Epidermal Growth Factor Receptor Gene Amplification Research Articles

Value of peripheral blood rare cell EGFR gene amplification detection in the evaluation of benign and malignant pulmonary nodules

Objective: To explore the value of detection of epidermal growth factor receptor (EGFR) gene amplification in peripheral blood rare cells in the assessment of benign and malignant pulmonary nodules. Methods: A total of 262 patients with pulmonary nodules were selected as the retrospectively study subjects from the Second Affiliated Hospital of Army Military Medical University and Peking Union Medical College Hospital from July 2022 to August 2023. There were 98 males and 164 females, with the age range from 16 to 79 (52.1±12.1) years. The EGFR gene amplification testing was performed on the rare cells enriched from patients' peripheral blood, and the clinical manifestations, CT imaging features, histopathological and/or pathological cytological confirmed results of patients were collected. The receiver operating characteristic (ROC) curve was used to determine the optimal cut-off value of the method of detection of EGFR gene amplification in peripheral blood rare cells, and its diagnostic efficacy was evaluated. Results: Among the 262 patients, 143 were malignant pulmonary nodules and 119 were benign pulmonary nodules. The differences between malignant pulmonary nodules and benign pulmonary nodules in nodule diameter and nodule density were statistically significant (both P<0.001), while the differences in age, gender and nodule number were not statistically significant (all P>0.05). The number [M (Q1, Q3)] of EGFR gene amplification positive rare cells in patients with malignant pulmonary nodule was 8 (6, 11), which was higher than that in patients with benign pulmonary nodule [2 (1, 4), P<0.001]. The ROC curve results showed that when the optimal cut-off value was 5 (that was, the number of EGFR gene amplification positive rare cells was>5), the area under the curve (AUC) of the detection of EGFR gene amplification in peripheral blood rare cells for discrimination of benign and malignant pulmonary lesions was 0.816 (95%CI: 0.761-0.870), with a sensitivity of 83.2%, a specificity of 80.7%, and an accuracy of 82.1%. Based on the analysis of the diameter of the nodules, the AUC for distinguishing between benign and malignant pulmonary nodules with diameter 5-9 mm and 10-30 mm was 0.797 (95%CI: 0.707-0.887) and 0.809 (95%CI: 0.669-0.949), respectively, with sensitivity, specificity and accuracy reached 75% or above. Based on the analysis of nodule density, the AUC for distinguishing between benign and malignant solid nodule and subsolid nodule was 0.845 (95%CI: 0.751-0.939) and 0.790 (95%CI: 0.701-0.880), respectively, with sensitivity, specificity and accuracy reached 75% or above. Based on the analysis of nodule number, the AUC for distinguishing between benign and malignant solitary pulmonary nodule and multiple pulmonary nodule was 0.830 (95%CI: 0.696-0.965) and 0.817 (95%CI: 0.758-0.877), respectively, with sensitivity, specificity and accuracy reached 80% or above. Conclusion: The detection of EGFR gene amplification in peripheral blood rare cells contributes to the evaluation of benign and malignant pulmonary nodules, and can be used in the auxiliary diagnosis of benign and malignant pulmonary nodules.

Abstract A040: Characterization of a humanized monoclonal antibody targeting cancer-expressed EGFR

Abstract The epidermal growth factor receptor (EGFR), an established oncology target, is a transmembrane protein highly expressed on the surface of malignant tumors such as glioblastoma, lung, liver, bladder and breast cancers. In addition to overexpression, EGFR mutants are frequently associated with malignancy. For example, EGFR variant III (EGFRvIII), deletion of exons 2-7, is only expressed on cancer cells and is associated with poor prognosis and chemotherapy resistance. To target cancer-expressed EGFR, including EGFRvIII, we generated the 40H3 monoclonal antibody which is reactive with the 287-302 loop that is fully exposed on the extracellular domain of EGFRvIII. The 40H3 antibody also showed strong reactivity for cells expressing gene-amplified EGFR but little or no reactivity for cells expressing normal levels of wild type EGFR. When conjugated with toxic payloads (either tesirine or deruxtecan), the resulting antibody-drug conjugates (ADCs) exhibited antitumor activity in several xenograft models. To improve its utility as a clinical candidate, we humanized the variable portions of the heavy and light chains of 40H3, without changing the original CDRs. The variable regions of 40H3, heavy and light chains, were compared to the closest human immunoglobulin families and then framework residues were altered to produce candidate humanized antibodies. The humanized variable chains were then fused with a human IgG1 heavy chain and a kappa light chain to generate full-sized humanized antibodies. A total of 9 humanized antibodies were generated by pairing 3 variable humanized heavy chains (VH1, VH2, VH3) with 3 variable humanized light chains (VL1, VL2, VL3). To evaluate the binding activity of humanized antibodies, we used either immobilized EGFRvIII (by ELISA or Octet) or various cancer cells expressing either EGFR or EGFRvIII (by flow cytometry). All humanized antibodies retained binding activity to immobilized EGFRvIII but bound with different apparent affinities to cells overexpressing EGFR or cells transfected with EGFRvIII. The ‘A10’ antibody was the best candidate. Mutations introduced in A10 preserved the binding affinity of the 40H3 antibody but exhibited an increased affinity compared to the corresponding chimeric antibody constructed with the original mouse VH and VL. The other eight antibodies retained EGFRvIII binding activity, albeit with a lower cell binding affinity. Specifically, A10 showed improved binding to cells with EGFR gene amplification including two TNBC lines, MDA-MB-468 and BT-20, epithelial cancer cells, A431, head and neck squamous carcinoma, UMSCC17B, as well to cells expressing EGFRvIII, F98npEGFRvIII. No binding of A10 was detected to cell lines expressing wild type EGFR, WI-38 or U87MG. In summary, humanized candidates of the 40H3 monoclonal antibody retained EGFR binding. Of 9 antibodies, A10 exhibited the best cell binding activity elevating it to our top clinical candidate for the production of ADCs or similar antibody-based therapeutics. Citation Format: Tamara G Fernandes Costa, Robert Sarnovsky, Maria Teresa Bilotta, Antonella Antignani, David FitzGerald. Characterization of a humanized monoclonal antibody targeting cancer-expressed EGFR [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A040.

