Epidermal Growth Factor Receptor Variant III Expression Research Articles

Formation of EGFRwt/EGFRvIII homo- and hetero-dimers in glioblastoma cells as detected by single molecule localization microscopy.

Super-resolution microscopy has been used to show the formation of receptor clusters and adapted lipid organization of cell membranes for many members of the ErbB receptor family. The clustering behaviour depends on the receptor size and shape, possibly ligand binding or expression activity. Using single molecule localization microscopy (SMLM), we also showed this typical clustering for the epidermal growth factor receptor variant III (EGFRvIII) in glioblastoma multiforme (GBM) cells. EGFRvIII is co-expressed with the wild type (EGFRwt) and both receptors are assumed to preferentially form hetero-dimers leading to transactivation and elevated oncogenic EGFR-signalling in GBM cells. Here, we analysed EGFRvIII and EGFRwt co-localization using our already described model system of the glioblastoma cell line DKMG, displaying endogenous EGFRvIII expression. Using EGFRvIII and EGFRwt specific antibodies, EGFR localization and their potential for dimerization in a given membrane cluster were analysed by dual colour SMLM supported by novel approaches of mathematic evaluations including Ripley statistics, persistent homology and similarity algorithms. Surprisingly, cluster analysis, Ripley point-to-point distance statistics for cluster geometry and persistent homology comparing cluster topology, revealed that both EGFRvIII and EGFRwt do primarily not form hetero-dimers but the results support the hypothesis that they tend to form homo-dimers. The ratio of homo-dimers obtained by this calculation was significantly higher (>5σ, standard deviation) than expected from randomly arranged points. In comparison, hetero-dimer formation was only slightly increased. We confirmed these data by immunoprecipitation, which show no co-precipitation of EGFRvIII and EGFRwt. Furthermore, we showed that the topology of the clusters was more similar among the same type than among the different types of receptors. Taken together, these data indicate that EGFRvIII does induce oncogenic signalling by homo-dimerisation and not preferentially by hetero-dimer formation with EGFRwt. These data offer a new perspective on EGFRvIII signalling which will lead to a better understanding of this tumour associated receptor variant in GBM.

Ceramide Is Involved in Temozolomide Resistance in Human Glioblastoma U87MG Overexpressing EGFR.

Glioblastoma multiforme (GBM) is the most frequent and deadly brain tumor. Many sphingolipids are crucial players in the regulation of glioma cell growth as well as in the response to different chemotherapeutic drugs. In particular, ceramide (Cer) is a tumor suppressor lipid, able to induce antiproliferative and apoptotic responses in different types of tumors including GBM, most of which overexpress the epidermal growth factor receptor variant III (EGFRvIII). In this paper, we investigated whether Cer metabolism is altered in the U87MG human glioma cell line overexpressing EGFRvIII (EGFR+ cells) to elucidate their possible interplay in the mechanisms regulating GBM survival properties and the response to the alkylating agent temozolomide (TMZ). Notably, we demonstrated that a low dose of TMZ significantly increases Cer levels in U87MG cells but slightly in EGFR+ cells (sensitive and resistant to TMZ, respectively). Moreover, the inhibition of the synthesis of complex sphingolipids made EGFR+ cells sensitive to TMZ, thus involving Cer accumulation/removal in TMZ resistance of GBM cells. This suggests that the enhanced resistance of EGFR+ cells to TMZ is dependent on Cer metabolism. Altogether, our results indicate that EGFRvIII expression confers a TMZ-resistance phenotype to U87MG glioma cells by counteracting Cer increase.

Increased replication stress and R-loop accumulation in EGFRvIII-expressing glioblastoma present new therapeutic opportunities.

BackgroundThe oncogene epidermal growth factor receptor variant III (EGFRvIII) is expressed in approximately one-third of all glioblastomas (GBMs). So far it is not clear if EGFRvIII expression induces replication stress in GBM cells, which might serve as a therapeutical target.MethodsIsogenetic EGFRvIII− and EGFRvIII+ cell lines with endogenous EGFRvIII expression were used. Markers of oncogenic and replication stress such as γH2AX, RPA, 53BP1, ATR, and CHK1 were analyzed using western blot, immunofluorescence, and flow cytometry. The DNA fiber assay was performed to analyze replication, transcription was measured by incorporation of EU, and genomic instability was investigated by micronuclei and CGH-Array analysis. Immunohistochemistry staining was used to detect replication stress markers and R-loops in human GBM samples.ResultsEGFRvIII+ cells exhibit an activated replication stress response, increased spontaneous DNA damage, elevated levels of single-stranded DNA, and reduced DNA replication velocity, which are all indicative characteristics of replication stress. Furthermore, we show here that EGFRvIII expression is linked to increased genomic instability. EGFRvIII-expressing cells display elevated RNA synthesis and R-loop formation, which could also be confirmed in EGFRvIII-positive GBM patient samples. Targeting replication stress by irinotecan resulted in increased sensitivity of EGFRvIII+ cells.ConclusionThis study demonstrates that EGFRvIII expression is associated with increased replication stress, R-loop accumulation, and genomic instability. This might contribute to intratumoral heterogeneity but may also be exploited for individualized therapy approaches.

