Programmed Cell Death Ligand 1 Expression in Osteosarcoma

Abstract

Programmed cell death 1 ligand 1 (PD-L1, B7H1) is a cell-surface protein that suppresses the cytotoxic CD8+ T cell-mediated immune response. PD-L1 expression and its clinical relevance in sarcomas are not well understood. Therefore, we sought to measure RNA expression levels for PD-L1 in 38 clinically annotated osteosarcoma tumor samples, and aimed to determine if PD-L1 expression correlates with clinical features and tumor-infiltrating T-lymphocytes (TILs). Quantitative real-time RT-PCR for PD-L1 was optimized in 18 cell lines, of which 5 were osteosarcoma-derived. qRT-PCR results were validated via flow cytometry and immunohistochemistry (IHC) in select cell lines. Total RNA was isolated from 38 human osteosarcoma samples for qRT-PCR analysis. Clinical data were sorted and significance was determined by Student t-test. TILs were examined in patient samples by tissue microarray (TMA) hematoxylin-eosin (HE) staining. We confirmed the constitutive PD-L1 mRNA expression in cell lines by qRT-PCR, flow cytometry, and IHC. Across human osteosarcoma samples, PD-L1 mRNA gene expression ranged over four-log (>5000-fold difference). Relative expression levels were evaluated against clinical factors such as age/gender, metastasis, recurrence, chemotherapy, percent necrosis, and survival; no significant associations were identified. The presence of TILs was associated with high PD-L1 expression (R2=0.37, P=0.01). In summary, we developed an RNA-based assay to determine PD-L1 expression levels, and we show for the first time that high levels of PD-L1 are expressed in a subset of osteosarcoma, and PD-L1 expression is positively correlated with TILs. There are multiple agents targeting PD-1/PD-L1 in clinical development, and this may be a novel immunotherapeutic strategy for osteosarcoma clinical trials.

PDL1 expression is an independent prognostic factor in localized GIST

“…These limitations led to the use of alternative methods, such as mRNA analysis based on in situ hybridization (ISH), 42 DNA microarrays, 43 or quantitative real-time polymerase chain reaction (qRT-PCR). 44 A positive relationship between protein and mRNA expression has been reported. 42 One limitation of DNA microarray-or qRT-PCR-based measurements is that they quantify expression level of both tumor and non-tumor cells present in the sample.…”

PDL1 expression is a poor-prognosis factor in soft-tissue sarcomas

“…For example, the "Blueprint PD-L1 IHC assay comparison project" was launched last year by a consortium of pharmaceutical companies, diagnostic companies, and academic associations 54 to compare four PD-L1 IHC diagnostic assays developed in conjunction with four PD-1/ PD-L1 immune checkpoint inhibitors used in lung cancer clinical trials. This current limitation of IHC led to use alternative methods, such as mRNA analysis based on in situ hybridization (ISH), 55 DNA microarrays, 56 or qRT-PCR, 57 all the more that a positive relationship exists between protein and mRNA expression. 50,55,57,58 The mRNA analysis based on DNA microarrays and RNA sequencing allowed not only to avoid the limitations of IHC, but also to work on a large series of samples and to search for associations with other genes.…”

The contribution of immune infiltrates and the local microenvironment in the pathogenesis of osteosarcoma

“…Similarly, osteosarcoma cells express B7-H3 which is correlated with the aggressiveness of the disease and metastatic capacity [85,86]. Program Death Ligand-1 (PDL-1) and PDL-2 were also detectable in all osteosarcoma cases [89][90][91][92][93][94].…”