Detecting EGFR gene amplification using a fluorescence in situ hybridization platform based on digital microfluidics

Signal transduction mediated by epidermal growth factor receptor (EGFR) gene affects the proliferation, invasion, metastasis, and angiogenesis of tumor cells. In particular, non-small cell lung cancer (NSCLC) patients with increased in copy number of EGFR gene are often sensitive to tyrosine kinase inhibitors. Despite being the standard for detecting EGFR amplification in the clinic, fluorescence in situ hybridization (FISH) traditionally involves repetitive and complex benchtop procedures that are not only time consuming but also require well-trained personnel. To address these limitations, we develop a digital microfluidics-based FISH platform (DMF-FISH) that automatically implements FISH operations. This system mainly consists of a DMF chip for reagent operation, a heating array for temperature control and a signal processing system. With the capability of automatic droplet handling and efficient temperature control, DMF-FISH performs cell digestion, gradient elution, hybridization and DAPI staining without manual intervention. In addition to operational feasibility, DMF-FISH yields comparable performance with the benchtop FISH protocol but reducing the consumption of DNA probe by 87 % when tested with cell lines and clinical samples. These results highlight unique advantages of the fully automated DMF-FISH system and thus suggest its great potential for clinical diagnosis and personalized therapy of NSCLC.

P06.10.A CAR-ENGINEERED PRIMARY NK CELLS SHOW PROMISING EFFECTS AS TARGETED THERAPY AGAINST EGFR-POSITIVE GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma (GB) is the most frequent primary brain tumor and despite intensive treatment regimens including surgery, radiotherapy, and chemotherapy, it is still characterized by a dismal prognosis. Immunotherapies such as T cells or natural killer cells (NK) that carry tumor-specific chimeric antigen receptors (CAR) hold promise for the development of more effective treatment strategies. CAR-engineered derivatives of the human NK cell line NK-92 targeting EGFR or EGFRvIII showed potent antitumor activity against glioblastoma cell lines in vitro and in vivo. Nevertheless, NK-92 has some limitations compared to primary NK cells, such as the required irradiation prior to application in humans. MATERIAL AND METHODS To overcome this limitation, we evaluated the suitability of peripheral blood derived human primary NK cells (pNK) as carriers for a CAR targeting the common GB antigen epidermal growth factor receptor (EGFR) and the tumor-specific EGFR variant EGFRvIII. pNK cells from healthy donors were transduced with a lentiviral vector encoding the cetuximab-derived 225.28.z CAR (CAR-pNK) which recognizes an epitope common to both receptors. Specific tumor cell lysis by the CAR-pNK cells was then investigated in FACS-based cytotoxicity assays with GB cells expressing EGFR, EGFRvIII, or both target antigens. In addition, we tested in vivo feasibility of the CAR-pNK cells by single time intravenous injection in a subcutanous LNT-229/EGFRvIII glioblastoma xenograft model. RESULTS In comparison to non-transduced pNK cells, CAR-pNK cells displayed enhanced lysis of LNT-229 GB cells expressing EGFR and EGFRvIII. Likewise, markedly increased cytotoxicity of CAR-pNK was observed against GB cell lines such as SKMG-3 and BS153 with intrinsic EGFR gene amplification or expression of EGFRvIII, respectively. Furthermore, in a subcutaneous LNT-229/EGFRvIII glioblastoma xenograft model we observed delayed tumor growth after a single intravenous injection of CAR-pNK cells. CONCLUSION In summary, these results demonstrate promising efficacy of primary CAR-NK cells targeting EGFR and EGFRvIII both in vitro and in vivo. In ongoing work we are evaluating CAR-pNK cells in an orthotopic LNT-229 glioblastoma xenograft mouse model, which may provide the basis to translate this approach to early clinical testing.

