Steroid Hormone Receptor-positive Breast Cancer Research Articles

Thyroid hormone enhances estrogen-mediated proliferation and cell cycle regulatory pathways in steroid receptor-positive breast Cancer.

Estrogen receptor (ER) α expression and associated signaling is a major driver of over two-thirds of all breast cancers (BC). ER targeting strategies are typically used as a first-line therapy in patients with steroid receptor positive (SR+) disease. Secondary resistance to anti-estrogenic agents may occur with clonal expansion and disease progression. Mechanisms underlying hormone resistance are an expanding field of significant translational importance. Cross-talk with other nuclear hormones, receptors, and signaling pathways, including thyroid hormones (TH) and their receptors (THRs), have been shown to promote endocrine therapy resistance in some studies. We have shown that TH replacement therapy (THRT) was independently and significantly associated with higher rates of relapse and mortality in SR positive (+), node-negative (LN-) BC patients, whereas it showed no association with outcomes in SR negative (-) patients. LN-, SR+ patients receiving THRT and tamoxifen had the worst outcomes, suggesting a pro-carcinogenic interaction that significantly and independently shortened survival and increased mortality. Using in vivo and in vitro models, we previously showed hormonal cross-talk, altered gene signaling, target gene activation, and resistance to tamoxifen in the presence of TH. In this report, we show TH ± E2 ± tamoxifen inhibits cell cycle control signaling, reduces apoptosis, and enhances cell proliferation, tumor growth, tamoxifen resistance, and clonal expansion. Mechanistically these changes involve numerous genes and pathways, including critical cell cycle regulatory proteins and genes identified using various molecular methods. These studies facilitate a greater mechanistic understanding of the biological and molecular impact of TH on SR+ BC.

Progesterone Receptor-Mediated Regulation of Cellular Glucose and 18F-Fluorodeoxyglucose Uptake in Breast Cancer.

Positron emission tomography imaging with 2-deoxy-2-[18F]-fluoro-D-glucose (FDG) is used clinically for initial staging, restaging, and assessing therapy response in breast cancer. Tumor FDG uptake in steroid hormone receptor-positive breast cancer and physiologic FDG uptake in normal breast tissue can be affected by hormonal factors such as menstrual cycle phase, menopausal status, and hormone replacement therapy. The purpose of this study was to determine the role of the progesterone receptor (PR) in regulating glucose and FDG uptake in breast cancer cells. PR-positive T47D breast cancer cells treated with PR agonists had increased FDG uptake compared with ethanol control. There was no significant change in FDG uptake in response to PR agonists in PR-negative MDA-MB-231 cells, MDA-MB-468 cells, or T47D PR knockout cells. Treatment of T47D cells with PR antagonists inhibited the effect of R5020 on FDG uptake. Using T47D cell lines that only express either the PR-A or the PR-B isoform, PR agonists increased FDG uptake in both cell types. Experiments using actinomycin D and cycloheximide demonstrated the requirement for both transcription and translation in PR regulation of FDG uptake. GLUT1 and PFKFB3 mRNA expression and the enzymatic activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were increased after progestin treatment of T47D cells. Thus, progesterone and progestins increase FDG uptake in T47D breast cancer cells through the classical action of PR as a ligand-activated transcription factor. Ligand-activated PR ultimately increases expression and activity of proteins involved in glucose uptake, glycolysis, and the pentose phosphate pathway.

Thyroid Hormone and Estrogen Promote Endocrine Resistance, Proliferation, Dedifferentiation, and Cancer Stem Cells in Steroid Receptor-Positive Breast Cancers

