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Strong Programmed Death Ligand 1 Expression Predicts Poor Response and De Novo Resistance to EGFR Tyrosine Kinase Inhibitors Among NSCLC Patients With EGFR Mutation

Open ArchivePublished:July 26, 2018DOI:https://doi.org/10.1016/j.jtho.2018.07.016

      Abstract

      Introduction

      This study evaluated whether tumor expression of programmed death ligand 1 (PD-L1) could predict the response of EGFR-mutated NSCLC to EGFR tyrosine kinase inhibitor (TKI) therapy.

      Methods

      We retrospectively evaluated patients who received EGFR-TKIs for advanced NSCLC at the Guangdong Lung Cancer Institute between April 2016 and September 2017 and were not enrolled in clinical studies. The patients' EGFR and PD-L1 statuses were simultaneously evaluated.

      Results

      Among the 101 eligible patients, strong PD-L1 expression significantly decreased objective response rate, compared with weak or negative PD-L1 expression (35.7% versus 63.2% versus 67.3%, p = 0.002), and shortened progression-free survival (3.8 versus 6.0 versus 9.5 months, p < 0.001), regardless of EGFR mutation type (19del or L858R). Furthermore, positive PD-L1 expression was predominantly observed among patients with de novo resistance rather than acquired resistance to EGFR-TKIs (66.7% versus 30.2%, p = 0.009). Notably, we found a high proportion of PD-L1 and cluster of differentiation 8 (CD8) dual-positive cases among patients with de novo resistance (46.7%, 7 of 15). Finally, one patient with de novo resistance to EGFR-TKIs and PD-L1 and CD8 dual positivity experienced a favorable response to anti–programmed death 1 therapy.

      Conclusions

      This study revealed the adverse effects of PD-L1 expression on EGFR-TKI efficacy, especially in NSCLC patients with de novo resistance. The findings indicate the reshaping of an inflamed immune phenotype characterized by PD-L1 and CD8 dual positivity and suggest potential therapeutic sensitivity to programmed death 1 blockade.

