Nancy E. Davidson

Detection of breast cancer cells in ductal lavage fluid by methylation-specific PCR

If detected early, breast cancer is curable. We tested cells collected from the breast ducts by methylation-specific PCR (MSP). Methylated alleles of Cyclin D2, RAR-beta, and Twist genes were frequently detected in fluid from mammary ducts containing endoscopically visualised carcinomas (17 cases of 20), and ductal carcinoma in situ (two of seven), but rarely in ductal lavage fluid from healthy ducts (five of 45). Two of the women with healthy mammograms whose ductal lavage fluid contained methylated markers and cytologically abnormal cells were subsequently diagnosed with breast cancer. Carrying out MSP in these fluid samples may provide a sensitive and powerful addition to mammographic screening for early detection of breast cancer.

Role of Estrogen Receptor Gene Demethylation and DNA Methyltransferase·DNA Adduct Formation in 5-Aza-2′deoxycytidine-induced Cytotoxicity In Human Breast Cancer Cells

The cytosine analog 5-aza-2′-deoxycytidine is a potent inhibitor of DNA methyltransferase. Its cytotoxicity has been attributed to several possible mechanisms including reexpression of growth suppressor genes and formation of covalent adducts between DNA methyltransferase and 5-aza-2′-deoxycytidine-substituted DNA which may lead to steric inhibition of DNA function. In this study, we use a panel of human breast cancer cell lines as a model system to examine the relative contribution of two mechanisms, gene reactivation and adduct formation. Estrogen receptor-negative cells, which have a hypermethylated estrogen receptor gene promoter, are more sensitive than estrogen receptor-positive cells and underwent apoptosis in response to 5-aza-2′-deoxycytidine. For the first time, we show that reactivation of a gene silenced by methylation, estrogen receptor, plays a major role in this toxicity in one estrogen receptor-negative cell line as treatment of the cells with anti-estrogen-blocked cell death. However, drug sensitivity of other tumor cell lines correlated best with increased levels of DNA methyltransferase activity and formation DNA·DNA methyltransferase adducts as analyzed in situ. Therefore, both reexpression of genes like estrogen receptor and formation of covalent enzyme· DNA adducts can play a role in 5-aza-2′-deoxycytidine toxicity in cancer cells.

Methyl-group dietary intake and risk of breast cancer among African-American women: a case–control study by methylation status of the estrogen receptor alpha genes

Objectives: Recent molecular studies show that the absence of estrogen receptor (ER) α gene expression in breast cancer is associated with methylation of the CpG island located in the 5′ region and the first exon of the ER α gene. Because CpG island methylation is an early event in carcinogenesis and because a methyl-deficient diet may lead to abnormal DNA methylation including CpG island methylation, we hypothesized that a methyl-deficient diet is more likely to be associated with breast cancer with methylated ER α gene CpG islands. This study aimed to test this hypothesis in African-American women using a case–control design. Methods: Cases were 304 African-American women pathologically diagnosed with breast cancer during 1995–1998 who lived in three Tennessee counties. Controls were 305 African-American women without breast cancer, who were selected through random-digit dialing and frequency matched to cases by 5-year age range and county of residence. Information on dietary intake and other risk factors was collected through telephone interviews. Dietary methyl-components were defined based on folate and methionine intakes and alcohol consumption. Tumor tissue samples were collected for measuring methylation status of the ER α gene. Results: Our results showed that the odds ratio (OR) estimates for lower dietary folate intake were 2.0 (95% confidence interval, CI: 0.8–4.8) for cases with a methylated ER α gene, 0.6 (95% CI: 0.3–1.5) for cases with an un-methylated ER α gene, and 1.6 (95% CI: 0.7–3.8) for cases with unknown methylation status (presumably including cases with both methylated and un-methylated genes). However, low methionine intake appeared more likely to be associated with tumors with unknown methylation status and high level of alcohol consumption seemed more likely to be related to tumors with un-methylated genes. Conclusions: These results did not show a pattern consistent with the study hypothesis that methyl-deficient diets are more likely related to breast cancer with a methylated ER gene.

Multiparametric Magnetic Resonance Imaging, Spectroscopy and Multinuclear (23Na) Imaging Monitoring of Preoperative Chemotherapy for Locally Advanced Breast Cancer

RATIONALE AND OBJECTIVES The aim of this prospective study was to investigate using multiparametric and multinuclear magnetic resonance imaging during preoperative systemic therapy for locally advanced breast cancer. MATERIALS AND METHODS Women with operable stage 2 or 3 breast cancer who received preoperative systemic therapy were studied using dynamic contrast-enhanced magnetic resonance imaging, magnetic resonance spectroscopy, and ²³Na magnetic resonance. Quantitative metrics of choline peak signal-to-noise ratio, total tissue sodium concentration, tumor volumes, and Response Evaluation Criteria in Solid Tumors were determined and compared to final pathologic results using receiver-operating characteristic analysis. Hormonal markers were investigated. Statistical significance was set at P < .05. RESULTS Eighteen eligible women were studied. Fifteen responded to therapy, four (22%) with pathologic complete response and 11 (61%) with pathologic partial response. Three patients (17%) had no response. Among estrogen receptor-positive, HER2-positive, and triple-negative phenotypes, observed frequencies of pathologic complete response, pathologic partial response, and no response were 2, 5, and 0; 1, 4, and 0; and 1, 1, and 3, respectively. Responders (pathologic complete response and pathologic partial response) had the largest reductions in choline signal-to-noise ratio (35%, from 7.2 ± 2.3 to 4.6 ± 2; P < .01) compared to nonresponders (11%, from 8.4 ± 2.7 to 7.5 ± 3.6; P = .13) after the first cycle. Total tissue sodium concentration significantly decreased in responders (27%, from 66 ± 18 to 48.4 ± 8 mmol/L; P = .01), while there was little change in nonresponders (51.7 ± 7.6 to 56.5 ± 1.6 mmol/L; P = .50). Lesion volume decreased in responders (40%, from 78 ± 78 to 46 ± 51 mm³; P = .01) and nonresponders (21%, from 100 ± 104 to 79.2 ± 87 mm³; P = .23) after the first cycle. The largest reduction in Response Evaluation Criteria in Solid Tumors occurred after the first treatment in responders (18%, from 24.5 ± 20 to 20.2 ± 18 mm; P = .01), with a slight decrease in tumor diameter noted in nonresponders (17%, from 23 ± 19 to 19.2 ± 19.1 mm; P = .80). CONCLUSIONS Multiparametric and multinuclear imaging parameters were significantly reduced after the first cycle of preoperative systemic therapy in responders, specifically, choline signal-to-noise ratio and sodium. These new surrogate radiologic biomarkers maybe able to predict and provide a platform for potential adaptive therapy in patients.

