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Presently, three generations of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are approved against oncogene addicted EGFR-mutant non-small cell lung cancer (NSCLC). Patients with actionable EGFR mutations invariably develop resistance. This resistance can be intrinsic (primary) or acquired (secondary).This was a retrospective study carried out between January 2016 and April 2021 analysing 486 samples of NSCLC for primary and secondary resistance to first- (erlotinib, gefitinb), second- (afatinib) and/or third-generation (osimertinib) TKIs in EGFR-mutant NSCLCs by next generation sequencing (NGS). Tissue NGS was carried out using the Thermofischer Ion Torrent™ Oncomine™ Focus 52 gene assay; liquid biopsy NGS was carried out using the Oncomine Lung Cell-Free Total Nucleic Acid assay. All cases were previously tested for a single EGFR gene with the Therascreen® EGFR RGQ PCR kit.The results were divided into four groups: (i) group 1: primary resistance to first- and/or second-generation TKIs. This group, with 21 cases, showed EGFR exon 20 insertions, dual, complex mutations and variant of unknown significance, de novo MET gene amplification besides other mutations. (ii) Group 2: primary resistance to third-generation TKIs. This group showed two cases, with one showing dual EGFR mutation (L858R and E709A) and EGFR gene amplification. (iii) Group 3: secondary resistance to first- and second-generation TKIs. This group had 27 cases, which were previously reported negative for EGFR T790M by single gene testing. Significant findings were MET gene amplification in four cases, with one also showing MET exon 14 skipping mutation. Three cases showed small cell change and one showed loss of primary mutation. (iv) Group 4: secondary resistance to third-generation TKIs. The latter group was further subgrouped into group 4A: secondary resistance to osimertinib (third-generation TKI) when offered as second-line therapy after first- and second-generation TKIs on detection of T790M mutation. This group had 15 cases. EGFR T790M mutation was lost in 10 (10/15; 67%) cases and was retained in five cases. Patients with T790M loss experienced early resistance (6.9 months versus 12.6 months mean, P = 0.0024) compared with cases that retained T790M. Two cases gained MET amplification as the resistance mechanisms. Other mutations that were found when EGFR T790M was lost were in FGFR3, KRAS, PIK3CA, CTNNB1, BRAF genes. One case had EML4-ALK translocation. Two cases showed driver EGFR deletion 19, retained T790M and C797S mutation in Cis form. Group 4B: secondary resistance to osimertinib (when given as first-line therapy) in EGFR-mutant NSCLC. This group had three cases. The duration of osimertinib treatment ranged from 11 to 17 months. Two patients showed additional C797S mutation along with primary EGFR mutation.This study shows the wide spectrum of primary and secondary EGFR resistance mechanisms to first, second and third generation of TKIs and helps us to identify newer therapeutic targets that could carry forward the initial advantage offered by EGFR TKIs.

The epidermal growth factor receptor (EGFR) is a pleiotropic glycoprotein which plays a role in regulating cell proliferation, migration and differentiation. However, the genetic diversity of EGFR in crustaceans as well as its function, such as whether it is involved in immune regulation, remains obscure. In this study, two EGFR genes, including EGFR1 and EGFR2, and three transcripts were identified and characterized in Scylla Paramamosain for the first time. To our knowledge, this is the first time that more than one EGFR gene was identified in a single species. The complete open reading frames (ORFs) of SpEGFR1, SpEGFR2a and SpEGFR2b were 4377 bp, 4404 bp and 4341 bp encoding deduced proteins of 1458 amino acids (aa), 1467 aa and 1446 aa, respectively. All EGFR had a signal peptide region and two Recep_L_domain region, followed by a transmembrane region and a conserved tyrosine kinase domain (TyrKc), and phylogenetic analysis demonstrated three SpEGFRs clustered together with invertebrate EGFR branch. Tissue specific expression analysis depicted that all SpEGFRs presented similar transcription patterns. The expression levels of SpEGFR1 and SpEGFR2s in hepatopancreas and gills were significantly altered after the stimulation of bacterial and viral pathogens including Staphylococcus aureus, Vibrio alginolyticus, White spot syndromre virus and Polycytidylinic acid. The in vivo RNA interference assays demonstrated that expression levels of SpIKK, two members of NF-κB (SpRelish and SpDorsal) and six antimicrobial peptide (AMP) genes (SpCrustin and SpALF1-5) were significantly reduced when SpEGFR1 or SpEGFR2 was silenced, respectively. The transcription patterns of SpIKK, SpRelish, SpDorsal and AMPs exhibited similar down- or up-regulation trend when the primary cultured hemocytes were treated with EGFR antagonist or agonist for 24 h. These results suggested that SpEGFR might play an important role in innate immune responses to bacterial and viral infections by regulating the NF-κB pathway. It also provided a better understanding of the origin or evolution of EGFR in crustaceans and even invertebrates.

Introduction : Osimertinib is a standard first-line treatment for non–small cell lung cancer (NSCLC) harboring mutations of the epidermal growth factor receptor gene (EGFR). However, tumors with the L858R mutation appear to be less sensitive to EGFR–tyrosine kinase inhibitors (TKIs) than those with exon-19 deletions, and subgroup analysis of the FLAURA study revealed that osimertinib did not significantly prolong overall survival (OS) compared with gefitinib or erlotinib in patients with the L858R. The RELAY study revealed a similar high efficacy of combination therapy with erlotinib plus ramucirumab (E+RAM) in patients with L858R and in those with exon-19 deletions. Patients who acquire the TKI resistance–associated T790M mutation during E+RAM treatment can also expect to receive benefit from second-line osimertinib. We have therefore planned a phase 3 study to evaluate the clinical efficacy of E+RAM compared with osimertinib monotherapy for untreated patients with advanced NSCLC harboring L858R. Patients and Methods : A total of 230 patients will be enrolled. The primary end point is time to failure of strategy (TFS), which is defined for this study as the time from randomization of treatment until disease progression or death on osimertinib, or the time from randomization until first disease progression or death of the primary treatment when osimertinib is not administered in the E+RAM group. Secondary end points include OS and progression-free survival. Conclusions : This is the first phase 3 clinical trial to target only NSCLC patients with the L858R mutation. Its results may establish an optimal treatment for such individuals.

