EGFR-TKIs In Non-small Cell Lung Cancer Research Articles

Chemical Scaffolds for the Clinical Development of Mutant-Selective and Reversible Fourth-Generation EGFR-TKIs in NSCLC.

In nonsmall cell lung cancer (NSCLC), as well as in other tumors, the targeted therapy is mainly represented by tyrosine kinase inhibitors (TKIs), small molecules able to target oncogenic driver alterations affecting the gene encoding the epidermal growth factor receptor (EGFR). Up to now, several different TKIs have been developed. However, cancer cells showed an incredible adaptive tumor response to the inhibition of the sequentially mutated EGFR (EGFRM+), triggering the need to explore novel pharmacochemical strategies. This Review summarizes the recent efforts in the development of new reversible next-generation EGFR TKIs to fight the resistance against T790M and C797S mutations. Specifically, after giving an overview of the role of the EGFR's signaling pathways in cancer progression, we are going to discuss the most relevant approved drugs and drug candidates in terms of chemical structure, binding modalities, and their potency and selectivity against the mutated EGFR over the wild-type form. This could provide important guidelines and rationale for the discovery and iterative development of new drugs.

Abstract 6484: Overcoming resistance to EGFR-TKI in NSCLC by simultaneously inhibition of anti-apoptotic proteins

Abstract Lung cancer is the second most common cancer worldwide with 1.8 million deaths in 2020 being the leading cause of cancer related death. With 86% of cases, non-small cell lung cancer (NSCLC) is the most prominent subtype. The survival rate improved over the past 20 years by application of targeted therapies. Driver mutations in protein kinases such as the epidermal growth factor receptor (EGFR) are frequent and have evolved as therapeutic targets in anti-cancer therapy. Inhibitors of receptor tyrosine kinase (RTKIs), prominently EGFR-TKIs, are effective in anti-cancer therapy but tumor evolution inevitably generates resistance. Thus, strategies for the treatment of patients with acquired resistance to RTKIs remains an urgent need. Resistance in solid tumors is overcome by a combination of bortezomib with the BH3-mimetic ABT-199 (Muenchow et al. 2020). Also, overcoming ALK-TKI resistance in NSCLC was described by Tanimoto et al. (2021) by proteasome inhibiton that mediates induction of NOXA. Furthermore, the novel described BCL-2 independent activity of ABT-199 to transactivate NOXA expression (Weller et al. 2022) represents a generally active principle by combining drugs with independent mechanism of action. We hypothesize, that combined treatment of EGFR-TKI Osimertinib (OSI) with BH3-mimetics enhances sensitivity to apoptosis induction. To explore this hypothesis, we investigated cell death induction by combined administration of OSI and BH3-mimetics or PIs in NSCLC differing in their mutational status. In addition, we analyzed the expression of BCL-2 family proteins and their relevance and regulation during RTKI-induced cell death by RNA sequencing and Western Blot. These analyses will be performed in cellular model systems of NSCLC with specific resistance mediating mutations of EGFR or activated parallel survival pathways. We found that BCL-2 inhibitors enhance efficacy of OSI in NSCLC, resulting in synergistic cell death induction. Additionally, the sensitivity to BCL-2 inhibitors tends to increase in OSI-resistant NSCLC. Western Blot analysis and RNA sequencing data of OSI-resistant NSCLC identified BCL-2 overexpression and an EMT signature as resistance-mediating mechanisms, which can be overcome by combination of OSI with BCL-2 inhibitors. Indeed, our data suggest that combination of OSI with BCL-2 inhibitors disrupts resistance to OSI in NSCLC. We propose that BCL-2 inhibitors sensitize OSI-resistant NSCLC to effectively induce cell death. Thus, studying the molecular mechanism of synergistic efficacy of RTKI/BCL-2 inhibitor combination therapies, should prompt future efforts in the clinical evaluation of combinatorial regimens. We hypothesize that a general therapeutic concept which relies on simultaneous blocking of driver mutations and survival-promoting BCL-2 proteins represents a generally active principle of a tumor-agnostic mechanism of action. Citation Format: Sandra Weller, Tobias Beigl, Frank Essmann, Annika Harsch, Hans-Georg Kopp, Tom Ekstrom. Overcoming resistance to EGFR-TKI in NSCLC by simultaneously inhibition of anti-apoptotic proteins [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 6484.

