Photochemical enhancement of PD-L1-SAP immunotoxin efficacy in non-small cell lung cancer cell lines

BackgroundThoracic SMARCA4-deficient undifferentiated tumors (SMARCA4-UT) are aggressive neoplasms. Data linking BAF alterations with tumor microenvironment (TME) and efficacy of immune checkpoint inhibitors (ICI) are contradictory. The TME of SMARCA4-UT and their response to ICI are unknown.Materials and MethodsPatients diagnosed with SMARCA4-UT in our institution were included. Immunostainings for tertiary lymphoid structures (TLS), immune cell markers, and checkpoints were assessed. Validation was performed using an independent transcriptome dataset including SMARCA4-UT, non–small cell lung cancers (NSCLC) with/without SMARCA4 mutations, and unclassified thoracic sarcomas (UTS). CXCL9 and PD-L1 expressions were assessed in NSCLC and thoracic fibroblast cell lines, with/without SMARCA4 knockdown, treated with/without interferon gamma.ResultsNine patients were identified. All samples but one showed no TLS, consistent with an immune desert TME phenotype. Four patients received ICI as part of their treatment, but the only one who responded, had a tumor with a TLS and immune-rich TME. Unsupervised clustering of the validation cohort using immune cell scores identified 2 clusters associated with cell ontogeny and immunity (cluster 1 enriched for NSCLC independently of SMARCA4 status (n = 9/10; P = .001); cluster 2 enriched for SMARCA4-UT (n = 11/12; P = .005) and UTS (n = 5/5; P = .0005). SMARCA4 loss-of-function experiments revealed interferon-induced upregulation of CXCL9 and PD-L1 expression in the NSCLC cell line with no effect on the thoracic fibroblast cell line.ConclusionSMARCA4-UT mainly have an immune desert TME with limited efficacy to ICI. TME of SMARCA4-driven tumors varies according to the cell of origin questioning the interplay between BAF alterations, cell ontogeny and immunity.

Immune-Related Adverse Events and Outcomes in Patients with Advanced Non–Small Cell Lung Cancer: A Predictive Marker of Efficacy?

Lung cancer remains one of the leading causes of cancer‒related mortality worldwide, underscoring the urgent need for innovative diagnostic and therapeutic approaches. The advent of immune checkpoint inhibitors(ICIs)has significantly transformed the treatment landscape, particularly for non‒small cell lung cancer(NSCLC). However, predictive biomarkers for ICI efficacy remain limited, with PD‒L1 expression, tumor mutational burden(TMB), and microsatellite instability providing only partial insight into therapeutic response. Recent attention has focused on tertiary lymphoid structures(TLS), ectopic lymphoid aggregates resembling secondary lymphoid organs that form in the tumor microenvironment. TLS are composed of B cells, T cells, dendritic cells, and other immune components, and play a central role in coordinating local antitumor immune responses. In NSCLC, the presence of TLS has been associated with favorable prognosis and improved response to ICIs, independent of PD‒L1 or TMB status. Moreover, emerging evidence suggests that the quality of TLS-such as the degree of maturation and the nature of infiltrating immune cells-may further influence immunotherapeutic outcomes. This review outlines the clinical significance of TLS in lung cancer, discussing their structure, function, and potential as novel biomarkers for stratifying patients undergoing immunotherapy. We also explore future directions including therapeutic strategies aimed at promoting TLS formation, such as vaccines or immune adjuvants, as well as the application of artificial intelligence and spatial omics technologies for the standardized evaluation and in‒depth characterization of TLS. As the integration of TLS analysis into clinical oncology evolves, a more precise and personalized approach to lung cancer immunotherapy may be realized.