Abstract CT156: MCLA-158 (petosemtamab), an IgG1 bispecific antibody targeting EGFR and LGR5, in advanced gastric/esophageal adenocarcinoma (GEA)

Abstract The epidermal growth factor receptor (EGFR) is a known driver of cancer growth and the leucine-rich, repeat-containing, G-protein coupled receptor 5 (LGR5) is a transmembrane receptor expressed on cancer stem cells. Petosemtamab is a human, common light chain, IgG1 bispecific antibody with enhanced ADCC activity targeting EGFR and LGR5, which has shown potent antitumor activity in patient (pt)-derived xenograft models of gastric and esophageal cancer. The dose escalation part of an ongoing phase 1/2 study is completed (JCO 2021:39.3 Sup 62). In the expansion part, petosemtamab is being investigated at the RP2D (1500 mg Q2W, 4-week cycle) in pts with selected advanced solid tumors, including GEA. Primary objective (expansion): investigator-assessed ORR per RECIST 1.1. Secondary objectives: ORR, DOR, PFS (per investigator and central review), OS, and safety and tolerability. Key eligibility criteria: advanced/metastatic GEA, prior exposure to ≥2 lines of standard therapy, ECOG PS 0-1, measurable disease (RECIST 1.1), available baseline tumor biopsy. At the data cutoff date of 24 October 2022, 14 GEA pts were treated at the RP2D. Median age was 63 years (range 40-80), ECOG PS 0/1: 3/11 pts, and 79% of pts were male. Primary tumor locations were esophagus (57%), stomach (36%), and gastroesophageal junction (7%), and all were adenocarcinoma histology. Pts received a median of 3 lines of prior systemic therapy (range 1-4) including platinum-based chemotherapy (100%) and checkpoint inhibitors (14%). 3/14 pts had EGFR overexpression (H score ≥200). A median of 2 treatment cycles (range 1-24) was administered, with 1 pt continuing therapy at the cutoff. Among the 14 pts evaluable for efficacy (≥2 cycles and ≥1 postbaseline scan, or early progression) limited activity was seen, however 1 pt with tumor EGFR protein overexpression and gene copy number amplification (CNA) showed a confirmed sustained PR (67% tumor reduction; response ongoing after 24 cycles) and 3 pts had SD (1 with EGFR overexpression and gene CNA; 2 not evaluable for IHC), with tumor reductions of 2%, 17%, and 40%. Among 78 pts treated at the RP2D (escalation and all expansion cohorts; cutoff 28 November 2022), the most frequent AEs regardless of causality (all grades/G3-4) were rash (33%/0%), hypotension (26%/6%), dyspnea (26%/4%), nausea (26%/1%), dermatitis acneiform (24%/1%), blood magnesium decreased (19%/5%), erythema (19%/0%), diarrhea (19%/0%); IRRs (composite term) were reported in 74%/21% of pts, mostly at the first infusion, and all resolved. 5 pts (6%) discontinued treatment due to IRRs on Day 1. 1 esophageal cancer pt died due to unrelated G5 GI bleeding. Petosemtamab demonstrated promising clinical efficacy among patients with pretreated GEA having EGFR gene amplification and/or overexpression, with a manageable safety profile. Citation Format: Marc Díez García, Antoine Hollebecque, Rocio Garcia-Carbonero, Christiane Jungels, Elisabeth Smyth, Shumei Kato, Guillem Argilés, Carlos Gomez Martin, Marina Magin, Yu-Ming Shen, Renée de Leeuw, Mohamed Bekradda, Eduardo Pennella, Ernesto Wasserman, Viktoriya Stalbovskaya, Jeroen Lammerts van Bueren, Aline Engbers, Andrew Joe, Josep Tabernero. MCLA-158 (petosemtamab), an IgG1 bispecific antibody targeting EGFR and LGR5, in advanced gastric/esophageal adenocarcinoma (GEA) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT156.

Genetic alterations shaping tumor response to anti-EGFR therapies.

The Epidermal Growth Factor Receptor (EGFR) has been targeted through the development of selective tyrosine kinase inhibitors (TKIs) and monoclonal antibodies (mAb). These molecules have shown effectiveness in a subset of patients with specific genetic alterations (i.e. gain-of-function EGFR mutations or EGFR gene amplification) and have been approved for their use in non-small-cell lung cancer (NSCLC), colorectal cancer (CRC), pancreatic cancer and head and neck cancer. In addition, extensive research is being performed in many other tumour types hoping for a future approval. However, the majority of the patients show no benefit from these molecules due to primary mechanisms of resistance, already present before treatment or show disease progression upon the acquisition of drug resistance mechanisms during the treatment. At present, the majority of patients display resistance due to alterations in genes related to the EGFR signalling pathway that eventually circumvent EGFR inhibition and allow cancer progression. Thus, in this review article we focus on the molecular mechanisms underlying drug resistance via genetic alterations leading to resistance to all anti-EGFR drugs approved by the FDA and/or EMA. We also discuss novel approaches to surmount these chemoresistance modalities.

