Mutant Epidermal Growth Factor Research Articles

The Agonistic Activity of the Human Epidermal Growth Factor is Reduced by the D46G Substitution.

Resistance to anti-tumor agents targeting the epidermal growth factor receptor (EGFR) reduces treatment response and requires the development of novel EGFR antagonists. Mutant epidermal growth factor (EGF) forms with reduced agonistic activity could be promising agents in cancer treatment. EGF D46G affinity to EGFR domain III was assessed with affinity chromatography. EGF D46G acute toxicity in Af albino mice at 320 and 3200 μg/kg subcutaneous doses was evaluated. EGF D46G activity in human epidermoid carcinoma cells at 10 ng/mL concentration in serum-free medium and in subcutaneous Ehrlich ascites carcinoma mice model at 320 μg/kg dose was studied. The D46G substitution decreases the thermal stability of EGF complexes with EGFR domain III by decreasing the ability of the C-terminus to be released from the intermolecular β- sheet. However, with remaining binding sites for EGFR domain I, EGF D46G effectively competes with other EGF-like growth factors for binding to EGFR and does not demonstrate toxic effects in mice. EGF D46G inhibits the proliferation of human epidermoid carcinoma cells compared to native EGF. A single subcutaneous administration of EGF D46G along with Ehrlich carcinoma cells injection inhibits the proliferation of these cells and delays tumor formation for up to seven days. EGF D46G can be defined as a partial EGFR agonist as this mutant form demonstrates reduced agonistic activity compared to native EGF. The study emphasizes the role of the EGF C-terminus in establishing interactions with EGFR domain III, which are necessary for EGFR activation and subsequent proliferation of cells.

Minor (≤ 10%) Ground-Glass Opacity Component in Clinical Stage I Non-Small Cell Lung Cancer: Associations With Pathologic Characteristics and Clinical Outcomes.

BACKGROUND. The presence of a ground-glass opacity (GGO) component is a favorable prognostic factor in non-small cell lung cancer (NSCLC), although the prognostic impact of a very small GGO component remains poorly investigated. OBJECTIVE. The purpose of this article is to investigate the impact of a minor (≤ 10%) GGO component on the prognosis of clinical stage I NSCLC in comparison with pure-solid nodules. METHODS. This retrospective study included 382 patients (mean age, 60.8 years; 210 men, 172 women) who underwent surgical resection between January 1, 2015, and December 31, 2015, for clinical stage I NSCLC appearing on preoperative chest CT as a nodule with a consolidation-to-tumor ratio (CTR) of 0.9 to 1.0. Two radiologists independently assigned nodules to groups as either minor GGO (CTR, ≥ 0.9 and < 1.0) or pure solid (CTR = 1.0). Recurrence-free survival (RFS) and cancer-specific survival (CSS) were assessed by Kaplan-Meier curves and compared between groups using log-rank tests. Cox proportional hazards models were used to assess associations with outcomes. RESULTS. The two radiologists agreed for all nodules' classification into the minor-GGO (n = 106) or pure-solid (n = 276) groups. The mean CTR of the minor-GGO group was 0.93 ± 0.02 (SD) (range, 0.90-0.97). Minor-GGO nodules, in comparison with pure-solid nodules, showed greater solid-component diameter (2.68 vs 2.16 cm; p < .001) and total nodule diameter (2.89 vs 2.16 cm; p < .001). The minor-GGO group, in comparison with the pure-solid group, showed lower frequencies of visceral pleural invasion (6.6% vs 17.0%, p = .009) and pathologic lymph node involvement (4.7% vs 20.3%, p < .001), and EGFR mutation (71.6% vs 39.9%; p < .001). The minor-GGO group, in comparison with the pure-solid group, showed better 5-year RFS (83.4% vs 55.0%; p < .001) and higher frequency of better 5-year CSS (92.4% vs 76.4%, p = .004). In multivariable analysis adjusting for patient, imaging, pathologic, and genetic factors, a minor-GGO component was independently associated with a decreased likelihood of recurrence (HR = 0.37, p = .001) but not with the likelihood of CSS. CONCLUSION. Among patients with clinical stage I NSCLC, cancers with a minor-GGO component were associated with a better prognosis versus those with a pure-solid appearance. CLINICAL IMPACT. Radiologists encountering predominantly solid nodules on CT should carefully assess images for even a minor-GGO component given the favorable prognosis.

Clinical characteristics, outcomes and prognostic factors in KRAS mutant lung cancers: experience from a tertiary care cancer center in India.

