Comprehensive analysis of PD‐L1 in non‐small cell lung cancer with emphasis on survival benefit, impact of driver mutation and histological types, and archival tissue

Abstract Background The aim of the study was to assess programmed death‐ligand‐1 (PD‐L1) expression in different histological types and gene mutation status of patients with non‐small cell lung cancer (NSCLC). Methods A total of 4062 pathology‐confirmed lung cancer patients were retrospectively screened at Kaohsiung Chang Gung Memorial Hospital from November 2010 to June 2017. There were 699 NSCLC patients with confirmed PD‐L1 expression level retrospectively enrolled for analysis. Results There was a trend of higher PD‐L1 expression in squamous cell carcinoma and adenosquamous cell carcinoma than in adenocarcinoma (p = 063). Significant higher PD‐L1 expression in EGFR wild‐type was noted (p < 0.001). No significant differences in PD‐L1 expression were found between ALK wild‐ and mutant types, but there seem was a trend of high PD‐L1 level noted in ALK mutation patients (p = 0.069). In EGFR mutation patients, a higher time to treatment failure (TTF) duration was observed in no PD‐L1 expression (p = 0.011). Longer tumor tissue storage time correlated with lower PD‐L1 expression in lung cancer (p < 0.001 for linear trend). Conclusions There were a trend or significant differences in PD‐L1 expression between different histological types in NSCLC, different EGFR and ALK status, and different tumor tissue storage time. A higher survival benefit was observed in no PD‐L1 expression than with PD‐L1 expression in adenocarcinoma, EGFR and ALK mutation patients. We recommend that PD‐L1 assay should be performed as early as possible if tissue is available.


INTRODUCTION
The immune system defends the body against infection and disease (including cancer). Certain inhibitory checkpoints Chao-Cheng Huang and Meng-Chih Lin contributed equally in this study compared with the corresponding author. drugs can help the immune system fight cancer. The immune system, by driven T lymphocytes and close regulation between inhibitory checkpoints and activating signals, plays an important role in controlling and eliminating cancer. [1][2][3][4][5][6][7] Cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death-1 (PD-1) are the two main immune checkpoint receptors that when binding their ligand B7 and programmed death-ligand-1 (PD-L1), respectively, determine the downregulation of the T cell effector functions, thus contributing to the maintenance of the tolerance to tumor cells. 3,8 The immune checkpoint inhibitors of PD-1/ PD-L1 (anti-PD-1/PD-L1) are currently changing the approach to treatment of patients with advanced non-small cell lung cancer (NSCLC). The FDA has released the approval of nivolumab (Opdivo, Bristol-Myers Squibb), pembrolizumab (Keytruda, Merck Sharp and Dohme), and atezolizumab (Tecentriq, Genentech Oncology) for advanced NSCLC in patients previously treated with platinum-based chemotherapy. [9][10][11][12] In addition, durvalumab (MEDI4736, AstraZeneca) and avelumab (MSB0010718C, Merck KGaA and Pfizer) are being investigated for the treatment of NSCLC. [13][14][15] The expression of PD-L1 has been reported in a number of human malignancies including NSCLC. 7,16 Immunoassays using different primary antibodies, assay formats, and scoring approaches have been reported to assess the prevalence of PD-L1 positivity and the efficacy of treatment in NSCLC. [9][10][11][12]17,18 Reports in the literature have clearly shown that immune checkpoint inhibitors might represent an important therapeutic option for NSCLC patients. However, in spite of exciting overall treatment outcomes, a considerable number of patients failed to achieve long-term clinical benefit. [9][10][11][12]14,18,19 In the clinical trial of KEYNOTE-001 and KEYNOTE-010, Pembrolizumab had better efficacy in NSCLC patients with PD-L1 expression level ≥ 50%. 11,18 Therefore, based on pembrolizumab series trials in NSCLC patients, PD-L1 expression level might be a predictive biomarker for using pembrolizumab in NSCLC patients. 7,16 Although there have been previous reports regarding PD-L1 expression in different histological and gene types of lung cancer, there are still few comprehensive studies on PD-L1 expression of lung cancer in an endemic area with high epidermal growth factor receptor (EGFR) mutation such as Taiwan. In this study, we retrospectively reviewed the medical records of patients histologically or cytologically diagnosed with non-small cell lung cancer from November 2010 to June 2017 at Kaohsiung Chang Gung Memorial Hospital (KCGMH) in Taiwan to assess PD-L1 expression in different histological types and gene types of lung cancer.

