Specific Epidermal Growth Factor Receptor Research Articles

Specificity of U2 and GOL1 aptamers to EGFR-positive human glioblastoma cells in vitro

Overexpression of the epidermal growth factor receptor (EGFR) or its mutations mediate signaling pathways leading to proliferation, invasion of tumor cells, as well as to an increase in their survival. Despite the success of the clinical use of antibodies against EGFR in patients with colorectal cancer and squamous cell carcinoma of the head and neck, their low effectiveness in glioblastoma has been shown. Therefore, for the treatment of gliomas, a specific EGFR drug is needed, capable of penetrating into the tumor focus in the brain, and having low immunogenicity. In this work, aptamers – single-stranded DNA oligonucleotides specific to EGFR, U2 and Gol1 are presented as such a preparation. In this study, we obtained a cellular model of human glioma with EGFR and EGFRvIII overexpression, which showed the specificity of U2 and Gol1 aptamers to these receptors using classical methods, as well as the method of aptaimmunocytochemistry. A study of the effect of binding of the Gol1 aptamer to the EGFRvIII receptor on the next steps of the signaling pathway showed a change in the expression levels of genes associated with cell proliferation and survival (JUN, FOS, CCND1, PI3K and AKT3), while the U2 aptamer did not demonstrate a significant effect on cells in vitro. These results showed that the Gol1 aptamer has therapeutic potential against human glioblastoma tumor cells overexpressing the EGFRvIII mutant type receptor.

Real world treatment patterns, healthcare resource use and costs in patients with advanced or metastatic non-small cell lung cancer by EGFR mutation type

Aims This study described treatment patterns, healthcare resource utilization (HRU) and costs among advanced or metastatic non-small cell lung cancer (a/mNSCLC) patients with different epidermal growth factor receptor (EGFR) mutation types. Materials and methods This retrospective study leveraged NeoGenomics NeoNucleus linked with IQVIA PharMetrics Plus between 01 January 2016 to 30 April 2021 (study period). Patients with evidence of a/mNSCLC between 01 July 2016 to 31 March 2021 (selection window) with EGFR test results indicating exon 19 deletion (exon19del), exon 21 L858R (L858R), or exon 20 insertion (exon20i) mutations were included; date of first observed evidence of a/mNSCLC was the index date. Treatment patterns, all-cause HRU and costs during ≥1 month follow-up were reported for each cohort (exon19del, L858R, and exon20i). Results A total of 106 exon19del, 75 L858R, and 13 exon20i patients met the study criteria. The prevalence of hospitalization was highest in the exon20i cohort (76.9%), followed by L858R (62.7%) and exon19del (55.7%) cohorts. A higher proportion of patients had evidence of hospice/end-of-life care in the exon20i (30.8%) and L858R (29.3%) cohorts relative to the exon19del cohort (22.6%). The exon20i cohort had higher median total healthcare costs per patient per month ($27,069) relative to exon19del ($17,482) and L858R ($17,763). EGFR tyrosine kinase inhibitors (TKI) were the most frequently observed treatment type for exon19del and L858R cohorts, while chemotherapy was the most observed treatment in exon20i cohort. Limitations The sample size for the study cohorts was small, thus no statistical comparisons were conducted. Conclusions This is one of the first real-world studies to describe HRU and costs among a/mNSCLC patients by specific EGFR mutation type. HRU and costs varied between EGFR mutation types and were highest among exon20i cohort, potentially reflecting higher disease burden and unmet need among patients with this mutation.

Nanoenabled intracellular zinc bursting for efficacious reversal of gefitinib resistance in lung cancer.

