Epidermal Growth Factor Receptor Fusion Research Articles

Correlation between ALK, ROS1 Biomarkers and EGFR Oncogene Mutations in Lung Tumours: Our Observations in an Apex Oncopathology Laboratory

Introduction: Analysis of Anaplastic lymphoma receptor tyrosine kinase gene (ALK), Repressor of Silencing 1 (ROS1) gene are determined by immunohistochemistry (IHC) and it is an easily applicable, cost-effective assay for potential treatment with crizotinib. Mutations of Epidermal growth factor receptor (EGFR) genes are evaluated by IHC/Multiplex RT-PCR. The purpose of this study is to assess the frequencies of ALK, ROS1 and their association with EGFR fusion gene mutations in a spectrum of lung tumours. Materials and methods: A total of 202 cases of lung tumours reported at our Center for Oncopathology from September 1st 2020 to 31st August 2021, were retrospectively analyzed for ALK, ROS1 and EGFR fusion genes based on Immunohistochemistry (IHC) and Multiplex PCR findings. ALK was tested using D5F3 clone, and ROS1 was analyzed using Cell Signalling’s D4D6 clone on the Ventana immunohistochemistry platform. EGFR status was analyzed using EGFR mutation test V2 real-time multiplex PCR assay on Roche Cobas Z480. Results: 202 biopsy samples and cellblocks of fluid aspirates were analyzed. 175/202 were histologically and immunologically proved as Non-small cell lung carcinoma (Primary pulmonary adenocarcinoma) and its metastases. 09/199 (5.23%) were Positive for ALK IHC and 03/199 (1.74%) cases had equivocal results. 06/179 (3.85%) cases were Positive for ROS1 IHC and 03/179 (1.92%) cases had equivocal results. Other histo-morphological diagnoses i.e., adenosquamous, squamous, small cell, mucinous carcinoma etc (27 cases) were all ALK and ROS1 Negative. 188/202 tumours were analyzed for EGFR mutation status, which showed 70/188 (37.23%) had specific EGFR mutations. 118/188 (62.76%) cases were EGFR wildtype. Conclusion: We observed that age related incidence of EGFR mutations was highest in elderly females, of 61 to 70 years. ALK gene mutations occurred in 6.03% and ROS1 gene mutations occurred in 5.02% of lung tumours and their metastases. EGFR-mutations were associated with ROS1 mutated lung adenocarcinomas. There are no coexistent ALK-EGFR or ALK-ROS1 mutations. All ALK IHC positive pulmonary adenocarcinomas are ROS1 negative and are mutually exclusive.

Rapid separation and binding configuration prediction of the components in Danshen decoction to endothelin A receptor using affinity chromatography and molecular dynamics simulation.

As a famous traditional Chinese formula, Danshen Decoction has the potential to relieve the pain of pulmonary arterial hypertension patients, however, the functional components remain unknown. Herein, we reported a method to screen the functional components in Danshen Decoction targeting endothelin receptor A, an accepted target for the treatment of the disease. The receptor was functionalized on the macroporous silica gel through an epidermal growth factor receptor fusion tag and its covalent inhibitor. Using the affinity gel as the stationary phase, the bioactive compound was identified as salvianolic acid B by mass spectrometry. The binding kinetic parameter (dissociation rate constants kd ) of salvianolic acid B with the receptor was determined via peak profiling. Using the specific ligands of the receptor as probes, the binding configuration prediction of salvianolic acid B with the receptor was performed by molecular dynamics simulation. Our results indicated that salvianolic acid B is a potential bioactive compound in Danshen Decoction targeting the receptor. This work showed that receptor chromatography in combination with molecular dynamics simulation is applicable to predicting the binding kinetics and configuration of a ligand to a receptor, providing crucial insight for the rational design of drugs that recognize functional proteins.