Abstract P134: Novel EGFRvIII-selective antibody-drug conjugate REGN3124-PBD is strongly efficacious against orthotopic glioblastoma multiforme patient derived xenografts

Abstract Glioblastoma Multiforme (GBM) is a highly aggressive cancer with few specific molecular targets and poor therapeutic outcome. Epidermal Growth Factor Receptor variant III (EGFRvIII) is a promising target for the treatment of GBM due to its exclusive expression in GBM. However, prior therapeutics have failed in part due to the development of EGFRvIII negative tumors cells from heterogenous tumor populations. We therefore developed a potent antibody-drug conjugate with bystander killing capabilities to target heterogeneous EGFRvIII-expressing GBM tumors. REGN3124 is a fully human EGFRvIII-selective antibody that also demonstrates some binding to amplified wild-type EGFR. REGN3124 was conjugated to the pyrrolobenzodiazepine (PBD) linker-payload SG-3249 to form REGN3124-PBD, and the cytotoxicity and cellular bystander killing activity was characterized in vitro. Initial in vivo activity of REGN3124-PBD was assessed in subcutaneous (s.c.) U251/EGFRvIII and U87/EGFRvIII cell line xenografts and then in s.c. EGFRvIII-positive patient derived xenograft (PDX) models (GBM6 and GBM59). Immunohistochemistry demonstrated heterogenous expression of EGFRvIII in GBM59 tumors. Lastly, efficacy was assessed in animals with established intracranial GBM6 or GBM59 tumors to examine the activity of REGN3124-PBD in an orthotopic setting. REGN3124-PBD demonstrated sub-nM cytotoxicity in vitro and clear bystander killing of EGFRvIII negative U251 cells following targeting of U251/EGFRvIII cells. A single dose of 0.38 mg/kg REGN3124-PBD (3.4 drug: antibody ratio) induced sustained regression of both s.c. U251/EGFRvIII and U87/EGFRvIII xenografts. A single dose of 0.53 mg/kg REGN3124-PBD induced complete regression of s.c. GBM6 PDX tumors and sustained regression of GBM59 tumors. Single dose of 0.53 mg/kg REGN3124-PBD significantly prolonged survival of mice with established intracranial GBM6 or GBM59 tumors, with 5/8 and 7/8 animals surviving >90 days post-treatment, respectively. The high unmet need for effective therapies combined with the potent anti-tumor activity observed, including in those with heterogenous expression of EGFRvIII, support continued assessment of REGN3124-PBD as a novel therapy for treatment of GBM. Citation Format: Marcus P. Kelly, Sosina Makonnen, Carlos Hickey, Shu Mao, Feng Zhao, Arthur Kunz, Frank Delfino, Thomas Nittoli, Dangshe Ma, William C. Olson, Gavin Thurston, Jessica R. Kirshner. Novel EGFRvIII-selective antibody-drug conjugate REGN3124-PBD is strongly efficacious against orthotopic glioblastoma multiforme patient derived xenografts [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P134.

ANO1 regulates the maintenance of stemness in glioblastoma stem cells by stabilizing EGFRvIII

Glioblastoma multiforme (GBM) or glioblastoma is the most deadly malignant brain tumor in adults. GBM is difficult to treat mainly due to the presence of glioblastoma stem cells (GSCs). Epidermal growth factor receptor variant III (EGFRvIII) has been linked to stemness and malignancy of GSCs; however, the regulatory mechanism of EGFRvIII is largely unknown. Here, we demonstrated that Anoctamin-1 (ANO1), a Ca2+-activated Cl- channel, interacts with EGFRvIII, increases its protein stability, and supports the maintenance of stemness and tumor progression in GSCs. Specifically, shRNA-mediated knockdown and pharmacological inhibition of ANO1 suppressed the self-renewal, invasion activities, and expression of EGFRvIII and related stem cell factors, including NOTCH1, nestin, and SOX2 in GSCs. Conversely, ANO1 overexpression enhanced the above phenomena. Mechanistically, ANO1 protected EGFRvIII from proteasomal degradation by directly binding to it. ANO1 knockdown significantly increased survival in mice and strongly suppressed local invasion of GSCs in an in vivo intracranial mouse model. Collectively, these results suggest that ANO1 plays a crucial role in the maintenance of stemness and invasiveness of GSCs by regulating the expression of EGFRvIII and related signaling molecules, and can be considered a promising therapeutic target for GBM treatment.