      Keywords

      Introduction

      Tumor cells develop multiple mechanisms to escape immune surveillance, such as the activation of oncogenes.
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      Upregulation of PD-L1 by EML4-ALK fusion protein mediates the immune escape in ALK positive NSCLC: implication for optional anti–PD-1/PD-L1 immune therapy for ALK-TKIs sensitive and resistant NSCLC patients.
      • Ota K.
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      Induction of PD-L1 expression by the EML4-ALK oncoprotein and downstream signaling pathways in non–small cell lung cancer.
      • Chen N.
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      • Zhan J.
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      Upregulation of PD-L1 by EGFR activation mediates the immune escape in EGFR-driven NSCLC: implication for optional immune targeted therapy for NSCLC patients with EGFR mutation.
      Various studies have shown that mutations in driver genes mediate immune escape by upregulating the expression of immune checkpoint molecules. Furthermore, several recent studies have indicated that in melanoma, the immune microenvironment may contribute to acquired resistance to mitogen-activated protein kinase inhibitors.
      • Ebert P.J.R.
      • Cheung J.
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      MAP kinase inhibition promotes T cell and anti-tumor activity in combination with PD-L1 checkpoint blockade.
      • Reddy S.M.
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      Influences of BRAF inhibitors on the immune microenvironment and the rationale for combined molecular and immune targeted therapy.
      • Smith M.P.
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      • et al.
      The immune microenvironment confers resistance to MAPK pathway inhibitors through macrophage-derived TNFalpha.
      • Kakavand H.
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      • et al.
      PD-L1 Expression and immune escape in melanoma resistance to MAPK inhibitors.
      Notably, Koha et al.
      • Koh J.
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      • et al.
      EML4-ALK enhances programmed cell death-ligand 1 expression in pulmonary adenocarcinoma via hypoxia-inducible factor (HIF)-1 alpha and STAT3.
      reported that strong expression of programmed death-ligand 1 (PD-L1) was significantly associated with improved clinical outcomes after crizotinib treatment in patients with anaplastic lymphoma kinase (ALK)–derived pulmonary adenocarcinomas. These results suggest that the immune phenotype can influence the effects of targeted therapy in certain cancers and may serve as a novel source of resistance to small molecule inhibitors. However, most studies have been limited to cellular and animal models, and few have examined the relationships between immune mechanisms and targeted therapy in human cancers. Therefore, there is an urgent need to determine whether immune surveillance contributes to resistance to targeted therapy among patients harboring specific oncogenes and cancer types.
      Since the identification of EGFR as a key driver gene, the EGFR signaling pathway has become a major target in the successful treatment of NSCLC.
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      Dacomitinib versus gefitinib as first-line treatment for patients with EGFR-mutation-positive non–small-cell lung cancer (ARCHER 1050): a randomised, open-label, phase 3 trial.
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      Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma.
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      Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR.
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      Icotinib versus whole-brain irradiation in patients with EGFR-mutant non–small-cell lung cancer and multiple brain metastases (BRAIN): a multicentre, phase 3, open-label, parallel, randomised controlled trial.
      Nevertheless, some patients with NSCLC do not respond to EGFR inhibitors. Currently, the cause of de novo resistance to EGFR–tyrosine kinase inhibitors (TKIs) is not fully understood beyond genomic mechanisms such as the T790M mutation or MNNG HOS Transforming gene (c-MET) amplification.
      • Lee J.K.
      • Shin J.Y.
      • Kim S.
      • et al.
      Primary resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in patients with non–small-cell lung cancer harboring TKI-sensitive EGFR mutations: an exploratory study.
      • Nanjo S.
      • Arai S.
      • Wang W.
      • et al.
      MET copy number gain is associated with gefitinib resistance in leptomeningeal carcinomatosis of EGFR-mutant lung cancer.
      • Zhong J.
      • Li L.
      • Wang Z.
      • et al.
      Potential resistance mechanisms revealed by targeted sequencing from lung adenocarcinoma patients with primary resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs).
      Given that immune mechanisms are known contributors to resistance to targeted therapy and that increased PD-L1 expression has been detected in the context of acquired resistance to EGFR-TKIs,
      • Han J.J.
      • Kim D.W.
      • Koh J.
      • et al.
      Change in PD-L1 expression after acquiring resistance to gefitinib in EGFR-mutant non-small-cell lung cancer.
      • Haratani K.
      • Hayashi H.
      • Tanaka T.
      • et al.
      Tumor immune microenvironment and nivolumab efficacy in EGFR mutation-positive non–small-cell lung cancer based on T790M status after disease progression during EGFR-TKI treatment.
      one might to assume that PD-L1 expression on tumor and immune cells could predict a poor response to targeted therapy in NSCLCs with EGFR mutation. However, previous studies regarding this topic have yielded conflicting results as a consequence of differences in the sample sizes, prior treatment status, assays, antibody clones, and grading cutoff standards.
      • D'Incecco A.
      • Andreozzi M.
      • Ludovini V.
      • et al.
      PD-1 and PD-L1 expression in molecularly selected non–small-cell lung cancer patients.
      • Gainor J.F.
      • Shaw A.T.
      • Sequist L.V.
      • et al.
      EGFR mutations and ALK rearrangements are associated with low response rates to PD-1 pathway blockade in non-small cell lung cancer: a retrospective analysis.
      • Tang Y.
      • Fang W.
      • Zhang Y.
      • et al.
      The association between PD-L1 and EGFR status and the prognostic value of PD-L1 in advanced non–small cell lung cancer patients treated with EGFR-TKIs.
      • Soo R.A.
      • Kim H.R.
      • Asuncion B.R.
      • et al.
      Significance of immune checkpoint proteins in EGFR-mutant non–small cell lung cancer.
      Therefore, the present work aimed to determine whether immune surveillance affects the response to targeted therapy for NSCLC. Specifically, 101 patients with newly diagnosed advanced NSCLC with an EGFR mutation were retrospectively analyzed to assess the predictive effects of PD-L1 expression on EGFR-TKI responsiveness. The hypothesis was set to determine the correlation of PD-L1 expression with de novo resistance to EGFR-TKIs. Accordingly, the immune phenotypes of patients with EGFR-mutant NSCLC and de novo resistance to EGFR-TKIs were further explored.