Background: lung cancer is the primary cause of cancer deaths worldwide. Activation of epidermal growth factor receptor (EGFR) leads to lung cancer progression and poor prognosis while involuntary weight loss remains a major problem. Tumour-derived parathyroid hormone-related protein (PTHrP) emerged as a potential mediator of cachexia. Here, we investigated the modulatory role of EGFR signalling in PTHrP (encoded by Pthlh) gene expression and the impact of this relationship on cancer cachexia. Methods: global gene expression profiles of Lewis lung carcinoma (LLC) cells were analysed. Pthlh mRNA levels were measured by qRT-PCR in LLC cells treated with EGFR ligands and tyrosine kinase inhibitors (TKIs). LLC tumour-bearing mice received EGFR TKI erlotinib for 7 days via intraperitoneal injection or oral gavage. Tumour Pthlh mRNA, weight of fat/muscle tissue, and grip strength were assessed. RNA-seq data from The Cancer Genome Atlas and gene expression analysis tools were used to characterize expression profiles of PTHLH and EGFR along with correlation analysis of PTHLH with EGFR and transforming growth factor alpha (TGFA) in human lung cancer and head and neck squamous carcinoma (HNSC). Survival of lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD) patients with EGFR gene alterations was analysed in regard to PTHLH expression. Results: expression of EGFR ligands, EGFR itself, and PTHrP co-clusters in LLC cells. Activation of EGFR signalling with its ligands significantly increases (3.8-fold, P < 0.0005) while EGFR TKIs significantly decrease (90%, P < 0.0005) Pthlh mRNA levels in LLC cells. Pthlh mRNA drops 65-75% (P < 0.0005) in tumours upon treatment of LLC tumour-bearing mice with erlotinib while their muscle mass and grip strength increase (9.2% P < 0.05, 23% P < 0.005, respectively) compared with tumour-bearing control mice. PTHLH is overexpressed in tumours of LUSC (45.8-fold, P < 0.05) and HNSC (17.5-fold, P < 0.05) compared with normal tissue. PTHLH expression correlates with EGFR and its ligand TGFA in both cancers (LUSC: n = 745, R = 0.32, P R = 0.51, P n = 545, R = 0.34, P R = 0.50, P < 0.001, respectively). High PTHLH mRNA associates with poor overall survival in LUAD patients with activating EGFR mutations (n = 40, log-rank test, P = 0.0451). Conclusions: epidermal growth factor receptor signalling regulates expression of cachexia mediator PTHrP. EGFR inhibition reduces PTHrP expression in LLC tumours and ameliorates cachexia in LLC tumour-bearing mice., Scientific and Technological Research Council of Turkey (TÜBİTAK); European Molecular Biology Organization (EMBO)

Phosphohistidine phosphatase 1 (PHPT1) is an oncogene that has been reported to participate in multiple tumorigenic processes. As yet, however, the role of PHPT1 in lung cancer development remains uncharacterized.RNA sequencing assay and 18 pairs of tumor and normal tissues from patients were analyzed to reveal the upregulation of PHPT1 in lung cancer, followed by confirming the biological function in vitro and in vivo. Next, Gene Set Enrichment Analysis, lung cancer samples, apoptosis assay, mass spectrometry experiments and western blotting were used to investigate the molecular mechanism underlying PHPT1 driven progression in epidermal growth factor receptor (EGFR)-mutant lung cancer. Finally, we performed cellular and animal experiments to explore the tumor suppressive function of F-box protein 32 (FBXO32).We found that PHPT1 is overexpressed in lung cancer patients and correlates with a poor overall survival. In addition, we found that the expression of PHPT1 is elevated in EGFR-mutant lung cancer cells and primary patient samples. Inhibition of PHPT1 expression in EGFR mutant lung cancer cells significantly decreased their proliferation and clonogenicity, and suppressed their in vitro tumor growth. Mechanistic studies revealed that activation of the ERK/MAPK pathway is driven by PHPT1. PHPT1 is required for maintaining drug resistance to erlotinib in EGFR mutant lung cancer cells. We found that FBXO32 acts as an E3 ubiquitin ligase for PHPT1, and that knockdown of FBXO32 leads to PHPT1 accumulation, activation of the ERK/MAPK pathway and promotion of the proliferation, clonogenicity and growth of lung cancer cells.Our findings indicate that PHPT1 may serve as a biomarker and therapeutic target for acquired erlotinib resistance in lung cancer patients carrying EGFR mutations.

Epidermal growth factor receptor (EGFR) mutations were frequently found with concomitant genetic alterations in lung adenocarcinoma (LUAD). This study aimed to investigate the profile of concomitant alterations of EGFR-mutant LUAD ≤3 cm in size and its prognostic effect on recurrence.From January 2018 to December 2018, patients with resected LUAD ≤3 cm in size in Shanghai Chest Hospital were identified. All patients underwent capture-based targeted next-generation sequencing (NGS) with a panel of 68 lung cancer-related genes and were found with EGFR mutation. Clinicopathological and molecular characteristics and recurrence-free survival (RFS) were analyzed.A total of 637 patients were enrolled in this study. The top three frequent co-mutational genes were TP53 (179 of 637, 28.1%), PIK3CA (27 of 637, 4.2%), and ATM (22 of 637, 3.5%). The most common amplified genes were EGFR (37 of 637, 5.8%), followed by CDK4 (37 of 637, 5.8%) and MYC (12 of 637, 2.0%). Only TP53 mutation and EGFR amplification were adverse prognostic factors for RFS (all p 0.001) in univariate analysis. Multivariable analysis further demonstrated that TP53 mutation and EGFR amplification were independent risk factors for RFS [(hazard ratio (HR) 2.07, 95% confidence interval (CI) 1.07-4.00, p = 0.030; HR 3.09, 95% CI 1.49-6.40, p = 0.002, respectively].Concomitant TP53 mutation and EGFR amplification were poor prognostic factors for RFS in patients with EGFR-mutant resected LUAD. Our findings provide valuable understanding of the impact of concurrent alterations and implication for better implementation of precision therapy for patients.

