The National Surgical Adjuvant Breast and Bowel Project Trial Breast Cancer Prevention Trial (P1) showed a 50% reduction in estrogen-sensitive breast cancer in premenopausal high-risk women who took tamoxifen chemoprevention (33
). Importantly, tamoxifen decreased risk of invasive breast cancer in women with lobular carcinoma in situ
and atypical hyperplasia by 56% and 86%, respectively (33
). These findings provide evidence that women with lobular carcinoma in situ
and atypia benefit most from tamoxifen (33
). As a result, tamoxifen chemoprevention is an option for premenopausal women with mammary atypia. Although tamoxifen has been shown to have significant benefit in many women, not all women benefit from tamoxifen prevention and there are significant side effects associated with tamoxifen treatment. Progress in breast cancer prevention is currently limited by our lack of biological markers to identify which women will respond to prevention therapies such as tamoxifen, as well as to identify women with tamoxifen-resistant atypia. Here, we tested whether ESR1
promoter hypermethylation predicted persistent atypia in high-risk women who were treated with tamoxifen chemoprevention. Recent reports show that ESR1
promoter hypermethylation outdid hormone receptor status as a predictor of clinical response to tamoxifen hormonal therapy in women with invasive breast cancer (20
). In contrast to these prior studies in invasive breast cancer, neither ESR1
promoter hypermethylation nor low ER expression predicted persistent atypia at 12 months in this study. These studies highlight potential differences between ER+ breast cancer and mammary atypia.
Although the presence of atypia has been shown to increase breast cancer risk, it is currently unknown if the presence of persistent atypia in RPFNA can be used as a surrogate marker of breast cancer risk or as a marker of resistance to tamoxifen chemoprevention. We are unable to make conclusions relative to women who have a loss of atypia as this may be due to sampling error; further studies are necessary. However, in women with persistent atypia, it is clear that tamoxifen did not result in the elimination of atypia; yet the long term implications of these observations must be interpreted with caution. It is possible that women who have persistent atypia on RPFNA will still have a risk reduction benefit. Currently we are unable to identify whether an individual woman with either atypia on excisional biopsy or cytologic atypia on RPFNA will progress to develop breast cancer. Further longitudinal studies are ongoing to determine whether women with persistent atypia on RPFNA are at increased short-term breast cancer risk relative to women who have had disappearance of atypia. It is important to recognize that this study is limited by being a small, single institution study. Although these results are interesting, further validation in a larger multi-institution study is required before our findings can be generalized to a larger, high-risk population.
Although the molecular mechanism of tamoxifen action in ER+ breast cancer is well-studied, there is little information on how tamoxifen may act in noncancerous human breast tissue that normally expresses low levels of ER (ER “poor”). Prevention models for tamoxifen action in ER-poor, noncancerous breast tissue have been primarily based on observations in ER+ breast cancer cells. Normal mammary tissue is composed of a heterogeneous population of cells. Greater than 90% of normal mammary epithelial cells express low ER, and only 5% to 10% of normal mammary epithelial cells express moderate ER (26
). If normal breast tissue is evaluated solely by ER expression, it would be classified as ER(−). This classification system, however, may not be adequate for describing normal mammary tissue. Although normal mammary tissue exhibits low ER, it responds to estrogen and, therefore, is not equivalent to ER(−), estrogen-resistant invasive breast cancer.
There are a number of possible explanations why ER-poor mammary atypia may respond to tamoxifen chemoprevention therapy. First, in vivo
atypical mammary epithelial cells may be more sensitive to extragenomic effects of tamoxifen. In our in vitro
cellular models of early mammary carcinogenesis, we observed that immediately after the acute loss of p53 function, primary human mammary epithelial cells exhibit a narrow window of tamoxifen-induced apoptosis sensitivity (34
). It is possible that loss of p53 or other tumor suppressors early in mammary carcinogenesis (38
) sensitizes cells to tamoxifen chemoprevention, leading to elimination of these atyplical cells in high-risk women.
Although inhibition of ER transcriptional activity and signaling is the predominate effect of tamoxifen in the breast, not all of the effects of tamoxifen can be directly attributed to competitive interactions with ER. Tamoxifen induces apoptosis in cholangiocarcinoma cells and inhibits angiogenesis in fibrosarcomas (40
). Tamoxifen also has a wide variety of other pharmacologic activities including stimulation of transforming growth factor-β, blockade of various chloride channels (42
), inhibition of protein kinase C (43
), and antagonism of calmodulin activity (44
). Furthermore, tamoxifen-binding sites, independent of ER, have been identified. For example, Sutherland, et al. (45
) reported a high-affinity antiestrogen binding site in human and rat uterine cells, as well as in other tissues. Tamoxifen also directly inhibits calmodulin in a calcium-dependent manner (44
). Because therapeutic concentrations of tamoxifen are several orders of magnitude higher than required to saturate ER (46
), these “extragenomic” effects of tamoxifen may play an important role in ER-poor normal breast tissue.
The molecular mechanisms for tamoxifen resistance in ER(+) invasive breast cancer is an area of intense investigation. It is possible that similar mechanisms for tamoxifen resistance in invasive breast cancer may exist during early mammary carcinogenesis. For example, ER/progesterone receptor–positive invasive breast cancer is more likely to respond to antiestrogen therapy than ER(+)/progesterone receptor(−) breast cancer. The present study did not examine progesterone receptor status. Growth factor signaling pathways downstream of ErbB family members, such as the Akt/mammalian target of rapamycin pathway, are often up-regulated in tamoxifen-resistant ER(+) breast cancers. Therefore, if these pathways are up-regulated in high-risk women with mammary atypia, it is possible that these women would be resistant to tamoxifen chemoprevention. Furthermore, the ER coactivators AIB1/SRC-3 and MNAR/PELP1 have both been shown to promote tamoxifen resistance in models of ER(+) breast cancer, both dependent and independent of ER signaling (47
). Finally, alterations in tamoxifen metabolism may also predict resistance. Production of tamoxifen metabolites occurs in the liver via cytochrome P450 CYP2D6
). Several polymorphisms in CYP2D6
that result in a decrease in tamoxifen metabolism have been identified (51
). Although the effect of these polymorphisms on the efficacy of tamoxifen is still controversial (53
), it has been reported that specific mutations in CYP2D6
results in a higher risk of disease relapse but a decrease in the incidence of tamoxifen side effects (55
). Although there is no clinical data associated with tamoxifen chemoprevention, it is reasonable to hypothesize that women with CYP2D6
polymorphisms may be resistant to tamoxifen chemoprevention.
In this study, we found that ESR1 hypermethylation or low ER expression was not able to predict persistent atypia in response to tamoxifen treatment in our high-risk cohort. Additionally, our results indicate that, similar to invasive breast cancer, not all mammary atypia is responsive to tamoxifen; 53% of women in this cohort showed a loss of atypia at 12 months. Although the implications of persistant atypia relative to breast cancer risk are still unclear, further analysis of RPFNA samples from ayptia that is tamoxifen responsive versus tamoxifen resistant may be useful to determine biomarkers of tamoxifen sensitivity, as well as to increase our understanding of the early events underlying mammary carcinogenesis and the different tumor types that arise in the breast.