EGFR-mutant Tumors Research Articles

Developmental mosaicism underlying EGFR-mutant lung cancer presenting with multiple primary tumors.

Although the development of multiple primary tumors in smokers with lung cancer can be attributed to carcinogen-induced field cancerization, the occurrence of multiple tumors at presentation in individuals with EGFR-mutant lung cancer who lack known environmental exposures remains unexplained. In the present study, we identified ten patients with early stage, resectable, non-small cell lung cancer who presented with multiple, anatomically distinct, EGFR-mutant tumors. We analyzed the phylogenetic relationships among multiple tumors from each patient using whole-exome sequencing (WES) and hypermutable poly(guanine) (poly(G)) repeat genotyping as orthogonal methods for lineage tracing. In four patients, developmental mosaicism, assessed by WES and poly(G) lineage tracing, indicates a common non-germline cell of origin. In two other patients, we identified germline EGFR variants, which confer moderately enhanced signaling when modeled in vitro. Thus, in addition to germline variants, developmental mosaicism defines a distinct mechanism of genetic predisposition to multiple EGFR-mutant primary tumors, with implications for their etiology and clinical management.

Updated overall survival and ctDNA analysis in patients with EGFR T790M-positive advanced non-small cell lung cancer treated with lazertinib in the phase 1/2 LASER201 study.

Lazertinib is a potent, irreversible, third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) with significant efficacy in patients with EGFR T790M-mutated non-small cell lung cancer (NSCLC). This is the final overall survival (OS) report from the phase 1/2 LASER201 study in patients with advanced NSCLC with disease progression on or after prior EGFR TKI therapy. Eligible patients were aged ≥ 20years, with advanced EGFR-mutated NSCLC and previous therapy with EGFR TKI. Patients in this integrated analysis received oral lazertinib 240mg/day. Endpoints included efficacy and safety; exploratory analyses included associations between circulating EGFR-mutant tumor DNA (ctDNA) and efficacy parameters. This integrated analysis included 78 patients in Korea who received second- or later-line lazertinib. The median OS was 38.9months; estimated survival rates at 12, 24, and 36months were 89.5%, 73.9%, and 52.8%, respectively. The cumulative 12-month incidence of central nervous system progression was 9.4%. EGFR-mutant ctDNA was detected in 46 patients (62.2%) at baseline. The presence of ctDNA at baseline significantly predicted progression-free survival (PFS), disease control rate (DCR), and OS. PFS, response rate, and DCR were significantly associated with EGFR-mutant ctDNA clearance at cycle 3; PFS and OS were significantly associated with ctDNA clearance at cycle 5. The safety profile of lazertinib 240mg/day was consistent with previous findings. Lazertinib is a promising treatment option for patients with EGFR T790M-positive NSCLC following disease progression on prior EGFR-directed TKIs. Patients in LASER201 experienced prolonged OS, regardless of their EGFR mutation, brain metastases, or prior brain radiation status. Clearance of plasma EGFR mutations after lazertinib was associated with patient outcomes. ClinicalTrials.gov identifier NCT03046992.

Tumor Cell Spatial Organization Directs EGFR/RAS/RAF Pathway Primary Therapy Resistance through YAP Signaling.

Non-small cell lung cancers (NSCLC) harboring common mutations in EGFR and KRAS characteristically respond transiently to targeted therapies against those mutations, but invariably, tumors recur and progress. Resistance often emerges through mutations in the therapeutic target or activation of alternative signaling pathways. Mechanisms of acute tumor cell resistance to initial EGFR (EGFRi) or KRASG12C (G12Ci) pathway inhibition remain poorly understood. Our study reveals that acute response to EGFR/RAS/RAF-pathway inhibition is spatial and culture context specific. In vivo, EGFR mutant tumor xenografts shrink by > 90% following acute EGFRi therapy, and residual tumor cells are associated with dense stroma and have increased nuclear YAP. Interestingly, in vitro EGFRi induced cell cycle arrest in NSCLC cells grown in monolayer, while 3D spheroids preferentially die upon inhibitor treatment. We find differential YAP nuclear localization and activity, driven by the distinct culture conditions, as a common resistance mechanism for selective EGFR/KRAS/BRAF pathway therapies. Forced expression of the YAPS127A mutant partially protects cells from EGFR-mediated cell death in spheroid culture. These studies identify YAP activation in monolayer culture as a non-genetic mechanism of acute EGFR/KRAS/BRAF therapy resistance, highlighting that monolayer vs spheroid cell culture systems can model distinct stages of patient cancer progression.