Background Mesenchyme homeobox-2 (MEOX2)-mediated regulation of glioma-associated oncogene-1 (GLI1) has been associated with poor overall survival, conferring chemoresistance in lung cancer. However, the role of MEOX2/GLI1 in resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs)-based therapy remains unexplored in human lung cancer. Methods Functional assays using genetic silencing strategy by short hairpin RNAs, as well as cytotoxic (tetrazolium dye MTT) and clonogenic assays, were performed to evaluate MEOX2/GLI1-induced malignancy capacity in lung cancer cells. Further analysis performed includes western blot, qPCR and ChIP-qPCR assays to identify whether MEOX2/GLI1 promote EGFR/AKT/ERK activation, as well as EGFR overexpression through epigenetic mechanisms. Finally, preclinical tumour progression in vivo and progression-free disease interval analyses in patients treated with EGFR-TKI were included. Results Overexpressed MEOX2/GLI1 in both EGFR wild-type and EGFR/KRAS-mutated lung cancer cells were detected and involved in the activation/expression of EGFR/AKT/ERK biomarkers. In addition, MEOX2/GLI1 was shown to be involved in the increased proliferation of tumour cells and resistance capacity to cisplatin, EGFR-TKIs (erlotinib and AZD9291 ‘osimertinib’), AZD8542-SMO, and AZD6244-MEKK1/2. In addition, we identified that MEOX2/GLI1 promote lung tumour cells progression in vivo and are clinically associated with poorer progression-free disease intervals. Finally, both MEOX2 and GLI1 were detected to be epigenetically involved in EGFR expression by reducing both repressive markers polycomb-EZH2 and histone H3K27me3, but, particularly, increasing an activated histone profile H3K27Ac/H3K4me3 at EGFR-gene enhancer-promoter sequences that probably representing a novel EGFR-TKI-based therapy resistance mechanism. Conclusion MEOX2/GLI1 promote resistance to cisplatin and EGFR-TKI-based therapy in lung cancer cells, modulating EGFR/AKT/ERK signalling pathway activation, as well as inducing an aberrant epigenetic modulation of the EGFR-gene expression in human lung cancer.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs) are standard of care in the first-line (1L) setting for patients with metastatic non-small cell lung cancer (mNSCLC) with activating EGFR mutations. EGFR-activating mutations are a predictive factor for response to EGFR-TKIs. Meta-analyses have shown that patients with exon 21_L858R mutations exhibit reduced sensitivity to EGFR-TKIs, resulting in inferior patient outcomes compared to those with exon 19 deletion mutations, with worse overall survival, progression-free survival, objective response, and disease control rates. Clinical activity observed with 1L therapy with first-generation (1G), second-generation (2G), and third-generation (3G) EGFR-TKIs is not permanent, and resistance inevitably develops in all cases, supporting the importance of overall treatment planning. The introduction of the 3G EGFR-TKI, osimertinib, provides an opportunity to overcome T790M-mediated resistance to 1G and 2G EGFR-TKIs. Additionally, with the use of osimertinib, fewer T790M mutations are being detected as T790M is not a reported resistance mechanism to 3G EGFR-TKIs. However, there are currently no approved targeted therapies after 3G EGFR-TKIs. In order to further improve patient outcomes, there is a need to explore additional options for the overall treatment strategy for patients, including 1L and beyond. Combination of vascular endothelial growth factor (VEGF) inhibitors and EGFR-TKIs or chemotherapy and EGFR-TKIs may be a potential therapeutic approach in the 1L setting. This review discusses current treatment options for mNSCLC with activating EGFR mutations based on tumor, patient, and treatment characteristics and how an overall treatment plan may be developed.

Lung cancer ranks first in the incidence and mortality of cancer in the world, of which more than 80% are non-small cell lung cancer (NSCLC). The majority of NSCLC patients are in stage IIIB~IV when they are admitted to hospital and have no opportunity for surgery. Compared with traditional chemotherapy, specific targeted therapy has a higher selectivity and fewer adverse reactions, providing a new treatment direction for advanced NSCLC patients. Tyrosine kinase inhibitors of epidermal growth factor receptor (EGFR-TKIs) are the widely used targeted therapy for NSCLC patients. Their efficacy and prognosis are closely related to the mutation status of the EGFR gene. Clinically, detecting EGFR gene mutation is often limited by difficulty obtaining tissue specimens, limited detecting technology, and economic conditions, so it is of great clinical significance to find indicators to predict EGFR gene mutation status. Clinicopathological characteristics, tumor markers, liquid biopsy, and other predictors are less invasive, economical, and easier to obtain. They can be monitored in real-time, which is supposed to predict EGFR mutation status and provide guidance for the accurate, individualized diagnosis and therapy of NSCLC patients. This article reviewed the correlation between the clinical indicators and EGFR gene mutation status in NSCLC patients.

Summary Knockoff-based methods have become increasingly popular due to their enhanced power for locus discovery and their ability to prioritize putative causal variants in a genome-wide analysis. However, because of the substantial computational cost for generating knockoffs, existing knockoff approaches cannot analyze millions of rare genetic variants in biobank-scale whole-genome sequencing and whole-genome imputed datasets. We propose a scalable knockoff-based method for the analysis of common and rare variants across the genome, KnockoffScreen-AL, that is applicable to biobank-scale studies with hundreds of thousands of samples and millions of genetic variants. The application of KnockoffScreen-AL to the analysis of Alzheimer disease (AD) in 388,051 WG-imputed samples from the UK Biobank resulted in 31 significant loci, including 14 loci that are missed by conventional association tests on these data. We perform replication studies in an independent meta-analysis of clinically diagnosed AD with 94,437 samples, and additionally leverage single-cell RNA-sequencing data with 143,793 single-nucleus transcriptomes from 17 control subjects and AD-affected individuals, and proteomics data from 735 control subjects and affected indviduals with AD and related disorders to validate the genes at these significant loci. These multi-omics analyses show that 79.1% of the proximal genes at these loci and 76.2% of the genes at loci identified only by KnockoffScreen-AL exhibit at least suggestive signal (p < 0.05) in the scRNA-seq or proteomics analyses. We highlight a potentially causal gene in AD progression, EGFR, that shows significant differences in expression and protein levels between AD-affected individuals and healthy control subjects.

The present study aimed to investigate the protective effects and molecular mechanisms of Dendrobium officinale polysaccharides on gastric mucosal injuries. Following one week of continuous intragastric administration, a gastric mucosal injury model was established using intragastric administration of anhydrous ethanol. The area of gastric ulcer was measured, the contents of interleukin- 6 (IL-6), epidermal growth factor receptor (EGFR), and thyroid transcription factor 1 (TFF-1) in serum were detected by enzyme linked immunosorbent assay (ELISA), and the expressions of EGFR, TFF-1, IL-6, Raf-2, MAP kinase kinase 1 (MEK1), MEK2, and ERK1 in the gastric tissue were determined utilizing qPCR, Western blotting and immunohistochemistry. Simultaneously, Dendrobium officinale polysaccharides and anhydrous ethanol were added to the gastric mucosal cells (GES1) cultured in vitro, and the protective effects of Dendrobium officinale polysaccharides on cell viability was detected using Cell Counting Kit (CCK)-8. The addition of Dendrobium officinale polysaccharides markedly improved the gastric epithelial defect, inflammatory cell infiltration, and redness and swelling stemmed from gastric mucosal injuries and greatly reduced the area of gastric ulcer. The inhibition rates of gastric ulcer were 48.12 ± 2.98, 42.95 ± 1.52, and 27.96 ± 2.05% in the high, medium, and low concentration Dendrobium officinale polysaccharide groups, respectively. Dendrobium officinale polysaccharides could increase the expressions of EGFR and TFF-1 and decrease the expressions of IL-6, Raf-2, MEK1, MEK2, and ERK1. Dendrobium officinale polysaccharides could reduce the level of inflammatory factors and protect gastric mucosa by inhibiting the expression of MAPK pathway genes and proteins., Graphical abstract

