BackgroundClinically relevant predictive biomarkers to tailor anti-angiogenic therapies to breast cancer (BRC) patient subpopulations are an unmet need.MethodsWe analyzed tumor vascular density and VEGFR2 protein expression in various subsets of primary human BRCs (186 females; Mean age: 59 years; range 33–88 years), using a tissue microarray. Discrete VEGFR2+ and CD34+ tumor vessels were manually scored in invasive ductal, lobular, mixed ductal-lobular and colloid (N = 139, 22, 18, 7) BRC cores.ResultsThe observed CD34+ and VEGFR2+ tumor vascular counts in individual cases were heterogeneous. Mean CD34+ and VEGFR2+ tumor vessel counts were 11 and 3.4 per tumor TMA core respectively. Eighty-nine of 186 (48%) cases had >10 CD34+ tumor vessels, while 97/186 (52%) had fewer CD34+ vessels in each TMA core. Of 169 analyzable cores in the VEGFR2 stained TMA, 90 (53%) showed 1–5 VEGFR2+ tumor vessels/TMA core, while 42/169 (25%) cores had no detectable VEGFR2+ tumor vessels. Thirteen of 169 (8%) cases also showed tumor cell (cytoplasmic/membrane) expression of VEGFR2. Triple-negative breast cancers (TNBCs) appeared to be less vascular (Mean VD = 9.8, range 0–34) than other breast cancer subtypes. Overall, VEGFR2+ tumor vessel counts were significantly higher in HER2+ as compared to HR+ (p = 0.04) and TNBC (p = 0.02) tissues. Compared to HER2− cases, HER2+ breast cancers had higher VEGFR2+ tumor vessel counts (p = 0.007).ConclusionCharacterization of pathologic angiogenesis in HER2+ breast cancer provides scientific rationale for future investigation of clinical activity of agents targeting the VEGF/VEGFR2 axis in this clinically aggressive breast cancer subtype.

Ewing’s sarcoma (ES) is a rare but aggressive cancer typically occurring in the bone or soft tissue of children and adolescents. Current treatment regimens composed of surgery, radiation, and high-dose chemotherapy have improved outcomes for localized disease cases; however, ES continues to present a clinical challenge in the recurrent or metastatic setting. ES is often characterized by a chromosomal translocation that results in a chimeric EWS/ETS transcription factor. This fusion protein may contribute to tumorigenesis through modulation of downstream targets leading to cell cycle dysregulation, due in part to the overexpression of Cyclin D1. In addition, ES also has a high degree of DNA methylation when compared to other tumor types, which can lead to epigenetic silencing of key tumor suppressor genes. Abemaciclib is a small-molecule inhibitor of cyclin-dependent kinases 4 and 6 (CDK4 and 6) currently under clinical evaluation for metastatic breast cancer, non-small cell lung cancer, and other adult solid tumors. Abemaciclib shows antitumor activity in preclinical models of human adult and pediatric cancer with functional Rb and high cyclin D expression, and has been reported to elicit an immune response in addition to its cell cycle effects in breast cancer. This study was designed to assess abemaciclib-induced antitumor effects, in addition to epigenetic and immunogenic changes, in preclinical ES models. Six ES cell lines were treated with abemaciclib and the resulting changes in protein expression and methylation were evaluated by Western blot and DNA promoter methylation analysis, respectively. In addition, tumor-secreted factors from conditioned media were measured by ELISA and activation of the interferon (IFN) pathway was assessed through Western blot, qRT-PCR, and RNAseq analysis. In vivo studies of abemaciclib as a single agent or in combination with ES standard-of-care (SOC) in multiple mouse xenograft models were also performed to measure abemaciclib-mediated antitumor effects and epigenetic and immunogenic changes. Single-agent abemaciclib elicited tumor growth delay or tumor stasis, while the combination with SOC was generally superior to either monotherapy arm, often resulting in objective responses. Abemaciclib induced global DNA demethylation through the Rb-E2F-DNMT1 axis, and a ligand-independent type III IFN response. In addition, abemaciclib increased expression of the tumor cell antigen presentation protein MHC-1, and this effect was more durable than abemaciclib-dependent cell cycle effects. These data demonstrate that abemaciclib activity in ES goes beyond cell cycle arrest to include a measurable and durable immunogenic response. Therefore, the multipronged antitumor effects of abemaciclib, namely CDK4/6 inhibition, global DNA demethylation, and resulting immunogenic changes, may be a viable therapeutic option for ES patients when used in combination with appropriate chemotherapy, and additional studies to better understand this unique biology are ongoing. Citation Format: Michele Dowless, Caitlin Lowery, Matthew Renschler, Wayne Blosser, Jennifer Stephens, Robert Flack, Kelly Credille, Mia Chen, Frank Dorsey, Lillian Sams, Philip Ebert, Jonathan Olsen, Terry Shackleford, Peter Houghton, Louis Stancato. The CDK4 and 6 inhibitor abemaciclib induces both epigenetic and immunogenic responses in preclinical models of Ewing’s sarcoma [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B43.

