Overexpression Of Epidermal Growth Factor Receptor Research Articles

The anticancer and EGFR-TK/CDK-9 dual inhibitory potentials of new synthetic pyranopyrazole and pyrazolone derivatives: X-ray crystallography, in vitro, and in silico mechanistic investigations

Treatment options for the management of breast cancer are still inadequate. This inadequacy is attributed to the lack of effective targeted medications, often resulting in the recurrence of metastatic disorders. Cumulative evidence suggests that epidermal growth factor receptor (EGFR-TK) and cyclin-dependent kinases-9 (CDK-9) overexpression correlates with worse overall survival in breast cancer patients. Pyranopyrazole and pyrazolone are privileged options for the development of anticancer agents. Inspired by this proven scientific fact, we report here the synthesis of two new series of suggested anticancer molecules incorporating both heterocycles together with their characterization by IR, 1H NMR, 13C NMR, 13C NMR-DEPT, and X-ray diffraction methods. An attempt to get the pyranopyrazole-gold complexes was conducted but unexpectedly yielded benzylidene-2,4-dihydro-3H-pyrazol-3-one instead. This unexpected result was confirmed by X-ray crystallographic analysis. All newly synthesized compounds were assessed for their anti-proliferative activity against two different human breast cancer cells, and the obtained results were compared with the reference drug Staurosporine. The target compounds revealed variable cytotoxicity with IC50 at a low micromolar range with superior selectivity indices. Target enzyme EGFR-TK and CDK-9 assays showed that compounds 22 and 23 effectively inhibited both biological targets with IC50 values of 0.143 and 0.121 µM, respectively. Molecular docking experiments and molecular dynamics simulation were also conducted to further rationalize the in vitro obtained results. Communicated by Ramaswamy H. Sarma

Detection of Early Esophageal Neoplastic Barrett Lesions with Quantified Fluorescence Molecular Endoscopy Using Cetuximab-800CW.

Esophageal adenocarcinoma causes 6% of cancer-related deaths worldwide. Near-infrared fluorescence molecular endoscopy (NIR-FME) uses a tracer that targets overexpressed proteins. In this study, we aimed to investigate the feasibility of an epidermal growth factor receptor (EGFR)-targeted tracer, cetuximab-800CW, to improve detection of early-stage esophageal adenocarcinoma. Methods: We validated EGFR expression in 73 esophageal tissue sections. Subsequently, we topically administered cetuximab-800CW and performed high-definition white-light endoscopy (HD-WLE), narrow-band imaging, and NIR-FME in 15 patients with Barrett esophagus (BE). Intrinsic fluorescence values were quantified using multidiameter single-fiber reflectance and single-fiber fluorescence spectroscopy. Back-table imaging, histopathologic examination, and EGFR immunohistochemistry on biopsy samples collected during NIR-FME procedures were performed and compared with invivo imaging results. Results: Immunohistochemical preanalysis showed high EGFR expression in 67% of dysplastic tissue sections. NIR-FME visualized all 12 HD-WLE-visible lesions and 5 HD-WLE-invisible dysplastic lesions, with increased fluorescence signal in visible dysplastic BE lesions compared with nondysplastic BE as shown by multidiameter single-fiber reflectance/single-fiber fluorescence, reflecting a target-to-background ratio of 1.5. Invisible dysplastic lesions also showed increased fluorescence, with a target-to-background ratio of 1.67. Immunohistochemistry analysis showed EGFR overexpression in 16 of 17 (94%) dysplastic BE lesions, which all showed fluorescence signal. Conclusion: This study has shown that NIR-FME using cetuximab-800CW can improve detection of dysplastic lesions missed by HD-WLE and narrow-band imaging.

Overexpression of EGFR in esophageal squamous cell carcinomas - A new biological target in cancer therapy.

Esophageal squamous cell carcinoma (ESCC) is the predominant type of esophageal cancer in the Asian belt. These cancers show poor prognosis with an overall 5-year survival rate less than 19%. Exploring new molecular therapeutic targets such as epidermal growth factor receptor (EGFR) could be the corner stone of new curative treatment. The present study was done to analyze the overexpression of EGFR in different grades of ESCC and explore its role as a diagnostic and theranostic marker in ESCC. In this retrospective study, 50 formalin-fixed paraffin-embedded blocks of ESCCs diagnosed from 2014 to 2019 were retrieved. The biopsies were subjected to immunohistochemistry staining of EGFR. The intensity of the membrane staining was reviewed and scored. Compared with various intrinsic and extrinsic factors using Chi-square test, scores more than 2+ were considered as overexpression. Majority (84%) specimens demonstrated overexpression of EGFR where high-grade ESCCs had greater overexpression rates compared to low-grade ESCC (P < 0.05). By targeting the EGFR molecules, anti-EGFR drugs could block their signals and stop the growth and spread of ESCCs especially high-grade tumors while harming the normal cells as little as possible. A clinical trial using anti-EGFR monoclonal antibodies will help in the long run to develop immunotherapy drugs.