Commentary: Another win for immunotherapy

e20101 Background: The RUNX1T1 gene, also known as ETO, was first identified in the fusion transcript AML1/ETO, generated by the translocation between chromosome 8 and 21, which occurs in 12%-15% of AML. We recently discovered that RUNX1T1 was specifically amplified only in the small cell lung cancer (SCLC) but not the non-small cell lung cancer (NSCLC) component of combined SCLC tumors. Here, we investigated if RUNX1T1 expression plays a role in transforming NSCLC to SCLC in combined SCLC tumor formation. Methods: We examined tumors from 92 of our SCLC patients by targeted exome and CNV analyses. RUNX1T1 mRNA expression was determined in various cancers by analysis of the CCLE and TCGA databases. RUNX1T1 mRNA expression was detected by RNAscope analysis of another 22 patient SCLC tumors. Stable overexpression of RUNX1T1 in NSCLC and SCLC cell lines used commercial RUNX1T1 lentiviral constructs. The resulting stable cell lines were analyzed by RT-qPCR, western blotting, Affymetrix gene array, and E2F-luciferase assays. Results: We found that 2 of 2 patients with combined SCLC demonstrated RUNX1T1 amplification in the SCLC component. Notably, only 2 of the remaining 90 patients with pure SCLC demonstrated RUNX1T1 amplification. RUNX1T1 mRNA is highly expressed in SCLC compared to the majority of other cancers, including NSCLC, using either CCLE or TCGA databases. Western blots confirmed higher RUNX1T1 protein expression in SCLC vs NSCLC cell lines, and RNAscope analysis confirmed greater RUNX1T1 mRNA expression in SCLC vs NSCLC patient tumors. When we stably overexpressed RUNX1T1 in NSCLC cell lines, there was no increase in neuroendocrine protein expression, however we did find an increased sensitivity to etoposide. Bioinformatic analysis of microarray data demonstrated significantly altered E2F pathway activity, and we could show a significant increase in E2F-luciferase activity after RUNX1T1 overexpression. Interestingly, this was associated with decreased p21 expression, which is a putative suppressor of E2F activity. Conclusions: RUNX1T1 expression is specifically upregulated in SCLC compared to NSCLC. E2F pathway activity is elevated after RUNX1T1 overexpression, consistent with an increased SCLC phenotype.

Lung Cancer in Germany

11552 Background: SMARCA4-deficient thoracic sarcomas (SDS) are rare and aggressive sarcomas characterized by inactivating SMARCA4 mutations, with no approved treatment to date. Previous data linking SWI/SNF deficiency with tumor immune microenvironment (TME) are contradictory. While an immunogenic microenvironment and efficacy of immune checkpoint inhibitors (ICI) were described in SMARCA4-deficient small cell carcinoma of the ovary, the TME phenotype of SDS is unknown; in addition, response of patients to ICI is lacking. Methods: All consecutive patients diagnosed with SDS between 2016 and 2019 in Strasbourg University Hospital were included and clinical outcomes collected. Immunostainings for immune cell markers, immune checkpoints and tertiary lymphoid structures (TLS) were assessed on available samples. Validation was performed using an independent transcriptomes dataset of SDS (n = 12), not otherwise specified (NOS) non-small cell lung cancer (NSCLC) with/without SMARCA4 mutations (n = 14) and undifferentiated thoracic sarcoma (n = 5). Finally, chemokines (CXL9 and CXCL10) and PD-L1 expressions were assessed in NSCLC and thoracic fibroblast cell lines, treated with/without interferon gamma (IFNG). Results: Nine patients were identified and had all metastatic disease at presentation, with a median overall survival of 1.8 months (0.3-NR). Among them, 4 received ICI as part of their treatment. Out of 11 evaluated tumors samples, all but one case showed no TLS, consistent with an immune desert TME phenotype charted by low densities of CD3+ T-cells, CD8+ T-cells, CD20+ B-cells from one side and high density of CD68+ macrophage-cells from the other side. Conversely, the unique tumor with TLS aggregate showed an immune-rich TME phenotype associated with high mutational tumor burden. While the patient with TLS harboring tumor showed an exceptional long-lasting response, the 3 remaining patients without TLS had progressive disease at best response to ICI. Using an independent cohort, unsupervised clustering using immune cell scores identified two clusters tightly associated with cell ontogeny of cancer subtypes and immunity; while cluster 1 (C1) was enriched for NOS NSCLC independently from SMARCA4 status (n = 9/10; 90%) (p = 0.001), C2 was enriched for SDS (n = 11/12; 91.7%) (p = 0.005) and undifferentiated thoracic sarcomas (n = 5/5; 100%) (p = 0.0005). Finally, SMARCA4 loss of function experiments revealed upregulation of chemokines (CXL9 and CXCL10) and PD-L1 expression in the NSCLC cell line with no effect on thoracic fibroblast cell line. Conclusions: SDS harbor an immune desert TME phenotype with limited efficacy to ICI, similar to other sarcomas. Our data suggest that TME of SMARCA4-driven tumors might vary according to the cell of origin. Further studies are needed to understand the interplay between SWI/SNF mutations, cell ontogeny and immunity.