Platelet-derived growth factor receptor alpha (PDGFRα) is implicated in several types of adult and pediatric malignancies, where its aberrant expression and/or activation in tumor cells and/or tumor-associated stromal cells promote primary tumor growth and metastasis. Therefore, PDGFRα signaling may regulate disease progression via autocrine and paracrine modes of activation and facilitating crosstalk between the tumor and stroma. Olaratumab is a fully humanized monoclonal antibody that selectively binds human PDGFRα and blocks signaling initiated by ligand binding. We evaluated the efficacy of olaratumab and 1E10, a high affinity anti-mouse PDGFRα antibody in preclinical metastatic models of human osteosarcoma. A metastatic derivative of the PDGFRα-positive human osteosarcoma cell line HuO9 (HuO9-H3) was implanted in the gastrocnemius of mice. Olaratumab/1E10 was administered twice weekly once tumors grew to ~ 150 mm3. When tumors reached an average of volume of 1600 mm3, tumor-bearing limbs were amputated and four separate treatment cohorts were evaluated. These cohorts were as follows: (1) continuous IgG control antibody treatment, (2) continuous olaratumab/1E10 treatment prior to and after amputation, (3) olaratumab/1E10 treatment pre-amputation followed by IgG administration post-amputation, and (4) IgG treatment pre-amputation followed by olaratumab treatment post-amputation. A statistically significant and prolonged overall survival (OS) benefit (p<0.001) was observed in the continuous olaratumab/1E10 treatment group only, and correlated with a reduced tumor burden in the lung as determined by histologic evaluation. An olaratumab/1E10-dependent statistically significant OS benefit (p<0.001) was also observed in the HuO9-H3 and PDGFRα/PDGFRβ-positive MG63.3 human osteosarcoma cell lines introduced via tail vein injection. Interestingly, histologic review shows reduced tumor burden in the lung and lung pleura/mediastinum of these models. Mouse-specific bright field in situ hybridization (BRISH) showed increased expression of PDGFRA, PDGFRB, associated ligands, and VEGFA in the murine lung stroma prior to histologically apparent metastases. These data indicate that olaratumab/1E10-mediated PDGFRα blockade significantly increases OS in preclinical mouse models of human osteosarcoma and suggest a novel role for the PDGFRα pathway in the pathogenesis of metastatic osteosarcoma lung lesions. Citation Format: Amy K. LeBlanc, Gerard J. Oakley, Caitlin D. Lowery, Arnulfo Mendoza, Ling Ren, Timothy Holzer, Kelly Credille, Cynthia Winings, Amanda Estelle, Mia Chen, Patrick Finnegan, Wayne Blosser, Andrew Schade, Symantha Melemed, Louis F. Stancato. The anti-platelet-derived growth factor receptor α antibody olaratumab (Lartruvo) increases overall survival in metastatic mouse models of human osteosarcoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-B01.