Oukseub Lee

Oral low dose and topical tamoxifen for breast cancer prevention: modern approaches for an old drug

Tamoxifen is a drug that has been in worldwide use for the treatment of estrogen receptor (ER)-positive breast cancer for over 30 years; it has been used in both the metastatic and adjuvant settings. Tamoxifens approval for breast cancer risk reduction dates back to 1998, after results from the Breast Cancer Prevention Trial, co-sponsored by the National Cancer Institute and the National Surgical Adjuvant Breast and Bowel Project, showed a 49% reduction in the incidence of invasive, ER-positive breast cancer in high-risk women. Despite these positive findings, however, the publics attitude toward breast cancer chemoprevention remains ambivalent, and the toxicities associated with tamoxifen, particularly endometrial cancer and thromboembolic events, have hampered the drugs uptake by high-risk women who should benefit from its preventive effects. Among the strategies to overcome such obstacles to preventive tamoxifen, two novel and potentially safer modes of delivery of this agent are discussed in this paper. Low-dose tamoxifen, expected to confer fewer adverse events, is being investigated in both clinical biomarker-based trials and observational studies. A series of systemic biomarkers (including lipid and insulin-like growth factor levels) and tissue biomarkers (including Ki-67) are known to be favorably affected by conventional tamoxifen dosing and have been shown to be modulated in a direction consistent with a putative anti-cancer effect. These findings suggest possible beneficial clinical preventive effects by low-dose tamoxifen regimens and they are supported by observational studies. An alternative approach is topical administration of active tamoxifen metabolites directly onto the breast, the site where the cancer is to be prevented. Avoidance of systemic administration is expected to reduce the distribution of drug to tissues susceptible to tamoxifen-induced toxicity. Clinical trials of topical tamoxifen with biological endpoints are still ongoing whereas pharmacokinetic studies have already shown that appropriate formulations of drug successfully penetrate the skin to reach breast tissue, where a preventive effect is sought.

A Randomized Phase II Presurgical Trial of Transdermal 4-Hydroxytamoxifen Gel versus Oral Tamoxifen in Women with Ductal Carcinoma In Situ of the Breast

Purpose: Local transdermal therapy to the breast may achieve effective target-organ drug delivery, while diminishing systemic effects. We conducted a randomized, double-blind, placebo-controlled phase II trial comparing transdermal 4-hydroxytamoxifen gel (4-OHT) to oral tamoxifen (oral-T) in women with ductal carcinoma in situ (DCIS). Methods: Twenty-seven pre- and postmenopausal women were randomized to 4-OHT (4 mg/day) or oral-T (20 mg/day) for 6 to 10 weeks before surgery. Plasma, nipple aspirate fluid, and breast adipose tissue concentrations of tamoxifen and its major metabolites were determined by liquid chromatography/tandem mass spectrometry. The primary endpoint was Ki67 labeling in DCIS lesions, measured by immunohistochemistry. In plasma, insulin-like growth factor-1 (IGFI), sex hormone–binding globulin (SHBG), and coagulation protein concentrations were determined. Results: Posttherapy Ki67 decreased by 3.4% in the 4-OHT and 5.1% in the oral-T group (P ≤ 0.03 in both, between-group P = 0. 99). Mean plasma 4-OHT was 0.2 and 1.1 ng/mL in 4-OHT and oral groups, respectively (P = 0.0003), whereas mean breast adipose tissue concentrations of 4-OHT were 5.8 ng/g in the 4-OHT group and 5.4 ng/g in the oral group (P = 0.88). There were significant increases in plasma SHBG, factor VIII, and von Willebrand factor and a significant decrease in plasma IGFI with oral-T, but not with 4-OHT. The incidence of hot flashes was similar in both groups. Conclusions: The antiproliferative effect of 4-OHT gel applied to breast skin was similar to that of oral-T, but effects on endocrine and coagulation parameters were reduced. These findings support the further evaluation of local transdermal therapy for DCIS and breast cancer prevention. Clin Cancer Res; 20(14); 3672–82. ©2014 AACR.