Background: Breast cancer (BC) and thyroid disease are well-recognized comorbidities. Hyperthyroidism and supraphysiologic thyroid hormone (TH) have been shown to promote BC incidence. We recently reported that thyroid hormone replacement therapy (THRT) was significantly and independently associated with shortened disease-free and overall survival, as well as endocrine resistance only in patients with steroid receptor-positive (SR+) BC (1). TH markedly upregulated estrogen and cell cycle signaling in vivo and in vitro and promoted dedifferentiation to basaloid and pre-stem phenotypes. Metformin (Met) attenuated this shift. Mechanisms of TH-mediated endocrine therapy resistance in ER+ BC are the focus of this report. Design: Two clinical cohorts of early-stage lymph node-negative (LN-) SR+ BC patients (n=820 and n=160) were used to determine the effect of THRT on overall survival using Kaplan-Meier methods. Bi-directional cross-talk between TH and E2 was tested using different BC cell lines, ER+ PDX in vivo models, in vitro methods, and publically available in silico data for modeling. Results: Our results show that E2+TH increases cell proliferation, enhances cell cycle, and hormone-associated oncogenic signaling in SR+/ER+ BC. Given that high expression of THRA is associated with poor prognosis in SR+ BC, knockdown of THRA and ESR1 reduced cell proliferation in ER+ BC cells. ER+ PDX tumors were implanted into NSG mice containing E2 pellet and subsequently treated with TH, Tamoxifen (Tam), Fulvestrant (ICI) or Met. Our data show that TH-mediated endocrine resistance only in the E2+TH+Tam treated tumors (P<0.0001 vs E2+Tam alone). Both ICI and Met provided significant attenuation of tumor growth in vivo. RNAseq analysis of E2+TH+Tam tumors show an increase in pro-oncogenic signaling (Wnt/Fizzled, MMPs, and TCL/LEFT). Our data suggest that the use of Tam did not dampen tumor growth whereas a full ER-antagonist (ICI) or Met attenuated E2-TH mediated cross-talk and tumor growth. Conclusions: These findings suggest that TH+Tam may enhance oncogenic signaling and is associated with a significantly increase in mortality risk in ER+/SR+ BC tumors. Exogenous TH adversely affects SR+ BC and not SR- BC. Understanding the mechanism of cross-talk between TH and E2 allows us to define novel therapeutic strategies that will facilitate rapid clinical application for ER+ BC patients currently taking THRT and anti-estrogen treatments. Reference: (1) Wahdan-Alaswad et. al. Clin Cancer Res October 23 2020 DOI: 10.1158/1078-0432.CCR-20-264.

Exogenous Thyroid Hormone Is Associated with Shortened Survival and Upregulation of High-Risk Gene Expression Profiles in Steroid Receptor-Positive Breast Cancers.

Thyroid disease is a frequent comorbidity in women with breast cancer, and many require thyroid hormone replacement therapy (THRT). We postulated that THRT has a deleterious clinical effect mechanistically through hormonal interactions, nuclear receptor cross-talk, and upregulation of high-risk breast cancer genes. Observational studies of patients with lymph node-negative (LN-) breast cancer (n = 820 and n = 160) were performed to test interactions between THRT and clinical, histologic, outcome, and treatment variables. Differences between the two cohorts include but are not limited to patient numbers, decades of treatment, duration of follow-up/treatment, tumor sizes, incidence, and type and dose/regimen of antihormonal and/or chemotherapeutic agents. In vivo and vitro models, in silico databases, and molecular methods were used to study interactions and define mechanisms underlying THRT effects. THRT significantly and independently reduced disease-free and breast cancer-specific overall survival of only the steroid receptor (SR)-positive (as compared with SR-negative) node-negative patients in both long-term observational studies. Patients with SR+ LN- breast cancer who received THRT and tamoxifen experienced the shortest survival of all treatment groups. A less potent interaction between THRT and aromatase inhibitors was noted in the second patient cohort. Using in vivo and in vitro models, TH administration enhanced estrogen and TH-associated gene expression and proliferation, nuclear colocalization of estrogen receptor and thyroid hormone receptor, and activation of genes used clinically to predict tumor aggression in SR+ breast cancer, including the IGF-IR, WNT, and TGFβ pathways. We show clinically significant adverse interactions between THRT, estrogenic, and oncogenic signaling in patients with SR+ LN- breast cancer.

Abstract B44: Clinical utility of semiquantitative evaluation of progesterone receptor immunohistochemistry in neuroendocrine tumors of the pancreas

Abstract Purpose: Progesterone receptor (PR) is expressed strongly in a subset of neuroendocrine cells in the pancreas. Tamoxifen has long been used in adjuvant endocrine therapy of steroid hormone receptor–positive breast cancer, and currently clinical trials are assessing its use in pancreatic neuroendocrine tumors (NET) that show variable PR expression. Prior studies have reported receptor status as “positive” or “negative,” using 1% or 10% positive nuclei as a cutoff. In this study, we assessed PR immunohistochemical expression in a cohort of NET and other pancreatic nonexocrine carcinomas. Theoretically, tumors with highest PR expression are more likely to respond to adjuvant tamoxifen. Experimental Design: Immunohistochemical staining for PR was performed on tissue microarrays of 54 well-differentiated neuroendocrine tumors of the pancreas and 8 poorly differentiated carcinomas of the pancreas, including 4 acinar cell carcinomas, 2 neuroendocrine carcinomas, and 2 small-cell neuroendocrine carcinomas. A minimum of 4 separate 2-mm tumor cores were sampled on each case. Results: PR expression in non-neoplastic pancreatic islets was used for positive control. PR staining was evaluated using a semiquantitative H-score method. Positive PR staining (defined as greater than 1%) was identified in 61% (33 of 54 cases) of well-differentiated NET (mean H-score 100) and 0% (0 of 8 cases) of poorly differentiated carcinomas of the pancreas. Of the 33 PR positive cases of well-differentiated NET, 3 cases had less than 10% nuclear staining (mean H-score 7), 8 cases had 10% to less than 50% nuclear staining (mean H-score 44), and 22 cases had 50% or greater nuclear staining (mean H-score 229). Conclusions: Based on these findings, we suggest analysis of NET tumor PR expression should include a quantitative or semiquantitative score rather than dichotomized immunohistochemical evaluation of hormone receptors, particularly in the evaluation of patients’ response to clinical trials with tamoxifen. High-grade endocrine and acinar carcinomas showed no PR expression and are less likely to derive benefit from adjuvant endocrine therapy. Citation Format: Sonya Purushothaman, Simona De Michele, Dorothy Low, Helen E. Remotti. Clinical utility of semiquantitative evaluation of progesterone receptor immunohistochemistry in neuroendocrine tumors of the pancreas [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B44.

