Background: Breast cancer and its treatments are associated with a detrimental effect on bone health. Here we report the results of an exploratory analysis assessing changes in levels of biomarkers of bone metabolism in patients enrolled in the phase IIIb 4EVER study. Methods: The 4EVER trial investigated everolimus in combination with exemestane in postmenopausal women with hormone receptor-positive, human epidermal growth factor receptor 2-negative locally advanced or metastatic breast cancer. In this prespecified exploratory analysis, changes in biomarkers of bone turnover were assessed in patients from baseline to weeks 4, 12, and 24. The serum bone markers assessed were procollagen type 1 N-terminal propeptide (P1NP), C-terminal cross-linking telopeptide of type 1 collagen (CTX), osteocalcin, parathyroid hormone (PTH), and 25-hydroxyvitamin D (25-OH-vitamin D). On-treatment changes in bone markers over time were described per subgroup of interest and efficacy outcomes. Results: Bone marker data were available for 241 of 299 enrolled patients. At the final assessment, P1NP, osteocalcin, PTH, 25-OH-vitamin D (all P < 0.001), and CTX (P = 0.036) were significantly decreased from baseline values per the Wilcoxon signed-rank test. At the last assessment (24 weeks or earlier), levels of serum CTX and PTH were significantly lower (P = 0.009 and P = 0.034, respectively) among patients with vs. without prior antiresorptive treatment (ART). Serum CTX levels were significantly lower (P < 0.001), and 25-OH-vitamin D concentrations significantly higher (P = 0.029), at the last postbaseline assessment in patients receiving concomitant ART vs. those without ART. Changes from baseline in PTH and 25-OH-vitamin D concentrations to the final assessment were significantly smaller in patients with prior ART. Lower baseline serum concentrations of osteocalcin and PTH were associated with clinical response (partial vs. non-response) at 24 weeks. High serum levels of CTX and P1NP at baseline were risk factors for progression at 12 weeks. Conclusions: These exploratory analyses support use of everolimus plus exemestane for the treatment of postmenopausal women with hormone receptor-positive, human epidermal growth factor receptor 2-negative advanced breast cancer, and add to the body of evidence suggesting a potentially favorable impact of everolimus on bone turnover.

In BOLERO-2, adding everolimus to exemestane resulted in a twofold increase in median progression-free survival (PFS) vs exemestane in postmenopausal women with hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) advanced breast cancer (aBC) after progression on a non-steroidal aromatase inhibitor (NSAI). Here, we report on the open-label, single-arm, phase IIIB 4EVER trial (NCT01626222). This trial evaluated the clinical effectiveness of everolimus plus exemestane in postmenopausal women with HR+, HER2- aBC who had progressed on or after an NSAI, but with no restrictions on the time of progression after NSAI, prior chemotherapy for advanced disease or previous exemestane. The primary endpoint was overall response rate (ORR; i.e. the percentage of patients with a best overall response of complete or partial response per RECIST 1.1) within the first 24 weeks of treatment. Secondary endpoints included PFS, overall survival, safety and health-related quality of life. Between June 2012 and November 2013, 299 patients were enrolled at 82 German centers: 281 patients were evaluable for efficacy and 299 for safety. The ORR was 8.9% (95% confidence interval [CI]: 5.8-12.9%). Median PFS was 5.6 months (95% CI: 5.4-6.0 months). The most frequent grade 3/4 adverse events were stomatitis (8.4%), general physical health deterioration (6.7%), dyspnea (4.7%) and anemia (4.3%). The ORR in 4EVER was lower than in BOLERO-2, likely due to inclusion of patients with more advanced disease and extensive pretreatment. These data confirm the clinical benefits and known safety profile of everolimus plus exemestane in postmenopausal women with HR+, HER2- aBC.

Protean agonists are of great pharmacological interest as their behavior may change in magnitude and direction depending on the constitutive activity of a receptor. Yet, this intriguing phenomenon has been poorly described and understood, due to the lack of stable experimental systems and design strategies. In this study, we overcome both limitations: First, we demonstrate that modulation of the ionic strength in a defined experimental set-up allows for analysis of G protein-coupled receptor activation in the absence and presence of a specific amount of spontaneous receptor activity using the muscarinic M-2 acetylcholine receptor as a model. Second, we employ this assay system to show that a dualsteric design principle, that is, molecular probes, carrying two pharmacophores to simultaneously adopt orthosteric and allosteric topography within a G protein-coupled receptor, may represent a novel approach to achieve protean agonism. We pinpoint three molecular requirements within dualsteric compounds that elicit protean agonism at the muscarinic M-2 acetylcholine receptor. Using radioligandbinding and functional assays, we posit that dynamic ligand binding may be the mechanism underlying protean agonism of dualsteric ligands. Our findings provide both new mechanistic insights into the still enigmatic phenomenon of protean agonism and a rationale for the design of such compounds for a G protein-coupled receptor.

