Blood Of Non-small Cell Lung Cancer Patients Research Articles

Immune checkpoint expression as prognostic biomarker candidates in non-small cell lung carcinoma patients.

Cancer immunotherapy has had an important role in oncologic therapeutics for patients with non-small cell lung cancer (NSCLC) using checkpoint inhibitors. We will explore the possible prognosis biomarker candidates such as: soluble OX40 (sOX40), OX40L (sOX40L), Glucocorticoid-induced tumor necrosis factor receptor family-related receptor (GITR), and their ligand (GITRL), 4-1BB or tumor necrosis factor receptor superfamily 9 (TNFRS9) and inducible T cell co-stimulator (ICOS) in peripheral blood of NSCLC patients. Fifty-eight patients were diagnosed with advanced NSCLC between January 2019 and March 2020. High sOX40 and low s4-1BB levels in smokers compared non-smoker NSCLC patients. Lower sOX40L levels were found in the male than female (p < 0.05). High sOX40 and sGITRL in stage III compared to the stage IV (p < 0.05). With follow-up at 21.4 months, 44.1% and 91.1% were alive in the sGITRhigh and sGITRlow groups, respectively (p = 0.02), and 73.3% and 27.7% were alive in the sGITRLhigh and sGITRLlow groups, respectively (p = 0.02). At 22 months, 38.7% and 92.3% were alive in the sOX40Lhigh and sOX40Llow groups, respectively (p = 0.01). sGITR, sGITRL, and sOX40L levels were potential prognostic biomarkers and could have an important role as new targets of immunotherapy in NSCLC patients. sGITR, sGITRL, sOX40L, and sOX40 levels were associated with smoking, sex, stage, and age in NSCLC.

Role of Peripheral Blood T Helper 17 Cells in Non-Small Cell Lung Cancer of Different Clinical Stages, Pathological Types and Differentiation Degrees.

We aimed to explore the roles of T helper 17 (Th17) cells and cytokines interleukin 17 (IL-17), IL-6, IL-4, transforming growth factor β (TGF-β) and γ-interferon (IFN-γ) in the peripheral blood of patients with non-small cell lung cancer (NSCLC) of different clinical stages, pathological types, and differentiation degrees. A total of 120 NSCLC patients admitted and 80 healthy people receiving physical examinations from June 2019 to October 2021 were enrolled as the case group (TNM stage: I, II, III and IV; pathological type: adenocarcinoma and squamous cell carcinoma; differentiation degree: low and moderate-high) and the control group, respectively. The serum levels of IL-17, IL-6, IL-4, TGF-β and IFN-γ were measured using an enzyme-linked immunosorbent assay. Th17 cells were counted by flow cytometry. The case group had higher levels of Th17 cells, IL-17, IL-6, IL-4, and TGF-β and lower IFN-γ level in the peripheral blood than those of the control group (p < 0.05). NSCLC patients with different TNM stages had significantly different levels of Th17 cell, IL-17, IL-6, TGF-β, and IFN-γ (p < 0.05). The expression levels of Th17 and IL-6 in patients with lowly differentiated NSCLC were lower than those of patients with moderately and highly differentiated NSCLC, while the IFN-γ expression level followed an opposite trend (p < 0.05). The count of Th17 cells in the peripheral blood of NSCLC patients had significantly positive correlations with IL-17, IL-6, IL-4, and TGF-β levels, but negative correlation with IFN-γ level (p < 0.05). The count of Th17 cells increases significantly in NSCLC patients and has correlations with TNM stage and differentiation degree.

The importance of alterations in innate lymphoid cell subsets in patients with non-small cell lung cancer and their role in tumorigenesis

Objective. Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related morbidity and mortality. Diverse functions of innate lymphoid cells (ILCs) and NK cell subsets are investigated thoroughly in cancer immunotherapy. ILC and recently described NK cell subsets in NSCLC patients’ blood samples and tumor draining lymph nodes were investigated.&#x0D; Methods. The study included chemotherapy and/or radiotherapy-naive NSCLC patients with clinical stage T1-4N0-2M0 who underwent video-assisted mediastinal lymphadenectomy and 14 healthy controls. Mononuclear cells were isolated from peripheral blood of both groups and mediastinal lymph nodes of NSCLC patients. NK cells and ILC subsets were analyzed by flow cytometry.&#x0D; Results. Total NK cells are shown to be increased in peripheral blood of NSCLC patients compared to lymph nodes while the ratio of CD56dimCD16- exhausted NK cells is higher in lymph nodes than in blood samples of NSCLC patients. Compared to control group, peripheral blood ILC1 cells were lower in NSCLC patients, however ILC2 and ILC3 cells were significantly increased. However, mediastinal lymph nodes of NSCLC patients had decreased ratio of ILC2 and increased ratio of ILC3 cells than in peripheral blood of patients. NSCLC patients had significantly increased ratio of NKp44-ILC3 cells and decreased ratio of NKp44+ILC3 in lymph nodes. &#x0D; Conclusion. Decreased ratio of ILC1 cells is an important indicator of impaired anti-tumoral response. Increased in the ratio of NKp44-ILC3 cells in NSCLC patients may potentially contribute to tumor progression. These findings highlight the distinct roles of ILCs, which play a pivotal role in the pathogenesis of lung cancer.