This prespecified subanalysis of the global, randomized controlled phase III KEYNOTE‐024 study of pembrolizumab vs chemotherapy in previously untreated metastatic non‐small‐cell lung cancer without EGFR/ALK alterations and a programmed death ligand 1 (PD‐L1) tumor proportion score of 50% or higher evaluated clinical outcomes among patients enrolled in Japan. Treatment consisted of pembrolizumab 200 mg every 3 weeks (35 cycles) or platinum‐based chemotherapy (four to six cycles). The primary end‐point was progression‐free survival; secondary end‐points included overall survival and safety. Of 305 patients randomized in KEYNOTE‐024 overall, 40 patients were enrolled in Japan (all received treatment: pembrolizumab, n = 21; chemotherapy, n = 19). Median progression‐free survival was 41.4 (95% confidence interval [CI], 4.2‐42.5) months with pembrolizumab and 4.1 (95% CI, 2.8‐8.3) months with chemotherapy (hazard ratio [HR], 0.27 [95% CI, 0.11‐0.65]; one‐sided, nominal P = .001). Median overall survival was not reached (NR) (95% CI, 22.9‒NR) and 21.5 (95% CI, 5.2‐35.0) months, respectively (HR, 0.39 [95% CI, 0.17‐0.91]; one‐sided, nominal P = .012). Treatment‐related adverse events occurred in 21/21 (100%) pembrolizumab‐treated and 18/19 (95%) chemotherapy‐treated patients; eight patients (38%) and nine patients (47%), respectively, had grade 3‐5 events. Immune‐mediated adverse events and infusion reactions occurred in 11 pembrolizumab‐treated patients (52%) and four chemotherapy‐treated patients (21%), respectively; four patients (19%) and one patient (5%), respectively, had grade 3‐5 events. Consistent with results from KEYNOTE‐024 overall, first‐line pembrolizumab improved progression‐free survival and overall survival vs chemotherapy with manageable safety among Japanese patients with metastatic non‐small‐cell lung cancer without EGFR/ALK alterations and a PD‐L1 tumor proportion score of 50% or higher. The trial is registered with Clinicaltrials.gov: NCT02142738., This prespecified subanalysis of the global, randomized controlled phase III KEYNOTE‐024 study of pembrolizumab vs chemotherapy in previously untreated metastatic non‐small‐cell lung cancer without EGFR/ALK alterations and a PD‐L1 tumor proportion score of 50% or higher evaluated clinical outcomes among patients enrolled in Japan. Consistent with results from KEYNOTE‐024 overall, first‐line pembrolizumab improved progression‐free survival and overall survival vs chemotherapy with manageable safety among 40 Japanese patients in the study.

Targeted-therapy failure in treating nonsmall cell lung cancer (NSCLC) frequently occurs because of the emergence of drug resistance and genetic mutations. The same mutations also result in aerobic glycolysis, which further antagonizes outcomes by localized increases in lactate, an immune suppressor. Recent evidence indicates that enzymatic lowering of lactate can promote an oncolytic immune microenvironment within the tumour. Here, we review factors relating to lactate expression in NSCLC and the utility of lactate oxidase (LOX) for governing therapeutic delivery, its role in lactate oxidation and turnover, and relationships between lactate depletion and immune cell populations. The lactate-rich characteristic of NSCLC provides an exploitable property to potentially improve NSCLC outcomes and design new therapeutic strategies to integrate with conventional therapies Refereed/Peer-reviewed

Background The genetic changes underlying carcinogenesis in patients with risk factors of gallbladder carcinoma (GBC) remains controversial, especially in patients with pancreaticobiliary maljunction (PBM). This study aimed to clarify the association between risk factors of GBC and genetic changes using next-generation sequencing (NGS). Methods We retrospectively analyzed resected tissues of 64 patients who were diagnosed with GBC (n = 26), PBM [with GBC (n = 8), without GBC (n = 20)], and chronic cholecystitis, used as a control group (n = 10). DNA was extracted from tumors and their surrounding tissues, which were precisely separated by laser-capture microdissection. Gene alterations of 50 cancer-related genes were detected by NGS and compared with clinical information, including PBM status. Results The most frequent gene alterations in GBC tissues occurred in TP53 (50%), followed by EGFR (20.6%), RB1 (17.6%), and ERBB2 (17.6%). Gene alterations that were targetable by molecular targeted drugs were detected in 20 cases (58.8%). Statistical analysis of gene alterations and risk factors revealed that TP53 alteration rate was higher in GBC patients with PBM than those without PBM (p = 0.038), and the TP53 mutation rates in the epithelium of control patients, epithelium of PBM patients without GBC, peritumoral mucosa of GBC patients with PBM, and tumor tissue of GBC patients with PBM were 10, 10, 38, and 75%, respectively (p Conclusions TP53 alteration more than KRAS mutation was revealed to underlie carcinogenesis in patients with PBM.

Purpose To investigate the survival benefit with first/second generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR‐TKIs) and osimertinib in different treatment sequences. Methods We retrospectively screened 3807 patients diagnosed with cancer between 2013 and 2019 at Kaohsiung Chang Gung Memorial Hospital. In total, 76 patients with EGFR T790M mutation who received osimertinib after re‐biopsy or liquid biopsy were enrolled for the analysis. Results The median progression‐free survival (PFS), median overall survival (OS), and median OS2 of the 76 patients were 11.93, 66.53, and 29.57 months, respectively. A significant difference was observed in the disease control rate between those who received osimertinib treatment after chemotherapy (group A) and those who received osimertinib immediately following EGFR‐TKI therapy (group B) (34 [94.4%] vs. 31 [77.5%], p = 0.036). In addition, chronic obstructive pulmonary disease tended to be a poor prognostic factor for PFS and OS. Conclusion This real‐world analysis revealed that previous chemotherapy could affect the treatment outcomes of patients with non‐small cell lung cancer treated with osimertinib. Osimertinib treatment following first/second generation EGFR‐TKI treatment or chemotherapy resulted in improved survival benefit., Osimertinib treatment after one line of chemotherapy (group A) had a better response rate and a better PFS than osimertinib treatment immediately following treatment with first/second generation EGFR‐TKIs (group B). COPD tended to a poor prognostic factor for PFS and OS2 in patients with osimertinib use.

Background Extraosseous benign notochordal cell tumor is extremely rare, and there are only five reported cases worldwide. The presented case of pulmonary primary benign notochordal cell tumor is the sixth case, but the first to report the deletion mutation of EGFR gene exon 19. Case presentation The patient was a 50-year-old asymptomatic woman, who had been followed up for 3 years for a nodule in the right lung. After ten months of the wedge resection, the patient is alive without evidence of recurrence or metastasis. The tumor was 7 mm in diameter and was well demarcated. The tumor was consisted of a sheet of large round vacuolated cells with small and bland nuclei. No connective tissue containing blood vessels or inflammatory cell infiltration was detected in the stroma. The tumor was positive for CK AE1/AE3, Vimentin, S100 and Brachyury. EGFR gene mutation and amplification were not detected. Conclusions We firstly reported the positive immunohistochemical staining for EGFR and the negative molecular results of EGFR gene of pulmonary primary benign notochordal cell tumor. Due to the rarity of this tumor, more reports are needed to explore pathological characteristics, especially the molecular characteristics, in order to better understand the nature of tumors.