Abstract Introduction: The BOLERO-2 study showed significant doubling of PFS benefit with the addition of EVE to EXE in postmenopausal women with hormone receptor–positive advanced breast cancer progressing after non-steroidal aromatase inhibitor (NSAI) therapy. The BOLERO-2 bone sub study indicated an immediate positive influence of EVE on bone health after 6 and 12 weeks of treatment. To further investigate the longer term effect of mTOR inhibition on bone health, we included this exploratory objective in the phase IIIb, multi-center, open label study 4EVER for postmenopausal women with hormone receptor positive advanced breast cancer treated with EVE +EXE. Methods: From May 2012 to November 2012 bone biomarker samples of 247 postmenopausal women with metastatic or locally advanced, hormone receptor positive, HER2 negative breast cancer refractory to NSAI were collected within this phase IIIb study. Here, we report the results of the planned exploratory analysis of biomarkers of bone turnover. The objective of the exploratory biomarker analysis of bone resorption and formation was to assess the effect of EVE on changes of biomarker levels of bone metabolism (CTX, P-I-NP, osteocalcin, vitamin D, testosterone, estradiol, DHEAS, SHBG, PTH,TSH and FSH) from day 1 to weeks 4, 12 and 24 to confirm potential protective effects of EVE on bone via inhibition of bone osteoclast resorption. Descriptive statistics were used to summarize the single bone resorption markers by visit and difference to baseline. Results: Trial data base lock will occur in late June 2014, therefore, the final biomarker analysis of bone resorption and formation and their influence on ORR, PFS and safety will be presented at SABCS 2014. The preliminary analysis on the changes of bone marker levels from baseline to week 24 included 247 patients. The measured changes from baseline to week 24 in biomarker levels revealed increasing levels of testosterone, DHEAS and FSH, while SHBG and PTH were significantly decreased from baseline to week 24. The combination of EVE to EXE resulted in stabilization of bone health as documented by a decrease of absolute P-I-NP levels at week 24 compared to baseline as well as a stabilization of bone resorption as measured by CTX. Conclusion: Our first preliminary results (data cut off 15 Nov 2013) on the changes in bone marker levels supported the hypothesis of EVE having direct impact on bone health, suggesting a decrease of bone turnover and a reversal of the increase in bone resorption associated with aromatase inhibitor therapy. The final analysis of the 4EVER sub study on bone marker levels in correlation with the efficacy data will provide more detailed insights into possible benefits of the combination of EVE plus EXE. Citation Format: Peyman Hadji, Hans Tesch, Oliver Stoetzer, Thomas Decker, Christian M Kurbacher, Romy Neumeister, Frederik Marmé, Andreas Schneeweiss, Christoph Mundhenke, Andrea Distelrath, Peter A Fasching, Micheal P Lux, Diana Lüftner, Wolfgang Janni, Mathias Muth, Julia Kreuzeder, Claudia Weiss, Eva-Maria Grischke. 4EVER: The impact of mTOR inhibition on bone health in postmenopausal women with hormone receptor positive (HR+) advanced breast cancer treated with everolimus (EVE) in combination with exemestane (EXE) [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 P3-06-04.