Audit of Molecular Mechanisms of Primary and Secondary Resistance to Various Generations of Tyrosine Kinase Inhibitors in Known Epidermal Growth Factor Receptor-Mutant Non-small Cell Lung Cancer Patients in a Tertiary Centre

AimsPresently, three generations of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are approved against oncogene addicted EGFR-mutant non-small cell lung cancer (NSCLC). Patients with actionable EGFR mutations invariably develop resistance. This resistance can be intrinsic (primary) or acquired (secondary). Materials and methodsThis was a retrospective study carried out between January 2016 and April 2021 analysing 486 samples of NSCLC for primary and secondary resistance to first- (erlotinib, gefitinb), second- (afatinib) and/or third-generation (osimertinib) TKIs in EGFR-mutant NSCLCs by next generation sequencing (NGS). Tissue NGS was carried out using the Thermofischer Ion Torrent™ Oncomine™ Focus 52 gene assay; liquid biopsy NGS was carried out using the Oncomine Lung Cell-Free Total Nucleic Acid assay. All cases were previously tested for a single EGFR gene with the Therascreen® EGFR RGQ PCR kit. ResultsThe results were divided into four groups: (i) group 1: primary resistance to first- and/or second-generation TKIs. This group, with 21 cases, showed EGFR exon 20 insertions, dual, complex mutations and variant of unknown significance, de novo MET gene amplification besides other mutations. (ii) Group 2: primary resistance to third-generation TKIs. This group showed two cases, with one showing dual EGFR mutation (L858R and E709A) and EGFR gene amplification. (iii) Group 3: secondary resistance to first- and second-generation TKIs. This group had 27 cases, which were previously reported negative for EGFR T790M by single gene testing. Significant findings were MET gene amplification in four cases, with one also showing MET exon 14 skipping mutation. Three cases showed small cell change and one showed loss of primary mutation. (iv) Group 4: secondary resistance to third-generation TKIs. The latter group was further subgrouped into group 4A: secondary resistance to osimertinib (third-generation TKI) when offered as second-line therapy after first- and second-generation TKIs on detection of T790M mutation. This group had 15 cases. EGFR T790M mutation was lost in 10 (10/15; 67%) cases and was retained in five cases. Patients with T790M loss experienced early resistance (6.9 months versus 12.6 months mean, P = 0.0024) compared with cases that retained T790M. Two cases gained MET amplification as the resistance mechanisms. Other mutations that were found when EGFR T790M was lost were in FGFR3, KRAS, PIK3CA, CTNNB1, BRAF genes. One case had EML4-ALK translocation. Two cases showed driver EGFR deletion 19, retained T790M and C797S mutation in Cis form. Group 4B: secondary resistance to osimertinib (when given as first-line therapy) in EGFR-mutant NSCLC. This group had three cases. The duration of osimertinib treatment ranged from 11 to 17 months. Two patients showed additional C797S mutation along with primary EGFR mutation. ConclusionThis study shows the wide spectrum of primary and secondary EGFR resistance mechanisms to first, second and third generation of TKIs and helps us to identify newer therapeutic targets that could carry forward the initial advantage offered by EGFR TKIs.

What do we know about the molecular and biological features of EGFR in glioblastomas and non-small cell lung cancer?

The EGFR mutation is one of the most common mutations in malignant neoplasms. The epidermal growth factor receptor (EGFR) is a growth factor receptor that induces cell differentiation and proliferation when activated by binding one of its ligands. The receptor is located on the cell surface, where ligand binding activates a tyrosine kinase in the intracellular region of the receptor. The tyrosine kinase phosphorylates a number of intracellular substrates and further activates pathways leading to cell growth, DNA synthesis and oncogene expression. Gene amplification is a process characterized by an increase in the copy number of a restricted region in the chromosome shoulder, which is associated with overexpression of the corresponding amplified gene. Amplification of the EGFR gene is detected in about 40% of glioblastoma cases. It should be noted that EGFR gene amplification is accompanied by the acquisition of many mutations, which include intragenic deletions and point mutations. The most common EGFR mutation in glioblastomas of the brain is a deletion in exon 2-7 (EGFRvIII) frame, which occurs in 50% of all cases of EGFR-amplified glioblastoma. Despite great advances in molecular biology and targeted therapies, patients with non-small cell lung cancer (NSCLC) and glioblastoma still lead in mortality. Most of them have “classical” EGFR mutations (deletions in exon 19 and 21), but 15-20% of patients have rare mutations, which most often include point mutations, deletions and insertions in exon 18 and 25. Thus, rare EGFR mutations are a promising diagnostic and therapeutic target in cancer. This review summarizes data on the role of EGFR in the carcinogenesis of NMPL and glioblastoma. The literature search was performed using the Pubmed database.

