P53 and NLR could recognize distinct subtypes of lung adenocarcinoma with high expression of PD-L1 (TPS≥50%) defined by 22C3

P53 and NLR could recognize distinct subtypes of lung adenocarcinoma with high expression of PD-L1 (TPS≥50%) defined by 22C3 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article P53 and NLR could recognize distinct subtypes of lung adenocarcinoma with high expression of PD-L1 (TPS≥50%) defined by 22C3 Ming Li, Mengqi Huang, Yuchen Huang, Xiaoliang Zhang, Haibo Wu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7186339/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Background: Lung adenocarcinoma (LuAD) with high expression of PD-L1 (TPS≥50%) is a distinct subset of NSCLC and a potential candidate for immunotherapy. Improved understanding of the heterogeneity of PD-L1 high LuAD and recognizing novel biomarkers combined with PD-L1 would facilitate more precise use of PD-1 inhibitors in clinical practice. Methods: Surgically resected tumor tissues from 69 LuAD patients with PD-L1 TPS≥50% defined by 22C3 were tested for p53 and IFN-γ protein expression using immunohistochemistry. The densities of CD8 and Granzyme B-positive T cells were evaluated by immunohistochemistry. EGFR mutation was detected by RT-PCR, ALK rearrangement was examined by fluorescence in situ hybridization (FISH) assay and TP53 gene mutation was sequenced using Sanger sequencing. Results: The differences of clinicopathological features, including age and spread through air space (STAS), were observed among PD-L1 higher-expression (TPS≥50%，<70%) and highest-expression (TPS≥70%) patients. LuAD with PD-L1 TPS≥70%, aberrant expression of p53 protein or NLR<5 had more expression of IFN-γ protein and higher densities of CD8-positive tumor infiltrating lymphocytes (TILs) as well as Granzyme B -positive T cells comparing with TPS＜70%, p53 protein normal expression or NLR≥5 patients. Conclusions: The present study firstly revealed that distinct subsets could be identified in PD-L1 high LuAD according to p53 protein expression and NLR level, presenting as different characteristics of tumor microenvironment associated with response to PD-1 inhibitors. Lung adenocarcinoma PD-L1 p53 NLR Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Lung cancer is the main cause of tumor-related death worldwide. The development of targeted therapeutics against driver oncogenes has already improved prognosis of non-small cell lung cancer (NSCLC) patients. However, a fair percentage of NSCLC do not harbor targetable mutations. Antitumor immunotherapy targeting immune checkpoints, such as programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1), has become a successful therapeutic modality for NSCLC without oncogene mutation 1 . The expression of PD-L1 on tumor cells currently is considered as an important predictive biomarker for treatment with PD-1/PD-L1 inhibitors. High expression of PD-L1, defined as tumor proportion score (TPS) ≥ 50% when detecting by 22C3 antibody, is proved to be associated with improved responses to PD-1 inhibitors in NSCLC [ 1 , 2 ] . Nevertheless, a substantial proportion of PD-L1 high patients do not have best responses to anti-PD-1 therapy [ 1 , 2 ] . Therefore, PD-L1 positivity alone cannot be considered a rock-solid predictive factor for response to PD-1/PD-L1 blockade in NSCLC [ 3 , 5 ] . It is of clinical relevance to recognize the subgroup in PD-L1 high NSCLC which would be better responders to PD-1 inhibitors. Then it is essential to identify other biomarkers beyond PD-L1. Our work was performed in LuAD with high expression of PD-L1. The density of CD8-positive TILs and Granzyme B (GZMB)-positive T cells as well as expression level of IFN-γ protein, that were associated with efficacy of PD-1 inhibitors, were observed significantly different among two subsets of PD-L1 high LuAD. The two distinct subsets could be recognized according to expression pattern of p53 protein and neutrophil-to-lymphocyte ratio (NLR) level. These results provided a new perspective to better understand the heterogeneity of LuAD with PD-L1 TPS ≥ 50%. Moreover, our work firstly revealed that p53 and NLR could make an easy-to-practice approach that were able to identify potential better responders to PD-1 blockers in PD-L1 high LuAD patients. MATERIALS AND METHODS Patients and Samples 69 patients included in the present study underwent curative-intent surgical resection of stage I-III LuAD in the thoracic surgery department of The Second Affiliated Hospital of Anhui Medical University and The First Affiliated Hospital of USTC (University of Science and Technology of China), between 2019 and 2021. Inclusion criteria were: pathological diagnosis of primary LuAD and PD-L1 TPS≥50% detected with 22C3 antibody, adequate formalin-fixed paraffin-embedded (FFPE) blocks with tumor tissue available for further evaluation. All patients had not received any treatment prior to the surgery. Clinical data were retrospectively collected from medical records. Median age of 69 patients was 63 years (range: 28-79 years). The patients were composed of 52.17% male and 47.83% female. 67% of 69 patients were never-smokers. According to the 8th version of the Union for International Cancer Control (UICC) consortium TNM staging, the stages in the LuAD cohort were stage I 34.78%, stage II 33.33% and stage Ⅲ 31.89%. All tumor tissue sections were reviewed by two pathologists (ML and ZZF), and classified according to the WHO 2015 classification of LuAD 6 . The study was approved by the Ethics Committee of The Second Affiliated Hospital of Anhui Medical University (Ethics Code: YX2025-092). All participants provided written informed consent prior to enrolment in the study and this study was conducted in accordance with the Declaration of Helsinki. Immunohistochemical staining All FFPE tumor samples were subjected to immunohistochemical staining using primary antibodies against CD8 (1:200, clone# SP16, GT211229; GeneTech Inc., Washington, DC, USA), Granzyme B (1:100, clone# EP230, ZA-0599; ZSGB-BIO Co., Ltd., Beijing, China), IFN-γ (1:300, Polyclonal antibody, cat#DF6045; Affinity Biosciences, Jiangsu, China) and p53 (1:100, clone#DO-07, IR616; Dako, CA, USA). Tissue sections (4μM) were deparaffinized, rehydrated, and subjected to microwave antigen retrieval in an EDTA buffer (pH=9) for 20 min followed by cooling at room temperature. After blocking with 3% hydrogen peroxide for 10 min, the slides were washed with phosphate-buffered saline (PBS) for 15 min and incubated with primary antibodies for 12 h at 4 ℃. Immunoreactivity was visualized using the DAB method (GK600705; Genetech Inc.) after the slides were incubated with horseradish peroxidase (HRP)-linked secondary antibodies. Slides stained with PBS instead of primary antibody were considered as the negative control. Detection of EGFR, ALK and TP53 gene status EGFR mutations were detected using the Cobas IVD method (Cobas EGFR test, Roche Molecular systems, Inc., Branchburg, NJ, USA) and ALK rearrangement was examined by the ALK break-apart fluorescence in situ hybridization (FISH) assay. FISH was performed using a break-apart probe (Vysis LSI ALK Dual Color, break- apart rearrangement probe; Abbott Molecular, Abbott Park, IL, USA) according to the manufacturer’s instructions. FISH signals were observed using an Olympus BX61 fluorescence microscope and the results were evaluated according to previous study [ 7] . As for TP53 gene, exons 1 and 3-8 were sequenced using Sanger sequencing. The evaluation of immunohistochemical staining Expression of PD-L1 The expression of PD-L1 in 69 patients was confirmed by two experienced pathologists (ML and ZZF). PD-L1 expression was determined as the percentage of tumor cells with positive membranous staining using the 22C3. Densities of CD8 and Granzyme B-positive T cells To evaluate the CD8+ T cells, tissue sections were firstly viewed at a low power, and four independent areas containing the greatest abundance of CD8+ cells (termed as hot spot area) were selected. Subsequently, three views were randomly selected from each hot spot area at a higher power. CD8+ T cells in the tumor island as well as the tumor stroma adjacent to parenchyma were counted in 12 high-power fields (HPFs). The average amount from 12 high-power fields was calculated as the density of CD8+ T cells per HPF [8] . The density of Granzyme B-positive T cells was also assessed according to the above-mentioned method. Expression of IFN-γ protein Immunohistochemistry was used to detect IFN-γ protein in previous study and H-score was used to evaluate its expression level in tumors [9] . H-score was determined by multiplying the percentage of positive cells by an intensity score. Expression of p53 protein Expression pattern of p53 protein shown as diffusely strong positive or complete absence were interpreted as abnormal/aberrant/mutation-type [10,11 ] . The normal/wild- type expression pattern of p53 protein was characterized by an admixture of negative cells with weakly as well as strongly positive cells [10,11] . The three expression patterns of p53 protein and their interpretation requires the presence of a positive internal control with staining of lymphocytes, fibroblasts and other nonneoplastic cells. Statistical analysis Categorical data were compared using c2 test or Fisher’ s exact test when it was appropriate. Quantitative data were presented as means±SD and were analyzed using Student t test and ANOVA test according to data distribution. A p value less than 0.05 was considered as statistically significant. All analyses were performed using IBM SPSS software program (IBM Corp., Armonk, N.Y., USA), version 26.0. RESULTS 1. Baseline clinical and pathologic characteristics in LuAD with higher- expression (TPS≥50%, <70%) and highest-expression (TPS ≥70% ) of PD-L1 Of the 69 patients, 37 (53.62%) had a higher-expression of PD-L1 and 32 (46.38%) had a highest-expression of PD-L1. PD-L1 TPS≥70% was observed more frequently in patients with older age (≥65 years old, P <0.05) as well as in patients without spread through air space (STAS) of tumor cells ( P <0.05). PD-L1 TPS≥70% seemed to be more common in LuAD with histological subtype of solid, papillary or micropapillary comparing with lepidic and acinar subtypes. 