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Background It is uncertain if the addition of OFS to adjuvant endocrine therapy with T improves outcomes in premenopausal HR+ BC. The SOFT trial was designed to determine the value of OFS in women who remain premenopausal and are treated with T (OFS question) and also to test whether an AI improves outcome in premenopausal women with HR+ BC treated with OFS (AI question). In the combined analysis of the TEXT and SOFT trials (AI question), the group treated with the AI exemestane+OFS had a significantly better disease-free survival (DFS) than those with T+OFS. This abstract presents the results of the OFS question (n=2,045). Patients and Methods Between November 2003 and January 2011, the SOFT phase 3 trial randomized 3066 premenopausal women with HR+ BC to 5 years of adjuvant endocrine therapy with exemestane+OFS versus T+OFS versus T alone, with OFS initiated by choice of GnRH agonist triptorelin, oophorectomy or ovarian irradiation. Prior chemotherapy was allowed, provided women had confirmed premenopausal estradiol levels within 8 months of completing chemotherapy. The primary end point was DFS which included invasive local, regional, distant and contralateral breast events, second non-breast malignancies and deaths without prior cancer. Because of a lower-than expected overall event rate and to ensure results within a reasonable time-frame, a protocol amendment in 2011 changed the originally event-driven analysis plan. The amendment (1) designated the comparison of T+OFS versus T alone as the primary analysis for SOFT (n=2045), recognizing that the statistical power for the original three pairwise comparisons would be substantially reduced and (2) specified that the primary analysis of T+OFS versus T be undertaken when median follow-up was at least 5 years. The comparison would be tested at the 2-sided 0.05 level with no interim analysis. Based on the projected number of events at the lower rates, the estimated power to detect hazard ratios of 0.75, 0.70, and 0.66 for the comparison would be 52%, 69%, and 80%. Results The SOFT trial completed planned patient enrollment with an international collaboration involving 426 centres from BIG and NABCG led by the International Breast Cancer Study Group (IBCSG). Numbers of patients randomized, randomization strata, and pt age are summarized in the Table. The database lock for the primary analysis of the OFS question will occur in September 2014 and the final analysis will be completed by October 15, 2014. SOFT Patients (%)Tamoxifen alone1021 (33%)Tamoxifen+OFS1024 (33%)Exemestane+OFS1021 (33%)* Prior Chemotherapy54%Lymph node positive35%Median age (% < 40 years)43 years (30%)*Patients randomized to Exemestane+OFS are not part of the current analysis Conclusions We will present the primary analysis of outcomes and toxicities by treatment for women randomized to receive T+OFS versus T and address the value of OFS in addition to T as adjuvant endocrine therapy for premenopausal women with HR+ BC. Citation Format: Aron Goldhirsch, Richard D Gelber, Prudence A Francis, Meredith M Regan, Gini F Fleming, Istvan Lang, Eva M Ciruelos, Meritxell Bellet, Herve Bonnefoi, Miguel A Climent, Lorenzo Pavesi, Harold J Burstein, Silvana Martino, Nancy E Davidson, Charles E Geyer Jr, Barbara A Walley, Robert E Coleman, Pierre Kerbrat, Manuela Rabaglio-Poretti, Alan S Coates. Randomized comparison of adjuvant tamoxifen (T) plus ovarian function suppression (OFS) versus tamoxifen in premenopausal women with hormone receptor-positive (HR+) early breast cancer (BC): Analysis of the SOFT trial [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 S3-08.

