Non-small Cell Lung Cancer Specimens Research Articles

Hsa_circ_0109320 Serves as a Novel Circular RNA Biomarker in Non-small Cell Lung Cancer by Promoting Metastasis.

Non-small cell lung cancer (NSCLC), including squamous cell carcinoma and adenocarcinoma, ranks among the top 10 cancers worldwide in terms of prevalence and mortality. NSCLC, a highly malignant tumor, exhibits distant invasion and migration as well as an unfavorable prognosis. As an innovative circular RNA, hsa _circ_0109320 (circ_0109320) has been recognized as a promising cancer modulator. However, our understanding of the influence of circ_0109320 in NSCLC remains insufficient. Our research explored the clinical significance and effects of circ_0109320 on oncogenic non-small cell lung cancer (NSCLC) phenotypes. Microarray analysis and qPCR indicated that circ_0109320 expression in NSCLC specimens increased relative to that in adjacent normal tissues and was further elevated in metastatic lymph nodes. The specimens acquired from 25 patients confirmed these findings. Additionally, circ_0109320 indicated a good score (AUC = 0.688, P = 0.013) on the ROC curves, which suggests its suitability as a promising biomarker for lung cancer. Meanwhile, circ_0109320 was noticeably upregulated in lung cancer (LC) cell lines compared to human bronchial epithelial cells. Next, we performed loss- and gain-of-function experiments to examine the role of circ_0109320 in the tumor phenotypes of the cell lines. We observed that depletion or overexpression of circ_0109320 did not alter cell viability. However, the ectopic removal of circ_0109320 repressed the migration and invasion of A549 and SK-MES-1 cells, whereas circ_0109320 overexpression promoted cell migration and invasion. Furthermore, the examination of epithelial-mesenchymal transition (EMT) markers indicated that circ_0109320 elevates cell EMT activity. In conclusion, circ_0109320 level was highly associated with increased tumor cell proliferation and metastasis. circ_0109320 could be a promising predictor of clinical outcomes and a reliable target to treat NSCLC by inhibiting metastasis.

UCHL1 Overexpression Is Related to the Aggressive Phenotype of Non-small Cell Lung Cancer.

Ubiquitin C-terminal hydrolase L1 (UCHL1), which encodes thiol protease that hydrolyzes a peptide bond at the C-terminal glycine residue of ubiquitin, regulates cell differentiation, proliferation, transcriptional regulation, and numerous other biological processes and may be involved in lung cancer progression. UCHL1 is mainly expressed in the brain and plays a tumor-promoting role in a few cancer types; however, there are limited reports regarding its role in lung cancer. Single-cell RNA (scRNA) sequencing using 10X chromium v3 was performed on a paired normal-appearing and tumor tissue from surgical specimens of a patient who showed unusually rapid progression. To validate clinical implication of the identified biomarkers, immunohistochemical (IHC) analysis was performed on 48 non-small cell lung cancer (NSCLC) tissue specimens, and the correlation with clinical parameters was evaluated. We identified 500 genes overexpressed in tumor tissue compared to those in normal tissue. Among them, UCHL1, brain expressed X-linked 3 (BEX3), and midkine (MDK), which are associated with tumor growth and progression, exhibited a 1.5-fold increase in expression compared to that in normal tissue. IHC analysis of NSCLC tissues showed that only UCHL1 was specifically overexpressed. Additionally, in 48 NSCLC specimens, UCHL1 was specifically upregulated in the cytoplasm and nuclear membrane of tumor cells. Multivariable logistic analysis identified several factors, including smoking, tumor size, and high-grade dysplasia, to be typically associated with UCHL1 overexpression. Survival analyses using The Cancer Genome Atlas (TCGA) datasets revealed that UCHL1 overexpression is substantially associated with poor survival outcomes. Furthermore, a strong association was observed between UCHL1 expression and the clinicopathological features of patients with NSCLC. UCHL1 overexpression was associated with smoking, tumor size, and high-grade dysplasia, which are typically associated with a poor prognosis and survival outcome. These findings suggest that UCHL1 may serve as an effective biomarker of NSCLC.

