Low Epidermal Growth Factor Receptor Research Articles

Impact of epidermal growth factor single‐nucleotide polymorphism on recurrence of hepatocellular carcinoma after hepatectomy in patients with chronic hepatitis C virus infection

Epidermal growth factor (EGF) gene single-nucleotide polymorphism (SNP) is associated with an increased risk of hepatic tumors. The study aimed to elucidate the impact of EGF SNP and EGF receptor (EGFR) expression on the recurrence of hepatocellular carcinoma (HCC) after hepatectomy. To examine the impact of EGF SNP and EGFR on recurrent HCC, we retrospectively analyzed 141 HCC patients with chronic hepatitis C virus infection who underwent curative hepatectomy. The EGF *61 GG allele was present in 69 patients (48.9%), AG in 56 (39.7%) and AA in 16 (11.4%). The AA group had a significantly lower rate of intrahepatic metastasis (0% vs 16.5%, P = 0.02), lower serum EGF concentration (26.3 ± 15.9 pg/mL vs 43.4 ± 30.5 pg/mL, P = 0.02) and lower proportion of early recurrence (≤2 years; 28.6% vs 71.2%, P = 0.03) than the AG/GG group. The AA group had significantly higher recurrence-free survival than the AG/GG group (P = 0.04), but there was no significant difference in overall survival between these two groups (P = 0.97). High versus low EGFR expression analyzed by immunohistochemical staining in cancer cells was not significantly associated with overall survival (P = 0.37) or recurrence-free survival (P = 0.39). Therefore, EGF *61 AA was associated with a lower risk of recurrence after curative hepatectomy for HCC in patients with hepatitis C virus infection than other genotypes, but EGFR expression in cancer cells was not significantly associated with prognosis.

Relationship Between EGFR Expression, EGFR Mutation Status, and the Efficacy of Chemotherapy Plus Cetuximab in FLEX Study Patients with Advanced Non–Small-Cell Lung Cancer

The phase III FLEX study (NCT00148798) in advanced non-small-cell lung cancer indicated that the survival benefit associated with the addition of cetuximab to cisplatin and vinorelbine was limited to patients whose tumors expressed high levels of epidermal growth factor receptor (EGFR) (immunohistochemistry score of ≥200; scale 0-300). We assessed whether the treatment effect was also modulated in FLEX study patients by tumor EGFR mutation status. A tumor mutation screen of EGFR exons 18 to 21 included 971 of 1125 (86%) FLEX study patients. Treatment outcome in low and high EGFR expression groups was analyzed across efficacy endpoints according to tumor EGFR mutation status. Mutations in EGFR exons 18 to 21 were detected in 133 of 971 tumors (14%), 970 of which were also evaluable for EGFR expression level. The most common mutations were exon 19 deletions and L858R (124 of 133 patients; 93%). In the high EGFR expression group (immunohistochemistry score of ≥200), a survival benefit for the addition of cetuximab to chemotherapy was demonstrated in patients with EGFR wild-type (including T790M mutant) tumors. Although patient numbers were small, those in the high EGFR expression group whose tumors carried EGFR mutations may also have derived a survival benefit from the addition of cetuximab to chemotherapy. Response data suggested a cetuximab benefit in the high EGFR expression group regardless of EGFR mutation status. The survival benefit associated with the addition of cetuximab to first-line chemotherapy for advanced non-small-cell lung cancer expressing high levels of EGFR is not limited by EGFR mutation status.

Predictive Efficacy of Low Burden EGFR Mutation Detected by Next-Generation Sequencing on Response to EGFR Tyrosine Kinase Inhibitors in Non-Small-Cell Lung Carcinoma

Direct sequencing remains the most widely used method for the detection of epidermal growth factor receptor (EGFR) mutations in lung cancer; however, its relatively low sensitivity limits its clinical use. The objective of this study was to investigate the sensitivity of detecting an epidermal growth factor receptor (EGFR) mutation from peptide nucleic acid-locked nucleic acid polymerase chain reaction (PNA-LNA PCR) clamp and Ion Torrent Personal Genome Machine (PGM) techniques compared to that by direct sequencing. Furthermore, the predictive efficacy of EGFR mutations detected by PNA-LNA PCR clamp was evaluated. EGFR mutational status was assessed by direct sequencing, PNA-LNA PCR clamp, and Ion Torrent PGM in 57 patients with non-small cell lung cancer (NSCLC). We evaluated the predictive efficacy of PNA-LNA PCR clamp on the EGFR-TKI treatment in 36 patients with advanced NSCLC retrospectively. Compared to direct sequencing (16/57, 28.1%), PNA-LNA PCR clamp (27/57, 47.4%) and Ion Torrent PGM (26/57, 45.6%) detected more EGFR mutations. EGFR mutant patients had significantly longer progressive free survival (14.31 vs. 21.61 months, P = 0.003) than that of EGFR wild patients when tested with PNA-LNA PCR clamp. However, no difference in response rate to EGFR TKIs (75.0% vs. 82.4%, P = 0.195) or overall survival (34.39 vs. 44.10 months, P = 0.422) was observed between the EGFR mutations by direct sequencing or PNA-LNA PCR clamp. Our results demonstrate firstly that patients with EGFR mutations were detected more frequently by PNA-LNA PCR clamp and Ion Torrent PGM than those by direct sequencing. EGFR mutations detected by PNA-LNA PCR clamp may be as a predicative factor for EGFR TKI response in patients with NSCLC.

