Programmed Death Ligand 1 ( PD-L 1 ) Expression in Malignant Mesenchymal Tumors

PD-L1, CD274 or B7 homolog 1 (B7-H1) is a 40 kD type 1 transmembrane protein found on activated T cells, B cells and myeloid lineage members plays an important role in regulation of cellular immune response (1). Activation of PD-1/PD-L1 complex leads to inhibition of antigen specific T cell proliferation and induces apoptosis of these antigen specific T cells. It is hypothesized that PD-L1 expression in neoplastic cells enhances its disguise ability to escape from the immune system. Many studies have shown increased expression of PD-L1 ligand in various cancers arising from the lung, skin, ovary, cervix, esophagus, breast, bladder, brain, bone, kidney, and liver (2-7).

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 HyperScript TM 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 Log 10 2 (threshold cycle of b-actin -threshold cycle of PDL1) .relative expression was scored on a log10 scale (0-2).

DIsCussIoN
With this sarcoma series investigating PD-L1 expression with 222 cases, we have shown that a small subset (~15%) of sarcomas express PD-L1 in neoplastic cells.The highest PD-L1 expression among sarcomas was reported from the 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).
Quantitative real-time PCr revealed that 16 of 34 (47%) cases showed significant (>1 log) but low-level PD-L1  (8,9).It is known that antibodies against PD-L1 are not identical and the use of some clones are not recommended (10).High levels of PD-L1 expression from korea may also indicate the possible impact of race on differential expression of immunomodulatory mechanisms.Similar to our results, expression prevalence seems to be lower in the Western population.In a comprehensive immunohistochemical analysis of PD-L1 in 5536 cases, 59 of 986 (6%) demonstrated PD-L1 expression over 5% of tumor cells (11) Antibody with 5% cut-off, respectively.We believe that a relatively lower figure of 6% by Inaguma et al. is mainly due to the absence of cases diagnosed as undifferentiated pleomorphic sarcomas and dedifferentiated liposarcomas in their series (11).
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 PD1and 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-L1 (14).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 PD-L1 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 phenotype ( 14).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 osteosarcomas (7).In their study, 9 samples had high-level expression (24%) with over 3 log fold increase, 19 samples (50%) had
. D'Angelo et al. (12) and Matthew et al. (13) from USA reported that 12% of 50 sarcomas and 20% of 85 sarcomas were positive for PD-L1, respectively.Similarly, Paydas et al. from Turkey recorded a PD-L1 positivity of 29% of 65 cases (14).Although we used tissue microarrays unlike many of the previous studies, our result of 15.3% is comparable with previous results of D'Angelo et al., Matthew et al., and Paydas et al., that used DAkO antibody with 1% cutoff, Cell Signaling antibody with 5% cut-off, and Acris
PD-L1 expression in tumor and accompanying inflammatory cells

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

table II :
PD-L1 expression among various sarcomas (16)rmediate 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 prognosis(16).In our series, immunohistochemically PD-L1 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.