Material and Method: Tissue microarrays of 3-4 mm diameter were composed from paraffin blocks of 222 various sarcomas. Slides prepared from microarrays were stained for PD-L1 antibody (Cell Signaling, E1L3N®) using Leica Bond Autostainer. Any membranous staining over 5% of the cells was regarded as positive. Quantitative real-time PCR with TaqMan gene expression assays for PDL1 was performed using whole sections from FFPE tissue of PD-L1 positive cases, by normalizing absolute values to β-actin. Relative expression level of mRNA of PDL1 was calculated and scored using Log102(threshold cycle of b-actin - threshold cycle of PDL1).

Results: Immunohistochemically, PD-L1 expression was present in 34 of 222 (15%) sarcomas. 5/13 (39%) undifferentiated pleomorphic sarcomas, 6/18 (33%) malignant peripheral nerve sheath tumors, 5/16 (31%) dedifferentiated liposarcomas, 4/19 (21%) rhabdomyosarcomas, 2/16 (13%) epithelioid sarcomas, 2/15 (13%) leiomyosarcomas, 3/26 (12%) synovial sarcomas, 1/18 (6%) myxoid liposarcoma, 1/2 (50%) extraskeletal myxoid chondrosarcoma, 1/3 (33%) alveolar soft part sarcoma, 1/3 (33%) parachordoma/myoepithelioma, 1/5 (20%) pleomorphic liposarcoma, 1/7 (14%) angiosarcoma, 1/8 (13%) Ewing sarcoma showed PD-L1 expression. Cases of solitary fibrous tumor/hemangiopericytoma (18), desmoplastic round cell tumor (14), Ewing-like sarcoma (6), epithelioid hemangioendothelioma (5), clear cell sarcoma (4), myxofibrosarcoma (4), low grade fibromyxoid sarcoma (2) were all negative. Tumor-infiltrating hematopoietic cells were positive for PD-L1 in 32 cases (15%) with only 2 cases overlapping with PD-L1 staining in tumoral cells. Sixteen of 34 (47%) immunohistochemically PD-L1 positive cases showed significant but low-level PD-L1 mRNA overexpression.

Conclusion: We have shown PD-L1 expression in a subset of sarcomas, both at the protein and mRNA level. High-grade pleomorphic sarcomas tend to show more frequent PD-L1 expression. Clinical trials are necessary to further assess the effect of anti PD-L1 drugs on sarcomas showing PD-L1 expression.

Management of primary sarcoma is basically surgery, while chemotherapy or radiotherapy has only marginal effect on metastatic sarcomas. Immunotherapy with anti PD-L1 monoclonal antibodies is a promising treatment option in the management of metastatic sarcoma. Therefore, in this study we investigated the presence of PD-L1 expression among sarcomas at both the protein and RNA expression level.

Representative areas were selected and 3-4 mm diameter tissue microarrays were composed from paraffin blocks. Microarray slides were stained for PD-L1 antibody (Cell Signaling, E1L3N®, 1/400) using Leica Bond Autostainer, according to the manufacturer’s instructions. Antigen retrieval was performed with EDTA pretreatment for 20 minutes. Any membranous staining over 5% of the cells was regarded as positive. PD-L1 expression in tumor infiltrating inflammatory cells was also noted.

Quantitative real-time PCR with Fast-Plus EvaGreen Master Mix (Cat No:31020, Biotium) and gene expression assays for CD274 was performed using whole sections from FFPE tissue of CD274 positive cases, by normalizing absolute values to β-actin. Total RNA was extracted using Hybrid-R RNA purification kit (GeneAll Biotechnology Co. Ltd.) according to the manufacturer protocol. RNA samples were quantified using the highly sensitive Qubit quantitation assays (Thermo Fisher Scientific). Equal amounts of RNA were reverse transcribed using a HyperScriptTM Reverse Transcriptase (GeneAll Biotechnology Co. Ltd.) with oligod (T) primers according to the manufacturer’s instruction (Table I) PCR was performed with Fast-Plus EvaGreen gene expression master mix, cDNA, nuclease free water, forward and reverse primers in a 20μl final reaction volume. The amplification cycles were performed by the Rotor Gene - Q System (Qiagen) as follows, hold at 95°C for 3 min, followed by amplification for 40 cycles, each cycle consisting of denaturation at 95°C for 5 s, annealing at 55°C for 30 s. The housekeeping gene β-actin (Table I) expression level was used as an internal control to evaluate the integrity of each sample. Relative expression level of mRNA of PDL1 was calculated and scored using Log₁₀2^{(threshold cycle of b-actin - threshold cycle of PDL1)}. Relative expression was scored on a log10 scale (0–2).

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Table I: The forward and reverse primers used in the real-time polymerase chain reaction analyses for CD274 and β-Actin genes.

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Table II: PD-L1 expression among various sarcomas

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Figure 1: Examples of PD-L1 expression in various sarcomas.

PD-L1 expression in tumor infiltrating inflammatory cells was present in 32 of 222 (14.4%) sarcomas (Figure 2A-C). Only 4 of 34 (11.8%) PD-L1 positive sarcomas, 3 of which were dedifferentiated liposarcomas, also had PD-L1 positive inflammatory cells within the neoplasm (Table III).