Abstract 2781: De novo molecular mechanisms of resistance to mobocertinib

Abstract Mobocertinib is an oral tyrosine kinase inhibitor recently approved by the FDA for the treatment of patients (pts) with locally advanced or metastatic non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) exon 20 insertion mutations who have progressed on or after platinum-based chemotherapy. To understand the impact of co-occurring genetic alterations on the therapeutic response to mobocertinib, tumors from a subset of 72 platinum-pretreated pts with EGFR exon 20 insertions from the phase 1/2 study (NCT02716116) were collected before treatment and analyzed by next-generation sequencing. More than 200 co-occurring hotspot genetic alterations were detected in 43 pts, with some alterations detected with greater than 20% frequency, such as PTCH1 (67%), HNF1A (58%), TP53 (58%), and GNAQ (35%). In addition to EGFR exon 20 insertion mutations, co-occurring EGFR substitutions were also observed in the exon 20 loop following the C-helix (amino acid positions 770, 771, 772) in 4 pts and L858Q in 1 pt. The pt with the L858Q mutation had a confirmed partial response as assessed by an independent review committee, while the 4 pts with exon 20 substitutions were non-responders (2 had stable disease and 2 had progressive disease as best response). An abnormally high level of mutational burden was seen in 4 pts with EGFR exon 20 insertions, along with alterations in the mismatch repair gene MSH6 and/or the elongation factor MLLT3. Association analysis of mutations reported increased odds of association between the presence of PIK3CA, TP53, and EGFR alterations and a lack of mobocertinib response. Gene amplifications were observed in 12 pts (16%) affecting EGFR (6 pts), MYC (4 pts), CDK4 (3 pts), and CCND1 (2 pts). When assessed for association with lack of response (P values from the Fisher exact test), odds ratios were 2.47 (P=0.66) and 6.34 (P=0.085) for EGFR amplification and any-gene amplification, respectively. When combined, the presence of either PIK3CA mutations or any-gene amplification was associated with a lack of response to mobocertinib treatment with an odds ratio of 12.93. Following adjustment for selected clinical covariates in a multivariate model, the presence of either PIK3CA mutation or gene amplification was an independent predictor of lack of response to mobocertinib (P value of 0.047). Given the retrospective nature of these analyses and the insufficient control of statistical multiplicity, this finding should be considered exploratory. These data suggest that intrinsic genetic alterations, present at baseline in patients with NSCLC harboring an EGFR exon 20 insertion mutation, might impact the response to mobocertinib treatment. The presence of PIK3CA mutations, EGFR gene amplification, or cell cycle regulator MYC-CDK4-CCND1 amplification is associated with lack of response to mobocertinib, which might guide rational combinations for future clinical investigation. Citation Format: Sylvie Vincent, Zhenqiang Su, Adarsh Joshi, Kalyani Penta, Pingkuan Zhang. De novo molecular mechanisms of resistance to mobocertinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2781.

Potential for Using EGFR Expression in Rhabdomyosarcoma, Osteosarcoma and Ewing’s Sarcoma: Clinicopathological and Prognostic Significance

Sarcomas are a heterogeneous group of malignant tumors that arise from mesenchymal cells and can arise anywhere in the body, whether it is soft tissue or bone. Epidermal growth factor receptor (EGFR) expression in pediatric sarcomas is explored in the current study. A key feature of EGFRs is their general involvement in signal transduction and oncogenesis, making them one of the most studied receptor protein-tyrosine kinase families. The study included 104 archived formalin-fixed paraffin-embedded blocks assessed using immunohistochemical stains for EGFR expression in rhabdomyosarcoma, osteosarcoma, and Ewing's sarcoma. EGFR gene copy number was analyzed by dual silver in situ hybridization (DISH). EGFR was positively expressed in 56.7% of pediatric sarcoma. Immunostaining for EGFR was significantly associated with deep large tumors, stage, and histologic grade. Significantly, lower chances of overall survival were observed with elevated levels of EGFR five years post-diagnosis. EGFR staining identified independent risk factors for poor patient outcomes. The results of in situ hybridization did not indicate EGFR gene amplification in any of the cases assessed. EGFR overexpression was an independent predictor of pediatric sarcoma outcome, which is highly associated with histologic grade and stage. Results indicate EGFR inhibitors should be potentiated and directed against pediatric sarcoma. KEYWORDS EGFR, Ewing’s sarcoma, osteosarcoma, rhabdomyosarcoma

Comprehensive analysis of the ErbB receptor family in pediatric nervous system tumors and rhabdomyosarcoma.