Efficacy of osimertinib against EGFRvIII+ glioblastoma.

Epidermal Growth Factor Receptor variant III (EGFRvIII) is an active mutant form of EGFR that drives tumor growth in a subset of glioblastoma (GBM). It occurs in over 20% of GBMs, making it a promising receptor for small molecule targeted therapy. We hypothesize that poor penetration of the blood-brain barrier by previously tested EGFR-tyrosine kinase inhibitors (EGFR-TKIs) such as afateninb, erlotinib, gefitinib, and lapatinib played a role in their limited efficacy. The present study examined the effects of osimertinib (previously known as AZD9291) on EGFRvIII+ GBM models, both in vitro and in vivo. Therefore, a panel of six GBM stem cells (GSCs) expressing EGFRvIII+ was evaluated. The EGFRvIII+ GSC differed in the expression of EGFRvIII and other key genes. The GSC line D317, which expresses high levels of EGFRvIII and has robust tyrosine kinase activity, was selected for assessing osimertinib’s efficacy. Herein, we report that osimertinib inhibits the constitutive activity of EGFRvIII tyrosine kinase with high potency (<100 nM) while also inhibiting its downstream signaling. Further, osimertinib inhibited D317’s growth in vitro and in both heterotopic and orthotopic xenograft models. Additional preclinical studies are warranted to identify EGFRvIII+ GBM’s molecular signature most responsive to osimertinib.

Tailored therapy for recurrent glioblastoma: report of a personalized molecular approach.

Failure of clinical trials with targeted therapies in glioblastoma (GBM) is probably related to the enrollment of molecularly unselected patients. In this study we report the results of a precision medicine protocol in recurrent GBM. We prospectively evaluated 34 patients with recurrent GBM. We determined the expression of vascular endothelial growth factor (VEGF), epidermal growth factor receptor variant III (EGFRvIII), and phosphatase and tensin homolog (PTEN). According to the molecular pattern we administered bevacizumab alone in patients with VEGF overexpression, absence of EGFRvIII, and normal PTEN (group A; N.=16); bevacizumab + erlotinib in patients with VEGF overexpression, expression of EGFRvIII, and normal PTEN (group B; N.=14); and bevacizumab + sirolimus in patients with VEGF overexpression and loss of PTEN, irrespective of the EGFRvIII status (group C; N.=4). We evaluated the response rate, the clinical benefit rate, the 6-month progression-free survival (PFS-6), the 12-month PFS (PFS-12) and the safety profile of the treatment. Moreover, we compared our results with the ones of EORTC 26101 trial. Response rate was 50% in the whole cohort with the highest rate in group C (75%). Clinical benefit rate was 71% with the highest rate in group C (75%). PFS-6 was 56% in the whole cohort with the highest rate in group B (64%). PFS-12 was 21% in the whole cohort with the highest rate in group B (29%). When comparing our results with those from the combination arm of the EORTC 26101 trial we found a significantly higher PFS-6 and PFS-12 in our cohort. The precision medicine protocol for recurrent GBM is feasible and leads to improved results if compared with studies lacking molecular selection.

EGFRvIII upregulates DNA mismatch repair resulting in increased temozolomide sensitivity of MGMT promoter methylated glioblastoma

