Tumor Epidermal Growth Factor Receptor Research Articles

Quantitative cell-free circulating EGFR mutation concentration is correlated with tumor burden in advanced NSCLC patients

ObjectivesDroplet digital polymerase chain reaction (ddPCR) has shown sufficient concordance in detecting plasma epidermal growth factor receptor (EGFR) status in non-small cell lung cancer (NSCLC), compared to tumor tissues. However, the clinical significance of the quantitative plasma mutated EGFR concentration remains unknown. The purpose of this study was to explore the relationship of plasma mutated EGFR concentration with tumor burden in advanced NSCLC patients. Materials and methodsUsing ddPCR, plasma DNA samples prior to administration of therapies from 113 consecutive NSCLC patients were analyzed for EGFR L858R substitution and deletion of exon19 (ex19del). Plasma EGFR status was compared to tumor EGFR status to determine concordance. Then, we assessed the correlation of plasma mutated EGFR concentrations with tumor burden and other tumor characteristics. Results and conclusionCompared to tumor EGFR, the concordance rate of plasma and tissue EGFR status was 86.73%. Of the 64 patients who harbored tumor EGFR mutation, plasma mutated EGFR concentrations significantly correlated with number of metastatic sites (Spearman’s r=0.4954, p<0.0001), number of lesions (Spearman’s r=0.4484, p=0.0002), and sum of measurable lesions’ diameters (Spearman’s r=0.3539, p=0.0048). Number of metastatic sites was independently associated with mutated EGFR concentration in multiple linear regression. Besides, plasma mutated EGFR concentrations were significantly higher in those with extensive tumor burden (median concentration, 386.9 vs. 13.4copies/mL; p<0.0001) and stage IV disease (median concentration, 244.2 vs. 0copies/mL; p=0.0252). In conclusion, mutated plasma EGFR concentration determined by ddPCR analysis significantly correlated with tumor burden.

EGFR in Tumor-Associated Myeloid Cells Promotes Development of Colorectal Cancer in Mice and Associates With Outcomes of Patients

Background & AimsInhibitors of the epidermal growth factor receptor (EGFR) are the first-line therapy for patients with metastatic colorectal tumors without RAS mutations. However, EGFR inhibitors are ineffective in these patients, and tumor level of EGFR does not associate with response to therapy. We screened human colorectal tumors for EGFR-positive myeloid cells and investigated their association with patient outcome. We also performed studies in mice to evaluate how EGFR expression in tumor cells and myeloid cells contributes to development of colitis-associated cancer and ApcMin-dependent intestinal tumorigenesis.MethodsWe performed immunohistochemical and immunofluorescent analyses of 116 colorectal tumor biopsies to determine levels of EGFR in tumor and stroma; we also collected information on tumor stage and patient features and outcomes. We used the Mann-Whitney U and Kruskal-Wallis tests to correlate tumor levels of EGFR with tumor stage, and the Kaplan-Meier method to estimate patients’ median survival time. We performed experiments in mice lacking EGFR in intestinal epithelial cells (Villin-Cre; Egfrf/f and Villin-CreERT2; Egfrf/f mice) or myeloid cells (LysM-Cre; Egfrf/f mice) on a mixed background. These mice were bred with ApcMin/+ mice; colitis-associated cancer and colitis were induced by administration of dextran sodium sulfate (DSS), with or without azoxymethane (AOM), respectively. Villin-CreERT2 was activated in developed tumors by administration of tamoxifen to mice. Littermates that expressed full-length EGFR were used as controls. Intestinal tissues were collected; severity of colitis, numbers and size of tumors, and intestinal barrier integrity were assessed by histologic, immunohistochemical, quantitative reverse transcription polymerase chain reaction, and flow cytometry analyses.ResultsWe detected EGFR in myeloid cells in the stroma of human colorectal tumors; myeloid cell expression of EGFR associated with tumor metastasis and shorter patient survival time. Mice with deletion of EGFR from myeloid cells formed significantly fewer and smaller tumors than the respective EGFR-expressing controls in an ApcMin/+ background as well as after administration of AOM and DSS. Deletion of EGFR from intestinal epithelial cells did not affect tumor growth. Furthermore, tamoxifen-induced deletion of EGFR from epithelial cells of established intestinal tumors in mice given AOM and DSS did not reduce tumor size. EGFR signaling in myeloid cells promoted activation of STAT3 and expression of survivin in intestinal tumor cells. Mice with deletion of EGFR from myeloid cells developed more severe colitis after DSS administration, characterized by increased intestinal inflammation and intestinal barrier disruption, than control mice or mice with deletion of EGFR from intestinal epithelial cells. EGFR-deficient myeloid cells in the colon of DSS-treated LysM-Cre; Egfrf/f mice had reduced expression of interleukin 6 (IL6), and epithelial STAT3 activation was reduced compared with controls. Administration of recombinant IL6 to LysM-Cre; Egfrf/f mice given DSS protected them from weight loss and restored epithelial proliferation and STAT3 activation, compared with administration of DSS alone to these mice.ConclusionsIncreased expression of EGFR in myeloid cells from the colorectal tumor stroma associates with tumor progression and reduced survival time of patients with metastatic colorectal cancer. Deletion of EGFR from myeloid cells, but not intestinal epithelial cells, protects mice from colitis-induced intestinal cancer and ApcMin-dependent intestinal tumorigenesis. Myeloid cell expression of EGFR increases activation of STAT3 and expression of survivin in intestinal epithelial cells and expression of IL6 in colon tissues. These findings indicate that expression of EGFR by myeloid cells of the colorectal tumor stroma, rather than the cancer cells themselves, contributes to tumor development.