Physiologic estrogen receptor alpha signaling in non-tumorigenic human mammary epithelial cells.

SummaryCurrently, a number of breast cancer cell lines exist that serve as models for both estrogen receptor alpha (ERα) positive and ERα negative disease. Models are also available for pre-neoplastic breast epithelial cells that do not express ERα; however, there are no ideal systems for studying pre-neoplastic cells that are ERα positive. This has been largely due to the inability to establish an estrogen growth stimulated, non-tumorigenic breast epithelial cell line, as most human breast epithelial cells engineered to overexpress ERα have been found to be growth inhibited by estrogens. We have developed independently derived clones from the non-cancerous MCF-10A human breast cell line that express ERα and are growth stimulated by 17-beta-estradiol (E2) in the absence of epidermal growth factor (EGF), a cytokine normally required for MCF-10A cell proliferation. This effect is blocked by the selective estrogen receptor modulator (SERM), Tamoxifen and the selective estrogen receptor downregulator, ICI 182,780 (Faslodex, Fulvestrant). Exposure of these cells to EGF and E2 results in a growth inhibitory phenotype similar to previous reports. These data present a reconciling explanation for the previously described paradoxical effects of ERα overexpression, and provide a model for examining the carcinogenic effects of estrogens in non-tumorigenic human breast epithelial cells.

Induction of spermidine/spermine N1-acetyltransferase in breast cancer tissues treated with the polyamine analogue N1,N11-diethylnorspermine

PurposeThe polyamine analogue, N1,N11-diethylnorspermine (DENSpm), is currently being evaluated in clinical trials for the treatment of solid tumors. The response of solid tumors to this drug has been associated with superinduction of the polyamine catabolic enzyme, spermine/spermidine N1-acetyltransferase (SSAT). Therefore, to estimate the response of breast cancers to DENSpm, we measured induction of SSAT in breast cancer explants treated in vitro with this polyamine analogue.Experimental designExpression of SSAT protein was evaluated by immunohistochemistry in tissue explants from 38 invasive breast cancer tumors incubated in vitro in the presence (or absence) of DENSpm. In addition, SSAT enzymatic activity was measured in tissue explants from four tumors with high cellularity.ResultsSSAT expression was significantly increased in 30 of 38 tumor samples treated with DENSpm compared to untreated controls. This induction of SSAT protein expression was found specifically in neoplastic cells of the treated samples, and was seen in all histologic patterns (ductal, lobular, and mucinous) of breast cancer examined. In tumor samples evaluated for changes in SSAT enzymatic activity, these changes correlated closely with changes in protein expression.ConclusionsImmunohistochemical staining for induction of SSAT correlates with measures of enzymatic activity in a small sample where measurements were possible and suggests that immunohistochemistry may be used for predicting response of breast cancers to DENSpm. A high proportion of breast cancers induced SSAT in response to DENSpm, supporting the continued consideration of this class of agents for treatment of breast cancer.

Apoptosis and Breast Cancer

Apoptosis is an integral part of normal mammary gland development, differentiation, and function. Numerous descriptive and mechanistic studies of programmed cell death pathways in normal and malignant mammary cells have been presented. This chapter reviews the evidence that established breast cancer treatments act via apoptotic pathways, and how ongoing laboratory work defining these pathways might lead to the development of new therapeutic ap proaches. Also, the possibility that evaluation of apoptotic-related molecules or events might serve as prognostic or predictive factors is discussed.

Use of SERMs for the adjuvant therapy of early-stage breast cancer

Abstract: Tamoxifen was the first in a class of drugs now commonly referred to as selective estrogen receptor modulators or SERMs. SERMs exhibit tissue‐specific estrogenic agonist/antagonist activity through their ability to bind to the estrogen receptor α (ER) protein and interact with coregulatory proteins, thereby modulating transcription of estrogen target genes. Since its first approval by the United States Food and Drug Administration (FDA) in 1977, tamoxifen has been found to (a) lower the risk of recurrence and death for women with early‐stage hormone receptor‐positive breast cancer, irrespective of menopausal and node status or use of adjuvant chemotherapy; (b) reduce the risk of invasive breast cancer following breast conservation in women with ductal carcinoma in situ (DCIS); and (c) reduce the risk of breast cancer in high‐risk women. Toremifene is the only other SERM approved by the FDA for breast cancer treatment. However, it offers no clear clinical advantage over tamoxifen in the adjuvant or metastatic settings. Several other SERMs are in various phases of clinical development. In addition, strategies to combine SERMs with other endocrine therapy like ovarian suppression or aromatase inhibitors are active areas of investigations. At present, SERMs are recognized as the first targeted and relatively nontoxic medical therapy for women with high‐risk or steroid hormone receptor‐positive breast cancer.