The oncogene epidermal growth factor receptor variant III (EGFRvIII) is frequently expressed in glioblastomas (GBM) but its impact on therapy response is still under controversial debate. Here we wanted to test if EGFRvIII influences the sensitivity towards the alkylating agent temozolomide (TMZ). Therefore, we retrospectively analyzed the survival of 336 GBM patients, demonstrating that under standard treatment, which includes TMZ, EGFRvIII expression is associated with prolonged survival, but only in patients with O6-methylguanine-DNA methyltransferase (MGMT) promoter methylated tumors. Using isogenic GBM cell lines with endogenous EGFRvIII expression we could demonstrate that EGFRvIII increases TMZ sensitivity and results in enhanced numbers of DNA double-strand breaks and a pronounced S/G2-phase arrest after TMZ treatment. We observed a higher expression of DNA mismatch repair (MMR) proteins in EGFRvIII+ cells and patient tumor samples, which was most pronounced for MSH2 and MSH6. EGFRvIII-specific knockdown reduced MMR protein expression thereby increasing TMZ resistance. Subsequent functional kinome profiling revealed an increased activation of p38- and ERK1/2-dependent signaling in EGFRvIII expressing cells, which regulates MMR protein expression downstream of EGFRvIII. In summary, our results demonstrate that the oncoprotein EGFRvIII sensitizes a fraction of GBM to current standard of care treatment through the upregulation of DNA MMR.

EGFRvIII: An Oncogene with Ambiguous Role.

Epidermal growth factor receptor variant III (EGFRvIII) seems to constitute the perfect therapeutic target for glioblastoma (GB), as it is specifically present on up to 28–30% of GB cells. In case of other tumor types, expression and possible role of this oncogene still remain controversial. In spite of EGFRvIII mechanism of action being crucial for the design of small active anticancer molecules and immunotherapies, i.e., CAR-T technology, it is yet to be precisely defined. EGFRvIII is known to be resistant to degradation, but it is still unclear whether it heterodimerizes with EGF-activated wild-type EGFR (EGFRWT) or homodimerizes (including covalent homodimerization). Constitutive kinase activity of this mutated receptor is relatively low, and some researchers even claim that a nuclear, but not a membrane function, is crucial for its activity. Based on the analyses of recurrent tumors that are often lacking EGFRvIII expression despite its initial presence in corresponding primary foci, this oncogene is suggested to play a marginal role during later stages of carcinogenesis, while even in primary tumors EGFRvIII expression is detected only in a small percentage of tumor cells, undermining the rationality of EGFRvIII-targeting therapies. On the other hand, EGFRvIII-positive cells are resistant to apoptosis, more invasive, and characterized with enhanced proliferation rate. Moreover, expression of this oncogenic receptor was also postulated to be a marker of cancer stem cells. Opinions regarding the role that EGFRvIII plays in tumorigenesis and for tumor aggressiveness are clearly contradictory and, therefore, it is crucial not only to determine its mechanism of action, but also to unambiguously define its role at early and advanced cancer stages.

EGFRvIII epigenetically regulates ARHI to promote glioma cell proliferation and migration

Epidermal growth factor receptor variant III (EGFRvIII) is a tumor-specific mutation widely expressed in glioma. However, its role and molecular mechanism in glioma have not been completely elucidated. Immunohistochemistry analyses of EGFRvIII, enhancer of zeste homolog 2 (EZH2) and aplysia ras homolog I (ARHI) were performed in tumor tissues from patients with glioma. Regulatory mechanisms among EGFRvIII, EZH2 and ARHI were examined by western blot and chromatin immunoprecipitation (ChIP). Cell proliferation and migration of glioma cells were examined. EGFRvIII and EZH2 expression were upregulated, while ARHI was downregulated in glioma tissues. EZH2 knockdown increased ARHI expression in glioma cell lines. ChIP assay suggested that EZH2 was enriched in the ARHI promoter. Furthermore, ectopic expression of EGFRvIII upregulated EZH2, suppressed ARHI expression, and promoted glioma cell proliferation. Additionally, treatment with 3-deazaneplanocin A (DZNep, an inhibitor of EZH2) inhibited expression of EZH2, increased protein level of ARHI, and partially abrogated the promoting effects of ARHI knockdown on glioma cell proliferation and migration. In summary, EGFRvIII-mediated epigenetic suppression of ARHI promoted glioma cell proliferation and migration via upregulating EZH2.

CBMT-16. EGFRvIII EXPRESSION CONFERS CHEMOSENSITIVITY BY INCREASING DNA MISMATCH REPAIR PROTEIN EXPRESSION AND REPLICATION STRESS