Immunohistochemical Expression of Epidermal Growth Factor Receptor in Head and Neck Squamous Cell Carcinoma.

To determine the frequency of expression of epidermal growth factor receptor (EGFR) by immunohistochemistry in head and neck squamous cell carcinoma (HNSCC). Cross-sectional study. Armed Forces Institute of Pathology (AFIP), Rawalpindi, from September 2015 to March 2016. Fifty-two cases of head and neck squamous cell carcinoma diagnosed on H&E stain were included in the study. Patients' gender and age were noted. Immunohistochemistry for EGFR was applied and the results were recorded. The data were analyzed by using computer software program SPSS version 19. Descriptive statistics, frequencies and percentages were calculated. Out of the 52 patients of HNSCC, 37 patients were males and 15 females. The age of the patients was between 21 and 80 years with an average age of 58.58 ±12.63. Out of 52 cases, 45 cases (86.53%) were positive for EGFR while 7 cases (13.46%) were negative for EGFR. Significant statistical association was not seen between the tumour grade and EGFR expression (p=0.162). The high expression of EGFR in head and neck cancers among Pakistani patients suggests its value as a therapeutic target. EGFR inhibitors have become well-known part of HNSCC treatment; therefore, patients with EGFR positive HNSCC can be benefitted from the therapy.

EGFR Induces E2F1-Mediated Corticotroph Tumorigenesis.

The epidermal growth factor receptor (EGFR), expressed in adrenocorticotrophic hormone (ACTH)–secreting pituitary adenomas causing Cushing disease, regulates ACTH production and corticotroph proliferation. To elucidate the utility of EGFR as a therapeutic target for Cushing disease, we generated transgenic (Tg) mice with corticotroph-specific human EGFR expression (corti-EGFR-Tg) using a newly constructed corticotroph-specific promoter. Pituitary-specific EGFR expression was observed by 2.5 months, and aggressive ACTH-secreting pituitary adenomas with features of Crooke’s cells developed by 8 months with 65% penetrance observed. Features consistent with the Cushing phenotype included elevated plasma ACTH and corticosterone levels, increased body weight, glucose intolerance, and enlarged adrenal cortex. Gefitinib, an EGFR tyrosine kinase inhibitor, suppressed tumor POMC expression and downstream EGFR tumor signaling, and ACTH and corticosterone levels were attenuated by 80% and 78%, respectively. Both E2F1 and phosphorylated Ser-337 E2F1 were increased in corti-EGFR-Tg mice and also colocalized with human POMC (hPOMC) in human pituitary corticotroph tumor samples. EGFR inhibition reversed E2F1 activity in vivo, whereas E2F1 inhibition suppressed POMC and ACTH in cultured human pituitary tumor cells. The corti-EGFR-Tg phenotype recapitulates ACTH-secreting pituitary adenomas and Cushing disease, validating the relevance of EGFR to corticotroph tumorigenesis. E2F1 is identified as a promising corticotroph-specific target for ACTH-dependent Cushing disease.