The landscape of EGFR mutation in Chinese patients with glioma.

e14028 Background: Epidermal growth factor receptor (EGFR), a trans-membrane receptor tyrosine kinase, consists of an extracellular ligand-binding domain (ECD), a single transmembrane domain (STMD) and an intracellular kinase domain (ICD). Glioma is the most common primary brain tumor, and variations of EGFR are frequently reported in glioma patients. This study aimed to analysis the landscape of EGFR mutation subtype in Chinese patients with glioma. Methods: We retrospectively collected 1779 patients with glioma. In all patients enrolled, the comprehensive genomic profiling containing EGFR gene was detected using tumor tissue, including WES, 539 tumor related-gene panel or 131+4 gene panel (Simceredx). And the somatic mutations including SNV, INDEL, CNV, fusion were analyzed correspondingly. Results: Among 1779 patients with glioma, EGFR mutations were identified in 392 (22.03%) patients. The age was significantly higher in patients with EGFR-mutant group than in patients without EGFR-mutant group, with median age 54 vs. 45 (p = 4.81e-19). The sex didn’t reach statistical significance in two group. The incidence rate of different EGFR mutation types among all patients with EGFR-mutant were amplification (314,80.10%), SNV(198,50.51%), Indel(24,6.12%) and fusion(44,11.22%) respectively. 162 (46.69%) had two or more mutation types simultaneously. The main partner gene of EGFR fusion was LOC100996654, followed by ELDR and SEC61G, and the main EGFR breakpoints were in intron24, intron7, exon7. Within 347 SNV and Indel mutations, exon7, 15, 20, 6 and 8 were detected in 103(29.68%), 50(14.41%), 28 (8.07%), 25(7.20%) and 18 (5.19%) respectively. The top three mutaton subtypes within exon7 were account for 19.31%, which included p.A289V,p.A289T,p.T263P. Meanwhile, the mutation frequency within ICD encoded by exons 18-24 was 17.00%, in which exon20 mostly distributed (8%). In exon20, the most common mutation subtypes were p.V774M,p.H773dup,p.T790M, while in the second proportion exon18 (3.46%), the main subtype were p.G719A/D. Conclusions: EGFR amplification was the most common mutation type. There were 122 SNV and Indel subtypes altogether and occurred throughout almost the entire exon region, in which exon7 contained maximum 21 different subtypes and the p.A289V was the main type. The distribution of subtypes in ECD were much more than in ICD. The relationship between subtypes occurrence and cancer will be further studied in the future.

113P The analysis of EGFR fusions characteristics in Chinese solid tumor patients

Oncogenic mutations in the epidermal growth factor receptor (EGFR) serve as important predictive biomarkers in non-small cell lung cancer (NSCLC). The constitutive activity and sensitivity to EGFR tyrosine kinase inhibitors (TKIs) in lung cancer with small in-frame deletions in exon 19, mutations in exon 21 in the EGFR tyrosine kinase domain had been conferred. Atypical EGFR aberrations, including EGFR kinase domain duplications (KDD) and EGFR fusions (EGFR-RAD51, EGFR-SEPT14, KIF5B-EGFR) had been identified.

Clinical Characteristics, Treatments, and Concurrent Mutations in Non–Small Cell Lung Cancer Patients With NF1 Mutations

ObjectivesMetastatic non–small cell lung cancer (mNSCLC) is characterized by complex genomic alterations. NF1 mutations may confer distinct clinical characteristics within NSCLC, and real-world evidence on concurrent mutations, treatment patterns, and health outcomes is lacking. Materials and MethodsThis retrospective study was performed in patients with mNSCLC treated in the Flatiron Health network who underwent the FoundationOne tumor-sequencing. Anticancer therapies, concurrent mutations, real-world progression-free survival (rwPFS), and overall survival (OS) were assessed. ResultsOf the 1663 patients, 103 patients were identified with NF1 mutation. Concurrent mutations with Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (16.5%) and epidermal growth factor receptor fusion (6.8%) were the most frequent. In patients with NF1 mutation only (n = 57), 42% were women, 86% patients had smoking history, and 70% had non-squamous cell carcinoma type. Most (51%) of the patients with NF1 mutations received a single line of therapy versus other mutations and the overall treated population (44%). Platinum-based chemotherapy was the predominant first-line therapy, with programmed cell death-1/programmed cell death-ligand-1 inhibitors as subsequent lines of therapy. The NF1 mutation only group had numerically the shortest median rwPFS (82 days) than other mutation groups. Median OS for the NF1 mutation group in first, second, and third lines of therapy was 321, 498, and 210 days, respectively. ConclusionsNF1 mutations confer distinct clinical characteristics in patients with mNSCLC. These patients may have different trajectories for progression and survival than seen for other mutations, experience less systemic therapy after first-line therapy, and may have shorter survival.

