Non-small Cell Lung Cancer Specimens Research Articles

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.

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.

Regulation of hPCL3 isoforms' ubiquitination by TRIM21 in non-small cell lung cancer progression.

Non-small cell lung cancer (NSCLC) is the main subtype of lung cancer. The role of hPCL3 isoforms, hPCL3S and hPCL3L, remains ambiguous. This study examines the functional implications of these isoforms in NSCLC, using lung cancer cell lines A549 and NCI-H226c for in vivo and in vitro analyses. The results indicate that elevated expression of both hPCL3S and hPCL3L correlates with diminished overall survival, although only hPCL3S levels are augmented in clinical NSCLC specimens. Inhibition of either isoform leads to reduced cell proliferation, invasion, and migration, with hPCL3S knockdown displaying superior effectiveness. Moreover, the findings reveal that TRIM21 interacts with both isoforms and mediates hPCL3S degradation through K48-linked ubiquitination in NSCLC cells. Conversely, TRIM21 does not facilitate hPCL3L degradation, despite forming K63-linked polyubiquitin chains. These observations highlight the divergent roles of hPCL3 isoforms in NSCLC and underscore the potential therapeutic value of targeting hPCL3S.

Comprehensive analysis of the clinical and prognostic significance of SFRP1 and PRKCB expression in non-small cell lung cancer: a retrospective analysis.

Secreted frizzled-related protein 1 (SFRP1) and protein kinase C-B (PRKCB) contribute to cancer progression and angiogenesis. This study intended to detect SFRP1 and PRKCB expression in non-small-cell lung cancer (NSCLC) patients and analyze its association with clinicopathological features. A total of 108 NSCLC patients who underwent surgical resection in our hospital between 2012 and 2017 were retrospectively analyzed. SFRP1 and PRKCB expression was detected using immunohistochemical staining. The relationships between SFRP1 and PRKCB expression and clinicopathological data were analyzed using the chi-square method. Kaplan-Meier analysis was used to investigate survival probability over time. The potential risk of NSCLC morbidity associated with SFRP1 and PRKCB levels was analyzed using univariate and multivariate Cox proportional risk models. SFRP1 and PRKCB expression was negative in 114 and 109 of the 180 NSCLC specimens, respectively. SFRP1 expression was significantly associated with TNM stage ( P < 0.001) and tumor diameter ( P < 0.001). PRKCB expression was significantly associated with the TNM stage ( P < 0.001). The correlation between SFRP1 and PRKCB expression was evident ( P = 0.023). SFRP1(-) or PRKCB(-) patients shows lower survival rates than SFRP1(+) or PRKCB(+) patients ( P < 0.001). SFRP1(-)/PRKCB(-) patients had the worst prognosis ( P < 0.001). Furthermore, the mortality of SFRP1(-) or PRKCB(-) patients was significantly higher than that of SFRP1(+) or PRKCB(+). SFRP1 and PRKCB expression can be used to predict prognosis in patients with NSCLC.

Clinicopathological characteristics of Non-Small Cell Lung Cancer (NSCLC) patients with c-MET exon 14 skipping mutation, MET overexpression and amplification

PurposeMET exon 14 skipping is one of the rare mutations in non-small cell lung cancer (NSCLC), involving its pathogenesis and progression. The performances of several MET inhibitors in clinical trials have been validated based on NGS, immunohistochemistry (IHC), and gene copy number assessments. Thus, a detailed understanding of the relationship between these markers and prognosis is required.MethodsThis study has recruited patients (n = 17) with MET exon 14 skipping mutation and initially screened genes (n = 10) by polymerase chain reaction (PCR) from 257 specimens of NSCLC, including small biopsies and surgical resection. Further, the IHC analysis detected MET overexpression and recorded the score using the MetMAb trial (rial ( recruited patients (n = 17) with MET exstainings). Finally, the fluorescence in situ hybridization (FISH) resulted in the MET amplification with a MET copy number initially screened genes (n = 10) by p.ResultsPCR results indicated strong MET staining ( 3+) in more than 50% of tumor cells. Among the recruited 17 cases of MET exon 14 skipping, 9 cases presented MET amplification, and 10 cases with MET overexpression. These attributes were not correlated to the clinicopathological characteristics and overall survival. In addition, 4 cases showed gene amplification, and 3 cases presented polyploidy condition. The correlation analysis showed a significant relationship between MET amplification and MET overexpression (Pearson’s r2 = 0.4657, P < 0.005).ConclusionTogether, these findings indicated a significant correlation between MET overexpression and MET amplification in NSCLC patients but no correlation to prognosis.

Restin protein expression in non-small cell lung cancer.