Anti-Müllerian hormone type II receptor protein expression in non-small cell lung cancer and the effect of AMH/AMHR2 signaling on cancer cell proliferation.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths worldwide despite advances in cancer therapeutics. In several gynecological cancers, anti-Müllerian hormone receptor type 2 (AMHR2) mediates AMH-induced growth inhibition and is expressed at high levels. Furthermore, 5%-8% of NSCLCs exhibit high AMHR2 expression, suggesting that AMH may inhibit the progression of some lung cancers. However, the clinical relevance of AMHR2 expression and its role in lung cancer is not fully clarified. Immunostaining was performed on 79 surgical specimens of NSCLC. The Cancer Genome Atlas RNA-seq data for lung adenocarcinoma were analyzed, and gene ontology and gene set enrichment analyses were performed. In cellular experiments, AMHR2-overexpressing NSCLC cell lines were established, and the role of the AMH-AMHR2 pathway in cell proliferation with recombinant human AMH protein treatment was examined. A total of 13 cases (16.5%) were positive for immunostaining in lung adenocarcinoma tissues; no positive signals were detected in lung squamous carcinoma tissues. Gene expression variation analysis using The Cancer Genome Atlas data showed that the expression of genes related to the cell cycle was downregulated in the AMHR2-high group. Cellular experiments showed that activation of the AMH-AMHR2 pathway suppressed cell proliferation. In lung adenocarcinoma tissues with high expression of AMHR2, activation of the AMH-AMHR2 pathway may suppress cell proliferation.

WNT5B promotes the malignant phenotype of non-small cell lung cancer via the FZD3–DVL3–RAC1–PCP–JNK pathway

The WNT5B ligand regulates the non-canonical wingless-related integration site (WNT)–planar cell polarity (PCP) pathway. However, the detailed mechanism underlying the activity of WNT5B in the WNT–PCP pathway in non-small cell lung cancer (NSCLC) is unclear. In this study, we assessed the clinicopathological significance of WNT5B expression in NSCLC specimens. WNT5B-overexpression and -knockdown NSCLC cell lines were generated in vivo and in vitro, respectively. WNT5B overexpression in NSCLC specimens correlates with advanced tumor node metastasis (TNM) stage, lymph node metastasis, and poor prognosis in patients with NSCLC. Additionally, WNT5B promotes the malignant phenotype of NSCLC cells in vivo and in vitro. Interactions were identified among WNT5B, frizzled3 (FZD3), and disheveled3 (DVL3) in NSCLC cells, leading to the activation of WNT–PCP signaling. The FZD3 receptor initiates DVL3 recruitment to the membrane for phosphorylation in a WNT5B ligand-dependent manner and activates c-Jun N-terminal kinase (JNK) signaling via the small GTPase RAC1. Furthermore, the deletion of the DEP domain of DVL3 abrogated these effects. Overall, we demonstrated a novel signal transduction pathway in which WNT5B recruits DVL3 to the membrane via its DEP domain through interaction with FZD3 to promote RAC1–PCP–JNK signaling, providing a potential target for clinical intervention in NSCLC treatment.

MiR-3074-5p suppresses non-small cell lung cancer progression by targeting the YWHAZ/Hsp27 axis

Non-small cell lung cancer (NSCLC) accounts for more than 80% of lung cancer cases, and the 5-year survival rate of patients remains unsatisfactory. MicroRNAs (miRNAs) are small endogenous noncoding RNAs that are considered essential posttranscriptional regulators of tumorigenesis, including NSCLC. In this study, we aimed to investigate the biological role of miR-3074-5p in NSCLC cells and the underlying molecular mechanisms. We showed that miR-3074-5p expression was decreased in human NSCLC specimens and cell lines. Moreover, miR-3074-5p overexpression inhibited cell proliferation, migration and invasion and induced apoptosis and cell cycle arrest. In addition, miR-3074-5p overexpression not only suppressed tumor growth but also enhanced the antitumor effect of paclitaxel (PTX) on NSCLC cells in vitro and in vivo. A transcriptome sequencing assay revealed genes that were differentially expressed after miR-3074-5p overexpression, and among the genes whose expression levels were most significantly decreased, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ) was a target of miR-3074-5p. The regulatory effect of miR‐3074-5p on YWHAZ expression was verified by Western blotting and dual‐luciferase reporter assays. The inhibition of A549 cell growth, migration and invasion was reversed by YWHAZ overexpression. Furthermore, we showed that PTX stimulated the expression of the YWHAZ and Hsp27 proteins and promoted the phosphorylation of Hsp27 (at S15 and S78). YWHAZ was confirmed to interact with Hsp27 in A549 cells, and downregulating YWHAZ expression promoted the degradation of the Hsp27 protein. Taken together, these results suggest that the miR‐3074-5p/YWHAZ/Hsp27 axis may be a novel therapeutic target for NSCLC treatment.

Young adults (YA) with non-small cell lung cancer (NSCLC): Snapshot of the oncogenic drivers and immune landscapes.