Antiproliferative Effects of Hinute‐AM in Human Breast Cancer Cell Lines

Breast cancer is a debilitating disease that affects women worldwide. In the United States, breast cancer is the second leading cause of cancer‐related deaths in women. Diet is an important factor in the development of numerous diseases, including breast cancer. We reported earlier that the consumption of soy protein controlled not only the proliferation of dimethylbenz [a] anthracene (DMBA)‐induced breast cancer cells in rats but also its aggressiveness. Soy protein is hydrolyzed in the gut to small peptides which are easily absorbed and are involved in various cellular functions. Fuji Oil Company, Osaka, Japan, has commercially marketed a soy protein hydrolysate, termed Hinute‐AM, composed of primarily di‐ and tripeptides, to be used as a health food. This product was found to have anti‐inflammatory property. It is possible that the antiproliferative effects of soy protein on breast cancer in rats may be mediated by its hydrolysis in the small intestine to small peptides. Hence, we hypothesized that the beneficial effect of soy protein in breast cancer development may be mediated by its hydrolysis product as peptides. Several normal (MCF 10A, 12A, and 184 A1), triple negative (MDA–MB‐231, BT‐549 and HCC‐1806) and steroid receptor positive (T47D and MCH 7) breast cancer cell lines were treated with different concentrations of Hinute‐AM for 24 h. Hinute‐AM at both low and high concentrations did not have any significant effect on proliferation of normal cells. However, while it inhibited proliferation of all breast cancer cell lines at low concentration (0.02–0.10 mg/ml), at high concentration (0.5–2.0 mg/ml), it had no effect. Furthermore, while Hinute‐AM had no effect on the levels of Ki‐67 and cyclin D1 levels in normal cells, it significantly decreased their levels in all breast cancer cell lines. The mechanism by which Hinute‐AM inhibits proliferation of breast cancer cell lines is under investigation.Support or Funding Information(Supported by grants from Fuji Oil Company, Osaka, Japan and NIH 5U54MD007593)

Abstract P3-04-11: Thyroid hormone up-regulates estrogenic and pro-carcinogenic signaling, inducing a basaloid and more motile phenotype in only steroid receptor positive breast cancer cells