      Methods

      Study Population

      We reviewed the medical records of 1,042 patients diagnosed with NSCLC at the Guangdong Lung Cancer Institute between April 2016 and September 2017. EGFR-activating mutations were defined as mutations associated with EGFR-TKIs sensitivity, including the exon 19 deletion, G719X, L858R, and L861Q. We excluded 823 patients with EGFR wild-type, 22 patients who did not receive EGFR-TKIs, 88 patients without baseline data, and 8 patients who were diagnosed with stage I–III disease. Finally, 101 patients with EGFR-TKI treatment-naïve NSCLC were included in this study (Fig. 1). The characteristics of the enrolled patients at the baseline (i.e., before EGFR-TKI treatment) are summarized in Supplement Data 1. This study was approved by the Institutional Review Board of Guangdong General Hospital (Guangzhou, China), and informed consent was obtained from all patients before the use of biopsy tissues for genetic analysis.
      Figure thumbnail gr1
      Figure 1Flow chart of the study. PD-L1, programmed death ligand 1; GLCI, Guangdong Lung Cancer Institute; TKI, tyrosine kinase inhibitor.

      PD-L1 and CD8 Analysis

      Tumor specimens from the 101 included patients were immunohistochemically (IHC) evaluated to detect the expression of PD-L1 (SP142; Spring Bioscience Inc., Pleasanton, California) and cluster of differentiation 8 (CD8) (C8/144B, Gene Tech Co., Ltd., Shanghai, China). The stained tissue sections were independently scored by two pathologists who were blinded to the patients’ clinical characteristics and outcomes.
      PD-L1 expression on tumor and immune cells was evaluated using a 3-tiered grading system wherein strong expression (TC3/IC3) was defined as ≥50% for tumor cells (TCs) or ≥10% for immune cells (ICs), weak expression (TC1–2/IC1–2) was defined as 5% to 49% for TC or 5% to 9% for IC, and negative expression was defined as less than 5% for TC or IC. CD8 expression on lymphocytes was reported as the proportion of positive cells among all nucleated cells in the stromal compartments of each core, and scoring was recorded as negative (less than 10%) or positive (10% or more).

      EGFR, KRAS, MET, ALK, Human Epidermal Growth Factor Receptor 2 (HER2), and Bcl-2-Interacting Mediator of Cell Death (BIM) Analysis

      EGFR gene mutations were detected using an amplification refractory mutation system and a kit that could detect 29 mutations (DxS EGFR mutation test kit; Amoy Diagnostics, Xiamen, China). Mutations in exons 2 and 3 of KRAS were evaluated by polymerase chain reaction amplification using the BigDye 3.1 Kit (Applied Biosystems, Foster City, California) according to the manufacturer’s protocol.
      The level of MET amplification was evaluated in deparaffinized 4-μm sections using dual-color fluorescence in situ hybridization and a c-MET/CEN7q Dual Color FISH Probe (Vysis; Abbott Laboratories, Abbott Park, Illinois). Amplification was considered present in cases with an MET/CEN7 ratio of greater than 2.0, an average MET gene copy number greater than 6.0 per cell, or greater than 10% of the tumor cells containing greater than 15 MET signals.
      • Cappuzzo F.
      • Marchetti A.
      • Skokan M.
      • et al.
      Increased MET gene copy number negatively affects survival of surgically resected non–small-cell lung cancer patients.
      The ALK fusion status was detected using IHC with a monoclonal rabbit anti-ALK primary antibody (D5F3; Ventana/Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions.
      • Wynes M.W.
      • Sholl L.M.
      • Dietel M.
      • et al.
      An international interpretation study using the ALK IHC antibody D5F3 and a sensitive detection kit demonstrates high concordance between ALK IHC and ALK FISH and between evaluators.
      • Ying J.
      • Guo L.
      • Qiu T.
      • et al.
      Diagnostic value of a novel fully automated immunochemistry assay for detection of ALK rearrangement in primary lung adenocarcinoma.
      Sanger sequencing was used to evaluate BIM splicing from exon 4 to exon 3 and HER2 mutations, based on a previously reported primer sequence.
      • Ng K.P.
      • Hillmer A.M.
      • Chuah C.T.
      • et al.
      A common BIM deletion polymorphism mediates intrinsic resistance and inferior responses to tyrosine kinase inhibitors in cancer.
      Labeling was performed using the BigDye 3.1 Kit according to the manufacturer’s protocol.

