Epidermal Growth Factor Receptor DNA Research Articles

An ultrasensitive DNA-enhanced amplification method for detecting cfDNA drug-resistant mutations in non-small cell lung cancer with selective FEN-assisted degradation of dominant somatic fragments.

Blood cell-free DNA (cfDNA) can be a new reliable tool for detecting epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) patients. However, the currently reported cfDNA assays have a limited role in detecting drug-resistant mutations due to their deficiencies in sensitivity, stability, or mutation detection rate. We developed an Archaeoglobus fulgidus-derived flap endonuclease (Afu FEN)-based DNA-enhanced amplification system of mutated cfDNA by designing a pair of hairpin probes to anneal with wild-type cfDNA to form two 5'-flaps, allowing for the specific cleavage of wild-type cfDNA by Afu FEN. When the dominant wild-type somatic cfDNA fragments were cleaved by structure-recognition-specific Afu FEN, the proportion of mutated cfDNA in the reaction system was greatly enriched. As the amount of mutated cfDNA in the system was further increased by PCR amplification, the mutation status could be easily detected through first-generation sequencing. In a mixture of synthetic wild-type and T790M EGFR DNA fragments, our new assay still could detect T790M mutation at the fg level with remarkably high sensitivity. We also tested its performance in detecting low variant allele frequency (VAF) mutations in clinical samples from NSCLC patients. The plasma cfDNA samples with low VAF (0.1 and 0.5 %) could be easily detected by DNA-enhanced amplification. This system with enhanced amplification of mutated cfDNA is an effective tool used for the early screening and individualized targeted therapy of NSCLC by providing arapid, sensitive, and economical way for the detection of drug-resistant mutations in tumors.

Abstract A034: Synthesis and biological activities of a novel series of “combi-molecules” designed to delay metabolic dealkylation prior to crossing the blood brain barrier in the context of optimizing their potency against glioblastoma multiforme

Abstract Introduction. Glioblastoma multiforme (GBM) is the most aggressive form of primary brain tumor in adults with a survival of only 12-15 months. GBM stem cells contribute to resistance to the DNA-damaging first-line chemotherapy, temozolomide, which accounts for tumor recurrence and treatment failure. Amplification of epidermal growth factor receptor (EGFR) is one of the most common genetic alterations associated with GBM aggressiveness. Despite the evidence that EGFR-induced pathway represents an attractive therapeutic target in GBM, gefitinib, an orally active selective EGFR-tyrosine kinase inhibitor, showed only a limited potency in clinical trials. We previously showed that ZR2002, an aminoquinazoline “combi-molecule” designed to possess mixed EGFR tyrosine kinase inhibition and DNA damaging properties, induced submicromolar cytotoxic activity against GBM stem cells resistant to temozolomide and gefitinib. Significantly increased survival of mice harboring an intracranially implanted aggressive temozolomide-resistant tumour by comparison with the clinical treatment gefitinib. Furthermore, mass spectrometry imaging provided evidence of its ability to cross the blood–brain barrier but metabolites devoid of its critical 2-chloroethyl DNA alkylating function were observed. Given that the loss of DNA alkylating species may decrease efficacy in vivo, here we show a structure-activity relationship study toward structures with increased stability to metabolic dealkylation. Materials and methods. Three glioblastoma cells line (U87-wt, U87-EGFR and U87-EGFRvIII) were used to determine IC50 of the new molecules with SRB assay. CD1 mice were treated with a dose of 100mg/kg by oral gavage and analyzed after 3h. Drug metabolism was measured by LC-MS. Results. In vitro growth inhibitory showed that the new analogues have the same cytotoxicity profile as ZR2002. In vivo analysis in mice administered p.o. with the new analogues showed that our newly synthesized molecules were less metabolized than ZR2002 using the ratio of intact structure levels/over metabolites as a parameter that we defined as extent of metabolism. Furthermore, they were found to maintain good brain penetration. Conclusions. These results proved that we were able to design and synthesize combi-molecules that can escape metabolism and preserve the 2-chloroethyl in vivo, a group that is required for the dual activity of the combi-molecules. Importantly, the combi-molecules, despite the structural change, remain very water soluble and available orally. Citation Format: Ana Belen Fraga-Timiraos, Caterina Facchin, Anne-Laure Larroque-Lombard, Bertrand Jacques Jean-Claude. Synthesis and biological activities of a novel series of “combi-molecules” designed to delay metabolic dealkylation prior to crossing the blood brain barrier in the context of optimizing their potency against glioblastoma multiforme [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A034.