METHODS
The study was approved by the Institutional Review Board of Chang Gung Memorial Hospital, and the requirements for patient consent were waived (IRB: 201601146B0).
We retrospectively reviewed the medical records of patients histologically or cytologically diagnosed with lung cancer from November 2010 to June 2017 at Kaohsiung Chang Gung Memorial Hospital (KCGMH). KCGMH is a 2500 bed medical facility serving as a primary care and tertiary referral center in Kaohsiung, Taiwan. More than 600 new lung cancer patients each year have been documented and have received treatment in this hospital.
Data including basic demographic information, tumor histological type, epidermal growth factor receptor (EGFR) status, anaplastic lymphoma kinase (ALK) status, programmed death-ligand 1 (PD-L1) expression status, and formalin-fixed paraffin-embedded (FFPE) tumor tissue storage status were collected and analyzed. EGFR status was performed by EGFR RGQ PCR Kit (Qiagen). Automated immunohistochemical (IHC) study for ALK expression was performed in a Benchmark XT staining module (Ventana Medical Systems) on 5-μm thick FFPE sections with D5F3 rabbit anti-human CD246 monoclonal antibody. The anti-PD-L1 antibody clone 22C3 (Merck) and a prototype IHC assay with a Dako Autostainer Link 48 platform (Agilent Technologies) was used to determine the PD-L1 tumor proportion score (TPS). The PD-L1 TPS was divided into no expression (<1%), low expression (1%-49%), and high expression (≥50%). 11 Furthermore, tumor tissue storage time was also collected for analysis for archival tumor samples. The tumor tissue storage time was divided into four groups: <0.5 year, 0.5-2 years, 2-3.5 years, and ≥3.5 years. Staging was based on the American Joint Committee on Cancer (AJCC) seventh lung cancer TNM classification and staging system. Time to treatment failure (TTF) and overall survival (OS) were calculated to evaluate their efficacy. The TTF duration was defined as the interval from initiation of first-line treatment to its discontinuation, and it could occur due to various reasons such as cancer progression, adverse events, patient choice, or patient death. Furthermore, the OS duration was calculated as the duration from osimertinib treatment initiation until patient death.

Statistical analysis
Data (including age, sex, nodal stage, and EGFR mutation subtypes) were collected and analyzed using SPSS for Windows version 15.0 (SPSS Inc.). In descriptive statistics, data are presented as n (%) or median (interquartile range: Q1, Q3). Statistical significance of univariate analysis was determined by the Mann-Whitney U test and Kruskal-Wallis test for continuous variables and chi-square test for dichotomous variables. The log-rank test was used to compare the survival distributions. Differences were considered significant when p-value was <0.05.

RESULTS
A total of 4062 pathology-confirmed lung cancer patients were retrospectively screened at Kaohsiung Chang Gung Memorial Hospital from November 2010 to June 2017. There were 853 lung cancer patients assessed for PD-L1 expression based on their specimens with anti-PD-L1 antibody clone 22C3 IHC assay. A total of 731 cell lung cancer patients with PD-L1 expression status available were retrospectively screened, and the remaining 122 were excluded due to inadequate tumor tissue (less than 100 tumor cells) for PD-L1 analysis. Among the 731 lung cancer patients, there were 32 (4.4%) small cell lung cancer (SCLC) patients and 699 (95.6%) NSCLC patients. A total of 699 NSCLC patients were retrospectively enrolled for analysis in this study; there were 539 (77.1%) with adenocarcinoma, 66 (9.4%) with squamous cell carcinoma, 17 (2.4%) with adenosquamous carcinoma, and 77 (11.0%) with others ( There was a trend of significant difference in PD-L1 expression between tumor histologic types in NSCLC, showing higher PD-L1 expression in squamous cell carcinoma and adenosquamous cell carcinoma than in adenocarcinoma (p = 063) ( Table 2). Patients were predominantly female (p < 0.001), non-smokers (p < 0.001), and received first-line treatment with TKIs (p < 0.001) for adenocarcinoma than squamous cell carcinoma.
