Epidermal Growth Factor Receptor Staining Research Articles

Patho-morphological and Immunohistochemical Studies on Bovine Horn Core Carcinoma

Background: An investigation was carried out on twelve clinical cases of neoplasm of horn in Durg, Dhamtari and Rajnandgaon districts of Chhattisgarh, suspected of bovine horn core carcinoma (squamous cell carcinoma) revealed the cytology, pathomorphology and immunohistochemical (IHC) expression of Pan-cytokeratin (Pan- CK), p53 gene, epidermal growth factor receptor (EGFR) and p16 gene in tumourous growth at horn in bovines. Methods: In this field-laboratory investigation conducted during January to June 2022, we explicate the cytological, pathological and immunohistochemical alterations in bovine horn core carcinoma from 12 tissue samples. Cytological studies includes special papaniculaou staining and immunohistochemistry was performed through Benchmark automated staining system. Result: Eight out of 12 cases (66.66%) were confirmed as SCC of horn on the basis of histopathological and immunohistochemical analysis. Papanicolaou staining revealed variation in shape and size of cells and altered nuclear details. Grossly unilateral large cauliflower like neoplastic growth at the base of the horn was observed. Differentiation of tumours were based on histopathology and Immunohistochemistry. Well differentiated SCCs (n=4; 50%) were characterized by severe keratinization of horn epithelium with concentric arrangement forming keratin pearls also called as “cell nests”. Moderately differentiated SCCs (n=2; 25%) characterized by small keratin pearl formations and mitotic figures. Poorly differentiated SCCs of horn (n=2; 25%) revealed absence of distinctive keratin pearls although deep invasion from primary site was observed. Tissue samples revealed strong immunohistochemical staining of Pan-CK, p53 and EGFR and negative to p16. Highest immunohistochemical expression was observed in Pan-CK which confirmed the tumours were of epithelial origin and EGFR immunoexpression was confirmatory for malignancy and degree of metastasis.

Investigating the novel CD16A and epidermal growth factor receptor (EGFR) bispecific innate cell engager, AFM24, to leverage the innate immune system: Interim results from the colorectal cancer (CRC) cohort.

e14508 Background: AFM24 is a tetravalent, bispecific innate cell engager that binds EGFR on solid tumors and CD16A on natural killer (NK) cells. AFM24 redirects NK cells to EGFR-expressing (EGFR+) solid tumors, enhancing antibody-dependent cellular cytotoxicity. AFM24 induced lysis of EGFR+ tumor cell lines in vitro independent of EGFR expression level and mutation status. The dose escalation phase (Phase 1) of the presented study (NCT04259450) previously established the recommended Phase 2 dose at 480 mg based on tolerability, receptor occupancy, and pharmacokinetics. Correlative science data revealed an activation and enrichment of immune cells in the tumor microenvironment. Here we present preliminary data from the Phase 2a CRC expansion cohort. Methods: The Phase 2a expansion phase is evaluating 480 mg AFM24 in patients with histologically or cytologically confirmed microsatellite stable, RAS wild type EGFR+ CRC. Patients have relapsed or refractory disease to ≥2 prior lines of therapy including an EGFR targeted therapy. EGFR+ is defined as positive staining for EGFR in ≥1% of tumor cells per local immunohistochemistry assay. The study follows Simon’s two-stage design. The primary endpoint is the overall response rate per RECIST v1.1 by investigator assessment; secondary endpoints include safety and tolerability; efficacy per iRECIST is exploratory. AFM24 is administered intravenously once weekly in a 28-day cycle. Tumor assessments are performed at screening, cycles 2, 4, 6, 8, and every 3 cycles thereafter. Results: As of December 2022, 16 patients with Stage 4 CRC were enrolled and treated with 480 mg AFM24 in the study. The median (range) age was 66 years (51–77); 11 males and 5 females; 9 Asian, 7 White). The median number of prior therapies was 4 (2–7) and the median treatment duration was 5.1 weeks (1.0–22.0). Treatment-related adverse events (TRAEs) were reported in 14/16 patients; infusion-related reactions (IRRs, 12), pruritus (3), lymphopenia (3), and anemia (2) were the most common. AFM24 TRAEs of Grade ≥3 occurred in 6 patients and included lymphopenia (3), decreased neutrophil count (1), increased blood bilirubin (1) and IRRs (1). To date, no objective responses were observed. Seven patients remain on treatment including 1 patient with confirmed stable disease ongoing for 5 months exhibiting a tumor reduction of –17%. Final interim results will be presented. Conclusions: The novel mechanism of action of AFM24 may provide an alternative treatment approach for patients with EGFR+ solid tumors. In this study, AFM24 monotherapy was adequately tolerated in a heavily pretreated population of patients with EGFR+ CRC. AFM24 is also being investigated in combination with atezolizumab and autologous NK cells in various EGFR+ solid tumors. Clinical trial information: NCT04259450 .