RANKL expression in normal and malignant breast tissue responds to progesterone and is up-regulated during the luteal phase

The receptor activator of nuclear factor-κB ligand (RANKL) acts as a paracrine factor in progesterone-induced mammary epithelial proliferation and tumorigenesis. This evidence comes mainly from mouse models. Our aim was to examine whether RANKL expression in human normal and malignant breast is under the control of progesterone throughout the menstrual cycle. Breast epithelial samples were obtained by random fine needle aspiration (rFNA) of the contralateral unaffected breasts (CUB) of 18 breast cancer patients, with simultaneous serum hormone measurements. Genes correlated with serum progesterone levels were identified through Illumina microarray analysis. Validation was performed using qRT-PCR in rFNA samples from CUB of an additional 53 women and using immunohistochemistry in tissue microarrays of 61 breast cancer samples. Expression of RANKL, DIO2, and MYBPC1 was correlated with serum progesterone in CUB, and was significantly higher in luteal phase. RANKL and MYBPC1 mRNA expression were highly correlated between CUB and matched tumor samples. RANKL protein expression was also significantly increased in the luteal phase and highly correlated with serum progesterone levels in cancer samples, especially in hormone receptor positive tumors. The regulatory effects of progesterone on the expression of RANKL, DIO2, and MYBPC1 were confirmed in three-dimensional cultures of normal breast organoids. In normal breast and in breast cancer, RANKL mRNA and protein expression fluctuate with serum progesterone with highest levels in the luteal phase, suggesting that RANKL is a modulator of progesterone signaling in normal and malignant breast tissue and a potential biomarker of progesterone action and blockade.

Patterns of Sex Steroid Hormones in Nipple Aspirate Fluid during the Menstrual Cycle and after Menopause in Relation to Serum Concentrations

Previous studies have shown that progesterone concentrations in serum and nipple aspirate fluid (NAF) are significantly correlated in premenopausal women, but estradiol concentrations are not. We therefore sought to ascertain the patterns of both steroids in NAF throughout the menstrual cycle and in postmenopausal women. Simultaneous samples of blood and NAF were obtained from 40 premenopausal and 16 postmenopausal women. Premenopausal samples were backdated from the following menstrual period. Steroids were purified by high-performance liquid chromatography before quantification by immunoassays. Serum steroids and NAF progesterone followed the expected pattern across the menstrual cycle, with a midcycle peak of estradiol and a midluteal peak of progesterone. However, the estradiol peak in NAF occurred about a week after the serum peak in the midluteal phase, when serum estradiol had declined to less than half the value at midcycle. NAF estrone was also elevated at the midluteal phase. Potential estrogen precursors androstenedione, estrone sulfate, and dehydroepiandrosterone sulfate declined in NAF from midcycle to the midluteal phase as NAF estradiol was increasing. Progesterone concentrations were significantly lower in NAF in postmenopausal women than in premenopausal women, but estrogen concentrations were not. This is the first description of the temporal relationships of sex steroids in NAF and serum relative to the menstrual cycle. These results provide insights into the lack of correlation of NAF and breast tissue estrogens with serum estrogens, and generate new hypotheses. Cancer Epidemiol Biomarkers Prev; 19(1); 275–9

Lipid metabolism genes in contralateral unaffected breast and estrogen receptor status of breast cancer

Risk biomarkers that are specific to estrogen receptor (ER) subtypes of breast cancer would aid the development and implementation of distinct prevention strategies. The contralateral unaffected breast of women with unilateral breast cancer (cases) is a good model for defining subtype-specific risk because women with ER-negative (ER−) index primaries are at high risk for subsequent ER-negative primary cancers. We conducted random fine needle aspiration of the unaffected breasts of cases. Samples from 30 subjects [15 ER-positive (ER+) and 15 ER− cases matched for age, race and menopausal status] were used for Illumina expression array analysis. Findings were confirmed using quantitative real-time PCR (qRT-PCR) in the same samples. A validation set consisting of 36 subjects (12 ER+, 12 ER− and 12 standard-risk healthy controls) was used to compare gene expression across groups. ER− case samples displayed significantly higher expression of 18 genes/transcripts, 8 of which were associated with lipid metabolism on gene ontology analysis (GO: 0006629). This pattern was confirmed by qRT-PCR in the same samples, and in the 24 cases of the validation set. When compared to the healthy controls in the validation set, significant overexpression of 4 genes (DHRS2, HMGCS2, HPGD and ACSL3) was observed in ER− cases, with significantly lower expression of UGT2B11 and APOD in ER+ cases, and decreased expression of UGT2B7 in both subtypes. These data suggest that differential expression of lipid metabolism genes may be involved in the risk for subtypes of breast cancer, and are potential biomarkers of ER-specific breast cancer risk. Cancer Prev Res; 6(4); 321–30. ©2013 AACR.

in vitro human skin permeation of endoxifen: potential for local transdermal therapy for primary prevention and carcinoma in situ of the breast