P3.02b-002 Treatment Outcome Comparison between Exon 19 and 21 EGFR Mutations after Second-Line TKIs in Advanced NSCLC Patients: Topic: EGFR Biomarkers

Although superior clinical benefits of first-line epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in the treatment of advanced non-small-cell lung cancer (NSCLC) had been reported with different sensitivity. The sensitivity of second-line TKIs in NSCLC patients with different EFGR mutations was unknown. The purpose of this study is to investigate the clinical outcome of NSCLC patients with and without brain and/or bone metastases in different EGFR tumor genotypes receiving EGFR-TKIs as a second-line treatment. The treatment outcomes of 166 NSCLC patients harboring either the exon 19 deletion or the L858R point mutation of EGFR treated by second-line TKIs were retrospectively reviewed. The disease control rate (DCR) and objective response rate (ORR) of enrolled NSCLC patients were 77.7% and 11.4%, respectively. The exon 19 deletion group had a significantly longer median progression-free survival (PFS) (6.7 vs. 4.5 months, P=0.002) and overall survival (OS) (13.7 vs. 11.7 months, P=0.02) compared with the L858R mutation group for NSCLC patients, as well for patients with brain metastasis [PFS: (6.7 vs. 3.9 months, p<0.001), OS: (13.7 vs. 7.9 months, p=0.006)]. The median PFS and OS for NSCLC patients with bone metastasis were 4.8 months (95% Cl, 4.0-5.6 months) and 12.9 months (95% Cl, 8.7-17.1 months), respectively. No significant difference was observed for patients with bone metastasis for different mutations. EGFR genotype and ECOG PS were independent predictors of PFS for both NSCLC patients with and without brain metastasis. Never smoking (P=0.001), exon 19 deletion (P=0.03), EGOC PS (0-1) (P<0.001) and no brain metastasis (p=0.01) were correlated with longer OS for all NSCLC patients. For patients with brain metastasis, age at disease progression (p=0.009), genotype (p=0.02) and EGOC PS (p<0.001) were independent predictors of OS. For patients with bone metastasis, EGOC PS (p<0.001) was an independent predictor for both PFS and OS. Female (P=0.01), never smoking (P=0.005), number of bone metastases (P=0.03) and EGOC PS (0-1) (P=0.002) were related to a longer PFS. EGOC PS (0-1) (P<0.001) was associated with a longer OS. Rash, fatigue, and anorexia were the three most frequent side effects observed No significant side effects difference between exon 19 and 21 mutation groups were observed. NSCLC patients harboring exon 19 deletion achieved better PFS and OS than those with L858R mutation, indicating that EGFR mutations are significant prognostic factors for advanced NSCLC patients with and without brain metastasis receiving second-line EGFR-TKIs treatment.

P3.02b-019 TTF-1 Expression as a Predictor of Response to EGFR-TKIs in Patients with Lung Adenocarcinoma: Topic: EGFR Biomarkers

Several studies have shown that overexpression of thyroid transcription factor (TTF-1) may be associated with the presence of epidermal growth factor receptor (EGFR) gene mutations and predict survival in patients with non-small cell lung cancer (NSCLC). Nevertheless, the potential significance of TTF-1 immunostaining as a predictor of response to EGFR tyrosine kinase inhibitors (TKIs) has received limited research attention thus far. The aim of this study was to further explore the potential association between TTF-1 immunohistochemical expression and response to EGFR TKIs in patients with lung adenocarcinoma. The medical records of 129 patients with stage IV lung adenocarcinoma, treated at the Oncology Unit of “Sotiria” Athens General Hospital between January 2011 and December 2014, were retrospectively reviewed. All patients had received treatment with EGFR TKIs (erlotinib or gefitinib) and had a known TTF-1 immunohistochemical expression status in formalin-fixed, paraffin-embedded tumor tissue. Demographic and clinicopathological features (age, gender, smoking status and performance status), TTF-1 immunohistochemistry and EGFR mutation status results were correlated to each other as well as with the response rate (RR) to EGFR TKIs, using univariate and multivariate regression analysis. Median age of our study population was 68 years (range 26-88 years), while the majority were male (79/129 cases, 61.2%). Patients with EGFR mutant tumors had significantly higher response rates to EGFR TKIs compared to patients with wild-type EGFR tumors (p=0.001). TTF-1 positive staining was weakly associated with the presence of EGFR mutations, without reaching the level of statistical significance (p=0.053). Most importantly, TTF-1 positive staining was not significantly correlated with RR to EGFR TKIs, when gender, age, smoking status, EGFR mutation status and PS were included in the multivariate model. The present results failed to demonstrate any independent association between TTF-1 overexpression and the RR to EGFR TKIs in patients with advanced-stage lung adenocarcinoma. Prospective data from larger cohorts of patients are needed to clarify the exact predictive value of TTF-1 immunostaining in this setting.