Leveraging innate immunity with AFM24, a novel CD16A and epidermal growth factor receptor (EGFR) bispecific innate cell engager: Interim results for the non-small cell lung cancer (NSCLC) cohort.

2533 Background: Patients with EGFR mutant NSCLC who progress on or after small molecule EGFR inhibitors have only limited treatment options. AFM24 is a tetravalent, bispecific innate cell engager that binds EGFR on solid tumor cells and CD16A on NK cells to redirect and enhance antitumor antibody-dependent cellular cytotoxicity, thus acting independently of the EGFR downstream signaling pathway and EGFR expression levels. The Phase 1 dose escalation phase of the presented study (NCT04259450) established the recommended Phase 2 dose of AFM24 monotherapy at 480 mg based on tolerability, receptor occupancy, and pharmacokinetics (PK). Correlative science data revealed an activation and enrichment of immune cells in the tumor microenvironment. Methods: The Phase 2a expansion phase is evaluating 480 mg AFM24 in patients with EGFR mutant NSCLC, relapsed or refractory to ≥1 prior lines of therapy; EGFR-positivity is defined as positive staining for EGFR in ≥1% of tumor cells per local immunohistochemistry assay. The study follows Simon’s two-stage design. The primary endpoint is overall response rate per local RECIST v1.1; secondary endpoints include safety, PK, and immunogenicity. Efficacy per iRECIST is an exploratory endpoint. AFM24 is administered intravenously once weekly in a 28-day cycle. Tumor assessments are performed at initial screening, cycles 2, 4, 6, 8, and every 3 cycles thereafter. Results: As of December 2022, 14 patients (median [range] age 55 [37–82]; 12 male; 11 Asian, 3 White) were enrolled; the median number of prior therapies was 2.5 (1–12). Median treatment duration with AFM24 was 6.7 weeks (1.0–26.1). Best objective response in 10 evaluable patients was confirmed partial response in 1 patient (change from baseline –45%); 4 patients exhibited stable disease, 1 was confirmed. Tumor shrinkage occurred in 2 patients with stable disease (–6%, –18%). The tumor control rate was 50%. Treatment-related adverse events (TRAEs) were reported in 13 patients; infusion related reactions (IRRs, 12), dermatitis acneiform, neutrophil count decreased, decreased appetite, and myalgia (2 each) were most common. Grade ≥3 TRAEs occurred in 4 patients and included decreased neutrophil count (2), lymphopenia, and IRR (1 each). Grade 5 pneumonitis was observed in 1 patient with disease progression and multiple comorbidities; relation to AFM24 could not be ruled out. As of January 2023, enrollment is ongoing; results of the interim analysis will be presented. Conclusions: AFM24 demonstrated clinical activity and acceptable safety in heavily pretreated patients with EGFR mutant NSCLC. The novel mechanism of action of AFM24 could add to the therapeutic options for patients with EGFR expressing tumors and is under clinical evaluation as a single agent and in combination with atezolizumab or autologous NK cells. Clinical trial information: NCT04259450 .

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

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

Prognostic Impact of Low-Level p53 Expression on Brain Astrocytomas Immunopositive for Epidermal Growth Factor Receptor.

Although the expression of p53 and epidermal growth factor receptor (EGFR) is associated with therapeutic resistance and patient outcomes in many malignancies, the relationship in astrocytomas is unclear. This study aims to correlate p53 and EGFR expression in brain astrocytomas with overall patient survival. Eighty-two patients with astrocytomas were enrolled in the study. Semi-quantitative p53 and EGFR immunohistochemical staining was measured in tumor specimens. The mean follow-up after astrocytoma surgery was 18.46 months. The overall survival rate was 83%. Survival was reduced in EGFR-positive patients compared with survival in EGFR-negative patients (p < 0.05). However, no significant differences in survival were detected between patients with high and low p53 expression. In patients with low p53 expression, positive EGFR staining was associated with significantly worse survival compared with patients with negative EGFR staining (log-rank test: p < 0.001). Survival rates in positive and negative EGFR groups with high p53 protein expression were similar (log-rank test: p = 0.919). The IC50 of an EGFR inhibitor was higher in GBM cells with high p53 protein expression compared with the IC50 in cells with low p53 expression. Combined EGFR and p53 expression may have prognostic significance in astrocytomas.