While thousands of breast cancer cells disseminate and home to bone marrow (BM) until primary surgery, usually less than a handful will succeed in establishing manifest metastases months to years later. Signals and mechanisms determining failure or success of disseminated cancer cells (DCCs) are largely unknown and there is no in vivo model available to study the spontaneous progression and genomic evolution from early bone marrow infiltration to manifestation of bone metastasis, as spontaneous or transgenic mouse models do not generate bone metastases. We therefore profiled DCCs from BM of breast cancer patients long before manifestation of metastasis by RNAseq to identify signals supporting survival or outgrowth of DCCs and identified IL6/PTEN/PI3K signaling as candidate pathway for DCC activation. Since early DCCs often display close-to-normal genomes we used mammary epithelial cells ex vivo isolated from reduction mammoplasties and immortalized pre-malignant breast cancer cell lines as model for functional testing in vitro. Using specific activators and inhibitors of IL6 signaling revealed that IL6 trans, but not classical signaling, regulates stemness of mammary epithelial cells. Moreover, knock-down of PTEN revealed that PI3K/PTEN pathway activation renders cells independent of IL6 trans-signaling. Interestingly, gp130 expression, a pre-requisite for IL6 trans-signaling was found to be down-regulated by bone marrow stromal and endosteal, but not vascular niche cells, and as a consequence the number of cells with stem-like ability was significantly reduced. Consistent with a bottleneck function of microenvironmental DCC control, we found PIK3CA mutations highly associated with late-stage metastatic DCCs and CTCs while generally absent in early DCCs. Our data suggest that the initial steps of metastasis formation depend on microenvironmental signals and are not cancer cell-autonomous. Citation Format: Melanie Werner-Klein, Ana Grujovic, Milan Obradovic, Martin Hoffmann, Xin Lu, Stefan Kirsch, Steffi Treitschke, Cäcilia Köstler, Kathrin Weidele, Christoph Irlbeck, Catherine Botteron, Christian Werno, Bernhard Polzer, Miodrag Guzvic, Stefan Buchholz, Petra Rümmele, Norbert Heine, Stefan Rose-John, Christoph A. Klein. Interleukin 6 transsignaling is a candidate mechanism to drive progression of human DCCs during periods of clinical latency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-312.

Introduction: Intra-vaginal estradiol (E2) is effective to treat symptomatic vaginal atrophy. However, in postmenopausal (PM) women on aromatase inhibitors (AI), this increases serum E2 levels. We here report pharmacokinetic (PK), efficacy and safety results of a phase I study using vaginal tablets with lyophilized lactobacilli and 0.03 mg estriol (E3) (Gynoflor®) in PM breast cancer (BC) survivors on AI reporting atrophic vaginitis. Patients and Methods: In this open label phase I PK study, women being treated for ≥6 months with a non-steroidal AI and reporting non-hormone treatment-resistant vaginal atrophy were recruited. The primary objective was absorption and serum concentration of estrone (E1), E2, E3, FSH, LH and SHBG during initial therapy with one vaginal tablet of Gynoflor® daily for 28 days and during subsequent maintenance therapy of a daily tablet 3x per week for another 8 weeks. The secondary objective was to evaluate clinical symptoms, changes in vaginal pH, epithelium and microflora. Patients were assessed 6 times for endpoints. Blood was taken pre-insertion (baseline samples) at study entry, week 2, 4, 8, 12. At entry and week 4, multiple PK blood samples were drawn 0.5 hours pre-insertion and 0.5, 1, 2, 3, 4, 6, 8, 24 hours post-insertion. Serum samples were assayed by highly sensitive GC/MS. Results: The interim results of 8 of 16 enrolled patients who have completed the 12 week study period are presented. Basline estrogen concentrations of all women were below the levels of quantitation (E3: < 10–20 pg/ml; E2: < 1 pg/ml; E1: < 2 pg/ml). After first insertion, E3 serum levels transiently increased in 7 of 8 women; the single maximum concentration of all women was 132 pg/ml and highest levels (12–132 pg/ml) were observed usually 3 hours post-insertion. After 4 weeks of daily application, PK results showed an increase in E3 levels as compared to baseline in only 5 of 8 women. In addition, the single maximum concentration of all women was lower, 44 pg/ml, which was only reached after 8 hours. The baseline E3 concentrations did not change over the complete period of 12 weeks and were in the range of the limit of quantitation. The serum concentrations of E2 and E1 did not increase at any time point in any of the women. Clinical symptoms, vaginal pH and maturation index improved already after 2 weeks of therapy. Global efficacy and tolerability were rated by the investigators in at least 75% of the cases as (very) good, and by at least 87.5 % of the women. No serious adverse events were observed. Conclusions: Gynoflor® vaginal tablet is an effective and safe option to treat vaginal atrophy in PM patients with early BC on AI. Our data showed only a transient increase in E3 serum concentration after first application, and this increase was lower after 4 weeks. Baseline E3 concentrations did not increase demonstrating that no accumulation occurred. Serum E2 and E1 concentrations did not change and were always below limit of quantitation. The clinical significance of transient vaginal E3 absorption is unknown, however the absence of elevated E2 and E1 serum levels is compatible with the desired effect for BC patients taking an AI. *The first and second author have shared first authorship. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-12-06.