Cell-Free RNA Content in Peripheral Blood as Potential Biomarkers for Detecting Circulating Tumor Cells in Non-Small Cell Lung Carcinoma

Circulating tumor cells (CTCs) have been implicated in tumor progression and prognosis. Techniques detecting CTCs in the peripheral blood of patients with non-small cell lung carcinoma (NSCLC) may help to identify individuals likely to benefit from early systemic treatment. However, the detection of CTCs with a single marker is challenging, owing to low specificity and sensitivity and due to the heterogeneity and rareness of CTCs. Herein, the probability of cell-free RNA content in the peripheral blood as a potential biomarker for detecting CTCs in cancer patients was investigated. An immunomagnetic enrichment of real-time reverse-transcription PCR (RT-PCR) technology for analysis of CTCs in NSCLC patients was also developed. The mRNA levels of four candidate genes, cytokeratin 7 (CK7), E74-like factor 3 (ELF3), epidermal growth factor receptor (EGFR), and erythropoietin-producing hepatocellular carcinoma receptor B4 (EphB4) that were significantly elevated in tumor tissues and peripheral blood mononuclear cells (PBMCs) were determined. The expression of CK7 and ELF3 in tumor tissues and EGFR in PBMCs was associated with lymph node metastasis (all p < 0.05). The expression of CK7 in PBMCs was correlated with age and EphB4 in PBMCs correlated with histopathological type, respectively (all p < 0.05). The expression of all four genes in tumor tissues and PBMCs was significantly correlated with the clinical stage (all p < 0.01). Survival analysis showed that the patients with enhanced expression of CK7, ELF3, EGFR, and EphB4 mRNA in PBMCs had poorer disease-free survival (DFS) and overall survival (OS) than those without (all p < 0.0001). The present study showed that this alteration of cell-free RNA content in peripheral blood might have clinical ramifications in the diagnosis and treatment of NSCLC patients.

527P - Impact of tumor epidermal growth factor receptor (EGFR) status on the outcomes of first-line FOLFOX-4 ± cetuximab in patients (pts) with RAS-wild-type (wt) metastatic colorectal cancer (mCRC) in the randomized phase 3 TAILOR trial

Cetuximab in combination with chemotherapy (either FOLFIRI or FOLFOX) is a standard-of-care first-line treatment for pts with RAS-wt, EGFR-expressing mCRC. In this prospective subgroup analysis of the randomized phase 3 TAILOR trial, we evaluate the impact of EGFR status on the efficacy of FOLFOX-4 ± cetuximab in the first-line treatment of Chinese pts with RAS-wt mCRC. TAILOR is a randomized phase 3 trial that includes a modified intention-to-treat (mITT) population of 393 Chinese pts with RAS-wt mCRC treated with FOLFOX-4 ± cetuximab. The primary endpoint of TAILOR is progression-free survival (PFS); secondary endpoints include overall survival (OS) and overall response rate (ORR). Tumor EGFR detectability was assessed in evaluable pts within the mITT population via immunohistochemistry. Within the mITT population, 193 pts with RAS-wt mCRC were randomized and treated with FOLFOX-4 + cetuximab and 200 pts received FOLFOX-4. Adding cetuximab to FOLFOX-4 significantly improved median PFS (9.2 vs 7.4 mo; HR [95% CI] = 0.691 [0.536-0.891]; p = .004), preliminary assessment of median OS (20.7 vs 17.8 mo; HR [95% CI] = 0.763 [0.607-0.958]; p = .020), and ORR (61.1% vs 39.5%; odds ratio [95% CI] = 2.410[1.607-3.614]; p < .001). Among 354 EGFR-evaluable pts in the mITT population, efficacy gains in PFS and ORR were independent of tumor EGFR status (Table).Tabled 1EGFR statusFOLFOX-4 + cetuximab (n = 173)FOLFOX-4 (n = 181)PFS, median (mo) FOLFOX-4 + cetuximab vs FOLFOX-4 (HR [95% CI])ORR, % FOLFOX-4 + cetuximab vs FOLFOX-4 (odds ratio [95% CI])# of pts# of ptsPercentage of positively staining cells0%85779.2 vs 7.9 (0.62 [0.41-0.92])67.1 vs 35.1 (3.77 [1.97-7.23])> 0%-10%354811.3 vs 7.4 (0.62 [0.35-1.11])71.4 vs 41.7 (3.50 [1.38-8.88])> 10%-20%15149.3 vs 8.1 (0.45 [0.15-1.39])73.3 vs 42.9 (3.67 [0.77-17.4])> 20%-35%9147.5 vs 9.2 (1.34 [0.47-3.86])44.4 vs 64.3 (0.44 [0.08-2.46])> 35%29287.0 vs 5.2 (0.73 [0.40-1.35])44.8 vs 35.7 (1.46 [0.50-4.24]) Open table in a new tab Irrespective of tumor EGFR status, the addition of cetuximab to first-line FOLFOX-4 clearly improved PFS and ORR in Chinese pts with RAS-wt mCRC. The TAILOR study data confirm cetuximab in combination with chemotherapy as a standard-of-care first-line treatment regimen for pts with RAS-wt mCRC, independent of tumor EGFR status.