EGFR signaling in the HGG-02 glioblastoma cell line with an unusual loss of EGFR gene copy

Epidermal growth factor receptor (EGFR) gene amplification and the overexpression of EGFR are described as common features of glioblastoma multiforme (GBM). Nevertheless, we previously reported the loss of EGFR gene copy in a GBM specimen from a patient with an unusually favorable course of the disease, and the HGG-02 cell line with this aberration was successfully derived from this tumor. Here, we present a detailed analysis of changes in gene expression and cell signaling in the HGG-02 cell line; the GM7 reference cell line with a standard EGFR gene copy number derived from a very aggressive GBM was used as a control. We confirmed the downregulation of EGFR expression and signaling in HGG-02 cells using different methods (RTK analysis, gene profiling and RT-PCR). Other changes that may have contributed to the non-aggressive phenotype of the primary tumor were identified, including the downregulated phosphorylation of the Axl and Trk receptors, as well as increased activity of JNK and p38 kinases. Notably, differences in PDGF signaling were detected in both of these cell lines; HGG-02 cells preferentially expressed and signaled through PDGFRα, and PDGFRβ was strongly overexpressed and phosphorylated in the GM7 reference cell line. Using expression profiling of cancer-related genes, we revealed the specific profile of HGG-02 cells that included upregulated tumor-suppressors as well as downregulated genes associated with the extracellular matrix. This study represents the first comprehensive analysis of gene expression and cell signaling in glioblastoma cells with lower EGFR gene dosage. As indicated by our results, the TAM receptors, Trk receptors and PDGFRs need to be investigated further since their regulation appears to be important for glioblastoma biological features as well as the clinical course of the disease.

Prognostic role of epidermal growth factor receptor in head and neck cancer: A meta-analysis

To evaluate the predicting value of the epidermal growth factor receptor (EGFR) for survival in patients with head and neck cancer (HNC). Data were collected from studies comparing overall survival (OS) or progression-free survival (PFS) in patients with higher or lower EGFR levels. Studies were pooled and combined hazard ratios (HRs) of EGFR for survival were calculated. A total of 68 studies involving 6,781 patients were included for meta-analysis. Either EGFR protein expression or gene copy number had prognostic value in HNC patients. EGFR overexpression could predict worse outcome, with HRs of 1.65 (95% CI: 1.45, 1.86) for OS and 1.27 (95% CI: 1.17, 1.37) for PFS. Increased EGFR copy number was also associated with reduced survival, with HRs of 1.5 (95% CI: 1.15, 1.96) for OS and 1.35 (95% CI: 1.14, 1.61) for PFS. Furthermore, EGFR overexpression could predict poorer OS in both eastern and western countries. Particularly, EGFR was considered a strong predictor in laryngeal squamous cell cancer (HR > 2). Elevated EGFR expression and gene copy number could predict poor survival in HNC patients.

Evaluation of EGFR protein expression by immunohistochemistry using H-score and the magnification rule: Re-analysis of the SATURN study

IntroductionThe phase III SATURN study demonstrated that first-line maintenance erlotinib extended progression-free survival (PFS) and overall survival (OS) versus placebo in patients with advanced non-small cell lung cancer (NSCLC). Analysis of epidermal growth factor receptor (EGFR) expression by immunohistochemistry (IHC) found no significant interaction between EGFR IHC status and PFS (p=0.63) or OS (p=0.52). The FLEX study of first-line cetuximab plus chemotherapy demonstrated that EGFR IHC expression was predictive of improved OS with cetuximab when assessed by H-score with a magnification rule. This novel method was used to reassess samples from SATURN. MethodsThe H-score method assigned a score of 0–300 to each patient, based on the percentage of cells stained at different intensities viewed at various magnifications. The discriminatory threshold was set at 200, per the FLEX study, and existing samples were re-read and classed as low (H-score<200) or high (≥200) EGFR expression. PFS and OS were re-analyzed based on these new classifications. ResultsIn the overall and EGFR wild-type populations, erlotinib provided a consistent survival benefit versus placebo. Hazard ratios (HRs) in the overall population were similar between EGFR IHC-positive and -negative patients for median PFS (HR 0.68 [95% confidence interval (CI) 0.53–0.86] and 0.76 [95% CI 0.62–0.93], respectively) and OS (HR 0.80 [95% CI 0.62–1.05] and 0.80 [95% CI 0.64–1.01] for IHC-positive and IHC-negative, respectively). In the EGFR wild-type population, HRs were again similar between EGFR IHC-positive and -negative subpopulations for PFS (HR 0.69 [95% CI 0.51–0.95] and 0.84 [95% CI 0.63–1.12], respectively) and OS (HR 0.78 [95% CI 0.55–1.10] and 0.76 [95% CI 0.55–1.05], respectively). ConclusionsThese data suggest that EGFR IHC does not have value as a marker to predict erlotinib benefit in the first-line maintenance setting for advanced NSCLC.