For the correlation between EGFR mutation status and PD-L1 expression in NSCLC, there were 292 (58.4%) out of 500 patients with EGFR mutation in our study cohort. Significantly higher PD-L1 expression in EGFR wild-type than in EGFR mutation was noted (p < 0.001) ( Table 3), but no significant difference in PD-L1 expression was found among different EGFR mutant forms (p = 0.207) ( Table 4). In addition, there were 450 patients with available ALK status, and 23 (5.1%) patients had ALK rearrangement identified. No significant differences in PD-L1 expression were found between ALK wild-type and mutant type, but there was a trend of high PD-L1 level noted in ALK mutation patients (p = 0.069) ( Table 3).
The correlation of PD-L1 expression and survival benefit of 699 NSCLC patients are described in Table 5. There were significant differences in TTF (p < 0.001) and OS (p = 0.029) observed between different PD-L1 expression levels in all patients. In EGFR mutation patients, a higher TTF duration was observed in no PD-L1 expression than in low PD-L1 expression (p = 0.007) and high PD-L1 expression (p = 0.011); on the contrary, no significant difference in TTF was observed between different PD-L1 expression level in ALK mutation patients (p = 0.266). Furthermore, no significant difference in OS was observed between different PD-L1 expression level in both EGFR and ALK mutation patients.
Finally, we also assessed the impact of tumor tissue storage time to PD-L1 expression in lung cancer. There was significant change in PD-L1 expression between different tumor tissue storage time in lung cancer (p < 0.001 for linear trend), showing a lower PD-L1 expression with prolonged tumor tissue storage (Table 6).

DISCUSSION
In this study, we evaluated PD-L1 expression on 655 enrolled lung cancer patients with anti-PD-L1 antibody clone 22C3 IHC assay. Two previous clinical trials, KEYNOTE-001 and KEYNOTE-010, also analyzed PD-L1 expression with the same platform. The KEYNOTE-001 trial had enrolled 824 patients and the KEYNOTE-010 enrolled 2222 patients for PD-L1 testing. 11,18 There was a significant difference in PD-L1 expression between our study and KEYNOTE-010 (p < 0.001) and between KEYNOTE-001 and KEYNOTE-010 (p = 0.005) ( Table 7). These results may have been caused by a difference in terms of tissue storage between prospective clinical trials and our retrospective study, distinct PD-L1 expression in different populations or genetic background, and selection bias in screen procedures for clinical trials.
There was significant difference in PD-L1 expression detected between different tumor tissue storage time for lung cancer in our study (p < 0.001 for linear trend, Table 6). The new tissue sample appeared to have higher PD-L1 expression than archival tissue sample ( Table 6). As our hospital is located in a subtropical area in a humid, warm to hot climate, we routinely keep our archival FFPE tissue blocks in an air-conditioned room of 24-26 C. However, long-term storage might decrease immunoreactivity of tissue for IHC study. A small series of PD-L1 study in 58 NSCLC has shown fading with time of PD-L1 immunoreactivity, which is in agreement with our results. 20 Another issue of concern is tissue availability for current personalized medicine. For most lung cancer patients, the tumor tissue samples for diagnosis are usually small with a limited number of tumor cells obtained from either fibrobronchoscopy or chest CT-guided biopsy. For such a small biopsy sample, tissue conservation strategies are crucial for further molecular analysis and PD-L1 evaluation. 21 Accordingly, to prevent tissue exhaustion and increase detection sensitivity, we recommend that PD-L1 assay should be performed as early as possible if the tissue is available. In addition, a comparison of the prevalence of PD-L1 protein positivity in a renal cancer cohort with fresh frozen tissue versus in FFPE tissue demonstrated a higher PD-L1 positivity rate in the cohort with fresh frozen tissue (37% vs. 24%, respectively). [22][23][24] The decreased positivity rate in the FFPE tissue may be caused by PD-L1 protein denaturation with formalin fixation and a loss in PD-L1 antigenicity. 