Introduction: The BOLERO-2 study showed significant doubling of PFS benefit with the addition of EVE to EXE in postmenopausal women with hormone receptor–positive advanced breast cancer progressing after non-steroidal aromatase inhibitor (NSAI) therapy. The BOLERO-2 bone sub study indicated an immediate positive influence of EVE on bone health after 6 and 12 weeks of treatment. To further investigate the longer term effect of mTOR inhibition on bone health, we included this exploratory objective in the phase IIIb, multi-center, open label study 4EVER for postmenopausal women with hormone receptor positive advanced breast cancer treated with EVE +EXE. Methods: From May 2012 to November 2012 bone biomarker samples of 247 postmenopausal women with metastatic or locally advanced, hormone receptor positive, HER2 negative breast cancer refractory to NSAI were collected within this phase IIIb study. Here, we report the results of the planned exploratory analysis of biomarkers of bone turnover. The objective of the exploratory biomarker analysis of bone resorption and formation was to assess the effect of EVE on changes of biomarker levels of bone metabolism (CTX, P-I-NP, osteocalcin, vitamin D, testosterone, estradiol, DHEAS, SHBG, PTH,TSH and FSH) from day 1 to weeks 4, 12 and 24 to confirm potential protective effects of EVE on bone via inhibition of bone osteoclast resorption. Descriptive statistics were used to summarize the single bone resorption markers by visit and difference to baseline. Results: Trial data base lock will occur in late June 2014, therefore, the final biomarker analysis of bone resorption and formation and their influence on ORR, PFS and safety will be presented at SABCS 2014. The preliminary analysis on the changes of bone marker levels from baseline to week 24 included 247 patients. The measured changes from baseline to week 24 in biomarker levels revealed increasing levels of testosterone, DHEAS and FSH, while SHBG and PTH were significantly decreased from baseline to week 24. The combination of EVE to EXE resulted in stabilization of bone health as documented by a decrease of absolute P-I-NP levels at week 24 compared to baseline as well as a stabilization of bone resorption as measured by CTX. Conclusion: Our first preliminary results (data cut off 15 Nov 2013) on the changes in bone marker levels supported the hypothesis of EVE having direct impact on bone health, suggesting a decrease of bone turnover and a reversal of the increase in bone resorption associated with aromatase inhibitor therapy. The final analysis of the 4EVER sub study on bone marker levels in correlation with the efficacy data will provide more detailed insights into possible benefits of the combination of EVE plus EXE. Citation Format: Peyman Hadji, Hans Tesch, Oliver Stoetzer, Thomas Decker, Christian M Kurbacher, Romy Neumeister, Frederik Marme, Andreas Schneeweiss, Christoph Mundhenke, Andrea Distelrath, Peter A Fasching, Micheal P Lux, Diana Luftner, Wolfgang Janni, Mathias Muth, Julia Kreuzeder, Claudia Weiss, Eva-Maria Grischke. 4EVER: The impact of mTOR inhibition on bone health in postmenopausal women with hormone receptor positive (HR+) advanced breast cancer treated with everolimus (EVE) in combination with exemestane (EXE) [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 P3-06-04.

Introduction BRAWO is a German non-interventional study of 3000 patients (pts) with advanced or metastatic, hormone-receptor-positive, HER2-negative breast cancer treated with everolimus (EVE) and exemestane (EXE). We report results of the 2nd preplanned interim analysis (IA) with a data cut-off on 8 th July 2014. Methods BRAWO collects data on the routine clinical treatment with EVE and EXE at about 400 sites. Main objectives are to extend the knowledge on a) the efficacy in the clinical routine and the impact of physical activity on efficacy and quality of life, b) prophylaxis and management of stomatitis, and c) the sequence of therapy in the clinical routine. The 2 nd IA was defined to take place 12 months after the inclusion of the 500. patient into the documentation. Results Efficacy data will be reported for the first 500 documented pts. Apart from data on the total study population we will present the PFS results for subgroups including pts with or without prior EXE therapy, with or without prior chemotherapy for the advanced setting, with or without visceral metastasis and with regard to the line of treatment in the advanced setting and the extent of physical activity. The respective summary of adverse events for these patients will be presented as well as results on treatment compliance and dosing. Baseline characteristics and medical history as well as insights into treatment sequences before EVE and EXE will further be shown for all enrolled patients with valid baseline documentation (approx. 1200). Conclusion This subgroup analysis will provide insights into the treatment efficacy in the clinical routine and will add to a more comprehensive understanding of the treatment with EVE/EXE. Citation Format: Christian Jackisch, Eva-Maria Grischke, Andreas Schneeweiss, Thomas Decker, Christoph Uleer, Frank Forster, Oliver Tome, Pauline Wimberger, Christian M Kurbacher, Bettina Mueller, Nadia Harbeck, Christoph Mundhenke, Sherko Kuemmel, Mathias Muth, Julia Kreuzeder, Wilhelm Bloch, Hans Tesch, Diana Luftner, Florian Schutz, Peter A Fasching. Subgroup analysis on efficacy in the routine treatment - Results of the 2nd interim analysis of BRAWO, the non-interventional trial Cancer Treatment with Everolimus and Exemestane for HR+ Women [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 P5-19-12.

Abstract Introduction BRAWO is a German non-interventional study of 3000 patients (pts) with advanced or metastatic, hormone-receptor-positive, HER2-negative breast cancer treated with everolimus (EVE) and exemestane (EXE). We report results of the 2nd preplanned interim analysis (IA) with a data cut-off on 8th July 2014. Methods BRAWO collects data on the routine clinical treatment with EVE and EXE at about 400 sites. Main objectives are to extend the knowledge on a) the efficacy in the clinical routine and the impact of physical activity on efficacy and quality of life, b) prophylaxis and management of stomatitis, and c) the sequence of therapy in the clinical routine. The 2nd IA was defined to take place 12 months after the inclusion of the 500. patient into the documentation. Results Efficacy data will be reported for the first 500 documented pts. Apart from data on the total study population we will present the PFS results for subgroups including pts with or without prior EXE therapy, with or without prior chemotherapy for the advanced setting, with or without visceral metastasis and with regard to the line of treatment in the advanced setting and the extent of physical activity. The respective summary of adverse events for these patients will be presented as well as results on treatment compliance and dosing. Baseline characteristics and medical history as well as insights into treatment sequences before EVE and EXE will further be shown for all enrolled patients with valid baseline documentation (approx. 1200). Conclusion This subgroup analysis will provide insights into the treatment efficacy in the clinical routine and will add to a more comprehensive understanding of the treatment with EVE/EXE. Citation Format: Christian Jackisch, Eva-Maria Grischke, Andreas Schneeweiss, Thomas Decker, Christoph Uleer, Frank Förster, Oliver Tomé, Pauline Wimberger, Christian M Kurbacher, Bettina Mueller, Nadia Harbeck, Christoph Mundhenke, Sherko Kuemmel, Mathias Muth, Julia Kreuzeder, Wilhelm Bloch, Hans Tesch, Diana Lüftner, Florian Schütz, Peter A Fasching. Subgroup analysis on efficacy in the routine treatment - Results of the 2nd interim analysis of BRAWO, the non-interventional trial "Breast Cancer Treatment with Everolimus and Exemestane for HR+ Women" [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 P5-19-12.