Abstract Background: Co-morbidity of thyroid disease and breast cancer has been recognized for over a century. Up to two-thirds of women with breast cancer have clinical or occult thyroid disease, as compared to one fifth of unaffected females. Thyroid disease typically proceeds breast cancer by at least a decade, and many (nearly half) are on long term thyroid hormone supplementation at the time of diagnosis. The exogenous administration of thyroid hormone, for hypothyroidism or status post thyroidectomy for neoplasia, has been associated with an increased risk of breast cancer and in some studies, a worse prognosis. Causality, however, has been controversial. Results: We studied over 800 consecutive node negative invasive breast cancer patients diagnosed between 1976 and 1993 from a single institution. Of these, 92% were chemo-naive and only 14% received tamoxifen, reducing interactions with treatment for the study design. We showed untoward significant interactions with both univariate and multivariate analyses, between thyroid hormone supplementation, disease free and disease specific survival only in patients with steroid receptor positive tumors. These interactions were strongest in pre-menopausal, as compared to post-menopausal patients. We thus hypothesized that thyroid hormone (TH), either indirectly or directly, promoted carcinogenesis via the steroid receptors (SR) for estrogen and/or progesterone and have interrogated mechanisms of these interactions in vitro. Estrogen (E2) induce significant cellular proliferation, whereas TH alone (at concentrations of T4 1 x 10-6:T3 at 2.5 x 10-7 M) induced only mild cell proliferation in SR+ (MCF-7 andT47D), but not SR- breast cancer cell lines (MDA-MB-468 and SKBR3). In contrast, TH induced significant proliferation in SR + cells at significantly lower doses (down to T4 1 x 10-12: T3 2.5 x 10-13 M) if administered with low dose estrogen (E2: 1 x 10 -10). These mitogenic effects were demonstrated by MTS and DNA quantification assays, as well as flow cytometry, which revealed the induction of G2/M arrest and an increase in the percentage of cells in S phase with TH alone (at higher doses) or TH + E2 (at lower doses). Tamoxifen partially mitigated the pro-growth effects of TH + E2, whereas ICI 182,780 completely abrogated these effects. Using Western blots we have shown that that TH (with or without E2) upregulated ERa expression and activation, Cyclin A, B,D1, and E, E2F1, MAPK/ERK1/2 but not AKT signaling. TH +/- E2 also promoted clonogenicity, motility, a more basal phenotype (CD24+/CD44+) and mammosphere formation in cells grown under non-adherent conditions. Conclusions: We have demonstrated in Stage I breast cancer patients with minimal systemic treatment, that the administration of TH was associated with a significantly shortened disease free and disease specific survival, only in patients whose tumors expressed SR. TH administered to SR+ cell lines, particularly when co-administered with E2 at therapeutic/physiologic doses, upregulates and activates ER, cell proliferation, a more aggressive and motile basaloid phenotype. Citation Format: Ann D Thor, Zeying Fan, Reema Wahdan-Alaswad, Susan M Edgerton. Thyroid hormone up-regulates estrogenic and pro-carcinogenic signaling, inducing a basaloid and more motile phenotype in only steroid receptor positive breast cancer cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-04-11.

Progesterone stimulates proliferation and promotes cytoplasmic localization of the cell cycle inhibitor p27 in steroid receptor positive breast cancers.

Progestins are reported to increase the risk of more aggressive estrogen receptor positive, progesterone receptor positive (ER+ PR+) breast cancers in postmenopausal women. Using an in vivo rat model of ER+ PR + mammary cancer, we show that tumors arising in the presence of estrogen and progesterone exhibit increased proliferation and decreased nuclear expression of the cell cycle inhibitor p27 compared with tumors growing in the presence of estrogen alone. In human T47D breast cancer cells, progestin increased proliferation and decreased nuclear p27 expression. The decrease of nuclear p27 protein was dependent on activation of Src and PI3K by progesterone receptor isoforms PRA or PRB. Importantly, increased proliferation and decreased nuclear p27 expression were observed in invasive breast carcinoma compared with carcinoma in situ. These results suggest that progesterone specifically regulates intracellular localization of p27 protein and proliferation. Therefore, progesterone-activated pathways can provide useful therapeutic targets for treatment of more aggressive ER+ PR+ breast cancers.

Progesterone decreases levels of the adhesion protein E-cadherin and promotes invasiveness of steroid receptor positive breast cancers.

Progestins are reported to increase the risk of invasive breast cancers in postmenopausal women receiving hormone therapy with estrogen plus progestin. We report here that estrogen and progesterone receptor positive (ER+PR+) rat mammary tumors arising in the presence of estrogen and progesterone exhibit increased invasiveness and decreased expression of E-cadherin protein compared with tumors growing in the presence of estrogen alone. A similar decrease of E-cadherin expression was observed in human ER+PR+ invasive ductal carcinoma compared with ductal carcinoma in situ. In agreement with findings in the rat, estrogen plus progestin R5020 treatment decreased E-cadherin expression in vitro in T47D human breast cancer cells. Decrease of E-cadherin protein was mediated by progesterone receptor B (PRB) and dependent on the activation of the Wnt pathway. These results suggest that progesterone signaling via PRB contributes to tumor invasiveness and can provide an important therapeutic target for treatment of invasive ER+PR+ breast cancers.