Clinicopathological Parameters and Immunohistochemical Expression of EGFR in Colorectal Carcinoma: A Cross-sectional Study

Introduction: Colorectal Carcinoma (CRC) is the third most common type of cancer worldwide and is one of the leading causes of cancer-related death. Adenocarcinoma is the most common type. Histopathological examination is necessary to determine the type and extent of the tumour, which is essential for patient management and prognosis. Molecular markers play a major role in CRC, among which Epidermal Growth Factor Receptor (EGFR) has prognostic significance. Aim: The aim of this study was to evaluate the immunohistochemical expression of EGFR in cases of colonic carcinoma and analyse its relationship with various histological and clinical parameters. Materials and Methods: An observational (cross-sectional) study was conducted in the Department of Pathology, Sree Mookambika Institute of Medical Sciences, Kulasekharam, Kanyakumari district, Tamil Nadu, India. Data from colonic carcinoma patients confirmed by Haematoxylin and Eosin (H&amp;E) staining were collected over a three-year period from January 2019 to December 2021. A total of 58 cases were included. The characteristics studied included age, sex, Carcinoembryonic Antigen (CEA) levels, tumour site, size, degree of histological differentiation (well, moderate, poor), TNM staging, and lymph node status. The EGFR score was obtained by multiplying the grade (% positive cells) by the intensity, and a composite score ranging from 0 to 9 was obtained. This score was used to define low EGFR expression (&lt;6) or high EGFR expression (&gt;6). Analysis was performed using the Chi-square test in Statistical Package for the Social Sciences (SPSS) version 20.0. Results: Among the 58 patients included in the study, the most common age group affected by CRC was 61 to 70 years (28 cases; 48.3%), with a mean age of 63.32±14.36 years. The most common histological variant was low-grade adenocarcinoma (47 cases; 81.03%). EGFR reactivity was positive in 50 patients (86.20%). EGFR overexpression was significantly associated with histopathological type (low-grade adenocarcinoma) and tumour stage, with p-values of 0.03 and 0.001, respectively. Conclusion: Immunohistochemical overexpression of EGFR in CRC is associated with histopathological type and tumour staging in this study. This overexpression is associated with poor prognosis and can be used as a predictive marker for patients who yield positive results with chemotherapy.

Evaluation of EGFR in prognostic characterization in histopathological variants of oral squamous cell carcinoma.

Epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor of the Erb B family contributing to proliferation, invasion, and metastasis. EGFR overexpression is frequently associated with poor clinical outcome in malignant neoplasms.• To evaluate and compare immunoexpression of EGFR in histopathological variants of oral squamous cell carcinoma (OSCC).• To hypothesize the role of EGFR in determining biological behavior and prognostic course of histopathological variants of OSCC. The study comprised a total of 40 cases including 10 cases each of Squamous cell carcinoma, Verrucous carcinoma, Adenosquamous cell carcinoma, and Adenoid squamous cell carcinoma. EGFR immunoexpression was observed qualitatively as low (1), moderate (2) and strong (3) and quantitatively as score 1 for <10%, 2 for 10%-50%, and 3 for >50% positive cells. The resulting data were analyzed using SPSS software version 19. Data have been expressed as mean and standard deviation. Differences between the different variables were analyzed using ANOVA, and Pearson's Chi-square. (p ≤ 0.05). The study results revealed that the EFGR immunoexpression was highest in adenosquamous cell carcinoma followed by adenoid squamous cell carcinoma then conventional squamous cell carcinoma followed by lowest immunoexpression in verrucous carcinoma. The results were statistically significant. (p ≤ 0.05). Expression of EGFR could be established as a valuable biomarker with significant association in predicting aggressive potential and treatment response in various histopathological variants of OSCC. Further studies where EGFR could be linked to predictive indicators and tumor prognosis could be undertaken.

The epidermal growth factor receptor in healthy pregnancy and preeclampsia.