25 The correlation of types of specimens with tumor tissue storage time is described in Table 8. There was high proportion tissue obtained by chest CT-guided biopsy or video-assisted thoracoscopic surgery (VATS). Our finding that 18.6% of adenocarcinoma (Table 2) had a high PD-L1 expression (TPS ≥ 50%) is lower than previous reports which used the same antibody and platform in larger cohorts. A PD-L1 TPS of at least 50% was reported in 24.9% to 30.2% of advanced NSCLCs in the phase I to III trials (KEYNOTE-001, KEYNOTE-010, and KEYNOTE-024) of pembrolizumab. 11,18,26 Our data is also lower than another study of Beth Israel Deaconess Medical Center, Harvard Medical School) which showed 29.6% of adenocarcinoma had a PD-L1 TPS of at least 50%. 27 These differences might be due to the different EGFR mutation distribution between East Asia and West areas. 28 There was a higher EGFR mutation rate in East Asia area than in West area, as shown in our study cohort that the EGFR mutation rate was 58.4%, and there was higher PD-L1 expression in EGFR wild-type than EGFR mutation type. 26,27 It was partly demonstrated by the increased percentage of high PD-L1 expression (TPS ≥ 50%) up to 29.8% of NSCLC with wildtype EGFR in our study (Table 3). In addition, our study cohort was a retrospective study with longer tissue storage in a part of archival tissue samples, and a prolonged tissue storage could lower PD-L1 expression as previously mentioned.
In one of the largest published screening cohorts for PD-L1 using anti-PD-L1 antibody clone 22C3 IHC assay to date   Log-rank test.
(i.e., that in the KEYNOTE-024 trial), the frequency of overlap between common driver oncogene aberrations (i.e., in EGFR or ALK) and a PD-L1 expression level of at least 50% was just 6% (30 of 500). 26 There was higher PD-L1 expression level in EGFR wild-type than EGFR mutation type in our study (p < 0.001, Table 3). However, there was no significant difference in PD-L1 between ALK wild-type and ALK mutation type (p = 0.069, Table 3). The small sample size might not reflect the real condition of PD-L1 expression level in ALK mutation patients in our study. PD-L1 expression in lung cancer could be heterogeneous and dynamic. Therefore, the consistency, reliability and feasibility to test PD-L1 expression on a single biopsy specimen as a reference for immuno-oncology treatment remains controversial.
It has been reported that EGFR mutation status is related to PD-L1 expression, with lower PD-L1 expression level noted in adenocarcinoma patients with EGFR mutation. 29,30 In another study, the author found that EGFR-TKIs directly inhibit tumor cell vitality, and also indirectly strengthen antitumor immunity by downregulating PD-L1. 31 This could explain why there was better TTF duration in adenocarcinoma patients with EGFR mutation in our study.
Furthermore, our retrospective study has several limitations. First, this study was conducted at a single medical center, and the patient population may be biased by patient selection and referred pattern. Second, this study was a retrospective survey, which not only resulted in incomplete data for some patients, but also did not control for the clinical courses of all lung cancer patients. Hence, further prospective investigations should be conducted to further validate the findings. Despite these limitations, this study provides relatively valuable data regarding the different survival benefit between subgroup and the significant decrease in PD-L1 expression along with increase in tumor tissue storage time.
In conclusion, we have shown there was a trend or significant differences in PD-L1 expression between different histological types in NSCLC, different EGFR status, and different ALK status, and different tumor tissue storage time; a T A B L E 6 The correlation of PD-L1 expression with tumor tissue storage time in lung cancer higher survival benefit (TTF or OS) was observed in no PD-L1 expression than in with PD-L1 expression in adenocarcinoma, EGFR mutation, and ALK mutation patients. Furthermore, we recommend that PD-L1 assay should be performed as early as possible if the tissue is available.