Assessment of letrozole and tamoxifen alone and in sequence for postmenopausal women with steroid hormone receptor-positive breast cancer: the BIG 1-98 randomised clinical trial at 8·1 years median follow-up

Postmenopausal women with hormone receptor-positive early breast cancer have persistent, long-term risk of breast-cancer recurrence and death. Therefore, trials assessing endocrine therapies for this patient population need extended follow-up. We present an update of efficacy outcomes in the Breast International Group (BIG) 1-98 study at 8·1 years median follow-up. BIG 1-98 is a randomised, phase 3, double-blind trial of postmenopausal women with hormone receptor-positive early breast cancer that compares 5 years of tamoxifen or letrozole monotherapy, or sequential treatment with 2 years of one of these drugs followed by 3 years of the other. Randomisation was done with permuted blocks, and stratified according to the two-arm or four-arm randomisation option, participating institution, and chemotherapy use. Patients, investigators, data managers, and medical reviewers were masked. The primary efficacy endpoint was disease-free survival (events were invasive breast cancer relapse, second primaries [contralateral breast and non-breast], or death without previous cancer event). Secondary endpoints were overall survival, distant recurrence-free interval (DRFI), and breast cancer-free interval (BCFI). The monotherapy comparison included patients randomly assigned to tamoxifen or letrozole for 5 years. In 2005, after a significant disease-free survival benefit was reported for letrozole as compared with tamoxifen, a protocol amendment facilitated the crossover to letrozole of patients who were still receiving tamoxifen alone; Cox models and Kaplan-Meier estimates with inverse probability of censoring weighting (IPCW) are used to account for selective crossover to letrozole of patients (n=619) in the tamoxifen arm. Comparison of sequential treatments to letrozole monotherapy included patients enrolled and randomly assigned to letrozole for 5 years, letrozole for 2 years followed by tamoxifen for 3 years, or tamoxifen for 2 years followed by letrozole for 3 years. Treatment has ended for all patients and detailed safety results for adverse events that occurred during the 5 years of treatment have been reported elsewhere. Follow-up is continuing for those enrolled in the four-arm option. BIG 1-98 is registered at clinicaltrials.govNCT00004205. 8010 patients were included in the trial, with a median follow-up of 8·1 years (range 0-12·4). 2459 were randomly assigned to monotherapy with tamoxifen for 5 years and 2463 to monotherapy with letrozole for 5 years. In the four-arm option of the trial, 1546 were randomly assigned to letrozole for 5 years, 1548 to tamoxifen for 5 years, 1540 to letrozole for 2 years followed by tamoxifen for 3 years, and 1548 to tamoxifen for 2 years followed by letrozole for 3 years. At a median follow-up of 8·7 years from randomisation (range 0-12·4), letrozole monotherapy was significantly better than tamoxifen, whether by IPCW or intention-to-treat analysis (IPCW disease-free survival HR 0·82 [95% CI 0·74-0·92], overall survival HR 0·79 [0·69-0·90], DRFI HR 0·79 [0·68-0·92], BCFI HR 0·80 [0·70-0·92]; intention-to-treat disease-free survival HR 0·86 [0·78-0·96], overall survival HR 0·87 [0·77-0·999], DRFI HR 0·86 [0·74-0·998], BCFI HR 0·86 [0·76-0·98]). At a median follow-up of 8·0 years from randomisation (range 0-11·2) for the comparison of the sequential groups with letrozole monotherapy, there were no statistically significant differences in any of the four endpoints for either sequence. 8-year intention-to-treat estimates (each with SE ≤1·1%) for letrozole monotherapy, letrozole followed by tamoxifen, and tamoxifen followed by letrozole were 78·6%, 77·8%, 77·3% for disease-free survival; 87·5%, 87·7%, 85·9% for overall survival; 89·9%, 88·7%, 88·1% for DRFI; and 86·1%, 85·3%, 84·3% for BCFI. For postmenopausal women with endocrine-responsive early breast cancer, a reduction in breast cancer recurrence and mortality is obtained by letrozole monotherapy when compared with tamoxifen montherapy. Sequential treatments involving tamoxifen and letrozole do not improve outcome compared with letrozole monotherapy, but might be useful strategies when considering an individual patient's risk of recurrence and treatment tolerability. Novartis, United States National Cancer Institute, International Breast Cancer Study Group.

5016 POSTER DISCUSSION BIG 1-98 Update: Evaluating Letrozole and Tamoxifen Alone and in Sequence at 8 Years Median Follow-up for Postmenopausal Women With Steroid Hormone Receptor-Positive Breast Cancer

5016 POSTER DISCUSSION BIG 1-98 Update: Evaluating Letrozole and Tamoxifen Alone and in Sequence at 8 Years Median Follow-up for Postmenopausal Women With Steroid Hormone Receptor-Positive Breast Cancer

Adjuvant Endocrine Therapy for Breast Cancer: Don't Ditch the Switch!