The epidermal growth factor receptor (EGFR) is expressed robustly in the placenta, and critical processes of pregnancy such as placental growth and trophoblast fusion are dependent on EGFR function. However, the role that aberrant EGFR signaling might play in the etiology and/or maintenance of preeclampsia (PE) remains largely unexplored. Recently, we have shown that overexpression of EGFR in cultured uterine artery endothelial cells (UAEC), which express little endogenous EGFR, remaps responsiveness away from vascular endothelial growth factor receptor (VEGFR) signaling and toward EGFR, suggesting that endothelial EGFR expression may be kept low to preserve VEGFR control of angiogenesis. Here we will consider the evidence for the possibility that the endothelial dysfunction observed in PE might in some cases result from elevation of endothelial EGFR. During pregnancy, trophoblasts are known to synthesize large amounts of EGFR protein, and the placenta regularly releases syncytiotrophoblast-derived exosomes and microparticles into the maternal circulation. Although there are no reports of elevated EGFR gene expression in preeclamptic endothelial cells, the ongoing shedding of placental vesicles into the vascular system raises the possibility that EGFR-rich vesicles might fuse with endothelium, thereby contributing to the symptoms of PE by interrupting angiogenesis and blocking pregnancy-adapted vasodilatory function.

Phase I study of anti-epidermal growth factor receptor antibody-drug conjugate serclutamab talirine: Safety, pharmacokinetics, and antitumor activity in advanced glioblastoma.

Serclutamab talirine (Ser-T, formerly ABBV-321) is an antibody-drug conjugate consisting of an antibody (AM-1-ABT-806) directed against activated epidermal growth factor receptor (EGFR) and a pyrrolobenzodiazepine dimer. We investigated Ser-T monotherapy in a phase I, first-in-human, dose-escalation, and dose-expansion study in patients with advanced solid tumors associated with EGFR overexpression. Eligible patients (≥18 years) had advanced, histologically confirmed solid tumors associated with EGFR overexpression (centralized testing). Patients received Ser-T intravenously once every 4 weeks (Q4W; 5-50 μg/kg) in the dose-escalation phase. Herein, preliminary antitumor activity at the recommended phase II dose (RP2D) is reported only for patients with glioblastoma (n = 24); additional assessments included all treated patients. Sixty-two patients (median age: 58 years) were enrolled within the dose-escalation (n = 43) and dose-expansion (n = 19) phases. One dose-limiting toxicity, grade 3 aspartate aminotransferase and alanine aminotransferase elevation, occurred at 20 μg/kg during dose escalation. The Ser-T RP2D regimen of 50 μg/kg × 1 (loading dose) followed by 25 μg/kg Q4W (maintenance dose) was administered during dose expansion. Fatigue (37%) was the only treatment-emergent adverse event (AE) occurring in >25% of patients. Two patients (3%) reported mild treatment-related ocular AEs (eye pruritus). Responses in patients with glioblastoma included 1 partial response (~33 months), 6 stable disease, and 14 progressive disease (not evaluable: n = 3). Ser-T monotherapy at doses up to 50 μg/kg initial dose, followed by 25 μg/kg Q4W demonstrated a tolerable safety profile with minimal antitumor activity observed in patients with glioblastoma. The glioblastoma dose-expansion cohort was closed due to a lack of efficacy (NCT03234712).

STEM-22. EFFECT OF A SRC INHIBITORY PEPTIDE BASED ON CONNEXIN43 ON NEURAL STEM CELLS WITH GLIOMA-DRIVER MUTATIONS

Abstract Glioblastomas are one of the most malignant tumors. Glioma Stem Cells (GSCs) are a subpopulation of cells resistant to standard treatments, responsible for tumor recurrence. Strong evidence supports that Neural Stem Cells (NSCs) from the subventricular zone (SVZ) are the origin of GSCs. This transition frequently concurs with epidermal growth factor receptor (EGFR) overexpression or mutations, such as EGFRvIII. Our group designed a cell penetrating peptide based on connexin43 (TAT-Cx43266-283) that inhibits the oncoprotein c-Src and therefore targets GSCs, increasing survival in glioma-bearing mice. Because c-Src and EGFR signaling are closely related, we explored the effect of TAT-Cx43266-283 in the transition of NSCs to GSCs. We compared the effect of TAT-Cx43266-283, control peptides, temozolomide and erlotinib, a common EGFR inhibitor, in SVZ NSCs (Control-NSCs) and murine NSC and patient-derived GSCs with key glioma-driver mutations in Nf1, PTEN and EGFR. EGFR signaling pathway was analyzed by Western blot. Furthermore, we are analyzing the effect of TAT-Cx43266-283 in a mouse model where tumors originate from SVZ NSCs with glioma-driver mutations. Our results showed a strong effect of TAT-Cx43266-283 in cell viability of NSCs and patient-derived GSCs with EGFR amplification or active EGFRvIII, without significant effects in Control-NSCs. Importantly, we found a lower effect of temozolomide, erlotinib, and other control peptides in these cells. Western blot analyses suggest that TAT-Cx43266-283 decreases EGFR, EGFRvIII and c-Src activity. Preliminary in vivo results suggest that TAT-Cx43266-283 decreases tumor size and enhances survival in mice bearing gliomas initiated by altered NSCs. So far, our results show that TAT-Cx43266-283 impairs EGFR signaling pathway with the subsequent reduction in the proliferation and survival of NSCs or GSCs with EGFR alterations. These results stress the relevance of TAT-Cx43266-283 as a future therapy against glioblastoma, alone or in combination with other treatments.

