Biomarker For Non-small Cell Lung Cancer Research Articles

1162P Tissue and liquid biopsy utilization in advanced NSCLC in a large community US practice

Professional guidelines advise broad molecular profiling for at least 8 biomarkers in non-small cell lung cancer (NSCLC) that have either approved targeted therapies or available clinical trials. Up to 38% of patients with metastatic NSCLC have inadequate tumor samples for molecular analysis at diagnosis. Professional guidelines recommend that if there is insufficient tissue to allow for testing of all biomarkers, repeat biopsy and/or plasma testing should be done. While both plasma and tissue testing are currently being incorporated into the standard workup of patients with NSCLC, there is currently no standardized workflow for testing. We performed a retrospective chart review to assess real world liquid and tissue biopsy - based biomarker testing and reflex testing dynamics to understand the current NSCLC biomarker testing scenario in a large US community practice. Advanced NSCLC patients (n=361) who were diagnosed or received first-line treatment on or after 1/1/2020 were included. Data was extracted from the electronic medical records in participating practices as well as via abstraction of patient records when available. Patient-level data was anonymized and reported in aggregate. Data collected for this analysis was evaluated in accordance with regulatory requirements for electronic records. We examined patient journeys from biopsy to treatment, determined the percentage of liquid and tissue-based testing, test failure rate, reflex testing rate, and turnaround time. 43% of NSCLC patients received both liquid and tissue testing, while 41% received only tissue testing. The median TAT for tissue testing was 10 days and for liquid testing was 7 days. Reflex testing occurred in 56% of solid tumor test failures and 31% of liquid tumor tests with negative results. Patients with both a liquid and tissue test had results in hand prior to treatment more often than those who had either test alone. In this real-world study, 43% of NSCLC patients received both liquid and tissue testing. Patients with both a liquid and solid tumor test had results in hand prior to treatment more often than those who just had a solid or liquid tumor test. These results demonstrate the utilization of tissue and liquid testing in a large US community practice.

Pyroptosis-Related Gene Model Predicts Prognosis and Immune Microenvironment for Non-Small-Cell Lung Cancer.

Non-small-cell lung cancer (NSCLC) has a high incidence and mortality worldwide. Moreover, it needs more accurate means for predicting prognosis and treatments. Pyroptosis is a novel form of cell death about inflammation which was highly related to the occurrence and development of tumors. Despite having some studies about pyroptosis-related genes (PRGs) and cancer, the correlation has not been explored enough between PRGs and immune in NSCLC. In this study, we constructed a PRG model by WGCNA to access the prognosis value PRGs have. The testing cohort (n = 464) with four datasets from the GEO database conducted a survival analysis to confirm the stability of the prognostic model. The risk score and age are examined as independent prognostic factors. Based on the PRGs, we found multiple pathways enriched in immune in NSCLC. Separating samples into three subtypes by consensus cluster analysis, Cluster 3 was identified as immune-inflamed phenotype with an optimistic prognostic outcome. A three-gene PRG signature (BNIP3, CASP9, and CAPN1) was identified, and BNIP3 was identified as the core gene. Knockdown of BNIP3 significantly inhibited the growth of H358 cells and induced pyroptosis. In conclusion, the model construction based on PRGs provides novel insights into the prediction of NSCLC prognosis, and BNIP3 can serve as a diagnostic biomarker for NSCLC.

Prognostic factors for long-term survival following complete resection by lobectomy in stage I non-small cell lung cancer.

BackgroundThe aim of this study was to evaluate predictors for long‐term overall survival (OS) in patients with stage I non‐small cell lung cancer (NSCLC).MethodsAll patients undergoing complete resection by lobectomy for stage I NSCLC between October 2012 and December 2015 at a single center were included. Univariable and multivariable Cox regression analyses were performed to identify prognostic factors.ResultsA total of 92 patients were included. Univariable and multivariable Cox regression analyses revealed preoperative neutrophil to lymphocyte ratio (NLR, p = 0.005), preoperative diffusion capacity of the lungs for carbon monoxide (DLCO, p = 0.010) and forced expiratory volume in 1 second (FEV1, p = 0.041) as well as male gender (p = 0.026) as independent prognostic factors for OS. Combining the calculated cutoff values for FEV1 (<73.0%) and NLR (>3.49) into one parameter resulted in a highly significant difference in survival times when stratified by this variable.ConclusionsRecently, much emphasis has been put on the prognostic importance of blood biomarkers in NSCLC. In our study, NLR was an independent factor for OS, as were baseline characteristics such as DLCO, FEV1, and gender. Further studies on the association of biomarkers for systemic inflammation and lung function parameters with respect to patient survival are warranted.