59P Clinical utility of plasma cell-free DNA EGFR mutation analysis in treatment-naïve stage IV non-small cell lung cancer patients

Plasma cell-free DNA epidermal growth factor receptor (EGFR) mutation tests are widely used at initial diagnosis and at progression in stage IV non-small cell lung cancer (NSCLC). We analyzed the factors associated with plasma EGFR mutation detection and the effect of plasma EGFR genotyping on the clinical outcomes of the patients with treatment-naïve stage IV NSCLC. In this retrospective cohort study, we included subjects with treatment-naïve stage IV NSCLC who underwent plasma EGFR genotyping between 2018 and 2020. The presence of plasma EGFR mutation was determined by real-time polymeric chain reaction. The prevalence of EGFR mutation in this cohort was 52.7% (164/311). Among 164 EGFR mutant subjects, 34 (20.7%) were positive for the plasma EGFR mutation assay only. The diagnostic sensitivity of the plasma EGFR mutation assay (80.5%) was similar to that of the tissue EGFR mutation assay (79.3%, p = 0.787). In multivariable analysis, the detection of plasma EGFR mutation was significantly related to higher serum carcinoembryonic antigen levels, never-smoker status, N3 stage, and brain or intrathoracic metastasis. The time to treatment initiation (TTI) of the plasma EGFR mutation-positive group (14 days) was shorter than that of the plasma EGFR mutation-negative group (21 days, p < 0.001). More patients received the 1st line EGFR-TKI in the plasma positive group compared with the tissue positive group. Smoking status and the factors reflecting tumor burden were associated with detection of plasma EGFR mutation. The plasma EGFR mutation assay can shorten the TTI, and facilitate the 1st line EGFR-TKI therapy for patients with treatment-naïve stage IV NSCLC, especially in the region of high-prevalence of EGFR mutation.

EGFR DNA Methylation Correlates With EGFR Expression, Immune Cell Infiltration, and Overall Survival in Lung Adenocarcinoma.

BackgroundThe epidermal growth factor receptor (EGFR) is a primary target of molecular targeted therapy for lung adenocarcinoma (LUAD). The mechanisms that lead to epigenetic abnormalities of EGFR in LUAD are still unclear. The purpose of our study was to evaluate the abnormal methylation of EGFR CpG sites as potential biomarkers for LUAD.MethodsTo assess the differentially methylation CpG sites of EGFR in LUAD, we used an integrative study of Illumina HumanMethylation450K and RNA-seq data from The Cancer Genome Atlas (TCGA). We evaluated and compared EGFR multiple-omics data to explore the role of CpG sites located in EGFR promoter regions and gene body regions and the association with transcripts, protein expression levels, mutations, and somatic copy number variation. We calculated the correlation coefficients between CpG sites of EGFR and immune infiltration fraction (by MCPcounter and ESTIMATE) and immune-related pathways in LUAD. Finally, we validated the differential methylation of clinically and prognostically relevant CpG sites using quantitative methylation-specific PCR (qMSP).ResultsWe found that the methylation level of many EGFR CpGs in the promoter region was negatively correlated with the transcription level, protein expression, and SCNV, while the methylation at the gene body region was positively correlated with these features. The methylation level of EGFR CpGs in the promoter region was positively correlated with the level of immune infiltration and IFN-γ signature, while the opposite was found for methylation of the gene body region. The qMSP results showed that cg02316066 had a high methylation level, while cg02166842 had a low methylation level in LUAD. There was a high degree of co-methylation between cg02316066 and cg03046247.ConclusionOur data indicate that EGFR is an epigenetic regulator in LUAD acting through DNA methylation. Our research provides a theoretical basis for the further detection of EGFR DNA methylation as a predictive biomarker for LUAD survival and immunotherapy.

Giant Magnetoresistive Nanosensor Analysis of Circulating Tumor DNA Epidermal Growth Factor Receptor Mutations for Diagnosis and Therapy Response Monitoring.