8538 Background: Approximately 5% of NSCLC occurs in patients 50 years or younger (median age:71) representing distinct clinicopathological features. Reports characterizing genomic alterations are scarce. Using a large real-world (RW) dataset, we characterize oncogenic drivers, and immune landscapes to better understand YA with NSCLC. Methods: 42,326 NSCLC specimens were analyzed using next-generation sequencing of DNA (DNA-592 panel or whole exome) or RNA (whole transcriptome) at Caris Life Sciences (Phoenix, AZ). YA (<=50 years) were categorized into three groups (years): 18-30 (A1, n=61), 31-40 (A2, n=277) & 41-50 (A3, n=1,549); vs >50 (A4, n=40,439). Composition of tumor microenvironment (TME) was estimated from bulk RNA sequencing using QuanTIseq method. RW survival on Osimertinib (Osi-OS) was obtained from insurance claims and calculated from initiation of Osi treatment to last contact and was compared between ages 18-50 and >50 years. Hazard ratio (HR) was calculated using the Cox proportional hazards model. Statistical significance was determined using Chi-square, Fisher’s Exact, Mann-Whitney U and log-rank tests and corrected for multiple comparisons where applicable (q<0.05). Results: Consistent with previous reports, NSCLC in YA was more prevalent in females, non-smokers and was associated with adenocarcinoma histology. Among driver alterations, ALK(IHC+), ROS1, RET and NTRK1 fusion were enriched and KRAS mutations were reduced in YA. Interestingly, while the prevalence of KRASG12D from transition (non-smoker related) and EGFRE746_A750 decreased with age, KRASG12C from transversion increased with age in YA. YA were also associated with improved Osi-OS (HR: 0.79, median Osi-OS=37.4 months vs 32 months in >50 years old, p=0.019). High tumor mutation burden (>=10 mut/Mb), the prevalence of mutations in TP53, KMT2D, NF1, RBM10, NFE2L2 and NOTCH1 increased while GNAS decreased with age. The expression of immune checkpoint genes such as PDCD1LG2(A1/A4: 0.63, A3/A4: 0.89), LAG3(A2/A4: 0.73, A3/A4: 0.89) and IFNG (A1/A4: 0.32, A3/A4: 0.75) were reduced, while M2 macrophage (A2/A4: 1.2) and neutrophil (A2/A4: 1.2) were increased in YA (all q<0.05). Conclusions: We report an increased prevalence of RET and NTRK1 fusions in YA and key differences in distributions of frequent KRAS and EGFR mutations in YA (vs A4) along with decreased expression of immune related genes in the tumor microenvironment. The implications are under active investigation. [Table: see text]

Characterization of TP53 mutations in actionable driver-negative non-small cell lung cancer (NSCLC).

8621 Background: TP53 mutations are common in NSCLC and have been associated with poor prognosis in patients with actionable genomic drivers. Despite the increased prevalence in actionable driver-negative (DN) NSCLC, the role of TP53 mutations in this subtype is not fully explored. We characterize the association of TP53 mutations with the molecular and immune landscapes, as well as outcomes, in DN NSCLC histologic subtypes. Methods: 17,689 NSCLC specimens were analyzed using next-generation sequencing of DNA (DNA-592 panel or whole exome) or RNA (whole transcriptome) at Caris Life Sciences (Phoenix, AZ). Tumors with KRAS, EGFR, BRAF, MET, HER2, ALK, ROS1, NRG1, and NTRK 1-3 alterations were excluded. Specimens were grouped as TP53WT (wild type) or TP53MUT (pathogenic/likely pathogenic mutations). PD-L1 expression was analyzed by IHC (Dako 22c3; PD-L1 positive: TPS ≥1%). TMB was measured by counting all somatic mutations found per tumor (TMB-H: ≥10 mutations/Mb). Composition of the tumor microenvironment (TME) was estimated from bulk RNA sequencing using QuanTIseq method. Overall survival was extracted from insurance claims data and calculated from the time of tissue collection (OS) or initiation of Pembrolizumab treatment (Pembro-OS) to the last contact, using Kaplan-Meier estimates. Significance was determined using chi-square, Fisher’s Exact, Mann Whitney U and adjusted for multiple comparisons (p-value < 0.05). Results: Among DN tumors, the prevalence of TP53MUT was 86%. TP53MUT was more common in men (62% vs 38%), those with a smoking history (99% vs 1%), and those with squamous cell histology (46% vs33.6%, all p<0.05). Interestingly, histological stratification suggested that TP53MUT was associated with poor OS in adenocarcinoma (AC) (HR:1.3, p<0.05), and shorter Pembro-OS in squamous cell carcinoma (SCC) (HR:1.425, p<0.05). In AC, mutations in FAT1, ARID1A, SMARCA4, ARID2 and RBM10 were enriched, while in SCC, mutations in PIK3CA were less common with TP53MUT. Gene sets involved in cell cycle regulation such as G2M, MYC, and E2F targets were enriched in TP53MUT and to a greater extent in AC (1.22-1.29) vs SCC (1.06-1.08, both p<0.05). Looking at immunotherapy-related biomarkers, both AC and SCC TP53MUT tumors had a higher prevalence of TMB-H compared to TP53WT, while PD-L1 positivity was enriched in only AC TP53MUT tumors. Conversely, KEAP1 and STK11 mutations were enriched in AC TP53WT. M2 Macrophage (1.25-fold) and NK cell (1.3-fold) fractions were enriched and B cell fractions (1.25-fold) were less common in TP53MUT SCC TMEs (all p<0.05). Conclusions: TP53MUT were associated with poor OS and Pembro-OS exclusively in AC and SCC, respectively. The distinct molecular and immune landscapes associated with TP53MUT in AC and SCC potentially explain the differences in outcomes mentioned above. Further research is needed to better understand therapeutic implications of TP53 mutations in DN-NSCLC.