Following the identification of specific epidermal growth factor receptor (EGFR)-activating mutations, gefitinib, one of the first-generation tyrosine kinase inhibitors (TKIs), has proven efficacious in targeting NSCLC that is driven by specific EGFR-activating mutations. However, most patients who initially respond to gefitinib, develop acquired resistance. In the current study, we devised a novel strategy to enhance the efficacy of gefitinib. We developed a simple and effective, nano-interrupter termed zeolitic imidazolate framework-8@Gefitinib@hyaluraonic nanoparticle (ZIF-8@G@HA NP). This nanoparticle was prepared by loading gefitinib onto a ZIF-8 nanoplatform followed by coating with hyaluronic acid (HA). The burst of Zn2+ release triggered by pH-sensitive degradation of ZIF-8@G@HA NPs was shown to enhance the efficacy of gefitinib in parental lung carcinoma HCC827 cells and overcame acquired gefitinib resistance in gefitinib drug resistant (GDR) HCC827 cells. We found that when treated with ZIF-8@G@HA NPs, Zn2+ acts synergistically with gefitinib via increased apoptosis in both parental and GDR HCC827 cells. Consistently, this in vitro activity was correlated with in vivo tumor growth inhibition. Interestingly, GDR cells were more sensitive to Zn2+ when compared with parental cells. We further found that ZIF-8 NPs overcame gefitinib resistance by triggering reactive oxygen species (ROS) generation and consequent cell cycle arrest at the G2/M phase, resulting in cancer cell apoptosis. Zn2+ was also found to block P-gp activity, facilitating the accumulation of gefitinib in GDR cells, thus enhancing the anti-tumor efficacy of gefitinib resulting in reversal of gefitinib resistance. Thus, this study offers a novel and promising strategy to surmount acquired gefitinib resistance via cell cycle arrest at the G2/M phase by facilitating gefitinib accumulation in GDR cells.

Glucose upregulates amphiregulin in oral dysplastic keratinocytes: A potential role in diabetes-associated oral carcinogenesis.

Compelling evidence implicates diabetes-associated hyperglycemia as a promoter of tumor progression in oral potentially malignant disorders (OPMD). Yet, information on hyperglycemia-induced cell signaling networks in oral oncology remains limited. Our group recently reported that glucose-rich conditions significantly enhance oral dysplastic keratinocyte viability and migration through epidermal growth factor receptor (EGFR) activation, a pathway strongly linked to oral carcinogenesis. Here, we investigated the basal metabolic phenotype in these cells and whether specific glucose-responsive EGFR ligands mediate these responses. Cell energy phenotype and lactate concentration were evaluated via commercially available assays. EGFR ligands in response to normal (5 mM) or high (20 mM) glucose were analyzed by quantitative real-time PCR, ELISA, and western blotting. Cell viability and migration assays were performed in the presence of pharmacological inhibitors or RNA interference. When compared to normal keratinocytes, basal glycolysis in oral dysplastic keratinocytes was significantly elevated. In highly glycolytic cells, high glucose-activated EGFR increasing viability and migration. Notably, we identified amphiregulin (AREG) as the predominant glucose-induced EGFR ligand. Indeed, enhanced cell migration in response to high glucose was blunted by EGFR inhibitor cetuximab and AREG siRNA. Conversely, AREG treatment under normal glucose conditions significantly increased cell viability, migration, lactate levels, and expression of glycolytic marker pyruvate kinase M2. These novel findings point to AREG as a potential high glucose-induced EGFR activating ligand in highly glycolytic oral dysplastic keratinocytes. Future studies are warranted to gain more insight into the role of AREG in hyperglycemia-associated OPMD tumor progression.

The next generation of EGFR inhibitors: a patenting perspective of PROTACs based EGFR degraders

ABSTRACT Introduction Abnormal expression of epidermal growth factor receptor (EGFR) contributes to tumor development, especially in non-small cell lung cancer (NSCLC). Although multiple inhibitors have been developed to target diverse EGFR mutations and several have been approved, the inevitable drug resistance and side effect remain a challenge, which motivates novel strategies. Proteolysis-targeting chimeras (PROTACs) have been gaining momentum for their potential as novel therapeutics for human diseases by triggering protein degradation. To date, various potent and specific EGFR PROTACs have been discovered and some of them have entered clinical trials. Areas covered This review provides an overview of EGFR degraders in patents from 2016 to 2022. It provides an update of the discovery strategies, chemical structures, and molecular profiling of all available EGFR PROTACs. SciFinder, PubMed, Web of Science, EPO, and CNIPA databases were used for searching the literature and patents for EGFR PROTACs. Expert opinion By employing the PROTAC technology, highly potent and selective EGFR degraders based on four generation EGFR inhibitors have been developed, which offer a new strategy to target EGFR mutations and overcome the drug resistance. Despite the satisfactory result in vitro and in vivo studies, their therapeutic value awaits more rigorous preclinical testing and clinical investigation.