Abstract MGMT promoter methylation is the only accepted biomarker with prognostic role in GBM but its routine implementation is limited partly response to TMZ is heterogeneous, but also due to lack of effective alternative treatment options. Therefore, additional biomarkers are needed to enable better prediction of survival and to improve individualized treatment of GBM patients. A potential new biomarker is the epidermal growth factor receptor variant III (EGFRvIII). This constitutively activated deletion variant is present in approximately one third of all IDH wildtype GBM, but its relevance to treatment response is poorly understood. The aim of the present study was to analyze the impact of endogenous EGFRvIII expression on chemosensitivity and the mechanisms underlying any differential treatment response. EGFRvIII expression was associated with prolonged median overall survival but only for GBM patients with MGMT promoter methylated tumors. In line with this, we observed increased TMZ sensitivity of EGFRvIII+ and MGMT promoter methylated cells, which translated into improved survival in xenograft experiments. The increased TMZ sensitivity was associated with an elevated DNA damage induction accompanied by an increased expression of DNA mismatch repair (MMR) proteins in EGFRvIII+ cell lines and EGFRvIII+ GBM patient samples. Subsequently, only a moderate reduction in MMR protein expression resulted in a dramatic TMZ resistance, suggesting that EGFRvIII expression specifically sensitized MGMT deficient cells to TMZ treatment by upregulating MMR. Furthermore, EGFRvIII expression in GBM cell lines was accompanied by increased DNA damage, replication fork slowing, stalling and enhanced origin firing, implying replication stress. Targeting of EGFRvIII-dependent replication stress by irinotecan led to hypersensitivity of EGFRvIII+ cells. Taken together this study illustrates that EGFRvIII-induced upregulation of MMR and replication stress increases chemosensitivity thereby highlighting the vulnerability of EGFRvIII+ GBM to available treatments. These important data may also guide the development of new and more effective personalized strategies.

B7-H3 as a Novel CAR-T Therapeutic Target for Glioblastoma

Glioblastoma (GBM) remains one of the most malignant primary tumors in adults, with a 5-year survival rate less than 10% because of lacking effective treatment. Here, we aimed to explore whether B7-H3 could serve as a novel therapeutic target for GBM in chimeric antigen receptor (CAR) T cell therapy. In this study, a CAR targeting B7-H3 was constructed and transduced into T cells by lentivirus. Antitumor effects of B7-H3-specific CAR-T cells were assessed with primary and GBM cell lines both in vitro and in vivo. Our results indicated that B7-H3 was positively stained in most of the clinical glioma samples, and its expression levels were correlated to the malignancy grade and poor survival in both low-grade glioma (LGG) and GBM patients. Specific antitumor functions of CAR-T cells were confirmed by cytotoxic and ELISA assay both in primary glioblastoma cells and GBM cell lines. In the orthotropic GBM models, the median survival of the CAR-T-cell-treated group was significantly longer than that of the control group. In conclusion, B7-H3 is frequently overexpressed in GBM patients and may serve as a therapeutic target in CAR-T therapy.

Prognostic implications of epidermal growth factor receptor variant III expression and nuclear translocation in Chinese human gliomas.

ObjectiveTo determine the prognostic implications and clinical significance of epidermal growth factor receptor variant III (EGFRvIII) expression and EGFRvIII nuclear translocation in Chinese human gliomas.MethodsWe retrospectively examined EGFRvIII expression and EGFRvIII nuclear translocation using immunohistochemistry in specimens of 240 Chinese patients with glioma, including 84 World Health Organization (WHO) II gliomas, 84 WHO III gliomas and 72 glioblastomas (WHO IV). Factors that correlated with EGFRvIII and EGFRvIII nuclear translocation expression were analyzed by the Chi-square test. Kaplan-Meier methodology and Cox regression were used for the survival analysis.ResultsLog-rank tests showed that patient age, Karnofsky performance scale (KPS) score, tumor grade, EGFRvIII expression, EGFRvIII nuclear translocation, 1p/19q codeletion, isocitrate dehydrogenase (IDH) mutation, Ki-67 labeling index and O6-methylguanine-DNA methyltransferase (MGMT) status (P<0.05) were significantly correlated with overall survival (OS) time. Multivariate Cox regression analysis revealed that patient age, tumor grade, EGFRvIII nuclear translocation, 1p/19q codeletion, and IDH mutation (P<0.05) were significantly correlated with OS. Patients with a high level of EGFRvIII nuclear translocation (≥7%) had both significantly shorter OS [hazard ratio (HR): 1.920, 95% confidence interval (95% CI): 1.228−3.003, P=0.004] and progression-free survival (PFS) times (HR: 1.661, 95% CI: 1.116−2.471, P=0.012) than those with a low level of EGFRvIII nuclear translocation (<7%).ConclusionsA high level of EGFRvIII nuclear translocation in glioma is an independent factor indicating a poor prognosis, but EGFRvIII expression is not an independent clinical prognostic factor. The level of EGFRvIII nuclear translocation maybe a novel and crucial prognostic biomarker in glioma.