Sustaining the Activation of EGFR Signal by Inflammatory Cytokine IL17A Prompts Cell Proliferation and EGFR-TKI Resistance in Lung Cancer

Non-small-cell lung cancer (NSCLC) is a typical inflammation-associated cancer, and lung adenocarcinoma (LUAD) is the most common pathological subtype. Epidermal growth factor (EGF) receptor (EGFR) mutations are the most common driver mutations of LUAD, and they have been identified as important therapeutic targets by EGFR-tyrosine kinase inhibitors (TKIs). The proinflammatory cytokine, interleukin (IL)-17A, and IL-17A-producing cells were reported to be elevated in the tumor microenvironment and peripheral blood of NSCLC patients and to be correlated with tumor progression and poor prognoses. However, the pathophysiological role of IL-17A in NSCLC remains unclear, although some studies suggested its involvement in cancer cell invasion and metastasis. Herein, we observed that expressions of IL-17A and its receptor, IL-17 receptor C (IL-17RC), were elevated in LUAD tissues and were correlated with poor survival in different lung cancer cohorts. In LUAD cells with mutant EGFR, the IL-17A/IL-17RC axis was shown to enhance phosphorylation of EGFR and Met, thereby promoting proliferation and resistance to EGFR-TKIs such as afatinib. In LUAD cells with wild-type (WT) EGFR, we found that the IL-17A/IL-17RC axis enhanced EGF-induced EGFR activation and cell proliferation through causing impairment of EGF-induced EGFR lysosomal degradation. Collectively, our results indicated diverse impacts of the IL-17A/IL-17RC axis on EGFR activation in LUAD cells with WT and mutant EGFR and suggested that developing therapeutic strategies against IL-17A/IL-17RC would be valuable for LUAD treatment.

Correlation analysis of lung mucosa-colonizing bacteria with clinical features reveals metastasis-associated bacterial community structure in non-small cell lung cancer patients

BackgroundMicrobes colonizing lower airways can regulate the host immune profile and consequently participate in lung disease. Increasing evidence indicate that individual microbes promote lung cancer progression and are involved in metastasis incidence. To date, however, no study has revealed the community structure of lung bacteria in metastatic non-small cell lung cancer (NSCLC) patients.MethodsWe prospectively enrolled 50 healthy subjects and 57 NSCLC patients. All healthy subjects and NSCLC patients underwent bronchoscope procedures for brush specimen collection. The 16 S ribosomal RNA gene was sequenced to characterize the community structure of lung mucosa-colonizing bacteria. The peripheral blood of NSCLC patients was also measured for leukocytes and cancer markers.ResultsThe lung bacteria of healthy subjects and NSCLC patients were divided into four communities. All community 2 members showed increased abundance in NSCLC patients compared with healthy subjects, and most community 2 members showed increased abundance in the metastatic NSCLC patients compared with the non-metastatic group. These bacteria were significantly and positively correlated with eosinophils, neutrophils and monocytes in the metastatic NSCLC group. In addition, the correlation between lung bacteria and cancer markers differed between the metastatic and non-metastatic NSCLC patients. Furthermore, bronchoalveolar lavage fluid from lung adenocarcinoma patients directly promoted NSCLC cell migration.ConclusionsThe community structure of lung mucosa-colonizing bacteria was relatively stable, but changed from the healthy population to NSCLC patients, especially the metastatic group. This distinct community structure and specific correlation with immune cells and cancer markers could help to distinguish NSCLC patients with or without metastasis.