Kirsten rat sarcoma viral oncogene homologue (KRAS) mutations in lung cancers, long considered untargetable, have had a recent rise in interest due to promising data of agents targeting KRAS p.G12C. As Indian data are scarce, we sought to identify baseline clinical characteristics, prognostic factors and outcomes of lung cancer patients with KRAS mutations at our hospital. Patients with KRAS mutant lung cancers treated at our institute from 2016 to 2022 were analysed. 133 patients with KRAS mutant lung cancers were identified. Median age was 57 (interquartile range 28-78) years, and 58 (43.6%) were smokers. 17 (12.7%) had brain metastases. The commonest variant was p.G12C, seen in 53 (39.8%) patients. Six (4.5%) had programmed death ligand 1 (PDL-1) expression >50% by Ventana SP263 PDL-1 assay, and 13 (9.7%) had epidermal growth factor mutation. Of 92 patients with available treatment details, the majority received intravenous chemotherapy, nine (9.8%) received tyrosine kinase inhibitors and four (4.4%) received immunotherapy (pembrolizumab). Median progression-free survival (PFS) with first-line therapy was 6 (95% confidence interval (CI) 2.8-9.2) months and median overall survival (OS) was 12 (CI 9.2-14.8) months. The incidence of brain metastases was higher in patients with G12C mutations (p = 0.025). Brain metastases (HR: 3.57, p < 0.001), Eastern Cooperative Oncology Group performance status (PS) ≥ 2 (HR: 2.13, p = 0.002) and G12C mutation (HR: 1.84, p = 0.011) were associated with inferior PFS, while brain metastases (HR: 4.6, p < 0.001), PS ≥ 2 (HR: 2.33, p = 0.001) and G12C mutation (HR: 1.93, p = 0.01) were associated with inferior OS. This is the largest dataset of KRAS mutant lung cancers from India. Brain metastases were higher in patients with G12C mutations and associated with poorer PFS and OS. G12C mutation and PS ≥ 2 were also associated with inferior PFS and OS. Experience with targeted therapy for KRAS mutations remains an area of future exploration due to the unavailability of these agents in India.

The Epidermal Growth Factor, Anaplastic Lymphoma Kinase, and ROS Proto-oncogene 1 Mutation Profile of Non-Small Cell Lung Carcinomas in the Turkish Population: A Single-Center Analysis.

The management of non-small cell lung carcinomas (NSCLC) has changed with the identification of molecular pathways. We aimed to reveal the 3-year epidermal growth factor (EGFR), anaplastic lymphoma kinase (ALK), and ROS proto-oncogene 1 (ROS1) mutation profile in the Turkish population. The histopathological and molecular data of all NSCLC cases from our department between May 2019 and April 2022 were evaluated. Molecular testing was performed in 197 NSCLC cases, and results were obtained in 182 (92.4%) (M/F: 144/38, aged 39-86). Of these, 121 were diagnosed with adenocarcinoma, 36 with squamous cell carcinoma, and 25 with NSCLC-not otherwise specified. The EGFR mutation was seen in 21 (11.5%) cases (6 exon 19 deletions, 3 exon 18 [all codon 719], 2 exon 20, 8 exon 21 point mutations, 1 concurrent exon 19 deletion and exon 20 codon 790 M point mutation, and 1 concurrent exon 19 deletion and exon 21 point mutation). The double mutation rate of EGFR was 1.1%. The mean age of these patients was 63.4 (40-79), with 24% of all females (n = 9) and 8.3% of all males (n = 12). The ALK mutation was detected in 6 (3.3%) patients (M/F: 4/2, aged 45-82), whereas the ROS1 mutation was detected in 3 (1.7%) (M/F: 2/1, aged 40-64). It is well established in the literature that EGFR-activating mutation rates vary depending on regions and ethnic groups. We concluded that the EGFR-activating mutation rates of the Turkish population are similar to the European molecular data instead of the Asian. The ALK and ROS1 mutation rates also seem concordant with the literature.

Molecular Docking, Pharmacokinetic and Molecular Simulation Analysis of Novel Mono-Carbonyl Curcumin Analogs as L858R/T790M/C797S Mutant EGFR Inhibitors.

