ER mRNA within matched normal and breast tumor tissues, eighteen cases were selected in the Manitoba Breast Tumor Bank, which had well separated and histopathologically characterized normal and adjacent neoplastic components. Total RNA was extracted from frozen tissue sections and reverse-transcribed as described in the "Materials and Methods" section.

Relative expression of ERC4 mRNA in matched normal and breast tumor tissues

A recently described triple-primer PCR assay (TP-PCR assay) was used to compare the relative expression of ERC4 mRNA between adjacent normal and tumor components (9). In this assay, three primers are used simultaneously during the PCR: the upper primer is able to recognize both WT-ER and ERC4 cDNA sequences whereas the two lower primers are specific for each cDNA. Competitive amplification of two PCR products occurs, giving a final PCR-product ratio related to the initial input of target cDNAs. As shown in Figure 1, two PCR products were obtained, that migrated at the apparent size of 149 bp and 536 bp.

Fig. 1:Detection of ERC4 variant mRNA in matched normal (N) and tumor (T) human breast samples.

These products have been shown to correspond to WT-ER and ERC4 mRNAs, respectively. For each case, the mean of the ratios obtained in at least three independent PCR experiments, expressed in arbitrary units, is shown for both normal and tumor compartments (Fig. 2)

Fig. 2: Comparison of the relative expression of ERC4 variant mRNA between breast tumor (black bars) and adjacent matched normal (white bars) breast samples.

A general trend toward a higher clone 4 mRNA relative expression in the tumor compartment was observed (12 out of 18 cases, p=0.47, Wilcoxon signed rank test). When considering only the ER positive/PR positive subset (n=9), as measured by the ligand binding assay, a statistically higher ERC4 mRNA relative expression was found in the neoplastic components, as compared to matched adjacent normal tissues (p=0.019, Wilcoxon signed rank test).

Relative expression of ERD3 mRNA in matched normal and breast tumor tissues
A PCR assay, performed using primers annealing to sequences in exon 2 and 4, was used to investigate ERD3 mRNA expression relative to WT-ER in these 18 matched cases. We have previously shown that the co-amplification of WT-ER and an exon-deleted ER-a variant cDNA, resulted in the amplification of two PCR products, the relative signal intensity of which provided a previously validated measurement of exon-deleted ER-a variant expression (7). Two PCR products, that migrated with an apparent size of 354 bp and 237 bp, were obtained (Fig. 3).

Fig. 3:Detection of ERD3 variant mRNA in matched normal (N) and tumor (T) human breast samples.

These fragments were shown by subcloning and sequencing to correspond to WT-ER and ERD3 mRNAs. The relative ERD3 signal was measurable in the normal and the tumor compartments of 13 cases (see Fig. 4).

Fig. 4: Comparison of the relative expression of ERD3 variant mRNA between breast tumor (black bars) and adjacent matched normal (white bars) breast samples.

Out of these 13 cases, ERD3 mRNA expression was higher in the normal compartment of 10 cases. This difference, however, did not reach statistical significance (p=0.057, Wilcoxon signed rank test). A significantly higher expression of ERD3 mRNA in the normal compared to the adjacent neoplastic components was found when only the ER positive subset was considered (n=8, p=0.023, Wilcoxon signed rank test).

Relative expression of ERD5 mRNA in matched normal and breast tumor tissues
Using primers annealing to sequences in exon 4 and 6 of WT-ER, we also investigated the relative expression of ERD5 mRNA in these 18 matched cases. Two PCR products were obtained, that migrated at an apparent size of 483 bp and 344 bp and that have previously been shown to correspond to WT-ER and ERD5 cDNAs, respectively (Fig. 5).

Fig. 5:Detection of ERD5 variant mRNA in matched normal (N) and tumor (T) human breast samples.

As shown in Fig. 6, a statistically significant higher relative expression of ERD5 mRNA was observed in tumor components when this expression was measurable in both normal and adjacent tumor tissues (n=15, p=0.035, Wilcoxon signed rank test).

Fig. 6: Comparison of the relative expression of ERD5 variant mRNA between breast tumor (black bars) and adjacent matched normal (white bars) breast samples.

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