975P - Individual ADCC capability predict treatment outcome under cetuximab-based therapy in head and neck cancer?

Cetuximab is a IgG1 monoclonal antibody against epidermal growth factor receptor (EGFR) used for the treatment of local advanced-head and neck squamous cell carcinoma (LA-HNSCC). A working mechanism of cetuximab include the ability to develop antibody-dependent cell-mediated cytotoxicity (ADCC), triggered by the interaction of the Fc portion of the antibody with the Fc receptor on the immune cell, mainly on Natural Killer (NK) cells. Invariant NKT (iNKT) cells play an important role in the activation of immune cell subset, including NK cells, and their deficiency is related to poor clinical outcome in LA-HNSCC. The aim of this work was to investigate the predictive role of ADCC and iNKT in LA-HNSCC patients treated with cetuximab-based therapy. Twenty-eight LA-HNSCC patients treated with curative intent with cetuximab-based radio-chemotherapy were analysed. Peripheral blood samples were collected at start of therapy. ADCC was evaluated as ex vivo NK-dependent activity measuring LDH release as previously standardized in our laboratory. EGFR tumoral expression was analyzed by immunohistochemistry. ADCC activity above the median value (high) correlated with a better OS as compared to ADCC below the median (low) in LA-HNSCC (p = 0.033). We observed a correlation also between high ADCC and response rate (p = 0.038). By univariate analysis on OS there were ADCC activity tumor size and HPV status as significant pronostic indicators. According to multivariate log-rank analysis only ADCC remained a significant predictor for OS (p = 0.03). If we stratified the EGFR 3+ patients into high and low ADCC performers, we observed an impressive increased OS in those with high ADCC (p = 0.024). iNKT cells number didn't significantly correlate with OS: nonetheless, we observed a trend to a longer survival after 10 months in patients with high iNKT cells (above the median values). ADCC has a strong predictive value of cetuximab response in HNSCC. High tumoral EGFR expression and high ADCC confer a particularly long overall survival and suggest to combine this two biomarkers in order to predict clinical outcome in cetuximab-treated HNSCC patients.

L02 - Individual ADCC capability predict treatment outcome under cetuximab-based therapy in head and neck cancer?