Curcumin, an anticancer natural compound with multiple pharmacological activities, has a weak pharmacokinetic and instability due to diketone moiety. Curcumin's stability challenges can be overcome by removing the diketone moiety and shortening the 7-carbon chain, resulting in mono-carbonyl analogs. Cancer proliferation is caused by the activation of Epidermal Growth Factor (EGFR) pathways. Current available EGFR inhibitors have an issue of resistance. Thus, we aimed to design new mono-carbonyl curcumin derivatives and analyse their drug likeness properties. Further, to investigate them on three distinct crystal structures, namely two wild-type and L858R/T790M/C797S mutant generations for EGFR inhibitory activity. Ten New Molecular Entities (NME's) were designed using literature survey. These molecules were subjected to comparative molecular docking, on the EGFR crystal structures viz. wild-type (PDB: 1M17 and 4I23) and L858R/T790M/C797S mutant (PDB: 6LUD) using Schrodinger software. The molecules were also tested for Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) properties. The docked complex of the hit molecule was studied for molecular simulation. In molecular docking studies, NMEs 1, 2, and 3 were found to have good binding affinity with 1st , 2nd , and 3rd generation EGFR crystal structures and a greater dock score than standard curcumin. All molecules have shown a good ADMET profile. Since L858R/T790M/C797S is currently being explored more, we decided to take the best molecule, NME 3, for molecular dynamics with 6LUD, and the results were compared with those of the co-crystallized ligand S4 (Osimertinib). It was found that the Relative mean square standard deviation (RMSD) (1.8 Å), Relative mean standard Fluctuation (RMSF) (1.45 Å) and radius of gyration (4.87 Å) values of NME 3 were much lower than those of reference S4. All these confirm that our designed NME 3 is more stable than reference S4. NME 1 and NME 2 have shown better binding against wild type of EGFR. NME 3 have shown comparable binding and more stability as compared to Osimertinib against L858R/T790M/C797S mutated protein structure. The hit compound can be further explored for its Molecular mechanics with generalised Born and surface area solvation (MM-GBSA) and discrete Fourier transform (DFT) studies to find out the energy and atomic level study. In the future, this molecule could be taken for wet lab studies and can be tested for mutated EGFR inhibitory activity.

Molecular Status Predicts for Local Control in Patients with Non-Small Cell Lung Cancer Spinal Metastases Following Spine Stereotactic Body Radiotherapy

We report outcomes after spine stereotactic body radiotherapy (SBRT) in patients with metastatic non-small cell lung cancer (NSCLC), to determine the significance of programmed death-ligand 1 (PD-L1) status and epidermal growth factor (EGFR) mutation on local failure (LF) rate. A total of 165 patients and 389 spinal segments were retrospectively reviewed from 2009 to 2021. Baseline patient characteristics, treatment and outcomes were abstracted. Primary endpoint was LF and secondary outcomes included overall survival (OS) and vertebral compression fracture (VCF) rates. OS was estimated using the Kaplan-Meier method. Cumulative LF and VCF rates were calculated using competing risk analysis method. Multivariable analysis (MVA) evaluated factors predictive of LF and VCF. Median follow-up was 13 months (range, 0.5-95 months). Median OS was 18.4 months (95% CI 11.4-24.6). Median age was 67 years (range, 28.2-89.9). 52% were female, 76% had an adenocarcinoma histology and 61% had a smoking history. 49/165 (29%) had an EGFR mutation. PD-L1 status was analyzed in 109/165 (66%) patients with 16% PD-L1 ≥ 50%, 20% PD-L1 1-49% and 35% PD-L1 <1%. Of 389 segments, 79% were de novo and 21% were previously radiated. At baseline, 35% had a VCF, 27% had epidural disease, 27% had paraspinal extension, and 49% were Spinal Instability in Neoplasia Score (SINS) stable. 239/389 (61%) were treated with either 24 or 28 Gy in 2 SBRT fractions. Within 1 month of SBRT, 39/165 (24%) had a tyrosine kinase inhibitor, 27/165 (16%) immunotherapy (IO) with or without chemotherapy, and 31/165 (19%) chemotherapy alone. LF cumulative incidence at 1- and 2-years was 16.3% (95% CI 12.8-20.3%) and 25.4% (95% CI 20.9%-30%), respectively. EGFR positivity (p<0.0001), PD-L1≥50% (p = 0.013) and treatment with IO within 1 month of SBRT (p = 0.004) predicted for improved local control on MVA. The 1- and 2-year LF rate in EGFR-positive vs. negative patients were 12.9% vs. 16.6% and 17.7% vs. 28.8%, respectively, and in those PD-L1 ≥50% vs PD-L1<50% were 7.8% vs. 19.6% and 7.8% vs. 38.1% respectively. Cumulative incidence of VCF at 1- and 2-years were 6.6% (95% CI 4.4-9.4%) and 8.8% (95% CI 6.1-12.0%). MVA identified prior SBRT to the same treated segment (P<0.0001) and a baseline VCF (p<0.0001) as significant predictors. 18/389 (4.6%) had radiation-induced radiculopathy and no radiation myelopathy events detected. We identify the predictive utility of EGFR mutation and PD-L1 ≥50% status on local control in NSCLC patients with spinal metastases treated with spine SBRT, and a therapeutic benefit with peri-SBRT IO.

