(P 391) THE EXPRESSION OF PVR AND ITS CLINICOPATHOLOGICAL SIGNIFICANCE IN MPNST.

Objective: Malignant peripheral nerve sheath tumors (MPNST) is defined as a relatively rare malignant tumors (3-5% of all malignant soft tissue tumors) derived from nerve sheath cells. It frequently occurs in thigh, hip, and axilla in one’s 20s to 50s. The disease is treated multimodally with a combination of chemotherapy and radiation therapy, but the efficacy of each therapy is unknown. At present, there is a lack of effective molecular-targeted drugs, and the development of new therapeutic targets and strategies is required.
Transmembrane protein PVR (CD155) has been identified as a member of the Nectin family of intercellular adhesion molecules in epithelial cells and is involved in cell adhesion and differentiation in normal epithelia. On the other hand, it is reported to be aberrantly up-regulated in cancer cells and involved in cell proliferation and invasion. Resent establishment of cancer immunotherapy and the search for new therapeutic targets have focused on the immune checkpoint TIGIT-PVR as a promising therapeutic target. It has been reported that PVR is not only expressed on antigen-presenting cells but is also aberrantly up-regulated in cancer cells and suppresses cancer immunity.
In this study, we aim to clarify the expression pattern, significance and function of PVR in MPNSTs in order to assess its validity as a therapeutic target and to provide a basis for establishing new treatment strategies to the MPNST with limited therapeutic choices.We perform immunohistochemistry of PVR using MPNST surgical materials and analyze its expression pattern. Furthermore, we examine MPNST cell lines to determine whether and how PVR is involved in cancer malignant transformation.

Methods: < Patients and specimens>
　Surgical specimens obtained from 29 lesions in 22 cases of MPNT during the period from 2004 to 2020 were resected at Sapporo medical university Hospital. The control group consisted of 34 lesions in 34 neurofibroma (NF) cases.
< Immunohistochemistry>
Primary antibody：monoclonal rabbit anti-PVR antibody (CST), 1:200
Secondary antibody and detection: Dako REAL™ EnVision™ Detection System.

Intensity: negative (0), weak (1), moderate (2), or strong (3)
Area: 0 (no staining), 1 (1-10%), 2 (11-20%) ～ 8 (71-80%), 9 (81-90%), 10 (91-100%)
Immunoreactive score (IRS): i.e., intensity 3 x proportion 10 = immunoreactive score 30, scale of 0-30.

　Human MPNST cell line: SCC24
＜PVR KO cells＞
　PVR knock out cells were established using the CRISPR-Cas9 system.
＜Omics analysis＞
　Proteins were extracted from control and PVR KO cells, and peptide samples were prepared for comparative proteomic analysis.
＜Statistics＞
　Statestical analysis were performed using 、t-test、Fisher's exact test、Kruskal-Wallis test、Kaplan-Meier survival curve analysis. All statistical analyses were performed with EZR version 1.32(Saitama Medical Center, Jichi Medical University, Saitama, Japan).

Results: Immunohistochemistry of PVR using MPNST surgical material showed that PVR was highly expressed in MPNST compared to NF.
The group with high PVR staining had worse prognosis compared to that with low PVR, but there were no significant differences between them.
In PVR KO cell lines, proliferation, colony formation, migration, and invasive capacity were significantly decreased.
Comparative proteomic analysis showed that expression of an extracellular matrix (ECM) protein and cell-ECM adhesion-related proteins was significantly decreased in PVR KO cells.
As identified by comparative proteomic analysis, Western blotting confirmed expression of ECM and adhesion-related proteins was decreased in PVR KO cells .
ECM and adhesion-related proteins were also decreased in culture supernatants, indicating decreased secretion of the proteins from PVR KO cells　.
Adhesion ability was significantly decreased in PVR KO cells .

Conclusion: Increased expression of PVR was observed in MPNST surgical material. Cell biological analysis confirmed that PVR is involved in proliferation, colony formation, migration, and invasive ability of MPNST cells. It was also shown that PVR is involved in cell-ECM adhesion property including production of an ECM protein itself.
These results strongly suggest that PVR contributes to cancer malignant transformation independently of its function as an immune checkpoint.
Future analyses will focus on the interaction between cancer immune escape mechanisms and the production of ECM.