The meta-analysis has been an invaluable tool in the evaluation of adjuvant therapy for breast cancer and has supported the use of tamoxifen, anthracyclines, and taxanes via the compilation of results from small trials that individually yielded less than dramatic results. Indeed, recent results of the Early Breast Cancer Trialists’ Collaborative Group (EBCTCG) meta-analysis demonstrated the enduring benefit in 15-year recurrence-free and overall survival for 5 years of adjuvant tamoxifen for women of any age with earlystage steroid receptor–positive breast cancer (1). It also confirmed an age-related increase in the risks of thromboembolic events and endometrial cancer without an excess in non-breast cancer–related deaths. In recent years, the use of tamoxifen in postmenopausal women has been eclipsed by enthusiasm about aromatase inhibitors. Multiple studies have documented a greater reduction in breast cancer recurrence and a seemingly more favorable toxicity profile with aromatase inhibitors compared with tamoxifen, which has led to their widespread use over tamoxifen, despite the fact that an overall survival benefit with this approach has not been observed. These results have led to guidelines by entities such as the American Society of Clinical Oncology (2) and the National Comprehensive Cancer Network (3) stating that aromatase inhibitors should be considered in place of or in sequence with tamoxifen for most postmenopausal women with early-stage hormone receptor–positive breast cancer. The emergence of data from large trials of adjuvant aromatase inhibitors has enabled several meta-analyses to be performed, one of which is reported by Amir et al. (4) in this issue of the Journal. The authors hypothesized that the relative toxicity of aromatase inhibitors compared with tamoxifen could explain the discordant observation of their improved benefit in disease-free survival without enhanced overall survival benefit. Their meta-analysis was restricted to seven randomized controlled trials that enrolled 30 0 23 patients and compared 5 years of aromatase inhibitor, 5 years of tamoxifen, or a switch from one to the other as primary endocrine therapy. In addition to conventional breast cancer endpoints, they focused on six prespecified serious adverse events —cardiovascular disease, cerebrovascular disease, bone fracture, thromboembolic events, endometrial carcinoma, and other non-breast cancers. In large part, the analysis confirmed what we already know: Compared with tamoxifen, the use of aromatase inhibitors was associated with a statistically significant increase in bone fractures and decrease in endometrial cancer and thromboembolic events and no difference in the risks of second cancers. What we learned, however, was that there was no difference in the risk of cerebrovascular disease with tamoxifen compared with aromatase inhibitors, and the risk of cardiovascular disease was statistically significantly higher with aro matase inhibitors compared with tamoxifen, although the magnitude of the difference was small—an absolute difference in risk of 0.8% and a number needed to harm of 132. It is notable that use of up-front aromatase inhibitors was also associated with a nonstatistically significantly higher odds of death without breast cancer recurrence compared with the use of tamoxifen alone or switching from tamoxifen to aromatase inhibitors (odds ratio = 1.11, 95% confidence interval = 0.98 to 1.26, P = .09). Of particular interest was the finding that those treated with a switching strategy had a statistically significant reduction in the odds of death without breast cancer recurrence compared with those treated with 5 years of tamoxifen or 5 years of an aromatase inhibitor alone (odds ratio = 0.87, 95% 95% confidence interval = 0.77 to 0.99, P = .03). In absolute terms, the difference is exceedingly small—a 0.2% difference in absolute risk of death without breast cancer recurrence. From these findings, Amir et al. ( 4) concluded that the switching strategy allows patients to reap the positive benefits of the aromatase inhibitors while limiting potential toxicity.

Abstract P4-09-08: PTEN Protein Expression in Postmenopausal Steroid Receptor Positive Early Breast Cancer Patients Treated with Adjuvant Tamoxifen

Abstract Background: Since one of possible causes of resistance to antiestrogen therapy in SR+BC patients is an alteration of PTEN (Phosphatase and Tensin homolog deleted on chromosome 10) signaling pathways, the aim of this study was to determine the PTEN protein expression in postmenopaual patients with steroid receptor (SR)+ breast cancer (BC) treated with adjuvant tamoxifen, to investigate the association of PTEN protein expression with tumor histology, size and grade, estrogen receptor (ER), progesteron receptor (PR) and human epidermal growth receptor 2 (HER2) statuses and disease outcome, as well. Patients and methods: This was a retrospective analysis of 78 postmenopausal SR+BC patients treated for stage I/II with adjuvant tamoxifen. PTEN protein expression and ER, PR and HER2 status were determined using immunohistochemistry method. Results: The distribution of PTEN protein expression according to tumor histology was as follows: PTEN+ status in 27/43 (62,8%) patients with ductal and in 26/35 (74,3%) patients with lobular carcinomas and PTEN-status in 16/43 (37,2%) patients with ductal and in 9/35 (25,7%) patients with lobular carcinomas. Disease relapse was observed in 38/78 pateints: 14/53 (26.4%) of PTEN+BC subgroup and 24/25 (96%) of PTEN-BC subgroup. There were no significant association between PTEN protein expression and tumor histology, size and grade, and ER, PR and HER2 expression, as well. Patients with PTEN-BC had significantly shorter disease-free interval and overall survival (for both Log rank test, P&amp;lt;0.01) compared to PTEN+BC pateints. Conclusion: Our results suggest that PTEN protein expression might be of prognostic significance in postmenopausal SR+ BC patients treated with ajduvant Tamoxifen. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-09-08.