BIOMARKER STATUS PREDICTS FOR LOCAL CONTROL (LC) FOLLOWING SPINE STEREOTACTIC BODY RADIOTHERAPY (SBRT) IN NON-SMALL CELL LUNG CANCER (NSCLC) PATIENTS WITH SPINAL METASTASES

Abstract Report of spine SBRT outcomes in NSCLC patients and impact of death-ligand 1 (PD-L1) status and epidermal growth factor (EGFR) mutation on LC. METHODS: 165 patients and 389 spinal segments were retrospectively reviewed. Primary endpoint was LF and secondary outcomes included overall survival (OS) and vertebral compression fracture (VCF) rates. RESULTS: Median follow-up, OS and age was 13 months (range, 0.5-95 months), 18.4 months (95% CI 11.4-24.6), and 67 years (range, 28.2-89.9) respectively. 52% were female and 76% had adenocarcinoma. 29% had an EGFR mutation, 16% were PD-L1 ≥ 50%, 20% PD-L1 1-49% and 35% PD-L1 &amp;lt;1%. Of 389 segments, 79% were denovo. At baseline, 35% had VCF, 27% epidural disease and 27% paraspinal extension. 61% were treated with 24 or 28 Gy in 2 SBRT fractions. LF cumulative incidence (CI) at 2-years was 25.4% (95% CI 20.9%-30%). EGFR positivity (p&amp;lt;0.0001), PD-L1≥50% (p=0.013) and treatment with IO within 1 month of SBRT (p=0.004) predicted for LC on multivariable analysis (MVA). The 2-year LF rate in EGFR-positive vs. negative patients were 17.7% vs. 28.8%, and in those PD-L1 ≥50% vs PD-L1&amp;lt;50% were 7.8% vs. 38.1%. CI of VCF at 2-years were 8.8% (95% CI 6.1-12.0%). Prior SBRT to the same segment (P&amp;lt;0.0001) and baseline VCF (p&amp;lt;0.0001) were predictors on MVA. CONCLUSION: We identify predictability of EGFR mutation, PD-L1 ≥50% and peri-SBRT IO on LC following spine SBRT in NSCLC.

HPR41 Predictors of Epidermal Growth Factor (EGFR) Testing Among Patients with Metastatic Non-Small Cell Lung Cancer (MNSCLC) Treated in the Real-World Setting

Current guidelines recommend testing for driver mutations before treatment initiation for mNSCLC patients. EGFR mutations are the most common genetic aberrations seen in mNSCLC and treatment targeting these alterations has been shown to be associated with improved outcomes

Mechanistic investigation into selective cytotoxic activities of gold nanoparticles functionalized with epidermal growth factor variants.

Epidermal growth factor (EGF) gains unique selective cytotoxicity against cancer cells upon conjugation with gold nanoparticles (GNPs). We have previously developed several lysine-free EGF mutants for favorable interactions between the nanoparticle conjugates with EGF receptor (EGFR) and found one mutant (SR: K28S/K48R) showing stronger anticancer activities. However, the exact mechanisms for the selective cytotoxicity enhancement in the SR mutant remained unsolved. In this study, we analyzed how the nanoparticle conjugates of EGF variants interacted differently with A431 cancer cells, in terms of receptor binding, activation, and trafficking. Our results indicate that the essential feature of the SR-GNP conjugates in the cytotoxicity enhancement is their preferential activation of the clathrin-independent endocytosis pathway. It is suggested that we should focus on not only ligand-receptor binding affinity but also the selectivity of the receptor endocytic route to optimize the anticancer effects in this modality.

Usage of epidermal growth factor mutation testing and impact on treatment patterns in non-small cell lung cancer: An international observational study

ObjectivesEpidermal growth factor receptor (EGFR) mutations (EGFRm) are common oncogene drivers in non-small cell lung cancer (NSCLC). This real-world study explored treatment patterns and time to receive EGFRm test results in patients with advanced EGFRm NSCLC. MethodsA cross-sectional medical chart review was completed May–August 2020 in Australia, Canada, Germany, Italy, South Korea, Taiwan, UK, and USA. Eligible patients had advanced NSCLC and a positive EGFRm test result January–December 2017. Data were abstracted from NSCLC diagnosis to end of follow-up (31 March 2020) or patient’s death whichever occurred earlier. The index date was the date of EGFRm confirmation. Results223 physicians provided data for 1,793 patients. Patients’ mean age was 64.7 years, 54 % were male, 30.7 % had no history of smoking. Overall, 78 % of EGFRm test results were received ≤ 2 weeks after request (range of median 7–14 days across countries). Median time from advanced NSCLC diagnosis to EGFRm test result was 18 days (median range 10–22 days across countries). Over a third (37 %) of patients received a systemic treatment prior to EGFRm result; chemotherapy (25 %) and EGFR-TKI (15 %) were most commonly prescribed; post-EGFR test-result was EGFR-TKI (68 %); 80 % of patients initiated EGFR-TKI at any time point post-NSCLC diagnosis. Of those receiving a first-line EGFR-TKI post-EGFRm testing, 84 % received a TKI alone, 12 % in combination with chemotherapy, and 3 % with other treatments. Median time from first-line EGFR-TKI initiation post-EGFRm testing to first subsequent treatment was 19.8 months. ConclusionOver one-fifth of patients wait >14 days for their EGFRm test results, affecting their likelihood of receiving first-line EGFR-TKI with 20 % of patients never receiving EGFR TKI treatment. There was significant inter-country variability in the proportion of patients receiving EGFR TKIs. Our study highlights the need to improve EGFRm testing turnaround times and treatment initiation across countries.