Integrative transcriptome analysis reveals the molecular events underlying impaired T-cell responses in EGFR-mutant lung cancer

EGFR mutations are critical oncogenic drivers in lung adenocarcinoma (LUAD). However, the mechanisms by which they impact the tumor microenvironment (TME) and tumor immunity are unclear. Furthermore, the reasons underlying the poor response of EGFR-mutant (EGFR-MU) LUADs to immunotherapy with PD-1/PD-L1 inhibitors are unknown. Utilizing single-cell RNA (sc-RNA) and bulk RNA sequencing datasets, we conducted high-dimensional weighted gene coexpression network analysis to identify key genes and immune-related pathways contributing to the immunosuppressive TME. EGFR-MU cancer cells downregulated MHC class I genes to evade CD8+ cytotoxic T cells, expressed substantial levels of MHC class II molecules, and engaged with CD4+ regulatory T cells (Tregs). EGFR-MU tumors may recruit Tregs primarily through the CCL17/CCL22/CCR4 axis, leading to a Treg-enriched TME. High levels of MHC class II-positive cancer-associated fibroblasts and tumor endothelial cells were found within EGFR-MU tumors. Owing to the absence of costimulatory factors, they may inhibit rather than activate the tumor antigen-specific CD4+ T-cell response, contributing further to immune suppression. Multiplex immunohistochemistry analyses in a LUAD cohort confirmed increased expression of MHC class II molecules in cancer cells and fibroblasts in EGFR-MU tumors. Our research elucidates the highly immunosuppressive TME in EGFR-MU LUAD and suggests potential targets for effective immunotherapy.

EGFR mutations induce the suppression of CD8+ T cell and anti-PD-1 resistance via ERK1/2-p90RSK-TGF-β axis in non-small cell lung cancer

BackgroundNon-small cell lung cancer (NSCLC) patients with EGFR mutations exhibit an unfavorable response to immune checkpoint inhibitor (ICI) monotherapy, and their tumor microenvironment (TME) is usually immunosuppressed. TGF-β plays an important role in immunosuppression; however, the effects of TGF-β on the TME and the efficacy of anti-PD-1 immunotherapy against EGFR-mutated tumors remain unclear.MethodsCorresponding in vitro studies used the TCGA database, clinical specimens, and self-constructed mouse cell lines with EGFR mutations. We utilized C57BL/6N and humanized M-NSG mouse models bearing EGFR-mutated NSCLC to investigate the effects of TGF-β on the TME and the combined efficacy of TGF-β blockade and anti-PD-1 therapy. The changes in immune cells were monitored by flow cytometry. The correlation between TGF-β and immunotherapy outcomes of EGFR-mutated NSCLC was verified by clinical samples.ResultsWe identified that TGF-β was upregulated in EGFR-mutated NSCLC by EGFR activation and subsequent ERK1/2-p90RSK phosphorylation. TGF-β directly inhibited CD8+ T cell infiltration, proliferation, and cytotoxicity both in vitro and in vivo, but blocking TGF-β did not suppress the growth of EGFR-mutated tumors in vivo. Anti-TGF-β antibody combined with anti-PD-1 antibody significantly inhibited the proliferation of recombinant EGFR-mutated tumors in C57BL/6N mice, which was superior to their monotherapy. Mechanistically, the combination of anti-TGF-β and anti-PD-1 antibodies significantly increased the infiltration of CD8+ T cells and enhanced the anti-tumor function of CD8+ T cells. Moreover, we found that the expression of TGF-β1 in EGFR-TKI resistant cell lines was significantly higher than that in parental cell lines. The combination of anti-TGF-β and nivolumab significantly inhibited the proliferation of EGFR-TKI resistant tumors in humanized M-NSG mice and prolonged their survival.ConclusionsOur results reveal that TGF-β expression is upregulated in NSCLC with EGFR mutations through the EGFR-ERK1/2-p90RSK signaling pathway. High TGF-β expression inhibits the infiltration and anti-tumor function of CD8+ T cells, contributing to the “cold” TME of EGFR-mutated tumors. Blocking TGF-β can reshape the TME and enhance the therapeutic efficacy of anti-PD-1 in EGFR-mutated tumors, which provides a potential combination immunotherapy strategy for advanced NSCLC patients with EGFR mutations.Graphical

Phase II Trial of Afatinib in Patients With EGFR-Mutated Solid Tumors Excluding Lung Cancer: Results From NCI-MATCH ECOG-ACRIN Trial (EAY131) Subprotocol A.