Abstract Introduction: The phase III BOLERO-2 trial showed a significant doubling of PFS benefit with EVE + EXE over EXE alone in postmenopausal women with hormone receptor positive advanced BC progressing after non-steroidal aromatase inhibitor (NSAI) therapy. The 4EVER trial further evaluated the combination of EVE+EXE with regard to efficacy and safety, quality of life and health resources utilization in a broader patient population, i.e. without limitations as to the number of previous chemotherapy lines, the time point of progression after NSAI therapy, and the previous EXE therapy. Methods: From May 2012 to November 2012 a total of 299 postmenopausal women with metastatic or locally advanced, hormone receptor positive, HER2 negative breast cancer, refractory to NSAI were recruited to this phase IIIb study. Here we report the results of the planned analysis of the primary and secondary endpoints. The primary endpoint was the overall response rate (ORR) at week 24. The secondary endpoints included: Progression-free survival (PFS), ORR at week 48, overall survival (OS), and quality of life. This study includes a broad exploratory translational research program e.g. changes in serum bone turnover biomarkers, the correlation of Interleukin-6 with anxiety and depression, presence and molecular characteristics of circulating tumor cells, the correlation of response to EXE+EVE with pharmacogenomics. Results: Trial database lock will occur in late June 2014, therefore, the final data concerning the primary and secondary efficacy and safety endpoints will be presented at SACBS 2014. The preliminary baseline analysis included 299 patients (data cut off 15 Nov 2013): HR status: ER+/PgR+ 78.1%, ER+/PgR- 20.9%, 0.7% ER-/PgR+, 0.3% ER-/PgR-. Tissue for receptor status analysis: 71.0% primary tumor, 29.0% metastasis. The mean time since initial diagnosis was 9.6 years, the mean time since first relapse/metastasis was 4.3 years. The mean time since last relapse/metastasis was 2.8 months. 68.1% of patients had bone lesions. Last anti-neoplastic therapy had been administered in the adjuvant (23.9%) and metastatic setting (73.0%). 25.9% of patients had no prior antineoplastic therapy in the metastatic setting, 16.3% had one, 12.2% two and 47.4% three or more prior therapies. Conclusion: The final analysis of the 4EVER study provides more important information on disease patterns and benefits of the combined treatment with EVE and EXE. Citation Format: Hans Tesch, Oliver J Stoetzer, Thomas Decker, Christian M Kurbacher, Romy Neumeister, Frederik Marmé, Andreas Schneeweis, Christoph Mundhenke, Andrea Distelrath, Peter A Fasching, Michael P Lux, Diana Lueftner, Peyman Hadji, Wolfgang Janni, Mathias Muth, Julia Kreuzeder, Claudia Weiss, Diethelm Wallwiener. 4EVER - Final efficacy analysis of the phase IIIb, multi-center, open label study for postmenopausal women with estrogen receptor positive locally advanced or metastatic breast cancer (BC) treated with everolimus (EVE) in combination with exemestane (EXE) [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 P5-19-06.