EXTH-84. A NOVEL DUAL-SPECIFIC CHIMERIC ANTIGEN RECEPTOR T CELL WITH HIGH SPECIFICITY FOR EGFR AND EGFRVIII IMPROVES SURVIVAL IN EGFR EXPRESSING MEDULLOBLASTOMA

Abstract BACKGROUND Medulloblastoma is the most common malignant brain tumor in children 0-19 years of age and current treatment requires surgical resection, followed by high dose chemotherapy and radiotherapy. Therapy carries high morbidity, with late effects including neurocognitive decline, endocrine dysfunction, and subsequent malignancies. Chimeric antigen receptor (CAR) T cell therapy presents the potential for less toxic and more effective treatment for medulloblastoma. Epidermal growth factor (EGFR) is expressed in medulloblastoma derived cell lines and appears to serve an important role in the metastatic potential of this tumor type. D2C7 is a recombinant monoclonal antibody short chain variable fragment (scFv) with dual specificity, binding to wild type EGFR (EGFRwt) and its mutant EGFR variant III (EGFRvIII). We previously developed a novel, third-generation chimeric antigen receptor T-cell construct utilizing the D2C7 scFv and performed analysis on both glioblastoma and medulloblastoma tumor models. METHODS U87 and U87vIII glioblastoma and DAOY medulloblastoma cell lines were characterized by flow cytometry to evaluate for EGFRwt and EGFRvIII expression. Cytotoxicity assays were performed utilizing flow cytometry on serially diluted effector: target ratios of U87, U87vII, and DAOY mixed with D2C7 CAR. DAOY was orthotopically implanted into the frontal lobe of NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. After tumor implantation, D2C7 CAR was introduced within the tumor bed. RESULTS EGFR was detected in U87, U87vIII, and DAOY on flow analysis. EGFRvIII was expressed only on U87vIII. D2C7 CAR demonstrated effective in vitro cytotoxicity against all three cell lines with &amp;gt;90% cytotoxicity achieved at an effector: target ratio of 2.5:1. In vivo, D2C7 CAR treatment significantly prolonged survival compared to treatment with mock CAR. CONCLUSION D2C7 CAR demonstrates efficacy in vitro and in mouse models of medulloblastoma and glioblastoma. Future steps in our work will set out to assess the D2C7 CAR in NSG mice with DAOY in the posterior fossa.

Identification of a novel nitroflavone-based scaffold for designing mutant-selective EGFR tyrosine kinase inhibitors targeting T790M and C797S resistance in advanced NSCLC

The inhibition of the Epidermal Growth Factor (EGFR) represents one of the most promising strategies in non-small cell lung cancer (NSCLC) therapy. The recently identified C797S mutation causes resistance of EGFRL858R/T790M against osimertinib, the latest approved third generation EGFR inhibitor. The identification of small molecules capable of selectively inhibiting the T790M mutations also in the late-onset C797S mutation is a desirable strategy and novel chemical structures might provide new insight in the overcoming resistance mechanisms. Here we report the identification of a novel mutant-selective privileged molecular core; guided by a structure-based drug design, a flavone skeleton has been rationally modified, and a virtual library generated. Reversible EGFR inhibitors targeting both L858R/T790M and L858R/T790M/C797S mutations with a higher affinity with respect to the wild type one are discovered via a three-track virtual screening. Selected hits were synthesized and tested in an activity-based enzyme assay against wild-type EGFR, L858R/T790M, as well as L858R/T790M/C797S. The results showed that a nitroflavone-based compound inhibits the phosphorylation of EGFR mutants at low-micromolar concentration showing selectivity over the wild type ones. Structurally similar flavone analogues have been synthesized and the following inhibition assays underlied the importance of both the presence and position of the nitrophenoxy moiety.

Luteolin enhances erlotinib's cell proliferation inhibitory and apoptotic effects in glioblastoma cell lines.

The epidermal growth factor (EGFR) receptor is frequently overexpressed in glioblastoma multiforme IV (GBM). Increased expression of EGFR leads to increased proliferation, decreased apoptosis, and increased resistance to chemotherapeutic agents. A small molecule called erlotinib inhibits EGFR receptors by binding to their adenosine triphosphate (ATP) binding sites. It is FDA approved to treat a variety of EGFR-mediated cancers. Several clinical trials have explored a combination of erlotinib with other agents to treat glioblastoma since it is believed that erlotinib would benefit patients with GBM with EGFR mutations or expression. Luteolin, a natural flavonoid, inhibits cell growth and induces apoptosis in cancer cells. We investigated the combined effects of erlotinib and luteolin on proliferation and apoptosis on glioblastoma cell lines overexpressing EGFR or glioma cells expressing truncated EGFR (ΔEGFR). In a concentration-dependent fashion, the combination of luteolin and erlotinib reduced cell proliferation (p < 0.05) and induced apoptosis by cleaving PARP and increasing caspase expression. In addition, the combination of luteolin and erlotinib reduced the phosphorylation of downstream EGFR cell signaling molecules such as Akt, NF kappa B, and STAT3 in a concentration-dependent manner. These findings suggest that combining luteolin with erlotinib offers a potential treatment strategy for glioblastoma multiforme IV.