Epidermal growth factor receptor expression in laryngeal cancer predicts the effect of hypoxia modification as an additive to accelerated radiotherapy in a randomised controlled trial

Accelerated radiotherapy (AR) improves the poor prognosis associated with epidermal growth factor receptor (EGFR) overexpression frequently seen in head and neck carcinomas. Combining AR with carbogen and nicotinamide (ARCON) counteracts enhanced tumour cell proliferation- and hypoxia-related radioresistance. The purpose of this study was to investigate if EGFR expression levels are associated with response to ARCON in patients with carcinoma of the larynx.Patients (N=272) with advanced stage larynx carcinoma were randomised between AR alone and ARCON. Paraffin-embedded biopsies from these patients were processed for immunohistochemical staining of EGFR. EGFR fraction was quantitated by automated image analysis and related to clinical outcome.A large variation was observed in EGFR fraction between tumours with expression levels ranging from 0 to 0.93 (median fraction 0.4). No difference in 5-year locoregional control was found between low and high EGFR expressing tumours in the AR arm (69% versus 75%), which is in line with the established effect of AR in EGFR overexpressing tumours. There was, however, a significant association in the ARCON arm: patients with low EGFR levels had a better 5-year locoregional control (88% versus 72% p=0.02) and disease-specific survival (92% versus 77% p=0.01). ARCON improved locoregional control relative to AR only in patients with low EGFR expression (hazard ratio (HR) 0.34 p=0.009).In conclusion, only in tumours with a low EGFR fraction, adding hypoxia modification to AR has an additive beneficial effect on outcome. EGFR expression is a predictive biomarker for the selection of patients that will or will not respond to ARCON.

EGFR mutations in patients with lung adenocarcinoma in southwest China: are G719S/A and L861Q more likely detected in tumors derived from smokers?

The clinical characteristics of epidermal growth factor receptor (EGFR) hotspot mutations, such as deletions in exon 19, substitution of L858R in exon 21, and mutations in exon 20, have been widely reported in nonsmall cell lung cancer. However, the clinical features of other low frequency EGFR mutations in these four exons (especially the relationship with smoking history), eg, substitutions of G719S/A/C in exon 18 and L861Q in exon 21, remain unclear. This study investigated the relationship between G719S/A/C and L861Q mutations (in exon 18 and 21) and smoking history. Specimens from 194 patients with lung adenocarcinoma were analyzed for EGFR mutations in exons 18-21 by high-resolution melting curve analysis and amplification refractory mutation technology to establish the relationship between G719S/A/C and L861Q mutations and smoking history. Ninety-six of 194 tumors (49.5%) were confirmed to be EGFR mutation-positive. Among these mutations, 71 of 104 (68.3%) were from never smokers, six of 17 (35.3%) were from former smokers, and 19 of 73 (26.0%) were from current smokers (P < 0.001). The mutation rate in heavy smokers (5/23, 21.7%) was significantly lower than in light smokers (20/67, 29.9%) and never smokers (71/104, 68.3%, P < 0.001). Seven low frequency EGFR mutations (four substitutions of G719S, and G719 A, respectively, and three of L861Q in exon 21) were identified. Five of these mutations were derived from smokers (one former light smoker, one current heavy smoker, and three current light smokers). Four of these patients had been treated with tyrosine kinase inhibitors and all had a partial response, with median overall survival (14.5 months) and median progression-free survival (6.8 months), being longer than in patients with similarly staged lung adenocarcinoma without EGFR mutation or treatment with tyrosine kinase inhibitors (6.8 and 3.1 months, respectively, according to data from an as yet unpublished study at our institution). This study provides further evidence that smoking status, include years of smoking and number of cigarettes smoked per day, plays an important role in EGFR mutation in patients with lung adenocarcinoma. Five of seven specimens with G719S/A or L861Q mutations coming from smokers indicates that there may be a relationship between G719S/A or L861Q mutation and smoking history. However, regardless of the influence of smoking, the effectiveness of tyrosine kinase inhibitors was satisfactory in four patients harboring G719S/A and L861Q EGFR mutation.