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Figure 2: A) Both tumor cells and tumor infiltrating inflammatory cells were positive in a dedifferentiated liposarcoma (PD-L1; x200). B-C) In 13% of cases, only inflammatory cells were positive (PD-L1;x400).

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Table III: PD-L1 expression in tumor and accompanying inflammatory cells

Quantitative real-time PCR revealed that 16 of 34 (47%) cases showed significant (>1 log) but low-level PD-L1 mRNA overexpression (Figure 3). None of the cases showed >2 log mRNA expression.

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Figure 3: Quantitative PCR results of PD-L1 positive cases.

Malignant mesenchymal tumors showing predominantly pleomorphic morphology, such as undifferentiated pleomorphic sarcomas, malignant peripheral nerve sheath tumors, and dedifferentiated liposarcomas demonstrated more PD-L1 expression (over 30% of the cases) than other types of sarcomas. In line with this, in a study including 53 MPNSTs, 13% were shown to express PD-L1 in tumor cells^15, while in others 25% and 21% of MPNSTs were PD-L1 positive^11,14. Some studies also confirmed a significant correlation between PD-L1 expression and advanced clinicopathological parameters, such as higher clinical stage, presence of distant metastasis, as well as higher histological grade, poor differentiation of tumor, and tumor necrosis^8,9. Moreover, both PD1- and PD-L1 positivity were reported to be independent prognostic factors for overall survival by multivariate analysis; cases with a PD1+/PD-L1+ phenotype had the worst prognosis, which may benefit from PD1-based immunotherapy^8,9. Besides, few low-grade sarcomas, such as myxoid liposarcoma without a high-grade round cell component also showed PD-L1 expression. Knowing ththat chemotherapy and radiotherapy has very limited effect on low grade sarcomas, anti-PD-L1 treatment may be the best option to prevent progression in metastatic low grade sarcomas showing PD-L1 expression. Larger series are required to assess the prevalence of PD-L1 expression among extraskeletal myxoid chondrosarcomas, alveolar soft part sarcomas, and parachordoma/myoepitheliomas as PD-L1 was positive in a significant percent of the low number of cases. Unfortunately, various sarcoma types including solitary fibrous tumor/hemangiopericytoma, desmoplastic round cell tumor, Ewing-like sarcoma, epithelioid hemangioendothelioma, clear cell sarcoma, myxofibrosarcoma, and low-grade fibromyxoid sarcoma, many of which are high-grade by definition, did not express PD-L1, thus having less potential to benefit from anti-PD-L1 therapies.

Expression of PD-L1 in tumor-infiltrating lymphocytes and macrophages was previously documented in sarcomas in detail^8,12. Inflammatory cells are present in almost all of the sarcomas, and 30% of lymphocytes and 58% of macrophages expressed PD-L1 (12). Paydas et al. also recorded that 30% of cells forming tumor microenvironment expressed PD-L1¹⁴. Our result of 14.4% PD-L1 positive tumor-infiltrating inflammatory cells seems to be lower than expected, possibly due to the use of tissue microarrays instead of whole sections. We found a very low level (4%) of co-expression of PD-L1 in both tumor and tumor infiltrating inflammatory cells. In the series of Paydas et al., 21% of the sarcomas showed PDL1 expression in both the tumor microenvironment and tumor cells; and Kaposi sarcoma, not surprisingly, was the most common sarcoma with 80% of the cases showing this phenotype¹⁴. This high level of co-expression could be attributed to the phenomenal role of the immune system on the development and regression of this tumor.

Previously, PD-L1 expression was also demonstrated in both mRNA and protein level in a subset of osteosarcomas⁷. In their study, 9 samples had high-level expression (24%) with over 3 log fold increase, 19 samples (50%) had intermediate expression, 4 (10%) had low-level expression, and 6 (16%) were negative. More recently, only 5 of 89 (5.6%) non-small cell lung carcinomas were shown to have a PD-L1 gene amplification, which is associated with worse prognosis¹⁶. In our series, immunohistochemically PDL1 positive cases showed a low to intermediate level of PD-L1 mRNA expression by quantitative PCR. Our results indicate that overexpression of PD-L1 protein is mainly modulated by post-translational mechanisms rather than at the transcriptional level, and the mechanism of PD-L1 overexpression differs among different kind of tumors.

In conclusion, we have shown PD-L1 expression in a subset of sarcomas both on the protein and mRNA level. Highgrade sarcomas including undifferentiated pleomorphic sarcomas, dedifferentiated liposarcomas and MPNSTs tend to show more frequent PD-L1 expression. Quantitative PCR results indicate the influence of post-translational modifications rather than transcriptional level as the possible mechanisms of PD-L1 overexpression in sarcomas. The results of ongoing clinical trials are necessary to further assess the effect of anti PD-L1 drugs on sarcomas showing PD-L1 expression.

ACKNOWLEDGEMENTS
The authors thank to Elif Nihan Çetin for her technical support. This study was supported by the Hacettepe University Scientific Research Unit, Grant number: THD- 2015-5202.

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