Abstract 2344: The PD-1:PD-L1 pathway in the context of the osteosarcoma tumor microenvironment
Authors: Koirala Pratistha , Gill Jonathan , Roth Michael , Piperdi Sajida , Park Amy , Poon Vincent , Fremed Michael , Hoang Bang , Gorlick Richard 
Published: August 2015

Abstract
Background
PD-1 is a transmembrane protein found on immune cells including T cells, B cells, natural killer (NK) T cells, activated monocytes, and dendritic cells. Its interaction with its ligand, PD-L1, plays a role in the suppression of the immune system and is important in many processes such as autoimmunity and both central and peripheral tolerance. Cancer cells can hijack the PD-1:PD-L1 pathway in order to evade clearance by the immune system—a mechanism that is evident in many solid tumors, including breast, ovarian, renal cell carcinoma, colorectal, and lung cancers. Inhibition of the PD-1:PD-L1 pathway with both PD-1 and PD-L1 inhibitors have shown varying levels of efficacy, and provide proof of concept—that modulating the PD- 1:PD-L1 pathway can tip the balance of immune response in favor of the patient, working to eliminate the tumor. We seek to determine the expression of PD-L1 in osteosarcoma (OS), specifically in relation to the prescience of tumor infiltrating lymphocytes (TILs). By displaying expression of PD-L1 and the presence of TILs we hope to subsequently target the PD-1:PD-L1 pathway in OS.
Methods
Paraffin imbedded and sectioned patient primary OS tumor samples were provided by IRB approved protocols at Montefiore Medical Center and Memorial Sloan Kettering Cancer Center. Antibodies to identify various immune cells were first validated and optimized using immune tissues and subsequently used to stain tumor sample. Next, RNA was extracted from patient OS tumor samples and used to generate cDNA. PD-L1 expression was determined using quantitative real time PCR. Protein was also extracted and will be used to confirm PD-L1 expression using western blotting.
Results
A variety of TILs are found in OS patient tumor samples. We see expression of T cells (CD3) and T cell subsets—helper (CD4), cytotoxic (CD8), and natural killer (CD56) T cells. There is also robust expression of B cells (CD20) and macrophages (CD68). Furthermore, the OS tumors displayed varying degrees of PD-L1 positivity, with some tumors displaying greatly increased expression, some showing moderate elevation, and others showing low to no changes in PD-L1 expression.
Conclusions and Future Directions
OS tumors display varying levels of PD-L1 positivity, suggesting that the PD-1:PD-L1 pathway can be modulated for immunotherapeutic purposes. Importantly, we also demonstrated that a subset of immune cells, potentially expressing PD-1, is present in some tumors. Cell lines derived from tumors expressing low, moderate, and high levels of PD-L1 are being cultured. These cell lines will be used to test inhibition of the PD-1:PD-L1 pathway and its impact on their tumorigenic properties.
Citation Format: Pratistha Koirala, Jonathan Gill, Michael Roth, Sajida Piperdi, Amy Park, Vincent Poon, Michael Fremed, Bang Hoang, Richard Gorlick. The PD-1:PD-L1 pathway in the context of the osteosarcoma tumor microenvironment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2344. doi:10.1158/1538-7445.AM2015-2344