Overexpression of PVR and PD-L1 and its association with prognosis in surgically resected squamous cell lung carcinoma

Targeting T-Cell Immunoreceptor with Ig and ITIM domain-poliovirus receptor (PVR) pathway is a potential therapeutic strategy in lung cancer. We analyzed the expression of PVR and programmed death ligand-1 (PD-L1) in surgically resected squamous cell lung carcinoma (SQCC) and determined its prognostic significance. We collected archival surgical specimens and data of 259 patients with SQCC at Yonsei Cancer Center (1998–2020). Analysis of variance was used to analyze the correlations between PVR and PD-L1 expression and patient characteristics. Kaplan–Meier curves were used to estimate recurrence-free survival (RFS) and overall survival (OS). Most patients were male (93%); the majority were diagnosed with stage 1 (47%), followed by stage 2 (29%) and stage 3 (21%). Overexpression of PVR resulted in a significantly shorter median RFS and OS (P = 0.01). PD-L1 expression was not significant in terms of prognosis. Patients were subdivided into four groups based on low and high PVR and PD-L1 expression. Those expressing high levels of PVR and PD-L1 had the shortest RFS (P = 0.03). PVR overexpression is associated with a poor prognosis in surgically resected SQCC. Inhibition of PVR as well as PD-L1 may help overcome the lack of response to immune checkpoint monotherapy.


Scientific Reports
| (2021) 11:8551 | https://doi.org/10.1038/s41598-021-87624-x www.nature.com/scientificreports/ chemotherapy did not improve overall survival (OS) in patients with metastatic squamous or non-squamous NSCLC 11 . Other checkpoint receptor blockers that modulate immune cell activation are currently under investigation since a significant proportion of patients do not respond to immunotherapy 12 13,14 . Mouse models have shown that TIGIT/PVR binding induces immune evasion of tumor cells, one of the key hallmarks of cancer 15,16 . Levels of PVR are usually low (or PVR is not expressed) in normal tissues, but are high in tumor cells. PVR expression is associated with the invasion, migration, and proliferation of tumor cells 17 . Overexpression of PVR is observed in multiple malignancies, and tumors expressing high levels of PVR are also associated with poor prognosis [18][19][20] .

. Among checkpoint blockers, T-cell immunoglobulin and ITIM domain (TIGIT) binds to poliovirus receptor (PVR). TIGIT is enriched on T-cells, such as regulatory T-cells, type 1 regulatory T-cells, memory T-cells, exhausted CD8 + T-cells, natural killer T-cells, and natural killer cells
Recently, several clinical trials have evaluated the efficacy of blocking the TIGIT/PVR axis in solid cancers such as NSCLC 21 . Tiragolumab, an anti-TIGIT antibody, in combination with atezolizumab, an anti-PD-L1 agent, showed a promising treatment response and a manageable toxicity profile in first-line NSCLC treatment. Compared to monotherapy (atezolizumab), combination therapy improved the objective response rate (31.3% vs. 16.2%) and median progression-free survival (5.4 vs. 3.6 months). These results strongly suggest that the TIGIT/PVR axis may be a clinically useful target for treating patients with NSCLC.
Despite increasing evidence of the involvement of the TIGIT/PVR axis, few studies have addressed the prognostic role of PVR 22 . Therefore, we aimed to assess PVR expression in surgically resected SQCC tissues to determine its relationship with PD-L1. We evaluated the correlations of PVR and PD-L1 expression with clinicopathological factors and determined their prognostic implications for survival outcomes in SQCC (Fig. 1).
According to the TCGA data, PVR was expressed independently of PD-L1 expression in SQCC. We also analyzed the survival difference for both high expression and low expression of PD-L1 and PVR but found no significant differences in OS between the groups with high and low PD-L1 (P = 0.91) and PVR expression (P = 0.55; Fig. 2d). We further categorized patients into four groups depending on PD-L1 and PVR expression: PD-L1 hi / PVR lo , PD-L1 lo /PVR lo , PD-L1 hi /PVR hi , and PD-L1 lo /PVR hi (Fig. 2e). Similarly, no significant differences were found between the four subgroups using multivariate analyses. There was a trend for the PD-L1 hi /PVR hi group to have the worst survival rate, indicating that high expression of PD-L1 and PVR may contribute to poor prognosis.