Liquid biopsy circulating tumor DNA (ctDNA) mutational analysis holds great promises for precision medicine targeted therapy and more effective cancer management. However, its wide adoption is hampered by high cost and long turnaround time of sequencing assays, or by inadequate analytical sensitivity of existing portable nucleic acid tests to mutant allelic fraction in ctDNA. We developed a ctDNA Epidermal Growth Factor Receptor (EGFR) mutational assay using giant magnetoresistive (GMR) nanosensors. This assay was validated in 36 plasma samples of non-small cell lung cancer patients with known EGFR mutations. We assessed therapy response through follow-up blood draws, determined concordance between the GMR assay and radiographic response, and ascertained progression-free survival of patients. The GMR assay achieved analytical sensitivities of 0.01% mutant allelic fraction. In clinical samples, the assay had 87.5% sensitivity (95% CI = 64.0-97.8%) for Exon19 deletion and 90% sensitivity (95% CI = 69.9-98.2%) for L858R mutation with 100% specificity; our assay detected T790M resistance with 96.3% specificity (95% CI = 81.7-99.8%) with 100% sensitivity. After 2 weeks of therapy, 10 patients showed disappearance of ctDNA by GMR (predicted responders), whereas 3 patients did not (predicted nonresponders). These predictions were 100% concordant with radiographic response. Kaplan-Meier analysis showed responders had significantly (P < 0.0001) longer PFS compared to nonresponders (N/A vs. 12 weeks, respectively). The GMR assay has high diagnostic sensitivity and specificity and is well suited for detecting EGFR mutations at diagnosis and noninvasively monitoring treatment response at the point-of-care.

Secondary Structure Analysis by SHAPE‐MaP of the EGFR pre‐mRNA Transcript to Identify Target Regions for RNA Anti‐sense Therapies

Glioblastoma Multiforme (GBM) is the most common primary brain malignancy with a median survival of 10–14 months. A common aberration in GBM is the overexpression of epidermal growth factor receptor (EGFR), dysregulated in 57% of all GBM. Standard care increases survival to only 14–15 months indicating a necessity to generate novel therapeutics. Our strategy is to deliver a gene directly to the CNS using an adeno‐associated virus gene transfer vector encoding antisense RNA therapeutics. This therapy targets critical splicing elements surrounding Intron 10 of the EGFR pre‐mRNA transcript. Cryptic poly‐a signals within intron 10 have the potential to activate the expression of a therapeutic isoform of EGFR. Alternative splicing and polyadenylation is regulated by secondary structure of the pre‐mRNA transcript. To improve our therapeutic strategy, we have analyzed the EGFR secondary structure using selective 2′ hydroxyl acylation and primer extension followed by mutational profiling (SHAPE‐MaP). The SHAPE reagent 1M7 reacts with the 2′ hydroxyl of RNA molecules when the RNA molecule is in a conformationally flexible position creating a 2′ O‐adduct. The modified RNA is reverse transcribed, incorporating mismatches at the acylated positions; a comparison of unmodified to modified RNA allows us to determine RNA nucleotides that are involved in secondary structure, part of RNA‐binding‐protein complexes, or single stranded. The EGFR DNA sequence of Exon 10, Intron 10, Exon 11 was cloned into a plasmid. The plasmid was PCR amplified with a forward T7 promoter primer and reverse primer corresponding to exon 11, this generated the template for T7 RNA transcription. RNA was T7 transcribed and purified by RNA gel electrophoresis. RNA was treated with 1M7 (+) or DMSO (−), reverse transcribed under SHAPE conditions with Superscript II then converted to cDNA. Reverse transcription conditions for SHAPE experiments incorporate Mn++ as the divalent cation, which allows the enzyme to incorporate nucleotide mismatches. 3.5 million reads generated using Nanopore MinION sequencing corresponded to (+) and (−) conditions with a mean read length of 936 nucleotides and a quality score of 9.7. Sequencing output was run through the ShapeMapper2 pipeline. The median read depth at each nucleotide was at least 92K for all conditions after elimination of reads under quality control score of 15. SHAPE‐MaP reactivity profiles were generated using RNAfold. Splicing elements within the targeted region were identified and compared to reactivity profiles. SRp40 binding motifs show high accessibility upstream of the 5′ splice site. The exon‐intron junction of the 5′ splice site is structurally available while the 3′ intron‐exon junction shows moderate accessibility. We identified a putative poly‐a signal exists within intron 10 and the literature shows expression of this shortened EGFR isoform in various cell types. Importantly, this cryptic poly‐a signal shows no reactivity indicating high secondary structures blocking recognition by the poly‐a machinery. Based on these structural profiles, we generated RNA antisense molecules to unravel the hidden poly‐a signal to activate expression of the short isoform. In conjunction, we used eCLIP to identify target specific RNA‐binding proteins.