There is a paucity of knowledge regarding pediatric biomarkers, including the relevance of ErbB pathway aberrations in pediatric tumors. We investigated the occurrence of ErbB receptor aberrations across different pediatric malignancies, to identify patterns of ErbB dysregulation and define biomarkers suitable for patient enrichment in clinical studies. Tissue samples from 297 patients with nervous system tumors and rhabdomyosarcoma were analyzed for immunohistochemical expression or gene amplification of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2). Exploratory analyses of HER3/HER4 expression, and mRNA expression of ErbB receptors/ligands (NanoString) were performed. Assay validation followed general procedures, with additional validation to address Clinical Laboratory Improvement Amendments (CLIA) requirements. In most tumor types, samples with high ErbB receptor expression were found with heterogeneous distribution. We considered increased/aberrant ErbB pathway activation when greater than or equal to two EGFR/HER2 markers were simultaneously upregulated. ErbB pathway dysregulation was identified in ∼20%-30% of samples for most tumor types (medulloblastoma/primitive neuroectodermal tumors 31.1%, high-grade glioma 27.1%, neuroblastoma 22.7%, rhabdomyosarcoma 23.1%, ependymoma 18.8%), 4.2% of diffuse intrinsic pontine gliomas, and no recurrent or refractory low-grade astrocytomas. In medulloblastoma/primitive neuroectodermal tumors and neuroblastoma, this was attributed mainly to high EGFR polysomy/HER2 amplification, whereas EGFR gene amplification was observed in some high-grade glioma samples. EGFR/HER2 overexpression was most prevalent in ependymoma. Overexpression and/or amplification of EGFR/HER2 were identified as potential enrichment biomarkers for clinical trials of ErbB-targeted drugs.

Investigating the Structure of Epidermal Growth Factor Receptor (EGFR) Dimers and Oligomers in Cells

A crucial step in the evolution of multi-cellular organisms was the emergence of receptor tyrosine kinases (RTKs). Trafficked to the plasma membrane, RTKs transduce signals in response to agonist ligands. EGFR was the first RTK to be cloned and is of paramount importance to cell function and human health. EGFR mutations and gene amplification are observed in many human cancers. Structural insights from macro-molecular crystallography and nuclear magnetic resonance described the atomic resolution structure of inactive ligand-free EGFR monomers and active ligand-bound dimers, and how these might underpin receptor autophosphorylation.

Expression of EGFR and conformational forms of EGFR in malignant pleural mesothelioma and its impact on survival

AimConformational forms of the epidermal growth factor receptor (EGFR) are pro‐tumorigenic. The prevalence and impact of conformational forms of EGFR in malignant mesothelioma (MM) is unknown. We investigated expression of EGFR and conformational forms of EGFR by immunohistochemistry using EGFR-targeting monoclonal antibodies (mAb). In addition, EGFR gene amplification was investigated by fluorescent in-situ hybridization (FISH). Findings were correlated with survival. MethodsPatients treated between 1988 and 2014 were identified from the thoracic surgery database of the Austin Hospital, Melbourne, Australia. Tissue microarrays (TMAs) were constructed, subjected to wild type (wt) EGFR IHC staining and FISH analysis. Conformational and mutation forms of EGFR were detected by IHC using mAb806, and LMH-151 which detects EGFRVIII. `H-scores` were derived and EGFR expression correlated with survival by Kaplan-Meier and log rank analysis. ResultsWtEGFR expression was seen in 93 % (299/321) of cases with overexpression (defined as an H-score ≥200) seen in more than half of cases (64 %). EGFR overexpression in MM was seen more commonly in the epithelioid subtype. EGFR overexpression was not associated with true EGFR amplification, although multiple copies were appreciated in samples with polysomy. EGFR expression did not correlate with survival. A conformational form of EGFR associated with EGFR dysregulation was found in 8.2 % of cases, and patients with these tumors had a trend towards a poorer outcome. No cases of the EGFRVIII mutation were identified. ConclusionMM consistently demonstrated high expression of EGFR, with a subset of tumors showing conformational EGFR forms consistent with EGFR dysregulation, but withoutEGFR amplification or EGFR VIII mutation. wtEGFR expression did not influence survival. The impact of EGFR conformation on survival warrants further investigation.

Exploring Predictors of Response to Dacomitinib in EGFR-Amplified Recurrent Glioblastoma.

Despite the high frequency of EGFR genetic alterations in glioblastoma (GBM), EGFR-targeted therapies have not had success in this disease. To improve the likelihood of efficacy, we targeted adult patients with recurrent GBM enriched for EGFR gene amplification, which occurs in approximately half of GBM, with dacomitinib, a second-generation, irreversible epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that penetrates the blood-brain barrier, in a multicenter phase II trial. We retrospectively explored whether previously described EGFR extracellular domain (ECD)-sensitizing mutations in the context of EGFR gene amplification could predict response to dacomitinib, and in a predefined subset of patients, we measured post-treatment intratumoral dacomitinib levels to verify tumor penetration. We found that dacomitinib effectively penetrates contrast-enhancing GBM tumors. Among all 56 treated patients, 8 (14.3%) had a clinical benefit as defined by a duration of treatment of at least 6 months, of whom 5 (8.9%) remained progression free for at least 1 year. Presence of EGFRvIII or EGFR ECD missense mutation was not associated with clinical benefit. We evaluated the pretreatment transcriptome in circulating extracellular vesicles (EVs) by RNA sequencing in a subset of patients and identified a signature that distinguished patients who had durable benefit versus those with rapid progression. While dacomitinib was not effective in most patients with EGFR-amplified GBM, a subset experienced a durable, clinically meaningful benefit. Moreover, EGFRvIII and EGFR ECD mutation status in archival tumors did not predict clinical benefit. RNA signatures in circulating EVs may warrant investigation as biomarkers of dacomitinib efficacy in GBM.