Postoperative Prognostic Predictors of Bile Duct Cancers: Clinical Analysis and Immunoassays of Tissue Microarrays.

Cholangiocarcinoma frequently recurs even after curative resection. Expression levels of proteins such as epidermal growth factor receptor (EGFR), Snail, epithelial cadherin (E-cadherin), and interleukin-6 (IL-6) examined by immunohistochemistry have been studied as potential prognostic factors for cholangiocarcinoma. The aim of this study was to investigate significant factors affecting the prognosis of resectable cholangiocarcinoma. Ninety-one patients who underwent surgical resection at Samsung Medical Center for cholangiocarcinoma from 1995 to 2013 were included in this study. Expression levels of E-cadherin, Snail, IL-6, membranous EGFR, and cytoplasmic EGFR were analyzed by immunohistochemistry using tissue microarray blocks made from surgical specimens. Patients with high levels of membranous EGFR in tissue microarrays had significantly shorter overall survival (OS) and disease-free survival (DFS): high membranous EGFR (score 0-2) 38.0 months versus low membranous EGFR (score 3) 14.4 months (p=0.008) and high membranous EGFR (score 0-2) 23.2 months versus low membranous EGFR (score 3) 6.1 months (p=0.004), respectively. On the other hand, E-cadherin, Snail, cytoplasmic EGFR, and IL-6 did not show significant association with OS or DFS. Patients with distant metastasis had significantly higher IL-6 levels than those with locoregional recurrence (p=0.01). This study showed that overexpression of membranous EGFR was significantly associated with shorter OS and DFS in surgically resected bile duct cancer patients. In addition, higher IL-6 expression was a predictive marker for recurrence in cholangiocarcinoma patients with distant organ metastasis after surgical resection.

The anticancer effect of EGFR-targeting artificial microRNA controlled by SLPI promoter in nasopharyngeal carcinoma.

This study intends to use artificial microRNA (recombinant adenovirus vector) targeting epidermal growth factor receptor (EGFR) to inhibit the overexpressed EGFR in nasopharyngeal carcinoma, thereby inhibiting the proliferation and metastasis of nasopharyngeal carcinoma. The research group verified the expression of EGFR in nasopharyngeal carcinoma through databases, clinical tissues, and cellular pathways. The team first tested the transfection of the recombinant adenovirus by fluorescence microscopy. After adenovirus treatment with different multiplicity of infection (MOI), EGFR level and cell viability in cells were examined by Western blot and MTT assay. Next, the effects of adenovirus (Ad)-SLPI-EGFR on cell proliferation, migration, apoptosis, and related proteins were sequentially examined by EdU, scratch, Transwell, and Western blot. In vivo experiments were performed to evaluate the biological function of EGFR in nasopharyngeal carcinoma. All three validation pathways showed the increase in EGFR expression in nasopharyngeal carcinoma. Transfection tests showed that the SLPI promoter was specific in CNE2 cells. With the increase in MOI, the inhibition of EGFR expression and cancer cell viability by Ad-SLPI-EGFR was enhanced. Meanwhile, Ad-SLPI-EGFR effectively reduced the proliferation and metastasis of CNE2 cells and affected the expression of related proteins. Furthermore, Ad-SLPI-EGFR inhibited the invasion and metastasis of nasopharyngeal carcinoma in vivo. Ad-SLPI-EGFR inhibits the expression of EGFR in nasopharyngeal carcinoma cells, and finally achieves the purpose of inhibiting the proliferation and metastasis of cancer cells, which may provide novel targeted intervention for the treatment of nasopharyngeal carcinoma.