456 CD39 T Cells With Low PD-1 Expression are Markers for Durable Immunotherapy Response to Anti-PD-1 in Murine Glioblastoma

INTRODUCTION: Clinical trials investigating anti-PD-1 therapy for glioblastoma (GBM) have failed to demonstrate increased overall survival, unlike in other cancers such as non-small cell lung cancer (NSCLC). Despite the immunosuppressive characteristics of GBM, a small subset of patients in these trials have shown durable anti-PD-1 response. Recently, CD39+ T cells have been implicated as infiltrating lymphocytes that, once activated, demonstrate robust anti-tumor activity. Understanding the unique immunosuppressive milieu of GBM including this cell population may help elucidate why some patients show treatment response. METHODS: Mice orthotopically implanted with GL261 had blood collection prior to anti-PD-1 exposure with t-SNE mapping of T cells. The molecular profiles of mice with long-term survival were compared with those that did not. Due to a lack of GBM patient samples that have shown immunotherapy response, we examined whether these molecular patterns recapitulated in the blood of NSCLC patients responded to anti-PD-1. RESULTS: The blood of long-term survivor mice implanted with GBM demonstrated a significantly greater proportion of CD39+ CD8 T cells with low PD-1 expression (PD-1lo) with subsequent increased representation of this population in the tumor as well (P &lt; 0.05). This CD39+ CD8+ PD-1lo population was also highly represented in the blood of treatment-naÏve NSCLC patients who responded to anti-PD-1 (P &lt; 0.05). Furthermore, once exposed to anti-PD-1 in vivo, this CD39+ CD8+ PD-1lo population showed high immune activity with robust IFN-y expression (P &lt; 0.05). CONCLUSIONS: Mice with CD39+ CD8+ PD-1lo T cells are strongly associated with anti-PD-1 response. This has implications for exploring this cell population as a blood biomarker and an active anti-tumor population that may be mobilized with adoptive cell transfer and CAR-T cell strategies.

A systematic review and meta-analysis of the diagnostic value of circulating microRNA-17-5p in patients with non-small cell lung cancer.

nonSmall Cell Lung Cancer (NSCLC) is the most common type of lung cancer with high morbidity and mortality rates. Studies have shown that miR-17-5p levels are significantly increased in the circulating blood of NSCLC patients. This meta-analysis aimed to investigate the diagnostic value of miR-17-5p in NSCLC in China. A literature search was conducted for studies on the correlation between miR-25 and the diagnosis of NSCLC until October 2022 using English and Chinese databases. The Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) was adopted to evaluate the quality of studies in the literature. Numerical values for sensitivity and specificity were obtained from false negative (FN), false positive (FP), true negative (TN), and true positive (TP) rates, presented alongside graphical representations with boxes marking the values and horizontal lines showing the confidence intervals. Summary Receiver Operating Characteristic (SROC) curves were applied to assess the performance of the diagnostic tests. The data were processed using RevMan 5.3. Three studies (208 cases of NSCLC patients and 198 healthy controls) met our evaluation criteria. The sensitivity was 0.70 to 0.75, and the specificity value was 0.82 to 0.83. The Area Under the Curve (AUC) from the SROC curves was > 80%; therefore, it was classified as a good category. Our meta-analysis shows that miR-17-5p can be used for the diagnosis of NSCLC and may serve as a biomarker for the detection of early NSCLC in the Chinese population.

Peripheral Blood-Derived CircVPS35L as a Potential Diagnostic Biomarker for Non-Small Cell Lung Cancer.

Circular RNAs (circRNAs) are dysregulated in cancers and are stably expressed in body fluids such as blood. We therefore identified and evaluated the clinical value of a newly found circRNA VPS35L (circVPS35L) as a biomarker for the diagnosis of non-small cell lung cancer (NSCLC). Reverse-transcription quantitative PCR (RT-qPCR) was used to determine the expression levels of circVPS35L in tissues, whole blood, and cell lines. The actinomycin D assay and RNase R treatment were utilized to determine the stability of circVPS35L. Receiver operating characteristic (ROC) curve analysis was conducted to predict the diagnostic value of blood-derived circVPS35L in NSCLC. CircVPS35L was found to be downregulated in NSCLC tissues and cell lines. Interestingly, circVPS35L expression was significantly correlated with tumor size (p = 0.0269), histology type (p &lt; 0.0001), and TNM stage (p = 0.0437). Importantly, circVPS35L was poorly expressed in peripheral blood of NSCLC patients when compared with healthy controls and patients with benign lung disease. ROC analysis revealed a higher diagnostic value of circVPS35L than the three conventional tumor markers (CYFR21-1, NSE, and CEA) in patients with NSCLC. Moreover, circVPS35L was highly stable in peripheral blood when exposed to undesirable conditions. These findings demonstrate that circVPS35L has great potential as a novel biomarker for the diagnosis of NSCLC and can be used to distinguish NSCLC from benign lung disease.

Glucometer Readout for Portable Detection of Heterogeneous Circulating Tumor Cells in Lung Cancer Captured on a Dual Aptamer Functionalized Wrinkled Cellulose Hydrogel Interface.