National Cancer Institute-Molecular Analysis for Therapy Choice (NCI-MATCH) was a multicohort phase 2 trial that assigned patients with advanced pretreated cancers to molecularly targeted therapies on the basis of tumor genomic testing. NCI-MATCH Arm A evaluated afatinib, an EGFR tyrosine kinase inhibitor (TKI) approved for advanced non-small cell lung cancer, in patients with tumors other than lung cancer harboring EGFR mutations. Patients with advanced pretreated cancers other than lung cancer found to have selected actionable EGFR mutations were offered participation in Arm A. Previous therapy with an EGFR TKI was not allowed. Patients received afatinib 40 mg once daily continuously until disease progression or unacceptable toxicity. The primary end point was objective response rate (ORR). Secondary end points included progression-free survival (PFS), 6-month PFS, and overall survival (OS). Seventeen patients received protocol therapy. Tumor types included glioblastoma multiforme (GBM) (13), gliosarcoma (1), adenocarcinoma not otherwise specified (NOS) (2), and adenosquamous carcinoma of the breast (1). Fifty-nine percent of patients received ≥2 lines of previous therapy. The ORR was 11.8% (90% CI, 2.1 to 32.6), with one complete response lasting 16.4 months (GBM harboring a rare exon 18 EGFR-SEPT14 fusion) and one partial response lasting 12.8 months (adenocarcinoma NOS with the classic EGFR mutation, p.Glu746_Ala750del). Three patients had stable disease. The 6-month PFS was 15% (90% CI, 0 to 30.7); the median OS was 9 months (90% CI, 4.6 to 14.0). Rash and diarrhea were the most common toxicities. Afatinib had modest activity in a cohort of patients with heavily pretreated cancer with advanced nonlung, EGFR-mutated tumors, but the trial's primary end point was not met. Further evaluation of afatinib in GBM with EGFR exon 18 fusions may be of interest.

New Treatment Options for Patients With Oncogene-Addicted Non-Small Cell Lung Cancer Focusing on EGFR-Mutant Tumors.

Druggable oncogene-driven non-small cell lung cancer has led to innovative systemic treatment options, improving patients' outcome. This benefit is not only achieved in the metastatic setting but also in the postsurgical setting, such as in lung cancers harboring a common sensitizing EGFR mutation or ALK-rearrangement. To enhance the outcome of these patients, we need to understand the mechanisms of acquired resistance and evaluate the role of new drugs with novel mechanisms of action in the treatment landscape. In this chapter, we review treatment strategies of EGFR-mutant tumors in all stages, the mechanisms of acquired strategies, and novel therapies in this subset.

PD-L2 drives resistance to EGFR-TKIs: dynamic changes of the tumor immune environment and targeted therapy.

There is a lack of effective treatments to overcome resistance to EGFR-TKIs in EGFR mutant tumors. A deeper understanding of resistance mechanisms can provide insights into reducing or eliminating resistance, and can potentially deliver targeted treatment measures to overcome resistance. Here, we identified that the dynamic changes of the tumor immune environment were important extrinsic factors driving tumor resistance to EGFR-TKIs in EGFR mutant cell lines and syngeneic tumor-bearing mice. Our results demonstrate that the acquired resistance to EGFR-TKIs is accompanied by aberrant expression of PD-L2, leading a dynamic shift from an initially favorable tumor immune environment to an immunosuppressive phenotype. PD-L2 expression significantly affected EGFR mutant cell apoptosis that depended on the proportion and function of CD8+ T cells in the tumor immune environment. Combined with single-cell sequencing and experimental results, we demonstrated that PD-L2 specifically inhibited the proliferation of CD8+ T cells and the secretion of granzyme B and perforin, leading to reduced apoptosis mediated by CD8+ T cells and enhanced immune escape of tumor cells, which drives EGFR-TKIs resistance. Importantly, we have identified a potent natural small-molecule inhibitor of PD-L2, zinc undecylenate. In vitro, it selectively and potently blocks the PD-L2/PD-1 interaction. In vivo, it abolishes the suppressive effect of the PD-L2-overexpressing tumor immune microenvironment by blocking PD-L2/PD-1 signaling. Moreover, the combination of zinc undecylenate and EGFR-TKIs can synergistically reverse tumor resistance, which is dependent on CD8+ T cells mediating apoptosis. Our study uncovers the PD-L2/PD-1 signaling pathway as a driving factor to mediate EGFR-TKIs resistance, and identifies a new naturally-derived agent to reverse EGFR-TKIs resistance.

Abstract 1989: Intratumor heterogeneity in EGFR-mutant lung tumors mediates targeted therapy resistance and formation of drug tolerant microenvironment