Detection of Potential Mutated Genes Associated with Common Immunotherapy Biomarkers in Non-Small-Cell Lung Cancer Patients.

Microsatellite instability (MSI), high tumor mutation burden (TMB-H) and programmed cell death 1 ligand 1 (PD-L1) expression are hot biomarkers related to the improvement of immunotherapy response. Two cohorts of non-small-cell lung cancer (NSCLC) were collected and sequenced via targeted next-generation sequencing. Drug analysis was then performed on the shared genes using three different databases: Drugbank, DEPO and DRUGSURV. A total of 27 common genes were mutated in at least two groups of TMB-H-, MSI- and PD-L1-positive groups. AKT1, SMAD4, SCRIB and AXIN2 were severally involved in PI3K-activated, transforming growth factor beta (TGF-β)-activated, Hippo-repressed and Wnt-repressed pathways. This study provides an understanding of the mutated genes related to the immunotherapy biomarkers of NSCLC.

Assessment of plasma amino acids, purines, tricarboxylic acid cycle metabolites, and lipids levels in NSCLC patients based on LC-MS/MS quantification

Non-small cell lung cancer (NSCLC) is the most common type of malignant tumor of the lung with poor prognosis. Currently, there is still no effective strategy for diagnosing lung cancer from the perspective of multiple biomarkers containing both polar and nonpolar molecules. In order to explore the pathological changes of NSCLC at the endogenous molecule levels, and further establish the strategy for identifying and monitoring drug efficacy of NSCLC, targeted metabolomics and lipidomics studies were established with NSCLC patients. Polar metabolites including 21 amino acids, 7 purines, 6 tricarboxylic acid (TCA) cycle metabolites, and nonpolar lipids like phosphatidylcholine (PC), phosphatidylethanolamine (PE), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), sphingomyelin (SM), and ceramide (Cer), diacylglycerol (DG), triacylglycerol (TG), were quantitatively determined based on LC-MS/MS, taking into account their metabolism were significantly concerned with the occurrence of lung cancer in previous study. As a result, 14 polar metabolites and 16 lipids were prominently altered in the plasma of NSCLC patients, among which, after multivariate statistical analysis, LPC 18:0 (sn-2), L-Phenylalanine (Phe), oxaloacetic acid (OAA) and xanthine (XA) were screened out as potential small molecules and lipid biomarkers for NSCLC. Furthermore, a new strategy for formulating equation of NSCLC identification was proposed and clinical utility was successfully evaluated through Kangai injection treatment to NSCLC patients. Taking together, this study investigated the pathological changes of NSCLC from the perspective of endogenous polar and nonpolar molecules, and shed a light on identification of NSCLC.

Modelling reoxygenation effects in non-small cell lung cancer cell lines and showing epithelial-mesenchymal transition

PurposeCirculating tumour cells (CTCs) are a rare cell subpopulation regulated by the tumour microenvironment. In hypoxic conditions, CTCs are able to invade the lymphatic and circulatory systems leading to metastasis at distant sites.MethodsTo mimic in vivo oxygen variations and effects on CTCs, we have cultured five non-small cell lung cancer (NSCLC) cell lines under normoxic and hypoxic conditions, followed by a pulse of reoxygenation for 4 h.ResultsProliferation, spheroid-formation and colony formation ability under varying O2 levels were investigated. Proliferation rate was not altered when cells were cultured in 2D models under hypoxic conditions. However, we observed that hypoxia enhanced in vitro formation of tumour-spheres and accelerated clonogenicity of NSCLC cell lines. In addition, cells exposed to hypoxia and reoxygenation conditions showed altered expression of epithelial-mesenchymal transition (EMT) related genes in NSCLC cell lines both at mRNA (AKT1, CAMK2NH1, DESI1, VIM, MAP1B, EGFR, ZEB1, HIF1α) and protein levels (Vimentin, Pan-cytokeratin).ConclusionOur data suggest that when investigating CTCs as a prognostic biomarker in NSCLC, it is also essential to take into consideration EMT status to obtain a comprehensive overview of CTCs in circulation.