The rarity of circulating tumor cells (CTCs) poses a great challenge to their clinical application as reliable "liquid biopsy" markers for cancer diagnosis. Meanwhile, the epithelial-mesenchymal transition (EMT) led to a reduced efficiency in capturing cells with lost or downregulated epithelial cell adhesion molecule (EpCAM) expressions. In this study, we proposed an integrated, highly efficient strategy for heterogeneous CTC capture and portable detection from the blood of non-small-cell lung cancer (NSCLC) patients. First, the cellulose wrinkled hydrogel with excellent biocompatibility and high specific area was employed as the biointerface to capture heterogeneous CTCs with an improved capture efficiency in virtue of dual targeting against epithelial and mesenchymal ones. Meanwhile, the strategy of glucometer readout was introduced for the quantification of captured CTCs on the same hydrogel interface by a detection probe, Au-G-MSN-Apt, which was fabricated via entrapping glucose into the amino group functionalized mesoporous silica nanoparticle (MSN) framework sealed by l-cysteine modified gold nanoparticles (AuNPs) and then linked with dual aptamers of EpCAM and Vimentin. The number of captured CTCs on the hydrogel could be reflected according to the portable glucose meter (PGM) readings. Moreover, it was found that the captured cells maintained a higher viability on the hydrogel and could be in situ recultured without releasing from the substrate. Finally, this integrated strategy was successfully applied to inspect the correlations between the number of heterogeneous CTCs in the blood of NSCLC patients with disease stage and whether there was distant metastasis.

Single-Cell Transcriptomics of Immune Cells Reveal Diversity and Exhaustion Signatures in Non-Small-Cell Lung Cancer.

Understanding immune cell phenotypes in the tumor microenvironment (TME) is essential for explaining and predicting progression of non-small cell lung cancer (NSCLC) and its response to immunotherapy. Here we describe the single-cell transcriptomics of CD45+ immune cells from tumors, normal tissues and blood of NSCLC patients. We identified three clusters of immune cells exerting immunosuppressive effects: CD8+ T cells with exhausted phenotype, tumor-associated macrophages (TAMs) with a pro-inflammatory M2 phenotype, and regulatory B cells (B regs) with tumor-promoting characteristics. We identified genes that may be mediating T cell phenotypes, including the transcription factors ONECUT2 and ETV4 in exhausted CD8+ T cells, TIGIT and CTL4 high expression in regulatory T cells. Our results highlight the heterogeneity of CD45+ immune cells in the TME and provide testable hypotheses about the cell types and genes that define the TME.

Abstract 271: Monitoring of CCR2 and CCR5 expression on circulating myeloid derived suppressor cells (MDSCs) in non-small cell lung cancer as a correlate of minimum residual disease

Abstract Objectives: Myeloid derived suppressor cells (MDSCs) are immature cells that aid in cancer progression and dissemination via immune system suppression. Previous work has shown that CCR 2 (C-C chemokine receptor) and CCR 5 expression on MDSCs is increased in non-small cell lung cancer (NSCLC). We hypothesized that patients with lung cancer will have detectable peripheral MDSCs with CCR2 and CCR5 expression preoperatively, that it would decrease immediately postoperatively, and then increase longitudinally if tumor recurs. Materials &amp; Methods: As part of a prospective longitudinal study, whole blood samples were obtained from patients suspected to have primary lung cancer prior to surgery. Patients were excluded if they were minors, could not provider consent, had malignancy within the past 10 years or any immunosuppressive condition. Blood samples were obtained prior to surgery or at follow-up in clinic and processed within 1 hour of acquisition. We stained samples via 2 different methods: 1) whole blood and 2) peripheral blood mononuclear cells (PBMC) extracted from Ficoll density gradient and determined that whole blood staining had superior results. Samples were analyzed via flow cytometer and gated after defining MO (monocytic)-MDSCs as CD33+HLADRlow/-CD14+ and PMN-MDSCs as CD33+HLADR-CD15+. MDSCs were reported as a percentage of live leukocytes and means were reported with T-test performed for statistical analysis. Results: A total of 18 patients were recruited with a median age of 69 years (63.8-75) and 61% (11/18) females. Adenocarcinoma was present in 16, carcinoid tumor in 1 and both adenocarcinoma and carcinoid tumor in 1 patient. Stage I and Stage II were the most common (66.7% and 22.2%, respectively). Majority of the tumors were in the right upper lobe (55.6%). There were 7 healthy controls with a median age of 29 years (28-43) and 71% females. There was a significantly increased proportion of MO-MDSCs in NSCLC patients preoperatively compared to healthy controls (11.64% versus 5.02%, p = 0.02). CCR2+CCR5+ MO-MDSCs were 0.85% in patients versus 0.06% in controls (p=0.04). No differences were noted with PMN-MDSCs. Five patients had post-operative follow up (mean 152 days) with an average decrease of 63% in MO-MDSCs, 68% in CCR2+CCR5+ MO-MDSCs and no recurrence of tumor on CT scans. Conclusion: Early results of this on-going study demonstrate the detection of circulating CCR2+CCR5+ MO-MDSCs in the preoperative whole blood of NSCLC patients compared to healthy controls. Resection of the tumor is associated with a decrease of these MO-MDSCs after treatment. We are evaluating if any increase in CCR2+CCR5+ MO-MDSC in long term will allow us to use it as an adjuvant tool along with CT monitoring as a biomarker of residual or recurrent disease. Citation Format: Hamza Khan, Anas Awan, Maria Shishikura, Carley Blevins, Kristen Rodgers, Yuping Mei, Wasay Nizam, Shun Ishiyama, Yun Chen, Richard Battafarano, Errol Bush, Stephen Broderick, Stephen Yang, Hajime Orita, Peng Huang, Ada Tam, Jinny Ha, Franck Housseau, Malcolm Brock. Monitoring of CCR2 and CCR5 expression on circulating myeloid derived suppressor cells (MDSCs) in non-small cell lung cancer as a correlate of minimum residual disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 271.