58 patients (84.06%) had no classic driver oncogene mutations, including EGFR and ALK. Of the 11 cases harboring oncogene mutations, ALK fusion was more frequently observed than EGFR mutation (11.59% versus 4.35%), but the difference was not statistically significant. Oncogene status was found no significant difference among LuAD with higher and highest-expression of PD-L1. The data was shown in Table 1. STAS: spread through air space; *, P < 0.05. 2. Expression of p53 protein and TP53 gene aberrations in LuAD with higher and highest-expression of PD-L1 The undoubted abnormal expression of p53 protein was observed in 31 patients and equivocal immunohistochemical result was founded in one case. TP53 gene mutations were detected in 31 cases by Sanger sequencing and they were all presented as mutation-type expression of p53 protein. The equivocal one was proved to be TP53 mutation using gene sequencing and it was finally defined as abnormal expression of p53 protein. Wild-type TP53 gene was detected in 37 patients who had normal expression of p53 protein. The concordance between abnormal expression of p53 protein and TP53 gene mutation was nearly 100%. Abnormal expression of p53 protein was detected in 13.51% of the 37 patients with PD-L1<70%. It was more frequently observed in PD-L1≥70% LuAD, that was 84.38%, compared with PD-L1<70% group ( P <0.001, Fig. 1 and Fig. 2). 3. The densities of CD8-positive TILs and GZMB-positive T cells in LuAD with higher and highest-expression of PD-L1 The average amount of CD8-positive TILs was 24±15/ HPF (range: 0–74/ HPF) in PD-L1 TPS<70% group. It was 57±19/HPF (range: 0-80/HPF) in PD-L1 TPS≥70% patients. The significant difference of CD8+ TILs was observed among TPS<70% and TPS≥70% LuAD ( P <0.001, Fig. 3A, Fig. 4A-a and b). The average amount of GZMB-positive T cells was 18±26/HPF (range: 0-117/HPF) in TPS<70% group and it was 52±44/HPF (range: 0-148/HPF) in TPS≥70% group. LuAD with PD-L1 TPS≥70% had significantly more GZMB-positive T cells than TPS<70% ( P <0.001, Fig. 3A, Fig. 4B-a and b). 4. The densities of CD8-positive TILs and GZMB-positive T cells in PD-L1 high LuAD with or without mutation-type expression of p53 protein The average density of CD8-positive TILs was 24±14/HPF (range: 0-65/HPF) in LuAD with normal expression of p53 protein. It was 57±19/HPF (range: 0-80/HPF) in LuAD with mutation-type expression of p53 protein. It was found that the density of CD8+ TILs was significantly higher in p53 abnormal expression comparing with normal expression group ( P <0.001, Fig. 3B, Fig. 4A-c and d). The average density of GZMB+ T cells was 60±43/HPF (range: 0-148/HPF) in LuAD with abnormal expression of p53 protein and it was 11±14/HPF (range: 0-66/HPF) in normal expression group. The significant difference of GZMB+ T cell densities was observed among two subsets according to p53 protein expression ( P <0.001, Fig. 3B, Fig. 4B-c and d). 5. The densities of CD8-positive TILs and GZMB-positive T cells in PD-L1 high LuAD with NLR<5 or NLR≥5 The average density of CD8+ TILs was 51±23/HPF (range: 0-80/HPF) in LuAD with NLR<5 and it was 27±16/HPF (range: 1-74/HPF) in LuAD with NLR≥5. The density of CD8+ TILs was significantly higher in NLR<5 than NLR≥5 subset ( P <0.001, Fig. 3C, Fig. 4A-e and f). The average amount of GZMB+ T cells was 45±41/HPF (range: 0-148/HPF) in NLR<5 patients and it was 22±34/HPF (range: 0-133/HPF) in NLR≥5 cases. PD-L1 high LuAD had significantly more GZMB+ T cells in NLR<5 comparing with NLR≥5 subgroup ( P <0.05, Fig. 3C, Fig. 4B-e and f). 6. The expression of IFN-γ protein in PD-L1 high LuAD according to PD-L1 TPS level, p53 protein expression and NLR level IFN-γ protein was reliably detected in tumor cells of LuAD. The average H-score of IFN-γ protein was 68±80 (range: 0-270) in PD-L1 TPS<70% subgroup and it was 137±85 (range: 0-265) in TPS≥70% patients. The expression level of IFN-γ protein was significantly higher in TPS≥70% comparing with TPS<70% subsets ( P <0.01, Fig. 5). The average H-score was 68±55 (range: 0-270) in patients with mutation-type expression of p53 protein. However, it was just 11±14 (range: 0-66) in patients with normal expression of p53 protein. The expression of IFN-γ was significantly higher in p53 protein abnormal expression comparing with normal expression subsets ( P <0.001, Fig. 5). The average score was 121±93 (range: 2-270) in patients with NLR<5. The expressive score of IFN-γ in NLR<5 was significantly higher than that in patients with NLR≥5, which was 78±80 (range: 0-263) ( P <0.05, Fig. 5). DISCUSSION PD-L1 TPS ≥ 50% is not more frequently observed in primary LuAD [ 12 ] . According to clinical practice, PD-L1 expression is not an entirely reliable predictive marker for the efficacy of immunotherapy because the response rate to PD-1 inhibitors in advanced NSCLC with strongly positive PD-L1 expression was only 44.8% based on KEYNOTE-024 and other studies [1,4,5[ . Previous studies including patients with heterogeneous histological subtype and different PD-L1 expression level had explored some other biomarkers beyond PD-L1. The 69 patients in our study were all surgically resected LuAD with high expression of PD-L1. Our work firstly shown that p53 and NLR could recognize subsets of PD-L1 high LuAD which would have different responses to PD-1 inhibitors. Then our data may underlie clinical observation that not all PD-L1 high LuAD had robust response to PD-1 inhibitors [ 1 , 4 , 5 ] . There had limited studies shown that PD-L1 high LuAD could be further classified into PD-L1 higher and highest subset, but cut-off values were not consistent [ 13 , 14 ] . It was used TPS 70% as threshold in our work according to Frost’s study [ 14 ] . Highest expression of PD-L1 (TPS ≥ 70%) was more frequently observed in patients with older age than higher patients ( P < 0.05). In addition, PD-L1 TPS ≥ 70% was more often founded in tumors without STAS ( P < 0.05). These results suggested that patients with TPS ≥ 70% or TPS < 70% could be regarded as distinct subsets in PD-L1 high LuAD according to clinicopathological characteristics. A growing body of evidences supported that response to PD-1 inhibitors predominantly in NSCLC with a brisk tumor immune microenvironment, such as high density of CD8-positive TILs [ 3 , 15 – 17 ] . Moreover, anti-tumor effector T cells were termed as granzyme B (GZMB)-positive T cells and they were reliable predictor of better response to anti-PD-1 blockade [ 18 ] . Our data indicated that the density of CD8 + and GZMB + T cells were significantly higher in PD-L1 ≥ 70% compared with PD-L1 < 70% tumors. Then it was concluded that PD-L1 ≥ 70% and PD-L1 < 70% LuAD could be considered as different subsets based on tumor immune microenvironment. The present result also shown that PD-L1 ≥ 70% LuAD would likely be better responders to PD-1 inhibitors comparing with PD-L1 < 70% patients. TP53 is the most common mutated gene in NSCLC [ 19 ] . Expression pattern of p53 protein has been approved to be an accurate surrogate reflecting the underlying TP53 mutation status in human malignancies [ 20 , 21 ] . The concordance between p53 protein expression and TP53 gene status in our work was nearly 100%. It was shown that mutation-type expression of p53 protein was more frequently observed in PD-L1 ≥ 70% than PD-L1 < 70% LuAD ( p < 0.001). In addition, it was suggested that there had no significant difference of other driver oncogenes mutation among PD-L1 ≥ 70% and PD-L1 < 70% patients, including EGFR and ALK. Taken together, it further supported that LuAD with TPS ≥ 70% or TPS < 70% could be regarded as different subsets when according to expression pattern of p53 protein. The strong association has already been revealed among high density of CD8 + TIL and TP53 mutation in NSCLC [ 3 , 22 ] . Biton’s work found that GZMB-positive T cells were more abundant in TP53-mutated LuAD comparing with wild type group. However, the abundance of GZMB-positive T cells was indirectly evaluated by detection of GZMB gene expression in tumor tissues [ 22 ] . It was more accurate to consider the density of GZMB-positive cells as a better predictor associated with immune cell cytotoxicity. In addition, it was not evaluated in subsets with high expression of PD-L1 in Biton’s study. Our work was performed just in PD-L1 high LuAD patients. It was shown that both CD8 + T cells and effector T cells were more robust in PD-L1 high LuAD with mutation-type expression of p53 protein, when comparing to patients with p53 normal expression. Then it was suggested that expression of p53 protein could recognize two subsets with different immune cell cytotoxicity in PD-L1 