      Statistical Analysis

      All statistical analyses were performed using GraphPad Prism software (version 7.01; GraphPad, Inc., La Jolla, California) and IBM SPSS software (version 22.0; IBM Corp., Armonk, New York). The chi-square test was used to analyze the objective response rates according to PD-L1 expression. Kaplan-Meier curves and the log-rank test were used to analyze progression-free survival (PFS) according to PD-L1 expression. All reported p values were 2-tailed, and differences were considered statistically significant at p < 0.05.

      Results

      PD-L1 Expression is Associated With Poor Responses to EGFR-TKIs in EGFR Mutation–Positive NSCLCs

      The patients’ overall characteristics are shown in Supplement Data 1. Among the 84 patients with available evaluation data, strong PD-L1 expression was associated with a significantly decreased objective response rate, compared with weak or negative expression (35.7% versus 63.2% versus 67.3%, p = 0.001) (Supplementary Data 2).
      Furthermore, in the overall population, PD-L1 expression was associated with a significantly shorter median PFS in response to EGFR-TKI therapy compared with weak or negative PD-L1 expression (3.8 versus 6.0 versus 9.5 months, p < 0.001) (Fig. 2A). Among patients harboring exon 19 deletions, strong PD-L1 expression was also associated with a significantly shorter median PFS compared to weak or negative PD-L1 expression (3.0 versus 7.0 versus 12.0 months, p < 0.001) (Fig. 2B). Similarly, among patients harboring the L858R mutation, the median PFS associated with strong PD-L1 expression was significantly shorter than those associated with weak or negative PD-L1 expression (4.7 versus 6.0 versus 9.0 months, p = 0.024) (Fig. 2C).
      Figure thumbnail gr2
      Figure 2Kaplan–Meier survival analysis of associations between programmed death ligand 1 (PD-L1) expression and progression-free survival (PFS) among patients with NSCLC. (A), Overall patient population. (B) Patients with exon 19 deletion. (C) Patients with the L858R mutation. Patients were divided into three groups by PD-L1 expression: TC3/IC3, TC1-2/IC1-2, and TC0/IC0.
      HR, hazard ratio; CI, confidence interval.

      Exploration of De Novo Resistance to EGFR-TKIs

      The characteristics of 15 patients with de novo resistance to EGFR-TKIs are summarized in Table 1. With the exception of one patient who exhibited disease progression with symptomatic brain metastases during EGFR-TKI treatment, most patients experienced disease progression characterized by primary lesion enlargement. An evaluation of the genetic profiles of these 15 patients revealed c-MET amplification in 4 patients, KRAS G12D mutation in 1 patient, and negative results for HER2, T790M, and ALK mutations in all 15 patients. PD-L1 positivity was significantly more common among patients with de novo resistance than among those with acquired resistance to EGFR-TKIs (66.7% versus 30.2%, p = 0.009) (Fig. 3). Based on a recently proposed classification, we divided the de novo resistance cases into four categories: type I adaptive immune resistance (PD-L1+/CD8+), type II immune ignorance (PD-L1–/CD8–), type III intrinsic induction (PD-L1+/CD8–), and type IV immune tolerance (PD-L1–/CD8+).
      • Teng M.W.
      • Ngiow S.F.
      • Ribas A.
      • et al.
      Classifying cancers based on T-cell infiltration and PD-L1.
      This approach revealed a high proportion of PD-L1 and CD8 dual positivity among patients with de novo resistance (46.7%, 7 of 15), suggesting that these patients may likely benefit from PD-1 blockade therapy (Supplementary Data 3).
      Table 1Summary of the Genetic Profiles of Patients With De Novo Resistance to EGFR-TKIs
      Patient(s)TypePFS (m)PD-L1 ScoreCD8