Th22 Cells/IL-22 Serves as a Protumor Regulator to Drive Poor Prognosis through the JAK-STAT3/MAPK/AKT Signaling Pathway in Non-Small-Cell Lung Cancer.

The interaction of immune cells and cytokines in the tumor microenvironment affects the development and prognosis of tumors with an unclear potential regulatory mechanism. Recent studies have elucidated the protumor role of Th22 cells and its lineage-specific cytokine IL-22 in different human cancers. The present study is aimed at investigating the biological effect of Th22 cells/IL-22 and its molecular mechanism in the pathogenesis process of non-small-cell lung cancer (NSCLC). It was initially found that Th22 cells were enriched in the peripheral blood of NSCLC patients. The level of Th22 cells in peripheral blood mononuclear cells (PBMCs) was positively correlated with the TNM stage, lymph node metastasis, and clinical tumor biomarkers. Furthermore, IL-22 not only antagonized the apoptosis inducing and cell cycle arresting effect by chemotherapy and molecular targeted drugs on NSCLC cell lines but also promoted tumor cell proliferation and tumor tissue growth. Moreover, IL-22 activated the JAK-STAT3/MAPK/AKT signaling pathway, both in vitro and in vivo. Conclusively, the present results confirm that Th22 cells/IL-22 may serve as a negative immune regulator in lung cancer.

Using biological information to analyze potential miRNA-mRNA regulatory networks in the plasma of patients with non-small cell lung cancer

BackgroundLung cancer is the most common malignant tumor, and it has a high mortality rate. However, the study of miRNA-mRNA regulatory networks in the plasma of patients with non-small cell lung cancer (NSCLC) is insufficient. Therefore, this study explored the differential expression of mRNA and miRNA in the plasma of NSCLC patients.MethodsThe Gene Expression Omnibus (GEO) database was used to download microarray datasets, and the differentially expressed miRNAs (DEMs) were analyzed. We predicted transcription factors and target genes of the DEMs by using FunRich software and the TargetScanHuman database, respectively. The Database for Annotation, Visualization, and Integrated Discovery (DAVID) was used for GO annotation and KEGG enrichment analysis of downstream target genes. We constructed protein-protein interaction (PPI) and DEM-hub gene networks using the STRING database and Cytoscape software. The GSE20189 dataset was used to screen out the key hub gene. Using The Cancer Genome Atlas (TCGA) and UALCAN databases to analyze the expression and prognosis of the key hub gene and DEMs. Then, GSE17681 and GSE137140 datasets were used to validate DEMs expression. Finally, the receiver operating characteristic (ROC) curve was used to verify the ability of the DEMs to distinguish lung cancer patients from healthy patients.ResultsFour upregulated candidate DEMs (hsa-miR199a-5p, hsa-miR-186-5p, hsa-miR-328-3p, and hsa-let-7d-3p) were screened from 3 databases, and 6 upstream transcription factors and 2253 downstream target genes were predicted. These genes were mainly enriched in cancer pathways and PI3k-Akt pathways. Among the top 30 hub genes, the expression of KLHL3 was consistent with the GSE20189 dataset. Except for let-7d-3p, the expression of other DEMs and KLHL3 in tissues were consistent with those in plasma. LUSC patients with high let-7d-3p expression had poor overall survival rates (OS). External validation demonstrated that the expression of hsa-miR-199a-5p and hsa-miR-186-5p in peripheral blood of NSCLC patients was higher than the healthy controls. The ROC curve confirmed that the DEMs could better distinguish lung cancer patients from healthy people.ConclusionThe results showed that miR-199a-5p and miR-186-5p may be noninvasive diagnostic biomarkers for NSCLC patients. MiR-199a-5p-KLHL3 may be involved in the occurrence and development of NSCLC.