PURPOSE Oral tamoxifen, a triphenylethylene (TPE), is useful for breast cancer prevention, but its adverse effects limit acceptance by women. Tamoxifen efficacy is related to its major metabolites 4-hydroxytamoxifen (4-OHT) and N-desmethyl-4-hydroxytamoxifen (endoxifen [ENX]). Transdermal delivery of these to the breast may avert the toxicity of oral tamoxifen while maintaining efficacy. We evaluated the relative effciency of skin permeation of 4-OHT and ENX in vitro, and tested oleic acid (OA) as a permeation-enhancer. METHODS 4-OHT, ENX, and estradiol (E2) (0.2 mg/mL of 0.5 μCi (3)H/mg) were dissolved in 60% ethanol-phosphate buffer, ±OA (0.1%-5%). Permeation through EpiDerm™ (Matek Corp, Ashland, MA) and split-thickness human skin was calculated based on the amount of the agents recovered from the receiver fluid and skin using liquid scintillation counting over 24 hours. RESULTS In the EpiDerm model, the absorption of 4-OHT and ENX was 10%-11%; total penetration (TP) was 26%-29% at 24 hours and was decreased by OA. In normal human skin, the absorption of 4-OHT and ENX was 0.3%; TP was 2%-4% at 24 hours. The addition of 1% OA improved the permeation of ENX significantly more than that of 4-OHT (P < 0.004); further titration of OA at 0.25%-0.5% further improved the permeation of ENX to a level similar to that of estradiol. CONCLUSION The addition of OA to ENX results in a favorable rapid delivery equivalent to that of estradiol, a widely used transdermal hormone. The transdermal delivery of ENX to the breast should be further developed in preclinical and clinical studies.

Progesterone receptor antagonism inhibits progestogen-related carcinogenesis and suppresses tumor cell proliferation

PURPOSE Blockade of the progestogen-progesterone receptor (PR) axis is a novel but untested strategy for breast cancer prevention. We report preclinical data evaluating telapristone acetate (TPA), ulipristal acetate (UPA), and mifepristone. METHODS Tumors were induced with medroxyprogesterone acetate (MPA) plus 7,12-dimethylbenz[a]anthracene (DMBA) in mice, and MPA or progesterone plus N-methyl-N-nitrosourea (MNU) in rats. Mammary gland histology, tumor incidence, latency, multiplicity, burden and histology were evaluated, along with immunohistochemical labeling of pHH3 (proliferation), CD34 (angiogenesis), and estrogen and progesterone receptors (ER and PR). A concentration gradient of TPA, UPA, and mifepristone was tested for growth inhibition of T47D spheroids. RESULTS In mouse mammary glands, no tumors formed, but TPA opposed the pro-hyperplastic effects of MPA (p = 0.002). In rats, TPA decreased tumor incidence (p = 0.037 for MPA + TPA vs. MPA, and p = 0.032 for progesterone + TPA vs. progesterone) and tumor burden (p = 0.042 for progesterone + TPA vs. progesterone), with significant decreases in pHH3 and CD34 positive cells. TPA and UPA were superior to mifepristone in growth inhibition of T47D spheroids. CONCLUSION TPA has consistent anti-tumorigenic effects in several models, which are accompanied by decreases in cell proliferation, angiogenesis, and hormone receptor expression.

Novel routes for administering chemoprevention: local transdermal therapy to the breasts

Breast cancer prevention with pharmacologic agents requires that the breast be exposed to an effective drug; systemic exposure is unnecessary, and its harms lead many eligible women to decline preventive therapy. Local transdermal therapy (LTT) to the breast involves the application of active drugs to the breast skin, resulting in high concentrations in the breast but low systemic exposure. It is non-invasive, self-delivered, and not dependent on hepatic metabolism. Existing data on LTT include investigations demonstrating relief of mastalgia with topical 4-hydroxytamoxifen (4-OHT, an active tamoxifen metabolite). Two presurgical window trials in women with invasive breast cancer, and ductal carcinoma in situ (DCIS) demonstrate that LTT decreases proliferation of invasive and non-invasive cancer cells to a similar degree as oral tamoxifen, with low systemic levels, and no effect on coagulation proteins. These data are promising regarding the use of LTT for the primary prevention of breast cancer, and for therapy of DCIS, since systemic exposure is not required for either of these purposes. They also suggest that an LTT approach could be developed for any small, lipophilic molecule with good dermal permeation, thus greatly expanding the menu of drugs that could be tested for breast cancer prevention.