240 (PB020) - Antitumor activity of Antibody-Drug Conjugates targeting cancer-expressed EGFR in Preclinical Models

Background: The epidermal growth factor receptor (EGFR) plays a key role in the growth and survival of many human tumors of epithelial origin. The monoclonal antibody, 40H3, targeting overexpressed EGFR and the truncated form EGFR variant III (EGFRvIII) on tumor cells, was conjugated to small molecules with potent cytotoxic activity, generating five antibody-drug conjugates (ADCs). Their lethality for tumor cells was evaluated in vitro and in vivo. Materials and methods: ADC construction: Purified 40H3 antibody was conjugated to five different payloads: two tubulin inhibitors, monomethyl auristatin E (MMAE) and DM1, two topoisomerase inhibitors, SN38 and deruxtecan (DXd1) and a PBD dimer (SG-3199). Binding assay: The binding affinity constants of the various 40H3-based ADCs were determined against the His-tagged EGFR peptide loop (aa 287– 302) immobilized on Ni-NTA biosensors with the Octet Red96 analyzer. Bystander assay: Cocultured F98npEGFRvIII and F98 cells were treated with media, free payload (SG-3199), 40H3 antibody, 40H3-tesirine or IgG-tesirine at the indicated concentrations. After 48 hours, the cells were labeled with cetuximab-PE and SYTOXTM Red viability dye and then analyzed for bystander killing by flow cytometry. In vivo studies: MDA-MB-468 or BT-20 tumor xenografts were treated with unmodified 40H3 antibody or 40H3-tesirine. Tumor volumes and mouse weights were measured at least three times weekly. Results: The five ADCs retained antigen binding activity of the unmodified 40H3 antibody. They showed a potent cytotoxicity on a panel of EGFR-expressing cells including three triple negative breast cancer lines. Cell killing was correlated to the number of binding sites for the 40H3 antibody. The 40H3 conjugate with the PBD dimer (40H3-tesirine) was the most active killing agent and it also exhibited bystander killing of cells not expressing human EGFR. Moreover, in vivo, on two different models of tumor xenografts, treatment with 40H3-tesirine achieved complete remissions. Conclusions: The 40H3 antibody is a valid delivery agent for toxic payloads. Among the five ADCs, 40H3-tesirine showed the highest cytotoxic effect toward EGFR-expressing tumor cells in vitro and in vivo. No conflict of interest.

Engaging innate immunity for targeting the epidermal growth factor receptor: Therapeutic options leveraging innate immunity versus adaptive immunity versus inhibition of signaling.

The epidermal growth factor receptor (EGFR) is a key player in the normal tissue physiology and the pathology of cancer. Therapeutic approaches have now been developed to target oncogenic genetic aberrations of EGFR, found in a subset of tumors, and to take advantage of overexpression of EGFR in tumors. The development of small-molecule inhibitors and anti-EGFR antibodies targeting EGFR activation have resulted in effective but limited treatment options for patients with mutated or wild-type EGFR-expressing cancers, while therapeutic approaches that deploy effectors of the adaptive or innate immune system are still undergoing development. This review discusses EGFR-targeting therapies acting through distinct molecular mechanisms to destroy EGFR-expressing cancer cells. The focus is on the successes and limitations of therapies targeting the activation of EGFR versus those that exploit the cytotoxic T cells and innate immune cells to target EGFR-expressing cancer cells. Moreover, we discuss alternative approaches that may have the potential to overcome limitations of current therapies; in particular the innate cell engagers are discussed. Furthermore, this review highlights the potential to combine innate cell engagers with immunotherapies, to maximize their effectiveness, or with unspecific cell therapies, to convert them into tumor-specific agents.

Challenges for the application of EGFR-targeting peptide GE11 in tumor diagnosis and treatment