Abstract P066: Prognostic and predictive value of pre-treatment T-Cell receptors (TCR) repertoire in non-small cell lung cancer (NSCLC) patients treated with single agent immunotherapy

Abstract Background: TCR repertoire plays a key role on the orchestration of the immune response. In particular, reduced pre-treatment Shannon diversity, increase clonality and increase convergence of TCRs have been suggested to reflect clonal expansion of antigen-specific T-cells in the tumor microenvironment. These are thought to be correlated with better response rate, improved progression free survival (PFS) and overall survival (OS). Here we aim to explore the above TCR repertoire features in peripheral blood of NSCLC patients (with PDL1≥50%) treated with single agent pembrolizumab in the first line setting; and correlate them with overall response rate (ORR), PFS and OS. Methods: We prospectively collected baseline blood from 48 NSCLC patients treated with first line pembrolizumab. High quality DNA was extracted from white blood cells and used for TCR sequencing using the Oncomine TCR Beta-SR Assay (Thermo Fisher). TCR clonality and convergence were calculated for each individual and correlated with survival using Kaplan-Meier curves and survival statistics. Multivariate analysis was carried out controlling for other variable that may influence the association of TCR repertoire and outcomes such as age, sex, ECOG, smoking status and pre-treatment neutrophil to lymphocyte ratio (NLR). Results: Our data matured for 29 patients only with a follow-up of at least 6 months. We observed a trend towards increased pre-treatment TCR clonality in patients with objective response to pembrolizumab and statistically significant reduced Shannon diversity (P = 0.042). Convergence did not seem to affect ORR in our cohort. Moreover, there was a significantly longer PFS in patients with reduced number of pre-treatment clones (HR = 0.54, 95%CI 0.21-1.43, P = 0.037), reduced Shannon diversity (HR = 0.52, 95%CI 0.20-1.38, P = 0.047), reduced Evenness (HR = 0.41, 95%CI 0.14-1.19, P = 0.044) and elevated clonality (HR = 2.45, 95%CI 0.84-7.11, P = 0.044). Reduced rather than increased convergence was correlated with a trend towards improved PFS. None of these parameters were statically significant in relation to OS. Conclusions: Increased pre-treatment TCR clonality and reduced diversity are associated with improved ORR and PFS, but not OS in NSCLC patients with high PD-L1 treated with pembrolizumab monotherapy. Further maturation of this cohort will demonstrate whether the circulating pre-treatment TCR repertoire is a prognostic factor for immunecheckpoint inhibition. Citation Format: Afaf Abed, Leslie Calapre, Samantha Bowyer, Michael Millward, Elin Gray. Prognostic and predictive value of pre-treatment T-Cell receptors (TCR) repertoire in non-small cell lung cancer (NSCLC) patients treated with single agent immunotherapy [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P066.

Effects of Chemotherapy on Peripheral Blood NK Cell Receptor NKG2D and Related Immune Cytokines in Patients with Non-Small Cell Lung Cancer

Objective: To analyze the effect of chemotherapy on peripheral blood NK cell receptor NKG2D and related immune cytokines (IL-12, IL-15, IL-18) in patients with non-small cell lung cancer (NSCLC). Methods: A total of 48 patients with NSCLC who visited the Oncology Department of the Affiliated Hospital of Chengde Medical College from September 2018 to September 2019 were selected as the study subjects. Changes in the expression levels of NKG2D, IL-12, IL-15 and IL-18 in peripheral blood of patients at different time points (before chemotherapy, after the first chemotherapy and after the second chemotherapy) were analyzed to investigate the correlation between NKG2D and IL-12, IL-15 and IL-18 in peripheral blood at each time point. Results: The expression levels of NKG2D, IL-15, and IL-18 in the peripheral blood of the patient before chemotherapy, after the first chemotherapy, and after the second chemotherapy gradually decreased. After the first chemotherapy and the second chemotherapy, the peripheral blood IL-12 was significantly lower than before chemotherapy, and IL-12 in peripheral blood after the second chemotherapy was slightly increased compared with that after the first chemotherapy. The comparison of each factor at different time points was statistically significant (all P0.05). Pearson correlation analysis showed that after the first chemotherapy, NKG2D in peripheral blood was positively correlated with IL-18 (r = 0.342, P = 0.031); after the second chemotherapy, NKG2D in peripheral blood was positively correlated with IL-18 (r = 0.411, P = 0.023), negatively correlated with IL-15 (r = -0.451, P = 0.001). Conclusion: There was no significant change in the number of NK cells in the peripheral blood of NSCLC patients after chemotherapy, while NKG2D and related immune cytokines decreased, which may be one of the mechanisms for the suppression of immune function in patients, and this provides a potential target for immunotherapy in patients.