high LuAD. As an easily evaluated biomarker in routine clinical practice, expression of p53 protein would be an additional signal together with PD-L1 to predict efficacy of PD-1 inhibitors. The neutrophil-to-lymphocyte ratio (NLR) is regarded as a surrogate for tumor-associated inflammation and previous result suggested NLR likely represents the activity of myeloid-derived suppressor cells (MDSCs), that suppress T-cell proliferation and expansion [ 23 ] . There has study indicated that more accumulation of neutrophils would suppress T-cell activity in lung cancer [ 24 ] . Then it was concluded that the baseline NLR level would be a potential predictor related to efficacy of PD-1 inhibitors. Banna’s work suggested that the appropriate NLR cut-off for advanced NSCLC treated with PD-1 inhibitors was 5 [ 13 ] . It was shown that advanced PD-L1 high NSCLC with NLR < 5 would exhibit favorable outcome following pembrolizumab treatment [ 13 ] , but not underlie the clinical observation. Higher density of CD8 + and GZMB-positive T cells was observed in PD-L1 high LuAD with NLR < 5. Then it was concluded that the NLR < 5 subset would likely be better responders to PD-1 inhibitors thanks to brisk microenvironment. Although the exact predictive value of NLR was not clearly clarified, our work firstly supported NLR could recognize distinct subgroup with different immune microenvironment in PD-L1 high LuAD. Previous studies detected expression of either the gene encoding IFN-γ (IFNG) or IFN-γ downstream genes in NSCLC as a surrogate for the abundance of IFN-γ protein [ 25 , 26 ] . It was shown that NSCLC with high expression of IFNG mRNA would have more benefit from nivolumab compared to low group [ 25 ] . In addition, deficiency of IFN-γ signaling in tumor cells could be served as a mechanism of resistance to PD-1 inhibitors [ 27 – 29 ] . Expression of IFN-γ protein had been detected by immunohistochemistry in melanoma [ 9 ] . Our work also evaluated expression of IFN-γ protein by immunohistochemistry in PD-L1 high LuAD. The result indicated that increased expression of IFN-γ protein was more frequently observed in LuAD with PD-L1 TPS ≥ 70%, NLR < 5 and abnormal expression of p53 protein ( p < 0.001). It further suggested that p53 and NLR together with PD-L1 could recognize potential better responders to PD-1 inhibitors in PD-L1 high LuAD owing to robust expression of IFN-γ protein. Currently there had limited studies involving subclassification of LuAD with PD-L1 TPS ≥ 50% and exploring its clinical relevance. To the best of our knowledge, the present work firstly raised the possibility that distinct subsets could be recognized in PD-L1 high LuAD according to p53 and NLR. PD-L1 high LuAD with highest-expression of PD-L1, p53 protein aberrant expression or NLR < 5 might be potential better responders to PD-1 inhibitors. Our work provided practical biomarkers to help making subclassification of PD-L1 high LuAD and their validation will be performed in next prospective studies. Declarations Author contributions Study concept/design: ML, ZZF ; data collection: MQH, ML, XLZ, HBW ; data analysis and interpretation: MQH, YCH ; review and final approval of manuscript: ZZF, HBW, MQH, ML . ML and MQH contributed equally to this article . Ethical considerations This study was approved by the Ethics Committee (EC) of The Second Affiliated Hospital of Anhui Medical University (Ethics Code: YX2025-092). The EC at The Second Hospital of Anhui Medical University acted as the central EC, whose review was accepted by the other participating institutions’ EC. Informed consent to use histopathological samples for research purposes was obtained from all patients prior to surgery. This study was conducted in accordance with the Declaration of Helsinki. Consent to participate The study was approved by the Ethics Committee (EC) of The Second Affiliated Hospital of Anhui Medical University (Ethics Code: YX2025-092). All participants provided written informed consent prior to participating. Written informed consent was obtained from a legally authorized representative for anonymised patient information to be published in this article. Consent for publication All participants provided written informed consent for publication of this article. All authors of the manuscript have read and agreed to its content and are accountable for all aspects of the accuracy and integrity of the manuscript. Declaration of conflicting interest The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Natural Science Foundation Incubation Program of The Second Affiliated Hospital of Anhui Medical University [grant number 2021GMFY03]. Data availability statement The study was conducted in accordance with applicable regulations. Requests concerning the data supporting the findings of this study can be directed to [email protected] for consideration. References Reck M, Rodríguez-Abreu D, Robinson AG et al. Pembrolizumab versus chemotherapy for PD-L1-positive non-small-cell lung cancer. N Engl J Med 2016; 375(19): 1823-33. Mok TSK, Wu YL, Kudaba I et al. Pembrolizumab versus chemotherapy for previously untreated, PD-L1-expressing, locally advanced or metastatic non-small-cell lung cancer (KEYNOTE-042): a randomised, open-label, controlled, phase 3 trial. Lancet 2019; 393(10183): 1819-30. Shirasawa M, Yoshida T, Shimoda Y, et al. Differential immune-related microenvironment determines programmed cell death protein-1/programmed death-ligand 1 blockade efficacy in patients with advanced NSCLC. J Thorac Oncol 2021; 16 (12): 2078-90. Satouchi M, Nosaki K, Takahashi T, et al. First-line pembrolizumab vs chemotherapy in metastatic non-small-cell lung cancer: KEYNOTE-024 Japan subset. Cancer Sci 2021; 112(12):5000-10. Reck M, Rodríguez-Abreu D, Robinson AG, et al. Updated analysis of KEYNOTE-024:pembrolizumab versus platinum-based chemotherapy for advanced non-small-cell lung cancer with PD-L1 tumor proportion score of 50% or greater. J Clin Oncol 2019; 37(7):537-46. Travis WD, Brambilla E, Nicholson AG, et al. The 2015 World Health Organization Classification of Lung Tumors: Impact of Genetic, Clinical and Radiologic Advances Since the 2004 Classification. J Thorac Oncol 2015; 10(9): 1243-60. Lee J, Park CK, Yoon HK, et al. PD-L1 expression in ROS1-rearranged non-small cell lung cancer: A study using simultaneous genotypic screening of EGFR, ALK, and ROS1. Thorac Cancer 2019; 10(1): 103-10. Zeng C, Gao Y, Xiong J, et al. Tumor-infiltrating CD8+ T cells in ALK-positive lung cancer are functionally impaired despite the absence of PD-L1 on tumor cells. Lung Cancer 2020; 150: 139-44. Liang X, Yin Y, Li N. GOLM1 is related to the inflammatory/immune nature of uveal melanoma and acts as a promising indicator for prognosis and immunotherapy response. Front Genet 2022; 13: 1051168. Köbel M, Reuss A, du Bois A, et al. The biological and clinical value of p53 expression in pelvic high-grade serous carcinomas. J Pathol 2010; 222(2): 191-98. McCluggage WG, Soslow RA, Gilks CB. Patterns of p53 immunoreactivity in endometrial carcinomas: 'all or nothing' staining is of importance. Histopathology 2011; 59(4): 786-88. Schoenfeld AJ, Rizvi H, Bandlamudi C, et al. Clinical and molecular correlates of PD-L1 expression in patients with lung adenocarcinomas. Ann Oncol 2020; 31(5): 599-608. Banna GL, Signorelli D, Metro G, et al. Neutrophil-to-lymphocyte ratio in combination with PD-L1 or lactate dehydrogenase as biomarkers for high PD-L1 non-small cell lung cancer treated with first-line pembrolizumab. Transl Lung Cancer Res 2020; 9(4): 1533-42. Frost N, Kollmeier J, Vollbrecht C, et al. KRAS G12C /TP53 co-mutations identify long-term responders to first line palliative treatment with pembrolizumab monotherapy in PD-L1 high (≥50%) lung adenocarcinoma. Transl Lung Cancer Res 2021; 10(2): 737-52. Tumeh PC, Harview CL, Yearley JH, et al. PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature 2014; 515(7528): 568-71. Fumet JD, Richard C, Ledys F, et al. Prognostic and predictive role of CD8 and PD-L1 determination in lung tumor tissue of patients under anti-PD-1 therapy. Br J Cancer 2018; 119(8): 950-60. Peters S, Gettinger S, Johnson ML, et al. Phase II trial of atezolizumab as first-line or subsequent therapy for patients with programmed death ligand 1-selected advanced non-small-cell lung cancer (BIRCH). J Clin Oncol 2017; 35 (24): 2781-89. G Berke. Unlocking the secrets of CTL and NK cells. Immunol Today 1995; 16(7): 343-46. Albitar M, Sudarsanam S, Ma W, et al. Correlation of MET gene amplification and TP53 mutation with PD-L1 expression in non-small cell lung cancer. Oncotarget 2018; 9(17): 13682-93. Serra P, Petat A, Maury JM, et al. Programmed cell death-ligand 1 (PD-L1) expression is associated with RAS/TP53 mutations in lung adenocarcinoma. Lung Cancer 2018; 118: 62-68. Nana Matsumoto, Padmini Manrai, Douglas Rottmann, et al. Correlative assessment of p53 immunostaining patterns and TP53 mutation status by next-generation sequencing in high-grade endometrial carcinomas. Int J Gynecol Pathol 2023; 42(6): 567-75. Biton J, Mansuet-Lupo A, Pécuchet N, et al. TP53, STK11, and EGFR mutations predict tumor immune profile and the response to Anti-PD-1 in lung adenocarcinoma. Clin Cancer Res 2018; 24(22): 5710-23. Tavakkoli M, Wilkins CR, Mones JV, et al. A Novel Paradigm Between Leukocytosis, G-CSF Secretion, Neutrophil-to-Lymphocyte Ratio, Myeloid-Derived Suppressor Cells, and Prognosis in Non-small Cell Lung Cancer. Front Oncol 2019; 9: 295. Koyama S, Akbay EA, Li YY, et al. STK11/LKB1 deficiency promotes neutrophil recruitment and proinflammatory cytokine production to suppress T-cell activity in the lung tumor microenvironment. Cancer Res 2016; 76(5):999-1008. Karachaliou N, Gonzalez-Cao M, Crespo G, et al. Interferon gamma, an important marker of response to immune checkpoint blockade in non-small cell lung cancer and melanoma patients. Ther Adv Med Oncol 2018; 10: 1758834017749748. Qian J, Wang C, Wang B, et al. The IFN-γ/PD-L1 axis between T cells and tumor microenvironment: hints for glioma anti-PD-1/PD-L1 therapy. J Neuroinflammation 2018; 15(1): 290. Manguso RT, Pope HW, Zimmer MD, et al. In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target. Nature 2017; 547 (7664): 413-18. Gao JJ, Shi LZ, Zhao H, et al. Loss of IFN-gamma pathway genes in tumor cells as a mechanism of resistance to anti-CTLA-4 therapy. Cell 2016; 167(2):397-404. Zaretsky JM, Garcia-Diaz A, Shin DS, et al. Mutations associated with acquired resistance to PD-1 blockade in melanoma. N Engl J Med 2016; 375(9):819-29. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 24 Aug, 2025 Reviews received at journal 23 Aug, 2025 Reviews received at journal 22 Aug, 2025 Reviewers agreed at journal 17 Aug, 2025 Reviewers agreed at journal 08 Aug, 2025 Reviewers invited by journal 08 Aug, 2025 Editor invited by journal 29 Jul, 2025 Editor assigned by journal 24 Jul, 2025 Submission checks completed at journal 24 Jul, 2025 First submitted to journal 22 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7186339","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":498426946,"identity":"cb27f07a-de7b-4a73-a391-11c3e0270a8a","order_by":0,"name":"Ming Li","email":"","orcid":"","institution":"the Second Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ming","middleName":"","lastName":"Li","suffix":""},{"id":498426947,"identity":"6904aac8-e7ec-4aea-9a2c-a332b81af920","order_by":1,"name":"Mengqi Huang","email":"","orcid":"","institution":"the Second Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Mengqi","middleName":"","lastName":"Huang","suffix":""},{"id":498426948,"identity":"1acedf47-95b6-4a35-86ea-d55fe7c3d930","order_by":2,"name":"Yuchen Huang","email":"","orcid":"","institution":"the Second Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuchen","middleName":"","lastName":"Huang","suffix":""},{"id":498426949,"identity":"f782ac0b-0888-437f-aa79-769460140d02","order_by":3,"name":"Xiaoliang Zhang","email":"","orcid":"","institution":"the First Affiliated Hospital of USTC, University of Science and Technology of China","correspondingAuthor":false,"prefix":"","firstName":"Xiaoliang","middleName":"","lastName":"Zhang","suffix":""},{"id":498426950,"identity":"fcc73d8a-af8a-4991-a0e8-306831c0013d","order_by":4,"name":"Haibo Wu","email":"","orcid":"","institution":"the First Affiliated Hospital of USTC, University of Science and Technology of China","correspondingAuthor":false,"prefix":"","firstName":"Haibo","middleName":"","lastName":"Wu","suffix":""},{"id":498426951,"identity":"a035b74c-3dfb-4c5c-bad4-59ade35ce029","order_by":5,"name":"Zhenzhong Feng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/UlEQVRIiWNgGAWjYHACwwcVIEqCgfEBQ4EEUVqMDc4ASR4JBmYDBgPitJhJQLWwSTAYEKGef0byBomDbXfk7aWbj1X8MLBI3HC7gfHDxxzcWiRupBUYHGx7ZtgjcyztZo+BROKGOweYJWduw2PNjRyD5I9thxl7JHLMbvCAtNxIYGPmxaNFHqjlwMG2w/YgLYV/iNFicCPHsAGoJRGkhZkoWwzPPCtmOHDuWXLPjbRkaRkDCeOZNxKb8fpF7njy9h8Hyu7Yts9IPvjxTUWdbN+N5IMfPuLzvkACAwMj2wE437GBgbEBj3og4Aep/oPQYo9f+SgYBaNgFIxEAABZnVzITGSu3QAAAABJRU5ErkJggg==","orcid":"","institution":"the Second Affiliated Hospital of Anhui Medical University","correspondingAuthor":true,"prefix":"","firstName":"Zhenzhong","middleName":"","lastName":"Feng","suffix":""}],"badges":[],"createdAt":"2025-07-22 11:08:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7186339/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7186339/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89229987,"identity":"5a94e43d-16c1-40c7-a996-704905b86663","added_by":"auto","created_at":"2025-08-17 14:08:47","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":36174,"visible":true,"origin":"","legend":"\u003cp\u003eExpression pattern of p53 protein in LuAD with higher and highest- expression of PD-L1. (A) Percentage of patients with p53 protein normal and abnormal expression in PD-L1＜70% LuAD. (B) Percentage of patients with p53 protein normal and abnormal expression in PD-L1≥70% LuAD. Mutation-type expression of p53 protein was more frequently observed in PD-L1≥70% LuAD compared with PD-L1＜70% subset (84.38% versus 13.51%, p\u0026lt;0.001)\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7186339/v1/f497a071350b736a2869b526.jpg"},{"id":89229986,"identity":"748494d7-99bb-4afc-a86f-2e8d0c55bf86","added_by":"auto","created_at":"2025-08-17 14:08:47","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":126760,"visible":true,"origin":"","legend":"\u003cp\u003eDifferent expression pattern of p53 protein in PD-L1 high lung adenocarcinoma (IHC×100). (A) Immunohistochemical staining of p53 protein shown as diffuse overexpression was interpreted as abnormal/aberrant/mutation-type expression. (B) Immunohistochemical staining of p53 protein appeared as complete absence, which requires the presence of a positive internal control with staining of normal alveolar epithelium or nonneoplastic cells such as lymphocytes, was also interpreted as abnormal/mutation-type expression. (C) and (E) Lung adenocarcinoma showing normal wild-type pattern of p53 protein expression with proportion of tumor cells staining with variable intensity. (D) and (F) Wild-type staining of p53 protein is characterized by scattered positive tumor cells mixed with negative cells\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7186339/v1/929491476e8600746d145657.jpg"},{"id":89229985,"identity":"9fef4e00-8805-45e8-a866-f3e78967279b","added_by":"auto","created_at":"2025-08-17 14:08:46","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":120686,"visible":true,"origin":"","legend":"\u003cp\u003eThe densities of CD8+ TILs and GZMB+ T cells in different subsets of PD-L1 high LuAD. (A) according to PD-L1 expression level, (B) according to expression pattern of p53 protein and (C) according to NLR level. The density of cells was expressed as average amount per high-power field (HPF) and was presented as mean ± SD. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7186339/v1/9da4068836600a29036093d4.jpg"},{"id":89229957,"identity":"ee146c59-7f9e-47a0-a705-940bc8af9b12","added_by":"auto","created_at":"2025-08-17 14:08:44","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":171531,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA \u003c/strong\u003eRepresentative examples of CD8+ TILs in different subsets of PD-L1 high LuAD (IHC×200). (a and b) based on different PD-L1 expression, (e and f) based on different NLR level and (c and d) based on distinct p53 protein expression. (a) PD-L1 TPS＜70%, (b) PD-L1 TPS≥70%, (c) normal expression of p53 protein, (d) abnormal expression of p53 protein, (e) NLR \u0026lt;5, (f) NLR≥5\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB\u003c/strong\u003e Representative examples of GZMB+ T cells in different subsets of PD-L1 high LuAD (IHC×200). (a and b) with different PD-L1 expression, (e and f) with different NLR level and (c and d) with distinct p53 protein expression. (a) PD-L1 TPS＜70%, (b) PD-L1 TPS≥70%, (c) normal expression of p53 protein, (d) abnormal expression of p53 protein, (e) NLR \u0026lt;5, (f) NLR≥5\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7186339/v1/a6fbd7f25606cf23eed1ab10.jpg"},{"id":89229977,"identity":"9acfe7c4-1472-446a-9ce1-6bb1bf55bb79","added_by":"auto","created_at":"2025-08-17 14:08:45","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":50238,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of IFN-γ protein in different subsets of PD-L1 high LuAD. The results were depicted as a continuous variable in box plot form according to PD-L1 expression level\u003cstrong\u003e \u003c/strong\u003e(A), expression pattern of p53 protein\u003cstrong\u003e \u003c/strong\u003e(B)\u003cstrong\u003e \u003c/strong\u003eas well as NLR level\u003cstrong\u003e \u003c/strong\u003e(C). The data was expressed as H-score and was presented as mean ± SD, the range of H-score was from 0 to 300. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01; and ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7186339/v1/d18ed0756c8d023ae305542a.jpg"},{"id":89231050,"identity":"1f41f33e-9415-441b-b679-6481e4e9ea47","added_by":"auto","created_at":"2025-08-17 14:16:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1364193,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7186339/v1/73d591aa-91ce-4b79-9cd3-292821f87d80.