Abstract 7206: Inhibition of CAD, an enzyme of de novo pyrimidine synthesis, to overcome EGFR-TKI resistance in NSCLC

Abstract Given the fast-growing cancer cells, there is an increased demand for large amounts of energy and nucleotides for DNA and RNA synthesis. Thus, targeting nucleotide metabolism, specifically de novo nucleotide biosynthesis, is a promising strategy for cancer treatment. Hyperactivation of Carbamoyl-phosphate synthetase, aspartate transcarbamylase (CAD), a key enzyme catalyzing the first three steps of de novo pyrimidine synthesis, is a common event in tumors and has been associated with chemoresistance in tumors. Our analysis, using TCGA data, revealed that increased expression of CAD is associated with poor prognosis in non-small cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have demonstrated significant benefits to patients with NSCLC harboring EGFR-activating mutations; however, acquired resistance limits their long-term efficacy. Therefore, it remains urgent to discover the underlying mechanisms for discovering novel therapeutic strategies for overcoming TKI resistance. Our previous findings demonstrated that blockade of S6K1 overcomes acquired resistance to EGFR-TKIs in NSCLC. It was reported that S6K1 is involved in de novo pyrimidine biosynthesis. In this study, we aimed to investigate whether CAD mediates S6K1-induced EGFR-TKI resistance in NSCLC. Immunohistochemistry analysis on a tissue microarray of lung adenocarcinoma tissues indicated that increased co-expression of p-CAD and S6K1 proteins is associated with clinicopathological parameters and poor survival outcomes. Our in vitro results showed an increased expression level of CAD and p-CAD proteins in TKI-resistant cells compared with parental cells upon TKI treatment. Knockdown of CAD by siRNA led to sensitizing TKI-resistant cells to osimertinib, as indicated by BrdU cell proliferation assay. Inhibition of S6K1 by a siRNA or specific inhibitors PF-470867 and LY2584702 effectively reduced the phosphorylation levels of CAD protein in TKI-resistant cells, suggesting that S6K1 may serve as an upstream regulator of CAD for TKI resistance. The String database predicted a protein-protein interaction between S6K1 and CAD. Indeed, our co-immunoprecipitation (co-IP) results revealed a direct association between S6K1 and CAD proteins. Importantly, TKI treatment enhanced the binding between S6K1 and CAD. Taken together, these findings provide evidence that CAD may mediate S6K1-mediated EGFR-TKI resistance in NSCLC. Our ongoing and future work will assess whether inhibition of CAD sensitizes resistant cells to TKI in vivo and elucidate the associated mechanism. Citation Format: Leili Saeednejad Zanjani, Lei Zhao, Shaomin Wang, Margot Federoff, Xin Sun, Jun He. Inhibition of CAD, an enzyme of de novo pyrimidine synthesis, to overcome EGFR-TKI resistance in 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 7206.

Machine learning-based radiomics strategy for prediction of acquired EGFR T790M mutation following treatment with EGFR-TKI in NSCLC

The epidermal growth factor receptor (EGFR) Thr790 Met (T790M) mutation is responsible for approximately half of the acquired resistance to EGFR-tyrosine kinase inhibitor (TKI) in non-small-cell lung cancer (NSCLC) patients. Identifying patients at diagnosis who are likely to develop this mutation after first- or second-generation EGFR-TKI treatment is crucial for better treatment outcomes. This study aims to develop and validate a radiomics-based machine learning (ML) approach to predict the T790M mutation in NSCLC patients at diagnosis. We collected retrospective data from 210 positive EGFR mutation NSCLC patients, extracting 1316 radiomics features from CT images. Using the LASSO algorithm, we selected 10 radiomics features and 2 clinical features most relevant to the mutations. We built models with 7 ML approaches and assessed their performance through the receiver operating characteristic (ROC) curve. The radiomics model and combined model, which integrated radiomics features and relevant clinical factors, achieved an area under the curve (AUC) of 0.80 (95% confidence interval [CI] 0.79–0.81) and 0.86 (0.87–0.88), respectively, in predicting the T790M mutation. Our study presents a convenient and noninvasive radiomics-based ML model for predicting this mutation at the time of diagnosis, aiding in targeted treatment planning for NSCLC patients with EGFR mutations.

Monitoring of Plasma EGFR Mutations during Osimertinib Treatment for NSCLC Patients with Acquired T790M Mutation.

Osimertinib was first approved for the treatment of non-small cell lung cancer (NSCLC) in patients who have developed the epidermal growth factor receptor (EGFR) T790M mutation after treatment with EGFR tyrosine kinase inhibitors (TKIs). We routinely evaluated the plasma of NSCLC patients with the T790M mutation to more rapidly detect an increase in disease activity and resistance to treatment. Eligible patients received osimertinib after resistance to the first- or second-generation of EGFR-TKIs in NSCLC harboring T790M mutation detectable in tumor tissue or plasma. Plasma samples were collected every 8 weeks during osimertinib treatment. The plasma analysis was performed using an improved PNA-LNA PCR clamp method. We tested samples for a resistance mechanism, including EGFR-activating, T790M, and C797S mutations, and assessed the association between the mutations and osimertinib treatment. Of the 60 patients enrolled in the study, 58 were eligible for this analysis. In plasma collected before osimertinib treatment, activating mutations were detected in 47 of 58 patients (81.0%) and T790M was detected in 44 patients (75.9%). Activating mutations were cleared in 60.9% (28/46) and T790M was cleared in 93.0% (40/43). Of these, 71.4% (20/28) of activating mutations and 87.5% (35/40) of T790M mutation were cleared within 8 weeks of treatment. The total response rate (RR) was 53.4% (31/58). The median duration of treatment was 259 days, with a trend toward longer treatment duration in patients who experienced the clearance of activating mutations with osimertinib. At the time of disease progression during osimertinib treatment, C797S was detected in 3 of 37 patients (8.1%). Plasma EGFR mutation analysis was effective in predicting the effect of osimertinib treatment.