Study on the Changes and Correlation of Related Immune Factors before and after Chemotherapy in Non-Small Cell Lung Cancer

Objective: To investigate the changes of related immune cytokines (Dendritic Cells (DC) cells, CD4+, CD8+, Th17, IgG, IgM, IgA) in patients with non-small cell lung cancer (NSCLC) before and after chemotherapy. Methods: Eighty-five NSCLC patients who were treated in the Oncology Department of the Affiliated Hospital of Chengde Medical College from December 2018 to February 2021 were selected as the research objects, and the patients were analyzed at different time points (before chemotherapy, after the first chemotherapy, and after the second chemotherapy) Changes in the expression levels of DC cells, CD4+, CD8+, Th17, IgG, IgM, IgA in peripheral blood, and explore their correlation. Results: Before chemotherapy, after the first chemotherapy, and after the second chemotherapy, the peripheral blood CD4+ and CD8+ were significantly increased, and the Th17, IgG, IgM, and IgA levels gradually decreased. The difference was statistically significant. But there was no obvious change in DC cells. Conclusion: There is no significant change in DC cells in peripheral blood of NSCLC patients before and after chemotherapy. CD4+ and CD8+ are significantly increased, Th17, IgG, IgM, and IgA levels are all decreased, which is a manifestation of impaired immune function of patients after chemotherapy.

66 Prognostic and predictive value of pre-treatment T-cell receptors (TCR) repertoire in non-small cell lung cancer (NSCLC) patients treated with single agent immunotherapy

BackgroundTCR repertoire plays a key role on the orchestration of the immune response. In particular, reduced pre-treatment Shannon diversity, increase clonality and increase convergence of TCRs have been suggested to reflect clonal expansion of antigen-specific T-cells in the tumour microenvironment. These are thought to be correlated with better response rate, improved progression free survival (PFS) and overall survival (OS). Here we aim to explore the above TCR repertoire features in peripheral blood of NSCLC patients (with PDL1≥50%) treated with single agent pembrolizumab in the first line setting; and correlate them with overall response rate (ORR), PFS and OS.MethodsWe prospectively collected baseline blood from 48 NSCLC patients treated with first line pembrolizumab. High quality DNA was extracted from white blood cells and used for TCR sequencing using the Oncomine TCR Beta-SR Assay (Thermo Fisher). TCR clonality and convergence were calculated for each individual and correlated with survival using Kaplan-Meier curves and survival statistics. Multivariate analysis was carried out controlling for other variable that may influence the association of TCR repertoire and outcomes such as age, sex, ECOG, smoking status and pre-treatment neutrophil to lymphocyte ratio (NLR).ResultsOur data matured for 29 patients only with a follow-up of at least 6 months. We observed a trend towards increased pre-treatment TCR clonality in patients with objective response to pembrolizumab and statistically significant reduced Shannon diversity (P = 0.042). Convergence did not seem to affect ORR in our cohort. Moreover, there was a significantly longer PFS in patients with reduced number of pre-treatment clones (HR = 0.40, 95%CI 0.14–1.17, P = 0.031), reduced Shannon diversity (HR = 0.44, 95%CI 0.16–1.21, P = 0.041), reduced Evenness (HR = 0.31, 95%CI 0.11–0.94, P = 0.033) and elevated clonality (HR = 3.18, 95%CI 1.06–9.53, P = 0.033) (table 1). Reduced rather than increased convergence was correlated with a trend towards improved PFS. None of these parameters were statically significant in relation to OS (table 2).Abstract 66 Table 1TCR diversity and PFSAbstract 66 Table 2TCR diversity and OSConclusionsIncreased pre-treatment TCR clonality and reduced diversity are associated with improved ORR and PFS, but not OS in NSCLC patients with high PD-L1 treated with pembrolizumab monotherapy. Further maturation of this cohort will demonstrate whether the circulating pre-treatment TCR repertoire is a prognostic factor for immunecheckpoint inhibition.Ethics ApprovalThe proposed project has already received approval by the Human Research Ethics Committees and Research Governance at Fiona Stanley Hospital, Sir Charles Gairdner Hospital and Edith Cowan University [ECU (No. 18957) and SCGH (RGS0000003289)].ConsentWritten informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.

