Causal Relationship between Postoperative Recurrence and PD-L1 and EGFR Mutations in NSCLC: A Retrospective Cohort Study

Although information on the PD-L1 expression and EGFR mutations in non-small cell lung cancer (NSCLC) is important for determining therapeutic strategies, the causality between these two factors and postoperative recurrence and the association between each factor have remained unclear. We retrospectively assessed the PD-L1 expression and EGFR mutations in tumors of 280 NSCLC patients. The causality between the PD-L1 expression, EGFR mutations and postoperative recurrence were evaluated by a multivariate Cox proportional hazard analysis. The association between the PD-L1 expression and EGFR mutations was evaluated by a multinomial logistic regression analysis. The adjusted hazard ratio (HR) in cases with high ( ≥ 50%) PD-L1 expression was 4.83 (95% condence interval [CI]: 1.51–15.5). The adjusted HRs in cases with EGFR major and minor mutations were 0.42 (95% CI: 0.14–1.25) and 0.63 (95% CI: 0.18–2.15), respectively. The high expression of PD-L1 ( ≥ 50%) was signicantly associated with exon 21 L858R mutation (Ex21) of EGFR (adjusted odds ratio, 0.10; 95% CI, 0.01–0.87). The high expression of PD-L1 was an independent risk factor for postoperative recurrence in NSCLC, whereas EGFR mutations were not. The high expression of PD-L1 was negatively associated with Ex21. These ndings may help identify NSCLC patients with an increased risk of postoperative recurrence.


Introduction
In recent years, the importance of immune checkpoint inhibitors (ICIs) in the treatment of lung cancer has increased [1]. Because the use of ICIs has been suggested to be potentially effective in the pre-surgical setting for surgically resectable non-small cell lung cancer (NSCLC), the bene ts of ICIs in the surgical eld have also been attracting attention [2]. The programmed death-ligand 1 (PD-L1) expression in tumors has already been shown to be a predictor of the treatment e cacy of ICIs for NSCLC [3]. On the other hand, epidermal growth factor receptor gene (EGFR) mutations are important in the pathogenesis of lung adenocarcinoma and EGFR tyrosine kinase inhibitor treatment has been shown to signi cantly prolong survival of patients with lung adenocarcinoma with EGFR mutations [4]. Thus, the information on the PD-L1 expression and EGFR mutations in NSCLC are extremely important for therapeutic strategies.
The role of the PD-L1 expression and EGFR mutations in the postoperative prognosis of NSCLC and the association of the PD-L1 expression with EGFR mutations has been reported in several previous studies [5,6]. Recently, a large cohort study on postoperative patients with NSCLC suggested that the expression of PD-L1 might be a poor prognostic factor for recurrence-free survival (RFS) [7]. In addition, a large cohort study examining the association between the type of EGFR mutations and postoperative recurrence showed that patients with exon 19 deletion (Ex19) had signi cantly shorter postoperative RFS in comparison to those with exon 21 L858R mutation (Ex21) [8]. Regarding the association between the expression of PD-L1 and EGFR mutations in NSCLC, it has been reported that NSCLC with EGFR mutation was less likely to express PD-L1 and exhibited a poor response to ICIs in comparison to EGFR wild-type [9].
The multivariate analyses of postoperative RFS according to the expression of PD-L1 and the EGFR mutation status The results of multivariate Cox proportional hazards analysis of factors associated with RFS are shown in Tables 2 and 3. A signi cant differences in RFS was observed between the PD-L1-high group and the PD-L1-negative group (hazard ratio [HR], 4.83; 95% con dence interval [CI], 1.51-15.5) after adjustment for patient background factors including the EGFR mutation status (Table 2). RFS did not differ to a statistically signi cant extent in the PD-L1-low expression group (HR, 1.78; 95% CI, 0.59-5.43) ( Table 2). In contrast, in the multivariate Cox proportional hazards analysis adjusted for patient background factors including the PD-L1 expression status, RFS did not differ to a statistically signi cant extent in the major mutation group (HR, 0.42; 95% CI, 0.14-1.25) or the minor mutation group (HR, 0.63; 95% CI, 0.18-2.15) with reference to the wild-type group ( Table 3).
The associations between the expression of PD-L1 and EGFR mutations A multinomial logistic regression analysis was performed to examine the link between the expression of PD-L1 and EGFR mutations (