Acquired resistance to EGFR-TKIs in NSCLC mediates epigenetic downregulation of MUC17 by facilitating NF-κB activity via UHRF1/DNMT1 complex.

Treatment with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) has brought significant benefits to non-small cell lung cancer (NSCLC) patients with EGFR mutations. However, most patients eventually develop acquired resistance after treatment. This study investigated the epigenetic effects of mucin 17 (MUC17) in acquired drug-resistant cells of EGFR-TKIs. We found that GR/OR (gefitinib/osimertinib-resistance) cells enhance genome-wide DNA hypermethylation, mainly in 5-UTR associated with multiple oncogenic pathways, in which GR/OR cells exerted a pro-oncogenic effect by downregulating mucin 17 (MUC17) expression in a dose- and time-dependent manner. Gefitinib/osimertinib acquired resistance mediated down-regulation of MUC17 by promoting DNMT1/UHRF1 complex-dependent promoter methylation, thereby activating NF-κB activity. MUC17 increased the generation of IκB-α and inhibit NF-κB activity by promoting the expression of MZF1. In vivo results also showed that DNMT1 inhibitor (5-Aza) in combination with gefitinib/osimertinib restored sensitivity to OR/GR cells. Acquired drug resistance of gefitinib/osimertinib promoted UHRF1/DNMT1 complex to inhibit the expression of MUC17. MUC17 in GR/OR cells may act as an epigenetic sensor for biomonitoring the resistance to EGFR-TKIs.

Review of epidermal growth factor receptor-tyrosine kinase inhibitors administration to non-small-cell lung cancer patients undergoing hemodialysis.

Non-small-cell lung cancer (NSCLC) causes significant mortality worldwide. Patients with chronic renal failure have an increased risk of developing lung cancer. NSCLC Patients with chronic renal failure undergoing hemodialysis (HD) often exhibit poor performance, and chemotherapy is generally contraindicated. Oral epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) are effective treatment agents for NSCLC patients. However, the benefits and adverse effects of EGFR-TKIs in NSCLC undergoing HD are known. There are no clinical studies on the effects of EGFR-TKIs on NSCLC patients undergoing HD. We reviewed all previous case reports about EGFR-TKIs in NSCLC patients undergoing HD. It is difficult to design studies about the effects of EGFR-TKIs in patients undergoing HD, and this review is quite important. EGFR-TKIs are well tolerated in patients undergoing HD. The main routes of elimination of EGFR-TKIs are metabolism via the liver, and renal elimination is minor. The recommended doses and pharmacokinetics of these EGFR-TKIs for patients undergoing HD are similar to those for patients with normal renal function. The plasma protein binding of EGFR-TKIs is very high, and it is not necessary to adjust the dose after HD. In conclusion, EGFR-TKIs are effective and well tolerated in patients undergoing HD.

Bevacizumab Combined with Continuation of EGFR-TKIs in NSCLC Beyond Gradual Progression.

ObjectiveContinuation of epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) has shown potential in prolonging survival in patients with non-small cell lung cancer (NSCLC) harboring EGFR mutation who had gradual progression at initial targeting therapy. However, it remains unknown whether the combination of bevacizumab and continuation of EGFR-TKIs would benefit this subpopulation. This study retrospectively explored the effect of bevacizumab combined with EGFR-TKIs in NSCLC beyond gradual progression in the real-world setting.MethodsThe records of 48 patients were reviewed who received bevacizumab and continuation of EGFR-TKIs beyond gradual progression at initial targeting therapy. The response to the treatment and post progression survival (PPS) were reviewed and analyzed.ResultsThe median PPS was 11.4 months (95% CI 7.368–15.492) for all patients included at the median follow-up time of 17.3 months. The objective response rate (ORR) was 8.3%, and the disease control rate (DCR) was 86.1% (with 3 partial response and 28 stable disease) in 36 patients who were evaluable for response with at least one measurable lesion. Univariate Cox analysis showed that age <60, male sex, and EGFR exon19 deletion mutation were associated with longer PPS (P<0.05). Patients harboring EGFR exon19 deletion mutation had significantly longer PPS than those with EGFR exon21 L858R mutation, with an mPPS of 15.5 months and 5.7 months, respectively (HR=0.251, 95% CI 0.112–0.561, P=0.019). Multivariate Cox analysis indicated that age <60 and EGFR exon19 deletion mutation were associated with longer PPS (P<0.05).ConclusionContinuation of EGFR-TKI with the combination of bevacizumab is a reasonable strategy in NSCLC patients beyond gradual progression in previous EGFR-TKI treatment. Younger patients with EGFR exon19 deletion mutation may benefit more from the combination therapy.