      Density
      MET

      Ampl
      BIM DelT790MKRAS

      Mutation
      PTEN

      Mutation
      PIK3CA

      Mutation
      HER2

      Mutation
      ALK

      Rearrangement
      119DEL0.25TC1IC13%+-------
      219DEL1TC3IC310%+-------
      3L858R1TC3IC33%--------
      4L858R1TC3IC315%+---NA---
      5L858R1TC2IC215%--------
      619DEL1.5TC3IC31%---G12D----
      7L858R1.6TC0IC08%----NA---
      820 Ins2TC0IC020%--------
      9L858R2TC0IC08%--------
      1019DEL2TC2IC210%----NA---
      11L858R2.3TC3IC350%----NA---
      12L858R2.5TC3IC380%--------
      13L858R3TC0IC05%----NA---
      1419DEL3TC3IC320%--------
      15L861Q3TC0IC010%+---NA---
      ALK, anaplastic lymphoma kinase; HER2, human epidermal growth factor receptor-2; MET, mesenchymal epithelial transition; TKIs, tyrosine kinase inhibitors; BIM, Bcl-2-interacting mediator of cell death; Del, deletion; PTEN, phosphatase and tensin homolog; PFS, progression-free survival; PD-L1, programmed death ligand 1; CD8, cluster of differentiation 8; T790M, substitution of methionine for threonine at position 790; PIK3CA, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha; Ampl, amplification.
      Figure thumbnail gr3
      Figure 3The relationship between programmed death ligand 1 expression and progression-free survival (PFS) among patients who received EGFR–tyrosine kinase inhibitors for EGFR-mutant NSCLC.

      The Efficacy of a Checkpoint Inhibitor for One Patient With De Novo Resistance to Gefitinib

      One patient harboring the EGFR L858R mutation did not benefit from gefitinib treatment (PFS: 2.5 months). In this case, IHC revealed strong PD-L1 expression with a high proportion of CD8+ cells (>50%). Negative results were observed during genetic testing for T790M, c-MET, KRAS, and BIM. The patient subsequently completed 6 cycles of treatment with a checkpoint inhibitor (pembrolizumab) and achieved a partial response characterized by more than 5 months of control of the primary lung lesions (Fig. 4).
      Figure thumbnail gr4
      Figure 4Presentation of a case with EGFR L858R mutation. TKI, tyrosine kinase inhibitor; PR, partial response; PD-L1, programmed death ligand 1; c-MET, mesenchymal epithelial transition; T790M, substitution of methionine for threonine at position 790; CD8, cluster of differentiation 8.