MiR-19b-3p promotes tumor progression of non-small cell lung cancer via downregulating HOXA9 and predicts poor prognosis in patients.

MiR-19b-3p has been reported in several types of human cancer. Nevertheless, the expression profile and biological functions of miR-19b-3p remain unclear in non-small cell lung cancer (NSCLC). The expression level of miR-19b-3p was evaluated in NSCLC tissues and cell lines using qRT-PCR. Survival analysis was performed using Kaplan-Meier curves, while the prognostic significance of miR-19b-3p was analyzed using Cox regression analysis in 80 NSCLC patients. The effects of miR-19b-3p on cell proliferation and invasion capacities were analyzed using CCK-8, crystal violet, and transwell assays. Target genes of miR-19b-3p were assessed using luciferase reporter assay, qRT-PCR, Western blot and rescue experiments. MiR-19b-3p was found to be upregulated in human NSCLC tissues and cell lines. The expression of miR-19b-3p was observed to be closely associated with TNM stage and metastasis. High expression of miR-19b-3p was found to be capable of predicting poor clinical prognosis in NSCLC patients. Whilst overexpression of miR-19b-3p was demonstrated to promote the proliferation and invasion of NSCLC cells, knockdown of miR-19b-3p showed an opposite inhibitory effect. Bioinformatics analysis and luciferase reporter assays confirmed that HOXA9 is a direct target of miR-19b-3p. Functional assays demonstrated that NSCLC cell proliferation and invasion were promoted by miR-19b-3p via negative regulation of HOXA9. Finally, overexpression of HOXA9 was shown to partially reverse the tumor promoting effect of miR-19b-3p. This study indicates that miR-19b-3p is a crucial prognostic biomarker of NSCLC, and that targeting of the miR-19b-3p/HOXA9 axis may be a promising strategy in NSCLC therapy.

Validation and analysis of expression, prognosis and immune infiltration of WNT gene family in non-small cell lung cancer.

Early diagnosis and prognosis prediction of non-small cell lung cancer (NSCLC) have been challenging. Signaling cascades involving the Wingless-type (WNT) gene family play important biological roles and show prognostic value in various cancers, including NSCLC. On this basis, this study aimed to investigate the significance of WNTs in the prognosis and tumor immunity in NSCLC by comprehensive analysis. Expression and methylation levels of WNTs were obtained from the ONCOMINE, TIMER, and UALCAN. The dataset obtained from The Cancer Genome Atlas (TCGA) was utilized for prognostic analysis. cBioPortal was used to perform genetic alterations and correlation analysis of WNTs. R software was employed for functional enrichment and pathway analysis, partial statistics, and graph drawing. TRRUST was used to find key transcription factors. GEPIA was utilized for the analysis of expression, pathological staging, etc. Correlative analysis of immune infiltrates from TIMER. TISIDB was used for further immune infiltration validation analysis. Compared with that of normal tissues, WNT2/2B/3A/4/7A/9A/9B/11 expressions decreased, while WNT3/5B/6/7B/8B/10A/10B/16 expressions increased in lung adenocarcinoma (LUAD); WNT2/3A/7A/11 expressions were lessened, while WNT2B/3/5A/5B/6/7B/10A/10B/16 expressions were enhanced in squamous cell lung cancer (LUSC). Survival analysis revealed that highly expressed WNT2B and lowly expressed WNT7A predicted better prognostic outcomes in LUAD and LUSC. In the study of immune infiltration levels, WNT2, WNT9B, and WNT10A were positively correlated with six immune cells in LUAD; WNT1, WNT2, and WNT9B were positively correlated with six immune cells in LUSC, while WNT7B was negatively correlated. Our study indicated that WNT2B and WNT7A might have prognostic value in LUAD, and both of them might be important prognostic factors in LUSC and correlated to immune cell infiltration in LUAD and LUSC to a certain extent. Considering the prognostic value of WNT2B and WNT7A in NSCLC, we validated their mRNA and protein expression levels in NSCLC by performing qRT-PCR, western blot, and immunohistochemical staining on NSCLC pathological tissues and cell lines. This study may provide some direction for the subsequent exploration of the prognostic value of the WNTs and their role as biomarkers in NSCLC.

