The significance of programed cell death‐ligand 1 expression in vestibular schwannoma

Abstract Background The association between programed cell death‐ligand 1 (PD‐L1) and tumor‐infiltrating lymphocytes (TILs) in vestibular schwannoma (VS) has been investigated in a few studies. These published studies report a difference in the PD‐L1 positivity rate in malignant peripheral nerve sheath tumors. We examined PD‐L1 expression and lymphocyte infiltration in patients with VS who had undergone surgical resection and investigated the association between PD‐L1 expression and clinicopathological features. Methods The expression of PD‐L1, CD8, and Ki‐67 in 40 VS tissue specimens was investigated using immunohistochemistry, and a clinical review of the patients was performed. Results Of the 40 VS samples, 23 (57.5%) were positive for PD‐L1 and 22 (55%) were positive for CD8. No significant differences in age, tumor size, pure‐tone audiometry, speech discrimination, or Ki‐67 expression were observed between patients in the PD‐L1‐positive and PD‐L1‐negative groups. A higher level of CD8‐positive cell infiltration was observed in PD‐L1‐positive tumors than in PD‐L1‐negative tumors. Conclusion We demonstrated that PD‐L1 was expressed in VS tissues. Although no correlation was identified between clinical characteristics and PD‐L1 expression, the association between PD‐L1 and CD8 was confirmed. Thus, additional research on targeting PD‐L1 is necessary to improve immunotherapy for VS in the future.


Vestibular schwannoma (VS) is a benign tumor originating from the
Schwann cells of the vestibular nerve (DeLong et al., 2011). In 95% of the patients with VS, the condition occurs unilaterally, although in neurofibromatosis type 2 (NF2), it may be bilateral (Møller et al., 2015).
Also, in schwannomatosis, characterized by the development of multiple schwannomas, unilateral VS can be seen in patients with an LZTR1 mutation (Gonzalvo et al., 2011). Most patients with VS present hearing loss, tinnitus, or dizziness. A large tumor can cause headache, facial paralysis, and other cranial neuropathies, in addition to death from brainstem compression (Foley et al., 2017). Treatment of VS includes microsurgery, stereotactic radiosurgery, and conservative treatment through wait and scan. But there remains a need for effective pharmaceutical therapies. We have been conducting research on various mechanisms for the inhibition of VS tumor growth (Kim et al., 2015(Kim et al., , 2016(Kim et al., , 2022Lee et al., 2012).
Programed cell death 1 (PD-1) is a receptor expressed on the surface of T cells, which regulates the activation of T cells. This receptor interacts with PD-1-ligand 1 (PD-L1). This binding suppresses T-cell migration, proliferation, and secretion of cytotoxic mediators and restricts tumor cell killing (Akinleye & Rasool, 2019). Inhibitors of PD-1 and PD-L1 disrupt PD-1/PD-L1 axis, thereby reversing T-cell suppression and enhancing endogenous antitumor immunity to unleash long-term antitumor responses for patients with a wide range of cancers (Paterson et al., 2011). The expression of PD-L1 in the tumor microenvironment is often associated with tumor-infiltrating lymphocytes (TILs), among which CD8-positive (CD8+) TILs may be the most potent (Zhou et al., 2018). Many clinical trials are being conducted to determine the therapeutic potential of PD-L1 inhibitors in various tumors, either alone or in combination with other therapies (Balar & Weber, 2017;.
Few studies have investigated the association between PD-L1 and TILs in VS. The published studies are mostly related to NF2 or malignant peripheral nerve sheath tumors, and these studies report a difference in the PD-L1-positivity rate in these cancers (Perry et al., 2019;Shurell et al., 2016;. We examined the expression of PD-L1 protein and the infiltration of lymphocytes into the tumor tissue in VS patients who had undergone surgical resection and investigated the association between the expression of PD-L1 and clinicopathological features.

Patients and samples
Forty patients who underwent surgical resection for pathologically confirmed VS and for whom formalin-fixed, paraffin-embedded samples were available were included in this study. None of the patients received preoperative chemotherapy or radiotherapy. All clinical data were collected by retrospective review of medical charts and pathol-ogy records. The maximal linear diameter of the tumor was measured on a post-gadolinium-focused magnetic resonance image.