Association between PVR expression and survival.
In conjunction with the survival curves drawn using TCGA data of lung squamous cell carcinoma (Fig. 2c), we analyzed the median recurrence-free survival (mRFS) and median OS (mOS) of patients according to high or low expression levels of PD-L1 (CD274) and PVR ( Fig. 3). High PVR expression was associated with inferior mRFS and mOS. The mRFS was 4.8 months for high PVR expression and 10.0 years for low PVR expression (hazard ratio [HR]: 1.54, 95% confidence interval [CI]: 1.15-2.07; P = 0.01) (Fig. 3a). The corresponding mOS values were 5.6 and 11.2 years, respectively (HR: 1.45, 95% CI: 1.10-2.00; P = 0.01) (Fig. 3b). In contrast, high PD-L1 expression or high CD8 + expression was not associated with either poor mRFS or mOS (
The comparison of mRFS and mOS between these subgroups (depicted in Fig. 4) showed that the PD-L1 hi / PVR hi group had the shortest mRFS of 3.7 years, followed by the PD-L1 lo /PVR hi , PD-L1 lo /PVR lo , and PD-L1 hi / PVR lo groups (P = 0.03). The PD-L1 hi /PVR hi group had a tendency for the shortest mOS, although the result was not significant (P = 0.06). Subgroup analysis showed that the PD-L1 hi /PVR lo group had improved mOS compared to the PD-L1 lo /PVR hi group (HR: 2.43, 95% CI: 1.46-3.96; P = 0.04).      Table S2 online). With the exception of smoking status, these factors were also significant in multivariate analyses. Similarly, age ≥ 65 years (HR: 0.57, 95% CI: 0.42-0.77; P = 0.01), advanced stage (HR: 0.41, 95% CI: 0.30-0.57; P = 0.01), and PVR > 20 (HR: 0.68, 95% CI: 0.50-0.91; P = 0.01) were associated with worse OS. On multivariate analyses, we found that all these factors remained significant as independent factors (see Supplementary Table S3 online). Overall, RFS and OS of surgically resected SQCC were affected by age, stage, and PVR expression. Of note, PD-L1 was not a prognostic factor for both RFS and OS in univariate and multivariate analyses. In contrast, high PVR expression was associated with short RFS and OS.

Discussion
In our study, we found that the PD-L1 and PVR clusters were independently expressed as per TCGA RNAsequencing data. In our cohort, PD-L1 hi /PVR hi status was correlated with the worst RFS, signifying that both PD-L1 and PVR are important factors for the recurrence of surgically resected SQCC. Similarly, PD-L1 hi /PVR hi status was also correlated with the shortest OS, although the result was not statistically significant. Subgroup analysis also showed that the PD-L1 lo /PVR hi group had shorter OS than the PD-L1 hi /PVR lo group, emphasizing the importance of PVR for OS, regardless of PD-L1 expression. Furthermore, multivariate analysis showed that high level of PVR expression was associated poor RFS and OS However, PD-L1 expression levels did not affect RFS or OS.
Currently, immunohistochemistry for PD-L1 is used to identify patients most likely to respond to immunotherapeutic agents, such as anti-PD-1 or anti-PD-L1 agents 24 . PD-L1 signaling blocks the T-cell response necessary for cytokine secretion and regulates the antitumor response by inducing regulatory T-cell differentiation 25,26 . Although targeting the PD-1/PD-L1 pathway with anti-PD-1 agents has shown reliable and robust results in metastatic and recurrent SQCC, some patients are unresponsive to these treatments [8][9][10] . Therefore, there is an unmet need to effectively treat non-responders.
Recently, a phase 2 trial of administering 2 cycles of atezolizumab, an anti-PD-L1 monoclonal antibody in neoadjuvant setting for resectable stage IB-IIIB NSCLC, yielded clinically meaningful major pathologic response rate of 20% 27 . The role of PD-L1 as well as PVR in neoadjuvant setting is yet to be elucidated.
Accumulating evidence suggests that targeting single checkpoints, such as the PD-1/PD-L1 pathway is insufficient for eliciting an immune response 28,29 . A better understanding of T-cell activity in combination with other checkpoint inhibitors may help overcome the lack of response due to the inhibition of immune activation. One potential mechanism is to block TIGIT, which interacts with PVR 30 . PVR binds to TIGIT, which is highly expressed in natural killer cells, T-cells, and regulatory T-cells, and induces immune escape of tumor cells 31,32 . Studies using mouse models have shown that the TIGIT/PVR axis is associated with inhibition of the proliferation, differentiation, and activation of T-cells and the activation of inhibitory T-cells (regulatory T-cells) 13,33 . Thus, targeting the TIGIT/PVR pathway may be a potential mechanism to overcome resistance to checkpoint inhibitors. Tiragolumab, an anti-TIGIT antibody, is a promising agent, which can be combined with atezolizumab to treat naïve, PD-L1-positive NSCLC.
There are few limitations to our study. First, our study was conducted retrospectively in a single center. The sole inclusion of Asian patients and the retrospective study design further limited the generalizability of our results. Validation using larger, prospective cohorts is warranted to bolster our findings. Second, our study lacked patients treated with immunotherapy after a systemic relapse, preventing the evaluation of the predictive roles of both PD-L1 and PVR. Identifying these factors in patients treated with immunotherapy in metastatic SQCC settings may help select those who may benefit from a combination of immunotherapeutic agents. Third, PD-L1 may possibly be underrepresented in some of the archival FFPE tissue blocks that date back to 1998. Indeed, studies have addressed that fresh biopsy samples better represent PD-L1 status 34,35 . However, PD-L1 was not a prerequisite at the time of tissue collection for this cohort, and this study adequately represents long term follow-up data on RFS and OS in surgically resected SQCC. Studies with more recent tissue samples are needed to validate our findings.
To our knowledge, the prognostic role of PD-L1 in correlation with PVR in SQCC has not been validated in any other study. Our study focused on the independent expression of PD-L1 and PVR and showed that patients with both high PD-L1 and PVR expression had a short RFS. Since patients with surgically resected SQCC often experience both local and distant relapses, better systemic treatment options for recurrent SQCC should be available in the immunotherapy era 5 .
In conclusion, overexpression of PVR is associated with poor prognosis in terms of RFS in surgically resected SQCC. Simultaneously targeting PD-L1 and the TIGIT/PVR axis with combination treatments may help overcome the lack of treatment response to immune checkpoint monotherapy. www.nature.com/scientificreports/ Expression of immune checkpoint ligand and receptor genes. We retrieved data on SQCC from TCGA to conduct a correlative analysis of ICLs and immune checkpoint receptors 23 . We collected data of normalized fragments per kilobase of transcript per million mapped reads for analysis of the RNA-sequencing dataset and performed a correlation analysis of 26 ICLs using Spearman's correlation 36 . Using the hierarchical clustering method, we identified clusters of ICLs with strong co-expression patterns and selected two clusters that did not show correlations. Each ICL was categorized based on high (≥ 50th percentile) or low (< 50th percentile) expression of the target gene. The Kaplan-Meier method was used to calculate the OS, and Cox regression analysis was used for multivariate analysis.