Vascular endothelial growth factor receptor 2 and downstream p38 mitogen-activated protein kinase are possible candidate markers of intrinsic resistance to adjuvant endocrine treatment in steroid receptor positive breast cancer

A cross talk between tyrosine kinase receptors and mitogen-activated protein kinases (MAPKs) is proposed as involved in endocrine resistance. We wanted to investigate intratumoral levels of vascular endothelial growth factor receptor 2 (VEGFR2) and p38 MAPK in relation to relapse-free (RFS) and breast cancer corrected survival (BCCS) after adjuvant endocrine treatment, mainly tamoxifen for 2 or 5 years. We also wanted to investigate these markers in relation to early and late recurrences. VEGFR2 (n = 381) and p38 (n = 174) were determined by enzyme-linked immuno-sorbent assays in tumor homogenates from primary BC diagnosed 1993-1996. Wide ranges of VEGFR2 and p38 proteins were found; median 0.72 pg/μg DNA (range 0.0-11.66), and 0.04 pg/μg DNA (range 0.0-6.79), respectively. Detectable levels of p38 were registered in 65% and classified positive. Higher VEGFR2 were correlated to higher VEGF (P = 0.005), p38 MAPK (P = 0.018), negative ER (P = 0.008), larger tumors (P = 0.001), histopathological grade III (P = 0.018), distant metastasis (P = 0.044), shorter RFS (P = 0.013), and shorter BCCS (P = 0.017). Expression of p38 was significantly correlated with negative PgR (P = 0.044) and with early relapses (P = 0.021), while no difference was seen during the later follow-up period (P = 0.73). Higher VEGFR2 had a significant negative impact on both early (P = 0.029) and later recurrences (P = 0.018), while VEGF only predicted later relapses (P = 0.037). Our preliminary results suggest higher intratumoral levels of VEGFR2 and p38 MAPK as candidate markers of intrinsic resistance for adjuvant endocrine therapy.

CYP2D6-Tamoxifen

Tamoxifen is a standard endocrine therapy for the prevention and treatment of steroid hormone receptor-positive breast cancer. Tamoxifen efficacy depends on the formation of clinically active metabolites. Endoxifen is the major metabolite and the cytochrome P450 (CYP) enzyme 2D6 plays a key role in its formation. CYP2D6 variants and drug interaction by CYP2D6 inhibitors influence plasma endoxifen concentrations and adjuvant tamoxifen outcome. As compared to patients with full CYP2D6 function (EM) patients with non-functional (poor metabolizer, PM) and impaired (intermediate metabolizer, IM) CYP2D6 variants have higher recurrence rates (e.g. PM vs EM: HR=1.9, 95% CI 1.10-3.28). A study with more than 1,300 patients confirmed for PM a significantly higher (11.5%) and for EM lower (2%) relapse rate after 9 years follow-up when compared to the total patient cohort not stratified by genotype. It follows that up-front CYP2D6 genotyping for the prediction of CYP2D6 metabolizer status may aid in the individualization of endocrine treatment choice and improve outcome. Strong CYP2D6 inhibitors (e.g. paroxetine) should be avoided as co-medication and all efforts should be made to ensure tamoxifen adherence.

Targeting of tamoxifen to enhance antitumour action for the treatment and prevention of breast cancer: The ‘personalised’ approach?

Tamoxifen is a standard endocrine therapy for the treatment of steroid receptor positive breast cancer. Tamoxifen efficacy depends on the formation of clinically active metabolites 4-hydroxytamoxifen and endoxifen which have a greater affinity to the oestrogen receptor and ability to control cell proliferation as compared to the parent drug. The cytochrome P450 2D6 enzyme plays a key role in this biotransformation and lack of tamoxifen efficacy has been linked to low activity. There is now considerable mechanistic, pharmacologic and clinical pharmacogenetic evidence in support of the notion that CYP2D6 genetic variants and phenocopying effects through drug interaction by CYP2D6 inhibitors influence plasma concentrations of active tamoxifen metabolites and negatively impact tamoxifen outcome. These interrelations are particularly critical for patients with non-functional (poor metaboliser) and severely impaired (intermediate metaboliser) CYP2D6 variants, and, moreover, for patients in need of co-medication such as serotonin re-uptake inhibitors to control adverse effects such as hot flashes and other menopausal symptoms. Therefore, in the future, a personalised approach for an optimal tamoxifen benefit should consider a CYP2D6 genotype guided adjuvant endocrine treatment strategy and avoid non-adherence as well as strong CYP2D6 inhibitors such as co-medications.