Interleukin-11: A Potential Biomarker and Molecular Therapeutic Target in Non-Small Cell Lung Cancer

Non-small cell lung cancer (NSCLC) accounts for 85% of lung cancer and is a fast progressive disease when left untreated. Identification of potential biomarkers in NSCLC is an ongoing area of research that aims to detect, diagnose, and prognosticate patients early to optimize treatment. We review the role of interleukin-11 (IL11), a stromal-cell derived pleiotropic cytokine with profibrotic and cellular remodeling properties, as a potential biomarker in NSCLC. This review identifies the need for biomarkers in NSCLC, the potential sources of IL11, and summarizes the available information leveraging upon published literature, publicly available datasets, and online tools. We identify accumulating evidence suggesting IL11 to be a potential biomarker in NSCLC patients. Further in-depth studies into the pathophysiological effects of IL11 on stromal-tumor interaction in NSCLC are warranted and current available literature highlights the potential value of IL11 detection as a diagnostic and prognostic biomarker in NSCLC.

PD-1T TILs as a Predictive Biomarker for Clinical Benefit to PD-1 Blockade in Patients with Advanced NSCLC.

Durable clinical benefit to PD-1 blockade in non-small cell lung cancer (NSCLC) is currently limited to a small fraction of patients, underlining the need for predictive biomarkers. We recently identified a tumor-reactive tumor-infiltrating T lymphocyte (TIL) pool, termed PD-1T TILs, with predictive potential in NSCLC. Here, we examined PD-1T TILs as biomarker in NSCLC. PD-1T TILs were digitally quantified in 120 baseline samples from advanced NSCLC patients treated with PD-1 blockade. Primary outcome was disease control (DC) at 6 months. Secondary outcomes were DC at 12 months and survival. Exploratory analyses addressed the impact of lesion-specific responses, tissue sample properties, and combination with other biomarkers on the predictive value of PD-1T TILs. PD-1T TILs as a biomarker reached 77% sensitivity and 67% specificity at 6 months, and 93% and 65% at 12 months, respectively. Particularly, a patient group without clinical benefit was reliably identified, indicated by a high negative predictive value (NPV) (88% at 6 months, 98% at 12 months). High PD-1T TILs related to significantly longer progression-free (HR 0.39, 95% CI, 0.24-0.63, P < 0.0001) and overall survival (HR 0.46, 95% CI, 0.28-0.76, P < 0.01). Predictive performance was increased when lesion-specific responses and samples obtained immediately before treatment were assessed. Notably, the predictive performance of PD-1T TILs was superior to PD-L1 and tertiary lymphoid structures in the same cohort. This study established PD-1T TILs as predictive biomarker for clinical benefit to PD-1 blockade in patients with advanced NSCLC. Most importantly, the high NPV demonstrates an accurate identification of a patient group without benefit. See related commentary by Anagnostou and Luke, p. 4835.

A Cross-Comparison of High-Throughput Platforms for Circulating MicroRNA Quantification, Agreement in Risk Classification, and Biomarker Discovery in Non-Small Cell Lung Cancer.