Background: Small cell lung cancer (SCLC) is a highly lethal malignancy characterized by rapid growth, early metastasis and poor prognosis. SCLC shows distinct molecular and clinical features when compared to other lung cancer subtypes. Previous analyses by us and others have identified genomic and proteomic differences between SCLC and Non-Small Cell Lung Cancer (NSCLC). Epigenetic alterations are some of the earliest events that could also lead to oncogenic changes and thus play an essential role in tumor initiation and progression. However, epigenetic differences between SCLC and NSCLC contributing to the alterations in gene and protein expression patterns, distinct biological features and therapeutic response have not been well characterized. Here, we investigate the differences in the methylation patterns of SCLC and NSCLC to provide novel insights into epigenetic associated gene alterations to identify potential therapeutic targets in SCLC. Material and Methods: A genome-wide DNA methylation profiling of SCLC and NSCLC cell lines was used for this investigation. We correlated DNA methylation status with gene expression and protein expression levels in 31 SCLC and 73 NSCLC lines to identify the relationship of epigenetic with genomic and proteomic features distinguishing SCLC from NSCLC. Results: SCLC and NSCLC lines exhibited different methylation profiles and we identified 484 genes that had a significant inverse correlation between methylation status and mRNA expression levels (Rho ≤ -0.5 and FDR = 0.01), (“genes regulated by methylation,” GRM) that distinguished SCLC from NSCLC. Ingenuity pathway analysis of the 484 genes identified significant associations with neuregulin signaling, immune trafficking, integrin signaling, glioma invasiveness canonical pathways. Proteomic profiling by Reverse Phase Protein Array (RPPA) validated the different expression of some of the 484 genes identifying nine that were hypermethylated and downregulated at protein levels in SCLC compared to NSCLC lines (PTEN, CyclinD1, Caveolin, Notch3, TAZ, HSP27, STAT6 and both total and phosphorylated levels of receptor tyrosine kinases such Her2 and, MET). Conclusions: Genome wide methylation, mRNA expression, and detailed proteomic analyses have identified specific epigenic differences between SCLC and NSCLC that impact on important signaling pathways including widespread loss of PTEN function and receptor tyrosine kinase (RTK) expression in SCLCs which need to be considered in developing new rationale therapies for SCLC. Citation Format: Seema Mukherjee, Bonnie S. Glisson, John D. Minna, Robert J. Cardnell, Luc Girard, Adi Gazdar, Lixia Diao, Jing Wang, Lauren A. Byers. Characterization of methylation profiles reveals distinct epigenomic patterns in SCLC and NSCLC. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2961. doi:10.1158/1538-7445.AM2015-2961

BackgroundProfound advances in cancer patient treatments have come with the advent of immunotherapies directed at blocking immune-suppressive ligand-receptor interactions. However, while there has been success in the use of these...