Abstract 6261: Comparison of glycoprotein nonmetastatic melanoma protein b, epidermal growth factor receptor and vascular endothelial growth factor expression in head and neck squamous cell carcinoma, and untreated and residual lymph node metastases

Abstract Objective: To evaluate expression of the potential molecular imaging targets glycoprotein nonmetastatic melanoma protein B (GPNMB) and vascular endothelial growth factor (VEGF) in comparison to classical epidermal growth factor receptor (EGFR) in previously untreated head and neck cancer primary tumors and corresponding lymph node metastases, and in lymph node metastases after initial (chemo)radiotherapy. Methods: Primary tumors and lymph node metastases of 38 patients who underwent primary resection of head and neck squamous cell carcinoma (HNSCC) were selected. In addition, lymph node metastases from salvage surgery after (chemo)radiotherapy from 22 patients with HNSCC were selected. Immunohistochemical staining for EGFR, VEGF and GPNMB was performed, after which protein expression was scored using H-scores (percentage of positive tumor cells multiplied by staining intensity ranging from 0 to 2). Primary tumors and lymph node metastases with an H-score ≥5 were considered positive, after which sensitivity rates were calculated. Groups were compared using the Mann-Whitney U test for non-parametric data with post-hoc Bonferroni corrections. Results: In untreated primary HNSCC the sensitivity was 86.8% for EGFR (median H-score 60), 92.1% for VEGF (median 60), and 100% for GPNMB (median 100). In previously untreated lymph node metastases the sensitivity was 81.6% for EGFR (median 30), 86.8% for VEGF (median 65), and 100% for GPNMB (median 118). In lymph node metastases after initial (chemo)radiotherapy, sensitivity was 86.4% for EGFR (median 95) and 100% for GPMB (median 100). VEGF sensitivity could not reliably be evaluated in these lymph nodes due to nonspecific staining in all tissue slides. GPNMB expression was significantly higher compared to EGFR in untreated primary HNSCC (p = .009 and p &amp;lt; .001, respectively) and lymph node metastases (p &amp;lt; .001 for both). Conclusion: GPNMB shows better tumor-specific receptor expression in both sensitivity and H-scores compared to EGFR and VEGF in immunohistochemistry. VEGF performed comparable to EGFR in untreated HNSCC and lymph node metastases. GPNMB could therefore be a new potential imaging target based on these immunohistochemical results. Citation Format: Jeroen E. van Schaik, Bert van der Vegt, Saskia H. Hanemaaijer, Gyorgy B. Halmos, Max J. Witjes, Bernard F.A.M. van der Laan, Rudolf S. Fehrmann, Sjoukje F. Oosting, Boudewijn E. Plaat. Comparison of glycoprotein nonmetastatic melanoma protein b, epidermal growth factor receptor and vascular endothelial growth factor expression in head and neck squamous cell carcinoma, and untreated and residual lymph node metastases [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 6261.

The combination of CD16A/EGFR innate cell engager, AFM24, with SNK01 autologous natural killer cells in patients with advanced solid tumors.