Background EGFR and ALK alternations often contribute to human malignancies, including lung cancer. EGFR and ALK mutations are usually sensitive to EGFR-tyrosine kinase inhibitors (TKIs) and ALK-TKIs. Although generally mutually exclusive, these mutations do co-exist in rare cases. This study investigated the frequencies, clinical characteristics, therapeutic efficacies, and genetic profiles of lung cancer patients with EGFR and ALK co-mutations. Methods Patients with concurrent EGFR and ALK mutations were included in this study, which analyzed mutation profiles and treatment histories. SPSS20.0 were used for survival analysis. Results Among 271 ALK-positive (ALK-pos) and 2975 EGFR-positive (EGFR-pos) patients in our database, nine (2.6% of ALK-pos and 0.2% of EGFR-pos) patients had concurrent EGFR and ALK mutations (including three exon19 Indel + EML4-ALK, two exon19 Indel + STRN-ALK, two L858R + L1152R, one L858R + EML4-ALK, and one G719C + S768I + STRN-ALK). Eight patients had at least one type of EGFR-TKIs treatment. The median progression free survival (PFS) of these patients on first-generation EGFR-TKIs was 14.5 months (95% CI: 11 - NR). Of these eight patients, one who progressed on Gefitinib and subsequently on Osimertinib had a T790M + C797G. The other seven EGFR-TKIs resistance patients had no known resistance mutations. No patients had ALK mutations before treatment, so ALK mutations may have developed as resistance mechanisms during EGFR-TKIs therapies. EGFR-TKIs-treated patients with EGFR/ALK L1152R mutations generally had a shorter PFS than patients with other mutation combinations. Conclusions ALK and EGFR mutations coincide at a relatively low frequency in lung cancer patients. ALK mutations developed either synchronously or heterochronously with EGFR mutations. Two ALK mutations (L1152R and STRN-ALK) may co-exist with EGFR mutations at a higher frequency than others. Most EGFR/ALK co-alteration patients (other than the EGFR/ALK L1152R type) can benefit from first line EGFR-TKIs.

This study evaluated the survival effects of metronomic maintenance therapy with oral fluoropyrimidine in patients with stage III colorectal cancer (CRC) according to epidermal growth factor receptor (EGFR) expression. We enrolled 197 patients with stage III CRC who had undergone radical resection and FOLFOX regimen adjuvant chemotherapy. The clinicopathological features and effects of metronomic maintenance therapy with oral capecitabine (daily dose of 850 mg/m2, twice daily, on days 114 every 3 weeks for 6 months) on survival according to treatment group and EGFR expression were analyzed. By conducting an in vitro cell line study and in vivo study through knockout of theEGFRgene, we analyzed the capacities of cell proliferation and migration. Relapse and survival were significantly more common in the FOLFOX group. Metronomic maintenance therapy was a significantly independent associated factor of relapse and survival as well as a prognostic factor of disease-free survival and overall survival. Significant intergroup differences in survival were only observed in patients with positive EGFR expression. Thus, our findings suggest EGFR expression is a prognostic factor in patients with stage III CRC receiving metronomic maintenance therapy. Analysis of EGFR expression in these patients helps identify potential candidates who may receive the optimal survival benefit from metronomic maintenance therapy.

Background Quick and reliable testing of EGFR and KRAS is needed in non-small cell lung cancer (NSCLC) to ensure optimal decision-making for targeted therapy. The Idylla™ platform was designed for Formalin-Fixed Paraffin-Embedded (FFPE) tissue sections but recently several studies were published that evaluated its potential for cytological specimens. This study aimed to validate the Idylla™ platform for the detection of EGFR/KRAS mutations in cytological NSCLC samples prepared as cytoblocks using AGAR and paraffin embedding. Material and methods The KRAS Idylla™ test were performed on 11 specimens with a known KRAS mutation. The EGFR Idylla™ test was performed on 18 specimens with a known primary EGFR mutation and 7 specimens with a primary EGFR-EGFR T790M resistance mutation combination. Results Concordant KRAS and primary EGFR mutations were detected for both KRAS and primary EGFR mutations. Samples with a total CQ value of 26 could not be assessed (probability of false-negative). In specimens with a primary EGFR-EGFR T790M resistance mutation combination, 5/7 cases were not concordant. Conclusion Our results confirm the conclusion of recent reports that the Idylla™EGFR assay is not suitable in a resistance to EGFR TKI setting, also not in our cytological NSCLC samples prepared as cytoblocks using AGAR and paraffin embedding. KRAS and primary EGFR mutations were detected using the Idylla™ assays in virtually all cytological NSCLC samples. This analysis was rapid and time-saving compared to other mutation detection assays and may be useful if the amount of material is insufficient to perform a full set of molecular tests.

Background: Epidermal growth factor receptor and anaplastic lymphoma kinase play key roles in tumorigenesis and disease progression. Currently, targeted therapy is a better approach for cancer therapy compared with traditional chemotherapy. EGFR-based/ALK-based target therapies are key targets for drug development in cancer therapy. Objective: The objective of this study was to show a recent trend in research and development of EGFR-based/ALK-based targets and to better understand the Intellectual Property surrounding EGFR-based and ALK-based targets. Methods: EGFR-based and ALK-based targets were analyzed by comprehensive US patent analysis. US patents of EGFR-based/ALK-based targets were analyzed from September 2001 to September 2020. Results: The results indicated that the key technologies and methods of EGFR-based/ALK-based targets were developed by large global pharmaceutical companies or American companies/universities. Small molecular inhibitors showed a higher percentage in the number of patents of EGFRbased targets. In addition, the present study also showed recent small molecular targeted drugs approved by FDA. Conclusion: Global large pharmaceutical companies and American companies/universities have obvious advantages in the research and development of targeted drugs. EGFR-based target was still an attractive target for research and drug development in the past 10 years. Also, large global pharmaceutical companies prefer to complete key technology research and development by independent innovation instead of collaboration.