Role of Circulating Tumor DNA Profiling in Patients with Non-Small Cell Lung Cancer Treated with EGFR Inhibitor

Objectives: During targeted therapy, tumor heterogeneity can drive the evolution of multiple tumor subclones harboring unique resistance mechanisms. Sequential profiling of plasma cell-free DNA (cfDNA) provides a noninvasive method for early detection of patient progression. We investigated whether the genetic dynamics detected in cfDNA during treatment can act as a predictive or prognostic marker of outcome. Methods: Patients with advanced EGFR-mutated non-small cell lung cancer (NSCLC) were included for consecutive blood sampling during EGFR-tyrosine kinase inhibitor (TKI) treatment. Blood samples were serially collected from patients at baseline, first follow-up, and progression. Extracted cfDNA was analyzed with next-generation sequencing. Results: Serial plasma samples (n = 187) from 63 patients were analyzed, and 44 patients showed circulating tumor DNA (ctDNA). EGFR mutations were detected in 36 of the 44 patients at baseline (81.8%). EGFR mutations were no longer detected in 19 of 36 shedders (52.8%) at 2 months after EGFR-TKI treatment and rebounded with resistant EGFR mutations (T790M or C797S) at progression. Other driver mutations such as KRAS G12D and BRAF V600E were found at baseline regardless of tissue EGFR status, suggesting tumor heterogeneity. Detection of ctDNA (shedder) at baseline associated with poor overall survival (p = 0.04) compared to nonshedder. Furthermore, in patients showing EGFR mutations in plasma at baseline, the clearing rate of those during the first 8 weeks of treatment served as a positive predictor for clinical outcome. Conclusion: Longitudinal liquid biopsies capture spatial and temporal heterogeneity underlining resistance to EGFR-TKIs in NSCLC. Thus, ctDNA monitoring during EGFR-TKI treatment is useful for detecting resistance mutations or predicting response. Dense serial monitoring using blood enables early prediction of treatment failure and provides a window of opportunity for well-timed intervention.

Contribution of p53 in sensitivity to EGFR tyrosine kinase inhibitors in non-small cell lung cancer

The emergence of resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) with activating EGFR mutations is a major hindrance to treatment. We investigated the effects of p53 in primary sensitivity and acquired resistance to EGFR-TKIs in NSCLC cells. Changes in sensitivity to EGFR-TKIs were determined using p53 overexpression or knockdown in cells with activating EGFR mutations. We investigated EMT-related molecules, morphologic changes, and AXL induction to elucidate mechanisms of acquired resistance to EGFR-TKIs according to p53 status. Changes in p53 status affected primary sensitivity as well as acquired resistance to EGFR-TKIs according to cell type. Firstly, p53 silencing did not affect primary and acquired resistance to EGFR-TKIs in PC-9 cells, but it led to primary resistance to EGFR-TKIs through AXL induction in HCC827 cells. Secondly, p53 silencing in H1975 cells enhanced the sensitivity to osimertinib through the emergence of mesenchymal-to-epithelial transition, and the emergence of acquired resistance to osimertinib in p53 knockout cells was much slower than in H1975 cells. Furthermore, two cell lines (H1975 and H1975/p53KO) demonstrated the different mechanisms of acquired resistance to osimertinib. Lastly, the introduction of mutant p53-R273H induced the epithelial-to-mesenchymal transition and exerted resistance to EGFR-TKIs in cells with activating EGFR mutations. These findings indicate that p53 mutations can be associated with primary or acquired resistance to EGFR-TKIs. Thus, the status or mutations of p53 may be considered as routes to improving the therapeutic effects of EGFR-TKIs in NSCLC.

Interactions between epidermal growth factor receptor tyrosine kinase inhibitors and proton-pump inhibitors/histamine type-2 receptor antagonists in non-small cell lung cancer: a systematic review and meta-analysis.

BackgroundEpidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are increasingly used for advanced non-small cell lung cancer (NSCLC) as first-line therapy. The bioavailability and efficacy of oral EGFR-TKIs could be affected by acid suppression (AS) therapy such as PPIs and H2RAs which are reported to be over-prescribed. Hence, there is a need to investigate the effect of AS on the overall survival (OS), progression-free survival (PFS) and adverse effect profile in patients treated with EGFR TKIs.MethodsAn electronic database search of Medline and Embase was performed following PRISMA guidelines on 17 January 2021. Studies analyzing interactions between EGFR TKIs and PPIs/H2RAs in NSCLC patients were included. s, non-English or non-Japanese studies or studies using non-EGFR TKIs were excluded. Hazard ratios (HRs) were pooled using generic inverse variance random effects model. Effect sizes for dichotomous variables were pooled using Mantel-Haenszel random effects model. Significance was considered at P≤0.05. Heterogeneity was assessed with Cochran Q-test and I2 test. Publication bias was assessed with funnel plots. The assessment of quality and risk of bias of randomized and non-randomized studies were undertaken with RoB 2 and the ROBINS-I tool respectively.ResultsOut of 1,173 potentially relevant articles, 14 articles were included in the final analysis. The pooled prevalence of AS in patients taking EGFR TKI was 30.71% in 4,010 individuals. Patients who were treated only with EGFR TKI had significantly better OS (HR =1.46, 95% CI: 1.27–1.72; P<0.00001) and PFS (HR =1.63, 95% CI: 1.35–1.98; P<0.00001). The OS for EGFR mutation positive patients only was as similarly significant as the OS in all patients taking EGFR TKI, while the PFS in mutation positive patients was significantly worsened with AS. PPIs resulted in a significantly worsened OS and PFS but H2RAs did not produce significantly different OS and PFS between AS and non-AS users. There were no significant differences in the incidence of rash (OR =0.81, 95% CI: 0.50–1.32; P=0.40), diarrhoea (OR =1.03, 95% CI: 0.63–1.67; P=0.91) or other adverse effects.ConclusionsCo-administration of AS medications with first-generation EGFR-TKIs in NSCLC worsens survival outcomes. Physicians should only prescribe AS medications when absolutely clinically indicated.

A phase I/II clinical trial on the efficacy and safety of NKT cells combined with gefitinib for advanced EGFR-mutated non-small-cell lung cancer

BackgroundEpidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib, have achieved good efficacy in EGFR mutation-positive non-small-cell lung cancer (NSCLC) patients, but eventual drug resistance is inevitable. Thus, new TKI-based combination therapies should be urgently explored to extend the overall survival time of these patients. CD8 + CD56+ natural killer T (NKT) cells are a natural and unique subset of lymphocytes in humans that present characteristics of T and NK cells and exert cytotoxicity on tumour cells in a granzyme B-dependent manner. The aim of this trial was to explore the efficacy and safety of CD8 + CD56+ NKT cell immunotherapy combined with gefitinib in patients with advanced EGFR-mutated NSCLC.MethodsThe study was designed as a prospective, randomized, controlled, open-label, phase I/II trial that includes 30 patients with EGFR mutation-positive stage III/IV NSCLC. All patients will be randomized in blocks at a 1:1 ratio and treated with gefitinib 250 mg/day monotherapy or combination therapy with allogeneic CD8 + CD56+ NKT cell infusions twice per month for 12 cycles or until disease progression occurs. The effectiveness of this treatment will be evaluated based on by progression-free survival (PFS), the time to progression (TTP), overall response rate (ORR), disease control rate (DCR) and overall survival (OS). The safety of the trail is being assessed based on adverse events (AEs). Recruitment and data collection, which started in December 2017, are ongoing.DiscussionAlthough immunotherapy, including programmed death-1/programmed death-1 ligand (PD-1/PD-L1) immunotherapy, has been used for NSCLC treatment with or without EGFR-TKIs, its clear efficacy still has not been shown. Assessing the safety and therapeutic potential of allogeneic CD8 + CD56+ NKT killer cells in combination with EGFR-TKIs in NSCLC will be of great interest.Trial registrationThis trial (Phase I/II Trails of NKT Cell in Combination With Gefitinib For Non Small Cell Lung Cancer) was registered on 21 November 2017 with www.chictr.org.cn, ChiCTR-IIR-17013471.

Clonal Architecture of EGFR Mutation Predicts the Efficacy of EGFR-Tyrosine Kinase Inhibitors in Advanced NSCLC: A Prospective Multicenter Study (NCT03059641).

Clonal architecture is fundamental for the understanding of cancer biology and therapy; however, multiregional sampling in advanced-stage cancers is not always applicable. This prospective clinical trial was to investigate whether paired tissue and circulating tumor DNA (ctDNA) could describe the clonal architecture of advanced non-small cell lung cancer (NSCLC) and its association with clinical outcome (NCT03059641). Paired tumor and plasma ctDNA samples were sequenced by target-capture deep sequencing of 1,021 genes. Clonal dominance analysis was performed on the basis of PyClone. Overall, 300 treatment-naïve patients with stage IIIB-IV NSCLC were recruited from 14 centers. Of the 94 patients with available ctDNA data for EGFR clonal architecture analysis, 72 (76.6%) showed EGFR as the dominant clone. The median progression-free survival was longer for these patients than for the 22 patients whose EGFR was nondominant clone [11 vs. 10 months; HR, 0.46; 95% confidence interval (CI), 0.24-0.88; P = 0.02]. The difference was more significant if both tissue and ctDNA defined EGFR as dominant clone (n = 43) versus those not (n = 8; 11 vs. 6 months; HR, 0.13; 95% CI, 0.04-0.50; P = 0.003). Moreover, multivariate Cox proportional HR analysis demonstrated EGFR clonal architecture as an independent prognostic indicator of the efficacy of EGFR-tyrosine kinase inhibitors (TKIs). Paired tissue and ctDNA could be analyzed for clonal architecture in advanced cancer. EGFR mutations do not always make up a dominant clone in advanced NSCLC, which was associated with the efficacy of EGFR-TKIs in NSCLC.