TPS2675 Background: Natural killer (NK) cells are a critical component of the innate immune system involved in the eradication of transformed cells via antibody-dependent cellular cytotoxicity (ADCC). For treatment of solid tumors autologous NK cell transfer represents a promising treatment strategy, with ex vivo expansion and activation enhancing the specificity and anti-tumor activity of NK cells. The efficacy of this approach may be enhanced through the addition of tumor-targeting antibodies, augmenting NK cell-mediated ADCC. Innate Cell Engagers (ICE) are bispecific antibodies that target a tumor cell-surface antigen and bind to CD16A expressed on NK cells. AFM24 is a novel ICE that targets epidermal growth factor receptor (EGFR), which is often overexpressed in solid tumors. The Phase 1 study of AFM24 monotherapy showed patients had a manageable safety profile, and SNK01 monotherapy has also shown to be well-tolerated in patients with rapidly progressive solid tumors. This study seeks to investigate AFM24 in combination with SNK01 autologous NK cells in patients with advanced EGFR+ solid tumors. Methods: An ongoing Phase 1/2a open-label, non-randomized, multicenter, dose escalation (Phase 1) and dose expansion (Phase 2a) study was initiated in November 2021 (NCT05099549) to evaluate the safety, tolerability and efficacy of AFM24 in combination with SNK01 NK cells in EGFR+ solid tumors. The primary aim of the Phase 1 study is to determine the maximum tolerated dose and/or recommended Phase 2 dose (RP2D) of AFM24 in combination with SNK01 at a fixed dose NK cells using a standard 3+3 design. Eligible patients must have advanced or metastatic disease with positive immunohistochemical staining for EGFR in &gt;1% of tumor cells and be refractory to standard-of-care treatment. Treatment begins with a safety lead-in phase with a single dose of AFM24 7 days prior to combination therapy. AFM24 will be administered at an escalating dose as weekly intravenous (IV) infusions; the starting dose (160 mg) and dose escalations for each cohort are based on results from the ongoing AFM24 monotherapy trial (NCT04259450). SNK01 NK cells will be given at a fixed dose (4.0 x109 cells) as a weekly IV infusion concomitantly with AFM24. Patients will receive combination therapy until disease progression, intolerable toxicity, patient withdrawal or termination at the investigator’s discretion. Phase 2 will then establish the overall response rate (as per RECIST v1.1) of combination therapy in patients with treatment refractory, advanced or metastatic squamous cell carcinoma of the head and neck, non-small cell lung cancer, or colorectal cancer as the primary endpoint. Efficacy will also be assessed by assessing progression-free and overall survival. Secondary endpoints for both phases include treatment-emergent adverse events, serious adverse events, pharmacokinetics and immunogenicity. Clinical trial information: NCT05099549.

Frequency of Expression of BRAF V600E and Epidermal Growth Factor Receptor (EGFR) in Ameloblastoma

Objective: To determine the Immunohistochemical expression of BRAF V600E and Epidermal growth factor receptor (EGFR) in ameloblastoma and correlate the expression with age and gender of patients, and patterns and types of ameloblastoma. Material &amp; Methods: 39 cases were retrieved with their formalin-fixed, paraffin embedded blocks, trimmed and cut into 5 microns sections. They were mounted on slides after staining with routine hematoxylin and eosin followed by Immunohistochemical staining of BRAF V600E and EGFR. Mean and standard deviation were calculated for quantitative variables. Frequency and percentages were calculated for qualitative variables. Chi-square test was employed to assess the significance of difference. The p-value ≤ 0.05 was considered significant. Results: There were 19 (41.2%) males and 20 (48.7%) female patients. The mean age of patients at which they presented was 39.97 ± 15.505 (mean ± SD) with an age range between 12 to 65 years. 25 (64.1 %) cases showed positive expression of BRAF V600E and 14 (35.8 %) cases showed negative expression of BRAF V600E. 27 (69.2 %) cases showed positive expression of EGFR whereas 6 (15.3 %) cases showed negative expression of EGFR. The p-value was ≤ 0.05 for expression of BRAF V600E with respect to patterns of ameloblastoma and tumor site and expression of EGFR with respect to sub-types of ameloblastoma.Conclusion: There is positive expression of BRAF V600E (64.1%) and EGFR (74.4%) in different sub-types and patterns of ameloblastoma. Correct assessment with the help of these markers can lead to early diagnosis and use of adjuvant treatment protocols

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

Role of Epidermal Growth Factor Receptor in Gallbladder Carcinoma and its Association with Various Clinicopathological Parameters: A Cross-sectional Study

Introduction: Carcinoma of gallbladder is an aggressive disease with poor outcome. The Epidermal Growth Factor Receptor (EGFR) is a transmembrane receptor that regulate growth, proliferation and differentiation in cells. Increased EGFR receptor expression has been studied in various cancers like lung, colorectal, breast and pancreatic tumours and antiEGFR antibody has been used successfully for therapeutic and diagnostic purposes. Aim: To evaluate EGFR expression in gallbladder carcinoma and its association with clinicopathological factors to reveal its relation to prognosis. Methods and Methods: The present study was a crosssectional study in which 64 samples were collected of resected specimen of Gallbladder Carcinoma (GBC), from the Department of Pathology, King George’s Medical University, Lucknow, Uttar Pradesh, India, for a period of one year and six months. Haematoxylin-eosin-stained sections were evaluated for tumour type and histopathological grading and TNM staging was done. Immunohistochemistry (IHC) was performed and analysed prospectively using ready to use anti-EGFR as per manufacturer’s protocols. Association between EGFR expression and clinicopathological factors were statistically analysed using Statistical Package for the Social Sciences (SPSS) software version 21.0. Results: EGFR expression was positive in 81.25% (52/64) cases of GBC which showed a highly significant association between tumour grade and stage. About 60% of poorly differentiated GBC displayed strong (3+) staining intensity as compared to 30% of moderately differentiated (3+) and 10% of well differentiated tumours (3+). It suggests that with decreasing differentiation of tumour EGFR staining intensity increases (p&lt;0.001). Positivity rate of EGFR expression were also increased with increase of tumour TNM stage (stage I to stage IV). Strong EGFR expression was associated with decreased overall survival with significant p value (p=0.031, log rank test). Conclusion: EGFR expression is inversely related with tumour differentiation, and overall survival. EGFR expression increases with high TNM staging. So, it can be used as prognostic marker for gallbladder carcinoma and opening a hope towards the new therapeutic options