Methods
Patient selection. Data of 259 patients diagnosed with SQCC at the Yonsei Cancer Center between January 1998 and July 2020 were collected. The inclusion criteria for patient selection were surgically resected SQCC, archival tumor samples available for PVR and PD-L1 analysis, and availability of clinical data. Clinicopathological variables, such as age, sex, stage, pleural involvement, lymphovascular invasion, mutations in the EGFR and KRAS genes, smoking status, and adjuvant treatment, were evaluated. The American Joint Committee on Cancer (seventh edition) guidelines for tumor-node-metastasis classification were used to determine cancer stage 37 . An experienced pathologist (HSS) blinded to the clinical data reviewed and interpreted the pathology findings.
Tissue microarray. We selected two or three different areas per sample representing tumor areas and developed tissue microarrays. Using formalin-fixed paraffin-embedded blocks, we collected tissue cores (3 mm in diameter) and arranged them in recipient paraffin blocks using a trephine. All tissue microarray blocks contained tumor lesions representing > 50% of the core, based on hematoxylin-eosin staining.

Immunohistochemistry.
A Ventana Benchmark XT autostainer (Ventana Medical Systems, Tucson, AZ, USA) was used to perform immunohistochemistry on 4-µm tissue microarray sections. The sections were stained with an anti-PD-L1 (clone SP263, Ventana Medical Systems) rabbit monoclonal primary antibody using the OptiView DAB IHC Detection kit for the PD-L1 IHC assay 38 . PVR antibody (rabbit monoclonal, cloneD8A5G, Cell Signaling Technology) was diluted to 1:100, treated, and incubated at 37 °C for 32 min for the PVR IHC assay. Using ultraview universal DAB Detection Kit (Ventana Medical Systems), the signals for PD-L1 and PVR were detected. Both the PD-L1 and PVR scores were interpreted as TPS according to the methods described in previous studies 9 . PD-L1 high and low expression status were determined according to the median cut-off value of 5% staining of tumor cells (see Supplementary Fig. S1 online). PVR expression was classified as high (> 20% TPS) or low (≤ 20% TPS) based on the median value (see Supplementary Fig. S1 online). Patients were then categorized into four groups depending on PD-L1 and PVR expression: PD-L1 hi /PVR lo , PD-L1 lo /PVR lo , PD-L1 hi /PVR hi , and PD-L1 lo /PVR hi (Fig. 1a-d).
For the CD8 + analysis, the percentages of CD8 + lymphocytes (RTU, clone C8/144B (Dako, Glostrup, Denmark) compared with the total amount of nucleated cells in the stromal compartments were assessed 39 . CD8 + expression was also determined as high (≥ 10) or low (< 10) based on the median cutoff value of 10%. Statistical analysis. The correlations between variables were analyzed using Fischer's exact test for categorical variables and the sample t-test for continuous variables. We used the Kaplan-Meier method to calculate OS and RFS. RFS was defined as the time from the date of surgery to the date of recurrence, last follow-up, or death. OS was defined as the time between the date of diagnosis and the date of last follow-up or death. Cox regression with backward elimination method was used for multivariate analysis of OS. Statistical analyses were two-sided with P = 0.05 as the level of significance. All statistical analyses were performed using Statistical Package for the Social Sciences (version 25; IBM, Chicago, IL, USA) and GraphPad Prism 8.0 Software (GraphPad Software, Inc., San Diego, CA).

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.