The treatment of patients with non-small cell lung cancer carrying uncommon EGFR mutations, HER2 mutations, or brain metastases: a systematic review of pre-clinical and clinical findings for dacomitinib.

Accumulating evidence has shown that dacomitinib has potential activities for patients with non-small cell lung cancer (NSCLC) harboring uncommon epidermal growth factor receptor (EGFR) mutations, human epidermal growth factor receptor 2 (HER2) mutations, or central nervous system (CNS) metastases. This study aimed to give a systematic review on its potential applications in the above settings by searching MEDLINE/PubMed, Embase, Cochrane Library, American Society of Clinical Oncology.org, European Society for Medical Oncology.org, and ClinicalTrials.gov. The literature search yielded 649 publications in total. According to our findings, dacomitinib exhibited promising efficacy in patients with major uncommon EGFR mutations (including G719X, S768I, and L861Q). Both EGFR exon 20 insertional mutation (Ex20ins) and HER2 Ex20ins demonstrated significant internal heterogeneity in response to dacomitinib, among which specific subtypes (including EGFR D770delinsGY, A763_Y764insFQEA, and HER2 M774delinsWLV) were highly sensitive. Other uncommon EGFR mutations including 18del and L747P have also been shown responsive to dacomitinib. Interestingly, limited studies suggested dacomitinib application on certain first or third generation tyrosine kinase inhibitors (TKIs)' resistant secondary mutations. Last but not least, both pre-clinical and clinical data indicated that dacomitinib has an encouraging intracranial tumor control ability, regardless of uncommon mutations. Dacomitinib demonstrated good disease control on patients with NSCLC harboring major uncommon EGFR mutations and specific EGFR or HER2 mutation subtypes, and selective clinical application of dacomitinib is considerable in this setting, especially for those with intracranial metastases.

Multistep continuous flow synthesis of Erlotinib

Erlotinib is an orally administered, highly effective, specific epidermal growth factor receptor tyrosine kinase inhibitor, used to treat non-small cell lung cancer and pancreatic cancer. The traditional synthetic methods for Erlotinib exhibit long reaction time and safety concern. Herein, we describe a novel five-step route for the synthesis of Erlotinib in flow. These five steps comprise etherification, nitration, reduction, addition and cyclization reactions. All steps were optimized and converted to continuous flow process, which drastically reduces the reaction time and considerably improves the process safety as well as the total yield. Enabled by five continuous flow units, Erlotinib is efficiently afforded with an E-factor of 38, an overall yield of 83%, and a total residence time of 25.1 min. Majority steps in this process have been optimized for quantitative conversion, which offers the possibility of telescoping the entire process.

Clinical Validation of the Unparalleled Sensitivity of the Novel Allele-Discriminating Priming System Technology-Based EGFR Mutation Assay in Patients with Operable Non-Small Cell Lung Cancer.