MCM-2, Ki-67, and EGFR downregulated expression levels in advanced stage laryngeal squamous cell carcinoma

We present the conceptual study investigated the capacity of minichromosome maintenance-2 (MCM-2), Ki-67, and epidermal growth factor receptor (EGFR) to assess the severity and progression of laryngeal squamous cell carcinoma (LSCC) disease and to study the correlations among these markers. A total of 30 patients with LSCC with immunohistochemistry (IHC) staining for MCM-2, Ki-67 and EGFR were examined. Mean expression levels of the three markers were evaluated for comparing between early and advanced stages of LSCC. The mean MCM-2, Ki-67, and EGFR expression levels were significantly decreased in advanced-stage compared with early-stage LSCC. Pearson correlation analysis showed a statistically significant correlation between the MCM-2 and Ki-67. Regarding subgroup analyses, MCM-2, Ki-67, and EGFR showed significant differences between early- and advanced-stage LSCC with non-recurrence, while for the recurrent subgroup LSCC, only MCM-2 revealed a significant difference between early- and advanced-stage LSCC. Altogether, these results support the role for downregulation of MCM-2, Ki-67 and EGFR in advanced-stage LSCC and correlation of MCM-2 and Ki-67 expressions that would be a promising strategy to predict prognosis of LSCC including severity and progression. We contextualize our findings and advocate the position of the biological markers, especially MCM-2, as an emerging evaluation tool for LSCC disease.

Androgen Receptor Expression in Triple-negative Breast Cancer and its Relation with Epidermal Growth Factor Receptor, CD 105, and Clinicopathological Parameters

Background: Triple-negative breast cancers (TNBC) are the tumors lacking expression of estrogen receptors, progesterone receptors, and human epidermal growth factor 2. The highest level of androgen receptors (AR) expression belongs to the Luminal androgen receptor subtype. AR is expressed in 70 to 90% of primary breast cancers. The biological role of AR in breast cancer continues to emerge. The overexpression of epidermal growth factor receptor (EGFR) has been previously studied in TNBC, where it was found to be associated with poor prognosis. In the evaluation of neovascularization, CD105 (endoglin) was found to be superior to CD34 and CD31 owing to its greater affinity for endothelial cells in tumor-related angiogenic tissue. We conducted the present work to assess the expression profile of androgen receptor in TNBC cases and its correlation with other clinicopathological parameters, EGFR and CD 105, in order to evaluate its clinical significance. Method: This retrospective study included 50 histologically confirmed breast cancer patients who were proven to be triple-negative based on immunohistochemical study. Formalin-fixed tissue blocks with tumor were chosen for immunohistochemical staining for AR, EGFR, CD105, and Ki 67. Results: Positive AR expression was associated with older age, postmenopausal status, negative nodes, and grade II tumors. AR was inversely correlated with EGFR, while there was no correlation between AR and both Endoglin and Ki 67. Conclusion: AR-positive TNBC may be a subtype of breast cancer with unique characteristics that could make it ideal for antiandrogen endocrine therapy. EGFR and Endoglin's distinct expression indicated that they might be unique biomarkers for targeted therapy and prognosis.