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"P53 and NLR could recognize distinct subtypes of lung adenocarcinoma with high expression of PD-L1 (TPS≥50%) defined by 22C3","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eLung cancer is the main cause of tumor-related death worldwide. The development of targeted therapeutics against driver oncogenes has already improved prognosis of non-small cell lung cancer (NSCLC) patients. However, a fair percentage of NSCLC do not harbor targetable mutations. Antitumor immunotherapy targeting immune checkpoints, such as programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1), has become a successful therapeutic modality for NSCLC without oncogene mutation\u003csup\u003e1\u003c/sup\u003e. The expression of PD-L1 on tumor cells currently is considered as an important predictive biomarker for treatment with PD-1/PD-L1 inhibitors. High expression of PD-L1, defined as tumor proportion score (TPS)\u0026thinsp;\u0026ge;\u0026thinsp;50% when detecting by 22C3 antibody, is proved to be associated with improved responses to PD-1 inhibitors in NSCLC \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Nevertheless, a substantial proportion of PD-L1 high patients do not have best responses to anti-PD-1 therapy\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Therefore, PD-L1 positivity alone cannot be considered a rock-solid predictive factor for response to PD-1/PD-L1 blockade in NSCLC\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. It is of clinical relevance to recognize the subgroup in PD-L1 high NSCLC which would be better responders to PD-1 inhibitors. Then it is essential to identify other biomarkers beyond PD-L1.\u003c/p\u003e\u003cp\u003eOur work was performed in LuAD with high expression of PD-L1. The density of CD8-positive TILs and Granzyme B (GZMB)-positive T cells as well as expression level of IFN-γ protein, that were associated with efficacy of PD-1 inhibitors, were observed significantly different among two subsets of PD-L1 high LuAD. The two distinct subsets could be recognized according to expression pattern of p53 protein and neutrophil-to-lymphocyte ratio (NLR) level. These results provided a new perspective to better understand the heterogeneity of LuAD with PD-L1 TPS\u0026thinsp;\u0026ge;\u0026thinsp;50%. Moreover, our work firstly revealed that p53 and NLR could make an easy-to-practice approach that were able to identify potential better responders to PD-1 blockers in PD-L1 high LuAD patients.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003ePatients and Samples\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e69 patients included in the present study underwent curative-intent surgical resection of stage I-III LuAD in the thoracic surgery department of The Second Affiliated Hospital of Anhui Medical University and The First Affiliated Hospital of USTC (University of Science and Technology of China), between 2019 and 2021. Inclusion criteria were: pathological diagnosis of primary LuAD and PD-L1 TPS\u0026ge;50% detected with 22C3 antibody, adequate formalin-fixed paraffin-embedded (FFPE) blocks with tumor tissue available for further evaluation. All patients had not received any treatment prior to the surgery. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eClinical data were retrospectively collected from medical records. Median age of 69 patients was 63 years (range: 28-79 years). The patients were composed of 52.17% male and 47.83% female. 67% of 69 patients were never-smokers. According to the 8th version of the Union for International Cancer Control (UICC) consortium TNM staging, the stages in the LuAD cohort were stage I 34.78%, stage II 33.33% and stage Ⅲ 31.89%. All tumor tissue sections were reviewed by two pathologists (ML and ZZF), and classified according to the WHO 2015 classification of LuAD\u003csup\u003e6\u003c/sup\u003e. The study was approved by the Ethics Committee of The Second Affiliated Hospital of Anhui Medical University (Ethics Code: YX2025-092). All participants provided written informed consent prior to enrolment in the study and this study was conducted in accordance with the Declaration of Helsinki.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemical staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll FFPE tumor samples were subjected to immunohistochemical staining using primary antibodies against CD8 (1:200, clone# SP16, GT211229; GeneTech Inc., Washington, DC, USA), Granzyme B (1:100, clone#\u0026nbsp;EP230, ZA-0599; ZSGB-BIO Co., Ltd., Beijing, China), IFN-\u0026gamma; (1:300, Polyclonal antibody, cat#DF6045; Affinity Biosciences, Jiangsu, China) and p53\u0026nbsp;(1:100, clone#DO-07, IR616; Dako, CA, USA).\u0026nbsp;Tissue\u0026nbsp;sections (4\u0026mu;M) were deparaffinized, rehydrated, and subjected to microwave antigen retrieval in an EDTA buffer (pH=9) for 20 min followed by cooling at room temperature. After blocking with 3% hydrogen peroxide for 10 min, the slides were washed with phosphate-buffered saline (PBS) for 15 min and incubated with primary antibodies for 12 h at 4\u0026nbsp;℃. Immunoreactivity was visualized using the DAB method (GK600705; Genetech Inc.) after the slides were incubated with horseradish peroxidase (HRP)-linked secondary antibodies. Slides stained with PBS instead of primary antibody were considered as the negative control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of EGFR, ALK and TP53 gene status\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEGFR mutations were detected using the Cobas IVD method (Cobas EGFR test, Roche Molecular systems, Inc., Branchburg, NJ, USA) and ALK rearrangement was examined by the ALK break-apart fluorescence in situ hybridization (FISH) assay. FISH was performed using a break-apart probe (Vysis LSI ALK Dual Color, break- apart rearrangement probe; Abbott Molecular, Abbott Park, IL, USA) according to the manufacturer\u0026rsquo;s instructions. FISH signals were observed using an Olympus BX61 fluorescence microscope and the results were evaluated according to previous study\u003csup\u003e[\u003c/sup\u003e\u003csup\u003e7]\u003c/sup\u003e. As for TP53 gene, exons 1 and 3-8 were sequenced using Sanger sequencing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe evaluation of immunohistochemical staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression of PD-L1\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe expression of PD-L1 in 69 patients was confirmed by two experienced pathologists (ML and ZZF). PD-L1 expression was determined as the percentage of tumor cells with positive membranous staining using the 22C3.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDensities of CD8 and Granzyme B-positive T cells\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo evaluate the CD8+ T cells, tissue sections were firstly viewed at a low power, and four independent areas containing the greatest abundance of CD8+ cells (termed as hot spot area) were selected. Subsequently, three views were randomly selected from each hot spot area at a higher power. CD8+ T cells in the tumor island as well as the tumor stroma adjacent to parenchyma were counted in 12 high-power fields (HPFs). The average amount from 12 high-power fields was calculated as the density of CD8+ T cells per HPF\u003csup\u003e[8]\u003c/sup\u003e.\u0026nbsp;The density of Granzyme B-positive T cells was also assessed according to the above-mentioned\u0026nbsp;method.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression of IFN-\u0026gamma; protein\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunohistochemistry was used to detect IFN-\u0026gamma; protein in previous study and H-score was used to evaluate its expression level in tumors\u003csup\u003e[9]\u003c/sup\u003e.\u0026nbsp;H-score was determined by multiplying the percentage of positive cells by an intensity score.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression of p53 protein\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExpression pattern of p53 protein shown as\u0026nbsp;diffusely strong positive or complete absence were interpreted as abnormal/aberrant/mutation-type\u003csup\u003e[10,11\u003c/sup\u003e\u003csup\u003e]\u003c/sup\u003e. The normal/wild- type expression pattern of p53 protein was characterized by an admixture of negative cells with weakly as well as strongly positive cells\u003csup\u003e[10,11]\u003c/sup\u003e. The\u0026nbsp;three expression patterns of p53 protein\u0026nbsp;and their interpretation\u0026nbsp;requires the presence of a positive internal control with staining of lymphocytes, fibroblasts and other nonneoplastic cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCategorical data were compared using\u0026nbsp;c2\u0026nbsp;test or Fisher\u0026rsquo; s exact test when it was appropriate. Quantitative data were presented as means\u0026plusmn;SD and were analyzed using Student t test and ANOVA test according to data distribution. A \u003cem\u003ep\u003c/em\u003e value less than 0.05 was considered as statistically significant. All analyses were performed using IBM SPSS software program (IBM Corp., Armonk, N.Y., USA), version 26.0.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003e1. Baseline clinical and pathologic characteristics in LuAD with higher- expression (TPS\u0026ge;50%, \u0026lt;70%) and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ehighest-expression (TPS\u003c/strong\u003e\u003cstrong\u003e\u0026ge;70%\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;of PD-L1\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOf the 69 patients, 37 (53.62%) had a higher-expression of PD-L1 and 32 (46.38%) had a highest-expression of PD-L1. PD-L1 TPS\u0026ge;70% was observed more frequently in patients with older age (\u0026ge;65 years old, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05) as well as in patients without spread through air space (STAS) of tumor cells (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05). PD-L1 TPS\u0026ge;70% seemed to be more common in LuAD with histological subtype of solid, papillary or micropapillary comparing with lepidic and acinar subtypes. 