Purpose: To date, studies regarding the use of prophylactic cranial irradiation (PCI) versus standard of care (SoC) for patients with non-small cell lung cancer (NSCLC) have not shown a significant effect in terms of overall survival (OS). Additionally, the effect of PCI among high-risk patients has been scarcely studied. The objective of this randomized phase 2 study was to evaluate the role of PCI in a population of patients at high risk for development of brain metastases (BM). Methods and Materials: Eligible patients had histologically confirmed NSCLC without baseline BM, harboring epidermal growth factor receptor mutations, anaplastic lymphoma kinase rearrangements, or elevated carcinoembryonic antigen levels at the time of diagnosis. Participants received systemic therapy according to molecular status, those without progressive disease were then assigned to receive SoC or SoC thorn PCI (25 Gy in 10 fractions). The primary outcome was cumulative incidence of brain metastases (CBM). The secondary endpoints included progression-free survival and OS. Quality of life and neurocognitive function are discussed in a separate article (Clinicaltrials.gov: NCT01603849). Results: From May 2012 to December 2017, 84 patients were enrolled in the study, with 41 patients allocated to receive PCI and 43 received SoC. Patients allocated to receive PCI had a CBM at 24 months of 7% versus 38% in those allocated to the SoC arm. PCI was associated with a hazard ratio of 0.12 (95% confidence interval, 0.035-0.42) for developing BM. A benefit in OS was also observed (64.5 vs 19.8 months; hazard ratio: 0.41 (95% confidence interval, 0.22-0.78; P = .007). Conclusions: Among a selected population at high risk for developing BM, PCI significantly decreased CBM in addition to increasing progression-free survival and OS. To our knowledge, this is the first study to evaluate PCI in epidermal growth factor receptor mutations, anaplastic lymphoma kinase rearrangements, or elevated carcinoembryonic antigen levels in patients with NSCLC, showing a significant improvement in CBM. This relevant information should be of particular importance in the context of patients without access to third-generation targeted agents. Further studies are warranted to ascertain this effect. (C) 2021 Elsevier Inc. All rights reserved.

RNA interference (RNAi) offers the potential to treat disease at the earliest onset by selectively turning off the expression of target genes, such as intracellular oncogenes that drive cancer growth. However, the development of RNAi therapeutics as anti-cancer drugs has been limited by both a lack of efficient and target cell-specific delivery systems and the necessity to overcome numerous intracellular barriers, including serum/lysosomal instability, cell membrane impermeability, and limited endosomal escape. Here, we combine two technologies to achieve posttranscriptional gene silencing in tumor cells: Centyrins, alternative scaffold proteins binding plasma membrane receptors for targeted delivery, and small interfering RNAs (siRNAs), chemically modified for high metabolic stability and potency. An EGFR Centyrin known to internalize in EGFR-positive tumor cells was site-specifically conjugated to a beta-catenin (CTNNb1) siRNA and found to drive potent and specific target knockdown by free uptake in cell culture and in mice inoculated with A431 tumor xenografts (EGFR amplified). The generalizability of this approach was further demonstrated with Centyrins targeting multiple receptors (e.g., BCMA, PSMA, and EpCAM) and siRNAs targeting multiple genes (e.g., CD68, KLKb1, and SSB1). Moreover, by installing multiple conjugation handles, two different siRNAs were fused to a single Centyrin, and the conjugate was shown to simultaneously silence two different targets. Finally, by specifically pairing EpCAM-binding Centyrins that exhibited optimized internalization profiles, we present data showing that an EpCAM Centyrin CTNNb1 siRNA conjugate suppressed tumor cell growth of a colorectal cancer cell line containing an APC mutation but not cells with normal CTNNb1 signaling. Overall, these data demonstrate the potential of Centyrin-siRNA conjugates to target cancer cells and silence oncogenes, paving the way to a new class of anticancer drugs.

Background Epidermal growth factor receptor‐tyrosine kinase inhibitor (EGFR‐TKI) has been considered as an effective treatment in epidermal growth factor receptor‐mutant (EGFR‐mutant) advanced non‐small cell lung cancer (NSCLC). However, most patients develop acquired resistance eventually. Here, we compared and analyzed the genetic alterations between tissue assay and circulating tumor DNA (ctDNA) and further explored the resistance mechanisms after EGFR‐TKI treatment. Methods and Materials Amplification refractory mutation system‐polymerase chain reaction (ARMS‐PCR), Cobas® ARMS‐PCR and next‐generation sequencing (NGS) were performed on tissue samples after pathological diagnosis. Digital droplet PCR (ddPCR) and NGS were performed on plasma samples. The association between genetic alterations and clinical outcomes was analyzed retrospectively. Results Thirty‐seven patients were included. The success rate of re‐biopsy was 91.89% (34/37). The total detection rate of EGFR T790M was 62.16% (23/37) and the consistency between tissue and ctDNA was 78.26% (18/23). Thirty‐four patients were analyzed retrospectively. For tissue re‐biopsy, 24 patients harbored concomitant mutations. Moreover, tissue re‐biopsy at resistance showed 21 patients (21/34, 61.76%) had the concomitant somatic mutation. The three most frequent concomitant mutations were tp53 (18/34, 52.94%), MET (4/34, 11.76%), and PIK3CA (4/34, 11.76%). Meanwhile, 21 patients (21/34, 61.76%) with EGFR T790M mutation. Progression‐free survival (PFS) and overall survival (OS) were better in patients with T790M mutation (p = 0.010 and p = 0.017) or third‐generation EGFR‐TKI treatment (p, It is feasible to use multi‐platforms to perform detection of EGFR T790M mutation on EGFR‐TKI resistant re‐biopsy tissue and blood samples. The detection consistency of EGFR T790M between tissue and blood samples is high. Concomitant genetic alterations may lead to a worse response to treatment and affect decisions of the sequential therapy strategy.

The reproductive division of labour is a hallmark of eusocial Hymenoptera. Females are either reproductive queens or non-reproductive workers. In ants, workers often display further task specialisation that is associated with variation in size and/or morphology. Because female polyphenism is typically under environmental control, it is thought epigenetic mechanisms (such as DNA methylation) play a central role since they mediate gene-by-environment interactions. Methylation of the growth-promoting geneepidermal growth factor receptor(egfr) was indeed shown to control worker size in a highly polymorphic ant. However, it remains unknown ifegfrmethylation could also regulate worker size in monomorphic species. By combining experimental pharmacology and molecular biology, we show that worker size is associated withegfrmethylation in two monomorphic ants. Furthermore, we functionally demonstrate that EGFR signalling affects worker size. These results indicate that worker size regulation byegfrmethylation has been mechanistically conserved in ants but remains unexploited in monomorphic species.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are widely used for the treatment of EGFR mutation positive advanced nonsmall cell lung cancer (NSCLC); however, acquired resistance is known to develop during these treatments. Among these mechanisms, histological transformation is seldom encountered. Although platinum based chemotherapy has been reported to be effective in the treatment of patients with small cell lung cancer transformation, there is a lack of information on the treatment of patients with squamous cell carcinoma (SQ) transformation.An 80-year-old nonsmoking woman was referred to our hospital because of an abnormal shadow on her chest radiograph. Diagnostic bronchoscopy was performed and pathological examination revealed adenocarcinoma. Mutation analysis of the EGFR gene revealed deletion of E746-A750 in exon 19. She refused both surgical treatment and radiation therapy, and preferred periodic radiologic follow-up. Unfortunately, approximately a year and a half after the initial diagnosis, the primary lesion enlarged, and many pleural nodules were newly detected (clinically T4N2M1a, stage IVA).Based on EGFR mutation analysis, a reduced dose of daily erlotinib was prescribed, which achieved a partial response and 34 months of progression-free survival (PFS). A repeated biopsy with an endobronchial cryoprobe was performed on the enlarged primary lesion. Pathological examination revealed SQ harboring an identical EGFR mutation with a secondary EGFR T790M mutation. Osimertinib 80 mg once a day was started as second line therapy, which resulted in 8 months of PFS and 15 months of survival.The literature review and our report suggest that osimertinib is a promising treatment for NSCLC regardless of histology if T790M is present as an acquired mutation.