Hormonal Determinants of Nipple Aspirate Fluid Yield among Breast Cancer Cases and Screening Controls

Background: Nipple aspiration fluid (NAF) use as a biosample is limited by the variable yield across studies. We investigated the endocrine determinants of yield in an ongoing breast cancer case–control study. Methods: One-hundred and eighteen women yielding ≥2 μL NAF and 120 non-yielders were included; serum hormones were measured; differences in median hormones were assessed using the Wilcoxon rank-sum test. ORs and 95% confidence intervals (95% CI) for yielder status relative to hormone levels were estimated using logistic regression, adjusting for parity and lactation, and, in premenopausal women, menstrual cycle phase (MCP). Results: Prolactin concentrations were higher in yielders than non-yielders (premenopausal: 7.6 and 2.5 ng/mL, P < 0.01; postmenopausal 5.3 and 2.2 ng/mL; P < 0.01). Among premenopausal-yielders, estradiol was lower (64.3 vs. 90.5 pg/mL, MCP-adjusted P = 0.02). In separate menopausal status and parity-adjusted models, significant case–control differences persisted in prolactin: case OR 1.93 (95% CI, 1.35–2.77), control OR 1.64 (95% CI, 1.17–2.29). Premenopausal control yielders had higher progesterone (OR, 1.70; 95% CI, 1.18–2.46) and sex-hormone binding-globulin (OR, 2.09; 95% CI, 1.08–4.05) than non-yielders. Among parous women, further adjustment for lactation suggested a stronger positive association of serum prolactin with yield in cases than controls. Conclusion: NAF-yielders show higher prolactin than non-yielders, regardless of menopause and parity; implications of this and other endocrine differences on NAF biomarkers of breast cancer risk deserve further study. Impact: NAF yield is associated with a distinct endocrine environment that must be considered in studies of NAF-based breast cancer risk markers. Cancer Epidemiol Biomarkers Prev; 22(12); 2277–84. ©2013 AACR.

Abstract P5-14-01: Telapristone acetate abrogates PR-dependent paracrine-mediated mammary cell proliferation

Background: Blockade of the progesterone (P)-progesterone receptor (PR) axis is a novel but untested strategy for breast cancer prevention. We report preclinical data evaluating telapristone acetate (TPA) compared with mifepristone (MFP), the prototype PR-antagonist. We hypothesize that the progesterone-PR blockade by TPA will inhibit PR-dependent paracrine expression (RANKL, WNT4, ID4, and calcitonin) attenuating cell proliferation and abrogating side branching and alveoli formation of mammary glands induced by hormones similar in extent or superior to MFP. Methods: Adult virgin FVB mice at 12 weeks of age were randomized to four treatment groups: no treatment control, EP (0.3 mg E + 30 mg P), EP + TPA (30mg), and EP + MFP (30mg). Hormone and drug pellets were subcutaneously implanted in flank area between the neck and shoulder. After 28-day treatments, the mice were euthanized to collect mammary glands, and processed as mammary whole mounts and as formalin-fixed paraffin embedding (FFPE) specimens. We evaluated cell proliferation (Ki67) by immunohistochemistry. Total RNA was extracted from FFPE specimens and the Nanostring nCounter assay was utilized to assess paracrine gene expression. The Mann Whitney test was used to calculate statistical significance (p Results: We observed a considerable increase in side branches and alveoli in EP treatment group compared to the controls. The growth of the mammary gland stimulated by EP treatment was corroborated by a significant increase in Ki67 compared to control mice (median 41% vs 24%, respectively, p Rankl expression was 18 fold increased by EP treatment but completely inhibited by TPA and MFP treatment (p Rank expression was increased two-fold by EP treatment (p Wnt4 , and Calca expression by EP treatment compared to controls (p Id4 expression showed the same trend but the change was non-significant. The addition of TPA completely opposed the hormone-stimulated increase of three paracrine molecules ( Wnt4, Calca, and Id4 ) to a level below control group (p Conclusions : We have demonstrated that TPA abrogates cell proliferation induced by exogenous EP hormones in an ovary intact mouse model. The blockade of PR-P binding was evident by complete inhibition of paracrine expression (not only RANKL/RANK expression but also WNT4, Calcitonin, and ID4 expression). TPA was efficacious as MFP in opposing paracrine-induced mammary cell proliferation, and warrants further testing in a breast cancer prevention trial. Citation Format: Lee O, Sun L, Karavites LC, Clare SE, Khan SA. Telapristone acetate abrogates PR-dependent paracrine-mediated mammary cell proliferation [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-14-01.