Genetic alterations in epidermal growth factor receptor-tyrosine kinase inhibitor-naïve non-small cell lung carcinoma.

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) are an approved first-line therapy against unresectable or advanced non-small cell lung cancer (NSCLC) harboring EGFR gene activating mutations. However, the majority of tumors develop acquired resistance against EGFR-TKIs and some tumors exhibit natural resistance. A number of resistance mechanisms against the latest third-generation EGFR-TKIs have been reported, including tertiary EGFR C797S mutation and several gene alterations activating EGFR or other signaling pathways. The current study aimed to identify the frequency of natural EGFR-TKI resistance in pretreatment NSCLC and to predict the therapeutic effect of EGFR-TKIs. A total of 246 EGFR-TKI-naïve NSCLC patients harboring known EGFR gene mutations were identified. The presence of EGFR C797S and T790M mutations were determined using the peptide nucleic acid-locked nucleic acid PCR clamp method. ERBB2, MET, EGFR, ALK, BRAF, FGFR1, MYC, RET, CCND1, CCND2, CDK4, CDK6, MDM2 and MDM4 gene amplification, which can lead to resistance against any generation EGFR-TKIs, was determined using the multiplex ligation-dependent probe amplification assay. No concurrent C797S mutation with known EGFR mutations were identified. T790M mutation was identified in 12 patients (4.9%). ERBB2 or MET gene amplification was found in some patients (0.0-0.4%). MDM2 gene amplification was associated with tumor recurrence and shorter progression-free survival (PFS) for first- or second-generation EGFR-TKIs. De novo EGFR C797S mutation was not identified. Other resistance mechanisms against EGFR-TKIs were indicated in some patients with EGFR-TKI-naïve NSCLC. MDM2 gene amplification, which can lead to altered cell cycle, was associated with tumor recurrence and shorter PFS in EGFR-TKI therapy.

Reduction of nuclear Y654-p-β-catenin expression through SH3GL2-meditated downregulation of EGFR in chemotolerance TNBC: Clinical and prognostic importance.

Triple negative breast cancer (TNBC) originates from a less differentiated ductal cell of breast, which is less sensitive to chemotherapy. The chemotolerance mechanism of TNBC has not yet been studied in detail. For this reason, molecular profiles (expression/genetic/epigenetic) of Y654-p-β-catenin (active) and its kinase epidermal growth factor receptor (EGFR) along with SH3GL2 (regulator of EGFR homeostasis) were compared between neoadjuvant chemotherapy treated (NACT) and pretherapeutic TNBC samples. Reduced nuclear expression of Y654-p-β-catenin protein with low proliferation index and CD44 prevalence showed concordance with reduced expression of EGFR/Y1045-p-EGFR proteins in the NACT samples than the pretherapeutic TNBC samples. Infrequent messenger RNA expression, gene amplification (10-32.5%), and mutation (1%) of EGFR were seen in the TNBC samples irrespective of therapy, suggesting the importance of EGFR protein stabilization in this tumor. The upregulation of SH3GL2 seen in the NACT samples in contrast to the pretherapeutic samples might be due to its promoter hypomethylation, as seen in the quantitative methylation assay. A similar trend of upregulation of SH3GL2 and downregulation of EGFR, Y1045-p-EGFR, Y654-p-β-catenin were seen in the MDA-MB-231 cell line using antharacycline antitumor drugs (doxorubicin/nogalamycin). The NACT patients with reduced expression of Y654-p-β-catenin and/or EGFR and high expression of SH3GL2 showed comparatively better prognosis than the pretherapeutic patients. Thus, our study showed that reduced nuclear expression of Y654-p-β-catenin in NACT samples due to downregulation of EGFR protein through promoter hypomethylation-mediated upregulation of SH3GL2, resulting in low proliferation index/CD44 prevalence with better prognosis of the NACT patients, might have an important role in the chemotolerance of TNBC.

Comparison between cryobiopsy and forceps biopsy in detection of epidermal growth factor receptor amplification in non-small-cell lung cancer

BackgroundNon-small-cell lung cancer (NSCLC) represents 85% of lung cancer cases. Genotyping is now considered as a cornerstone in proper management and better results of such cancers, especially with targeted therapy. Cryobiopsy is a promising tool in NSCLC to obtain larger samples, with well-preserved tissue sufficient for accurate histopathological and gene detection.AimTo compare cryobiopsy and ordinary forceps results in detection of epidermal growth factor receptor (EGFR) amplification in NSCLC.Materials and methodsSamples from 34 patients with proven NSCLC by cryobiopsy versus forceps biopsy were compared for size, quality, and diagnostic yield of EGFR gene amplification.ResultsThe samples obtained by cryoprobe had larger size and better artifact-free areas with more diagnostic yield of EGFR gene amplification (29.4%) versus with forceps biopsy (8.8%), with gene amplification showing higher statistical significance in younger patients, never smokers, and women (P<0.001).ConclusionCryobiopsy is an excellent tool for larger, better-quality sampling and for higher diagnostic yield of EGFR amplification in NSCLC.