PACIFIC: Time for a surgical IIIA uprising

Introduction: While there have been multiple studies identifying differences in phenotypes of small cell lung cancer (SCLC) compared to non-small cell lung cancer (NSCLC), identification of key signaling pathways that may differ and may represent new therapeutic targets for SCLC need to be specified. To this end, we have compared expression of a large number of proteins and genes involved in key cell regulatory pathways of SCLC with those in NSCLC using reverse phase protein array (RPPA) and genome-wide mRNA profiling. Methods: 34 SCLC and 74 NSCLC cell lines that have been previously characterized for oncogenotype mutations, copy number changes, and mRNA expression profiles (using Affymetrix U133A, U133B, U133 Plus 2.0, and Illumina arrays) were grown under 10% serum, serum-starved, and serum-stimulated conditions. Cell lysates from each media condition were harvested and 176 total and phosphoproteins representing multiple signaling pathways were quantified by RPPA. Differences in protein and gene expression levels between SCLC and NSCLC lines were assessed using unsupervised hierarchical clustering, t-test, and ANOVA. Results: Unsupervised hierarchical clustering separated SCLC from NSCLC cell lines based on their distinct protein expression patterns under all three media conditions. Two high-grade neuroendocrine NSCLC cell lines clustered with the SCLC cell lines, reflecting their SCLC-like protein expression. Using a false discovery rate of <1% to identify protein markers most differentially expressed between SCLC and NSCLC, we observed high levels of proteins in DNA repair pathways (ex., PARP1, XRCC1, ATM, Chk1, 53BP1) and in apoptosis pathways (ex., Bcl-2, BIM, BAX). In contrast, MAPK pathway activity was downregulated, as reflected by low levels of phospho-ERK1/2. Among SCLC lines, an inverse correlation was observed between cKit and cMyc protein levels such that one group overexpressed cKit but had relatively lower cMyc levels, while the other group (enriched for cMyc amplification) had high cMyc and low cKit. Gene expression analysis showed similar results, with good correlation between non-phosphorylated proteins and mRNA levels among the pathways differentially expressed in SCLC. Conclusions: Proteomic profiling identified increased DNA repair and apoptosis pathway activity and decreased MAPK pathway activity in SCLC. Among the proteins upregulated in SCLC, several have potential as therapeutic targets, such as PARP1. Although cKit over expression and cMyc amplification are both common in SCLC, we showed that they were mutually exclusive of each other at a protein expression level, reflecting the heterogeneity of SCLC and suggesting a rationale for testing targeted therapies specifically in subsets of patients whose tumors overexpress the targeted marker. Pathway activation will be further investigated in SCLC patient tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5556.

<div>Abstract<p><b>Purpose:</b> Therapies targeted to the immune checkpoint mediated by PD-1 and PD-L1 show antitumor activity in a subset of patients with non–small cell lung cancer (NSCLC). We have now examined PD-L1 expression and its regulation in NSCLC positive for the <i>EML4</i>–<i>ALK</i> fusion gene.</p><p><b>Experimental Design:</b> The expression of PD-L1 at the protein and mRNA levels in NSCLC cell lines was examined by flow cytometry and by reverse transcription and real-time PCR analysis, respectively. The expression of PD-L1 in 134 surgically resected NSCLC specimens was evaluated by immunohistochemical analysis.</p><p><b>Results:</b> The PD-L1 expression level was higher in NSCLC cell lines positive for <i>EML4</i>–<i>ALK</i> than in those negative for the fusion gene. Forced expression of EML4–ALK in Ba/F3 cells markedly increased PD-L1 expression, whereas endogenous PD-L1 expression in <i>EML4</i>–<i>ALK</i>–positive NSCLC cells was attenuated by treatment with the specific ALK inhibitor alectinib or by RNAi with ALK siRNAs. Furthermore, expression of PD-L1 was downregulated by inhibitors of the MEK–ERK and PI3K–AKT signaling pathways in NSCLC cells positive for either <i>EML4</i>–<i>ALK</i> or activating mutations of the EGFR. Finally, the expression level of PD-L1 was positively associated with the presence of <i>EML4</i>–<i>ALK</i> in NSCLC specimens.</p><p><b>Conclusions:</b> Our findings that both EML4–ALK and mutant EGFR upregulate PD-L1 by activating PI3K–AKT and MEK–ERK signaling pathways in NSCLC reveal a direct link between oncogenic drivers and PD-L1 expression. <i>Clin Cancer Res; 21(17); 4014–21. ©2015 AACR</i>.</p></div>