Introduction: The BOLERO-2 study showed significant doubling of PFS benefit with the addition of EVE to EXE in postmenopausal women with hormone receptor–positive advanced breast cancer progressing after non-steroidal aromatase inhibitor (NSAI) therapy. The BOLERO-2 bone sub study indicated an immediate positive influence of EVE on bone health after 6 and 12 weeks of treatment. To further investigate the longer term effect of mTOR inhibition on bone health, we included this exploratory objective in the phase IIIb, multi-center, open label study 4EVER for postmenopausal women with hormone receptor positive advanced breast cancer treated with EVE +EXE. Methods: From May 2012 to November 2012 bone biomarker samples of 247 postmenopausal women with metastatic or locally advanced, hormone receptor positive, HER2 negative breast cancer refractory to NSAI were collected within this phase IIIb study. Here, we report the results of the planned exploratory analysis of biomarkers of bone turnover. The objective of the exploratory biomarker analysis of bone resorption and formation was to assess the effect of EVE on changes of biomarker levels of bone metabolism (CTX, P-I-NP, osteocalcin, vitamin D, testosterone, estradiol, DHEAS, SHBG, PTH,TSH and FSH) from day 1 to weeks 4, 12 and 24 to confirm potential protective effects of EVE on bone via inhibition of bone osteoclast resorption. Descriptive statistics were used to summarize the single bone resorption markers by visit and difference to baseline. Results: Trial data base lock will occur in late June 2014, therefore, the final biomarker analysis of bone resorption and formation and their influence on ORR, PFS and safety will be presented at SABCS 2014. The preliminary analysis on the changes of bone marker levels from baseline to week 24 included 247 patients. The measured changes from baseline to week 24 in biomarker levels revealed increasing levels of testosterone, DHEAS and FSH, while SHBG and PTH were significantly decreased from baseline to week 24. The combination of EVE to EXE resulted in stabilization of bone health as documented by a decrease of absolute P-I-NP levels at week 24 compared to baseline as well as a stabilization of bone resorption as measured by CTX. Conclusion: Our first preliminary results (data cut off 15 Nov 2013) on the changes in bone marker levels supported the hypothesis of EVE having direct impact on bone health, suggesting a decrease of bone turnover and a reversal of the increase in bone resorption associated with aromatase inhibitor therapy. The final analysis of the 4EVER sub study on bone marker levels in correlation with the efficacy data will provide more detailed insights into possible benefits of the combination of EVE plus EXE. Citation Format: Peyman Hadji, Hans Tesch, Oliver Stoetzer, Thomas Decker, Christian M Kurbacher, Romy Neumeister, Frederik Marme, Andreas Schneeweiss, Christoph Mundhenke, Andrea Distelrath, Peter A Fasching, Micheal P Lux, Diana Luftner, Wolfgang Janni, Mathias Muth, Julia Kreuzeder, Claudia Weiss, Eva-Maria Grischke. 4EVER: The impact of mTOR inhibition on bone health in postmenopausal women with hormone receptor positive (HR+) advanced breast cancer treated with everolimus (EVE) in combination with exemestane (EXE) [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 P3-06-04.

Introduction: The phase III BOLERO-2 trial showed a significant doubling of PFS benefit with EVE + EXE over EXE alone in postmenopausal women with hormone receptor positive advanced BC progressing after non-steroidal aromatase inhibitor (NSAI) therapy. The 4EVER trial further evaluated the combination of EVE+EXE with regard to efficacy and safety, quality of life and health resources utilization in a broader patient population, i.e. without limitations as to the number of previous chemotherapy lines, the time point of progression after NSAI therapy, and the previous EXE therapy. Methods: From May 2012 to November 2012 a total of 299 postmenopausal women with metastatic or locally advanced, hormone receptor positive, HER2 negative breast cancer, refractory to NSAI were recruited to this phase IIIb study. Here we report the results of the planned analysis of the primary and secondary endpoints. The primary endpoint was the overall response rate (ORR) at week 24. The secondary endpoints included: Progression-free survival (PFS), ORR at week 48, overall survival (OS), and quality of life. This study includes a broad exploratory translational research program e.g. changes in serum bone turnover biomarkers, the correlation of Interleukin-6 with anxiety and depression, presence and molecular characteristics of circulating tumor cells, the correlation of response to EXE+EVE with pharmacogenomics. Results: Trial database lock will occur in late June 2014, therefore, the final data concerning the primary and secondary efficacy and safety endpoints will be presented at SACBS 2014. The preliminary baseline analysis included 299 patients (data cut off 15 Nov 2013): HR status: ER+/PgR+ 78.1%, ER+/PgR- 20.9%, 0.7% ER-/PgR+, 0.3% ER-/PgR-. Tissue for receptor status analysis: 71.0% primary tumor, 29.0% metastasis. The mean time since initial diagnosis was 9.6 years, the mean time since first relapse/metastasis was 4.3 years. The mean time since last relapse/metastasis was 2.8 months. 68.1% of patients had bone lesions. Last anti-neoplastic therapy had been administered in the adjuvant (23.9%) and metastatic setting (73.0%). 25.9% of patients had no prior antineoplastic therapy in the metastatic setting, 16.3% had one, 12.2% two and 47.4% three or more prior therapies. Conclusion: The final analysis of the 4EVER study provides more important information on disease patterns and benefits of the combined treatment with EVE and EXE. Citation Format: Hans Tesch, Oliver J Stoetzer, Thomas Decker, Christian M Kurbacher, Romy Neumeister, Frederik Marme, Andreas Schneeweis, Christoph Mundhenke, Andrea Distelrath, Peter A Fasching, Michael P Lux, Diana Lueftner, Peyman Hadji, Wolfgang Janni, Mathias Muth, Julia Kreuzeder, Claudia Weiss, Diethelm Wallwiener. 4EVER - Final efficacy analysis of the phase IIIb, multi-center, open label study for postmenopausal women with estrogen receptor positive locally advanced or metastatic breast cancer (BC) treated with everolimus (EVE) in combination with exemestane (EXE) [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 P5-19-06.