      Discussion

      Recent studies have revealed an epidemiological relationship between EGFR mutation and PD-L1 expression, leading to intense interest in the immunomodulatory mechanisms underlying responses to EGFR-TKI treatment.
      • Dong Z.Y.
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      Potential biomarker for checkpoint blockade immunotherapy and treatment strategy.
      • Li X.
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      Interactions between EGFR and PD-1/PD-L1 pathway: Implications for treatment of NSCLC.
      • Soo R.A.
      • Lim S.M.
      • Syn N.L.
      • et al.
      Immune checkpoint inhibitors in epidermal growth factor receptor mutant non–small cell lung cancer: current controversies and future directions.
      Therefore, the present study aimed to clarify the effects of immune surveillance in treatment-naïve NSCLC patients treated with EGFR-TKIs. Our retrospective analysis revealed that strong PD-L1 expression was associated with limited responses to EGFR-TKIs in patients with advanced NSCLC. Furthermore, patients with de novo resistance to EGFR-TKIs exhibited increased PD-L1 expression and a high frequency of PD-L1 and CD8 dual positivity. To the best of our knowledge, this is the first study to explore the immunophenotypic signature of patients with de novo resistance to EGFR-TKIs, and the results suggested that this population may be benefit from programmed death 1 (PD-1) blockade therapy.
      Various studies have indicated that tumor expression of PD-L1 predicts overall survival, PFS, and treatment responses in patients receiving immune therapy for advanced NSCLC.
      • Dong Z.Y.
      • Zhang C.
      • Li Y.F.
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      Genetic and immune profiles of solid predominant lung adenocarcinoma reveal potential immunotherapeutic strategies.
      • Dong Z.Y.
      • Zhong W.Z.
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      Potential predictive value of TP53 and KRAS mutation status for response to PD-1 blockade immunotherapy in lung adenocarcinoma.
      • Wu S.P.
      • Liao R.Q.
      • Tu H.Y.
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      Stromal PD-L1-positive regulatory T cells and PD-1–positive CD8-positive T cells define the response of different subsets of non-small cell lung cancer to PD-1/PD-L1 blockade immunotherapy.
      Similarly, we found that strong PD-L1 expression predicted a poor response to EGFR-TKI therapy, consistent with a recent report by Soo et al.
      • Soo R.A.
      • Kim H.R.
      • Asuncion B.R.
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      Significance of immune checkpoint proteins in EGFR-mutant non–small cell lung cancer.
      However, other investigators have drawn conflicting conclusions. Lin et al.
      • Lin C.
      • Chen X.
      • Li M.
      • et al.
      Programmed death-ligand 1 expression predicts tyrosine kinase inhibitor response and better prognosis in a cohort of patients with epidermal growth factor receptor mutation-positive lung adenocarcinoma.
      reported an association of PD-L1 expression with better PFS in EGFR mutation-positive patients treated with EGFR-TKIs (16.5 versus 8.6 months (PD-L1–negative), p = 0.001). Another subgroup analysis of 54 EGFR-mutant cases revealed a significantly longer time to progression after gefitinib or erlotinib treatment among PD-L1–positive patients, compared to their PD-L1–negative counterparts (13.1 versus 8.5 months, p = 0.01).
      • D'Incecco A.
      • Andreozzi M.
      • Ludovini V.
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      PD-1 and PD-L1 expression in molecularly selected non–small-cell lung cancer patients.
      In contrast, Tang et al.
      • Tang Y.
      • Fang W.
      • Zhang Y.
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      The association between PD-L1 and EGFR status and the prognostic value of PD-L1 in advanced non–small cell lung cancer patients treated with EGFR-TKIs.
      found no significant correlation of PD-L1 expression with the efficacy of EGFR-TKIs in patients with EGFR-mutated advanced NSCLC. There are several possible explanations for the differences between our findings and those of previous reports. First, we evaluated a larger sample size, which might have enhanced the analytical power and affected the final results. Second, PD-L1 expression is a dynamic biomarker that can be affected by previous treatment. Accordingly, our study focused on treatment-naïve patients to avoid the confounding effects of this factor. Finally, the use of different assays, antibody clones, and scoring cutoffs may have affected the results and conclusions and could explain why the existing data are ambiguous with respect to whether tumor PD-L1 expression can predict the response to EGFR-TKI treatment. Given the inconsistent findings regarding the relationship between PD-L1 expression and responses to EGFR-TKIs, further validation should be conducted using standardized methods.
