Abstract 2471: Evaluation of CD47 expression and effects of CD47-SIRPα fusion protein in patients with osteosarcoma
Authors: Piperdi Sajida , Roth Michael , Morriss Nick , Zinone Christian , Zhang Wendong , Koirala Pratistha , Geller David , Hoang Bang , Yang Rui , Gill Jonathan , Gorlick Richard 
Published: July 2016

Abstract
CD47 is a commonly expressed trans-membrane protein which binds to signal regulatory protein α (SIRPα) expressed on the surface of the macrophages resulting in inhibition of phagocytosis and cell clearance. Several malignancies demonstrate high expression of CD47 and recent studies suggest interfering with the CD47-macrophage interaction may be therapeutic. A prior expression analysis performed on osteosarcoma (OS) patient samples reported marked expression of CD47. The current study evaluated the utility of targeting CD47 in Osteosarcoma by determining the prevalence of CD47 expression and the prognostic value of CD47 expression in patients with Osteosarcoma. An immunofluorescence phagocytosis assay is being performed to directly assess the therapeutic potential of the CD47-SIRPα fusion protein.
A previously created and described tissue microarray (TMA) was stained with a murine monoclonal anti-CD47 (B6H12) antibody. The intensity and location of tissue staining were assessed by a comparison between the positive and negative control slides. 81 specimens were evaluated on the TMA. Overall, CD47 was expressed in 87.7% of specimens, with 28.4%, 27.2%, and 32.1% demonstrating high, intermediate, and low expression, respectively. Almost all metastatic tumor specimens (85.7%) expressed CD47. To evaluate CD47 expression quantitatively, a real time PCR was performed on OS patient-derived primary samples, and cell lines. 71.4% of patient derived cell lines (n = 15) showed higher level of CD47 expression compared to that of mesenchymal stem cells and an osteoblast cell line. Furthermore, flow cytometry was performed on human OS xenografts and cell lines to determine the surface expression level of CD47 prior to treatment with CD47-SIRPα fusion protein. 85% of the OS cell lines (n = 13) showed levels of CD47 expression comparable to that of the positive control, leukemia cell line HL60. Survival analyses suggested that increased expression of CD47 may be associated with poorer 5 year event free survival (69.3% vs 42.4%, p = 0.6 and 49.5% vs. 33.6%, p = 0.16) when using thresholds of intermediate/high expression of CD47 and high expression of CD47, respectively. Results of an in-vitro phagocytosis assay assessing cytotoxicity of a CD47-SIRPα fusion protein on OS cell lines using laser scanning cytometry at present show some inhibition of phagocytosis upon adding 500 ug/ml of CD47-SIRPα fusion protein, but it does not reach the statistical significance. These findings demonstrate that CD47 is highly prevalent in Osteosarcoma and suggest CD47 has potential utility as a novel target for anti-cancer therapy in Osteosarcoma. Although the immunofluorescence assays have not demonstrated marked augmentation of activity with the CD47-SIRPα fusion protein, this may be attributable to limitations of the method with clinical trials necessary to define its true level of activity in treating patients with Osteosarcoma.
Citation Format: Sajida Piperdi, Michael Roth, Nick Morriss, Christian Zinone, Wendong Zhang, Pratistha Koirala, David Geller, Bang Hoang, Rui Yang, Jonathan Gill, Richard Gorlick. Evaluation of CD47 expression and effects of CD47-SIRPα fusion protein in patients with osteosarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2471.