Abstract Introduction: Non-small cell lung cancers (NSCLC) harboring EGFR mutations are commonly treated with EGFR tyrosine kinase inhibitors (TKIs), but the emergence of drug resistance often leads to treatment failure. While various acquired resistance mechanisms have been identified, they are not always actionable. Furthermore, since drug resistance is typically studied after cancer relapse, little is known about the cellular or microenvironmental taking place during the formation of resistance at the stage of minimal residual disease (MRD). Therefore, it is necessary to understand the mechanisms leading to resistance during MRD and develop new ways to prevent drug resistance in EGFR-mutant cancers. Methods: In this study, we discovered pre-existing intratumor heterogeneity in EGFR expression within EGFR-mutant NSCLCs and found that patient tumors contain clones with low mutant EGFR expression. To study the effect of this intratumor heterogeneity on drug response, we isolated low and high expressing cells and conducted longitudinal drug challenge assays in ex vivo and in vivo models. To dissect how the observed heterogeneity affects drug resistance and the tumor microenvironment during MRD, we employed patient-derived tumor cells and used novel tumor-on-a-chip co-culture approaches. Results: Our findings reveal that EGFR-low cells are more tolerant to drug treatment and can survive the initial TKI insult, making them more likely to contribute to drug resistance in EGFR-mutant NSCLCs. Additionally, the surviving EGFR-low cells were able to modify the tumor microenvironment leading to fibroblast accumulation and immune suppression, contributing to the drug-tolerant tumor microenvironment. Importantly, we found that pharmacological induction of EGFR using epigenetic inhibitors was able to sensitize the drug tolerant cells to EGFR inhibition, suggesting a potential strategy to overcome TKI resistance in NSCLC patients. Conclusions: In conclusion, our study sheds light on the critical role of intratumoral heterogeneity in treatment resistance and offers valuable insights into potential strategies to overcome this challenge. Our findings suggest that combination therapies that can alter the cellular phenotype during MRD have the potential to prevent intrinsic drug resistance. This underscores the importance of gaining a better understanding of the mechanisms underlying drug-tolerant microenvironment and developing more effective treatment strategies for patients with EGFR-mutant NSCLCs. Citation Format: Heidi M. Haikala, Bassel Alsaed, Jieun Son, Linh Lin, Prafulla Gokhale, Pasi Jänne. Intratumor heterogeneity in EGFR-mutant lung tumors mediates targeted therapy resistance and formation of drug tolerant microenvironment [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 1989.

Abstract 5351: Tumor PKC-Delta induces immune exclusion in non-small cell lung cancer

Abstract The recruitment of a sufficient number of immune cells to induce an inflamed tumor microenvironment (TME) is a prerequisite for effective response to cancer immunotherapy. The immunological phenotypes in the TME of EGFR-mutated lung cancer were characterized as non-inflamed, for which immunotherapy is largely ineffective. To orchestrate host immunity against lung tumors, we proposed that PKCδ, a gatekeeper of immune homeostasis with kinase activity, is responsible for the un-inflamed phenotype in EGFR-mutated lung tumors. It promotes tumor progression by stimulating extracellular matrix (ECM) and PD-L1 expression which leads to immune exclusion and assists cancer cell escape from T cell surveillance. Ablation of PKCδ enhances the intratumoral penetration of T cells and suppresses the growth of tumors. Furthermore, blocking PKCδ significantly sensitizes the tumor to immune checkpoint blockade (ICB) therapy (αPD-1) in vitro and in vivo models. These findings revealed that PKCδ is a critical switch to induce inflamed tumors and consequently enhances the efficacy of ICB therapy in EGFR-mutated lung cancer. This opens a new avenue for applying immunotherapy against recalcitrant tumors. This work was funded by the FDCT project grant to Dr. Xing-Xing Fan (Grant no. 0058/2020/A2). Citation Format: Xing Xing Fan. Tumor PKC-Delta induces immune exclusion in non-small cell lung cancer [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 5351.

Abstract 5829: Elucidating the significance of amphiregulin and its implications for EGFR inhibition strategies in LKB1-mutant NSCLC

Abstract LKB1-mutant non-small cell lung cancer (NSCLC) is aggressive and treatment-refractory, underscoring the need for new therapeutic strategies. Our comparative study involving mouse lung tumors driven by oncogenic KrasG12D and LKB1 loss as well as tumors driven solely by KrasG12D, revealed that LKB1 loss upregulated amphiregulin (Areg), a pro-growth EGFR ligand. Therefore, this study investigates how the inactivation of LKB1 enhances AREG expression, the significance of elevated AREG in sustaining lung cancer growth, and the responses of AREG-high lung cancer to EGFR inhibitors. We first evaluated the potential clinical significance of AREG using The Cancer Genome Atlas (TCGA) lung adenocarcinoma dataset and the Cancer Cell Line Encyclopedia (CCLE) database. We observed that LKB1-null tumors with high AREG levels were associated with shorter patient survival and exhibited transcriptional signatures similar to EGFR-mutant tumors. High AREG expression also correlated positively in NSCLC cell lines with LKB1 mutations and Western blot analysis confirmed that elevated AREG levels in lung cancer cells were associated with constitutive EGFR signaling. We next explored the impact of LKB1 loss on AREG expression in lung cancer. Our prior RNA-seq and ChIP-seq investigations demonstrated CRTC2 and CREB enrichment on the AREG promoter in LKB1-null lung cancer cells. Upon LKB1 reintroduction, the enrichment diminished, indicating that CRTC-CREB transcriptionally induces AREG in LKB1-null lung cancer. To corroborate, reintroducing LKB1 or expressing a dominant-negative construct (dnCRTC), disrupting CREB/CRTC interaction, decreased AREG levels, as confirmed by RT-qPCR in LKB1-null lung cancer cells. This supports that AREG is a direct target of CRTC-CREB activation induced by LKB1 loss. To assess the impact of elevated AREG in LKB1-mutant lung cancer growth, we used RNA interference and antibodies to inhibit AREG. Both interventions decreased cell growth and colony formation, underscoring AREG is critical for LKB1-mutant lung cancer growth. Moreover, we investigated the potential correlation between AREG and the response to the EGFR tyrosine-kinase inhibitor Erlotinib in NSCLC cell lines, utilizing AREG expression data from CCLE and Erlotinib response data from the Genomics of Drug Sensitivity in Cancer (CDSC) databases. Stratifying NSCLC cell lines into sensitive, intermediate, and resistant categories revealed a potential positive association between AREG expression and Erlotinib sensitivity. Notably, LKB1-null lung cell lines with high AREG expression demonstrated heightened sensitivity compared to those with low AREG expression. Our study revealed that LKB1 loss increases AREG, triggering aberrant EGFR signaling critical for lung cancer growth, and AREG expression might impact the Erlotinib response in LKB1-inactive cells. Citation Format: Xzaviar Kaymar Solone, Xin Zhou, Mu Yu, Jennifer W. Li, Finn Rimay, Frederic Kaye, Lizi Wu. Elucidating the significance of amphiregulin and its implications for EGFR inhibition strategies in LKB1-mutant NSCLC [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 5829.

