Abstract PD9-07: Mdm2 gene amplification in estrogen receptor-positive breast cancer cells is associated with enhanced solid tumor growth and pronounced metastatic potential in humanized tumor mice (HTM) and a poor outcome of patients with luminal breast cancer
Authors: Wege Anja Kathrin , Vladimirova Valentina , Solbach Christine , Rom-Jurek Eva-Maria , Blohmer Jens-Uwe , Jank Paul , Sinn Bruno , Trumpp Andreas , Marangoni Elisabetta , Engels Knut , Weichert Wilko , Pfarr Nicole , Irlbeck Christoph , Polzer Bernhard , Ortmann Olaf , van Mackelenbergh Marion , Denkert Carsten , Loibl Sibylle , Brockhoff Gero 
Published: March 2022

Abstract
Background: Luminal, i.e., estrogen receptor (ER)-positive breast cancer (BC) is a heterogeneous disease in terms of tumor progression, therapy response, and relapse. Additional biomarkers with a prognostic and predictive impact would facilitate advanced patient stratification and can reveal advanced therapeutic options for individual patients suffering from luminal BC subtype. First objective of this study was the hypothesis driven validation and identification of biomarkers associated with successful engraftment, augmented tumor growth, and enhanced metastasis upon xenotransplantation into HTM and non-humanized tumor mice. Second objective was the retrospective validation and correlation of aforementioned markers with clinical outcomes (disease free-survival [DFS] and overall survival [OS]) of luminal BC patients after neoadjuvant chemotherapy within the GeparTrio trial. Methods: Immunodeficient NSG mice with and without immunological humanization were transplanted with primary, ER-positive BC tissues and cells. Engraftment rates, tumor progression, and metastasis were monitored as a function of marker expression and genomic alterations, considered to be associated with tumor cell stemness and tumor initiating capacity. Functional assays were applied to demonstrate the impact of identified markers on tumor cell viability, growth and migration in-vitro. Dual color fluorescence-in-situ-hybridization to monitor mdm2 gene and the cen12 region was applied to 502 pretherapeutic ER-positive tissue specimens of BC patients treated with anthracycline/taxane based neoadjuvant chemotherapy within the GeparTrio trial. Mdm2 gene expression was analyzed in the entire luminal BC cohort as well as in luminal-A and luminal-B samples classified based on a Ki-67 cut-off of 20% (St. Gallen guideline). Associations with survival outcomes were studied by Cox regression models. Results: We observed an elevated CD44 and c-MET expression in metastatic cells compared to the primarily growing xenotransplantants. Moreover, we found mdm2 gene amplification was associated with tumor growth and pronounced metastatic potential in NSG mice. Functional assays unveiled a reduced viability, proliferation, and migration capacity of inherently mdm2 positive breast cancer cells upon mdm2 knock-down or anti-mdm2 targeting. Validation of mdm2 gain in luminal BC cohort within the GeparTrio trial revealed a significant association of mdm2 amplification with worse DFS (HR=1.80 [95%CI 1.16-2.79], log rank p=0.008) and OS (HR=1.75 [95%CI 1.00-3.05], log-rank p=0.047). This association was even stronger in luminal-A BC: DFS (HR=2.56 [95%CI 1.40-4.71], log rank p=0.002 and OS (HR=3.27 [95%CI 1.51-7.09], log-rank p=0.001). Conclusions: Mdm2 gene amplification facilitates ER-positive BC engraftment and progression in a preclinical in-vivo xenograft humanized NSG mouse model. Targeting mdm2 in-vitro reduced malignant cell propagation and growth. In addition, an increased mdm2 gene dose is strongly associated with an unfavorable outcome of luminal BC. Prospective studies are required to verify the suitability of mdm2 for advanced luminal BC stratification and therapeutic targeting.
Citation Format: Anja Kathrin Wege, Valentina Vladimirova, Christine Solbach, Eva-Maria Rom-Jurek, Jens-Uwe Blohmer, Paul Jank, Bruno Sinn, Andreas Trumpp, Elisabetta Marangoni, Knut Engels, Wilko Weichert, Nicole Pfarr, Christoph Irlbeck, Bernhard Polzer, Olaf Ortmann, Marion van Mackelenbergh, Carsten Denkert, Sibylle Loibl, Gero Brockhoff. Mdm2 gene amplification in estrogen receptor-positive breast cancer cells is associated with enhanced solid tumor growth and pronounced metastatic potential in humanized tumor mice (HTM) and a poor outcome of patients with luminal breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD9-07.