Introduction: Cetuximab is a IgG1 monoclonal antibody against epidermal growth factor receptor (EGFR) used for the treatment of local advanced-head and neck squamous cell carcinoma (LA-HNSCC). The working mechanism of cetuximab include the ability to develop antibody-dependent cell-mediated cytotoxicity (ADCC), triggered by the interaction of the Fc portion of the antibody with the Fc receptor on the immune cell, mainly on Natural Killer (NK) cells. Invariant NKT (iNKT) cells play an important role in the activation of immune cell subset, including NK cells, and their deficiency is related to poor clinical outcome in LA-HNSCC. The aim of this work was to investigate the predictive role of ADCC and iNKT in LA-HNSCC patients treated with cetuximab-based therapy. Patients and methods: Twenty-eight LA-HNSCC patients treated with curative intent with cetuximab-based radio-chemotherapy were analysed. Peripheral blood samples were collected at start of therapy. ADCC was evaluated as ex vivo NK-dependent activity measuring LDH release as previously standardized in our laboratory (Monteverde et al., 2015). EGFR tumoral expression was analyzed by immunohistochemistry. Results: ADCC activity above the median value (high) correlated with a better OS as compared to ADCC below the median (low) in LA-HNSCC (p = 0.033). We observed a correlation also between high ADCC and response rate (p = 0.038). By univariate analysis on OS there were ADCC activity, tumor size and HPV status as significant prognostic indicators. According to multivariate log-rank analysis only ADCC remained a significant predictor for OS (p= 0.03). If we stratified the EGFR 3+ patients into high and low ADCC performers, we found that patients characterized by both high EGFR expression and high ADCC activity showed 100% OS at 44.2+ months compared to a median OS of 23.9 months (range 2.1-53.7) for patients with high EGFR but low ADCC (p = 0.024). iNKT cells number didn't significantly correlate with OS: nonetheless, we observed a trend to a longer survival after 10 months in patients with high iNKT cells (above the median values). Conclusion: ADCC has a strong predictive value in cetuximab-treated LA-HNSCC patients. High tumoral EGFR expression and high ADCC confer a particularly long overall survival and suggest to combine this two biomarkers in order to predict clinical outcome in cetuximab-treated LA-HNSCC patients.

Brain metastasis features and association with tumor epidermal growth factor receptor mutation in patients with adenocarcinoma of the lung.

Epidermal growth factor receptor (EGFR) mutations are frequent in pulmonary adenocarcinoma patients. The association between tumor EGFR mutation and characteristics of brain metastasis (BM) is still unclear. We retrospectively reviewed pulmonary adenocarcinoma patients with and without BMs, and characteristics of BM to analyze the association between tumor EGFR mutation and characteristics of BM. Of 374 cases, 239 had EGFR mutations; 69 had BM at initial diagnosis, and 82 with BMs after treatment. All eligible patients received EGFR-tyrosine kinase inhibitors treatment. Older patients (≥70 years old) were less likely to have BMs than younger patients (25.8% vs 48%, P < 0.001). Patients with higher N stage had higher proportion of BMs (P = 0.006). Patients with exon 19 deletion were more likely to have BMs than those without EGFR mutation (48.1% vs 34.1%, P = 0.021). Patients with exon 19 deletion didn't have significantly higher chance of BMs at initial diagnosis but had higher chance to develop BM after treatment than those without EGFR mutation (35.6% vs 21.2%, P = 0.019). Patients with exon 19 deletion survived longer than those without EGFR mutation (1-year survival rate 95.8% vs 78.7%, P = 0.003). Thus, longer survival may lead to higher proportion of BM occurrence in patients with exon 19 deletion than those without EGFR mutation. In pulmonary adenocarcinoma, there is no significant difference in frequency of BMs at initial diagnosis between patients with EGFR mutation and wild type. However, after treatment, patients with EGFR mutations are significantly more likely to develop BM.

Correlation of EGFR-expression with safety and efficacy outcomes in SQUIRE: a randomized, multicenter, open-label, phase III study of gemcitabine–cisplatin plus necitumumab versus gemcitabine–cisplatin alone in the first-line treatment of patients with stage IV squamous non-small-cell lung cancer

SQUIRE demonstrated addition of necitumumab to gemcitabine and cisplatin significantly improved survival in patients with stage IV sq-NSCLC. Here, we report additional outcomes for the subpopulation of patients with tumor epidermal growth factor receptor (EGFR) protein expression. Patients with pathologically confirmed stage IV sq-NSCLC were randomized 1:1 to receive a maximum of six 3-week cycles of gemcitabine (1250 mg/m(2) i.v., days 1 and 8) and cisplatin (75 mg/m(2) i.v., day 1) chemotherapy with or without necitumumab (800 mg i.v., days 1 and 8). Patients in the chemotherapy plus necitumumab group with no progression continued on necitumumab alone until disease progression or intolerable toxicity. SQUIRE included mandatory tissue collection. EGFR protein expression was detected by immunohistochemistry (IHC) in a central laboratory. Exploratory analyses were pre-specified for patients with EGFR protein expressing (EGFR > 0) and non-expressing (EGFR = 0) tumors. A total of 982 patients [90% of intention-to-treat (ITT)] had evaluable IHC results. The large majority of these patients (95%) had tumor samples expressing EGFR protein; only 5% had tumors without detectable EGFR protein. Overall survival (OS) for EGFR > 0 patients was significantly longer in the necitumumab plus gemcitabine-cisplatin group than in the gemcitabine-cisplatin group {stratified hazard ratio (HR) 0.79 [95% confidence interval (CI) 0.69, 0.92; P = 0.002]; median 11.7 months (95% CI 10.7, 12.9) versus 10.0 months (8.9, 11.4)}. Additionally, an OS benefit was seen in all pre-specified subgroups in EGFR > 0 patients. However, OS HR for EGFR = 0 was 1.52. Adverse events of interest with the largest difference between treatment groups in EGFR > 0 patients (Grade ≥3) were hypomagnesemia (10% versus <1%) and skin rash (6% versus <1%). In line with SQUIRE ITT, addition of necitumumab to gemcitabine-cisplatin significantly prolonged OS and was generally well tolerated in the subpopulation of patients with EGFR-expressing advanced sq-NSCLC. The benefit from addition of necitumumab to chemotherapy was not apparent in this analysis for the small subgroup of patients with non-EGFR-expressing tumors. NCT00981058.