CTNI-42. FIRST-IN-HUMAN FLUORESCENCE GUIDED SURGERY OF HIGH-GRADE GLIOMAS USING PANITUMUMAB-IRDYE800

Abstract INTRODUCTION High-grade gliomas (HGGs) are malignant brain tumors with devastating prognosis. Extent of resection predicts survival in patients, but current neuroimaging approaches lack tumor specificity. The epidermal growth factor receptor (EGFR) is a biomarker heterogeneously expressed in HGGs. We assessed the feasibility of imaging HGGs using a near-infrared fluorescent anti-EGFR antibody. METHODS Nine patients with contrast enhancing HGGs on presurgical MRI scan were systemically infused with a flat dose of either 50mg (n = 4) or 100mg (n = 5) panitumumab-IRDye800, 1–5 days before surgery. Near-infrared fluorescence imaging of tumor and histologically normal brain tissues was performed intraoperatively and ex vivo. Fluorescence was measured as mean fluorescence intensity (MFI), and tumor-to-background ratio (TBR) were calculated by comparing MFIs of tumor and histologically uninvolved tissue. Immunohistopathological staining of EGFR was performed on formalin fixed paraffin embedded tissue sections. RESULTS Both MFI and TBR were positively correlated to panitumumab-IRDye800 dose per body weight (R2 = 0.59 and 0.07, P &amp;lt; 0.0001 and P = 0.046 respectively). The TBR was higher at a 100 mg dose than at 50 mg (2.1 vs. 1.5). The smallest detectable tumor volume in a closed-field setting was 21 mg with 50 mg of dye and 12 mg with 100 mg. On sections of paraffin embedded tissues, positive EGFR protein expression was observed in 88.9% ± 12.4% of tumor tissues and positively correlated with fluorescence. Sensitivity and specificity of tumor fluorescence for viable tumor detection was calculated and fluorescence was found to be highly sensitive (93%) and specific (81%) for viable tumor tissues while normal brain tissues showed minimal fluorescence. No adverse events related to the imaging probe was observed. CONCLUSION This first-in-human study demonstrates the feasibility and safety of antibody based imaging for contrast enhancing high-grade gliomas.

Prognostic impact of cytoplasmatic EGFR upregulation in patients with oral squamous cell carcinoma: A pilot study.

In various tumors, epidermal growth factor-receptor (EGFR) serves a role in tumorigenesis and has an impact on survival. Usually the EGF-receptor is located on the surface of the cell membrane and is involved in various signaling pathways. The dimerization of EGFR with other ErbB family proteins, such as HER2, is important for the tumor progression. Nevertheless, a second EGFR-associated signaling pathway appears to be important for tumor cells, which is cytoplasmic/nuclear EGFR. The present study examined the influence of membranous or cytoplasmic localized EGFR on the prognosis of patients with oral squamous cell carcinoma (OSCC). Slides from 45 OSCC tumor samples were stained against EGFR using immunohistochemistry and analysed by the Remmele score system. The association with histopathological parameters and survival data was analyzed. Cytoplasmatic EGFR localization was identified as an independent predictive biomarker for overall survival in the examined OSCC cohort according to multivariate Cox regression analysis. Positive cytoplasmatic EGFR staining was correlated with a higher risk of early death (RR=3.0; P=0.035), while membranous EGFR localization did not affect patient survival. To the best of our knowledge, the present study is the first study to demonstrate that cytoplasmatic-localized EGFR is an independent prognostic biomarker for the overall survival of patients with OSCC.

Congenital mesoblastic nephroma is characterised by kinase mutations including EGFR internal tandem duplications, the ETV6-NTRK3 fusion, and the rare KLHL7-BRAF fusion.

Congenital mesoblastic nephroma (CMN) is histologically classified into classic, cellular and mixed subtypes. The aims of this study were to characterise the clinical, pathological and molecular features of a series of CMNs, and to determine the utility of pan-Trk and epidermal growth factor receptor (EGFR) immunohistochemistry as surrogate markers for NTRK gene fusions and EGFR internal tandem duplications (ITDs). Twenty-two archival CMN cases (12 classic, five cellular, and five mixed) were tested for the ETV6-NTRK3 fusion and EGFR ITD transcripts by the use of reverse transcriptase polymerase chain reaction (PCR), and next-generation sequencing-based anchored multiplex PCR. All 12 classic CMNs had EGFR ITD. Of the five cellular CMNs, four had the ETV6-NTRK3 fusion and one had the KLHL7-BRAF fusion. Of the five mixed CMNs, four had EGFR ITD, and one had the ETV6-NTRK3 fusion. Pan-Trk immunoreactivity was 100% sensitive and 94.1% specific for the presence of NTRK rearrangement. However, EGFR staining was only 62.5% sensitive and 33.3% specific for EGFR ITD. EGFR ITD is a consistent genetic event in classic CMN. A majority of cellular CMNs have the ETV6-NTRK3 fusion. Rare cellular CMNs may harbour non-canonical mutations such as the KLHL7-BRAF fusion, which was found in one case. Mixed CMNs may have either EGFR ITD or the ETV6-NTRK3 fusion. Pan-Trk immunohistochemistry is a sensitive, albeit not perfectly specific, marker for NTRK rearrangement. EGFR immunohistochemistry is not helpful as a marker of EGFR ITD.