BackgroundCirculating microRNAs (ct-miRs) are promising cancer biomarkers. This study focuses on platform comparison to assess performance variability, agreement in the assignment of a miR signature classifier (MSC), and concordance for the identification of cancer-associated miRs in plasma samples from non‐small cell lung cancer (NSCLC) patients.MethodsA plasma cohort of 10 NSCLC patients and 10 healthy donors matched for clinical features and MSC risk level was profiled for miR expression using two sequencing-based and three quantitative reverse transcription PCR (qPCR)-based platforms. Intra- and inter-platform variations were examined by correlation and concordance analysis. The MSC risk levels were compared with those estimated using a reference method. Differentially expressed ct-miRs were identified among NSCLC patients and donors, and the diagnostic value of those dysregulated in patients was assessed by receiver operating characteristic curve analysis. The downregulation of miR-150-5p was verified by qPCR. The Cancer Genome Atlas (TCGA) lung carcinoma dataset was used for validation at the tissue level.ResultsThe intra-platform reproducibility was consistent, whereas the highest values of inter-platform correlations were among qPCR-based platforms. MSC classification concordance was >80% for four platforms. The dysregulation and discriminatory power of miR-150-5p and miR-210-3p were documented. Both were significantly dysregulated also on TCGA tissue-originated profiles from lung cell carcinoma in comparison with normal samples.ConclusionOverall, our studies provide a large performance analysis between five different platforms for miR quantification, indicate the solidity of MSC classifier, and identify two noninvasive biomarkers for NSCLC.

LncRNA ELFN1-AS1 predicts poor prognosis and promotes tumor progression of non-small cell lung cancer by sponging miR-497.

More novel biomarkers need to be discovered to improve the therapeutic efficiency of non-small cell lung cancer (NSCLC). lncRNA ELFN1-AS1 (ELFN1-AS1) was proved to play crucial roles in numerous diseases, its intention in NSCLC remains unclear. This study aimed to investigate the function of ELFN1-AS1 and its potential mechanism in NSCLC development. A total of 117 NSCLC patients were recruited and provided paired NSCLC tissues and normal tissues. The expression of ELFN1-AS1 was analyzed by PCR. The biological function of ELFN1-AS1 was estimated by CCK8 and Transwell assay. Additionally, the potential mechanism underlying the function of ELFN1-AS1 was explored by the dual-luciferase reporter assay and western blotting. The significant upregulation of ELFN1-AS1 was found in NSCLC tissues and cells, which was closely associated with the TNM stage, lymph node metastasis status, and overall survival of patients. The knockdown of ELFN1-AS1 was found to inhibit the cellular processes and EMT of NSCLC. Moreover, ELFN1-AS1 was found to serve as a sponge to binding with miR-497, and CCNE1 was demonstrated to be the downstream target of miR-497, which was speculated as the potential mechanism underlying the function of ELFN1-AS1. ELFN1-AS1 acts as an independent prognostic biomarker and tumor promoter of NSCLC by sponging miR-497/CCNE1 axis.

Increased serum levels of KiSS1-derived peptides in non-small cell lung cancer patient liquid biopsies and biological relevance

BackgroundThe secreted products of the metastasis suppressor gene KiSS1 may represent useful biomarkers in non-small cell lung cancer (NSCLC) but their levels in patients have remained poorly investigated. We previously found that forced expression of KiSS1 decreased the invasive capability of NSCLC drug-resistant cells and a pro-apoptotic role for KiSS1 has been proposed in head and neck cancer. Thus, we designed a translational investigation including a pilot study to analyze KiSS1 levels in liquid biopsies, and in vitro experiments to explore the biological relevance of KiSS1 modulation.MethodsKiSS1-derived peptide levels in liquid biopsies from 60 NSCLC patients were assayed by ELISA. Preclinical experiments were carried out using quantitative real time polymerase chain reaction (qRT-PCR), ELISA, annexin V-binding and caspase activation assays.Results We compared KiSS1 release in 3 different matrices (serum, plasma and urine) and the highest levels were detectable in serum (range, 0–4.5 ng/mL). We observed increased levels of seric KiSS1 in NSCLC patients as compared to healthy donors. KiSS1 serum concentrations, after surgical procedure and/or adjuvant therapy. We observed differences among disease stages in urine samples. In preclinical models, KiSS1 mRNA levels were increased by short term exposure to azacytidine, enhanced KiSS1 release was induced by the combination of azacytidine and cisplatin and KiSS1-derived peptides enhanced cisplatin-induced apoptosis. KiSS1 increase was observed upon exposure neurons-enriched cultures to tumor cell conditioned medium.ConclusionsOur results showing a peculiar modulation of KiSS1 levels in liquid biopsies of NSCLC patients and a regulation of cisplatin-induced apoptosis by KiSS1-derived peptides support an involvement of KiSS1 in cell response to treatment and highlight its promising features as a potential biomarker in NSCLC.