Background: Individual therapy efficiency of HER2−positive metastatic and pre-metastatic breast cancer patients varies significantly and spans from effectual responsiveness over acquired insensitivity to complete resistance from the outset. Thus no predictive information can be deduced from HER2 diagnostics so that molecular biomarkers indicative for sensitivity / resistance to Herceptin are needed to be identified. The HER2 related HER4-receptor has been shown to have ambivalent (pro-apoptotic or pro-proliferative) activity and consequently represents a prime candidate to affect HER2 activity under Herceptin treatment. We retrospectively analyzed potential her4 gene amplification and HER4 protein expression in HER2−positive, Herceptin treated patients. Patient's overall and recurrence free survival was evaluated as a function of HER2/HER4 expression. Methods: Using dual color Fluorescence in-situ Hybridization (FISH probes, Zytovision, Bremerhaven, Germany) and qPCR (LC480, Roche, Penzberg, Germany) we quantitatively investigated primary breast cancer tissues from nearly 50 (FISH) and 160 (PCR) patients who received Herceptin treatment. We quantified the her4 gene copy numbers and evaluated the protein expression profile of all four known HER4 isotypes (JM-a/CYT1, JM-a/CYT2, JM-b/CYT1, JM-b/CYT2). Results: FISH analysis revealed a positive and independent prognostic marker in Herceptin treated breast cancer patients with respect to overall survival. Moreover by quantitative PCR analysis we found a significant variability of HER4 protein expression (JM-a/CYT1 and JM-a/CYT2; no JM-b isotypes) in HER2 positive breast cancer tissues, whereas HER2/HER4 positive patients show a significant better recurrence free survival compared to HER2 positive but HER4 negative patients (p = 0,003). Conclusions: HER4 has been demonstrated to potentially exert tumor suppressing activity and in turn to have a favourable impact on the course of breast cancer disease. We show here that HER4 expression prolongs in particular recurrence free survival of Herceptin treated patients which indicates a functional integration of HER4 into anti-HER2 targeting. Complementing functional studies allowing for isotype specific function of HER4 will elucidate the special role of this receptor tyrosine kinase in the context of Herceptin treatment and might facilitate individualized anti-ErbB-receptor targeting with higher efficiency. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-12-23.

Introduction:Triple-negative breast cancers are characterized by aggressive growth and early metastasic spread. Some preliminary clinical data have demonstrated good anti-tumor activity of platinum derivatives. 3rd generation compound Lobaplatin* has shown a favorable toxicity profile as compared to Cisplatin. We therefore evaluated this compound in in vitro models of triple negative breast cancers.Study design:Triple-negative humane breast cancer cell lines HCC 1937 and HCC 1806 were treated with increasing concentrations of Lobaplatin and Cisplatin (0,16-10 μm). Cells were incubated for 24, 48 and 72 h with both substances in serum-free and serum-containing medium, in order to evaluate cytotoxic effects in proliferating as well as in metabolically inactive cells. Induction of apoptosis was tested by cleavage of PARP and Caspase 3 as demonstrated by western blot analysis. Subsequently cells were coincubated with Lobaplatin and multi-caspase inhibitor ZVAD-fmk, in order to check if cell death induced by Lobaplatin can be abrogated by inhibition of classical apoptosis. Cytotoxic effects were evaluated by crystal-violet assay. To investigate in which phase of the cell cycle cytotoxicity is most pronounced, cell cycle FACS analysis was performed after treatment with various concentrations of Lobaplatin.Results:Lobaplatin showed a somewhat better anti-tumor activity in both cell lines than Cisplatin. Lobaplatin Induced cleavage of PARP and procaspase 3 indicative for induction of classical apoptosis. However, cytotoxic effects of Lobaplatin were not decreased by multi-caspase inhibitor ZVAD-fmk. Cell-cycle FACS analysis showed, that sub-G0/1 fraction (indicactive for apoptotic/necrotic cells) was increased and G2 fraction was decreased dose-dependently by treatment with Lobaplatin.Conclusion:Lobaplatin showed a good anti-tumor activity in in vitro models of triple negative breast cancers which was at least equal to Cisplatin. Main mechanism of ytotoxicity was programmed cell death, which could not be abrogated by multi-caspase inhibition. Due to the good anti-tumor activity and the favorable toxicity profile, Lobaplatin should be considered for clinical studies in triple negative breast cancers.*Aeterna/Zentaris GmbH, Frankfurt/M Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6115.