IHC analysis
IHC staining results were evaluated by one pathologist (S.C.) who was blinded to all clinical information. PD-L1 staining was evaluated for partial or complete membrane expression in tumor cells, regardless of the staining intensity. The percentage of cells with membrane expression in the total number of tumor cells was calculated, and samples with >5% were considered positive (Bellmunt et al., 2015;Lipson et al., 2013;Xylinas et al., 2014). For CD8 staining, the number of intratumoral CD8+ lymphocytes was counted in 10 representative high-power microscopic fields (magnification, 400×), then the percentage of CD8+ lymphocytes in the total intratumoral lymphocytes was calculated. The tumor background (nonspecific staining, e.g., in perivascular spaces), areas of necrosis, areas of hemorrhage, and artifactual staining were excluded from evaluation. CD8+ lymphocyte density was evaluated using a semiquantitative scoring system. Five categorical scales of the following levels based on the percentage of CD8+ lymphocytes were used: <1%, 1%-5%, 5%-10%, 10%-20%, and >20%.

Statistical analysis
All data were analyzed using SPSS software version 26.0 (SPSS Inc., Chicago, IL, USA). Chi-square and Fisher's exact tests were used to identify a correlation between PD-L1 expression and the clinicopathological characteristics among the groups. A p-value of less than .05 was considered to be significant.

Patient characteristics
Forty patients with VS who underwent surgical resection were included in this study. The mean age of the patients with VS was 52.75 years. The patient clinical characteristics are shown in Table 1.

Association between the expression of PD-L1 and clinicopathologic characteristics
Patient characteristics were correlated with PD-L1-positive and PD-L1-negative staining in VS specimens (Table 3). There were no significant differences in age, tumor size, PTA, or SD between the two groups. There also was no difference in the presence of serviceable hearing and PD-L1 expression. We found a higher level of CD8+ cell infiltration in PD-L1-positive VS tumors than in PD-L1-negative tumors (p < .001). We also assessed the proliferation rate by Ki-67 expression.

DISCUSSION
The immune system provides a defense against antigens introduced from the outside and functions through an appropriate balance between activation and inhibition of the immune response (Naidoo et al., 2014). This immune response is responsible for carcinogene- We investigated the association between PD-L1 in VS and the clinicopathological characteristics of the cells and found no significant correlation between PD-L1 expression and age, tumor size, PTA, or SD. Archibald et al. (2010) reported that patients with poor hearing at the time of surgery tended to show higher levels of B7-H1 than patients with better hearing, although no significant difference between the groups was found. Our study revealed similar results. There was no significant correlation between PD-L1 and PTA or SD. Additionally, there was no difference between the presence of serviceable hearing and PD-L1 expression.
We also assessed the association between PD-L1 expression and tumor size, and there was no significant difference in PD-L1 expression between the two groups. Archibald et al. (2010) compared the tumor size and B7-H1 expression and found no significant correlation.
Recently, it has been reported that VS tumor volume or progressiveness is associated with the concentration of PD-L1 (Bi et al., 2020;Perry et al., 2019). Since our study investigated the correlation between the tumor size at the time of surgery and the expression of PD-L1, additional studies on the characteristics associated with tumor progression are needed. We assessed the expression of  in VS, which is marker of high degree of tumor cell proliferation. Ki-67 expression has been used to evaluate other tumors of the central nervous system. It has been reported that VS tumors with Ki-67 labeling showed high proliferative activity, short doubling time, and high recurrence or regrowth after resection (Bedavanija et al., 2003;Prueter et al., 2019;Yokoyama et al., 1996) The present study has a few limitations. This was a retrospective study with a small sample size, which may have led to bias in the data.
In addition, PD-L1 IHC was conducted using only one antibody. In measuring the size of VS, linear measurement rather than volumetric quantification was used, but the size measurement may not be accu-

CONFLICT OF INTEREST STATEMENT
The authors declare no conflicts of interest.

DATA AVAILABILITY STATEMENT
Data sharing not applicable as no data generated.