P11.08 Defining EGFR amplification status for clinical trial inclusion

Abstract BACKGROUND Precision medicine trials targeting the epidermal growth factor receptor (EGFR) in glioblastoma patients require selection for EGFR-amplified tumors. However, there is currently no golden standard in determining the amplification status of EGFR or EGFRvIII expression. Here, we aimed to determine which technique and which cut-offs are suitable to determine EGFR amplification status. MATERIAL AND METHODS We compared fluorescent in-situ hybridization (FISH) and RT-qPCR data from patients screened for trial inclusion into the Intellance 2 clinical trial, with data from a panel-based next generation sequencing (NGS) platform (both DNA and RNA). RESULTS By using data from &gt;1000 samples, we show which cut-offs are optimal to determine EGFR gene amplification by FISH. Our data also show that gene amplification (as determined by FISH) correlates with EGFR expression levels (as determined by RT-qPCR) with ROC analysis showing an under the curve area of up to 0.902. EGFR expression as assessed by RT-qPCR therefore may function as a surrogate marker for EGFR amplification. Our NGS data shows that EGFR copy numbers can strongly vary between tumors with levels ranging from 2 to more than 100 copies per cell. Levels exceeding 5 gene copies can be used to define EGFR-amplification by NGS; below this level FISH detects very few (if any) EGFR amplified nuclei and none of the samples express EGFRvIII. CONCLUSION Our data from central laboratories and diagnostic sequencing facilities, using material from patients eligible for clinical trial inclusion, help defining the optimal cut-off for various techniques to determine EGFR amplification for diagnostic purposes.

Genetic Alterations of Epidermal Growth Factor Receptor in Glioblastoma: The Usefulness of Immunohistochemistry.

Epidermal growth factor receptor (EGFR) amplification is one of the common alterations in IDH-wildtype glioblastoma. It is frequently associated with EGFRvIII mutation. To evaluate the correlation between EGFR overexpression, gene amplification, and EGFRvIII mutation, we performed immunohistochemical (IHC) analysis, fluorescence in situ hybridization by Vysis LSI EGFR/CEP7 dual color probe, and polymerase chain reaction studies in 76 patients diagnosed with glioblastomas (67 IDH-wildtype and 9 IDH-mutant). EGFR expression was scored ranging from 0 to 3+. Using formalin-fixed paraffin-embedded sections, real-time reverse transcription-polymerase chain reaction was carried out with primers specific for EGFRvIII and EGFR wildtype. In addition, we evaluated the impact of EGFR status on prognosis. EGFR gene amplifications and EGFRvIII mutations were identified in 30.3% and 15.5% of all cases, respectively. All the EGFR-amplified or EGFRvIII mutant cases were IDH-wildtype glioblastomas and tested positive with IHC. The sensitivity and specificity of EGFR IHC predicting EGFR gene amplification status were 100.0% and 46.5%, respectively. The EGFR-amplified cases tended to show more intense immunostaining (3+) in a considerable number of tumor cells (≥50%). Survival analyses of 37 IDH-wildtype glioblastoma patients revealed that none of the EGFR alterations significantly affected prognosis. EGFR IHC displayed high sensitivity and low specificity in predicting EGFR gene amplification, and interpretation of IHC results is a challenge. Therefore, EGFR IHC represents a possible screening tool for evaluation of EGFR gene amplification in clinical neuropathology, and both the intensity and proportion score facilitate interpretation of EGFR IHC.

Defining EGFR amplification status for clinical trial inclusion.

BackgroundPrecision medicine trials targeting the epidermal growth factor receptor (EGFR) in glioblastoma patients require selection for EGFR-amplified tumors. However, there is currently no gold standard in determining the amplification status of EGFR or variant III (EGFRvIII) expression. Here, we aimed to determine which technique and which cutoffs are suitable to determine EGFR amplification status. MethodsWe compared fluorescence in-situ hybridization (FISH) and real-time quantitative (RT-q)PCR data from patients screened for trial inclusion into the Intellance 2 clinical trial, with data from a panel-based next generation sequencing (NGS) platform (both DNA and RNA). ResultsBy using data from >1000 samples, we show that at least 50% of EGFR amplified nuclei should be present to define EGFR gene amplification by FISH. Gene amplification (as determined by FISH) correlates with EGFR expression levels (as determined by RT-qPCR) with receiver operating characteristics analysis showing an area under the curve of up to 0.902. EGFR expression as assessed by RT-qPCR therefore may function as a surrogate marker for EGFR amplification. Our NGS data show that EGFR copy numbers can strongly vary between tumors, with levels ranging from 2 to more than 100 copies per cell. Levels exceeding 5 gene copies can be used to define EGFR-amplification by NGS; below this level, FISH detects very few (if any) EGFR amplified nuclei and none of the samples express EGFRvIII. ConclusionOur data from central laboratories and diagnostic sequencing facilities, using material from patients eligible for clinical trial inclusion, help define the optimal cutoff for various techniques to determine EGFR amplification for diagnostic purposes.