OA12.04 Pre-Treatment T-Cell Receptors (TCR) Repertoire in Non-Small Cell Lung Cancer (NSCLC) Patients Treated With Single Agent Immunotherapy

TCR repertoire plays a key role on the orchestration of the immune response. In particular, reduced pre-treatment Shannon diversity, increase clonality and increase convergence of TCRs have been suggested to reflect clonal expansion of antigen-specific T-cells in the tumour microenvironment. These are thought to be correlated with better response rate, improved progression free survival (PFS) and overall survival (OS). Here we aim to explore the above TCR repertoire features in peripheral blood of NSCLC patients (with PDL1≥50%) treated with single agent pembrolizumab in the first line setting; and correlate them with overall response rate (ORR), PFS and OS.

P12.05 Neutrophil Elastase as Combined Immunotherapy Target for Treating NSCLC

Immunotherapy has emerged as a promising option for treating non-small cell lung cancer (NSCLC). However, immunotherapy is not equally effective for all patients. Drug resistance is also a big hurdle in treatment. Recently, cancer immune microenvironment has proved of great significance for immunotherapy. Neutrophil elastase, a protease able to degrade several extracellular matrix proteins, is encoded by ELANE gene. neutrophil elastase (NE) and neutrophil dominate airway inflammation. Sivelestat is the only approved drug working as a selective NE inhibitor. However, their direct impact on the tumor immune microenvironment is not clear. The goal of this study, therefore, was to investigate the the effects of ELANE on the NSCLC microenvironment, and the application potential of sivelestat on combination therapies to enhance the efficacy of immune checkpoint inhibitors. The relationship between ELANE and tumor-infiltrated immune cells was analyzed using TIMER. Immunohistochemistry was used to evaluate the density of, CD8+TILs, and CD163+ macrophages. NE from peripheral blood of NSCLC patients was determined by ELISA. The mouse model and flow cytometry were used to investigate the effects of sivelestat on the tumor-infiltrating immune cells and the efficacy of immune checkpoint inhibitors. ELANE was significantly positively correlated with the proportion of M2 macrophages, and negatively associated the proportion of CD8+TILs. NE levels in peripheral blood of NSCLC patients was significantly positively correlated with the density of CD163+ macrophages, and negatively associated the density of, CD8+TILs. Sivelestat, which had already been marketed as a selective NE inhibitor, could increase CD8+ infiltration and reduced M2 macrophage infiltration. It can also improve the efficacy of immune checkpoint inhibitors in the mouse model. NE levels in peripheral blood could be used as potential biomarker that may predict sensitivity to immunotherapy. Sivelestat，as a selective NE inhibitor already marketed，can potentially be harnessed in combination with immune checkpoint inhibitors to increase the efficacy of immunotherapy.

Expression and Clinical Significance of lncRNA OSER1-AS1 in Peripheral Blood of Patients with Non-Small Cell Lung Cancer

Non-small cell lung cancer (NSCLC) is a malignant cancer worldwide. Long non-coding RNAs (lncRNAs) have emerged as key players in the development and progression of NSCLC, and may be potential biomarkers of NSCLC. Here, we investigated the clinical significance of lncRNA oxidative stress responsive serine rich 1 antisense RNA 1 (OSER1-AS1) in peripheral blood of patients with NSCLC. OSER1-AS1 in peripheral blood of patients with lung squamous cell carcinoma (LUSC), lung adenocarcinoma (LUAD), and healthy subjects was detected, and the clinical diagnostic efficacy was analyzed. The correlation between OSER1-AS1 expression and clinicopathological features in patients with LUSC and LUAD was analyzed. The downstream mechanism of OSER1-AS1 was explored. The area under the ROC curve of lncRNA OSER1-AS1 and miR-1298-5p/CHSY3 in LUSC and LUAD was compared using the MedCalc analysis. OSER1-AS1 was low in peripheral blood of patients with LUSC and LUAD. The area under the ROC curve for predicting LUSC was 0.800. The area under the ROC curve for predicting LUAD was 0.728. In LUSC and LUAD, OSER1-AS1 deficiency was associated with tumor node metastasis stage, lymph node metastasis, distal metastasis, and poor prognosis. miR-1298-5p was highly expressed, while chondroitin sulfate synthase 3 (CHSY3) was lowly expressed in patients with LUSC and LUAD. miR-1298-5p had target relations with OSER1-AS1 and CHSY3. lncRNA OSER1-AS1 had a higher diagnostic value in patients with NSCLC than miR-1298-5p and CHSY3. Overall, low expression of OSER1-AS1 in peripheral blood of NSCLC patients has high clinical significance, which provides a certain reference value for NSCLC early diagnosis.