Prognostic Role of Human Epidermal Growth Factor Receptor in Gastric Cancer: A Systematic Review and Meta-analysis

Human epidermal growth factor receptor (EGFR) and HER2 (ErbB2) both belong to EGFR family, which are overexpressed in a significant proportion of cases of gastric cancer (GC). Various studies have evaluated the prognostic value of EGFR or HER level in GC. However, the overall test performance remains unclear. We undertook this study to perform a systematic review and meta-analysis of prognostic cohort studies evaluating the use of EGFR or HER2 as a predictor of survival time in patients with GC. Eligible studies were identified through multiple search strategies. Studies were assessed for quality using the Newcastle-Ottawa Tool. Data were collected comparing overall survival (OS) in patients with high and low EGFR or HER2 level. Studies were pooled and summary hazard ratios were calculated. Studies were listed twice if they provided overall survival data for both EGFR and HER2. Eight studies (seven for EGFR and eight for HER2) were included. Two distinct groups were pooled for analysis and revealed that high EGFR, HER2 levels predicted poor overall (HR = 1.66, 95% CI: 1.35-2.02) and (HR = 1.43, 95% CI: 1.09-1.88) survival. No publication bias was found. This meta-analysis result suggested that EGFR or HER2 should have significant predictive ability for estimating overall survival in GC patients and may be useful for defining prognosis of GC patients.

Expression of Epidermal Growth Factor Receptor Detected by Cetuximab Indicates Its Efficacy to Inhibit In Vitro and In Vivo Proliferation of Colorectal Cancer Cells

Cetuximab is a chimeric mouse–human monoclonal antibody that targets the human epidermal growth factor receptor (EGFR). However, EGFR expression determined by immunohistochemistry does not predict clinical outcomes of colorectal cancer (CRC) patients treated with cetuximab. Therefore, we evaluated the correlation between EGFR levels detected by cetuximab and drug sensitivities of CRC cell lines (Caco-2, WiDR, SW480, and HCT116) and the A431 epidermoid carcinoma cell line. We used flow cytometry (FCM) to detect EGFR-binding of biotinylated cetuximab on the cell surface. Subcloned cell lines showing the highest and lowest EGFR expression levels were chosen for further study. Cytotoxic assays were used to determine differential responses to cetuximab. Xenograft models treated with cetuximab intraperitoneally to assess sensitivity to cetuximab. Strong responses to cetuximab were specifically exhibited by subcloned cells with high EGFR expression levels. Furthermore, cetuximab inhibited the growth of tumors in xenograft models with high or low EGFR expression levels by 35% and 10%–20%, respectively. We conclude that detection of EGFR expression by cetuximab promises to provide a novel, sensitive, and specific method for predicting the sensitivity of CRC to cetuximab.

TH-C-141-02: Imaging of Targeted Cancer Therapy Using Multifunctional Liposomal Nanoprobes and MRI

Purpose: To develop a multifunctional liposomal nanoprobe that selectively binds to epidermal growth factor receptor (EGFR) over‐expressing tumor cells with an encapsulating a magnetic resonance (MR) imaging contrast agent, gadolinium (Gd) and conjugating an EGFR‐specific monoclonal antibody within liposome particles. The function of the probe is to target cancer overexpressing cells so that uptake of anti‐EGFR targeted therapeutic agents may be imaged. We tested our nanoprobe using a head and neck cancer cell line and Cetuximab, an anti‐EGFR drug. Methods: Encapsulation of Gd within liposome particles was optimized by combining Dipalmitoylphosphatidylcholine (DPPC), cholesterol, 1,2‐dioleoyl‐sn‐glycero‐3‐phospho‐ethanolamine‐N‐(biotinyl)(sodium salt) (DOPE‐Biotin), and Diethylenetriaminepentaacetic acid‐bis(stearylamide)‐Gd (DTPA‐BSA‐Gd). Avidin was included in the liposomal membrane to conjugate with biotin‐modulated anti‐EGFR antibody. The probes were characterized for size, surface potential and structural integrity with dynamic light scattering (DLS) and transmission electronic microscopy (TEM). In‐vitro targeting specificity of the probe was evaluated using fluorescence microscopy by comparing the binding capacity of a fluorescence‐labeled probe in a human head and neck cancer cell line, 15B (high EGFR overexpression) and a human embryonic kidney cell line, HEK293 (low EGFR control). Finally, the in vitro applicability of MRI contrast Gd within the probe was demonstrated by detecting cell‐bound particles with MR imaging. Results: Using TEM scanning, the probes were near spherical in shape and identical in particle size (∼100nm). The surface potential was +5.13mV. The EGFR‐specific targeting assay with fluorescence‐labeled probes showed significantly high occupancy of the probe on the surface of 15B cells in comparison with HEK293 cells. The T1 relaxation time of this EGFR‐specific probe attached to15B cells was measured to be 379.3 ms, shorter than that of control vector without anti‐EGFR conjugation (516.2 ms). Conclusion: We have demonstrated targeting specificity of a nanoprobe in discriminating high and low EGFR‐expressing cells. The incorporation of Gd revealed the cell‐targeting under MR imaging.