Background/Aim: Next-generation sequencing (NGS) has recently made it possible to investigate polar charged amino acids in compound mutations in the epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC). Several preclinical studies have suggested the involvement of polar charged amino acids in the treatment of EGFR mutations and EGFR-tyrosine kinase inhibitors (TKIs). With this background, a retrospective study was conducted aiming to clarify the prognostic significance of these amino acids in complex mutations in NSCLC patients with common EGFR mutations. Patients and Methods: EGFR gene mutations were investigated using nonoverlapping integrated read sequencing system (NOIR-SS) in pathological specimens of 20 EGFR-mutated NSCLC patients. For clinical information, the medical records were retrospectively investigated. We investigated prognostic significance of these amino acids in compound mutations in progression free survival (PFS) and overall survival (OS) in patients treated with first-line afatinib. Results: Among the 20 patients examined, 5 patients had polar charged amino acids in compound mutations and 15 had not. There were no statistically significant differences in the clinical background factors examined in these two groups of patients. In uni- and multivariate analysis, ‘poor performance status’ and ‘polar charged amino acids in compound mutations’ were significant favorable factors in OS. Conclusion: Patients with ‘polar charged amino acids in compound mutations’ might have favorable prognosis than those without them. Detailed examination of EGFR gene information might contribute to the understanding of TKI response duration.

Lung carcinoma is a foremost cause of cancer-related mortality worldwide. Variable genetic factors are associated with the development of lung cancer. This study was performed to evaluate the possible association of epidermal growth factor receptor (EGFR) gene polymorphisms with non small cell lung carcinoma (NSCLC) in Iraqi population.DNA samples were extracted from 100 patients and 100 controls. Four PCR fragments were designed to amplify four high-frequency variants within EGFR, namely rs1050171, rs2072454, rs2227984, and rs2227983. The PCR fragments were genotyped by single-strand conformation polymorphism (SSCP) method, and each genotype was subjected to direct sequencing.Genotyping experiments confirmed the variability of three targeted variants, and logistic regression analysis showed that two of these variants (rs1050171 and rs2227983) tend to exhibit a significant association with NSCLC. Individuals with rs1050171:GA genotype showed a possible association with the increased risk of NSCLC (P = 0.0110; OD 5.2636; ClOur collective data indicated that EGFR-rs1050171G/A and EGFR-rs2227983G/G SNPs tend to exert significant and separate associations with the increased risk of NSCLC. However, this study recommends using a broader spectrum of the investigated samples to get further details of both SNPs in terms of their association with the susceptibility to NSCLC.

The lack of reliable and practical method for detecting rare hot mutation of epidermal growth factor receptor (EGFR) in circulating tumor DNA (ctDNA) for lung cancer has remained a challenge for general clinical application due to excess wild type DNA in clinical samples. In this study, we developed a droplet digital PCR (ddPCR) platform, integrating a PDMS chip and double-layer glass reservoir. The duplex T-junction droplet generators in PDMS chip can produce about one million uniform droplets of 4.187 pL within ∼10 min, which were then stored in the glass reservoir. The double-layer glass reservoir can protect droplets from evaporation and breaking, solving the problem of instability during thermal-cycling. The quantitative capabilities of the ddPCR chip were evaluated by testing EGFR exon gene 21, with a good linear correlation in the wide range of 10

Background EGFR mutations are good predictive markers of efficacy of EGFR tyrosine kinase inhibitors (EGFR‐TKI), but whether comprehensive genomic analysis beyond EGFR itself with circulating tumor DNA (ctDNA) adds further predictive or prognostic value has not been clarified. Methods Patients with NSCLC who progressed after treatment with EGFR‐TKI, and with EGFR T790 M detected by an approved companion diagnostic test (cobas®), were treated with osimertinib. Plasma samples were collected before and after treatment. Retrospective comprehensive next‐generation sequencing (NGS) of ctDNA was performed with Guardant360®. Correlation between relevant mutations in ctDNA prior to treatment and clinical outcomes, as well as mechanisms of acquired resistance, were analyzed. Results Among 147 patients tested, 57 patients received osimertinib, with an overall response rate (ORR) of 58%. NGS was successful in 54 of 55 available banked plasma samples; EGFR driver mutations were detected in 43 (80%) and T790 M in 32 (59%). The ORR differed significantly depending on the ratio (T790 M allele fraction [AF])/(sum of variant AF) in ctDNA (p = 0.044). The total number of alterations detected in plasma by NGS was higher in early resistance patients (p = 0.025). T790 M was lost in 32% of patients (6 out of 19) after acquired resistance to osimertinib. One patient with RB1 deletion and copy number gains of EGFR, PIK3CA, and MYC in addition to T790 M, showed rapid progression due to suspected small cell transformation. Conclusions NGS of ctDNA could be a promising method for predicting osimertinib efficacy in patients with advanced NSCLC harboring EGFR T790 M., A retrospective analysis of comprehensive next‐generation sequencing (NGS) of ctDNA in a multi‐center, prospective observational cohort study conducted to examine the efficacy of liquid biopsy as a predictive marker for third generation treatment with the EGFR tyrosine kinase inhibitor (EGFR‐TKI), osimertinib is reported in this paper. We demonstrated that the number of genomic alterations was significantly higher in non‐responders than in responders to osimertinib, suggesting that plasma NGS could be a better method for predicting osimertinib efficacy in patients with advanced NSCLC.