Recently, we developed allele-discriminating priming system (ADPS) technology. This method increases the sensitivity of conventional quantitative polymerase chain reaction up to 100 folds, with limit of detection, 0.01%, with reinforced specificity. This prospective study aimed to develop and validate the accuracy of ADPS epidermal growth factor receptor (EGFR) Mutation Test Kit using clinical specimens. In total 189 formalin-fixed paraffin-embedded tumor tissues resected from patients with non-small cell lung cancer were used to perform a comparative evaluation of the ADPS EGFR Mutation Test Kit versus the cobas EGFR Mutation Test v2, which is the current gold standard. When the two methods had inconsistent results, next-generation sequencing-based CancerSCAN was utilized as a referee. The overall agreement of the two methods was 97.4% (93.9%-99.1%); the positive percent agreement, 95.0% (88.7%-98.4%); and the negative percent agreement, 100.0% (95.9%-100.0%). EGFR mutations were detected at a frequency of 50.3% using the ADPS EGFR Mutation Test Kit and 52.9% using the cobas EGFR Mutation Test v2. There were 10 discrepant mutation calls between the two methods. CancerSCAN reproduced eight ADPS results. In two cases, mutant allele fraction was ultra-low at 0.02% and 0.06%, which are significantly below the limit of detection of the cobas assay and CancerSCAN. Based on the EGFR genotyping by ADPS, the treatment options could be switched in five patients. The highly sensitive and specific ADPS EGFR Mutation Test Kit would be useful in detecting the patients who have lung cancer with EGFR mutation, and can benefit from the EGFR targeted therapy.

Effects of Arctigenin in Proliferation, Migration, and Invasion of Nasopharyngeal Carcinoma 5-8F Cells.

Nasopharyngeal carcinoma (NPC) is a malignant tumor of the nasopharynx. Here, we aimed to understand better the molecular basis for arctigenin (ARG)'s ability to promote NPC 5- 8F cell invasion. We tested the effects of several doses of ARG on 5-8F cells that had been cultured in vitro. We estimated the metabolic activity of cells by The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium assay. We examined the influence on cell invasion, and migration using Transwell Evaluation. Real-time polymerase chain reaction analysis was used to determine the relative amounts of epidermal growth factor receptor (EGFR), Janus kinase 2 (JAK2) , and transcriptional activator 3 (STAT 3) mRNA expression. Using western blotting, we looked at the level of phosphorylation of specific proteins like EGFR, phosphorylated EGFR, JAK2, and STAT 3. Our findings revealed that ARG inhibited NPC 5-8F cell development in a dose-and time-dependent manner. The invasiveness and mobility of 5-8F cells were significantly suppressed when ARG was overexpressed in a tumor development model. Expression levels of EGFR, JAK2, and STAT 3 mRNA were considerably low in the experimental group. As a consequence of being treated with ARG, lower levels of EGFR, p-EGFR, p-JAK2, and p-STAT3 expression were observed. These results suggest that ARG may prevent NPC 5-8F cells from proliferating, migrating, and invading other tissues. There are a few potential molecular pathways, two of which are the inhibition of EGFR phosphorylation and the reduction of levels of phospho-JAK2 and phospho-STAT3.

Predictive data-driven modeling of C-terminal tyrosine function in the EGFR signaling network.

The epidermal growth factor receptor (EGFR) has been studied extensively because of its critical role in cellular signaling and association with disease. Previous models have elucidated interactions between EGFR and downstream adaptor proteins or showed phenotypes affected by EGFR. However, the link between specific EGFR phosphorylation sites and phenotypic outcomes is still poorly understood. Here, we employed a suite of isogenic cell lines expressing site-specific mutations at each of the EGFR C-terminal phosphorylation sites to interrogate their role in the signaling network and cell biological response to stimulation. Our results demonstrate the resilience of the EGFR network, which was largely similar even in the context of multiple Y-to-F mutations in the EGFR C-terminal tail, while also revealing nodes in the network that have not previously been linked to EGFR signaling. Our data-driven model highlights the signaling network nodes associated with distinct EGF-driven cell responses, including migration, proliferation, and receptor trafficking. Application of this same approach to less-studied RTKs should provide a plethora of novel associations that should lead to an improved understanding of these signaling networks.