Abstract 5426: SMC2 orchestrates non-small cell lung cancer proliferation, invasion, and metastasis by modulating the ERK/AKT/NF-κB pathways

Abstract Chromatin instability has been associated with the expression of structural maintenance of chromosomes (SMC) family members and is a common feature in many tumors. SMC family members have been associated with poor outcomes in several cancer types. However, little is known about their functions and mechanisms in promoting cancer progression, particularly in non-small cell lung cancer (NSCLC). Here, we evaluated the expression of SMC family members in the tumor microenvironment of NSCLC specimens using single-cell RNA sequencing analyses. After a survival analysis, it was found that the sole factor associated with a poor prognosis for NSCLC was member SMC2 expression. Immunohistochemistry was used with data from the Human Protein Atlas, the Cancer Genome Atlas, and the Gene Expression Omnibus databases to confirm the overexpression of SMC2 in NSCLC. We demonstrated that SMC2 regulates not only the fluctuations in the critical states of tumor cells, which are defined by different transcriptome and metabolic properties but also coordinates the immunological components and cell-to-cell communications of the immune milieu, potentially through the pleiotrophin and the Galectin pathways. In vitro and in vivo evidence support the pro-metastatic role of SMC2 in NSCLC. Subsequent investigations showed that SMC2 increases NSCLC progression and metastasis via modulating the ERK/AKT/NF-κB pathway and the epithelial-mesenchymal transition process. Our research clarifies the role and potential mechanisms of SMC2 in NSCLC, providing new treatment options and valuable insights for the management of NSCLC. Citation Format: Yan He, Zhenyu Luo, Yiyao Wang, Shan Xiong, Rui Wan, Xue Zhang, Hua Bai, Jie Wang. SMC2 orchestrates non-small cell lung cancer proliferation, invasion, and metastasis by modulating the ERK/AKT/NF-κB pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5426.

EGFR and ERBB2 Exon 20 Insertion Mutations in Chinese Non-small Cell Lung Cancer Patients: Pathological and Molecular Characterization, and First-Line Systemic Treatment Evaluation.

Data from studies looking at both EGFR and ERBB2 exon 20 insertion mutations (-20ins) in the same cohort of patients with non-small cell lung cancer (NSCLC) are limited. The purpose of this study was to analyze EGFR/ERBB2-20ins in all-stage NSCLC patients to reveal their histological and molecular features, and to retrospectively evaluate the results of first-line real-world systemic treatments in patients with advanced-stage disease. We collected 13,920 formalin-fixed paraffin-embedded NSCLC specimens. Clinicopathological features were recorded and DNA-based next-generation sequencing was performed. First-line systemic treatment data were obtained via chart review. In total, 414 (2.97%) EGFR-20ins cases and 666 (4.78%) ERBB2-20ins cases were identified. Both were more common in women, non-smokers, and patients with adenocarcinoma. The incidence of EGFR/ERBB2-20ins in adenocarcinoma is inversely proportional to the degree of invasion; 77 and 26 variants were detected in EGFR-20ins and ERBB2-20ins cases, respectively. The most common concurrently mutated genes were TP53 and RB1. In invasive adenocarcinoma, lepidic components were more common in EGFR/ERBB2-20ins-alone cases than in those with other concurrent mutated genes. In EGFR-/ERBB2-20ins patients, there was no significant difference in progression-free survival (PFS) or treatment response to first-line systemic treatments in this study. There was no significant difference in PFS or treatment response among patients with different EGFR/ERBB2-20ins variants and those with or without concurrent mutated genes. EGFR/ERBB2-20ins is more common in early lung adenocarcinoma. EGFR-20ins had more variants. In both cohorts, the results for first-line systemic treatments showed no significant difference.