Background: Tamoxifen is the oldest selective estrogen receptor modulator (SERM) developed to antagonize the estrogen receptor function on human breast cancer cells. JMR 132 is a novel GHRH Antagonist proven to be potent inhibitor of tumor growth in estrogen receptor negative human breast cancer cell lines. A combination of both substances could induce a more potent growth inhibition, as compared to the single drugs.Methods: To determine inhibitory effects of the GHRH antagonist JMR 132 and Tamoxifen alone and in combination, estrogen receptor positive cell lines ZR 75 and T 47D were incubated with substances at concentrations of 2.5µM, 5µM and 10µM. The cell viability after a period of 72 h was measured by MTT assay. To assess if the treatment arrested cell growth at a specific phase of the cell cycle or simply caused cell death, we performed a flow cytometric DNA cell cycle analysis on ZR 75 cells exposed to JMR 132, Tamoxifen and their combination. In an in vivo model, the cell line ZR 75 was xenografted subcutaneously into nude mice. Treatment groups were control, JMR 132 at a dose of 10µM, Tamoxifen at a dose of 100µgr per mouse q3 per week and the combination of both substances. All given for a period of 4 weeks.Results: In vitro 1o µM JMR 132 and Tamoxifen significantly decreased the proliferation of the ZR 75 and T47D cell lines, as compared to controls (p< 0.05). The combination of 1o µM inhibited the growth of ZR 75 was significantly by 14%, as compared to JMR 132 alone. The combination also significantly suppressed the growth of T47D by 14% and 22% respectively, as compared to Tamoxifen and JMR 132. In in vivo treatment with the combination of JMR 132 and Tamoxifen induced a significant inhibition of tumor growth of ZR 75 xenografts after 14 days compared to the single agents groups and inhibition remained significant until the end of the study on day 28. The flow cytometric DNA cell cycle analysis revealed a cell cycle arrest in the S Phase which led to apoptosis of the cells.Conclusion: Treatment of ZR 75 and T47D estrogen receptor positive human breast cancer lines with GHRH antagonist JMR 132 and its combination with Tamoxifen induced potent inhibitory effects in vitro and in vivo and led to subsequent cell death. Further studies are being conducted to elucidate the mechanisms of action of these effects. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5073.

Abstract #2137 Introduction Agonists of LHRH are a treatment option in premenopausal women with estrogen receptor positive breast cancer. The treatment with those agonists decreases the levels of FSH and LH in the blood and thus reduces the levels of sex hormones. Patients with estrogen receptor negative breast cancers do not benefit from therapy with LHRH agonists. Antagonists of LHRH lower LH, FSH and sex steroid levels but without the initial flare up of FSH and LH. In addition to the endocrine effects, Cetrorelix also shows a direct inhibition of cell growth in LHRH receptor (LHRH-R) positive gynecological cancer in vitro where alterations of FSH, LH and sex hormone levels are clearly excluded. In view of these findings we decided to investigate Cetrorelix on the tumor growth in two estrogen receptor negative but LHRH-R positive breast cancer cell lines. Materials and Methods The mRNA expression of LHRH-R was detected by PCR. For proliferation assays cells were treated with increasing doses of Cetrorelix acetate in 0.5% FBS supplemented RPMI 1640 media. MTS assays were performed after 72h. Fluorescence microscopy using ethidium bromide staining was performed to show cell death. In the in vivo study, nude mice were treated with Cetrorelix pamoate preparation at a dose of 3mg s.c. releasing an estimated 100mg Cetrorelix/day. Results MX1 and MDA-MB-231 estrogen receptor negative human breast cancer cell lines were positive for the expression of mRNA for LHRH-R. The cell proliferation in vitro of MX1 and MDA-MB-231 lines treated with different concentrations of Cetrorelix was significantly inhibited in a dose dependent manner. Time and dose dependent cell death was observed by fluorescence staining. Experimental treatment in vivo with Cetrorelix pamoate preparation reduced a significant inhibition of tumor growth in MX1 xenografts as compared to the Controls after 14 days and the suppression remained significant until the end of the study on day 28. MDA-MB-231 xenografts treated with Cetrorelix also showed a significant inhibition of tumor growth compared to the Control as early as day 7 and the inhibition remained significant until the end of the study on day 28. Conclusion Our results indicate a direct inhibitory effect of Cetrorelix on LHRH-R positive breast cancers. Since LHRH-R are expressed in ER negative breast cancers, these findings suggest a new approach to the treatment of LHRH-R positive breast cancer patients independent of the estrogen receptor status. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2137.