The Correlation between EGFR Mutation Status and DNA Content of Lung Adenocarcinoma Cells in Pleural Effusion.

Objectives: Lung adenocarcinomas with or without epidermal growth factor receptor (EGFR) mutations have shown different drug effects against EGFR inhibitors. But it is not very clear if EGFR mutation status affects the biological behavior of lung adenocarcinoma, because tumor gene regulation is very complicated and can be affected by many factors. We aimed to explore if EGFR mutation status is related with tumor malignant degree by investigating the relevance of EGFR mutation status with DNA content and aneuploid peaks of lung adenocarcinoma cells in pleural fluids without using EGFR-TKIs.Materials and Methods: 591 cases of lung adenocarcinoma patients in Hebei Tumor Hospital who had undergone EGFR gene detection and DNA quantitative analysis were collected from January 2012 to August 2018.They were divided into two groups: EGFR mutant group and non-mutant group. EGFR mutations were detected by Amplification Refractory Mutation System (ARMS) and ABI 7500 Fluorescence quantitative PCR with pleural effusions. DNA content and aneuploid peaks were detected by LD DNA image cytometry (DNA-ICM). Rank-sum test of SPSS 16 was used for statistical analysis.Results: The maximum DI, the mean DI of the first 20 cells greater than 5C, the percentage of cells greater than 5C and the number of cells greater than 9C of the first 20 cells in the mutant group were all higher than those in the non-mutant group, having statistical significance (p<0.001); the peaks of aneuploid cells in the mutant group occurred more often than those in the non-mutant group, having statistical significance (p<0.001).Conclusions: Our study has shown that advanced lung adenocarcinomas with EGFR-mutations had higher DI values, more aneuploid cells and more frequent aneuploid peaks compared with those without EGFR-mutations, suggesting that advanced lung adenocarcinomas with EGFR mutations are more aggressive than those without EGFR mutations.

Involvement of adenosine A2B receptor in radiation-induced translocation of epidermal growth factor receptor and DNA damage response leading to radioresistance in human lung cancer cells

BackgroundAdenosine receptors are involved in tumor growth, progression, and response to therapy. Among them, A2B receptor is highly expressed in various tumors. Furthermore, ionizing radiation induces translocation of epidermal growth factor receptor (EGFR), which promotes DNA repair and contributes to radioresistance. We hypothesized that A2B receptor might be involved in the translocation of EGFR. MethodsWe investigated whether A2B receptor is involved in EGFR translocation and DNA damage response (γH2AX/53BP1 focus formation) of lung cancer cells by means of immunofluorescence studies. Radiosensitivity was evaluated by colony formation assay after γ-irradiation. ResultsA2B receptor was expressed at higher levels in cancer cells than in normal cells. A2B receptor antagonist treatment or A2B receptor knockdown suppressed EGFR translocation, γH2AX/53BP1 focus formation, and colony formation of lung cancer cell lines A549, calu-6 and NCI-H446, compared with a normal cell line (beas-2b). γ-Irradiation-induced phosphorylation of src and EGFR was also attenuated by suppression of A2B receptor expression. ConclusionActivation of A2B receptor mediates γ-radiation-induced translocation of EGFR and phosphorylation of src and EGFR, thereby promoting recovery of irradiated lung cancer cells from DNA damage. General significanceOur results indicate that A2B receptors contribute to radiation resistance in a cancer-cell-specific manner, and may be a promising target for radiosensitizers in cancer radiotherapy.

Nuclear Delivery of Radiosensitizing EGFR-Targeted Nanoparticles Is Abrogated in Isogenic Cervical Cancer Cell Lines Following Mutagenesis of the Ligand Binding Domain