Phosphorylation of AGO2 by TBK1 Promotes the Formation of Oncogenic miRISC in NSCLC.

Non-small-cell lung cancer (NSCLC) is a highly lethal tumor that often develops resistance to targeted therapy. It is shown that Tank-binding kinase 1 (TBK1) phosphorylates AGO2 at S417 (pS417-AGO2), which promotes NSCLC progression by increasing the formation of microRNA-induced silencing complex (miRISC). High levels of pS417-AGO2 in clinical NSCLC specimens are positively associated with poor prognosis. Interestingly, the treatment with EGFR inhibitor Gefitinib can significantly induce pS417-AGO2, thereby increasing the formation and activity of oncogenic miRISC, which may contribute to NSCLC resistance to Gefitinib. Based on these, two therapeutic strategies is developed. One is jointly to antagonize multiple oncogenic miRNAs highly expressed in NSCLC and use TBK1 inhibitor Amlexanox reducing the formation of oncogenic miRISC. Another approach is to combine Gefitinib with Amlexanox to inhibit the progression of Gefitinib-resistant NSCLC. This findings reveal a novel mechanism of oncogenic miRISC regulation by TBK1-mediated pS417-AGO2 and suggest potential therapeutic approaches for NSCLC.

Unveiling the STAT3-ACC1 axis: a key driver of lipid metabolism and tumor progression in non-small cell lung cancer.

Background and Objective: Lung cancer is a prevalent global malignancy, and investigating the metabolic reprogramming of tumor cells has significant therapeutic implications. This study aims to explore the molecular mechanism driving the progression of non-small cell lung cancer (NSCLC), with a specific emphasis on the STAT3-ACC1-FAS axis involved in fatty acid synthesis. Methods: The levels of Signal transducer and activator of transcription 3 (STAT3) and acetyl-CoA carboxylase 1 (ACC1) were determined in mouse NSCLC specimens and cell lines using Western blot and qPCR methods. Various assays such as CCK-8, colony formation, EDU, wound-healing, and transwell migration were employed to assess cancer cell proliferation, migration, and invasion. Additionally, a nude mouse xenograft model was utilized for in vivo tumor growth analysis. The interaction between STAT3 and ACC1 was examined through chromatin immunoprecipitation and dual-luciferase assays. Results: The study observed upregulation of STAT3 and ACC1 in NSCLC tissues. Notably, the suppression of STAT3 and ACC1 inhibited the in vitro progression and lipid synthesis of NSCLC cells. Furthermore, STAT3 enhanced lipid synthesis by upregulating ACC1 expression. Mechanistic assays revealed that this process occurred through direct activation of ACC1 transcription by STAT3. STAT3 played a vital role in regulating lipid metabolism and supporting NSCLC progression. Conclusion: The findings of this study underscore the significance of the STAT3-ACC1-FAS axis in NSCLC. The activation of ACC1 through STAT3-mediated transcription serves as a crucial mechanism for stimulating the progression of NSCLC tumors and promoting lipid synthesis. Consequently, targeting the STAT3-ACC1 axis may present a promising avenue for the diagnosis and treatment of NSCLC patients.

PD-1 expression in tumor infiltrating lymphocytes as a prognostic marker in early-stage non-small cell lung cancer.

Programmed death ligand - 1 (PD-L1) expression is a well-established predictive biomarker for immunotherapy in non-small cell lung cancer (NSCLC). Programmed death - 1 (PD-1) serves as the target protein to PD-L1 and their interaction serves as a crucial pathway for immune evasion. This study aimed to investigate the expression pattern of PD-1 on Tumor-infiltrating lymphocytes (TILs) in early-stage NSCLC, and its potential role as prognostic biomarker. PD-1 was evaluated in 474 surgical resected early-stage NSCLC specimens, using Tissue microarray and immunohistochemical staining. Expression was scored as negative (<1%) or positive. Positive PD-1 expression was further divided into low (<10%) and high (≥10%). None of the patients had received treatment with PD-1/PD-L1 inhibitors. PD-1 expression ≥1% in TILs was observed in 83.5% of cases and was associated with pT stage (p=0.02), grade 3 (p=0.004), and adenocarcinoma subtype (p=0.05). Individuals with high PD-1 expression (≥10%) experienced reduced 10-year overall survival (Log-Rank test = 0.005). In addition, high PD-1 expression emerged as an independent factor associated with reduced survival on multivariate analysis (HR: 1.328 (95% CI: 1.074-1.641). Patients with early-stage NSCLC who exhibited PD-1 expression of ≥10% on TILs had an unfavorable 10-year OS rate. These findings indicate that elevated PD-1 expression on TILs can be associated with immune evasion during the early stages of malignancy evolution in the NSCLC setting and further research is required to further delineate the role of PD-1/PD-L1 pathway on tumor immune senescence. These results underline the potential role of PD-1/PD-L1 inhibitors in the treatment of early-stage NSCLC.