Increased expression of FAT4 suppress metastasis of lung adenocarcinoma through regulating MAPK pathway and associated with immune cells infiltration.

FAT4 is an extremely large atypical cadherin with crucial roles in the control of planar cell polarity (PCP) and regulation of the Hippo signaling pathway. Our study aims to clarify the FAT4 expression patterns, as well as the significance of FAT4 in predicting the prognosis and cancer immunity to non-small cell lung cancer (NSCLC). FAT4 mRNA and protein expressions were both underregulated in NSCLC and associated with poor prognosis in both lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). In addition, overexpress FAT4 with jujuboside A (JUA) or knockdown FAT4 with siRNA regulated the metastasis of LUAD through MAPK pathways. Moreover, the FAT4 expression included multiple immunological components to promote an immunosuppressive tumor microenvironment (TME). Furthermore, a study of the TCGA-LUAD cohort's DNA methylation results showed that most FAT4 DNA CpG sites were typically hypermethylated in NSCLC relative to the normal lung tissue. The DNA CpG sites cg25879360 and cg26389756 of FAT4 were found to be strongly associated with FAT4 expression in LUAD through the correlation study. In conclusion, this is the first to report the potential function of FAT4 in NSCLC. Hence, FAT4 could be used as a promising prognostic and immunological biomarker for NSCLC.

The Relationship Between Systemic Immune Inflammatory Index and Prognosis of Patients With Non-Small Cell Lung Cancer: A Meta-Analysis and Systematic Review

BackgroundThe relationship between systemic immune inflammation index (SII) and the prognosis of cancer has always been a subject of intense interest. However, the prognostic value of SII in non-small cell lung cancer (NSCLC) patients remains a controversial topic.ObjectiveTo evaluate the effect of SII index on prognosis of NSCLC.MethodsWe conducted a comprehensive search of PubMed, EMBASE, and the Cochrane Library databases to determine correlation between SII index, clinicopathological features, overall survival (OS), and progression-free survival (PFS). Odds ratio (ORs) and 95% confidence interval (CIs) were used to assess the connection between SII and clinicopathological parameters, and HRs and 95% CIs were used to assess the connection between SII and survival.ResultsSeventeen studies with 8,877 cases were included in the analysis. Compared with NSCLC patients with low SII level, patients with NSCLC with high SII level had a poor OS (HR = 1.75, 95% CI, 1.50–2.00; P < 0.001) and had a poor PFS (HR = 1.61, 95% CI, 1.25–1.96; P < 0.001). In addition, patients with higher pathological stage (II–III) had higher SII levels (OR = 2.32, 95% CI, 2.06–2.62; P < 0.001).ConclusionsThe SII index is a promising prognostic biomarker for NSCLC and may help clinicians choose appropriate NSCLC treatments.

NMT1 Enhances the Stemness of NSCLC Cells by Activating the PI3K/AKT Pathway

Introduction: Abundant studies have disclosed that proteins can function as pivotal tumor promoters or suppressors in cancers’ progression. This work was planned to investigate the regulatory function of N-myristoyltransferase-1 (NMT1) on non-small cell lung cancer (NSCLC) and the underlying molecular mechanisms. Methods: The self-renewal abilities were assessed through a spheroid-formation assay. The tumorigenic abilities were examined through nude mice in vivo assay. The proteins’ expression was measured through Western blot. The NMT1 protein expression in tumor tissues was measured through an IHC assay. The cell migration and invasion was confirmed through a transwell assay. The IC50 was verified through a CCK-8 assay. The NMT1 mRNA expression in NSCLC tissues was detected through RT-qPCR. Results: It was demonstrated that NMT1 exhibited higher expression in spheroid cells. Additionally, NMT1 facilitated the stemness in NSCLC. It was also found that NMT1 accelerated NSCLC tumor metastasis and the resistance to cisplatin. Moreover, NMT1 activated the PI3K/AKT pathway to facilitate stemness in NSCLC. NMT1 was also higher in tumor tissues of NSCLC patients and resulted in a poor survival rate. Conclusion: NMT1 enhanced the stemness of NSCLC cells by activating the PI3K/AKT pathway. This discovery suggested that NMT1 may be a valid therapeutic biomarker for NSCLC.