Abstract 3491: Absolute quantification of EGFR activation and resistance mutations as well as copy number in circulating nucleic acids by droplet digital PCR.

Abstract Background: Non-small cell lung cancer (NSCLC) patients who harbor activating mutations in epidermal growth factor receptor (EGFR) such as L858R and the Exon 19 deletions (E746-A750) are treated with either erlotinib or gefitinib. However, a large percentage of patients acquire the gatekeeper resistance mutation, T790M. Limited access to post-treatment tumor tissue restricts monitoring options, and even when biopsy specimens are available, sampling bias may confound diagnosis of the resistance mechanism. Circulating, cell-free DNA (CNA) provides a solution to circumvent tissue access limitations. Low overall recovery of CNA requires assays be designed with increased sensitivity and specificity not currently available. We have developed and optimized a series of droplet digital PCR assays that can quantify both EGFR activating and resistance mutations as well as copy number. Methods: The ddPCR assays were developed on the BioRAD QX100 system. Primers and probes were designed to optimize quantification of EGFR L858R and T790M mutations, Exon 19 deletions and EGFR copy number. Genomic DNA from H1975 (L5858R and T790M mutations) and H1650 (Exon 19 deletion) cell lines were diluted into wild type gDNA to determine the assays’ limits of detection (LOD). Patient samples were analyzed as well. Finally, ddPCR data were compared with qPCR data using the Qiagen EGFR Rotor-Gene (RGQ) kit. Results: The ddPCR assays were designed to quantify EGFR activating and resistance mutations in relative to the wild type template. The T790M and L858R assays displayed linear dynamic ranges from ∼14000 copies to LODs of 9 and 6 copies, respectively. In addition, ddPCR assays were successfully multiplexed to an LOD of 0.2%, or 15 copies based upon a 95% confidence interval in 20ng gDNA. In healthy donor spiked plasma samples, the LOD was determined to be 1.2%, or 8 copies in 2.2ng purified CNA. By comparison, in plasma, the RGQ assays displayed an average LOD of 80 copies with a dynamic range of ∼8000 copies to 76 copies. Data on Exon 19 deletion and EGFR copy number ddPCR assays will be presented. Spiked normal plasma samples analyzed for the ratio of T790M to activating mutations such as L858R and Exon 19 deletion will be presented. Conclusion: Droplet digital PCR provides sensitive quantification of low abundance EGFR resistance and activating mutations in CNA isolated from plasma. These assays provide a useful method for selecting treatment, monitoring disease progression, and providing early detection of treatment failure as well as gene amplification associated with EGFR acquired resistance. Citation Format: Jessica Kristof, Eric Bruening, Stephane Wong, Sabita Sankar. Absolute quantification of EGFR activation and resistance mutations as well as copy number in circulating nucleic acids by droplet digital PCR. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3491. doi:10.1158/1538-7445.AM2013-3491

HMG box‐containing protein 1 (HBP1) as a downstream signaling effector of EGFR in oral cancer

Aberrant activation of the epidermal growth factor (EGF) receptor (EGFR) signaling in the squamous cell carcinomas of the head and neck (SCCHN) is often associated with inauspicious prognosis and low survival. Transcription factor HMG box‐containing protein 1 (HBP1) functions as a potential tumor repressor in various cancers; however, its role in oral cancer is still unclear. Analysis of human oral cancer specimens revealed that the mean HBP1 mRNA level in tumors was significantly lower than that of the normal tissues, and that the concurrent status of low HBP1 and high EGFR was associated with the invasiveness of oral cancer. To explore the potential link between EGFR signaling and HBP1, we treated EGFR‐overexpressing oral cancer cell lines, HSC‐3 and FaDu, with EGFR inhibitors, AG1478 and Erlotinib, or EGFR siRNA, and found that EGFR down‐regulation increased HBP1 expression. Administration of phosphatidylinositol 3‐kinase (PI3K) inhibitor, Ly294002, decreased Akt phosphorylation and increased HBP1 expression. Overall, the expression of Akt and HBP1 acted in a reciprocal manner. Moreover, HBP1 knockdown in HSC‐3 cells enhanced in vitro colony formation and in vivo xenograft tumor growth. HBP1 knockdown attenuated Erlotinib‐mediated cyclin D1 reduction and subsequent G1 arrest. Together, our data reveal a novel role of HBP1 as a downstream effector of the EGFR/Akt signaling pathway in oral cancer.