The development of targeted treatment regimens for cervical cancer remains a critically unexplored area of research. One such possible target is epidermal growth factor receptor (EGFR). Our laboratory has previously synthesized radiosensitizing nanoparticles (NPs) that specifically target cells that express EGFR. Here, we test the hypothesis that nuclear localization following treatment of EGFR-targeted NPs is preferentially achieved in cervical cancer cell lines with high levels of EGFR expression and is dependent upon an intact ligand binding domain. A panel of cervical cancer cell lines (HeLa, SiHa, CaSki, ME180, and C33A) with varying levels of endogenous and genetically manipulated EGFR expression were cultured and treated with Fe3O4@TiO2 NPs conjugated to EGF “mimic” (B-loop) peptide (EGFR-targeted NPs) or its scrambled version (scrambled non-targeted NPs). Transient overexpression and knockdown of EGFR was achieved with transfection of EGFR DNA or small interfering RNA (siRNA). Isogenic cell lines were expanded from single cell clones of the ME-180 cervical cancer cell line with heterozygous or homozygous mutations, introduced by CRISPR-Cas9 technology, of the critical amino acids Leucine-14 (Leu-14) and Tyrosine-45 (Tyr-45) located within the ligand binding domain of the EGFR protein. Immunocytochemistry with confocal microscopy and flow cytometry was utilized to assess EGF-binding ability of isogenic cell lines as well ascellular delivery, subcellular distribution, and receptor binding ability of EGFR-targeted NPs and scrambled non-targeted NPs in this panel of cell lines. All tested cervical cancer cell lines endogenously produced both full-length EGFR mRNA variant 1 (v1), as well as shorter mRNA variants 3 and 4 which encode soluble isoforms of EGFR, while expression of full-length EGFR (v1) varied dramatically across cell lines. Cell lines with high levels of endogenous or genetically upregulated EGFR expression experienced increased nuclear uptake of EGFR-targeted NPs compared to cell lines with low levels of endogenous EGFR expression or following siRNA-mediated knockdown, while uptake of scrambled non-targeted NPs was similar across all cell lines. Isogenic clones of the ME-180 cervical cancer cell line demonstrated impaired EGF-binding as well as nuclear localization of EGFR-targeted NPs but scrambled non-targeted NPs. Nuclear accumulation of EGFR-targeted radiosensitizing NPs, but not scrambled non-targeted NPs, occurred preferentially in cervical cancer cell lines with high expression of EGFR and was abrogated following mutation of the ligand binding domain. Further studies are required to demonstrate in vivo radiosensitization of cervical cancer following treatment with EGFR-targeted NPs.

Selective gene amplification to detect the T790M mutation in plasma from patients with advanced non-small cell lung cancer (NSCLC) who have developed epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) resistance.

The epidermal growth factor receptor (EGFR) T790M mutation is associated with resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in non-small cell lung cancer (NSCLC). However, tissues for the genotyping of the EGFR T790M mutation can be difficult to obtain in a clinical setting. The aims of this study were to evaluate a blood-based, non-invasive approach to detecting the EGFR T790M mutation in advanced NSCLC patients using the PointMan™ EGFR DNA enrichment kit, which is a novel method for the selective amplification of specific genotype sequences. Blood samples were collected from NSCLC patients who had activating EGFR mutations and who were resistant to EGFR-TKI treatment. Using cell-free DNA (cfDNA) from plasma, EGFR T790M mutations were amplified using the PointMan™ enrichment kit, and all the reaction products were confirmed using direct sequencing. The concentrations of plasma DNA were then determined using quantitative real-time PCR. Nineteen patients were enrolled, and 12 patients (63.2%) were found to contain EGFR T790M mutations in their cfDNA, as detected by the kit. T790M mutations were detected in tumor tissues in 12 cases, and 11 of these cases (91.7%) also exhibited the T790M mutation in cfDNA samples. The concentrations of cfDNA were similar between patients with the T790M mutation and those without the mutation. The PointMan™ kit provides a useful method for determining the EGFR T790M mutation status in cfDNA.

Inhibition of EGFR nuclear translocation attenuate radioresistance through decreased p-DNA-PK expression in cervical cancer cells.