Abstract 645: Heterogeneity of immunotherapy biomarkers in the TRACERx non-small cell lung cancer multi-region lung cancer cohort study

Abstract Immunotherapy containing regimens have become the standard of care first-line therapy for non-small cell lung cancer (NSCLC) in the metastatic setting and are being explored in the adjuvant and neo-adjuvant setting. However, as intratumor heterogeneity (ITH) is pervasive in cancer we investigated the impact of ITH on established and putative biomarkers predictive of immunotherapy response. We have used whole exome sequencing, RNA sequencing and PDL1 immunohistochemistry (IHC) derived from the multiregion treatment naïve TRACERx cohort to investigate this. Whole exome sequencing data was available for 432 primary tumours from 421 patients with greater than 1500 unique tumour regions. Tumour mutational burden (TMB) was calculated using best practice guidelines from the TMB harmonisation project. Using the FDA approved threshold of ≥ 10 mutations per megabase to define high TMB we found that 41% of patients with high TMB had at least one region with &amp;lt;10 mutation per megabase. This could result in misclassification of patients so we modelled increased sampling in this cohort and found that at least 5 spatially separated samples would be required to ensure no patients are misclassified. Contrary to the published literature we did not find TMB was confounded by tumour purity. Multi-region PDL1 IHC data using the FDA approved companion diagnostic assay (22c3) was available for 132 spatially separated tumour regions from 36 patients. Tumour regions were scored according to the established tumour proportion scoring system (≥50%, 49-1%, &amp;lt;1%). Using only a single sample 31% of patients would have been misclassified. Finally, we investigated several putative transcriptomic biomarkers including 10 single genes (e.g. CXCL9), 15 expression signatures (e.g cytolytic score, TIDE) and 2 tumour microenvironment classification systems. We found that 20-45% of these putative biomarkers were discordant when applied to multi-region data and this increased with the number of tumour regions per patients but was not impacted by tumour purity or sequencing depth. To investigate this further we compared the mean geneset RNA-ITH score between a geneset of all the immune biomarker genes with 100 randomly selected non-immune, but expressed, genesets. The immune biomarker gene set was subject to significantly more intra tumour heterogeneity of expression (Mean RNA-ITH Score 0.50 vs 0.31 p=6.4e-12). In conclusion we demonstrate significant heterogeneity of immune biomarkers in NSCLC and in particular a lack of robustness of expression-based predictors of response to immunotherapy. This highlights the need for biomarkers that are robust to intra-tumour heterogeneity and sampling bias or the need for more representative tumour sampling techniques. Citation Format: Crispin T. Hiley, Kevin Litchfield, Oriol Pich, David Moore, Cristina Naceur-Lombardelli, Selvaraju Veeriah, Maise Al Bakir, Simranpreet Summan, Kristiana Grigoriadis, Carlos Martinez Ruiz, Clare Puttick, Katey Enfield, Sophia Ward, Alexander Frankell, Dhruva Biswas, Rachel Rosenthal, Nicolai J. Birkbak, Mariam Jamal-Hanjani, Nicholas McGranahan, Charles Swanton, TRACERx Consortium. Heterogeneity of immunotherapy biomarkers in the TRACERx non-small cell lung cancer multi-region lung cancer cohort study [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 645.

UBE2T regulates FANCI monoubiquitination to promote NSCLC progression by activating EMT.