Detection of EGFR T790M mutation using liquid biopsy for non-small cell lung cancer: Utility of droplet digital polymerase chain reaction vs. cobas real-time polymerase chain reaction

BackgroundDigital platforms for mutation detection yield higher sensitivity than non-digital platforms but lack universal positive cut-off values that correlate with the outcome of osimertinib treatment. This study determined compared droplet digital polymerase chain reaction (ddPCR) to the standard cobas assay for epithelial growth factor receptor (EGFR) T790M mutation detection in patients with non-small cell lung cancer. MethodsStudy patients had EGFR-mutant tumours with disease progression on first/second generation EGFR tyrosine kinase inhibitors, and osimertinib treatment after T790M mutation detection. T790M status was tested by cobas assay using liquid biopsy, and only by ddPCR if an EGFR mutation was identified but T790M was negative. Clinical efficacy of osimertinib was compared between patients with T790M detected by cobas vs. only by ddPCR. A positive cut-off value for ddPCR was determined by assessing efficacy with osimertinib. Results61 patients had tumors with an acquired T790M mutation, 38 detected by cobas and an additional 23 only by ddPCR. The median progression-free survival (PFS) for the cobas- and ddPCR-positive groups was 9.5 and 7.8 months, respectively (p=0.43). For ddPCR, a fractional abundance (FA) of 0.1% was used as a cut-off value. The median PFS of patients with FA ≥0.1% and <0.1% was 8.3 and 4.6 months, respectively (p=0.08). FA ≥0.1% was independently associated with a longer PFS. ConclusionUsing ddPCR to follow up the cobas assay yielded more cases (38% of total) with a T790M mutation. A cut-off value of FA ≥0.1% identified patients who responded as well to osimertinib as those identified by cobas assay. This sequential approach should detect additional patients who might benefit from osimertinib treatment.

Functional Heterogeneity in MET Pathway Activation in PDX Models of Osimertinib-resistant EGFR-driven Lung Cancer.

Using a novel cohort of in vivo PDX models of MET pathway activation with acquired resistance to osimertinib in EGFR-mutant lung cancer, we demonstrate that phospho-MET may be a clinically relevant assay to guide treatment selection with osimertinib and savolitinib combination. In addition, our work shows that patients with MET polysomy tumors may have subclonal MET amplification and therefore require close follow up for the use of osimertinib and savolitinib combination.

The HIF-1α/PKM2 Feedback Loop in Relation to EGFR Mutational Status in Lung Adenocarcinoma

Objective Gene mutations in tumor cells can lead to several unique metabolic phenotypes, which are crucial for the proliferation of cancer cells. EGFR mutation (EGFR-mt) is the main oncogenic driving mutation in lung adenocarcinoma (LUAD). HIF-1 α and PKM2 are two key metabolic regulatory proteins that can form a feedback loop and promote cancer growth by promoting glycolysis. Here, the linkage between EGFR mutational status and HIF-1α/PKM2 feedback loop in LUAD were evaluated. Methods Retrospective study were performed on LUAD patients (n = 89) undergoing first-time therapeutic surgical resection. EGFR mutation was analyzed by real-time PCR. Immunohistochemistry was used to measure the expressions of HIF-1α and PKM2. Results We found that the protein expressions of HIF-1α and PKM2 were significantly higher in LUAD than normal lung tissues. In adenocarcinomas, the two protein expressions were both correlated with worse pTNM stage. Moreover, the correlation between the proteins of HIF-1α/PKM2 feedback loop and the EGFR mutational status were also analyzed. We found that EGFR-mt tumors showed higher HIF-1α and PKM2 proteins compared to tumors with EGFR wild-type. Meanwhile, HIF-1α expression was significantly correlated with higher pTNM stage, and PKM2 showed a similar trend, only in EGFR-mutated tumors. The expression of HIF-1α was positively correlated with PKM2 in LUAD, furthermore, this correlation was mainly in patients with EGFR-mt. Conclusion Different expression and clinical features of HIF-1α/PKM2 feedback loop was existed between LUAD and normal lung tissues, especially in EGFR mutational tumors, supporting the relationship between EGFR mutation and the key related proteins of aerobic glycolysis (HIF-1α and PKM2) in lung adenocarcinomas.