Factors Affecting the Treatment Success of Short-Term Regimen for Drug Resistant Tuberculosis (DR TB) Patients at Dr. Saiful Anwar General Hospital Malang

Background: Epidermal growth factor (EGFR) is a transmembrane receptor that plays an important role in the development of cancer phenotypes. Some patients with lung cancer have genetic mutations. Several studies have found a close correlation of EGFR gene mutation with 15-20% of lung adenocarcinoma cases. This study aimed to determine the EGFR profile in adenocarcinoma lung cancer patients at Haji Adam Malik General Hospital.Methods: This was a prospective cohort study conducted at Haji Adam Malik General Hospital, Medan. This study used patients data for 3 years, starting from January 1, 2014 to D1ecember 31, 2016. The sample size in this study was 34 patients. The data were then analyzed using SPSS.Results: All patients were adenocarcinoma lung cancer patients with positive EGFR mutation. There were 9 subjects with EGFR mutation in exon 19; 11 subjects with exon 21 L858R mutation; and 3 subjects with exon 21 L861Q mutation. Meanwhile, there were 3 subjects with uncommon EGFR mutations, namely exon 18 mutation. Majority of subjects with exon 19 mutation were male, aged &gt;60 years, smokers with mixed types of cigarettes and severe brinkmann index. In subjects with exon 21 L858R mutation, most of the patients were male, aged 50-60 years, smokers with mixed cigarette types and severe brinkmann index. The same characteristics were also observed in subjects with exon 18 mutation. However, for exon 21 L861Q mutation, the majority of subjects were female with varying ages, and were not smokers. Conclusion: Most of the study subjects profiles were male, aged over 60 years, smokers, with mixed types of cigarettes, and with severe Brinkman Index. The EGFR mutations most commonly occured in exon 21, followed by exon 19 (ins/del exon 19), exon 18, and a combination of 2 exons.

Leveraging Bulk and Single-Cell RNA Sequencing Data of NSCLC Tumor Microenvironment and Therapeutic Potential of NLOC-15A, A Novel Multi-Target Small Molecule.

Lung cancer poses a serious threat to human health and has recently been tagged the most common malignant disease with the highest incidence and mortality rate. Although epidermal growth factor (EGFR)-tyrosine kinase inhibitors (TKIs) have significantly improved the prognosis of advanced non-small cell lung cancer (NSCLC) patients with EGFR mutations, patients often develop resistance to these drugs. There is therefore a need to identify new drug candidates with multitarget potential for treating NSCLC. We hereby provide preclinical evidence of the therapeutic efficacy of NLOC-015A a multitarget small-molecule inhibitor of EGFR/mitogen-activated protein (MAP) kinase kinase 1 (MAP2K1)/mammalian target of rapamycin (mTOR)/yes-associated protein 1 (YAP1) for the treatment NSCLC. Our multi-omics analysis of clinical data from cohorts of NSCLC revealed that dysregulation of EGFR/MAP2K1/mTOR/YAP1 signaling pathways was associated with the progression, therapeutic resistance, immune-invasive phenotypes, and worse prognoses of NSCLC patients. Analysis of single-cell RNA sequencing datasets revealed that MAP2K1, mTOR, YAP1 and EGFR were predominantly located on monocytes/macrophages, Treg and exhaustive CD8 T cell, and are involved in M2 polarization within the TME of patients with primary and metastatic NSCLC which further implied gene’s role in remodeling the tumor immune microenvironment. A molecular-docking analysis revealed that NLOC-015A bound to YAP1, EGFR, MAP kinase/extracellular signal-related kinase kinase 1 (MEK1), and mTOR with strong binding efficacies ranging –8.4 to –9.50 kcal/mol. Interestingly, compared to osimertinib, NLOC-015 bound with higher efficacy to the tyrosine kinase (TK) domains of both T790M and T790M/C797S mutant-bearing EGFR. Our in vitro studies and sequencing analysis revealed that NLOC-015A inhibited the proliferation and oncogenic phenotypes of NSCLC cell lines with concomitant downregulation of expression levels of mTOR, EGFR, YAP1, and MEK1 signaling network. We, therefore, suggest that NLOC-015A might represent a new candidate for treating NSCLC via acting as a multitarget inhibitor of EGFR, mTOR/NF-κB, YAP1, MEK1 in NSCLC.