Discussion
The multivariate Cox proportional hazards analysis adjusted for EGFR mutation status, pathological stage, histological type, and adjuvant chemotherapy revealed that postoperative recurrence of lung cancer was ve times as likely to occur with a PD-L1-high expression status in comparison to PD-L1negative cases. The Cox proportional hazards analysis with adjustment for the expression of PD-L1, pathological stage, histological type and adjuvant chemotherapy revealed that EGFR mutations were not signi cantly associated with postoperative recurrence. A multinomial logistic regression analysis showed a signi cant negative association between the high expression of PD-L1 and the presence of the Ex21 mutation.
Previous studies have not shown consistent results regarding the prognosis associated with the expression of PD-L1 and EGFR mutations in NSCLC. One study showed that the low expression of PD-L1 was associated with signi cantly reduced RFS in NSCLC patients with EGFR mutations [10]. However, this study did not show a signi cant association between the high expression of PD-L1 and RFS. In addition, this study focused solely on the in uence of the presence or absence of EGFR mutations, and the interaction between the PD-L1 expression and various EGFR mutations was not considered in the analysis. Furthermore, another study showed that the patients with Ex21 had a signi cantly longer RFS in comparison to those with WT or Ex19 [8]. However, this study did not evaluate RFS, with the inclusion of the PD-L1 expression as a confounding factor in the multivariate analysis.
In the present study, we performed multivariate analyzes considering the interaction of different PD-L1 expression levels with EGFR mutations in NSCLC and revealed the following three novel points. First, patients with the high expression of PD-L1 had signi cantly shorter postoperative RFS, whereas those with the low expression of PD-L1 showed no signi cant difference. Second, the presence of EGFR mutations did not contribute signi cantly to postoperative RFS. Third, the high expression of PD-L1 showed a signi cant negative connection with Ex21.
These three points may be biologically plausible. First, the high expression of PD-L1 in NSCLC may affect the postoperative prognosis. Because cancer cells inactivate T cells via the PD-L1 expression [11], the higher the expression of PD-L1, the more the immune system is suppressed, which may lead to greater cancer progression than expected after surgery. Some studies have reported that the expression of PD-L1 was associated with the AKT-mTOR pathway, which has been shown to be necessary for cell proliferation [12]. Therefore, the expression of PD-L1 may be correlated with oncogenic signal, the high expression of which may be involved in high tumor progression. Second, EGFR mutations in NSCLC may not affect postoperative recurrence. An in vitro study using cell lines have reported that EGFR mutations promote the expression of PD-L1 [13]. However, in vivo, it has been reported that various cytokines in the tumor microenvironment affect the PD-L1 expression on cancer cells [14]. An experiment in which cell lines were stimulated with cytokines, such as interferon-γ due to mimicking of the in vivo environment demonstrated that the expression of PD-L1 is higher in cells with wild-type EGFR in comparison to those with EGFR mutations [15]. In the in vivo tumor microenvironment, EGFR mutation may not be an independent risk factor for recurrence because of attenuated immunosuppression in connection with the tendency for low PD-L1 expression levels in cells with EGFR mutations. Third, among the EGFR mutation subtypes, Ex21 may be negatively associated with the high expression of PD-L1 in NSCLC. The tumor mutation burden (TMB) has been known to be an effective predictor of an improved response to ICIs in NSCLC [16]. One study suggested that there was no relevant association between the expression of PD-L1 and a high TMB in tumors [17]. In addition, another study examining the association between EGFR mutations and the TMB reported that the TMB tended to be higher in cells with Ex21 than in those with Ex19 [18]. Thus, the high TMB in cancer cells with Ex21 may contribute to the suppression of the high expression of PD-L1.
The present study was associated with some limitations. First, because the number of variables that could be included in the multivariable Cox proportional hazards analysis was limited due to the small number of postoperative recurrence cases, it was not possible to incorporate all of other confounding factors into the model at once. A larger sample size using a model that simultaneously includes all variables is needed in order to evaluate the in uence of the PD-L1 expression and EGFR mutation status on the postoperative recurrence with higher accuracy. Second, our study was limited to surgical cases; we did not examine advanced cases that were managed without surgery. EGFR mutation positivity was reportedly associated with the expression of PD-L1 in stage IV [19]. This result is completely opposite to the results of our study. Therefore, the PD-L1 expression in advanced stage may behave differently than other stages. A prognostic analysis that analyzed the in uence of the PD-L1 expression and EGFR mutation status, including patients with advanced stage disease, may also be needed.
In summary, we performed multivariate analyses using a cohort of patients who had undergone complete surgical resection for lung cancer to compare postoperative RFS between groups categorized according to their PD-L1 expression and EGFR mutation status. We showed that ≥ 50% PD-L1 positivity within the resected tumor tissue was an independent risk factor for recurrence after lung cancer surgery, whereas the EGFR mutation status was not. We showed that Ex21 may be not a risk factor for postoperative recurrence due to the di culty in expressing a high level of PD-L1. Our ndings may contribute to the selection of patients who are eligible for adjuvant chemotherapy using ICIs, regardless of pathological stage.