Clinical utility of immunohistochemical subtyping in patients with small cell lung cancer

ObjectivesMolecular subtyping of small cell lung cancer (SCLC) tumors based on the expression of four transcription factors (ASCL1, NEUROD1, POU2F3, and YAP1) using immunohistochemical (IHC) staining has recently emerged as a proposed approach. This study was aimed to examine this subtyping method in Asian patients with SCLC and investigate its correlation with treatment efficacy. Materials and methodsSeventy-two tumor samples from patients with SCLC, including de novo cases and those transformed from EGFR-mutant tumors, were analyzed. IHC staining was used to measure the expression of the four transcription factors and conventional SCLC markers. Subtypes were defined based on relative expression levels. The treatment response and outcome of patients receiving immune checkpoint inhibitors and chemotherapy were also reviewed. ResultsASCL1 was the most common subtype, observed in 55.2 % of the samples, followed by NEUROD1 (26.9 %) and POU2F3 (9 %). No tumor exhibited predominant YAP1 positivity, while 41.8 % of the samples demonstrated positivity for two subtype markers. Approximately 50 % of the patients experienced a subtype switch after disease progression. Patients with the ASCL1/NEUROD1 (SCLC-A/N) subtype had similar progression-free survival (PFS) compared to non-SCLC-A/N patients after treatment with immune checkpoint inhibitors plus chemotherapy. Transformed SCLC patients had significantly worse PFS than de novo SCLC patients after chemoimmunotherapy. (2.1 vs. 5.4 months, P = 0.023) ConclusionsThis study revealed the challenges associated with using IHC alone for molecular subtyping, highlighting the frequent co-expression of subtypes and temporal changes following treatment. Further research is warranted to explore the prognostic and therapeutic implications of IHC subtyping in patients with SCLC.

Advancements in understanding cardiotoxicity of EGFR- TKIs in non-small cell lung cancer treatment and beyond.

Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitors (EGFR-TKIs) are a class of oral targeted anticancer drugs that have been demonstrated to significantly inhibit tumor progression and improve clinical prognosis in patients diagnosed with EGFR-mutated tumors, particularly in those with non-small cell lung cancer. However, the sustained usage of EGFR-TKIs may cause potential cardiotoxicity, thus limiting their applicability. The primary objective of this review is to systematically analyze the evolving landscape of research pertaining to EGFR-TKI-induced cardiotoxicity and elucidate its underlying mechanisms, such as PI3K signaling pathway inhibition, ion channel blockade, oxidative stress, inflammatory responses, and apoptosis. Additionally, the review includes an exploration of risk assessment for cardiotoxicity induced by EGFR-TKIs, along with management and response strategies. Prospective research directions are outlined, emphasizing the need for more accurate predictors of cardiotoxicity and the development of innovative intervention strategies. In summation, this review consolidates recent research advances, illuminates the risks associated with EGFR-TKI-induced cardiac toxicity and presents crucial insights for refining clinical dosage protocols, optimizing patient management strategies, and unraveling the intricate mechanisms governing EGFR-TKI-induced cardiotoxicity.

Abstract B099: Targeting Non Small Cell Lung Cancer EGFR-Mutant and EGFR-Inhibitor resistant cell lines by ferroptosis induction: A potential therapeutic approach