Classification and regression tree for estimating predictive markers to detect T790M mutations after acquired resistance to first line EGFR-TKI: HOPE-002.

Osimertinib as first-line treatment for patients with non-small cell lung cancer (NSCLC) harboring epidermal growth factor (EGFR) mutations remains controversial. Sequential EGFR-tyrosine kinase inhibitor (TKI) might be superior to the first line osimertinib in patients at risk of developing acquired T790M mutations. We enrolled consecutive patients with EGFR-mutated (deletion 19 or L858R) advanced NSCLC treated with first-line drugs and evaluated predictive markers using classification and regression tree (CART) for the detection of T790M mutations based on patient backgrounds prior to initial treatment. Patients without acquired T790M mutations had worse outcomes than those with T790M mutations (median OS: 798days vs. not reached; HR: 2.70; P < 0.001). CART identified three distinct groups based on variables associated with acquired T790M mutations (age, CYF, WBC, liver metastasis, and LDH; AUROC: 0.77). Based on certain variables, CART identified three distinct groups in deletion 19 (albumin, LDH, bone metastasis, pleural effusion, and WBC; AUROC: 0.81) and two distinct groups in L858R (age, CEA, and ALP; AUROC: 0.80). The T790M detection frequencies after TKI resistance of afatinib and first-generation EGFR-TKIs were similar (35.3% vs. 37.4%, P = 0.933). Afatinib demonstrated longer PFS (398 vs. 279days; HR: 0.67; P = 0.004) and OS (1053 vs. 956days; HR: 0.68; P = 0.051) than first-generation EGFR-TKIs. Identification of patients at risk of acquiring T790M mutations after EGFR-TKI failure may aid in choice of first-line EGFR-TKI. Furthermore, afatinib may be the more effective 1st-line EGFR-TKI treatment for patients at risk of developing T790M as initial EGFR-TKI resistance.

A novel cyclic NP1 reveals obstructionof EGFR kinase activity and attenuation of EGFR-driven cell lines.

Aberrations of the epidermal growth factor receptor (EGFR), for example, mutations and overexpression, play pivotal roles in various cellular functions, such as proliferation, migration, and cell differentiation. Approved small molecule-based inhibitors, including gefitinib and erlotinib, are used clinically to target the tyrosine kinase domain of EGFR (TK-EGFR). However, the severity of the side effects, off-target effects, and drug resistance is a concern. Cyclic peptides are a well-known peptide format with high stability and are promising molecules for drug development. Herein, the Ph.D.™-C7C phage display library was used to screen cyclic peptides against TK-EGFR. Biopanning, both with and without propagation methods, was performed to assess the highest capacity peptides using the enzymatic activity of TK-EGFR. Interestingly, NP1, a peptide selected during biopanning without propagation demonstrated an inhibitory effect against TK-EGFR at IC50 within the nanomolar range; this effect was better than that of P1 obtained using biopanning with propagation. Moreover, NP1 elicited EGFR with an affinity binding (KD ) value of 18.40 ± 5.50 µM by surface plasmon resonance (SPR). Introducing cell-penetrating peptides or Arginine-9 (Arg9) at the N-terminus of NP1 thus improves cell-penetrability and can lead to the inhibition of EGFR-driven cancer cell lines; however, it exhibits no hepatotoxicity. Furthermore, NP1 caused a decrease in phosphorylated EGFR after activation within cells. A docking model shows that NP1 interacted primarily with TK-EGFR via hydrogen bonding. Together, this suggests that NP1 is a novel EGFR peptide inhibitor candidate with specificity and selectivity toward TK-EGFR, and may be applied to targeted therapy.

Improved anti-cancer effect of epidermal growth factor-gold nanoparticle conjugates by protein orientation through site-specific mutagenesis

ABSTRACT Epidermal growth factor (EGF)-nanoparticle conjugates have the potential for cancer therapeutics due to the unique cytotoxic activity in cancer cells with EGF receptor (EGFR) overexpression. To gain its maximum activity, the EGF molecule should be immobilized on the nanoparticle surface in a defined orientation so as the bulky nanoparticle will not interfere EGF-EGFR interaction. Herein, we demonstrate successful enhancement of the anti-cancer activity of EGF-gold nanoparticle conjugates (EGF-GNPs) by controlling the EGF orientation on the surface of the nanoparticle through site-specific mutagenesis. Three lysine-free EGF variants (RR, RS, and SR) were designed, where two endogenous lysine residues were replaced with either arginine (R) or serine (S). The EGF mutants can be conjugated to the GNPs in a controlled orientation through the single amino group at the N-terminus. The ability of the mutants to induce extracellular signal-regulated kinase (ERK) phosphorylation was no different from wild type EGF (WT) in soluble form, rather lowered for one mutant (RR). However, after conjugated to GNPs, the SR mutants exhibited an enhanced biological activity than WT, in terms of ERK phosphorylation and growth inhibition of cancer cells. Further analysis of the binding constant of each mutant indicated the emergent enhanced activity of the GNP conjugates of the SR mutant was not solely contributed to the orientation, but to its higher binding activity to EGFR. These results validate the present genetic recombination strategy to improve the anticancer efficiency of EGF-GNPs.