ABCA8 Elevation Predicts the Prognosis and Exerts the Anti-oncogenic Effects on the Malignancy of Non-small Cell Lung Cancer via TCF21-Mediated Inactivation of PI3K/AKT.

The malignant growth and metastatic potential of non-small-cell lung cancer (NSCLC) are the major causes for its poor prognosis. ATP-binding cassette (ABC) subfamily A member 8 (ABCA8) exerts contradictive roles in the development of several cancers. Nevertheless, its role in NSCLC remains unclear. In this study, three GEO datasets and bioinformatics databases (GEPIA2 and UALCAN) revealed the obvious down-regulation of ABCA8 in NSCLC tissues and cells, and this expression was associated with cancer stages and lymph node metastasis. Low expression of ABCA8 predicted poor survival in NSCLC. ABCA8 elevation inhibited cell proliferation and induced cell apoptosis. Moreover, ABCA8 overexpression suppressed cancer cell invasion. Mechanistically, ABCA8 was associated with TCF21 in NSCLC specimens and its overexpression enhanced TCF21 expression. ABCA8 elevation inactivated the PI3K/AKT signaling, which was reversed after TCF21 knockdown. Additionally, targeting TCF21 overturned the anti-oncogenic effects of ABCA8 elevation on cell proliferation, apoptosis and invasion. Thus, the current findings highlight that ABCA8 may be a promising prognostic marker and may act as a suppressor gene to regulate the malignancy of NSCLC cells via TCF21-mediated inactivation of PI3K/AKT signaling, supporting a new promising target for the treatment of NSCLC.

Challenges to the effectiveness of next-generation sequencing in formalin-fixed paraffin-embedded tumor samples for non-small cell lung cancer

IntroductionNext-generation sequencing (NGS) of Formalin-Fixed and Paraffin-Embedded (FFPE) specimens is routine in precision oncology practice. However, results are not always conclusive, and it is important to identify which factors may influence FFPE tumor sequencing success. Materials and methodsHere, we evaluated the influence of pre-analytical factors on 705 samples of non-small cell lung cancer specimens that underwent NGS testing. Factors such as tumor site, tumor cell percentage, fragment size, primary tumor or metastasis, presence of necrosis, DNA purity, DNA concentration, sample origin and year of testing. ResultsThe overall NGS success rate was 84.9 % (n = 599). Bone site specimens had a very low success rate (42.1 %), differing from lung samples (79.8 %) (P < 0.05). Samples with tumor percentages <5 % (success rate of 44.4 %) represented 14.1 % of failed sequencings. Moreover, samples with tumor percentages >10 %–20 % (82 %) did not differ from those with >30 % (88.9 %) on sequencing outcomes (P = 0.086). Specimens that provided DNA concentrations >2.0 ng/uL, 1.0–2.0 ng/uL, 0.5–1.0 ng/uL and <0.5 ng/uL had success rates of 92 %, 77.1 %, 61.3 % and 20.4 %, respectively. Small fragments (≤0.2 cm2) had a success rate of 74.7 % and were more prevalent in the unsuccessful group (P < 0.05). ConclusionsOur results suggest that tumor percentage, fragment size, decalcified bone specimens, and DNA concentration are potential modifiers of NGS success rates. Interestingly, specimens with tumor percentages between 10 % and 20 % have the same sequencing outcome than specimens with >30 %. These results can strengthen the understanding of factors that lead to NGS success variability.

Abstract B030: Characterizing MAIT cells in lung cancer: Insights from NSCLC and murine models