Abstract The epidermal growth factor receptor (EGFR) is a crucial signaling protein involved in regulating cellular growth, survival, and proliferation. EGFR mutations, particularly in non-small cell lung cancer (NSCLC), have been extensively studied due to their role in driving tumor progression and resistance to conventional therapies. While EGFR-mutant tumors initially respond well to EGFR inhibitors, emergence of resistance remains a significant challenge. Ferroptosis is a recently discovered form of regulated cell death characterized by accumulation of lipid peroxides and oxidative stress. It has gained attention as a potential therapeutic strategy in combating drug resistance. However, the vulnerability of various EGFR-mutant and resistant NSCLC cells to ferroptosis induction remains poorly understood. To bridge the existing knowledge gap, we investigated the responsiveness of both EGFR-mutant and their resistant counterpart NSCLC cell lines to ferroptosis induction. Interestingly, NSCLC cell lines and patients’ tumors which harbor EGFR mutations are distinct from those carrying STK11 and KEAP1 mutations, which are known to contribute to resistance to ferroptosis. This observation prompted us to test whether ferroptosis inducers could effectively target this specific subset of NSCLC. In our experimental approach, we first utilized a panel of commercially available EGFR mutant cell lines (n=3), including one harboring T790M resistance mutation, which is a common mechanism of acquired resistance to first-generation inhibitors. We subjected the EGFR-mutant cell lines in vitro to a process of acquiring resistance against three generations of EGFR inhibitors (n=4). Subsequently, upon confirmation of resistance, we evaluated their susceptibility to our proprietary ferroptosis inducer. Exome sequencing data of EGFR-inhibitor resistant cell lines and their age-matched control cell lines revealed that the former acquired genetic alterations that have previously been found to confer resistance in EGFR-inhibitor resistant patients’ tumors, suggesting that our approach is valid for modeling relevant resistance mechanisms. Our results indicate that both EGFR-mutant and resistant cell lines exhibit sensitivity to ferroptosis induction. Furthermore, we observed that EGFR-mutant patient-derived cells, which are refractory to EGFR inhibitors of several generations, were strongly cytostatic to our ferroptosis inducer. These findings provide valuable insights into the interplay between EGFR mutations, drug resistance, and ferroptosis. Exploiting the vulnerability of EGFR-inhibitor resistant NSCLC cells to ferroptosis could offer novel therapeutic options for overcoming drug resistance in EGFR-driven cancers. Further investigations into the underlying molecular mechanisms of ferroptosis inducers in NSCLC are warranted for the development of effective therapies. Citation Format: Taronish Dubash, Maria Cristina Munteanu, Cristian Nunez, Shrouq Farah, Laurence Jadin, Branko Radetich, Francesco M Marincola, Darby Schmidt, Nadia Gurvich. Targeting Non Small Cell Lung Cancer EGFR-Mutant and EGFR-Inhibitor resistant cell lines by ferroptosis induction: A potential therapeutic approach [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B099.

Common driver mutations and programmed death-ligand 1 expression in advanced non-small cell lung cancer in smokers and never smokers

BackgroundIn non-small cell lung cancer (NSCLC), there may be a relationship between programmed death-ligand 1 (PD-L1) expression, driver mutations and cigarette smoking.MethodsIn this single-center retrospective study, the relationship between common driver mutations (EGFR mutation and ALK rearrangement) and PD-L1 expression in advanced NSCLC according to the patients’ smoking history was examined. Light, moderate and heavy smokers had smoked < 20, 20–39, and ≥ 40 pack-years, respectively. The level of PD-L1 expression, assessed using Ventana SP263 monoclonal antibody assay, was defined by the tumor proportion score (TPS) as high expression (TPS ≥ 50%), low expression (TPS 1%—49%) and no expression (TPS < 1%).Results101 (52.9%) of 191 advanced NSCLC patients were never smokers. EGFR mutations were more common in never smokers (64.4%) than in smokers (17.8%) with advanced NSCLC (P < 0.0001). A higher proportion of smokers (26.7%) had high PD-L1 expression compared to never smokers (13.9%) (P = 0.042). There was a trend for a higher proportion of male NSCLC patients [28 of 115 (24.3%)] than female patients [10 of 76 (13.2%)] to have high PD-L1 expression (P = 0.087]. High PD-L1 expression was seen in 32 of 110 (29.1%) patients with EGFR wild-type NSCLC but only in 6 of 81 (7.4%) patients with EGFR-mutant tumors (P < 0.0001). Among the 90 smokers with NSCLC, a higher proportion of heavy smokers (35.8%) than non-heavy smokers (13.5%) had high PD-L1 expression (P = 0.034). In patients with adenocarcinoma, high PD-L1 expression was seen in 25 of 77 (32.5%) patients with EGFR wild-type tumors but only in 4 of 70 (5.7%) patients with EGFR-mutant tumors (P < 0.0001). Among patients with adenocarcinoma, a significantly higher proportion of ever smokers (29.3%) than never smokers (13.5%) had high PD-L1 expression (P = 0.032). Among smokers with adenocarcinoma, a significantly higher proportion of heavy smokers (44.1%) than non-heavy smokers (8.3%) had high PD-L1 expression (P = 0.004). On multivariate analysis, after adjusting for gender and smoking status, heavy smoking and EGFR wild-type tumors remained significantly associated with high PD-L1 expression in NSCLCs and also in adenocarcinoma.ConclusionsHeavy smoking and EGFR wild-type tumors were significantly associated with high PD-L1 expression in NSCLCs and also in adenocarcinoma.