Patients
A total of 280 lung cancer patients who had undergone surgical lung resection at Kinki-Chuo Chest Medical Center (KCMC) from April 2017 to January 2020 were included in our study. We selected the patients who had undergone complete resection. Those who had received limited resection were excluded because a previous report showed that limited resection was associated with a higher rate of locoregional recurrence in comparison to lobectomy [20]. The histopathological diagnosis according to the current 2015 World Health Organization classi cation was performed by pathologists [21]. Adjuvant chemotherapy with a platinum-based regimen was administered to patients with indications and who had given their informed consent. Patients who received neoadjuvant therapy were excluded because it was reported that the PD-L1 expression status of cancer cells was altered after neoadjuvant chemotherapy [22]. Other clinicopathological features, including age, sex, smoking status, pathologic tumor-node-metastasis (TNM) classi cation (eighth edition) and the presence of postoperative recurrence were collected from medical records. The present study was approved by the Institutional Review Board of KCMC (Approval number: 725). Informed consent was obtained by an opt-out method using the website of our institution. All methods were performed in accordance with relevant guidelines and regulations.
Tumor PD-L1 immunohistochemistry and the EGFR mutation assay All viable cancer cells on the entire pathological tissue section of each tumor sample were evaluated. We used the PD-L1 clone 22C3 pharmDx kit and Dako Automated Link 48 platform (Agilent Technologies, Dako, Carpinteria, CA, USA) to measure the PD-L1 expression. The PD-L1 tumor proportion score (TPS) was calculated as the percentage of complete or partial membrane staining in a sample. The cut-off value for the expression of PD-L1 was set at 50% and 1% based on a previous clinical trial [23]. The tumor samples of each patient were separated into 3 groups based on the presence of positivity stained cells in specimen, as follows: <1% (negative), 1-49% (low expression), and ≥50% (high expression). All patients were subjected to an EGFR mutation assay by the testing laboratories (Cobas EGFR Mutation Test; Roche Molecular Diagnostics, Pleasanton, CA, USA).

Recurrence-free survival
The primary outcome of this study was recurrence-free survival (RFS), de ned as the time from the date of curative resection to the date on which disease relapse was diagnosed. The patients received blood tests and a chest X-ray after surgery every three or six months. Additional screenings were performed in cases where abnormal ndings were observed. The diagnosis of relapse was comprehensively determined based on the results of examinations.

Statistical analyses
Chi-squared tests were used to compare the proportions of categorical variables between each of the PD-L1 expression groups and EGFR mutation groups. The probability of RFS was assessed using the Kaplan-Meier method and log-rank tests. A multivariate Cox proportional hazards analysis was performed to estimate the hazard ratios (HRs) with adjustment by risk factors for recurrence. A multinomial logistic regression analysis was performed to assess the odds ratios (ORs) for each EGFR mutation status in the PD-L1-high and PD-L1-low groups, with the PD-L1-negative group as a reference. A multivariate Cox proportional hazards analysis and a multivariate logistic regression analysis can analyze the covariates of the number of cases with an outcome divided by 10 or 5 [24,25]. In this study, the number in the Cox proportional hazards analysis was 39 (i.e., the number of cases with relapse) divided by 10 or 5 (result: 4 or 8). And the number of cases included in the logistic regression analysis was 56 (i.e., the number of cases with the high expression of PD-L1) divided by 10 or 5 (coming to 5 or 11). We selected the following 8 factors in a multivariate Cox hazards analysis: the PD-L1 expression level (low and high   Tables   Table 1 Association between the PD-L1 expression and patient clinicopathological variables. PD-L1 programmed cell death-ligand 1, ADC adenocarcinoma, SCC squamous cell carcinoma, EGFR epidermal growth factor receptor gene, Ex21 exon 21 L858R mutation, Ex19 exon19 deletion mutation. Table 2 The Cox proportional hazards analysis of RFS according to the PD-L1 expression. RFS recurrence-free survival, PD-L1 programmed death-ligand 1, HR hazard ratio, CI con dence interval, EGFR epidermal growth factor receptor gene. Table 3 The Cox proportional hazards analysis of RFS according to the EGFR mutation status. RFS recurrence-free survival, EGFR epidermal growth factor receptor gene, HR hazard ratio, CI con dence interval, PD-L1 programmed death-ligand 1, Ex21 exon 21 L858R mutation, Ex19 exon19 deletion mutation. Table 4 The multinomial logistic regression analysis to investigate the association between the expression of PD-L1 and EGFR mutations. PD-L1 programmed death-ligand 1, EGFR epidermal growth factor receptor gene, OR odds ratio, CI con dence interval, Ex21 exon 21 L858R mutation, Ex19 exon19 deletion mutation.  Kaplan-Meier curve showing the probability of a recurrence-free survival among patients after lung cancer resection according to the EGFR mutation status.

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