EGFRvIII Is Expressed in Cellular Areas of Tumor in a Subset of Glioblastoma.

Epidermal growth factor receptor variant III (EGFRvIII) is a tumor-specific cell surface antigen often expressed in glioblastoma and has drawn much attention as a possible therapeutic target. We performed immunohistochemistry on histology sections of surgical specimens taken from 67 cases with glioblastoma, isocitrate dehydrogenase-wild type, and evaluated the morphological characteristics and distribution of the EGFRvIII-positive tumor cells. We then evaluated the localization of EGFRvIII-expression within the tumor and peritumoral areas. EGFRvIII immunopositivity was detected in 15 specimens taken from 13 patients, including two recurrent specimens taken from the same patient at relapse. Immunofluorescence staining demonstrated that EGFRvIII-positive cells were present in cells positive for glial fibrillary acidic protein (GFAP), and some showed astrocytic differentiation with multiple fine processes and others did not shown. The EGFRvIII-positive cells were located in cellular areas of the tumor, but not in the invading zone. In the two recurrent cases, EGFRvIII-positive cells were markedly decreased in one case and retained in the other. With regard to overall survival, univariate analysis indicated that EGFRvIII-expression in patients with glioblastoma was not significantly associated with a favorable outcome. Double-labeling immunofluorescence staining of EGFRvIII and GFAP showed that processes of large, well differentiated, GFAP-positive glia extend to and surround less differentiated, EGFRvIII-positive glial cells in cellular areas of tumor. However, in the tumor periphery, EGFRvIII-positive tumor cells were not observed. This finding suggests that EGFRvIII is involved in tumor proliferation, but that invading glioma cells lose their EGFRvIII expression.

Identification and suppression of epidermal growth factor receptor variant III signaling in fibroblast-like synoviocytes from aggressive rheumatoid arthritis by the mimotope

Epidermal growth factor receptor (EGFR) signaling has been reported to play a vital role in the pathogenesis of rheumatoid arthritis (RA). In current study, we sought to observe whether the active immunization induced by the mimotope could recognize EGFR, inhibit their signaling and disrupt the pathogenic behavior of fibroblast-like synoviocytes (FLS) from RA patients. We prepared a linked EGFR mimotope and performed series of experiments to detect whether the mimotope could induce the desired immune responses. To our surprises, we detected the expression of EGFR variant III (EGFRvIII), but not EGFR in the synovial tissues and FLS from patients with aggressive RA by the linked EGFR mimotope-induced antibodies (LEMIA). Meanwhile, LEMIA could inhibit the signaling caused by the autophosphorylation of EGFRvIII in the FLS. The proliferation, migration, invasion and anti-apoptosis capabilities of the EGFRvIII-expressed FLS were disrupted by LEMIA. These results suggest that EGFRvIII signaling may participate in the malignant behaviors of FLS from aggressive RA. Meanwhile, the linked EGFR mimotope could be used to detect the expression of EGFRvIII and developed to be a potential therapy agent against the aggressive FLS.

Epidermal Growth Factor Receptor Variant III (EGFRvIII) Positivity in EGFR-Amplified Glioblastomas: Prognostic Role and Comparison between Primary and Recurrent Tumors.

Purpose: Approximately 40% of all glioblastomas have amplified the EGFR gene, and about half of these tumors express the EGFRvIII variant. The prognostic role of EGFRvIII in EGFR-amplified glioblastoma patients and changes in EGFRvIII expression in recurrent versus primary glioblastomas remain controversial, but such data are highly relevant for EGFRvIII-targeted therapies.Experimental Design:EGFR-amplified glioblastomas from 106 patients were assessed for EGFRvIII positivity. Changes in EGFR amplification and EGFRvIII status from primary to recurrent glioblastomas were evaluated in 40 patients with EGFR-amplified tumors and 33 patients with EGFR-nonamplified tumors. EGFR single-nucleotide variants (SNV) were assessed in 27 patients. Data were correlated with outcome and validated in 150 glioblastoma patients from The Cancer Genome Atlas (TCGA) consortium.Results: Sixty of 106 EGFR-amplified glioblastomas were EGFRvIII-positive (56.6%). EGFRvIII positivity was not associated with different progression-free or overall survival. EGFRvIII status was unchanged at recurrence in 35 of 40 patients with EGFR-amplified primary tumors (87.5%). Four patients lost and one patient gained EGFRvIII positivity at recurrence. None of 33 EGFR-nonamplified glioblastomas acquired EGFR amplification or EGFRvIII at recurrence. EGFR SNVs were frequent in EGFR-amplified tumors, but were not linked to survival.Conclusions: EGFRvIII and EGFR SNVs are not prognostic in EGFR-amplified glioblastoma patients. EGFR amplification is retained in recurrent glioblastomas. Most EGFRvIII-positive glioblastomas maintain EGFRvIII positivity at recurrence. However, EGFRvIII expression may change in a subset of patients at recurrence, thus repeated biopsy with reassessment of EGFRvIII status is recommended for patients with recurrent glioblastoma to receive EGFRvIII-targeting agents. Clin Cancer Res; 23(22); 6846-55. ©2017 AACR.