ACCLAIM-1 dose escalation: Determination of quaratusugene ozeplasmid and osimertinib recommended phase 2 dose (RP2D).

e15081 Background: Expression of TUSC2, a tumor suppressor gene, is decreased in 82% of patients with NSCLC. Quaratusugene ozeplasmid, which restores TUSC2 expression, consists of a plasmid containing the TUSC2 gene encapsulated in lipid nanoparticles. Preclinical work in mouse xenografts demonstrated markedly improved control of EGFR mutant tumors when quaratusugene ozeplasmid was combined with osimertinib. A previous monotherapy study led to a RP2D of 0.06 mg/kg which was also used in a study in combination with erlotinib. The present study evaluated escalating doses of quaratusugene ozeplasmid with standard osimertinib dosing. Methods: Eligible patients were NSCLC patients with EGFR mutations and progression on osimertinib. Quaratusugene ozeplasmid was administered IV every 21 days in escalating doses and osimertinib 80 mg was administered PO daily. Dexamethasone, acetaminophen, and diphenhydramine were given prior to each treatment to prevent a post-infusion inflammatory syndrome. Efficacy was evaluated at baseline and after every even cycle of treatment using RECIST 1.1 criteria. Safety was evaluated using CTCAE v5, with dose limiting toxicities (DLTs) generally defined as ≥Gr 3 adverse events (AEs). Three dose levels (0.06, 0.09, and 0.12 mg/kg) of quaratusugene ozeplasmid were planned. A standard dose escalation with 3-6 patients/dose level was used. Results: Eight patients were enrolled (2M/6F), median age 59.5 years, with 3 at 0.06 mg/kg, 4 at 0.09 mg/kg, and 1 at 0.12 mg/kg. There were no DLTs. The most common AEs in preliminary data, regardless of relationship, in ≥ 20% were, in order of frequency, myalgia, pyrexia, chills, diarrhea, headache, influenza-like illness and pain. The only Grade 3/4 AEs were lymphopenia and neutropenia in 1 patient each. There was a post-infusion syndrome of myalgia, pyrexia, and chills that started 3-4 hours after quaratusugene ozeplasmid infusion and resolved over several hours with acetaminophen and diphenhydramine therapy. One patient at the 0.06 mg/kg dose level, previously treated with carboplatin, pemetrexed, and osimertinib, had a partial remission (PR) by investigator evaluation and treatment is ongoing after 11 cycles with PR. Another patient at the 0.09 mg/kg dose level, previously treated with osimertinib, has stable disease and is continuing treatment after 9 cycles. Conclusions: There was 1 PR with quaratusugene ozeplasmid and osimertinib combination therapy in the 8 patients enrolled in the Phase 1 dose escalation, all of whom had progressed on osimertinib. Quaratusugene ozeplasmid was generally well tolerated, and was associated with a post-infusion syndrome of fever and chills managed with steroids, acetaminophen and diphenhydramine. Based on these data, the RP2D of quaratusugene ozeplasmid in combination with osimertinib in patients with NSCLC progressing after osimertinib treatment is expected to be 0.12 mg/kg. Clinical trial information: NCT04486833 .

Phase II Randomized Trial of Carboplatin, Pemetrexed, and Bevacizumab With and Without Atezolizumab in Stage IV Nonsquamous Non–Small-Cell Lung Cancer Patients Who Harbor a Sensitizing EGFR Mutation or Have Never Smoked

IntroductionPatients with non–small-cell lung cancer (NSCLC) who have never smoked or have tumors with mutations in EGFR generally derive minimal benefit from single-agent PD-1/PD-L1 checkpoint inhibitors. Prior data indicate that adding PD-L1 inhibition to anti-VEGF and cytotoxic chemotherapy may be a promising approach to overcoming immunotherapy resistance in these patients, however prospective validation is needed. This trial in progress (NCT03786692) is evaluating patients with stage IV NSCLC who have never smoked or who have tumors with sensitizing EGFR alterations to determine if a 4-drug combination of atezolizumab, carboplatin, pemetrexed, and bevacizumab can improve outcomes compared to carboplatin, pemetrexed and bevacizumab without atezolizumab. MethodsThis is a randomized, phase II, multicenter study evaluating carboplatin, pemetrexed, bevacizumab with and without atezolizumab in 117 patients with stage IV nonsquamous NSCLC. Randomization is 2 to 1 favoring the atezolizumab containing arm. Eligible patients include: 1) those with tumors with sensitizing EGFR alterations in exons 19 or 21 or 2) patients who have never smoked and have wild-type tumors (ie, no EGFR, ALK or ROS1 alterations). Patients are defined as having never smoked if they have smoked less than 100 cigarettes in a lifetime. Patients with EGFR-mutated tumors must have disease progression or intolerance to prior tyrosine kinase inhibitor (TKI) therapy. The primary endpoint is progression-free survival (PFS). Secondary endpoints include overall survival (OS), response rate, duration of response, and time to response. ConclusionThis phase II trial is accruing patients at U.S. sites through the National Comprehensive Cancer Network (NCCN). The trial opened in August 2019 and accrual is expected to be completed in the Fall of 2024.