Abnormal regulation of cell signaling pathways on cell survival, proliferation and migration contributes to the development of malignant tumors. Among them, epidermal growth factor receptor (EGFR) is one of the most important biomarkers in many types of malignant solid tumors. Its over-expression and mutation status can be served as a biomarker to identify patients who can be benifit from EGFR tyrosine kinase inhibitors and anti-EGFR monocloncal antibody (mAb) therapy. For decades, researches on EGFR targeted ligands were actively carried out to identify potent candidates for cancer therapy. An ideal EGFR ligand can competitively inhibit the binding of endogenous growth factor, such as epidermal growth factor (EGF) and transforming growth factor-α(TGF-α) to EGFR, thus block EGFR signaling pathway and downregulate EGFR expression. Alternatively, conjugation of EGFR ligands on drug delivery systems (DDS) can facilitate targeting delivery of therapeutics or diagnostic agents to EGFR over-expression tumors via EGFR-mediated endocytosis. GE11 peptide is one of the potent EGFR ligand screened from a phage display peptide library. It is a dodecapeptide that can specifically binds to EGFR with high affinity and selectivity. GE11 has been widely used in the diagnosis and targeted delivery of drugs for radiotherapy, genetherapy and chemotherpy against EGFR positive tumors. In this review, the critical factors affecting the in vivo and in vitro targeting performance of GE11 peptide, including ligand-receptor intermolecular force, linker bond properties and physiochemical properties of carrier materials, are detailedly interpreted. This review provides a valuable vision for the rational design and optimization of GE11-based active targeting strategies for cancer treatment, and it will promote the translation studies of GE11 from lab research to clinical application.

Epidermal growth factor receptor (EGFR) expression in high grade glioma and relationship with histopathologic findings, a cross sectional study

IntroductionThere are some evidences for the relationship between the expression of epidermal growth factor receptor (EGFR) gene and the clinical and histopathological behavior of the high grade glioma. However, such a relationship can take quite different forms in different societies. We aimed to evaluate the EGFR expression status in high grade of glioma and its relationship with histopathological patterns in the tumor. MethodsWe studied the immunohistochemichal expression of EGFR in 30 brain surgical resection specimens with different high grades. All samples were from pathology department of Sina Hospital in Tehran-Iran from 2020 to 2021. ResultsEGFR positivity was reported in 56.6% of all tissue samples assessed. The assessment of relationship between EGFR expression and baseline and tumor characteristics showed no significant association of EGFR expression and patients’ gender, age, tumor location, tumor size, its type, grade, and also existence of necrosis (P value > 0.05). ConclusionEGFR expression can be found in about half of our patients suffering high grade glioma. The over-expression of EGFR seems to be independent to demographic characteristics. Also, over-expression of EGFR may not predict the presence of necrosis in tumor tissue.

Multivalent Ligand-Nanoparticle Conjugates Amplify Reactive Oxygen Species Second Messenger Generation and Enhance Epidermal Growth Factor Receptor Phosphorylation.

The epidermal growth factor (EGF) receptor (EGFR) is heterogeneously distributed on the cellular surface and enriched in clusters with diameters of tens of nanometers. Multivalent presentation of EGF ligand on nanoparticles (NPs) provides an approach for controlling and amplifying the local activation of EGFR in these clusters. Reactive oxygen species (ROS) have been indicated to play a role in the regulation of EGFR activation as second messengers, but the effect of nanoconjugation on EGF-mediated ROS formation and ROS-induced EGFR activation is not well established. The goal of this manuscript is to characterize the multivalent enhancement of EGF-induced ROS formation and to test its effect on EGFR phosphorylation in breast cancer cell models using gold (Au) NPs with a diameter of 81 ± 1 nm functionalized with two different EGF ligand densities (12 ± 7 EGF/NP (NP-EGF12) and 87 ± 6 EGF/NP (NP-EGF87)). In the EGFR overexpressing cell lines MDA-MB-231 and MDA-MB-468, NP-EGF87 achieved a measurable multivalent enhancement of ROS that peaked at concentrations c ROSmax ≤ 25 pM and that were EGFR and nicotinamide adenine dinucleotide phosphate oxidase (NOX) dependent. NP-EGF12 failed to generate comparable ROS levels as NP-EGF87 in the investigated NP input concentration range (0-100 pM). In cells with nearly identical numbers of bound NP-EGF87 and NP-EGF12, the ROS levels for NP-EGF87 were systematically higher, indicating that the multivalent enhancement is exclusively related not only to avidity but also to a stronger stimulation per NP. Importantly, the increase in EGF-induced ROS formation associated with EGF nanoconjugation at c ROSmax resulted in a measurable gain in EGFR phosphorylation, confirming that ROS generation contributes to the multivalent enhancement of EGFR activation in response to NP-EGF87.