Abstract Background: Mucosal-associated invariant T cells (MAIT) are unconventional αβT cells with a semi-invariant TCR that recognizes microbially-derived riboflavin metabolites presented by MHC-related protein 1 (MR1). Upon TCR engagement or cytokine stimulation, MAIT cells can rapidly secrete pro-inflammatory cytokines and cytotoxic molecules. The precise role of MAIT cells in the tumor-immune microenvironment remains unclear, as different reports have described them as either beneficial or detrimental to patient prognosis. Considering that MAIT cells constitute a notable proportion (2-4%) of the lung T cell repertoire in both humans and mice, the lung serves as an appropriate setting for ex vivo and in vivo MAIT cell characterization. Objective: To characterize the phenotype of MAIT cells in lung tumor and uninvolved tissue from non-small cell lung cancer (NSCLC) specimens and orthotopic murine models. Results: Surgical resections of primary tumor and normal adjacent tissue from 29 treatment-naïve NSCLC patients were profiled using a comprehensive 31-marker spectral flow cytometry panel. MAIT cells expressing activating and inhibitory receptors including CD69, PD-1, CTLA-4, and CD39, were enriched in tumor tissue. Conversely, cytotoxic MAIT cells expressing granzyme B, granulysin, and CD57, were enriched in normal adjacent tissue. For in vivo studies, lungs of B6-MAITCAST mice, which harbor higher frequencies of MAIT cells compared to conventional mouse strains, were orthotopically injected with CMT167 tumor cells or vehicle control. MAIT cells from lungs injected with CMT167 tumors also expressed higher levels of activating and inhibitory receptors, and decreased levels of granzyme B. Additionally, there was a slight decrease in IFNγ and IL-17A production upon stimulation in vitro with PMA/ionomycin compared to vehicle controls. Conclusion: In the context of lung cancer, MAIT cells are activated but may lose functionality within the tumor-immune microenvironment. Rationale: There is growing interest in the immunotherapeutic potential of unconventional T cells due to their rapid effector functions and non-MHC-restricted nature. By investigating the role of MAIT cells in the tumor microenvironment, we can comprehensively identify their biological advantages and limitations. Citation Format: Stephanie WY Wong, Carolina de Amat Herbozo, Adriana Vukosich-Pennell, Ben X Wang, Pamela S Ohashi, Adrian G Sacher. Characterizing MAIT cells in lung cancer: Insights from NSCLC and murine models [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B030.

FBXO38 mediates FGL1 ubiquitination and degradation to enhance cancer immunity and suppress inflammation

Upregulation of FGL1 helps tumors escape from immune surveillance, and therapeutic antibodies targeting FGL1 have potential as another immune checkpoint inhibitor. However, the underlying mechanism of high FGL1 protein level in cancers is not well defined. Here, we report that FBXO38 interacts with and ubiquitylates FGL1 to negatively regulate its stability and to mediate cancer immune response. Depletion of FBXO38 markedly augments FGL1 abundance, not only suppressing CD8+ Tcell infiltration and enhancing immune evasion of tumor but also increasing inflammation in mice. Importantly, we observe a negative correlation of FBXO38 with FGL1 and IL-6 in non-small cell lung cancer specimens. FGL1 and IL-6 levels positively correlate with TNM (tumor, lymph node, metastasis) stages, while FBXO38 and the infiltrating CD8+ Tcells negatively correlate with TNM stages. Our study identifies a mechanism regulating FGL1 stability and a target to enhance the immunotherapy and suggests that the combination of anti-FGL1 and anti-IL-6 is a potential therapeutic strategy for cancer immunotherapy.

High Expression of Fas-Associated Factor 1 Indicates a Poor Prognosis in Non-Small-Cell Lung Cancer.

Fas-associated factor 1 (FAF1) is a death-promoting protein identified as an interaction partner of the death receptor Fas. The downregulation and mutation of FAF1 have been reported in a variety of human tumors, but there have been few studies on lung cancer. Here, we investigated the prognostic significance of FAF1 expression in non-small-cell lung cancer (NSCLC), and whether aberrant FAF1 expression may be involved in the pathogenesis and prognosis of NSCLC. FAF1 expression was examined in NSCLC specimens as well as human lung cancer cell lines. In addition, changes in cell viability and apoptosis upon regulating FAF1 expression were investigated in lung cancer cell lines. As a result, high FAF1 expression was significantly associated with a poor prognosis in NSCLC. In lung cancer cell lines, FAF1 downregulation hindered cell viability and tended to promote early apoptosis. In conclusion, this is the first study of the clinical significance of FAF1 in NSCLC, showing that FAF1 overexpression is associated with a poor prognosis in NSCLC and that FAF1 acts as a dangerous factor rather than an apoptosis promoter in NSCLC.

Adequacy of cytology and small biopsy samples obtained with rapid onsite evaluation (ROSE) for predictive biomarker testing in non-small cell lung cancer