Molecular Determinants of Epidermal Growth Factor Binding: A Molecular Dynamics Study

The epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase family that plays a role in multiple cellular processes. Activation of EGFR requires binding of a ligand on the extracellular domain to promote conformational changes leading to dimerization and transphosphorylation of intracellular kinase domains. Seven ligands are known to bind EGFR with affinities ranging from sub-nanomolar to near micromolar dissociation constants. In the case of EGFR, distinct conformational states assumed upon binding a ligand is thought to be a determining factor in activation of a downstream signaling network. Previous biochemical studies suggest the existence of both low affinity and high affinity EGFR ligands. While these studies have identified functional effects of ligand binding, high-resolution structural data are lacking. To gain a better understanding of the molecular basis of EGFR binding affinities, we docked each EGFR ligand to the putative active state extracellular domain dimer and 25.0 ns molecular dynamics simulations were performed. MM-PBSA/GBSA are efficient computational approaches to approximate free energies of protein-protein interactions and decompose the free energy at the amino acid level. We applied these methods to the last 6.0 ns of each ligand-receptor simulation. MM-PBSA calculations were able to successfully rank all seven of the EGFR ligands based on the two affinity classes: EGF>HB-EGF>TGF-α>BTC>EPR>EPG>AR. Results from energy decomposition identified several interactions that are common among binding ligands. These findings reveal that while several residues are conserved among the EGFR ligand family, no single set of residues determines the affinity class. Instead we found heterogeneous sets of interactions that were driven primarily by electrostatic and Van der Waals forces. These results not only illustrate the complexity of EGFR dynamics but also pave the way for structure-based design of therapeutics targeting EGF ligands or the receptor itself.

Inactivation of HNSCC Cells by 90Y-Labeled Cetuximab Strictly Depends on the Number of Induced DNA Double-Strand Breaks

Radioimmunotherapy is considered to have great potential for efficient and highly specific treatment of tumors. The aim of this study was to determine the efficacy of radioimmunotherapy when using (90)Y-labeled cetuximab and to determine to what degree induction and repair of DNA double-strand breaks (DSBs) are decisive for this approach. This study was performed with 9 cell lines of squamous cell carcinoma of the head and neck (HNSCC) differing strongly in epidermal growth factor receptor (EGFR) expression. The radionuclide (90)Y was coupled by the chelator trans-cyclohexyl-diethylene-triamine-pentaacetic acid (CHX-A″-DTPA)/linker construct to the EGFR-directed antibody cetuximab to yield (90)Y-Y-CHX-A″-DTPA-cetuximab with a specific activity of approximately 1.2 GBq/mg. EGFR expression was determined by immunofluorescence and Western blotting, cetuximab binding by fluorescence-activated cell sorter analysis, the number of DSBs by immunofluorescence staining γH2AX/53BP1-positive repair foci, and cell survival by colony formation. For the 9 HNSCC cell lines, cetuximab binding correlated with the amount of EGFR present in the cell membrane (r(2) = 0.967, P < 0.001). When cells were exposed to (90)Y-Y-CHX-A″-DTPA-cetuximab, the number of induced DSBs increased linearly with time (r(2) = 0.968, P = 0.016). This number was found to correlate with the amount of membranous EGFR (r(2) = 0.877, P = 0.006). Most DSBs were repaired during incubation at 37°C, but the small number of remaining DSBs still correlated with the amount of membranous EGFR (24 h: r(2) = 0.977, P < 0.001; 48 h: r(2) = 0.947, P < 0.001). Exposure to (90)Y-Y-CHX-A″-DTPA-cetuximab also resulted in efficient cell killing, whereby the extent of cell killing correlated strongly with the respective number of remaining DSBs (r(2) = 0.989, P < 0.001) and with the amount of membranous EGFR (r(2) = 0.967, P < 0.001). No cell killing was observed for UTSCC15 cells with low EGFR expression, in contrast to the strong reduction of 86% measured for UTSCC14 cells showing a strong overexpression of EGFR. (90)Y-Y-CHX-A″-DTPA-cetuximab affected cell survival through the induction of DSBs. This treatment was especially efficient for HNSCC cells strongly overexpressing EGFR, whereas no effect was seen for cells with low levels of EGFR expression. Therefore, EGFR-directed radioimmunotherapy using (90)Y-Y-CHX-A″-DTPA-cetuximab appears to be a powerful tool that can be used to inactivate tumors with strong EGFR overexpression, which are often characterized by a pronounced radioresistance.