Exosomes as a biomarker platform for detecting epidermal growth factor receptor-positive high-grade gliomas.

OBJECTIVE High-grade glial brain tumors are often characterized by an elevated expression of the tumorigenic epidermal growth factor receptor variant III ( EGFRvIII). The authors sought to establish a clinically adaptive protocol as a noninvasive diagnostic tool for EGFRvIII detection through serum exosomes. METHODS Purity of serum exosome/RNA was confirmed by electron microscopy and flow cytometry and through an RNA bioanalyzer profile. EGFRvIII amplification was initially established by semiquantitative polymerase chain reaction in tumor tissues and exosomes. Diagnostic performance of EGFRvIII transcript in tissue versus exosome was determined using a 2 × 2 clinical table approach. Overall survival was determined using Kaplan-Meier analysis. RESULTS The EGFRvIII transcript was detected in 39.5% of tumor tissue samples and in 44.7% of their paired serum exosome samples; 28.1% of biopsy tumors coexpressed wild-type EGFR and EGFRvIII. Tissue EGFRvIII amplification served as the reference-positive control for its paired serum expression. The overall clinical sensitivity and specificity of semiquantitative exosome EGFRvIII polymerase chain reaction detection assay in serum were 81.58% (95% CI 65.67%-92.26%) and 79.31% (95% CI 66.65%-88.83%), respectively. Age, sex, tumor location, and side of the body on which the tumor was located had no effect on the detection rate of exosomal EGFRvIII transcript. EGFRvIII expression either in exosomes or tissue correlated with poor survival. CONCLUSIONS The authors established a serum-based method for detection of EGFRvIII in high-grade brain tumors that might serve as an optimal noninvasive method for diagnosing EGFRvIII-positive high-grade gliomas.

Aptamer-conjugated PEGylated quantum dots targeting epidermal growth factor receptor variant III for fluorescence imaging of glioma.

The extent of resection is a significant prognostic factor in glioma patients. However, the maximum safe resection level is difficult to determine due to the inherent infiltrative character of tumors. Recently, fluorescence-guided surgery has emerged as a new technique that allows safe resection of glioma. In this study, we constructed a new kind of quantum dot (QD)-labeled aptamer (QD-Apt) nanoprobe by conjugating aptamer 32 (A32) to the QDs surface, which can specially bind to the tumors. A32 is a single-stranded DNA capable of binding to the epidermal growth factor receptor variant III (EGFRvIII) specially distributed on the surface of glioma cells. To detect the expression of EGFRvIII in human brain tissues, 120 specimens, including 110 glioma tissues and 10 normal brain tissues, were examined by immunohistochemistry, and the results showed that the rate of positive expression of EGFRvIII in the glioma tissues was 41.82%, and 0.00% in normal brain tissues. Besides, the physiochemical properties of QD-Apt nanoparticles (NPs) were thoroughly characterized. Biocompatibility of the NPs was evaluated, and the results suggested that the QD-Apt was nontoxic in vivo and vitro. Furthermore, the use of the QD-Apt in labeling glioma cell lines and human brain glioma tissues, and target gliomas in situ was also investigated. We found that not only could QD-Apt specially bind to the U87-EGFRvIII glioma cells but also bind to human glioma tissues in vitro. Fluorescence imaging in vivo with orthotopic glioma model mice bearing U87-EGFRvIII showed that QD-Apt could penetrate the blood–brain barrier and then selectively accumulate in the tumors through binding to EGFRvIII, and consequently, generate a strong fluorescence, which contributed to the margins of gliomas that were visualized clearly, and thus, help the surgeons realize the maximum safe resection of glioma. In addition, QD-Apt can also be applied in preoperative diagnosis and postoperative examination of glioma. Therefore, these achievements facilitate the use of tumor-targeted fluorescence imaging in the diagnosis, surgical resection, and postoperative examination of glioma.