58 patients (84.06%) had no classic driver oncogene mutations, including EGFR and ALK. Of the 11 cases harboring oncogene mutations, ALK fusion was more frequently observed than EGFR mutation (11.59% versus 4.35%), but the difference was not statistically significant. Oncogene status was found no significant difference among LuAD with higher and highest-expression of PD-L1. The data was shown in Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003cimg width=\"553\" height=\"532\" src=\"data:image/png;base64,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\" alt=\"image\"\u003e\u003c/p\u003e\n\u003cp\u003eSTAS: spread through air space; *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Expression of p53 protein and TP53 gene aberrations in LuAD with higher and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ehighest-expression of PD-L1\u003c/strong\u003e \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe undoubted abnormal expression of p53 protein was observed in 31 patients and equivocal immunohistochemical result was founded in one case. TP53 gene mutations were detected in 31 cases by Sanger sequencing and they were all presented as mutation-type expression of p53 protein. The equivocal one was proved to be TP53 mutation using gene sequencing and it was finally defined as abnormal expression of p53 protein. Wild-type TP53 gene was detected in 37 patients who had normal expression of p53 protein. The concordance between abnormal expression of p53 protein and TP53 gene mutation was nearly 100%. Abnormal expression of p53 protein was detected in 13.51% of the 37 patients with PD-L1\u0026lt;70%. It was more frequently observed in PD-L1\u0026ge;70% LuAD, that was 84.38%, compared with PD-L1\u0026lt;70% group (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, Fig. 1 and Fig. 2). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. The densities of CD8-positive TILs and GZMB-positive T cells in LuAD with higher and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ehighest-expression of PD-L1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe average amount of CD8-positive TILs was 24\u0026plusmn;15/ HPF (range: 0\u0026ndash;74/ HPF) in PD-L1 TPS\u0026lt;70% group. It was 57\u0026plusmn;19/HPF (range: 0-80/HPF) in PD-L1 TPS\u0026ge;70% patients. The significant difference of CD8+ TILs was observed among TPS\u0026lt;70% and TPS\u0026ge;70% LuAD (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001,\u0026nbsp;Fig. 3A, Fig. 4A-a and b). The average amount of GZMB-positive T cells was 18\u0026plusmn;26/HPF (range: 0-117/HPF) in TPS\u0026lt;70% group and it was 52\u0026plusmn;44/HPF (range: 0-148/HPF) in TPS\u0026ge;70% group. LuAD with PD-L1 TPS\u0026ge;70% had significantly more GZMB-positive T cells than TPS\u0026lt;70% (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, Fig. 3A, Fig. 4B-a and b). \u0026nbsp;\u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. The densities of CD8-positive TILs and GZMB-positive T cells in PD-L1 high LuAD with or without mutation-type expression of p53 protein\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe average density of CD8-positive TILs was 24\u0026plusmn;14/HPF (range: 0-65/HPF) in LuAD with normal expression of p53 protein. It was 57\u0026plusmn;19/HPF (range: 0-80/HPF) in LuAD with mutation-type expression of p53 protein. It was found that the density of CD8+ TILs was significantly higher in p53 abnormal expression comparing with normal expression group (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, Fig. 3B, Fig. 4A-c and d). The average density of GZMB+ T cells was 60\u0026plusmn;43/HPF (range: 0-148/HPF) in LuAD with abnormal expression of p53 protein and it was 11\u0026plusmn;14/HPF (range: 0-66/HPF) in normal expression group. The significant difference of GZMB+ T cell densities was observed among two subsets according to p53 protein expression (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, Fig. 3B, Fig. 4B-c and d).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5. The densities of CD8-positive TILs and GZMB-positive T cells in PD-L1 high LuAD with NLR\u0026lt;5 or NLR\u0026ge;5\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe average density of CD8+ TILs was 51\u0026plusmn;23/HPF (range: 0-80/HPF) in LuAD with NLR\u0026lt;5 and it was 27\u0026plusmn;16/HPF (range: 1-74/HPF) in LuAD with NLR\u0026ge;5. The density of CD8+ TILs was significantly higher in NLR\u0026lt;5 than NLR\u0026ge;5 subset (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001,\u0026nbsp;Fig. 3C, Fig. 4A-e and f). The average amount of GZMB+ T cells was 45\u0026plusmn;41/HPF (range: 0-148/HPF) in NLR\u0026lt;5 patients and it was 22\u0026plusmn;34/HPF (range: 0-133/HPF) in NLR\u0026ge;5 cases. PD-L1 high LuAD had significantly more GZMB+ T cells in NLR\u0026lt;5 comparing with NLR\u0026ge;5 subgroup (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, Fig. 3C, Fig. 4B-e and f). \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6. The expression of IFN-\u0026gamma; protein in PD-L1 high LuAD according to PD-L1 TPS level, p53 protein expression and NLR level\u0026nbsp;\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIFN-\u0026gamma; protein was reliably detected in tumor cells of LuAD. The average H-score of IFN-\u0026gamma; protein was 68\u0026plusmn;80 (range: 0-270) in PD-L1 TPS\u0026lt;70% subgroup and it was 137\u0026plusmn;85 (range: 0-265) in TPS\u0026ge;70% patients. The expression level of IFN-\u0026gamma; protein was significantly higher in TPS\u0026ge;70% comparing with TPS\u0026lt;70% subsets (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01,\u0026nbsp;Fig. 5). The average H-score was 68\u0026plusmn;55 (range: 0-270) in patients with mutation-type expression of p53 protein. However, it was just 11\u0026plusmn;14 (range: 0-66) in patients with normal expression of p53 protein. The expression of IFN-\u0026gamma; was significantly higher in p53 protein abnormal expression comparing with normal expression subsets (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, Fig. 5). The average score was 121\u0026plusmn;93 (range: 2-270) in patients with NLR\u0026lt;5. The expressive score of IFN-\u0026gamma; in NLR\u0026lt;5 was significantly higher than that in patients with NLR\u0026ge;5, which was 78\u0026plusmn;80 (range: 0-263) (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, Fig. 5).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003ePD-L1 TPS\u0026thinsp;\u0026ge;\u0026thinsp;50% is not more frequently observed in primary LuAD\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. According to clinical practice, PD-L1 expression is not an entirely reliable predictive marker for the efficacy of immunotherapy because the response rate to PD-1 inhibitors in advanced NSCLC with strongly positive PD-L1 expression was only 44.8% based on KEYNOTE-024 and other studies\u003csup\u003e[1,4,5[\u003c/sup\u003e. Previous studies including patients with heterogeneous histological subtype and different PD-L1 expression level had explored some other biomarkers beyond PD-L1. The 69 patients in our study were all surgically resected LuAD with high expression of PD-L1. Our work firstly shown that p53 and NLR could recognize subsets of PD-L1 high LuAD which would have different responses to PD-1 inhibitors. Then our data may underlie clinical observation that not all PD-L1 high LuAD had robust response to PD-1 inhibitors\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThere had limited studies shown that PD-L1 high LuAD could be further classified into PD-L1 higher and highest subset, but cut-off values were not consistent\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. It was used TPS 70% as threshold in our work according to Frost\u0026rsquo;s study\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Highest expression of PD-L1 (TPS\u0026thinsp;\u0026ge;\u0026thinsp;70%) was more frequently observed in patients with older age than higher patients (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In addition, PD-L1 TPS\u0026thinsp;\u0026ge;\u0026thinsp;70% was more often founded in tumors without STAS (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These results suggested that patients with TPS\u0026thinsp;\u0026ge;\u0026thinsp;70% or TPS\u0026thinsp;\u0026lt;\u0026thinsp;70% could be regarded as distinct subsets in PD-L1 high LuAD according to clinicopathological characteristics.\u003c/p\u003e\u003cp\u003eA growing body of evidences supported that response to PD-1 inhibitors predominantly in NSCLC with a brisk tumor immune microenvironment, such as high density of CD8-positive TILs\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Moreover, anti-tumor effector T cells were termed as granzyme B (GZMB)-positive T cells and they were reliable predictor of better response to anti-PD-1 blockade\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Our data indicated that the density of CD8\u0026thinsp;+\u0026thinsp;and GZMB\u0026thinsp;+\u0026thinsp;T cells were significantly higher in PD-L1\u0026thinsp;\u0026ge;\u0026thinsp;70% compared with PD-L1\u0026thinsp;\u0026lt;\u0026thinsp;70% tumors. Then it was concluded that PD-L1\u0026thinsp;\u0026ge;\u0026thinsp;70% and PD-L1\u0026thinsp;\u0026lt;\u0026thinsp;70% LuAD could be considered as different subsets based on tumor immune microenvironment. The present result also shown that PD-L1\u0026thinsp;\u0026ge;\u0026thinsp;70% LuAD would likely be better responders to PD-1 inhibitors comparing with PD-L1\u0026thinsp;\u0026lt;\u0026thinsp;70% patients.