Effects of epidermal growth factor receptor and phosphatase and tensin homologue gene expression on the inhibition of U87MG glioblastoma cell proliferation induced by protein kinase inhibitors

The aim of the present study was to analyse the antiproliferative effects and mechanisms of action of protein kinase inhibitors (PKIs) in human glioblastoma multiforme (GBM) cells with different epidermal growth factor receptor (EGFR) and phosphatase and tensin homologue (PTEN) status. The GBM cell models were established by transfection of plasmids carrying wild-type EGFR, mutated EGFRvIII or PTEN and clonal selection in U87MG cells. Phosphatidylinositol 3-kinase (PI3-K)/AKT pathway-focused gene profiles were examined by real-time polymerase chain reaction-based assays, protein expression was evaluated by western blotting and the antiproliferative effects of PKI treatment were determined by the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay in GBM cells. The cell model with intact PTEN and low EGFR levels was the most sensitive to treatment with the EGFR inhibitor erlotinib, whereas the model with EGFRvIII was the most resistant to treatment with the mitogen-activated protein kinase kinase inhibitor U0126. The dual PI3-K and mammalian target of rapamycin (mTOR) inhibitor PI103 had the most potent antiproliferative effects against all GBM cells tested. Following simultaneous stimulation of AKT and extracellular signal-regulated kinase, rapamycin concentrations > 0.5 nmol/L failed to exhibit a further growth inhibitory effect. Concurrent inhibition of mTOR and ribosomal protein s6 activity may underlie the inhibition of GBM proliferation by PKI. In conclusion, overexpression of EGFR or EGFRvIII, accompanied by a loss of PTEN, contributed to the activation of multiple intracellular signalling pathways in GBM cells. Rigorous examination of biomarkers in tumour tissues before and after treatment may be necessary to determine the efficacy of PKI therapy in patients with GBM.

A Pilot Characterization of Human Lung NSCLC by Protein Pathway Activation Mapping

An understanding of the activated protein signaling architecture in non-small-cell lung cancer (NSCLC) is of critical importance to the development of new therapeutic approaches and identification of predictive and prognostic biomarkers for patient stratification. We used reverse-phase protein microarrays to map the activated protein signaling networks of 47 NSCLC tumors, 28 of which were node negative, which were subjected to tumor cellular enrichment using laser capture microdissection. The phosphorylation/cleavage levels of 111 key signaling proteins and total levels of 17 proteins were measured for broadscale signaling analysis. Pathway activation mapping of NSCLC revealed distinct subgroups composed of epidermal growth factor receptor (ERBB1), v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ERBB2), v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (ERBB3), v-erb-a erythroblastic leukemia viral oncogene homolog 4 (ERBB4), v-akt murine thymoma viral oncogene homolog 1- mammalian target of rapamycin (AKT-mTOR), protein kinase, AMP-activated, alpha 2 catalytic subunit (AMPK), and autophagy-related signaling, along with transforming growth factor-beta-signaling protein 1 (SMAD), insulin-line growth factor receptor (IGFR), rearranged during transfection proto-oncogene (RET), and activated CDC42-associated kinase (ACK) activation. Investigation of epidermal growth factor receptor (EGFR)-driven signaling identified a unique cohort of tumors with low EGFR protein expression yet high relative levels of phosphorylated EGFR and high EGFR total protein with low relative levels of phosphorylation. Last, mapping analysis of patients with NSCLC with N0 disease revealed a pilot pathway activation signature composed of linked epidermal growth factor receptor family (HER)-AMPK-AKT-mTOR signaling network along with focal adhesion kinase- LIM domain kinase-1 (FAK-LIMK) and janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathways that correlated with short-term survival and aggressive disease. Functional protein pathway activation mapping of NSCLC reveals distinct activation subgroups that are underpinned by important therapeutic targets and that patients with early-stage node negative disease and poor prognosis may be identified by activation of defined, biochemically linked protein signaling events. Such findings, if confirmed in larger study sets, could help select and stratify patients for personalized targeted therapies.

A Potential Peptide Therapeutic Derived from the Juxtamembrane Domain of the Epidermal Growth Factor Receptor

The epidermal growth factor receptor (EGFR) is involved in many cancers and EGFR has been heavily pursued as a drug target. Drugs targeting EGFR have shown promising clinical results for several cancer types. However, resistance to EGFR inhibitors often occurs, such as with KRAS mutant cancers, therefore new methods of targeting EGFR are needed. The juxtamembrane (JXM) domain of EGFR is critical for receptor activation and targeting this region could potentially be a new method of inhibiting EGFR. We hypothesized that the structural role of the JXM region could be mimicked by peptides encoding a JXM amino acid sequence, which could interfere with EGFR signaling and consequently could have anti-cancer activity. A peptide encoding EGFR 645–662 conjugated to the Tat sequence (TE-64562) displayed anti-cancer activity in multiple human cancer cell types with diminished activity in non-EGFR expressing cells and non-cancerous cells. In nude mice, TE-64562 delayed MDA-MB-231 tumor growth and prolonged survival, without inducing toxicity. TE-64562 induced non-apoptotic cell death after several hours and caspase-3-mediated apoptotic cell death with longer treatment. Mechanistically, TE-64562 bound to EGFR, inhibited its dimerization and caused its down-regulation. TE-64562 reduced phosphorylated and total EGFR levels but did not inhibit kinase activity and instead prolonged it. Our analysis of patient data from The Cancer Genome Atlas supported the hypothesis that down-regulation of EGFR is a potential therapeutic strategy, since phospho- and total-EGFR levels were strongly correlated in a large majority of patient tumor samples, indicating that lower EGFR levels are associated with lower phospho-EGFR levels and presumably less proliferative signals in breast cancer. Akt and Erk were inhibited by TE-64562 and this inhibition was observed in vivo in tumor tissue upon treatment with TE-64562. These results are the first to indicate that the JXM domain of EGFR is a viable drug target for several cancer types.