Correlation between ALK, ROS1 Biomarkers and EGFR Oncogene Mutations in Lung Tumours: Our Observations in an Apex Oncopathology Laboratory

Introduction: Analysis of Anaplastic lymphoma receptor tyrosine kinase gene (ALK), Repressor of Silencing 1 (ROS1) gene are determined by immunohistochemistry (IHC) and it is an easily applicable, cost-effective assay for potential treatment with crizotinib. Mutations of Epidermal growth factor receptor (EGFR) genes are evaluated by IHC/Multiplex RT-PCR. The purpose of this study is to assess the frequencies of ALK, ROS1 and their association with EGFR fusion gene mutations in a spectrum of lung tumours. Materials and methods: A total of 202 cases of lung tumours reported at our Center for Oncopathology from September 1st 2020 to 31st August 2021, were retrospectively analyzed for ALK, ROS1 and EGFR fusion genes based on Immunohistochemistry (IHC) and Multiplex PCR findings. ALK was tested using D5F3 clone, and ROS1 was analyzed using Cell Signalling’s D4D6 clone on the Ventana immunohistochemistry platform. EGFR status was analyzed using EGFR mutation test V2 real-time multiplex PCR assay on Roche Cobas Z480. Results: 202 biopsy samples and cellblocks of fluid aspirates were analyzed. 175/202 were histologically and immunologically proved as Non-small cell lung carcinoma (Primary pulmonary adenocarcinoma) and its metastases. 09/199 (5.23%) were Positive for ALK IHC and 03/199 (1.74%) cases had equivocal results. 06/179 (3.85%) cases were Positive for ROS1 IHC and 03/179 (1.92%) cases had equivocal results. Other histo-morphological diagnoses i.e., adenosquamous, squamous, small cell, mucinous carcinoma etc (27 cases) were all ALK and ROS1 Negative. 188/202 tumours were analyzed for EGFR mutation status, which showed 70/188 (37.23%) had specific EGFR mutations. 118/188 (62.76%) cases were EGFR wildtype. Conclusion: We observed that age related incidence of EGFR mutations was highest in elderly females, of 61 to 70 years. ALK gene mutations occurred in 6.03% and ROS1 gene mutations occurred in 5.02% of lung tumours and their metastases. EGFR-mutations were associated with ROS1 mutated lung adenocarcinomas. There are no coexistent ALK-EGFR or ALK-ROS1 mutations. All ALK IHC positive pulmonary adenocarcinomas are ROS1 negative and are mutually exclusive.

A new paradigm for epidermal growth factor receptor expression exists in PTC and NIFTP regulated by microRNAs.

Identification of molecular alterations associated with tumor behavior is necessary to guide clinical management. The 2022 WHO classification has organized the thyroid follicular cell-derived neoplasms into benign, low-risk and high-risk neoplasms, and emphasized the value of biomarkers that may provide differential diagnostic and prognostic information to avoid overtreatment of low risk neoplasms. This work aims to study the epidermal growth factor receptor (EGFR) expression, functional and spatial dynamics in relation to specific miRNAs alterations in papillary thyroid cancer (PTC) and in non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) considered as models of high-risk and low-risk thyroid tumors respectively. Primary thyroid cultured cells were used for miRNA gain/loss of function and luciferase reporter assays. Paraffin embedded tissues were used for real time PCR, immuno-fluorescence stain and confocal microscopy experiments. Our results showed that in PTC, EGFR mRNA is reduced as an effect of miR-146b-5p upregulation. The EGF expression is low and the ERK pathway is inhibited. The EGFR protein high cytoplasmic expression and colocalization with the endosomal/exosomal markers, ALIX and CD63, suggest the occurrence of stress-induced EGFR internalization, accumulation in endosomal vesicles and secretion via exosomes. In NIFTP EGFR transcription is increased in association with downregulation of miR-7-5p and the EGFR/ERK pathway is active indicating dependence on the canonical EGFR pathway for growth. Downregulation of transcript level along with cytoplasmic accumulation of undegraded protein is a new pattern of EGFR regulation associated with malignancy in thyroid. Further research is needed to elucidate the intracellular trafficking defects responsible for this specific EGFR dynamic in PTC.

Targeting EGFR and Monitoring Tumorigenesis of Human Lung Cancer Cells In Vitro and In Vivo Using Nanodiamond-Conjugated Specific EGFR Antibody.