Restin is a member of the melanoma-associated antigen (MAGE) superfamily. Its expression has been reported to be up- or downregulated in cancer. Preclinical data suggest it is a tumor suppressor. In this study, we aimed to evaluate restin expression and prognostic value in non-small cell lung cancer (NSCLC). Restin expression was analyzed by immunohistochemistry in three tissue microarrays consisting of formalin-fixed/paraffin-embedded NSCLC specimens from 113 patients, represented in triplicate. Restin staining H-score was the result of the staining intensity (0-no, 1-weak, 2-moderate, and 3-strong) multiplied by the percentage of stained tumor cells; it was defined as low if 1-100, moderate if 101-200, and strong if 201-300. Haverage-score was the average H-score in the triplicate. Restin Haverage-scores were tested for correlations with clinical and pathological characteristics and patient outcome. Restin expression was localized to the cytoplasm, with nuclear enhancement, of 112/113 (99.1%) NSCLCs. Restin Haverage-scores were 0 in 1/113 (0.88%), low in 15/113 (13.3%), moderate in 48/113 (42.5%), and strong in 49/113 (43.4%) NSCLCs. Restin Haverage-scores did not correlate with NSCLC histological subtype, disease stage, recurrence/progression-free, or overall survival. Restin is moderately to strongly expressed in the majority of NSCLC tumors but its expression has no prognostic value in patients with NSCLC.

Prognostic impact of LILRB4 expression on tumor-infiltrating cells in resected non-small cell lung cancer.

Leukocyte immunoglobulin-like receptor subfamily B member 4 (LILRB4/ILT3) is an up-and-coming molecule that promotes immune evasion. We have previously reported that LILRB4 facilitates myeloid-derived suppressor cells (MDSCs)-mediated tumor metastasis in mice. This study aimed to investigate the impact of the LILRB4 expression levels on tumor-infiltrating cells on the prognosis of non-small cell lung cancer (NSCLC) patients. We immunohistochemically evaluated the LILRB4 expression levels of completely resected 239 NSCLC specimens. Whether the blocking of LILRB4 on human PBMC-derived CD33+ MDSCs inhibited the migration ability of lung cancer cells was also examined using transwell migration assay. The LILRB4 high group, in which patients with a high LILRB4 expression level on tumor-infiltrating cells, showed a shorter overall survival (OS) (p = 0.013) and relapse-free survival (RFS) (p = 0.0017) compared to the LILRB4 low group. Multivariate analyses revealed that a high LILRB4 expression was an independent factor for postoperative recurrence, poor OS and RFS. Even in the cohort background aligned by propensity score matching, OS (p = 0.023) and RFS (p = 0.0046) in the LILRB4 high group were shorter than in the LILRB4 low group. Some of the LILRB4 positive cells were positive for MDSC markers, CD33 and CD14. Transwell migration assay demonstrated that blocking LILRB4 significantly inhibited the migration of human lung cancer cells cocultured with CD33+ MDSCs. Together, signals through LILRB4 on tumor-infiltrating cells, including MDSCs, play an essential role in promoting tumor evasion and cancer progression, impacting the recurrence and poor prognosis of patients with resected NSCLC.

Stimulator of interferon gene (STING) expression as a biomarker for overall survival in PDL1-negative, TMB-low non-small cell lung cancer (NSCLC) treated with immune checkpoint inhibitors (ICIs).

9120 Background: cGAS-STING (CGS) is an intracellular DNA sensing pathway that when activated, stimulates interferon and other immune-related signaling pathways. While increased expression of CGS-related genes has been associated with better survival in NSCLC, studies investigating this phenomenon in tumors less likely to respond to ICIs such as PDL1-negative, TMB-Low NSCLC are lacking. Here we report the role of CGS pathway in NSCLC tumors that are both PDL1-negative and TMB-Low. Methods: DNA (592-gene or whole exome) and RNA sequencing (whole transcriptome) was performed for NSCLC specimens (n=7264) submitted to Caris Life Sciences (Phoenix, AZ). PDL1 negativity was defined as &lt;1% TPS score (22C3) &amp; TMB-Low as &lt;10 mutations/Mb. Hierarchical Agglomerative Clustering (HAC) was performed on STING pathway genes (CXCL10, CCL5 and GZB) and tumors were grouped into STING-high (n=3070), STING-intermediate (n=1609) and STING-low (n=2585) groups. Microenvironmental cell populations (MCP)-counter analysis was used to estimate cellular composition of immune cells. Statistical significance was determined using chi-square/Fisher’s exact/Mann-Whitney U tests and adjusted for multiple comparisons (q&lt;0.05). Kaplan Meier method was used to estimate the overall survival (OS) derived from insurance claims of IO treated pts with NSCLC (n=400). Pts were stratified into groups above or below the median expression of the STING gene ( TMEM173). Results: Compared to STING-low, tumors that were STING-high had enrichment in immune cell infiltrates including CD8+ T cells (4.4-fold) and B-cells (5.9-fold); Additionally, STING-high cohort had significantly higher expression of immune checkpoint (IC) genes including CD274(2.5-fold), CD80 (2.7-fold, all q&lt;0.05). STING-high tumors also had a higher prevalence of TP53 mutations (mts) and a lower prevalence of STK11 and KEAP1 mts (Table). Median overall survival (OS) was 21.1 months in the STING-high cohort (n=200) vs 11.9 months in the STING-low cohort (n=199) in IO treated pts (HR = 0.62, p&lt;0.000.1) Conclusions: To our knowledge this is the largest real-world dataset indicating that enrichment of both the STING gene, and gene signatures associated with the STING pathway represent valuable transcriptomic biomarkers to stratify pts with PDL1-negative and TMB-Low NSCLC who benefit more from IO therapies. Conversely, novel IO combination strategies need to be employed in STING-low tumors to enhance outcomes. [Table: see text]