      A recent report stated that a lack of T-cell infiltration, a shrinking proportion of PD-L1+/CD8+ tumor infiltrating lymphocyte (TIL), and a significantly decreased mutation burden cause weak immunogenicity in patients with EGFR-mutant NSCLC and lead to an inferior response to PD-1 blockade.
      • Dong Z.Y.
      • Wu S.P.
      • Liao R.Q.
      • et al.
      Potential biomarker for checkpoint blockade immunotherapy and treatment strategy.
      However, we discovered that patients with poor responses to EGFR-TKIs, especially those with de novo resistance, exhibited a high level of immunogenicity, suggesting that these patients may benefit from immune therapy and that the immune microenvironment is an important factor in responses to targeted therapy. Currently, evidence increasingly suggests the existence of immune mechanisms of resistance to targeted therapy. For example, researchers found that in melanoma models, cells resistant to a BRAF inhibitor exhibited increased MAPK signaling and PD-L1 expression.
      • Jiang X.
      • Zhou J.
      • Giobbie-Hurder A.
      • et al.
      The paradoxical activation of MAPK in melanoma cells resistant to BRAF inhibition promotes PD-L1 expression that is reversible by MEK and PI3K inhibition.
      In patients with melanoma, increased expression of markers of T cell exhaustion and PD-L1 were detected after treatment with a BRAF inhibitor.
      • Frederick D.T. 1
      • Piris A.
      • Cogdill A.P.
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      BRAF inhibition is associated with enhanced melanoma antigen expression and a more favorable tumor microenvironment in patients with metastatic melanoma.
      Furthermore, Smith et al.
      • Smith M.P.
      • Sanchez-Laorden B.
      • O'Brien K.
      • et al.
      The immune microenvironment confers resistance to MAPK pathway inhibitors through macrophage-derived TNFalpha.
      identified the immune microenvironment in BRAF-mutant melanomas as a novel source of resistance to MAPK pathway inhibitors via macrophage-derived tumor necrosis factor α.
      • Smith M.P.
      • Sanchez-Laorden B.
      • O'Brien K.
      • et al.
      The immune microenvironment confers resistance to MAPK pathway inhibitors through macrophage-derived TNFalpha.
      Against this previous research backdrop, we supposed that PD-L1 expression might impair the effects of targeted therapy such that patients exhibiting strong PD-L1 expression would respond poorly to EGFR-TKIs. This speculation was consistent with a further analysis of 15 de novo resistance cases in which a high proportion of PD-L1/CD8 dual positivity was observed.
      The mechanism of de novo resistance to EGFR-TKIs is known to be highly heterogenous. Only a fraction of cases involving resistance can be attributed to known resistant mechanisms such as de novo T790M mutation and C-MET amplification. Recently, somatic single-nucleotide mutation patterns and coexisting truncal drivers were also reported to associate with primary resistance to EGFR-TKIs.
      • Zhong J.
      • Li L.
      • Wang Z.
      • et al.
      Potential resistance mechanisms revealed by targeted sequencing from lung adenocarcinoma patients with primary resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs).
      • Nahar R.
      • Zhai W.
      • Zhang T.
      • et al.
      Elucidating the genomic architecture of Asian EGFR-mutant lung adenocarcinoma through multi-region exome sequencing.
      In this study, we only identified four patients harboring a c-MET amplification, one with a KRAS mutation, and one with a phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) mutation; the remaining nine patients exhibiting de novo resistance did not exhibit any known resistant mechanisms. However, these 15 patients included a large proportion of patients with both strong PD-L1 expression and CD8 positivity. Given the findings of previous studies and our observation of impaired responses to EGFR-TKIs, we speculated that PD-L1 expression might be associated with de novo resistance to targeted therapy.
      Findings from a series of clinical studies suggest an impaired response of EGFR-mutant NSCLC to PD-1 blockade monotherapy, and several combination trials of EGFR-TKIs with immune checkpoint inhibitors reported no addictive or synergistic effects.
      • Dong Z.Y.
      • Zhang J.T.
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      • et al.