Nanoprobes provide advantages for real-time monitoring of tumor markers and tumorigenesis during cancer progression and development. Epidermal growth factor receptor (EGFR) is a key protein that plays crucial roles for tumorigenesis and cancer therapy of lung cancers. Here, we show a carbon-based nanoprobe, nanodiamond (ND), which can be applied for targeting EGFR and monitoring tumorigenesis of human lung cancer cells in vitro and in vivo. The optimal fluorescent intensities of ND particles were observed in the human lung cancer cells and nude mice under in vivo imaging system. The fluorescence signal of ND particles can be real-time detected in the xenografted human lung tumor formation of nude mice. Moreover, the ND-conjugated specific EGFR antibody cetuximab (Cet) can track the location and distribution of EGFR proteins of lung cancer cells in vitro and in vivo. ND-Cet treatment increased cellular uptake ability of nanocomposites in the EGFR-expressed cells but not in the EGFR-negative lung cancer cells. Interestingly, single ND-Cet complex can be directly observed on the protein G bead by immunoprecipitation and confocal microscopy. Besides, the EGFR proteins were transported to lysosomes for degradation. Together, this study demonstrates that ND-conjugated Cet can apply for targeting EGFR and monitoring tumorigenesis during lung cancer progression and therapy.

EGFR-Mutant Non-small Cell Lung Cancer: State-of-the-Art and Future Perspectives

EGFR mutations are the first identified targetable driver alterations in advanced non-small cell lung cancer (NSCLC), for which specific epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKI) have been developed. These small molecules, administered orally, changed the natural history of patients with EGFR-mutated NSCLC, reporting impressive response and survival data. Osimertinib, a third-generation EGFR-TKI, can be considered the standard first-line therapy for the ‘common’ EGFR mutations, which include the exon 19 deletion and Leu858Arg point mutation in exon 21, accounting for 90% of cases. The ‘uncommon’ EGFR mutations, highly heterogeneous and with a low frequency, seem to be more sensitive to afatinib and osimertinib, a second-generation EGFR-TKI, excluding the EGFR exon 20 insertions mutations, for which a platinum-based regimen should be recommended while waiting for specific targeted inhibitors to reach the market. However, after an initial activity to first-line EGFR-TKI treatment, a disease progression is reported due to the presence of an intrinsic resistance or the onset of an acquired resistance. The latter can be broadly grouped into EGFR-dependent or EGFR-independent mechanisms of resistance, for which several new drugs and strategic approaches are under investigation. This review focuses on the state-of-the-art EGFR-TKIs in the treatment of metastatic NSCLC harbouring EGFR mutations, and also discusses potential future perspectives.

A novel combination treatment of antiADAM17 antibody and erlotinib to overcome acquired drug resistance in non-small cell lung cancer through the FOXO3a/FOXM1 axis.

After the identification of specific epidermal growth factor receptor (EGFR)-activating mutations as one of the most common oncogenic driver mutations in non-small cell lung cancer (NSCLC), several EGFR-tyrosine kinase inhibitors (EGFR-TKIs) with different clinical efficacies have been approved by various health authorities in the last two decades in targeting NSCLC harboring specific EGFR-activating mutations. However, most patients whose tumor initially responded to the first-generation EGFR-TKI developed acquired resistance. In this study, we developed a novel combination strategy, "antiADAM17 antibody A9(B8) + EGFR-TKIs", to enhance the efficacy of EGFR-TKIs. The addition of A9(B8) was shown to restore the effectiveness of erlotinib and overcome acquired resistance. We found that when A9(B8) antibody was treated with erlotinib or gefitinib, the combination treatment synergistically increased apoptosis in an NSCLC cell line and inhibited tumor growth in vivo. Interestingly, the addition of A9(B8) could only reduce the survival of the erlotinib-resistant NSCLC cell line and inhibit the growth of erlotinib-resistant tumors in vivo but not gefitinib-resistant cells. Furthermore, we revealed that A9(B8) overcame erlotinib resistance through the FOXO3a/FOXM1 axis and arrested the cell cycle at the G1/S phase, resulting in the apoptosis of cancer cells. Hence, this study establishes a novel, promising strategy for overcoming acquired resistance to erlotinib through the FOXO3a/FOXM1 axis by arresting the cell cycle at the G1/S phase.