<div>Abstract<p><b>Purpose:</b> Therapies targeted to the immune checkpoint mediated by PD-1 and PD-L1 show antitumor activity in a subset of patients with non–small cell lung cancer (NSCLC). We have now examined PD-L1 expression and its regulation in NSCLC positive for the <i>EML4</i>–<i>ALK</i> fusion gene.</p><p><b>Experimental Design:</b> The expression of PD-L1 at the protein and mRNA levels in NSCLC cell lines was examined by flow cytometry and by reverse transcription and real-time PCR analysis, respectively. The expression of PD-L1 in 134 surgically resected NSCLC specimens was evaluated by immunohistochemical analysis.</p><p><b>Results:</b> The PD-L1 expression level was higher in NSCLC cell lines positive for <i>EML4</i>–<i>ALK</i> than in those negative for the fusion gene. Forced expression of EML4–ALK in Ba/F3 cells markedly increased PD-L1 expression, whereas endogenous PD-L1 expression in <i>EML4</i>–<i>ALK</i>–positive NSCLC cells was attenuated by treatment with the specific ALK inhibitor alectinib or by RNAi with ALK siRNAs. Furthermore, expression of PD-L1 was downregulated by inhibitors of the MEK–ERK and PI3K–AKT signaling pathways in NSCLC cells positive for either <i>EML4</i>–<i>ALK</i> or activating mutations of the EGFR. Finally, the expression level of PD-L1 was positively associated with the presence of <i>EML4</i>–<i>ALK</i> in NSCLC specimens.</p><p><b>Conclusions:</b> Our findings that both EML4–ALK and mutant EGFR upregulate PD-L1 by activating PI3K–AKT and MEK–ERK signaling pathways in NSCLC reveal a direct link between oncogenic drivers and PD-L1 expression. <i>Clin Cancer Res; 21(17); 4014–21. ©2015 AACR</i>.</p></div>

Cytotoxic chemotherapeutic agents and the EGFR-TKI osimertinib induce calreticulin exposure in non–small cell lung cancer

<div>AbstractPurpose:<p>The response to immune checkpoint inhibitors (ICI) often differs between genders in non–small cell lung cancer (NSCLC), but metanalyses results are controversial, and no clear mechanisms are defined. We aim at clarifying the molecular circuitries explaining the differential gender-related response to anti–PD-1/anti–PD-L1 agents in NSCLC.</p>Experimental Design:<p>We prospectively analyzed a cohort of patients with NSCLC treated with ICI as a first-line approach, and we identified the molecular mechanisms determining the differential efficacy of ICI in 29 NSCLC cell lines of both genders, recapitulating patients’ phenotype. We validated new immunotherapy strategies in mice bearing NSCLC patient-derived xenografts and human reconstituted immune system (“immune-PDXs”).</p>Results:<p>In patients, we found that estrogen receptor α (ERα) was a predictive factor of response to pembrolizumab, stronger than gender and PD-L1 levels, and was directly correlated with PD-L1 expression, particularly in female patients. ERα transcriptionally upregulated <i>CD274/PD-L1</i> gene, more in females than in males. This axis was activated by 17-β-estradiol, autocrinely produced by intratumor aromatase, and by the EGFR-downstream effectors Akt and ERK1/2 that activated ERα. The efficacy of pembrolizumab in immune-PDXs was significantly improved by the aromatase inhibitor letrozole, which reduced PD-L1 and increased the percentage of antitumor CD8<sup>+</sup>T-lymphocytes, NK cells, and Vγ9Vδ2 T-lymphocytes, producing durable control and even tumor regression after continuous administration, with maximal benefit in <i><sup>17-β-estradiol/ERα high</sup></i>female immune-xenografts.</p>Conclusions:<p>Our work unveils that 17-β-estradiol/ERα status predicts the response to pembrolizumab in patients with NSCLC. Second, we propose aromatase inhibitors as new gender-tailored immune-adjuvants in NSCLC.</p></div>