The canonical TGF-\u03b2/Smad signalling pathway is involved in PD-L1-induced primary resistance to EGFR-TKIs in EGFR-mutant non-small-cell lung cancer

BackgroundApproximately 30% of patients with epidermal growth factor receptor (EGFR)-activating mutations have no response to EGFR-tyrosine kinase inhibitors (TKIs) (primary resistance). However, little is known about the molecular mechanism involved in primary resistance to EGFR-TKIs in EGFR-mutant non-small cell lung cancer (NSCLC). Programmed death ligand-1 (PD-L1) plays important regulatory roles in intracellular functions and leads to acquired resistance to EGFR-TKIs in NSCLC. Here, we investigated the mechanistic role of PD-L1 in primary resistance to EGFR-TKIs in EGFR-mutant NSCLC cells.MethodsThe expression levels of PD-L1 and the sensitivity to gefitinib in H1975, HCC827 and PC-9 cells were determined by quantitative real-time PCR analysis (qRT-PCR) and Cell Counting Kit-8 (CCK-8) assays, respectively. Molecular manipulations (silencing or overexpression) were performed to assess the effect of PD-L1 on sensitivity to gefitinib, and a mouse xenograft model was used for in vivo confirmation. Western blotting and qRT-PCR were used to analyse the expression of epithelial-mesenchymal transition (EMT) markers. The effect of PD-L1 on migratory and invasive abilities was evaluated using the Transwell assay and mice tail intravenous injection.ResultsHigher expression of PD-L1 was related to less sensitivity to gefitinib in EGFR-mutant NSCLC cell lines. The overexpression or knockdown of PD-L1 presented diametrical sensitivity to gefitinib in vitro and in vivo. Furthermore, the overexpression of PD-L1 led to primary resistance to gefitinib through the induction of EMT, which was dependent on the upregulation of Smad3 phosphorylation. Moreover, in the mouse model, the knockdown of PD-L1 inhibited transforming growth factor (TGF)-β1-induced cell metastasis in vivo.ConclusionPD-L1 contributes to primary resistance to EGFR-TKI in EGFR-mutant NSCLC cells, which may be mediated through the induction of EMT via the activation of the TGF-β/Smad canonical signalling pathway.

Abstract 777: Down-regulation of miR-506 contributes to EGFR-TKI resistance through inducing sonic hedgehog signaling in non-small cell lung cancer

Abstract Background: Lung cancer is the leading cause of cancer related death in the United States. Epidermal Growth Factor Receptor (EGFR) mutation predicts response to a tyrosine kinase inhibitor (TKI) of EGFR in approximately 25% of patients. Non-small cell lung cancer (NSCLC) patients with EGFR mutation eventually develop resistance to EGFR-TKI. The mechanism of resistance is not fully elucidated but majority of the cases is related to emergence of clones with T790M mutation in EGFR, amplification of cMET and Epidermal Mesenchymal Transition (EMT). Sonic Hedgehog (SHh) signaling activation is involved in EMT through FGF, notch, TGFβ signaling and microRNA (miRs) networks. Several miRs have been shown to correlate with TKI resistance indicating that miRs may serve as novel targets and/or biomarkers for anti-EGFR therapy. MiR-506 is involved in Hedgehog signaling and it is abnormally expressed in several cancers. However, the role of miR-506 in modulating TKI resistance in NSCLC remains unexplored. In this study, we investigated the role of miR-506 in the regulation of SHh in EGFR-TKI-resistant lung cancer cell lines. Methods: To generate resistant cell lines, two EGFR-TKI sensitive NSCLC cells (HCC4006 and HCC827) were exposed to increasing concentrations of erlotinib up to 20 µM over 6 months. The resultant clones were verified for sensitivity to erlotinib using a crystal violet staining assay. The expression of miR-506 in parental and resistant clones was measured using Taqman microRNA assay. Invasive/migratory ability of parental and resistant clones was measured by Boyden chamber assays. EMT and stemness markers were evaluated by Western blotting. Results: The studies demonstrated that miR-506 levels were ~90% and 75% lower in most resistant clones of HCC4006 (HCC4006ER4) and HCC827 (HCC827ER3), respectively. These clones showed overexpression of SHh (5-fold in HCC4006ER4 and 2.5-fold in HCC827ER3 as compared to their respective parental cells). Ectopic expression of miR-506 in these resistant cells inhibits the expression of SHh by ~80% as well as its downstream mediator, GLI1 by 55%. Our results showed that the IC50 of erlotinib was 22.4 μM for ER cells transfected with ctrl-miR and 4.7 μM for cells with miR-506 mimics. Moreover, an epithelial marker, E-cadherin is upregulated (~2.7-fold) and mesenchymal/cancer stem cell markers (e.g., N-cadherin, vimentin, Sox-2, Notch1, CD44 and ALDH1A1) were significantly downregulated (75-90%, p-value&amp;lt;0.001) in miR-506 overexpressing cells. Conclusions: Our data showed that miR-506 downregulation and induction of SHh are associated with EGFR-TKI resistance in EGFR mutated NSCLC cells. MiR-506 interference and inhibition of SHh pathway may be potential therapeutic strategies to reverse resistance to EGFR-TKI in NSCLC with EGFR mutation. (This study was funded by the support from IRG-16-194-07(IH) and Godwin's lab). Citation Format: Inamul Haque, Mukut Sharma, Andrew K. Godwin, Chao H. Huang. Down-regulation of miR-506 contributes to EGFR-TKI resistance through inducing sonic hedgehog signaling in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 777.