Expression, intracellular localization, and mutation of EGFR in conjunctival squamous cell carcinoma and the association with prognosis and treatment

Conjunctival squamous cell carcinoma (SCC) is primarily treated with surgical resection. SCC has various stages, and local recurrence is common. The purpose of this study was to determine molecular localization of epidermal growth factor receptor (EGFR) and the possibility of EGFR as a biomarker for the management of conjunctival SCC. In this retrospective study, we performed immunohistochemistry to evaluate EGFR expression and localization in tumor cells, EGFR mutation-specific expression (E746-A750del and L858R), and human papillomavirus expression in a series of 29 conjunctival SCCs. All 29 tumors in our cohort were EGFR positive (100%). Twenty-one of 29 tumors (72%) showed focal EGFR staining, and seven (28%) showed diffuse EGFR staining. In addition, we calculated the percentages of the two most important mutations in EGFR (exon 19 746-A750del (8/29, 27.5%), exon 21 (L858R mutant (2/29, 6.8%)) in conjunctival SCCs. We observed that the translocation of EGFR from the membrane into the cytoplasm was related to clinical prognosis, as we detected correlations between EGFR cytoplasmic staining and final orbital exenteration and between decreased EGFR membrane staining and progression-free survival. EGFR is important in the pathology of ocular surface squamous neoplasia including SCC and is a prognostic factor. Increased understanding of EGFR mutations may have important implications for future treatment options.

Epidermal growth factor receptor as a molecular determinant of glioblastoma response to dopamine receptor D2 inhibitors.

There are ongoing clinical trials exploring the efficacy of dopamine receptor D2 (DRD2) inhibition against glioblastomas, the most common primary brain tumor. Here we examine potential molecular determinants of this efficacy. The Cancer Genome Atlas glioblastoma database and other published mRNA profiles were used to analyze the DRD2 and epidermal growth factor receptor (EGFR) expression pattern. In vitro and in vivo responses to DRD2 inhibitors were determined using patient-derived xenograft (PDX) glioblastoma models. Immunohistochemical studies were performed on clinically annotated glioblastoma samples derived from patients treated with ONC201. Analysis of clinical glioblastoma specimens derived from independent patient cohorts revealed an inverse correlation between EGFR and DRD2 mRNA expression, with implication that signaling mediated by these proteins shares overlapping functions. In independent panels of PDX glioblastoma lines, high EGFR expression was associated with poor in vitro and in vivo response to DRD2 inhibitors, including haloperidol and ONC201. Moreover, ectopic expression of a constitutively active EGFR, variant (v)III, suppressed glioblastoma sensitivity to ONC201. DRD2 expression positively correlated with expression of rate-limiting enzymes for dopamine synthesis as well as dopamine secretion, suggesting contribution of autocrine DRD2 signaling. Analysis of specimens from patients treated with ONC201 (n = 15) showed an inverse correlation between the intensity of EGFR staining and clinical response. The median overall survival for patients with high and low EGFR staining was 162 and 373 days, respectively (0.037). High EGFR expression is a determinant of poor glioblastoma response to DRD2. This finding should inform future clinical trial designs.

Epidermal growth factor receptor expression as a molecular determinant of glioblastoma response to the dopamine receptor D2 antagonist, ONC201.