e17011 Background: The commonly used treatment for cervical cancer is radiotherapy. However, the resistance to irradiation and metastasis at the advanced stage is a common reason for the poor prognosis and high mortality. This study was designed to elucidate the role of epidermal growth factor receptor (EGFR) nuclear translocation in radioresistance, and its correlation with DNA damage repair pathway in the cervical cancer cells. Methods: The dynamic expression of EGFR, DNA-dependent protein kinase (DNA-PK), PDK-1, PKN1 and their phosphorylation level in irradiated cervical cancer cell line CaSki at 0 10 20 40 minutes was determined by western blotting. Besides, nuclear localization signal (NLS) peptide inhibitor and control peptides was synthesized and treated cells before irradiation to elucidate the correlation between EGFR nuclear translocation and DNA damage repair after irradiation. Results: Expression of EGFR, protein kinase N1 (PKN1), and DNA-PK in nucleus was increased after irradiation in CaSki cells. Irradiation also enhanced the phosphorylation level of EGFR at Thr654, PKN1 at T774 and DNA-PK at T2609. Inhibition of EGFR nuclear translocation by NLS peptide decreased the expression level of EGFR and DNA-PK in the nucleus, and attenuated their phosphorylation process. Conclusions: EGFR nuclear translocation riggered by irradiation promoted DNA damage repair in irradiated cervical cancer cells. This work facilitated us to understand the possible molecular mechanism of the resistance to irradiation in the treatment of cervical cancer, providing a potentially potent clinical method to cancer therapy.

Detection of wild-type EGFR amplification and EGFRvIII mutation in CSF-derived extracellular vesicles of glioblastoma patients.

RNAs within extracellular vesicles (EVs) have potential as diagnostic biomarkers for patients with cancer and are identified in a variety of biofluids. Glioblastomas (GBMs) release EVs containing RNA into cerebrospinal fluid (CSF). Here we describe a multi-institutional study of RNA extracted from CSF-derived EVs of GBM patients to detect the presence of tumor-associated amplifications and mutations in epidermal growth factor receptor (EGFR). CSF and matching tumor tissue were obtained from patients undergoing resection of GBMs. We determined wild-type (wt)EGFR DNA copy number amplification, as well as wtEGFR and EGFR variant (v)III RNA expression in tumor samples. We also characterized wtEGFR and EGFRvIII RNA expression in CSF-derived EVs. EGFRvIII-positive tumors had significantly greater wtEGFR DNA amplification (P = 0.02) and RNA expression (P = 0.03), and EGFRvIII-positive CSF-derived EVs had significantly more wtEGFR RNA expression (P = 0.004). EGFRvIII was detected in CSF-derived EVs for 14 of the 23 EGFRvIII tissue-positive GBM patients. Conversely, only one of the 48 EGFRvIII tissue-negative patients had the EGFRvIII mutation detected in their CSF-derived EVs. These results yield a sensitivity of 61% and a specificity of 98% for the utility of CSF-derived EVs to detect an EGFRvIII-positive GBM. Our results demonstrate CSF-derived EVs contain RNA signatures reflective of the underlying molecular genetic status of GBMs in terms of wtEGFR expression and EGFRvIII status. The high specificity of the CSF-derived EV diagnostic test gives us an accurate determination of positive EGFRvIII tumor status and is essentially a less invasive "liquid biopsy" that might direct mutation-specific therapies for GBMs.

A Rapid and Sensitive Method for Detection of the T790M Mutation of EGFR in Plasma DNA.

Epidermal growth factor receptor (EGFR) T790M mutation is associated with resistance to EGFR tyrosine kinase inhibitors' (EGFR-TKIs) in non-small cell lung cancer (NSCLC). The aims of this study are to develop a blood-based, non-invasive approach to detecting the EGFR T790M mutation in advanced NSCLC patients, using PointMan™ EGFR DNA Enrichment Kit which is a novel method for selective amplification of genotype specific sequences.Pairs of blood samples and tumor tissues were collected from NSCLC patients with an EGFR activating mutation and who were resistant to EGFR-TKI treatment. EGFR T790M mutation in plasma DNA were detected using the PointMan™ EGFR DNA Enrichment Kit. The concentrations of plasma DNA were determined using quantitative real-time PCR.Of the 52 patients enrolled in this study, 41 of the patients' plasma samples were collected at post EGFR-TKIs. Nineteen (46.3 %) of the 41 patients had an EGFR T790M mutation in their plasma DNA as detected using the PointMan™ EGFR DNA Enrichment Kit after disease progression to EFGR-TKI. Of 11 cases with a detected T790M mutation from tumor tissues, 10 (90.9 %) also had a detectable T790M mutation in the plasma DNA. There was no difference in the progression-free survival between patients with T790M and those without T790M.The PointMan™ proved to be a useful method for determining plasma EGFR T790M mutation status.