      EGFR mutation correlates with uninflamed phenotype and weak immunogenicity, causing impaired response to PD-1 blockade in non–small cell lung cancer.
      • Ahn M.J.
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      • Yu H.
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      136O: osimertinib combined with durvalumab in EGFR-mutant non–small cell lung cancer: results from the TATTON phase Ib trial.
      • Gibbons D.L.
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      57O efficacy, safety and tolerability of MEDI4736 (durvalumab [D]), a human IgG1 anti-programmed cell death-ligand-1 (PD-L1) antibody, combined with gefitinib (G): a phase I expansion in TKI-naive patients (pts) with EGFR mutant NSCLC.
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      These results highlight the challenges associated with the use of immune therapy to manage patients harboring EGFR mutations. Possibly, these patients require more stringent selection criteria that consider the immune phenotype. In the present study, several patients exhibiting de novo resistance to EGFR-TKIs were also PD-L1 and CD8 dual-positive, which implies a state of immune activation. In a subsequent clinical case, we observed a partial response to PD-1 blockade therapy in a patient with EGFR mutation–positive NSCLC and PD-L1/CD8 dual positivity who failed to respond to first-line gefitinib therapy. Recently, Socinski et al.
      • Socinski M.A.
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      reported the subgroup analysis from IMpower 150 clinical trial that check-point inhibitor atezolizumab improved the overall survival in EGFR/ALK-positive patients (hazard ratio: 0.54, median OS not yet reached versus 17.5 months) especially in TC3 or IC3 positive patients. Therefore, our study suggests a novel nonchemotherapeutic strategy for patients exhibiting de novo resistance to EGFR-TKIs. These findings may be useful for the future design of clinical trials of PD-L1 inhibitors for patients with EGFR-mutant NSCLC.
      • Liu S.Y.
      • Wu Y.L.
      Ongoing clinical trials of PD-1 and PD-L1 inhibitors for lung cancer in China.
      However, the present study also had several limitations. First, we did not perform next-generation sequencing of samples from patients exhibiting de novo resistance to EGFR-TKIs, which may have obscured the potential effects of altered genes on the response to targeted therapy. Additionally, as yet unknown gene mutations might also contribute to de novo resistance. Second, we did not conduct a further investigation of the relationship between gene changes and PD-L1 expression. Third, we did not consider other factors that might affect the tumor immune environment, such as cellular factors and the tumor burden. Additional research is needed to address these issues. Fourth, in this study we used SP142, an Immunohistological dye, which detected fewer stained tumor cells with this agent relative to three other PD-L1 IHC assays (22C3, 28-8, and SP263).
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      • McElhinny A.
      • Stanforth D.
      • et al.
      PD-L1 immunohistochemistry assays for lung cancer: results from phase 1 of the “Blueprint PD-L1 IHC Assay Comparison Project.
      However, Professor Zhang recently reported that the IHC detection of phosphatase and tensin homolog (PTEN) expression could be used as a surrogate tissue quality marker. The PD-L1 prevalence in NSCLC detected via SP142 in PTEN-qualified samples is comparable to previous reports of Asian patients.
      • Zhang X.C.
      • Cao X.
      • Sun C.
      • et al.
      Characterization of PD-L1 expression in Chinese non–small cell lung cancer patients with PTEN expression as a means for tissue quality screening.
      Therefore, our results should be further confirmed using a standardized method.
      In conclusion, this retrospective study revealed that strong PD-L1 expression predicted a poor response to EGFR-TKI treatment among patients with EGFR-mutated NSCLC. The observation of strong PD-L1 expression in patients exhibiting de novo resistance to EGFR-TKIs suggests that these patients may benefit from treatment with a PD-1 inhibitor.

      Acknowledgments

      This study was supported by the National Key R&D Program of China (2016YFC1303800 to Q. Zhou), the Key Lab System Project of Guangdong Science and Technology Department – Guangdong Provincial Key Lab of Translational Medicine in Lung Cancer (2012A061400006 and 2017B030314120 to Y.L. Wu), the Special Fund of Public Interest by National Health and Family Control Committee (201402031 to Y.L. Wu), and a General Research Project of Guangzhou Science and Technology Bureau (No. 201607010391 to X. Zhang).

      Supplementary Data

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