e14552 Background: Durable response in glioblastoma patients have been reported in phase I/II clinical trials for the blood-brain penetrant dopamine receptor D2 (DRD2) antagonist, ONC201. Here we examine potential molecular determinants of response to DRD2 inhibition. Methods: The Cancer Genome Atlas (TCGA) glioblastoma database and other published mRNA profiles were used to analyze the DRD2 expression pattern. In vitro and in vivo responses to ONC201 were determined using patient derived xenograft glioblastoma models. Immunohistochemical studies were performed on clinically annotated glioblastoma samples derived from phase I/II clinical trials involving ONC201. Results: For the majority of clinical glioblastoma specimens in both the TCGA and non-TCGA dataset, epidermal growth factor receptor (EGFR) expression was inversely correlated with DRD2. This observation was recapitulated in a panel of patient-derived glioblastoma lines. In this panel of DRD2 expressing lines, high EGFR expression was associated with poor response to ONC201 in vitro and in vivo. Moreover, ectopic expression of EGFR reduced DRD2 expression and ONC201 sensitivity, suggesting functional redundancy between DRD2 and EGFR. In cell lines and clinical glioblastoma samples, DRD2 expression closely associated with the expression of rate-limiting enzymes for dopamine synthesis, suggesting dependency of a subset of glioblastomas on autocrine DRD2 signaling. Analysis of specimens from patients treated with ONC201 (n = 15) showed an inverse correlation between the intensity of EGFR staining and clinical response. The median overall survival for patients with high and low EGFR staining was 162 and 373 days, respectively (p = 0.037). All patients who exhibited progression free survival beyond 200 days showed low to no EGFR expression. Conclusions: Our results suggest EGFR expression as a determinant of response to ONC201 in glioblastoma patients and should inform the design of future clinical trials.

EGFR cut-off point for prognostic impact in laryngeal squamous cell carcinoma

Background: Various molecular biomarkers, including epidermal growth factor receptor (EGFR), have gained importance as predictors of head and neck cancer disease progression.Aims: This study aimed to investigate the ability of EGFR expression as a prognostic marker for laryngeal squamous cell carcinoma (LSCC).Materials and methods: A total of 31 patients with LSCC with immunohistochemistry (IHC) staining for EGFR were examined. Digital image processing was applied to analyze EGFR staining intensity and percent distribution, which were calculated as the H-score. We used a receiver operating characteristic (ROC) curve to identify the best cut-off point of EGFR expression, with H-score separated into high- and low-grade for cancer recurrence prediction.Results: The cut-off point of EGFR expression for high- and low-grades was an H-score of 170 with a sensitivity of 75% and a specificity of 66.7%. Using this cut-off, 14 (45.16%) and 17 (54.84%) patients were categorized as having high- and low-grades EGFR, respectively. The analysis showed a significant reverse correlation between the EGFR grade and LSCC recurrence (RR, 0.4; 95% CI, 0.17–0.98; p = .02).Conclusions: Our study demonstrated that EGFR grading using H-score with the generated cut-off point by the ROC curve might be further applied as a potential marker for LSCC prognostic prediction.

Prognostic significance of immunohistochemical staining for myoferlin in clear cell renal cell carcinoma and its association with epidermal growth factor receptor expression

ObjectivesTo reveal the prognostic significance of myoferlin expression in clear cell renal cell carcinoma (ccRCC) and its association with epidermal growth factor receptor (EGFR) expression and cytoplasmic features based on lipogenic metabolism. Materials and methodsImmunohistochemical (IHC) staining for myoferlin and EGFR was conducted on 450 resected ccRCC specimens using tissue microarray, which was measured semiquantitatively. MYOF and EGFR mRNA data were acquired from the TCGA database. The cytoplasmic characteristics of ccRCC based on lipogenic metabolism were retrieved from previous study. Survival analyses were carried out for progression-free, overall, and cancer-specific survival using Kaplan-Meier with the log-rank tests and Cox regression models. Discrimination of prognostic prediction was analyzed by Harrell's C-index. In addition, cross-correlation analyses of myoferlin expression with clinicopathological characteristics and EGFR expression were performed using Pearson's χ2, Mann-Whitney U, and Pearson's r tests. ResultsHigh expression of myoferlin, which was related to high WHO grade (P< 0.001) and high TNM stage (P= 0.074), was significantly associated with short progression-free (P< 0.001), overall (P< 0.001), and cancer-specific survival (P< 0.001) of ccRCC patients. In multivariate analysis, IHC staining for myoferlin was independently associated with progression-free (hazard ratio [HR] = 1.734, P= 0.021), overall (HR = 1.750, P= 0.004), and cancer-specific (HR = 1.723, P= 0.044) survival when adjusted for TNM stage and WHO grade, with significantly improved C-indices. Furthermore, the expression of myoferlin and EGFR showed positive relationship at the protein (P< 0.001) and at the mRNA levels (r = 0.478, P< 0.001). In addition, clear to light-granular cytoplasm, representative of low-risk lipogenic metabolism, was predominantly observed in myoferlin-low ccRCC (P= 0.004). ConclusionsIHC staining for myoferlin was a useful prognostic biomarker of ccRCC. We hypothesize that EGFR overexpression and lipogenic metabolism alteration underlie the protumorous functions of myoferlin on ccRCC.