Improved therapeutic effectiveness by combining recombinant p14ARF with antisense complementary DNA of EGFR in laryngeal squamous cell carcinoma

PurposeThe tumor suppressor p14ARF and proto-oncogene epidermal growth factor receptor (EGFR) play important roles in the development of laryngeal squamous cell carcinoma (LSCC). This study was aimed to determine whether combining recombinant p14ARF with antisense complementary DNA of EGFR could improve the therapeutic effectiveness in LSCC. Materials and methodsAfter human larynx cancer cells (Hep-2) were infected with recombinant adenoviruses (Ad-p14ARF and Ad-antisense EGFR) together or alone in vitro, the proliferation and cell cycle distribution of Hep-2 cells were detected by MTT assay and flow cytometer analysis, respectively. Furthermore, the antitumor effects of recombinant adenoviruses together or alone on Hep-2 xenografts were examined in vivo. The levels of p14ARF and EGFR expressed in Hep-2 cells and xenografts were determined by western blot assay. ResultsAd-p14ARF combining with Ad-antisense EGFR markedly inhibited the Hep-2 proliferation compared with alone (P=0.001, P=0.002 respectively). Combination of Ad-p14ARF and Ad-antisense EGFR led to the proportion of Hep-2 cells in G0/G1 phases increased by up to 86.9%. The down-expression of EGFR protein and overexpression of p14ARF protein were observed in vitro and in vivo, and this effect was preserved when Ad-p14ARF was combined with Ad-antisense EGFR. Besides, Ad-p14ARF plus Ad-antisense EGFR significantly (P<0.05) increased the antitumor activity against Hep-2 tumor xenografts comparing with Ad-p14ARF or Ad-antisense EGFR alone. ConclusionCombination Ad-p14ARF with Ad-antisense EGFR significantly increased the antitumor responses in LSCC. An effectively potential gene therapy to prevent proliferation of LSCC was provided.

Abstract 1509: A ARMS-PNA PCR assay for detection of EGFR mutations in FFPE tumor samples

Abstract The activating mutations of Epidermal growth factor receptor (EGFR) are the predictive markers for response to tyrosine kinase inhibitors gefitinib and erlotinib in non-small cell lung cancer(NSCLC). Therefore detection of EGFR mutations will guide effective therapeutical options for patients with NSCLC. Here we developed a new method for the rapid and reliable detection of EGFR mutations by using a combination of modified mutation-specific ARMS primers and peptide nucleic acid (PNA) clamping system, named ARMS-PNA assay. In this real-time PCR system, the wild-type EGFR DNA is completely blocked by PNA with a higher annealing temperature, and the mutant gene is selectively amplified by the modified ARMS primers and the amplicon extending signals are collected by the taqman-probe at the same time. The ARMS-PNA assay could detect 29 mutations on EGFR gene with a sensitivity of 0.5% under the background of a total of 10ng wild-type genomic DNA. By using this assay, we analyzed the EGFR mutations in 1033 Chinese NSCLC FFPE samples in comparison with Sanger sequencing. Our results showed that the positive rates were 50.1%, which were higher than that measured by Sanger sequencing (48.3%). The coincidence rates of the two methods were 99.0% for the positive samples and 95.5% for the negative samples. The total coincidence rate was 97.2%. In conclusion, our study provides a basis for the detection of somatic mutations in EGFR in a routine clinical setting. The ARMS-PNA PCR assay allows the detection of the activating EGFR mutations in FFPE samples with better sensitivity and feasibility than the current assays used in clinic.&amp;lt;!–EndFragment–&amp;gt; Citation Format: Shanshan Zhao, Xiao Guo, Ziwen Long, Dehua Yu. A ARMS-PNA PCR assay for detection of EGFR mutations in FFPE tumor samples. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1509. doi:10.1158/1538-7445.AM2014-1509

EGFR ligands and DNA repair genes: genomic predictors of complete response after capecitabine-based chemoradiotherapy in locally advanced rectal cancer.