Interactions between EGFR and PD-1/PD-L1 pathway: Implications for treatment of NSCLC

Immune checkpoint inhibitors targeting the programmed cell death receptor/ligand 1 (PD-1/PD-L1) pathway displayed striking and durable clinical responses in patients with non-small-cell lung cancer (NSCLC). However, it is still undefined about the efficacy of PD-1/PD-L1 inhibitors in NSCLC patients with EGFR activating mutations. Preclinical studies indicate the immune modulatory effect of EGFR signaling by regulating expression of MHC I/II and PD-L1 on tumor cells and activity of lymphocytes. Thus, it might be practicable for the use of PD-1/PD-L1 inhibitors as monotherapy or combined with EGFR-TKIs in patients with EGFR activating mutations. In this review, we discussed the regulation effect of EGFR signaling on PD-1/PD-L1 pathway and the potential mechanisms behind combing EGFR-TKIs with PD-1/PD-L1 inhibitors. We also reviewed current available data on PD-1/PD-L1 inhibitors as monotherapy or combined with EGFR-TKIs in NSCLC with EGFR activating mutations, and explored possible factors influence its efficacy, which would be important considerations for future clinical trial designs.

The clinical outcome of brain metastases in nonsmall cell lung cancer patients with epidermal growth factor receptor mutation- A retrospective single-institution analysis

Introduction: Nonsmall cell lung cancer (NSCLC) is the leading cause of cancer-related deaths in many countries. Brain metastases (BM) from NSCLC are poor prognosis, with a median survival of 6 months following whole-brain radiotherapy. NSCLC patients harboring activating epidermal growth factor receptor (EGFR) mutations have generally responded to afatinib with good objective response rate, progression-free survival (PFS), and overall survival (OS), and platinum-double chemotherapy. These findings demonstrate the potential effectiveness of afatinib for the treatment of EGFR-positive with BM. The aim of this retrospective study is to present data on the prognostic effect of afatinib versus gefitinib in an unselected population with newly diagnosed NSCLC with BM collected. Patients and Methods: Data were obtained from our electronic database from 48 patients with newly diagnosed EGFR mutative NSCLC with BM and treated from January 2007 to December 2017, in which data on EGFR mutation was all available at the time of diagnosis. Results: Thirteen of forty-eight were treated with afatinib, and the other was treated with gefitinib. There were seven Exon 19 deletion and six were Exon 21 mutation ([p.Leu858Arg, (c.2573T>G)]:4; [p.Leu861Gln, (c.2582T>A)]:2). There was 18 Exon 19 deletion, and 16 were Exon 21 mutation ([p.Leu858Arg, (c.2573T>G)]:16) and the other one was Exon 18 mutation [p.Gly719Ser (c.2155G>A)]. There was no significant difference between two groups included PFS (afatinib: 12.0 months vs. gefitinib: 10.1 months, P = 0.766) and OS (afatinib: 20.6 months vs. gefitinib: 14.5 months, P = 0.362). Conclusion: From the K–M survival curve evaluation, neither the PFS nor OS have the statistical significance between gefitinib and afatinib. We provided a real-world data to compare the efficacy of EGFR-TKI in NSCLC. Due to relative small sample size, we urged large clinical trial to provide more precise results.

Histone deacetylation, as opposed to promoter methylation, results in epigenetic BIM silencing and resistance to EGFR TKI in NSCLC.

