Abstract P6-10-02: Differential qRT-PCR-Based Gene Expression Signatures by Menopausal Status and Menstrual Phase in Random Fine-Needle Aspiration Samples from Healthy High-Risk Women
Authors: Lee O , Ivancic D , Heinz R , Helenowski IB , Chatterton RT , Khan SA. 
Published: April 2011

Background: The utility of archived breast tissue samples for risk-related research is limited by the fact that menopausal and menstrual status at the time of tissue sampling is often unknown. We evaluated the expression of a panel of genes in degraded RNA samples from women undergoing random fine-needle aspirate (rFNA) in preparation for a soy-isoflavone intervention. Our goal was to identify breast epithelial gene expression patterns related to menopause and menstrual phase.

Methods: Among 86 at high risk women (a 5-year Gail risk estimate &gt;1.6%, or a prior history of contralateral breast cancer but no systemic therapy), 46 were premenopausal (mean age 43.8 years) and 40 were postmenopausal (mean age 55 years). Stratification by menopause and menstrual phase was accomplished using menstrual dates and plasma estradiol (E2) and progesterone(P4) concentrations, measured by radioimmunoassay. 100 ng of RNA from baseline rFNA samples of the breast was reverse transcribed(mean RNA integrity 3.2). Amplicons of interest were linearly amplified to 10 cycles for 28 genes related to breast cancer and estrogen responsiveness. Assays were designed with small amplicons (&lt;100 bp) and qPCR reactions were carried out using the TaqMan Low Density Arrays (Applied Biosystems). For each gene of interest, expression levels were normalized to the average expression of GAPDH and HPRT1. The means, standard deviations, and 95% confidence intervals were calculated for plasma E2, P4, and gene expression difference between pre-and postmenopausal groups, and between the follicular and luteal phase within the premenopausal group were conducted using the unpaired t-test. P-values from gene expression difference were adjusted via the Benjamini-Hochberg (1995) approach.

Results: Within the premenopausal women group, there was no significant change in plasma E2 level between follicular and luteal phase groups (p=0.1756) while P4 level was higher in luteal phase (p=0.0474). The expression of five genes was significantly higher in the premenopausal than in the postmenopausal group: relative expression was 5-fold higher for progesterone receptor (PGR), 6-fold for pS2 (TFF1), 7-fold for gene regulated in breast cancer 1 protein (GREB1), 2-fold for prolactin receptor (PRLR) and 2-fold for CyclinD1 (CCND1) (adjusted p&lt; 0.026 for all). Within the premenopausal women group, the expression of ERα (ESR1) was one-third lower, and the signal peptide, CUB domain, EGF-like 2 (SCUBE2) was one-fifth lower in the luteal phase group compared to the follicular phase group (adjusted p=0.0423 for ESR1, p=0.0076 for SCUBE2).

Conclusion: Our findings suggest that the expression of genes related to estrogenic response in the breast epithelium of premenopausal women declines following menopause, whereas the relative suppression of ESR1 and SCUBE2 relate to effects of progesterone during the luteal phase of the menstrual cycle. These changes may allow the development of a model to date archived breast samples by menopausal and menstrual status.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-10-02.