1360P Identification of epidermal growth factor receptor (EGFR) fusions in a large Chinese lung cancer population

Next generation sequencing has identified some rare EGFR fusions in lung cancer, such as EGFR-RAD51 and EGFR-SEPT14, which are known to respond to EGFR-TKIs. However, prevalence of EGFR fusions in a large population is unknown yet. Herein, we analyzed a large-scale dataset comprehensively to explore the mutation characteristics of EGFR fusions. This study offered an estimate of the proportion of patients with EGFR fusion who could benefit from therapies.

Abstract 1055: Evaluation of immune checkpoint inhibitor efficacy in EGFR mutant tumors

Abstract Tyrosine Kinase Inhibitor (TKI) of Epidermal Growth Factor Receptor (EGFR) is a first-line therapy for non-small cell carcinoma lung cancer patients with EGFR mutations, but patients often develop resistance to such a target therapy. Recently several PD-1 and PD-L1 inhibitors were approved for treating NSCLC, however, it remains unclear whether EGFR mutant NSCLC would benefit from PD-1/PD-L1 treatment. PD-1 response in cancer patients is closely associated with oncogenic mutation status, tumor microenvironment, PD-L1 expression and tumor mutation burden (TMB); however, the lack of pre-clinical models limited the investigation of the checkpoint inhibitors in EGFR mutant tumors. Here we use human PBMC reconstitution system for immune-humanization, and established a series of IO-Xenograft models (IO-CDX and IO-PDX) with cancer cell lines (IO-CDX) or patient-derived tissues (IO-PDX) that contain various EGFR mutations, including EGFR 19 del, L858R, T790M, and EGFR over-expression, amplification and fusion. We treated these models with either EGFR TKI (erlotinib, afatinib or osimertinib) or nivolumab, a PD-1 inhibitor. The predictive biomarkers including PD-L1 expression and TMB were analyzed by whole exom sequencing (WES) and FACS for tumor infiltrating lymphocytes (TILs), respectively. We found that 3 TKI-resistant PDX models bearing EGFR T790M mutation exhibited lower TILs than those bearing EGFR wild type or exon 19del mutation. Of the 8 IO-CDX or IO-PDX humanized models we have examined, those exhibiting higher TILs achieved better responses to nivolumab treatment. The 3 most sensitive models are EGFR amplification (wt), EGFR over-expression (wt) and EGFR exon 19del. In contrast, the EGFR T790M model from H1975 cell line was observed with hyper-progressive disease after nivolumab treatment. We will also discuss the contribution of PD-L1 expression and TMB to nivolumab in other models. In summary, these well-established IO-CDX and IO-PDX models can provide a human-resembling immune system for of immune therapeutics and combination with targeted therapies, and help to facilitate the understanding of relationship among tumor microenvironment, driver oncogenic mutations and drug response. Citation Format: Xuzhen Tang, Li Yang, Hui Qi, Xianzhi Zhai, Fuyang Wang, Xiangnan Qiang, Jie Xu, Xiaoran Qin, Qingyang Gu, Shaoyu Yan, Qunsheng Ji. Evaluation of immune checkpoint inhibitor efficacy in EGFR mutant tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1055.

Effects of epidermal growth factor receptor fusion protein on the cytotoxic activity of SOCS1-silenced dendritic cells in vitro.