Among non-small cell lung cancer (NSCLC) patients with therapeutically targetable tumor mutations in epidermal growth factor receptor (EGFR), not all patients respond to targeted therapy. Combining circulating-tumor DNA (ctDNA), clinical variables, and radiomic phenotypes may improve prediction of EGFR-targeted therapy outcomes for NSCLC. This single-center retrospective study included 40 EGFR-mutant advanced NSCLC patients treated with EGFR-targeted therapy. ctDNA data included number of mutations and detection of EGFR T790M. Clinical data included age, smoking status, and ECOG performance status. Baseline chest CT scans were analyzed to extract 429 radiomic features from each primary tumor. Unsupervised hierarchical clustering was used to group tumors into phenotypes. Kaplan–Meier (K–M) curves and Cox proportional hazards regression were modeled for progression-free survival (PFS) and overall survival (OS). Likelihood ratio test (LRT) was used to compare fit between models. Among 40 patients (73% women, median age 62 years), consensus clustering identified two radiomic phenotypes. For PFS, the model combining radiomic phenotypes with ctDNA and clinical variables had c-statistic of 0.77 and a better fit (LRT p = 0.01) than the model with clinical and ctDNA variables alone with a c-statistic of 0.73. For OS, adding radiomic phenotypes resulted in c-statistic of 0.83 versus 0.80 when using clinical and ctDNA variables (LRT p = 0.08). Both models showed separation of K–M curves dichotomized by median prognostic score (p EGFR mutations.

DuringC. eleganslarval development, an inductive signal mediated by LET-23 EGFR (Epidermal Growth Factor Receptor) specifies three of six vulva precursor cells (VPCs) to adopt vulval cell fates. An evolutionarily conserved complex consisting of PDZ domain-containing scaffold proteins LIN-2 (CASK), LIN-7 (Lin7 or Veli), and LIN-10 (APBA1 or Mint1) (LIN-2/7/10) mediates basolateral LET-23 EGFR localization in the VPCs to permit signal transmission and development of the vulva. We recently found that the LIN-2/7/10 complex likely forms at Golgi ministacks or recycling endosomes; however, the mechanism through which the complex targets the receptor to the basolateral membrane remains unknown. Here we found that overexpression of LIN-10 or LIN-7 can compensate for loss of their complex components by promoting LET-23 EGFR signaling through previously unknown complex-independent and receptor-dependent pathways. In particular, LIN-10 can independently promote basolateral LET-23 EGFR localization, and its complex-independent function uniquely requires its PDZ domains that also regulate its localization to Golgi ministacks and recycling endosomes. These studies point to a novel complex-independent function for LIN-7 and LIN-10 that broadens our understanding of how this complex regulates targeted sorting of membrane proteins.

Liquid biopsy analyzes the current status of primary tumors and their metastatic regions. We aimed to develop an optimized protocol for single‐cell sequencing of floating tumor cells (FTCs) in pleural effusion as a laboratory test. FTCs were enriched using a negative selection of white blood cells by a magnetic‐activated cell sorting system, and CD45‐negative and cytokeratin‐positive selection using a microfluidic cell separation system with a dielectrophoretic array. The enriched tumor cells were subjected to whole‐genome amplification (WGA) followed by genome sequencing. The FTC analysis detected an EGFR exon 19 deletion in Case 1 (12/19 cells, 63.2%), and EML4‐ALK fusion (17/20 cells, 85%) with an alectinib‐resistant mutation of ALK (p.G1202R) in Case 2. To eliminate WGA‐associated errors and increase the uniformity of the WGA product, the protocol was revised to sequence multiple single FTCs individually. An analytical pipeline, accurate single‐cell mutation detector (ASMD), was developed to identify somatic mutations of FTCs. The large numbers of WGA‐associated errors were cleaned up, and the somatic mutations detected in FTCs by ASMD were concordant with those found in tissue specimens. This protocol is applicable to circulating tumor cells analysis of peripheral blood and expands the possibility of utilizing molecular profiling of cancers., An optimized protocol is established for single‐cell sequencing of floating tumor cells (FTCs) in pleural effusion as a laboratory test that could be utilized for patient care. The FTC analysis detected an EGFR exon 19 deletion in Case 1 (12/19 cells, 63.2%) and EML4‐ALK fusion in Case 2 (17/20 cells, 85%). A novel analytical pipeline, accurate single‐cell mutation detector (ASMD), was developed to identify somatic mutations in single cells accurately.

Background Pleural effusion from patients with advanced non‐small cell lung cancer (NSCLC) has been proved valuable for molecular analysis, especially when the tissue sample not available. However, simultaneous detection of multiple driver gene alterations especially the fusions is still challenging. Methods In this study, 77 patients with advanced NSCLC and pleural effusion were enrolled, 49 of whom had matched tumor tissues. Supernatants, cell sediments, and cell blocks were prepared from pleural effusion samples for detection of driver alterations by a PCR‐based 9‐gene mutation detection kit. Results Mutations in EGFR, KRAS, and HER2 were detected in DNA and cfDNA, fusions in ALK was detected in RNA and cfRNA. Compared with matched tumor tissue, the supernatant showed the highest overall sensitivity (81.3%), with 81.5% for SNV/Indels by cfDNA and 80% for fusions by cfRNA, followed by cell blocks (71.0%) and the cell sediments (66.7%). Within the group of treatment‐naïve patients or malignant cells observed in the cell sediments, supernatant showed higher overall sensitivity (89.5% and 92.3%) with both 100% for fusions. Conclusions CfDNA and cfRNA derived from pleural effusion supernatant have been successfully tested with a PCR‐based multigene detection kit. Pleural effusion supernatant seems a preferred material for detection of multigene alterations to guide treatment decision of advanced NSCLC., CfDNA and cfRNA derived from pleural effusion supernatant have been successfully tested with a PCR‐based multi‐gene detection kit. Pleural effusion supernatant seems a preferred material for detection of multi‐gene alterations to guide treatment decision of advanced NSCLC.

Purpose: TRAF3IP3 is involved in the maturation of immune cells, the development of immune tissues and the immune response of the body. TRAF3IP3 is shown to expressed in a variety of malignant tumor cell lines. Down-regulated expression of TRAF3IP3 in malignant melanoma can inhibit tumor growth. However, the role of TRAF3IP3 in glioma is still unknown. In this study, we aimed to study the relationship between TRAF3IP3 and glioma based on TCGA data.Method: We used the Wilcoxon rank sum test to compare the expression of TRAF3IP3 in glioma and normal tissues. Subsequently, Kruskal-Wallis test, Wilcoxon rank sum test, and logistics regression were used to evaluate the relationship between TRAF3IP3 and clinicopathological parameters of glioma patients. GSEA was used to verify the key signal pathways involved in TRAF3IP3. We used the ssGSEA method to analyze the relationship between the expression level of TRAF3IP3 and the immune infiltration in the glioma tumor microenvironment. Finally, we used Kaplan-Meier and COX regression to evaluate the prognostic value of TRAF3IP3.Results: TRAF3IP3 transcription level was highly expressed in gliomas(PConclusion: TRAF3IP3 may play an important role in the occurrence and development of glioma, and may be a potential biomarker for the diagnosis and prognosis of glioma.