Introduction: The BOLERO-2 study showed significant doubling of PFS benefit with the addition of EVE to EXE in postmenopausal women with hormone receptor–positive advanced breast cancer progressing after non-steroidal aromatase inhibitor (NSAI) therapy. The BOLERO-2 bone sub study indicated an immediate positive influence of EVE on bone health after 6 and 12 weeks of treatment. To further investigate the longer term effect of mTOR inhibition on bone health, we included this exploratory objective in the phase IIIb, multi-center, open label study 4EVER for postmenopausal women with hormone receptor positive advanced breast cancer treated with EVE +EXE. Methods: From May 2012 to November 2012 bone biomarker samples of 247 postmenopausal women with metastatic or locally advanced, hormone receptor positive, HER2 negative breast cancer refractory to NSAI were collected within this phase IIIb study. Here, we report the results of the planned exploratory analysis of biomarkers of bone turnover. The objective of the exploratory biomarker analysis of bone resorption and formation was to assess the effect of EVE on changes of biomarker levels of bone metabolism (CTX, P-I-NP, osteocalcin, vitamin D, testosterone, estradiol, DHEAS, SHBG, PTH,TSH and FSH) from day 1 to weeks 4, 12 and 24 to confirm potential protective effects of EVE on bone via inhibition of bone osteoclast resorption. Descriptive statistics were used to summarize the single bone resorption markers by visit and difference to baseline. Results: Trial data base lock will occur in late June 2014, therefore, the final biomarker analysis of bone resorption and formation and their influence on ORR, PFS and safety will be presented at SABCS 2014. The preliminary analysis on the changes of bone marker levels from baseline to week 24 included 247 patients. The measured changes from baseline to week 24 in biomarker levels revealed increasing levels of testosterone, DHEAS and FSH, while SHBG and PTH were significantly decreased from baseline to week 24. The combination of EVE to EXE resulted in stabilization of bone health as documented by a decrease of absolute P-I-NP levels at week 24 compared to baseline as well as a stabilization of bone resorption as measured by CTX. Conclusion: Our first preliminary results (data cut off 15 Nov 2013) on the changes in bone marker levels supported the hypothesis of EVE having direct impact on bone health, suggesting a decrease of bone turnover and a reversal of the increase in bone resorption associated with aromatase inhibitor therapy. The final analysis of the 4EVER sub study on bone marker levels in correlation with the efficacy data will provide more detailed insights into possible benefits of the combination of EVE plus EXE. Citation Format: Peyman Hadji, Hans Tesch, Oliver Stoetzer, Thomas Decker, Christian M Kurbacher, Romy Neumeister, Frederik Marme, Andreas Schneeweiss, Christoph Mundhenke, Andrea Distelrath, Peter A Fasching, Micheal P Lux, Diana Luftner, Wolfgang Janni, Mathias Muth, Julia Kreuzeder, Claudia Weiss, Eva-Maria Grischke. 4EVER: The impact of mTOR inhibition on bone health in postmenopausal women with hormone receptor positive (HR+) advanced breast cancer treated with everolimus (EVE) in combination with exemestane (EXE) [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 P3-06-04.

Aim: BRAWO is a German non-interventional study of 3000 patients (pts) with advanced or metastatic, hormone-receptor-positive and HER2-negative breast cancer treated with everolimus (EVE) and exemestane (EXE). Data is collected at about 400 sites. Main objectives are to extend the knowledge on a) the impact of physical activity on efficiency and quality of life, b) prophylaxis and management of stomatitis in clinical routine, and c) the sequence of therapy, when EVE is used in daily clinical practice. We report the results of the 2nd preplanned interim analysis (IA) which was defined to take place 12 months after the inclusion of the 500th patient into the documentation.