Drug resistance remains a major challenge in epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) therapy. Bcl-2-like protein 11 (BIM), a B-cell lymphoma 2 family pro-apoptotic protein, is a prime target for specific anti-cancer therapeutics. However, the epigenetic regulation of BIM in non-small cell lung cancer (NSCLC) cell lines and patients with NSCLC in association with EGFR-TKI resistance requires investigation. Methylation-specific PCR (MSP), pyrosequencing, and nested quantitative (q)-MSP were conducted to explore the methylation status of BIM in NSCLC cell lines. In addition, the methylation profile of BIM in patients with NSCLC was assessed by nested q-MSP using circulating free DNA. Cell lines, treated with methylation inhibitor 5-Aza-2'-deoxycytidine (AZA) or histone deacetylation inhibitor trichostatin A (TSA) prior to gefitinib treatment, were examined for BIM gene expression and resistance to gefitinib. All cell lines used in the present study presented with hypo-methylated BIM. Treatment with AZA had no effect on BIM RNA expression in PC9 cells or the gefitinib-resistant cell lines PC9/R and PC9/G2, nor did it reverse their resistance to gefitinib. In contrast, TSA treatment produced the opposite result. In the present study, 25 (78.1%) patients with hypo-methylated BIM and 7 patients (21.9%) with partial or hyper-methylated BIM were identified. The clinicopathological data revealed a random hypo-methylated BIM distribution amongst patients with NSCLC. In the overall study group and EGFR mutant group, hypo-methylated BIM carriers presented with no significant differences in progression free survival compared with patients with partial or hyper-methylated BIM. All cell lines in the present study and the majority of patients with NSCLC carried hypo-methylated BIM. Histone deacetylation, as opposed to promoter methylation, may contribute to the epigenetic silencing of BIM and lead to EGFR TKI resistance in NSCLC.

Association of mutant EGFR L858R and exon 19 concentration in circulating cell-free DNA using droplet digital PCR with response to EGFR-TKIs in NSCLC.

The present study aimed to determine the diagnostic concordance of plasma epidermal growth factor receptor (EGFR) mutation using droplet digital polymerase chain reaction (ddPCR) with tumor tissue samples and the predictive clinical significance of plasma EGFR mutation concentration. Plasma DNA samples from patients with non-small cell lung cancer (NSCLC) were analyzed for EGFR exon 21 codon 858 (L858R) mutation, deletion of exon 19 (ex19del) and exon 20 codon 790 (T790M) mutation using ddPCR. Firstly, the mutations in the plasma samples were compared with the matched tumor samples to determine the concordance. Secondly, image examination follow-ups were analyzed to assess the association between plasma EGFR mutation concentration and patients' response to EGFR-tyrosine kinase inhibitors (TKIs). A total of 51 patients with NSCLC were enrolled, including 48 newly diagnosed patients. Compared with tumor tissue samples, the sensitivity and specificity of ddPCR were 76.19% (16/21) and 96.55% (28/29) for mutant L858R, and 88.89% (8/9) and 100% (41/41) for ex19del, respectively. No patient exhibited the T790M mutation in the tumor tissue or plasma samples. Furthermore, 5 patients with the L858R mutation and 4 patients with ex19del in plasma and tumor tissue samples had been followed up with image examination for ≥3 months following EGFR-TKI treatment. The baseline mutant EGFR concentrations were positively correlated with a reduction in tumor burden (Spearman's r=0.7000, P=0.0358). When analyzed separately, ex19del concentrations (Spearman's r=1.0000, P<0.0001) were also positively correlated with the reduction, while mutant L858R concentrations were not (Spearman's r=0.7000, P=0.1881). In the present study, detection of plasma EGFR mutations using ddPCR exhibited sufficient concordance with tumor tissue sample results. Baseline plasma mutant EGFR and ex19del concentrations were significantly and positively correlated with response to EGFR-TKIs.

The role of epithelial to mesenchymal transition in resistance to epidermal growth factor receptor tyrosine kinase inhibitors in non-small cell lung cancer.

Inhibition of the epidermal growth factor receptor (EGFR) is an important strategy when treating non-small cell lung cancer (NSCLC) patients. However, intrinsic resistance or development of resistance during the course of treatment constitutes a major challenge. The knowledge on EGFR-directed tyrosine kinase inhibitors (TKIs) and their biological effect keeps increasing. Within the group of patients with EGFR mutations some benefit to a much higher degree than others, and for patients lacking EGFR mutations a subset experience an effect. Up to 70% of patients with EGFR mutations and 10-20% of patients without EGFR mutations initially respond to the EGFR-TKI erlotinib, but there is a severe absence of good prognostic markers. Despite initial effect, all patients acquire resistance to EGFR-TKIs. Multiple mechanisms have implications in resistance development, but much is still to be explored. Epithelial to mesenchymal transition (EMT) is a transcriptionally regulated phenotypic shift rendering cells more invasive and migratory. Within the EMT process lays a need for external or internal stimuli to give rise to changes in central signaling pathways. Expression of mesenchymal markers correlates to a bad prognosis and an inferior response to EGFR-TKIs in NSCLC due to the contribution to a resistant phenotype. A deeper understanding of the role of EMT in NSCLC and especially in EGFR-TKI resistance-development constitute one opportunity to improve the benefit of TKI treatment for the individual patient. Many scientific studies have linked the EMT process to EGFR-TKI resistance in NSCLC and our aim is to review the role of EMT in both intrinsic and acquired resistance to EGFR-TKIs.