\u003c/p\u003e\u003cp\u003eTP53 is the most common mutated gene in NSCLC\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Expression pattern of p53 protein has been approved to be an accurate surrogate reflecting the underlying TP53 mutation status in human malignancies\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. The concordance between p53 protein expression and TP53 gene status in our work was nearly 100%. It was shown that mutation-type expression of p53 protein was more frequently observed in PD-L1\u0026thinsp;\u0026ge;\u0026thinsp;70% than PD-L1\u0026thinsp;\u0026lt;\u0026thinsp;70% LuAD (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In addition, it was suggested that there had no significant difference of other driver oncogenes mutation among PD-L1\u0026thinsp;\u0026ge;\u0026thinsp;70% and PD-L1\u0026thinsp;\u0026lt;\u0026thinsp;70% patients, including EGFR and ALK. Taken together, it further supported that LuAD with TPS\u0026thinsp;\u0026ge;\u0026thinsp;70% or TPS\u0026thinsp;\u0026lt;\u0026thinsp;70% could be regarded as different subsets when according to expression pattern of p53 protein.\u003c/p\u003e\u003cp\u003eThe strong association has already been revealed among high density of CD8\u0026thinsp;+\u0026thinsp;TIL and TP53 mutation in NSCLC\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Biton\u0026rsquo;s work found that GZMB-positive T cells were more abundant in TP53-mutated LuAD comparing with wild type group. However, the abundance of GZMB-positive T cells was indirectly evaluated by detection of GZMB gene expression in tumor tissues\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. It was more accurate to consider the density of GZMB-positive cells as a better predictor associated with immune cell cytotoxicity. In addition, it was not evaluated in subsets with high expression of PD-L1 in Biton\u0026rsquo;s study. Our work was performed just in PD-L1 high LuAD patients. It was shown that both CD8\u0026thinsp;+\u0026thinsp;T cells and effector T cells were more robust in PD-L1 high LuAD with mutation-type expression of p53 protein, when comparing to patients with p53 normal expression. Then it was suggested that expression of p53 protein could recognize two subsets with different immune cell cytotoxicity in PD-L1 high LuAD. As an easily evaluated biomarker in routine clinical practice, expression of p53 protein would be an additional signal together with PD-L1 to predict efficacy of PD-1 inhibitors.\u003c/p\u003e\u003cp\u003eThe neutrophil-to-lymphocyte ratio (NLR) is regarded as a surrogate for tumor-associated inflammation and previous result suggested NLR likely represents the activity of myeloid-derived suppressor cells (MDSCs), that suppress T-cell proliferation and expansion\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. There has study indicated that more accumulation of neutrophils would suppress T-cell activity in lung cancer\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Then it was concluded that the baseline NLR level would be a potential predictor related to efficacy of PD-1 inhibitors. Banna\u0026rsquo;s work suggested that the appropriate NLR cut-off for advanced NSCLC treated with PD-1 inhibitors was 5\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. It was shown that advanced PD-L1 high NSCLC with NLR\u0026thinsp;\u0026lt;\u0026thinsp;5 would exhibit favorable outcome following pembrolizumab treatment\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e, but not underlie the clinical observation. Higher density of CD8\u0026thinsp;+\u0026thinsp;and GZMB-positive T cells was observed in PD-L1 high LuAD with NLR\u0026thinsp;\u0026lt;\u0026thinsp;5. Then it was concluded that the NLR\u0026thinsp;\u0026lt;\u0026thinsp;5 subset would likely be better responders to PD-1 inhibitors thanks to brisk microenvironment. Although the exact predictive value of NLR was not clearly clarified, our work firstly supported NLR could recognize distinct subgroup with different immune microenvironment in PD-L1 high LuAD.\u003c/p\u003e\u003cp\u003ePrevious studies detected expression of either the gene encoding IFN-γ (IFNG) or IFN-γ downstream genes in NSCLC as a surrogate for the abundance of IFN-γ protein\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. It was shown that NSCLC with high expression of IFNG mRNA would have more benefit from nivolumab compared to low group\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. In addition, deficiency of IFN-γ signaling in tumor cells could be served as a mechanism of resistance to PD-1 inhibitors\u003csup\u003e[\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Expression of IFN-γ protein had been detected by immunohistochemistry in melanoma\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Our work also evaluated expression of IFN-γ protein by immunohistochemistry in PD-L1 high LuAD. The result indicated that increased expression of IFN-γ protein was more frequently observed in LuAD with PD-L1 TPS\u0026thinsp;\u0026ge;\u0026thinsp;70%, NLR\u0026thinsp;\u0026lt;\u0026thinsp;5 and abnormal expression of p53 protein (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). It further suggested that p53 and NLR together with PD-L1 could recognize potential better responders to PD-1 inhibitors in PD-L1 high LuAD owing to robust expression of IFN-γ protein.\u003c/p\u003e\u003cp\u003eCurrently there had limited studies involving subclassification of LuAD with PD-L1 TPS\u0026thinsp;\u0026ge;\u0026thinsp;50% and exploring its clinical relevance. To the best of our knowledge, the present work firstly raised the possibility that distinct subsets could be recognized in PD-L1 high LuAD according to p53 and NLR. PD-L1 high LuAD with highest-expression of PD-L1, p53 protein aberrant expression or NLR\u0026thinsp;\u0026lt;\u0026thinsp;5 might be potential better responders to PD-1 inhibitors. Our work provided practical biomarkers to help making subclassification of PD-L1 high LuAD and their validation will be performed in next prospective studies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudy concept/design: \u003cstrong\u003eML, ZZF\u003c/strong\u003e; data collection: \u003cstrong\u003eMQH, ML, XLZ, HBW\u003c/strong\u003e; data analysis and interpretation:\u003cstrong\u003e\u0026nbsp;MQH, YCH\u003c/strong\u003e; \u0026nbsp;review and final approval of manuscript:\u003cstrong\u003e\u0026nbsp;ZZF, HBW, MQH, ML\u003c/strong\u003e. \u003cstrong\u003eML and MQH contributed equally to this article\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical considerations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Ethics Committee (EC) of The Second Affiliated Hospital of Anhui Medical University (Ethics Code: YX2025-092). The EC at The Second Hospital of Anhui Medical University acted as the central EC, whose review was accepted by the other participating institutions\u0026rsquo; EC. Informed consent to use histopathological samples for research purposes was obtained from all patients prior to surgery. This study was conducted in accordance with the Declaration of Helsinki.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the Ethics Committee (EC) of The Second Affiliated Hospital of Anhui Medical University (Ethics Code: YX2025-092). All participants provided written informed consent prior to participating. Written informed consent was obtained from a legally authorized representative for anonymised patient information to be published in this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll participants provided written informed consent for publication of this article. All authors of the manuscript have read and agreed to its content and are accountable for all aspects of the accuracy and integrity of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of conflicting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Natural Science Foundation Incubation Program of The Second Affiliated Hospital of Anhui Medical University [grant number 2021GMFY03].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was conducted in accordance with applicable regulations. Requests concerning the data supporting the findings of this study can be directed to [email protected] for consideration.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eReck M, Rodr\u0026iacute;guez-Abreu D, Robinson AG et al. Pembrolizumab versus chemotherapy for PD-L1-positive non-small-cell lung cancer. N Engl J Med 2016; 375(19): 1823-33.\u003c/li\u003e\n\u003cli\u003eMok TSK, Wu YL, Kudaba I et al. Pembrolizumab versus chemotherapy for previously untreated, PD-L1-expressing, locally advanced or metastatic non-small-cell lung cancer (KEYNOTE-042): a randomised, open-label, controlled, phase 3 trial. Lancet 2019; 393(10183): 1819-30. \u003c/li\u003e\n\u003cli\u003eShirasawa M, Yoshida T, Shimoda Y, et al. Differential immune-related microenvironment determines programmed cell death protein-1/programmed death-ligand 1 blockade efficacy in patients with advanced NSCLC. J Thorac Oncol 2021; 16 (12): 2078-90. \u003c/li\u003e\n\u003cli\u003eSatouchi M, Nosaki K, Takahashi T, et al. First-line pembrolizumab vs chemotherapy in metastatic non-small-cell lung cancer: KEYNOTE-024 Japan subset. Cancer Sci 2021; 112(12):5000-10. \u003c/li\u003e\n\u003cli\u003eReck M, Rodr\u0026iacute;guez-Abreu D, Robinson AG, et al. Updated analysis of KEYNOTE-024:pembrolizumab versus platinum-based chemotherapy for advanced non-small-cell lung cancer with PD-L1 tumor proportion score of 50% or greater. J Clin Oncol 2019; 37(7):537-46.