EXTH-53. IL-12 ARMORED CAR T CELL THERAPY FOR HETEROGENEOUS GLIOBLASTOMA

Abstract INTRODUCTION Chimeric antigen receptor (CAR) T cells specific for the glioblastoma (GBM)-specific epidermal growth factor receptor variant III (EGFRvIII; CARvIII) have successfully treated tumors homogeneously expressing EGFRvIII when combined with lymphodepletion, however this is not recapitulated clinically. We generated an “armored” CARvIII which constitutively secretes IL-12, a stimulatory cytokine that enhances T cell persistence and function, that is capable of treating orthotopic heterogeneous GBM in immune competent mice. METHODS C57Bl/6 mice were intracranially (IC) implanted with 5 x 105 of either homogenous (CT2AvIII) or heterogeneous (1:1 CT2AvIII and CT2A parental) tumor cells. Mice were treated with 2 x 106 CARvIII or IL-12 CARvIII seven days post tumor implant and monitored for survival. TCRα -/-, CD8-/- and C57Bl/6 mice were IC implanted with homogeneous or heterogeneous tumors and treated 7 days post implant with intracranial IL-12 CARvIII therapy to assess the role of endogenous T cells. T cells within the tumor microenvironment were characterized by flow cytometry days 8, 14, and 17 post tumor implantation. RESULTS IL-12 CARvIII cells were successfully generated, secreted IL-12, and were cytotoxic against EGFRvIII-expressing tumor cells in vitro. IL-12 CARvIII therapy was curative in homogenous CT2AvIII tumors (p< 0.0001) and conferred a long-term survival in 50% of mice with heterogeneous CT2AvIII:CT2A parental tumors in vivo (p< 0.0001). Furthermore, IL-12 CARvIII therapy successfully eradicated the homogeneous CT2AvIII tumors in TCRα -/- and CD8-/- mice but failed to produce any efficacy against the heterogeneous CT2AvIII:CT2A parental tumors (p=0.0002). Endogenous T cells in IL-12 CARvIII treated mice were found to be more migratory, and interestingly more exhausted than in CARvIII treated mice. CONCLUSIONS Our findings show that IL-12 CARvIII can effectively eradicate IC homogenous tumors without lymphodepletion. Surprisingly, IL12 CARvIII therapy also treated IC heterogeneous glioma. Heterogeneous tumor clearance required an endogenous CD8 T cell response.

CRISPR-Based Fluorescent Reporter (CBFR) Assay for Sensitive, Specific, Inexpensive, and Visual Detection of a Specific EGFR Exon 19 Deletion in NSCLC.

Epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein receptor with intracellular tyrosine kinase activity. Mutations in the EGFR gene, including deletions in exon 19 and the mutation L858R, induce responsiveness of non-small cell lung cancer (NSCLC) to a group of drugs known as tyrosine kinase inhibitors. Here, we report the development of the CRISPR-based fluorescent reporter (CBFR) assay including a two-step strategy combining PCR amplification and Cas12a-driven cleavage to detect the delE746_A750 subtype of EGFR exon 19 deletions. Sensitivity and specificity of the CBFR assay were analyzed with different concentrations of fluorescence reporter and different amounts of PCR product. The results demonstrated that increasing the fluorescent reporter to 4μM and the PCR product to 5μl enhanced sensitivity. The CBFR assay could detect EGFR exon 19 deletion even with a frequency of 1% in samples. In clinical NSCLC samples, optimized CBFR assay enabled visual detection of the delE746_A750 subtype in less than 1h. The CBFR assay provides a sensitive, specific, and simple strategy designed based on a straightforward and inexpensive process. We suggest that the CBFR assay could serve as a diagnostic approach to detect mutations, deletions, and pathogens in underequipped laboratories and promote personalized therapeutic approaches.