LOCL-12 OUTCOMES OF STEREOTACTIC RADIOSURGERY FOR BRAIN METASTASES OF BREAST CANCER IN RELATION TO MOLECULAR SUBTYPES

Abstract PURPOSE To determine intracranial outcomes following stereotactic radiosurgery (SRS) for brain metastases of breast cancer in relation to molecular subtypes of HER2 (human epidermal growth factor receptor) overexpression and estrogen receptor (ER) expression. METHOD From a retrospective institutional database of SRS using VMAT (volumetric modulated arc therapy) carried out between 2012 and 2019, patients with a primary diagnosis of breast cancer were identified. Outcomes following first course of SRS were analyzed using radiotherapy plans and electronic patient records. Systemic anticancer treatments were not registered. Time to intracranial progression (TTP) was calculated using the Kaplan Meier method as the time between SRS and recurrence, whereas dates of death or last follow up without recurrence were censored. The study was approved by the Danish Patient Safety Authority (nr. 31-1521-125). RESULTS The study included 159 patients. 132 patients (83%) underwent a brain MRI examination after SRS and, of these, 101 had recurrence, either local (41%), remote (43%) or both (16%). Tumors were HER2 positive in 50% of cases and 61% were ER positive and 15% were triple negative. The median intracranial TTP was for the whole study group 9.1 months. Median intracranial TTP for patients with the HER2+ / ER+ subtype was 15.4 mos. whereas patients with the triple negative subtype had a significantly shorter median intracranial TTP of 5.1 mos. (p=0.007). Patients with HER2+/ER- and HER2 normal/ER+ had similar median intracranial TTP times of 7.7 mos. and 8.4 mos., respectively (n.s.). CONCLUSION This study found high rates of intracranial recurrence following SRS and poor intracranial TTP that was highly dependent on molecular subtypes, reflecting either biological differences or intracranial effect of systemic treatments or both. The study confirms the universally poor prognosis for breast cancer patients with brain metastases and the high frequency of HER2 overexpression in this patient group.

Investigating the clinical significance of EGFR expression using machine learning in a series of Iraqi patients with triple-negative breast cancer.

Breast cancer is a heterogeneous disease with a distinct profile of the expression of each tumor. Triple-negative breast cancer (TNBC) is a molecular subtype of breast cancer characterized by an aggressive clinical behavior linked to loss or reduced expression of estrogen, progesterone, and Her2/neu receptors. The study's main objective was to investigate the clinical significance of epidermal growth factor receptor (EGFR) overexpression in a series of Iraqi patients with TNBC. The sectional analytic study involved immunohistochemical analysis of EGFR expression in randomly selected 53 formalin fixed paraffin embedded tissue blocks of TNBC cases out of 127 Iraqi patients with TNBC and correlated expression data with clinicopathological parameters including survival time. Machine learning (statistical tests and principal component analysis (PCA)) was used to predict the outcome of the patients using EGFR expression data together with clinicopathological parameters. EGFR was expressed in approximately 28% of TNBC cases. We estimated the risk of mortality and distant metastasis based on EGFR expression and clinicopathologic factors using the principal component analysis (PCA) model. We found a substantial positive correlation between clinical stage and distant metastasis, clinical stage and death, death and distant metastasis, and death and positive EGFR expression. Overall, EGFR expression was linked to a poor prognosis and increased mortality. A higher risk of distant metastasis and death was associated with an advanced clinical stage of the tumor. Furthermore, the existence of distant metastases increased the risk of death. These findings raise the possibility of using EGFR expression data with other clinicopathological parameters to predict the outcome of patients with TNBC.

Epidermal growth factor receptor (EGFR) expression in the serum of patients with triple-negative breast carcinoma: prognostic value of this biomarker.

BackgroundEpidermal growth factor receptor (EGFR) overexpression has been considered a poor prognostic factor in breast cancer.MethodologyA prospective study of 206 women with breast cancer analysed by stages (I, II, III and IV) and by immunohistochemical subtype (Luminal A, Luminal B, HER2+ and triple-negative (TN)); 89 healthy controls with normal recent mammography were included. The EGFR measured in the serum (sEGFR) was detected by the Enzyme-Linked Immunosorbent Assay (ELISA) method (R&D Systems kit DY231) collected by blood before any treatment in patients. Kaplan–Meier method and Cox regression were carried out to obtain the prognostic value, considering significance if p < 0.05.ResultsWith a median follow-up of 36.6 months, 47 deaths occurred. Multivariable Cox regression showed difference of overall survival (OS) associated with sEGFR levels (sEGFR ≤ or > 47.8 ng/mL) in patients with TN cancers, but not of Luminal A, Luminal B or HER2+ subtypes; adjusted by stage, the death risk increased by approximately 415% [hazard ratio (HR): 5.149 (1.900–13.955), p = 0.001] for patients with sEGFR > 47.8 ng/mL compared to patients with a lower sEGFR value. There was no significant correlation of sEGFR with staging, histological tumour grade (G1/G2/G3), Ki67 (< or ≥14%) or body mass index.ConclusionsIncreased sEGFR expression in patients with TN tumours is a significant predictor of lower OS and its quantification is inexpensive and straightforward.