Epidermal growth factor receptor variant III in head and neck squamous cell carcinoma is not relevant for targeted therapy and irradiation.

BackgroundThe epidermal growth factor receptor (EGFR) is an important regulator of cell growth and survival, and is highly variable in tumor cells. The most prevalent variation of the EGFR extracellular domain is the EGFR variant III (EGFRvIII). Some studies imply that EGFRvIII may be responsible for the poor response to the monoclonal EGFR-antibody Cetuximab, used therapeutically in head and neck squamous cell carcinoma (HNSCC). Due to inconsistent data in the literature regarding EGFRvIII prevalence and clinical relevance in HNSCC, especially its predictive value, we examined EGFRvIII-transfected cell lines and patient tissue samples.ResultsIn contrast to other recent publications, we were able to demonstrate EGFRvIII expression in HNSCC. However, we noted that the different detection methods yielded inconsistent results. Furthermore, our EGFRvIII transfected and EGFR wild type cell lines exhibited similar characteristics and response rates in the performed in vitro experiments.Materials and MethodsWe conducted various inhibition and combined irradiation experiments using three EGFRvIII-transfected cell lines. Moreover, a patient cohort of 149 cases consisting of formalin fixed and paraffin embedded (FFPE) and fresh-frozen specimens was assayed via reverse transcriptase PCR (rtPCR) with gel electrophoresis and sequencing for EGFRvIII prevalence. In the rtPCR assays, we used five previously published EGFRvIII primers and EGFRvIII-positive glioblastoma tissue as a positive control. In addition, immunohistochemical staining was conducted.ConclusionsEGFRvIII can be detected in HNSCC patient samples. Nevertheless, the low prevalence and similar response rates to targeted drugs and irradiation in vitro cast doubt regarding the clinical relevance of EGFRvIII in HNSCC.

Abstract 41: DCA bind to EGFR/EGFRvIII/PDK1 and affect the proliferation and growth in TMZ resistant glioblastoma model

Abstract Glioblastomas are characterized by amplification of epidermal growth factor receptor (EGFR). Approximately half of the GBM tumors with EGFR over-expression also express a constitutively active ligand independent EGFR variant III (EGFRvIII). This phenotype represents a tumor specific target correlating with a high growth potential. Despite advances in treatment, whether surgery, chemotherapeutics or molecular targeted therapies, very few have shown promise. Temozolomide (TMZ), as standard of care alkylation therapy, demonstrates improved survival when administered with concomitant radiotherapy. Unfortunately, acquired TMZ chemo-resistance is observed in more than 90% of recurrent GBMs. To understand the mechanisms in the context of TMZ resistance, we generated an in vitro TMZ resistant model by continuous exposure of U373 cells constitutively expressing EGFRvIII (U373vIII) to 150uM TMZ for 6 months (U373vIIIR). Dicholoroacetate (DCA) is a known metabolic inhibitor of pyruvate dehydrogenase kinase 1 (PDK1). Its application to cancer has recently been revived in the realm of metabolic oncology, where reversal of the Warburg effect has resulted in decreased tumor growth. Treatment of U373, U373vIII, and U373vIIIR GBM cell lines with DCA was found to induce cytotoxicity and reduced cell survival across all cell lines. Further, micro array studies conducted on U373vIII or U373vIIIR and their subsequent treatment with DCA revealed that PDK1 is the sole target in TMZ resistant tumors expressing EGFRvIII. Additionally, we demonstrated that 1mM DCA induced mitochondrial membrane potential change as evidenced in JC-1 staining and electron microscopic studies confirming mitochondrial apoptosis. Consistent with our previous findings that EGFR interacts with PDK1, our computational analysis revealed that DCA aligns itself to the binding sites of both EGFR and EGFRvIII with strong binding affinity apart from its binding to PDK1. Clinically, expression of EGFRvIII correlated with PDK1 when compared to EGFR in GBM surgical specimens. Collectively, our studies demonstrate that principles of metabolic oncology and DCA are active in GBM treatment despite resistance providing new insight into the development of alternative treatment in TMZ failure. Citation Format: Kiran Kumar Velpula, Kamlesh Sahu, Jack Tuszynski, Maheedhara R. Guda, Swapna Asuthkar, Sarah E. Martin, Justin D. Lathia, Andrew J. Tsung. DCA bind to EGFR/EGFRvIII/PDK1 and affect the proliferation and growth in TMZ resistant glioblastoma model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 41.