\u003c/li\u003e\n\u003cli\u003eTravis WD, Brambilla E, Nicholson AG, et al. The 2015 World Health Organization Classification of Lung Tumors: Impact of Genetic, Clinical and Radiologic Advances Since the 2004 Classification. J Thorac Oncol 2015; 10(9): 1243-60.\u003c/li\u003e\n\u003cli\u003eLee J, Park CK, Yoon HK, et al. PD-L1 expression in ROS1-rearranged non-small cell lung cancer: A study using simultaneous genotypic screening of EGFR, ALK, and ROS1. Thorac Cancer 2019; 10(1): 103-10. \u003c/li\u003e\n\u003cli\u003eZeng C, Gao Y, Xiong J, et al. Tumor-infiltrating CD8+ T cells in ALK-positive lung cancer are functionally impaired despite the absence of PD-L1 on tumor cells. Lung Cancer 2020; 150: 139-44. \u003c/li\u003e\n\u003cli\u003eLiang X, Yin Y, Li N. GOLM1 is related to the inflammatory/immune nature of uveal melanoma and acts as a promising indicator for prognosis and immunotherapy response. Front Genet 2022; 13: 1051168. \u003c/li\u003e\n\u003cli\u003eK\u0026ouml;bel M, Reuss A, du Bois A, et al. The biological and clinical value of p53 expression in pelvic high-grade serous carcinomas. J Pathol 2010; 222(2): 191-98. \u003c/li\u003e\n\u003cli\u003eMcCluggage WG, Soslow RA, Gilks CB. Patterns of p53 immunoreactivity in endometrial carcinomas: \u0026apos;all or nothing\u0026apos; staining is of importance. Histopathology 2011; 59(4): 786-88. \u003c/li\u003e\n\u003cli\u003eSchoenfeld AJ, Rizvi H, Bandlamudi C, et al. Clinical and molecular correlates of PD-L1 expression in patients with lung adenocarcinomas. Ann Oncol 2020; 31(5): 599-608.\u003c/li\u003e\n\u003cli\u003eBanna GL, Signorelli D, Metro G, et al. Neutrophil-to-lymphocyte ratio in combination with PD-L1 or lactate dehydrogenase as biomarkers for high PD-L1 non-small cell lung cancer treated with first-line pembrolizumab. Transl Lung Cancer Res 2020; 9(4): 1533-42. \u003c/li\u003e\n\u003cli\u003eFrost N, Kollmeier J, Vollbrecht C, et al. KRAS\u003csup\u003eG12C\u003c/sup\u003e/TP53 co-mutations identify long-term responders to first line palliative treatment with pembrolizumab monotherapy in PD-L1 high (\u0026ge;50%) lung adenocarcinoma. Transl Lung Cancer Res 2021; 10(2): 737-52. \u003c/li\u003e\n\u003cli\u003eTumeh PC, Harview CL, Yearley JH, et al. PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature 2014; 515(7528): 568-71. \u003c/li\u003e\n\u003cli\u003eFumet JD, Richard C, Ledys F, et al. Prognostic and predictive role of CD8 and PD-L1 determination in lung tumor tissue of patients under anti-PD-1 therapy. Br J Cancer 2018; 119(8): 950-60.\u003c/li\u003e\n\u003cli\u003ePeters S, Gettinger S, Johnson ML, et al. Phase II trial of atezolizumab as first-line or subsequent therapy for patients with programmed death ligand 1-selected advanced non-small-cell lung cancer (BIRCH). J Clin Oncol 2017; 35 (24): 2781-89. \u003c/li\u003e\n\u003cli\u003eG Berke. Unlocking the secrets of CTL and NK cells. Immunol Today 1995; 16(7): 343-46. \u003c/li\u003e\n\u003cli\u003eAlbitar M, Sudarsanam S, Ma W, et al. Correlation of MET gene amplification and TP53 mutation with PD-L1 expression in non-small cell lung cancer. Oncotarget 2018; 9(17): 13682-93. \u003c/li\u003e\n\u003cli\u003eSerra P, Petat A, Maury JM, et al. Programmed cell death-ligand 1 (PD-L1) expression is associated with RAS/TP53 mutations in lung adenocarcinoma. Lung Cancer 2018; 118: 62-68. \u003c/li\u003e\n\u003cli\u003eNana Matsumoto, Padmini Manrai, Douglas Rottmann, et al. Correlative assessment of p53 immunostaining patterns and TP53 mutation status by next-generation sequencing in high-grade endometrial carcinomas. Int J Gynecol Pathol 2023; 42(6): 567-75.\u003c/li\u003e\n\u003cli\u003eBiton J, Mansuet-Lupo A, P\u0026eacute;cuchet N, et al. TP53, STK11, and EGFR mutations predict tumor immune profile and the response to Anti-PD-1 in lung adenocarcinoma. Clin Cancer Res 2018; 24(22): 5710-23. \u003c/li\u003e\n\u003cli\u003eTavakkoli M, Wilkins CR, Mones JV, et al. A Novel Paradigm Between Leukocytosis, G-CSF Secretion, Neutrophil-to-Lymphocyte Ratio, Myeloid-Derived Suppressor Cells, and Prognosis in Non-small Cell Lung Cancer. Front Oncol 2019; 9: 295.\u003c/li\u003e\n\u003cli\u003eKoyama S, Akbay EA, Li YY, et al. STK11/LKB1 deficiency promotes neutrophil recruitment and proinflammatory cytokine production to suppress T-cell activity in the lung tumor microenvironment. Cancer Res 2016; 76(5):999-1008. \u003c/li\u003e\n\u003cli\u003eKarachaliou N, Gonzalez-Cao M, Crespo G, et al. Interferon gamma, an important marker of response to immune checkpoint blockade in non-small cell lung cancer and melanoma patients. Ther Adv Med Oncol 2018; 10: 1758834017749748. \u003c/li\u003e\n\u003cli\u003eQian J, Wang C, Wang B, et al. The IFN-\u0026gamma;/PD-L1 axis between T cells and tumor microenvironment: hints for glioma anti-PD-1/PD-L1 therapy. J Neuroinflammation 2018; 15(1): 290. \u003c/li\u003e\n\u003cli\u003eManguso RT, Pope HW, Zimmer MD, et al. In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target. Nature 2017; 547 (7664): 413-18. \u003c/li\u003e\n\u003cli\u003eGao JJ, Shi LZ, Zhao H, et al. Loss of IFN-gamma pathway genes in tumor cells as a mechanism of resistance to anti-CTLA-4 therapy. Cell 2016; 167(2):397-404.\u003c/li\u003e\n\u003cli\u003eZaretsky JM, Garcia-Diaz A, Shin DS, et al. Mutations associated with acquired resistance to PD-1 blockade in melanoma. N Engl J Med 2016; 375(9):819-29.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"dion","sideBox":"Learn more about [Discover Oncology](https://www.springer.com/12672)","snPcode":"","submissionUrl":"","title":"Discover Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Lung adenocarcinoma, PD-L1, p53, NLR","lastPublishedDoi":"10.21203/rs.3.rs-7186339/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7186339/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eLung adenocarcinoma (LuAD) with high expression of PD-L1 (TPS≥50%) is a distinct subset of NSCLC and a potential candidate for immunotherapy. Improved understanding of the heterogeneity of PD-L1 high LuAD and recognizing novel biomarkers combined with PD-L1 would facilitate more precise use of PD-1 inhibitors in clinical practice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eSurgically resected tumor tissues from 69 LuAD patients with PD-L1 TPS≥50% defined by 22C3 were tested for p53 and IFN-γ protein expression using immunohistochemistry. The densities of CD8 and Granzyme B-positive T cells were evaluated by immunohistochemistry. EGFR mutation was detected by RT-PCR, ALK rearrangement was examined by fluorescence in situ hybridization (FISH) assay and TP53 gene mutation was sequenced using Sanger sequencing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eThe differences of clinicopathological features, including age and spread through air space (STAS), were observed among PD-L1 higher-expression (TPS≥50%，\u0026lt;70%) and highest-expression (TPS≥70%) patients. LuAD with PD-L1 TPS≥70%,\u003c/p\u003e\n\u003cp\u003eaberrant expression of p53 protein or NLR\u0026lt;5 had more expression of IFN-γ protein and higher densities of CD8-positive tumor infiltrating lymphocytes (TILs) as well as Granzyme B -positive T cells comparing with TPS＜70%, p53 protein normal expression or NLR≥5 patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eThe present study firstly revealed that distinct subsets could be identified in PD-L1 high LuAD according to p53 protein expression and NLR level, presenting as different characteristics of tumor microenvironment associated with response to PD-1 inhibitors.\u003c/p\u003e","manuscriptTitle":"P53 and NLR could recognize distinct subtypes of lung adenocarcinoma with high expression of PD-L1 (TPS≥50%) defined by 22C3","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-17 14:08:21","doi":"10.21203/rs.3.rs-7186339/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-24T11:52:05+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-24T03:12:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-22T08:05:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"179301272715248628246131329581176235346","date":"2025-08-17T15:29:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"240857138091420004733406383446262662893","date":"2025-08-08T12:10:13+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-08T11:59:38+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-29T06:55:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-25T02:38:34+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-25T02:36:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Oncology","date":"2025-07-22T10:58:19+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"dion","sideBox":"Learn more about [Discover Oncology](https://www.springer.com/12672)","snPcode":"","submissionUrl":"","title":"Discover Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d049c22b-84ac-4d4c-aab2-4c38e99f89e5","owner":[],"postedDate":"August 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-14T12:38:45+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-17 14:08:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7186339","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7186339","identity":"rs-7186339","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}