Fanconi anemia complementation group I (FANCI) is a critical protein for maintaining DNA stability. However, the exact role of FANCI in tumors remains to be elucidated. The present study aimed to explore the role and potential mechanism of action of FANCI in non-small cell lung cancer (NSCLC). To quantify the expression levels of FANCI and ubiquitin-conjugating enzyme E2T (UBE2T) in NSCLC tissues, reverse-transcription quantitative PCR and western blotting were employed. Cell Counting Kit-8, wound healing and Transwell assays along with flow cytometry analysis and tumor xenograft were used to investigate the biological effects of FANCI in NSCLC in vitro and in vivo. The binding of FANCI with UBE2T was confirmed using a co-immunoprecipitation assay. Epithelial-to-mesenchymal transition (EMT) protein markers were quantified via western blotting. The results showed that FANCI expression level was higher in NSCLC tumor tissues, compared with adjacent tissues. In A549 and H1299 cells, knockdown of FANCI inhibited cell proliferation, migration, invasion, cell cycle and EMT in vitro. Tumor growth was repressed in vitro, upon downregulation of FANCI expression. UBE2T was observed to directly bind to FANCI and regulate its monoubiquitination. Overexpression of UBE2T reversed the effects induced by FANCI knockdown in NSCLC cells. Furthermore, it was noted that FANCI interacted with WD repeat domain 48 (WDR48). Overexpression of WDR48 reversed the effects of FANCI on cell proliferation, migration and EMT. In conclusion, FANCI was identified to be a putative oncogene in NSCLC, wherein FANCI was monouniubiquitinated by UBE2T to regulate cell growth, migration and EMT through WDR48. The findings suggested that FANCI could be used as a prognostic biomarker and therapeutic target for NSCLC.

MiR-1294, mediated by KLF4, negatively regulates cell proliferation and apoptosis by targeting MYH9 in Non-Small Cell Lung Cancer

Lung cancer, especially non-small cell lung cancer (NSCLC) is the most frequent cause of cancer-related mortality worldwide. MicroRNAs (miRNAs) represent a class of small non-coding RNA molecules. In recent years, many studies have confirmed that abnormal miRNAs expression in tumor can participate in many biological processes of NSCLC. However, whether miR-1294 is involved in the development of NSCLC remains unclear. In this study, miR-1294 was inhibited in NSCLC cell lines, and its expression was associated with tumor size and progression. MiR-1294 overexpression inhibited cell proliferation and cell cycle, conversely promoted cell apoptosis and senescence, and miR-1294 binding to MYH9 3’-UTR mediated suppression of it. Besides, three bioinformatics software were searched, and KLF4 was predicted as an upstream regulator of miR-1294. This study is the first to illuminate that miR-1294, mediated by KLF4, by targeting MYH9 to regulate NSCLC cell proliferation and apoptosis, and is a potential biomarker and therapeutic target for NSCLC.

Integrated microbiome, metabolome, and proteome analysis identifies a novel interplay among commensal bacteria, metabolites and candidate targets in non-small cell lung cancer.

BackgroundAccumulation of evidence suggests that the gut microbiome, its specific metabolites, and differentially expressed proteins (DEPs) are related to non‐small cell lung cancer (NSCLC) pathogenesis. We now report the influences of the gut microbiota, metabolites, and DEPs on the mediation of NSCLC's chronic inflammation and immune dysregulation.MethodsWe conducted 16S ribosomal RNA sequencing for the gut microbiome in healthy volunteers and NSCLC patients. Liquid chromatography–mass spectrometry (LC–MS) analysis was employed to explore differences between metabolites and DEPs in serum samples. Additionally, LC–MS‐based metabolomic analysis was conducted in 40 NSCLC tissues and 40 adjacent tissues. The omics data were separately analysed and integrated by using Spearman's correlation coefficient. Then, faecal microbiota transplantation (FMT) assay was used to assess the effects of the gut microbiome and specific metabolites in mice.ResultsFaecal microbiome analysis revealed gut microflora dysbiosis in NSCLC patients with Prevotella, Gemmiger, and Roseburia significantly upregulated at the genus level. Then, we identified that nervonic acid/all‐trans‐retinoic acid level was negatively related to Prevotella. Additionally, a total of core 8 DEPs were selected in the proteome analysis, which mainly participated in the production of IL‐8 and NF‐κB pathways. CRP, LBP, and CD14 were identified as potential biomarkers for NSCLC. Transplantation of faecal microbiota from patients with NSCLC or Prevotella copri‐colonized recipient in mice resulted in inflammation and immune dysregulation. In turn, nervonic acid/all‐trans‐retinoic acid treatment improved the phenotype of C57BL/6 mice bearing P. copri‐treated Lewis lung cancer (LLC).ConclusionsOverall, these results pointed out that P. copri‐nervonic acid/all‐trans‐retinoic acid axis may contribute to the pathogenesis of NSCLC.