Complete biomarker workup of non-small cell lung cancer (NSCLC) specimens is essential for appropriate and timely clinical management decisions. This can be challenging to achieve from small cytology and histology specimens, with increasing numbers of molecular and immunohistochemical biomarkers required. We conducted a 5 year retrospective audit of cases at our institution to assess the diagnostic and biomarker testing adequacy rates, particularly those specimens obtained with rapid onsite evaluation (ROSE), performed by a cytopathologist and a cytology scientist or pathology trainee, including all endobronchial ultrasound guided transbronchial needle aspirations (EBUS-TBNA), CT guided lung fine needle aspirations (FNA) and CT guided lung core biopsies. A total of 5,354 cases were identified, of which 92.2% had sufficient material for diagnosis. Of the 1506 cases identified with a recorded diagnosis of lung adenocarcinoma or NSCLC, not otherwise specified, 1001 (66.5%) had biomarker testing requested. Sufficient material was available in 89.5% of cases for a complete biomarker workup which included EGFR and KRAS mutational testing (all cases), ALK, ROS1 and PD-L1 immunohistochemistry (all cases), and ALK and ROS1 FISH (as required). For EGFR and KRAS mutational testing across both cytology and histology specimens, 99% of cases were sufficient. Of the samples in which a complete biomarker workup was unable to be performed, approximately half were only insufficient due to inadequate numbers of tumour cells for PD-L1 immunohistochemistry. Excluding PD-L1 IHC, 952 (95.1%) of samples obtained with ROSE were sufficient for the remainder of the testing requirements. Next generation sequencing using a 33 gene custom AmpliSeq panel was achieved in up to 72% of cases. In conclusion, small cytology and histology specimens obtained with ROSE are suitable for predictive biomarker testing in NSCLC, although attention needs to be paid to obtaining sufficient cells (>100) for PD-L1 immunohistochemistry.

Loose Tumor Cells in Pulmonary Arteries of Lung Adenocarcinoma Resection Specimens: No Correlation With Survival, Despite High Prevalence.

Loose tumor cells and tumor cell clusters can be recognized in the lumen of intratumoral pulmonary arteries of resected non-small cell lung cancer specimens. It is unclear whether these should be considered tumor-emboli, and as such could predict a worsened prognosis. To investigate the nature and prognostic impact of pulmonary artery intraluminal tumor cells. This multicenter study involved an exploratory pilot study and a validation study from 3 institutions. For the exploratory pilot study, a retrospective pulmonary resection cohort of primary adenocarcinomas, diagnosed between November 2007 and November 2010, were scored for the presence of tumor cells, as well as potentially other cells in the intravascular spaces, using hematoxylin-eosin and cytokeratin 7 (CK7) stains. In the validation part, 2 retrospective cohorts of resected pulmonary adenocarcinomas, between January 2011 and December 2016, were included. Recurrence-free survival (RFS) and overall survival (OS) data were collected. In the pilot study, CK7+ intravascular cells, mainly tumor cells, were present in 23 of 33 patients (69.7%). The 5-year OS for patients with intravascular tumor cells was 61%, compared with 40% for patients without intravascular tumor cells (P = .19). In the validation study, CK7+ intravascular tumor cells were present in 41 of 70 patients (58.6%). The 5-year RFS for patients with intravascular tumor cells was 80.0%, compared with 80.6% in patients without intravascular tumor cells (P = .52). The 5-year OS rates were, respectively, 82.8% and 71.6% (P = .16). Loose tumor cells in pulmonary arterial lumina were found in most non-small cell lung cancer resection specimens and were not associated with a worse RFS or OS. Therefore, most probably they represent an artifact.

Deficiency of circ_0103809 Attenuates Non-small Cell Lung Cancer Malignant Progression by Controlling miR-153-3p/HDAC1 Network.

Circular RNAs are vital players in tumorigenesis. We held the purpose to investigate the role and mechanism of circ_0103809 in non-small cell lung cancer (NSCLC).The expressions of circ_0103809, miR-153-3p and HDAC1 mRNA were determined using quantitative real-time PCR assay, and HDAC1 protein was quantified using western blot analysis. MTT, EdU, flow cytometry, tube-formation, wound healing and tube-formation assays were conducted for functional analysis. The predicted relationship among circ_0103809, miR-153-3p and HDAC1 was ascertained using dual-luciferase analysis, RIP assay and pull-down analysis. Animal models were further constructed to realize circ_0103809's role in vivo.Circ_0103809 was upregulated NSCLC specimens, cells and serum-derived exosomes. Serum exosomal circ_0103809 had the potency to be a diagnostic biomarker for NSCLC. Circ_0103809 silencing inhibited NSCLC cell growth, metastasis and angiogenesis and triggered cell cycle arrest and apoptosis. Circ_0103809 deficiency also suppressed the growth of transplanted tumors. Circ_0103809 acted as the miR-153-3p sponge, and the biological effects of circ_0103809 knockdown were relieved by miR-153-3p inhibition. HDAC1 was directly targeted by miR-153-3p, and miR-153-3p enrichment inhibited NSCLC cell malignant phenotypes by sequestering HDAC1.Circ_0103809 knockdown repressed NSCLC malignant progression partly by regulating miR-153-3p/HDAC1 signaling.