The aim of the present study was to observe the effects of cytokine signaling suppressor1(SOCS1)-silenced dendritic cells (DCs) pulsed with epidermal growth factor receptor (EGFR) fusion protein on the activation of Tlymphocyte and cytotoxic T-lymphocyte (CTL) activity against Hep-2 cells. DCs were derived from the medullary cells of mice and authenticated by flow cytometry (FCM). Recombinant glutathione-S-transferase (GST)-EGFR fusion protein was produced and purified. After being pulsed with it, DCs were modified by recombinant SOCS1-siRNA adenoviral to silence SOCS1 gene expression. The maturation of DCs was evaluated by FCM. The effects of modified DCs on T-cell proliferation were assessed by MTT assay. The killing effects against Hep-2 cells of CTL were assessed by lactate dehydrogenase (LDH) release assay. High-purity DCs from the medullary cells of mice were obtained. Compared with the control, EGFR-pulsed DCs displayed higher expression of cell surface molecules, including CD83, CD860 and HLA-DR. The MTT assay revealed that all of the EGFR-pulsed, SOCS1‑silenced and EGFR-pulsed plus SOCS1-silenced DCs had an enhanced capacity to stimulate T-lymphocyte proliferation. As expected, EGFR-pulsed plus SOCS1-silenced DCs had the strongest effects on T-cell proliferation. The splenic Tcells isolated from both EGFR-pulsed DC-immunized mice and EGFR-pulsed plus SOCS1-silenced DC-immunized mice enhanced the cytotoxicity against Hep-2 cells, while Tcells from EGFR‑pulsed plus SOCS1-silenced DC-immunized mice exhibited significantly higher cytotoxicity than those from EGFR-DC-immunized mice. The EGFR-pulsed SOCS1‑siRNA-silenced DCs had the strongest effects on activation of T-cell proliferation and the CTL activity against Hep-2 cells.

Frequency of EGFR T790M mutation and multimutational profiles of rebiopsy samples from non-small cell lung cancer developing acquired resistance to EGFR tyrosine kinase inhibitors in Japanese patients.

BackgroundThe majority of non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutation eventually develop resistance to EGFR tyrosine kinase inhibitors (TKIs). Minimal information exists regarding genetic alterations in rebiopsy samples from Asian NSCLC patients who develop acquired resistance to EGFR-TKIs.MethodsWe retrospectively reviewed the medical records of patients with NSCLC harboring EGFR mutations who had undergone rebiopsies after developing acquired resistance to EGFR-TKIs. We analyzed 27 practicable samples using a tumor genotyping panel to assess 23 hot-spot sites of genetic alterations in nine genes (EGFR, KRAS, BRAF, PIK3CA, NRAS, MEK1, AKT1, PTEN, and HER2), gene copy number of EGFR, MET, PIK3CA, FGFR1, and FGFR2, and ALK, ROS1, and RET fusions. Additionally, 34 samples were analyzed by commercially available EGFR mutation tests.ResultsSixty-one patients underwent rebiopsy. Twenty-seven samples were analyzed using our tumor genotyping panel, and 34 samples were analyzed for EGFR mutations only by commercial clinical laboratories. Twenty-one patients (34 %) had EGFR T790M mutation. Using our tumor genotyping panel, MET gene copy number gain was observed in two of 27 (7 %) samples. Twenty patients received continuous treatment with EGFR-TKIs even after disease progression, and 11 of these patients had T790M mutation in rebiopsy samples. In contrast, only 10 of 41 patients who finished EGFR-TKI treatment at disease progression had T790M mutation. The frequency of T790M mutation in patients who received continuous treatment with EGFR-TKIs after disease progression was significantly higher than that in patients who finished EGFR-TKI treatment at disease progression (55 % versus 24 %, p = 0.018).ConclusionsThe frequency of T790M mutation in this study was lower than that in previous reports examining western patients. These results suggest that continuous treatment with EGFR-TKI after disease progression may enhance the frequency of EGFR T790M mutation in rebiopsy samples.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2902-0) contains supplementary material, which is available to authorized users.