Abstract 2423: Discovery of ZN-e4, an irreversible EGFR-TKI with potent anti-tumor activity in EGFR mutant non-small-cell lung cancer

Abstract Small molecule tyrosine kinase inhibitors against the epidermal growth factor (EGFR) have made significant impact in the treatment of advanced non-small-cell lung cancer (NSCLC) with activating EGFR mutations. Although the first and second-generation EGFR inhibitors have remarkably improved survival in advanced EGFR mutant NSCLC patients, most of the patients develop acquired resistance. The gatekeeper mutation (T790M) is the most common acquired resistance mechanism to the first and second-generation EGFR inhibitors. To overcome the T790M acquired resistance, third-generation EGFR inhibitors, such as osimertinib, were developed. However, additional mutant selective EGFR inhibitors with improved toxicity profile are still needed as osimertinib treatment produced incidence of adverse events, such as diarrhea and skin rush. Here, we describe the identification and characterization of ZN-e4, an orally bioavailable, selective, irreversible third generation EGFR inhibitor. ZN-e4 selectively inhibited the kinase activity of several mutated forms of EGFR, including L858R, T790M/L858R, and Exon19 del in biochemical assay. In cell-based assays, ZN-e4 demonstrated a 20-40-fold selectivity ratio for mutant EGFR forms over the wild-type form thus minimizing the potential for on target toxicity. ZN-e4 potently inhibited the proliferation of NSCLC cell lines harboring EGFR activating and T790M mutation and induced tumor regression in small and large tumors with activating EGFR mutations in preclinical human NSCLC xenograft models. Taken together, our data suggest that ZN-e4 is a potent and selective third generation EGFR inhibitor. Currently, ZN-e4 is in a Phase I clinical trial in patients with EGFR mutant NSCLC showing clinical activity and encouraging toxicity profile. Citation Format: Jiali Li, Sunny Abraham, Jianhui Ma, Peter Q. Huang, Kevin D. Bunker, Fernando Doñate, Ahmed A. Samatar. Discovery of ZN-e4, an irreversible EGFR-TKI with potent anti-tumor activity in EGFR mutant non-small-cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2423.

Reporting of Incidence and Outcome of Bone Metastases in Clinical Trials Enrolling Patients with Epidermal Growth Factor Receptor Mutated Lung Adenocarcinoma—A Systematic Review

Simple SummaryAround 30–60% of the patients with lung cancer develop bone metastases, which are associated with decreased survival, bone pain, and skeletal-related events such as need for radiation. Patients with an epidermal growth factor mutation (EGFR), a subgroup of the patients with lung cancer, seem to develop more bone metastases than other patients with lung cancer. Due to prolonged survival of these patients, they live longer with bone metastases and/or skeletal-related events, therefore optimal management is warranted. The aim of our systematic review is to gain more insight in reporting of bone metastases, skeletal-related events, and bone-specific outcome of treatment in clinical trials enrolling patients with EGFR-mutated lung cancer. We found that data on bone metastases and bone-related outcomes are largely lacking in clinical trials. There should be more focus on reporting and preventing of skeletal-related events in these patients.Bone metastases, occurring in 30–60% of patients with non-small cell lung cancer (NSCLC), are associated with decreased survival, cancer-induced bone pain, and skeletal-related events (SREs). Those with an activating epidermal growth factor mutation (EGFR+) seem to be more prone to develop bone metastases. To gain more insight into bone metastases-related outcomes in EGFR+ NSCLC, we performed a systematic review on Pubmed (2006–2021). Main inclusion criteria: prospective, phase II/III trials evaluating EGFR-tyrosine kinase inhibitors, ≥10 EGFR+ patients included, data on bone metastases and/or bone-related outcomes available. Out of 663 articles, 21 (3176 EGFR+ patients) met the eligibility criteria; 4 phase III (one double blind), 17 phase II trials (three randomized) were included. In seven trials dedicated bone imaging was performed at baseline. Mean incidence of bone metastases at diagnosis was 42%; 3–33% had progression in the bone upon progression. Except for one trial, it was not specified whether the use of bone target agents was permitted, and in none of the trials, occurrence of SREs was reported. Despite the high incidence of bone metastases in EGFR+ adenocarcinoma, there is a lack of screening for, and reporting on bone metastases in clinical trials, as well as permitted bone-targeted agents and SREs.