Endocrine therapy (ET) is a key treatment modality in hormone receptor positive (HR+) early breast cancer (BC) patients. Although the anticancer activity of adjuvant ET + zoledronic acid (ZOL) has been investigated, the potential effects of ET ± ZOL on endocrine hormones in premenopausal women with HR+ early BC are not well understood. ProBONE II was prospective, double-blind, randomized controlled trial. Premenopausal patients with histologically confirmed invasive BC with no evidence of metastases and a T score >−2.5 received ET ± ZOL 4 mg every 3 months for 2 years. Serum levels of estradiol (E2), follicle-stimulating hormone, anti-muellerian hormone (AMH), inhibins A and B, sex hormone-binding globulin, parathyroid hormone, total testosterone, and vitamin D were evaluated at baseline and at every scheduled visit. Of 71 women enrolled, 70 were evaluable (n = 34, ZOL; n = 36, placebo). No statistically significant differences were observed in hormone levels, except E2 and AMH, which showed minor differences. These included decreases in serum E2 levels, which reached a nadir after 3 and 9 months in placebo and ZOL groups, respectively, and decrease in serum AMH levels throughout the study with ZOL, but remained constant with placebo after 6 months. Adverse events in ZOL-treated group were influenza-like illness (32.4 %), bone pain (32.4 %), chills (20.6 %), and nausea (23.5 %). ET ± ZOL was well tolerated. This study showed no influence of ZOL on hormonal level changes that accompany ET, supporting inclusion of ZOL in adjuvant therapy for premenopausal women with HR + BC.

BACKGROUND:The objective of this prospectively randomized phase II trial (Trial registration: EUCTR2004-004007-37-DE) was to compare the clinical response of primary breast cancer patients to neoadjuvant therapy with letrozole alone (LET) or letrozole and zoledronic acid (LET + ZOL).METHODS:Patients were randomly assigned to receive either LET 2.5 mg/day (n = 79) or the combination of LET 2.5 mg/day and a total of seven infusions of ZOL 4 mg every 4 weeks (n = 89) for 6 months. Primary endpoint was clinical response rate as assessed by mammogram readings. The study was terminated prematurely due to insufficient recruitment. We report here on an exploratory analysis of this data.RESULTS:Central assessment of tumor sizes during the treatment period was available for 131 patients (66 LET, 65 LET + ZOL). Clinical responses (complete or partial) were seen in 54.5% (95% CI: 41.8-66.9) of the patients in the LET arm and 69.2% (95% CI: 56.6-80.1) of those in the LET + ZOL arm (P = 0.106). A multivariate model showed an OR of 1.72 (95% CI: 0.83-3.59) for the experimental arm.CONCLUSION:No increase in the clinical response rate was observed with the addition of ZOL to a neoadjuvant treatment regimen with LET. However a trend towards a better reponse in the LET + ZOL arm could be observed. This trend is consistent with previous studies that have investigated the addition of ZOL to chemotherapy, and it may support the evidence for a direct antitumor action of zoledronic acid.

Background: Imatinib is a tyrosine kinase inhibitor of BCR-ABL, ABL, PDGFR-α and -β, KIT, and DDR. In solid tumors, it inhibits proliferation and invasiveness and facilitates higher intratumoral cytotoxic drug concentrations. Vinorelbine has good tolerability and efficacy in metastatic breast cancer (MBC). This study evaluates the safety and efficacy of imatinib and vinorelbine in combination. Methods: In a prospective, open-label, phase I/II trial, 400 mg imatinib p.o. daily (corrected from 600 mg) was combined with an escalating dose of vinorelbine i.v. weekly in four dose levels of 10, 15, 20, and 25 mg/m2 (each n ≥ 5) to treat patients with MBC (expressing PDGFR-α and/or -β, and/or KIT). The last patient of each level was treated for >28 days, before enrolment for the next dose level started. Study endpoints were feasibility and tolerability, incidence of hematological and nonhematological toxicity, and clinical efficacy (data cutoff: November 18, 2011). A total of 33 patients have been enrolled, and all dose levels have been fully recruited. One patient is still on study medication. A translational subprotocol is ongoing. Results: All 33 included patients are evaluable for safety (32 within the ITT population). Eleven patients were excluded early from the study (progressive disease, toxicity, and withdrawal of consent). Twenty-two patients participated in the study for >28 days (‘ITT >28'). Within the ITT population, the response rate [complete response (CR) and partial response (PR)] was 9.4% (n = 3), the clinical benefit rate (CBR; CR+PR+stable disease) 50% (n = 16), and the median time to progression (TTP) 155 days. A total of 21.3% of the patients were on study medication for >6 months, and 15.2% for >12 months (mean 140 days, range 15-643). Within ‘ITT >28', the response rate was 13.6%, CBR 72.7%, and median TTP 176 days. The response was independent of the receptor status (PDGFR-α, -β, and KIT). Toxicities were as follows (safety population): 21.6% severe leukopenia, 9.1% severe neutropenia (with 1 febrile neutropenia), 1 case of bowel perforation, 36% diarrhea (3% severe), 84.8% nausea (severe 15.2%), 48.5% vomiting (severe 9.1%), 27.3% infections (severe 6.1%), 12.1% peripheral neuropathy (severe 9.1%), and 36.4% dyspnea (3% severe). Four patients on trial died (nondrug-related). Conclusion: The combination of imatinib and vinorelbine in MBC appeared to be feasible and tolerable. A CBR of 50% (ITT) in pretreated patients suggests that this combination may be active. Although toxicities were frequent, they appeared to be manageable.