Lung Cancers with Concomitant EGFR Mutations and ALK Rearrangements: Diverse Responses to EGFR-TKI and Crizotinib in Relation to Diverse Receptors Phosphorylation

We investigated the incidence of concomitant epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) rearrangements in Chinese patients with non-small cell lung cancer (NSCLC), and assessed responses to EGFR tyrosine kinase inhibitors (EGFR-TKIs) and crizotinib in such tumors. We screened 977 consecutive patients with NSCLC for the presence of concomitant EGFR mutations and ALK rearrangements by rapid amplification of cDNA ends-coupled PCR sequencing and FISH. Immunohistochemistry (IHC) and Western blotting were used to correlate the activation of EGFR, ALK, and downstream proteins with responses to EGFR-TKIs and crizotinib. The overall frequency of concomitant EGFR mutations and ALK rearrangements was 1.3% (13/977). EGFR/ALK co-alterations were found in 3.9% (13/336) EGFR-mutant and 18.6% (13/70) ALK-rearranged patients. Ten tumors were treated with first-line EGFR-TKIs, with a response rate of 80% (8/10). Two tumors with high phospho-ALK levels and low phospho-EGFR levels achieved stable and progressive disease, respectively. Median progression-free survival was 11.2 months. Coexpression of mutant EGFR and ALK fusion proteins in the same tumor cell populations was detected by IHC. Two cases with high phospho-ALK levels treated with crizotinib achieved partial responses; two cases with low phospho-ALK levels had progressive or stable disease. ALK rearrangements and EGFR mutations could coexist in a small subgroup of NSCLC. Advanced pulmonary adenocarcinomas with such co-alterations could have diverse responses to EGFR-TKIs and crizotinib. Relative phospho-ALK and phospho-EGFR levels could predict the efficacy of EGFR-TKI and crizotinib.

Dendritic cells pulsed with GST–EGFR fusion protein: Effect in antitumor immunity against head and neck squamous cell carcinoma

Overexpression of epidermal growth factor receptor (EGFR) is common in head and neck squamous cell carcinoma (HNSCC). Targeting EGFR is an effective approach to treat EGFR-positive HNSCC. However, the clinical benefits of the present EGFR-targeting agents are still limited in HNSCC patients. Recombinant glutathione-S-transferase (GST)-EGFR fusion protein was produced and purified. Dendritic cells (DCs) of C3H mice were pulsed with fusion protein. Mice were challenged with HNSCC cells before or after vaccination with these DCs, and the cytotoxic T-lymphocyte (CTL) response, interferon-gamma (IFN-gamma) secretion, tumor growth, and survival of mice were assessed. Significant in vitro and in vivo antitumor activities were observed for mice immunized with DCs pulsed with GST-EGFR fusion protein, compared with the control groups (p < .05). The DCs pulsed with GST-EGFR fusion protein can provide not only preventive but also therapeutic antitumor activities against HNSCC in the animal model.

(-)-Epigallocatechin gallate causes internalization of the epidermal growth factor receptor in human colon cancer cells

We recently found that the inhibitory effect of (-)-epigallocatechin gallate (EGCG) on epidermal growth factor (EGF) binding to the epidermal growth factor receptor (EGFR) is associated with alterations in lipid organization in the plasma membrane of colon cancer cells. Since changes in lipid organizations are thought to play a role in the trafficking of several membrane proteins, in this study we examined the effects of EGCG on cellular localization of the EGFR in SW480 cells. Treatment of the cells for 30 min with as little as 1 microg/ml of EGCG caused a decrease in cell surface-associated EGFRs and this was associated with internalization of EGFRs into endosomal vesicles. Similar effects were seen with a green fluorescent protein (GFP)-EGFR fusion protein. As expected, the EGFR protein was phosphorylated at tyrosine residues, ubiquitinated and partially degraded when the cells were treated with EGF, but treatment with EGCG caused none of these effects. The loss of EGFRs from the cell surface induced by treating the cells with EGF for 30 min persisted for at least 2 h. However, the loss of EGFRs from the cell surface induced by temporary exposure to EGCG was partially restored within 1-2 h. These studies provide the first evidence that EGCG can induce internalization of EGFRs into endosomes, which can recycle back to the cell surface. This sequestrating of inactivated EGFRs into endosomes may explain, at least in part, the ability of EGCG to inhibit activation of the EGFR and thereby exert anticancer effects.