Liposome-based Nanoprobe With Antibody Conjugation Combined With MRI Contrast Encapsulation as a Potential Diagnostic and Therapeutic Agent for Human Head-and-Neck Cancer Cells

Purpose/Objective(s): To develop a tumor-targeting nanoprobe that selectively binds to epidermal growth factor receptor (EGFR) overexpressing tumor cells by encapsulating a magnetic resonance (MR) imaging contrast agent gadolinium (Gd) and conjugating an EGFR-specific monoclonal antibody within liposome particles. Materials/Methods: Encapsulation of Gd within liposome particles was optimized by combining different concentrations of Dipalmitoylphosphatidylcholine (DPPC), cholesterol, 1,2-dipalmitoyl-sn-glycero-3phosphoethanolamine-N-(biotinyl) (Biotinyl PE), and Diethylenetriaminepentaacetic acid-bis(stearylamide)-Gd (DTPA-BSA-Gd). Avidin was included in the liposomal membrane to conjugate with biotinmodulated anti-EGFR antibody. The resultant probes were characterized for their particle size, surface potential and structural integrity with dynamic light scattering (DLS), Zetasizer, and transmission electronic microscopy (TEM), respectively. In-vitro targeting specificity of the probe was evaluated with fluorescence microscopy by comparing the binding capacity of a fluorescence-labeled probe in a human head and neck cancer cell line, 15B (high EGFR overexpression) and a human embryonic kidney cell line, HEK293 (low EGFR control). The subsequent cytotoxicity toward these two cell lines was quantified by MTT assay. Finally, the in vitro applicability of MRI contrast Gd within the probe was demonstrated by detecting cellbound particles with MR imaging. Results: Using TEM scanning, our probe was observed to be spherical in shape and identical in particle size, approximately 100nm. The surface potential was +5.13mV. The EGFR-specific targeting assay with fluorescence-labeled probes showed significantly high occupancy of the probe on the surface of 15B cells in comparison with HEK293 cells. Furthermore, the T1 relaxation time of this EGFR-specific probe attached to15B cells was measured to be 379.3 ms, shorter than that of control vector without anti-EGFR conjugation (516.2 ms). Conclusion: We have demonstrate targeting specificity of a multi-functional nanoprobe in discriminating high and low EGFR-expressing cells. The incorporation of MRI contrast Gd successfully revealed the cell-targeted probe under MR imaging. These results suggest that this probe can function as a targeting tracer for tumor cells, potentiating it in improving treatment efficacy through MR imaging. Author Disclosure: Y. Kuo: None. C. Hung: None. S.R. Raghavan: None. W.D. D’Souza: None.

1692P - Correlation of wtEGFR Activation Assessed by mAB806 Binding and EGFR Kinase Mutations in Stage IIIA N2 NSCLC

ABSTRACT The epidermal growth factor receptor (EGFR) pathway is a regulator of cellular proliferation and tumour progression. EGFR overexpression occurs with gene amplification and activating EGFR kinase mutations (EGFRmt). Monoclonal antibody 806 (mAb806) is an anti-EGFR antibody which targets tumours with EGFR mutation variant III and overexpression but not cells with low EGFR expression (wild type EGFR, wtEGFR) and normal tissues. To characterise mAb806 binding, we correlated mAb806 expression with EGFRmt, EGFR immunohistochemical (IHC) expression and overall survival (OS). Formalin fixed paraffin embedded tissues were stained using the mAb806 (Ludwig Institute for Cancer Research) and EGFR (Dako, PharmDx kit). A H-score was obtained based on staining intensity and percentage of cells stained. mAb806 H-scores 0-50 were classified as negative (mAb806-) and ≥50 as positive expression (mAb806+). EGFR H-scores were calculated to stratify patients into low (Dako score Disclosure A. Scott: A.S is an inventor of a patent for mAb806, and a consultant to Life Science Pharmaceuticals which has the license for mAb806. All other authors have declared no conflicts of interest.