Photoredox catalysis may be a general mechanism in photodynamic therapy

Elucidating the underlying photochemical mechanisms of action (MoA) of photodynamic therapy (PDT) may allow its efficacy to be improved and could set the stage for the development of new classes of PDT photosensitizers. Here, we provide evidence that "photoredox catalysis in cells," wherein key electron transport pathways are disrupted, could constitute a general MoA associated with PDT. Taking the cellular electron donor nicotinamide adenine dinucleotide as an example, we have found that well-known photosensitizers, such as Rose Bengal, BODIPY, phenoselenazinium, phthalocyanine, and porphyrin derivatives, are able to catalyze its conversion to NAD+. This MoA stands in contrast to conventional type I and type II photoactivation mechanisms involving electron and energy transfer, respectively. A newly designed molecular targeting photocatalyst (termed CatER) was designed to test the utility of this mechanism-based approach to photosensitizer development. Photoexcitation of CatER induces cell pyroptosis via the caspase 3/GSDME pathway. Specific epidermal growth factor receptor positive cancer cell recognition, high signal-to-background ratio tumor imaging (SBRTI = 12.2), and good tumor growth inhibition (TGI = 77.1%) are all hallmarks of CatER. CatER thus constitutes an effective near-infrared pyroptotic cell death photo-inducer. We believe the present results will provide the foundation for the synthesis of yet-improved phototherapeutic agents that incorporate photocatalytic chemistry into their molecular design.

045 An EGFR ligand maintains scleroderma skin and lung fibrosis

Systemic sclerosis (SSc/scleroderma) is an autoimmune disease that causes skin and internal organ fibrosis. Molecular signals that regulate the persistence of fibrosis are poorly understood. In SSc skin, Epidermal Growth Factor Receptor (EGFR) ligand expression correlates with skin fibrosis severity. However, EGFR inhibition has shown inconsistent results in fibrosis mouse models. We hypothesized that an immune-mesenchymal signaling circuit underlies SSc-related skin and lung fibrosis, and that targeting a specific EGFR ligand may prevent receptor activation on pathologic fibroblasts.

A rare case of Exon 19 deletion transforming to Exon 20 insertion in a case of adenocarcinoma of lung

Epidermal growth factor receptor (EGFR) gene mutations play an important role in the presentation, prognosis, and management of non-small cell lung cancer (NSCLC) patients. Several clinical studies claimed the incidence of EGFR Exon 20 insertion mutations in NSCLC and have similar clinical characteristics to those with common EGFR mutations but poorer prognosis. Insertion mutations within the Exon 20 of the EGFR gene are typically located after the C-helix of the tyrosine kinase to result in the domain of EGFR and have been reported to increase the kinase activity of the protein. This eventually leads to the ligand-independent activation of several downstream pathways such as mitogen activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (Pi3K)-(mTOR) mammalian target of rapamycin pathways, which are involved in several cellular processes such as cell proliferation, metastasis, and migration while preventing apoptosis. Thus, EGFR exon insertion mutations are gain of function mutations. Specific EGFR and frame-in insertions occur in 3–7% of NSCLC and are known to predict primary resistance to treatment with all clinically available EGFR-tyrosine kinase inhibitors (TKIs). Many clinical and preclinical studies have reported significant antitumor activity with various first and second-generation EGFR TKIs such as Erlotinib, Gefitinib, and Afatinib and have led to prolonged survival in EGFR mutated patients, as compared to wild-type EGFR tumors with chemotherapy. In addition, third-generation EGFR TKIs, such as Osimertinib, have shown encouraging results in metastatic NSCLC patients harboring EGFR mutations including EGFR, p. Thr790Met-mutations, that have shown to confer resistance to first and second-generation EGFR TKIs and who have progressed on prior TKI therapy. Drugs such as Afatinib, Dacomitinib, Erlotinib, Gefitinib, and Osimertinib have already gained FDA approval for use in EGFR-mutated metastatic NSCLC patients. Here, we report the case of a 47-year-old man who is a non-smoker with an EGFR Exon 19 positive NSCLC treated with Gefitinib developed an EGFR Exon 20 insertion positive brain metastasis initiated on Afatinib as the targeted therapy and responded well to the treatment.