Inhibition of Epidermal Growth Factor Receptor Signaling by Antisense Oligonucleotides as a Novel Approach to Epidermal Growth Factor Receptor Inhibition.

We report a novel method to inhibit epidermal growth factor receptor (EGFR) signaling using custom morpholino antisense oligonucleotides (ASOs) to drive expression of dominant negative mRNA isoforms of EGFR by ASO-induced exon skipping within the transmembrane (16) or tyrosine kinase domains (18 and 21). In vivo ASO formulations induced >95% exon skipping in several models of nonsmall cell lung cancer (NSCLC) and were comparable in efficacy to erlotinib in reducing colony formation, cell viability, and migration in EGFR mutant NSCLC (PC9). However, unlike erlotinib, ASOs maintained their efficacy in both erlotinib-resistant subclones (PC9-GR) and wild-type overexpressing EGFR models (H292), in which erlotinib had no significant effect. The most dramatic ASO-induced phenotype resulted from targeting the EGFR kinase domain directly, which resulted in maximal inhibition of phosphorylation of EGFR, Akt, and Erk in both PC9 and PC9GR cells. Phosphoproteomic mass spectrometry confirmed highly congruent impacts of exon 16-, 18-, and 21-directed ASOs compared with erlotinib on PC9 genome-wide cell signaling. Furthermore, EGFR-directed ASOs had no impact in EGFR-independent NSCLC models, confirming an EGFR-specific therapeutic mechanism. Further exploration of synergy of ASOs with existing tyrosine kinase inhibitors may offer novel clinical models to improve EGFR-targeted therapies for both mutant and wild-type NSCLC patients.

Multi-Targeting Approach in Glioblastoma Using Computer-Assisted Drug Discovery Tools to Overcome the Blood-Brain Barrier and Target EGFR/PI3Kp110β Signaling.

Simple SummaryTreatment of glioblastoma is hampered by the activation of compensatory survival mechanisms by malignant cells that lead to drug resistance. Moreover, the blood–brain barrier (BBB) precludes the brain entrance of most drugs. We hypothesized that computer-assisted drug discovery tools would reveal novel multi-targeting drug candidates with BBB-permeant and favorable ADMET properties. We aimed to discover molecules with predicted ability to inhibit the EGFR/PI3Kp110β pathway and to validate their efficacy and safety in biological assays. We used quantitative structure–activity relationship models and structure-based virtual screening, and assessed ADMET properties, to identify BBB-permeant drug candidates. Moreover, we tested their anti-tumor efficacy and BBB safety and permeation in cell models. We found two EGFR, two PI3Kp110β, and, mostly, two dual inhibitors with anti-tumor effects. Among them, one EGFR and two PI3Kp110β inhibitors were able to cross the BBB endothelium without compromising it. These studies revealed novel drug candidates for glioblastoma treatment.The epidermal growth factor receptor (EGFR) is upregulated in glioblastoma, becoming an attractive therapeutic target. However, activation of compensatory pathways generates inputs to downstream PI3Kp110β signaling, leading to anti-EGFR therapeutic resistance. Moreover, the blood–brain barrier (BBB) limits drugs’ brain penetration. We aimed to discover EGFR/PI3Kp110β pathway inhibitors for a multi-targeting approach, with favorable ADMET and BBB-permeant properties. We used quantitative structure–activity relationship models and structure-based virtual screening, and assessed ADMET properties, to identify BBB-permeant drug candidates. Predictions were validated in in vitro models of the human BBB and BBB-glioma co-cultures. The results disclosed 27 molecules (18 EGFR, 6 PI3Kp110β, and 3 dual inhibitors) for biological validation, performed in two glioblastoma cell lines (U87MG and U87MG overexpressing EGFR). Six molecules (two EGFR, two PI3Kp110β, and two dual inhibitors) decreased cell viability by 40–99%, with the greatest effect observed for the dual inhibitors. The glioma cytotoxicity was confirmed by analysis of targets’ downregulation and increased apoptosis (15–85%). Safety to BBB endothelial cells was confirmed for three of those molecules (one EGFR and two PI3Kp110β inhibitors). These molecules crossed the endothelial monolayer in the BBB in vitro model and in the BBB-glioblastoma co-culture system. These results revealed novel drug candidates for glioblastoma treatment.