Abstract Background: Based on baseline bone mineral density (BMD), adjuvant chemotherapy or endocrine therapy for early breast cancer patients can lead to substantially increased fracture risk. A significant decrease of BMD &gt;10% after 2 years of chemotherapy (CT) and/or endocrine therapy (ET) has been reported. In recent studies, zoledronic acid (ZOL) produced an increase in BMD in premenopausal and postmenopausal patients with breast cancer (ABCSG-12, Z-FAST, ZO-FAST, etc). In addition, a significant increase in disease-free survival (DFS) with ZOL vs no ZOL was observed in most of these studies. Methods: The aim of 2 single-center, placebo-controlled, randomized studies—Probone I and Probone II—was to investigate the effect of adjuvant treatment with ZOL on BMD in premenopausal women with early breast cancer treated with CT and/or ET. Patients with hormone-receptor-negative (HR−) breast cancer (Probone I) were treated with (neo)adjuvant CT; patients with hormone-receptor-positive (HR+) breast cancer (Probone II) were treated with ET alone or in combination with (neo)adjuvant CT. Randomized patients received ZOL 4 mg or placebo IV every 3 months for 24 months. The primary objective was the change in BMD at the lumbar spine between baseline and month 24 (measured by dual-energy X-ray absorptiometry [DXA]). Secondary objectives included DFS; BMD at total hip, femur, and os calcis; quantitative ultrasonometry (QUS) at os calcis and phalanges; markers of bone turnover (C-telopeptide of type I collagen [CTX] and N-terminal propeptide of type I procollagen [P1NP]); endocrine hormones (follicle-stimulating hormone [FSH], estradiol, testosterone, sex hormone-binding globulin [SHBG], parathyroid hormone [PTH], vitamin D, anti-Müllerian hormone [AMH], inhibin A/B, etc); pathologic fractures; and safety and tolerability. Results: 70 HR+ and 11 HR− breast cancer patients have been enrolled into the studies. The last patient will have been treated for 24 months by the end of June 2011. Conclusions: The effects of ZOL on lumbar spine BMD at 24 months and secondary endpoints will be presented at the meeting. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-19-03.

We present a new concept of partial agonism at G protein-coupled receptors. We demonstrate the coexistence of two functionally distinct populations of the muscarinic M2 receptor stabilized by one dynamic ligand, which binds in two opposite orientations. The ratio of orientations determines the cellular response. Our concept allows predicting and virtually titrating ligand efficacy, which opens unprecedented opportunities for the design of drugs with graded activation of the biological system.

591 Background: The presence of circulating tumor cells (CTCs) has been shown to be of prognostic relevance for patients with early and advanced breast cancer (BC). The usefulness of CTC assessments depends on accurate cell counts and corresponding analysis of molecular targets. The aim of this sub study was to assess the feasibility of a novel, integrated CTC platform for automated cellular protein and nucleic acid analysis in a prospective multi-center study. Methods: The German 4EVER study included patients with postmenopausal, metastastic, ER positive, HER2 negative BC, who progressed after therapy with a non-steroidal aromatase inhibitor and were treated with exemestane and the mTOR inhibitor everolimus. Baseline blood samples (TransFix vaccum tubes, Cytomark, Caltag Medsystems Ltd.) were used for CTC analysis and processed on the modified Versant kPCR Sample Prep system using 8µm pore size Whatman Nuclepore track-etched membranes (GE Healthcare Piscataway, NJ). After CTC capture, immunostaining was performed for Cytokeratin 8/18/19 and CD45. CTCs were detected by image analytics after fluorescence scanning microscopy using a dedicated software solution implemented by Siemens. The data is summarized and correlated with baseline clinical characteristics. Results: CTC counts and clinical data were available for 111 blood samples (91.7%). CTCs were found in 75 patients (67,6%), 13 patients having 1 CTC, 38 having 2-9 CTCs and 24 patients having 10 or more CTCs. In an exploratory analysis the presence of CTCs was correlated with baseline patient and tumor characteristics. There were no associations with primary TNM stage, hormone receptor status or tumor type. Conclusions: CTC assessment with this novel filtration based method was feasible in a multi-center study setting. The CTC positivity rate was within the expected range. The follow-up of this study will give first insights, how the CTC measures of this platform can be used as a prognostic tool. As this CTC assessment platform was developed to perform additional automated cellular protein and nucleic acid analysis, the usefulness might derive from these analytic tools as well. Clinical trial information: NCT01626222.