0054 Tumor size rather than c-erbB-2 gene amplification significantly influence the disease outcome in postmenopausal steroid receptor-positive breast cancer patients treated with adjuvant tamoxifen

0054 Tumor size rather than c-erbB-2 gene amplification significantly influence the disease outcome in postmenopausal steroid receptor-positive breast cancer patients treated with adjuvant tamoxifen

The K303R ERα mutation and aromatase inhibitor resistance.

Abstract Abstract #602 Aromatase inhibitors (AIs) are rapidly becoming the first choice for the endocrine treatment of steroid receptor positive breast cancer in postmenopausal women. However, de novo and acquired resistance is a significant problem. We have previously identified, in premalignant and invasive breast cancers, a somatic mutation at nucleotide 908 of ERα, which was significantly associated with postmenopausal status, larger tumor size, and axillary lymph node positivity. This mutation results in a lysine to arginine transition at residue 303 (K303R). We demonstrated that the K303R ERα mutation generated an estrogen hypersensitive phenotype in vitro and in vivo. Thus, we hypothesized that the mutant could provide a continuous mitogenic stimulus to the breast even during phases of low circulating hormone, such as in postmenopausal women, affording a proliferative advantage, especially during treatment with AIs. As preclinical models to directly test whether the mutation confers resistance to AIs, we generated MCF-7 parental and MCF-7-K303R-overexpressing cells stably transfected with the aromatase gene. Cells were stimulated with the aromatase substrate, androstenedione (AD), with or without the AI anastrazole (Ana). We found using MTT assays that Ana decreased AD-stimulated growth of WT vector control cells by 47%, but had no effect on AD-stimulated growth of MCF-7-K303R cells. Furthermore, in soft agar assays, the basal non-stimulated colony number of the mutant cells was increased 10-fold, and the AI was unable to inhibit the formation of mutant colonies. Aromatase activity of both cells was reduced by 95% by Ana, suggesting that this drug resistance cannot be explained by an intrinsic insensitivity of the aromatase enzyme itself to Ana.&amp;#x2028; To examine the molecular mechanisms associated with the resistance of the mutant-expressing cells, we examined short-term nongenomic signaling, and found constitutively increased levels of phospho-IGF1R, IRS1 and AKT in the mutant cells. Interestingly, the hypersensitive mutant cells showed increased growth as well as pAkt activation with IGF1 treatment. Blockade of the IGF1R or PI3K/AKT pathways completely reversed the resistant mutant phenotype. To examine for effects on proliferation, immunoblot analyses were performed. K303R cells exhibited decreased levels of the CDK inhibitor p21 with AI treatment. Accordingly, apoptosis was decreased in the mutant cells with an increased ratio of bcl-2/Bax and decreased PARP cleavage.&amp;#x2028; We conclude that the mutation indeed confers resistance to an AI. Potential mechanisms of resistance include cellular strategies to evade apoptosis and increase proliferation, possibly through enhanced cross-talk with growth factor pathways and reception of downstream signaling networks, such as AKT. Our results suggest that the mutation might be a new predictive marker of response to AIs in mutation-positive breast tumors, and highlight the importance of novel approaches toward the development of signal transduction inhibitors for treating patients with tumors resistant to hormone therapy. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 602.

Integrated actions of progesterone receptor and cell cycle machinery regulate breast cancer cell proliferation

Multiple laboratories have investigated progesterone receptor (PR) involvement in breast cancer cell cycle progression. There is now a growing body of evidence demonstrating complex interactions between PR and cell cycle regulatory proteins. Here we review the current literature linking PR to cell cycle control and discuss gaps in the current knowledge. A more complete understanding of the relationships between PR and cell cycle regulatory molecules may reveal additional avenues for prevention and treatment of steroid receptor positive breast cancers.

Aromatase inhibitors for breast cancer

Endocrine therapy is a mainstay for the many women who develop in situ or invasive steroid receptor-positive breast cancer. The use of tamoxifen has reduced mortality in such women. Recently estrogen deprivation strategies have come under scrutiny. Here the use of aromatase inhibitors for treatment of postmenopausal endocrine-responsive breast cancer in the metastatic, adjuvant, and preoperative settings is reviewed.