A novel platform to enrich for tumor-specific T cells in non–small-cell lung cancer.

2556 Background: To induce robust cellular immunity against non-small cell lung cancer (NSCLC), the leading cause of cancer-related death in the US, we have developed an innovative cell-based therapeutic platform technology to generate polyclonal, neoantigen-reactive T cells in vitro. Methods: Surgically resected NSCLC tissue and peripheral matched blood were collected from 17 patients under an IRB-approved protocol. From 3 of the patient-derived NSCLC specimens, methods were tested to enhance immunogenicity and stimulate neoantigen-specific immune responses. We then use a novel, human vascularized microtumor (VMT) platform to assess the sensitivity of tumor cells to T-cell mediated attack at the individual patient level. Results: The therapeutic platform induced a significant increase in T cell proliferation, interferon-gamma secretion, and blast phenotype of lymphocyte populations compared to T cells primed by standard methods. Interestingly, for one patient, immunosuppression of the T cell response with concomitant increase in CD96, an immune checkpoint receptor implicated in lung cancer progression, was observed in the tumor. Importantly, using sequencing data from one patient, an analytical and machine learning pipeline detected putative neoantigens using our therapeutic platform technology and not with standard methods. Conclusions: The therapeutic platform can be used to directly generate cell-based immunotherapies and to facilitate neoantigen discovery and development of targeted cancer immunotherapies. Our goal is to advance novel approaches to effectively mobilize anti-tumor immunity and overcome treatment resistance in NSCLC.

Abstract 2502: The role of high mobility group box-1 protein (HMGB1) in the microenvironment of non-small cell lung cancer

Abstract Introduction: The tumor microenvironment (TME) is critical in determining tumor development and progression. Among stromal cells in TME, cancer associated fibroblasts (CAFs) play a central role and are involved in various signals through their interaction with cancer cells in most solid tumors, leading to cancer progression and acquisition of drug resistance. Under these TME conditions, tumor cell death such as necrosis occurs, which in turn, results in the ectopic release of bioactive molecules collectively termed as damage-associated molecular patterns (DAMPs), which has been reported to play an important role in cancer progression. The purpose of this study is to clarify the effects of cancer cell-derived HMGB1, a representative DAMPs, on CAFs and to investigate whether it could be a potential therapeutic target. Methods: We established CAFs from resected non-small cell lung cancer (NSCLC) specimens and investigated the biological activity of HMGB1 on CAFs using in vitro co-culture models with NSCLC cell lines. Furthermore, we assessed the therapeutic efficacy of targeting cancer cell-derived HMGB1 using anti-HMGB1 neutralizing antibody. Results: Intracellular HMGB1 was expressed more in NSCLC cells than in normal bronchial epithelial cells and CAFs, and released from cancer cells extracellularly upon treatment with anticancer drugs. The stimulation with recombinant HMGB1 promoted the proliferation of CAFs in a dose-dependent manner, but not lung cancer cells. Co-culture with CAFs promoted cancer cells’ migratory ability and drug resistance, and stimulation with recombinant HMGB1 further enhanced these promoting effects, while administration of anti-HMGB1 neutralizing antibody suppressed these effects. On the other hand, these inhibitory effects were not observed in the absence of CAFs, suggestingHMGB1 has effect on CAFs, but not direct on cancer cells. Conclusion: HMGB1 plays an important role in cancer progression and acquisition of therapeutic resistance by upregulating the CAFs’ proliferation and activity and could be a therapeutic target in NSCLC. Citation Format: Tomohiro Habu, Keiichi Date, Ken Suzawa, Mao Yoshikawa, Masayoshi Ohki, Kazuma Iwata, Naoki Matsuda, Yin Min Thu, Kazuhiko Shien, Hiromasa Yamamoto, Shinichi Toyooka. The role of high mobility group box-1 protein (HMGB1) in the microenvironment of non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2502.