Epidermal growth factor receptor (EGFR) activation by radiation leads to increased cell proliferation and acts as a radioresistance mechanism. Neoadjuvant chemoradiation is the standard of care for locally advanced rectal cancer, and to date, no biomarkers of response have been found. We analyzed polymorphisms in the EGFR and its ligands, DNA repair genes and the thymidylate synthase in 84 stages II and III rectal cancer patients treated with neoadjuvant capecitabine plus radiotherapy. The rs11942466 polymorphism in the amphiregulin (AREG) gene region was associated with a pathological complete response (ypCR) (odds ratio: 0.26; 95% confidence interval: 0.06-0.79; P=0.014). The rs11615 C>T polymorphism in the ERCC1 gene also correlated with the ypCR as no patients with a C/C genotype achieved ypCR; P=0.023. This is the first work to propose variants within the AREG and the ERCC1 genes as promising predictive biomarkers of ypCR in rectal cancer.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Targeted Therapy Resistance Mediated by Dynamic Regulation of Extrachromosomal Mutant EGFR DNA

Intratumoral heterogeneity contributes to cancer drug resistance, but the underlying mechanisms are not understood. Single-cell analyses of patient-derived models and clinical samples from glioblastoma patients treated with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) demonstrate that tumor cells reversibly up-regulate or suppress mutant EGFR expression, conferring distinct cellular phenotypes to reach an optimal equilibrium for growth. Resistance to EGFR TKIs is shown to occur by elimination of mutant EGFR from extrachromosomal DNA. After drug withdrawal, reemergence of clonal EGFR mutations on extrachromosomal DNA follows. These results indicate a highly specific, dynamic, and adaptive route by which cancers can evade therapies that target oncogenes maintained on extrachromosomal DNA.

Detection of EGFR Mutation in Tissue Samples of Non–small-cell Lung Cancer by a Fluorescence Polarization Assay

It is important to identify epidermal growth factor receptor (EGFR) mutations, which have a good value for the individualized management of patients with non-small-cell lung cancer. A novel method for detecting the mutations on exons 19, 21 of EGFR in primary carcinoma samples by a fluorescence polarization (FP) assay was developed in this research. Firstly, 2 pairs of general primers of exons 19, 21 of EGFR were, respectively, used to amplify the target regions in each exon in 2 reactions. Then, 2 probes specific for wild or mutation exons 19, 21 of EGFR were labeled with tetramethyl 6-carboxyrhodamine or 6-carboxyfluorescein hybridized, respectively, with their target amplicons, and the hybridization resulted in an increase in the FP values. Exon 19 deletion and exon 21 missense mutation were determined by the analysis of the FP values. EGFR mutations in 372 non-small-cell lung cancer samples were analyzed in parallel with an FP assay and a sequencing assay. There was no significant difference between the mutation status results obtained with the FP assay and the results obtained with the sequencing assay. The minimum detection level established with this assay was 40 copies/uL. Reliable results could be obtained when more than 30 ng of DNA was tested by a FP assay. An FP assay was able to detect the mutation DNA of EGFR even when its content was as low as 10%. An FP assay allowed the semiautomated detection of EGFR mutations in solution, and it was much simpler and cost effective than the traditional methods.

Enhancement of the cytotoxic potential of the mixed EGFR and DNA-targeting ‘combi-molecule’ ZRBA1 against human solid tumour cells by a bis-quinazoline-based drug design approach

ZRBA1 is a quinazoline-based molecule termed 'combi-molecule' designed to block the epidermal growth factor receptor (EGFR) and further degrade to FD105, an EGFR inhibitor plus a DNA-alkylating agent. To augment the potency of ZRBA1, we designed JDE52, a bistriazene that, following degradation, was 'programmed' to yield higher concentrations of the free inhibitor FD105 and a more cytotoxic bifunctional DNA-damaging species. The results indicated that JDE52 was capable of inducing significant blockade of EGFR phosphorylation, DNA strand breaks and interstrand cross-links in human cells. The fluorescent property of FD105, the secondary inhibitor that both JDE52 and ZRBA1 are capable of releasing, has permitted the analysis of its levels in tumour cells by ultraviolet flow cytometry. It was found that JDE52 was indeed capable of significantly releasing higher levels of fluorescence (P<0.05) in human tumour cells when compared with ZRBA1. Apoptosis was triggered by JDE52 at a faster rate than ZRBA1 and led to higher levels of cell killing. The results in toto suggest that the superior potency of JDE52, when compared with ZRBA1, may be imputed to mechanisms associated with the generation of higher intracellular concentrations of FD105 and to the induction of DNA cross-links. These combined mechanisms (blockade of EGFR-tyrosine kinase and induction of cross-links) contributed to an accelerated rate of apoptosis by JDE52. This study conclusively demonstrated that designing molecules as prodrugs of high levels of quinazoline inhibitors of EGFR and bifunctional DNA cross-linking species is a valid strategy to enhance the potency of mixed EGFR-DNA-targeting combi-molecules.