Effects of different ligands on epidermal growth factor receptor (EGFR) nuclear translocation

The epidermal growth factor receptor (EGFR) is activated through binding to specific ligands and generates signals for proliferation, differentiation, migration, and cell survival. Recent data show the role of nuclear EGFR in tumors. Although many EGFR ligands are upregulated in cancers, little is known about their effects on EGFR nuclear translocation. We have compared the effects of six EGFR ligands (EGF, HB-EGF, TGF-α, β-Cellulin, amphiregulin, and epiregulin) on nuclear translocation of EGFR, receptor phosphorylation, migration, and proliferation. Cell fractionation and confocal immunofluorescence detected EGFR in the nucleus after EGF, HB-EGF, TGF-α and β-Cellulin stimulation in a dose-dependent manner. In contrast, amphiregulin and epiregulin did not generate nuclear translocation of EGFR. EGF, HB-EGF, TGF-α and β-Cellulin showed correlations between a higher rate of wound closure and increased phosphorylation of residues in the carboxy-terminus of EGFR, compared to amphiregulin and epiregulin. The data indicate that EGFR is translocated to the nucleus after stimulation with EGF, HB-EGF, TGF-α and β-Cellulin, and that these ligands are related to increased phosphorylation of EGFR tyrosine residues, inducing migration of SkHep-1 cells.

Frequent co-expression of EGFR and NeuGcGM3 ganglioside in cancer: it's potential therapeutic implications.

Interaction between epidermal growth factor receptor (EGFR) signaling with GM3 ganglioside expression has been previously described. However, little is known about EGFR and NeuGcGM3 co-expression in cancer patients and their therapeutic implications. In this paper, we evaluate the co-expression of EGFR and NeuGcGM3 ganglioside in tumors from 92 patients and in two spontaneous lung metastasis models of mice (Lewis lung carcinoma (3LL-D122) in C57BL/6 and mammary carcinoma (4T1) in BALB/c). As results, co-expression of EGFR and NeuGcGM3 ganglioside was frequently observed in 63 of 92 patients (68 %), independently of histological subtype. Moreover, EGFR is co-expressed with NeuGcGM3 ganglioside in the metastasis of 3LL-D122 and 4T1 murine models. Such dual expression appears to be therapeutically relevant, since combined therapy with mAbs against these two molecules synergistically increase the survival of mice treated. Overall, our results suggest that NeuGcGM3 and EGFR may coordinately contribute to the tumor cell biology and that therapeutic combinations against these two targets might be a valid strategy to explore.

Evaluation of EGFR as a prognostic and diagnostic marker for head and neck squamous cell carcinoma patients.

Approximately 90% of all head and neck tumors are squamous cell carcinomas. The overall survival of patients with head and neck squamous cell carcinoma (HNSCC) is low (≤50%). A non-invasive marker of disease progression is sorely required. The present study focused on the plasmatic levels of epidermal growth factor receptor (EGFR) in HNSCC patients (N=92) compared with healthy (N=29) and diabetic [type 2 diabetes mellitus (T2DM); N=26] controls. Enzyme-linked immunosorbent assay using antibodies against the extracellular region of EGFR (L25-S645) was performed. No significant changes were observed between diabetic and healthy controls. However, there were significantly higher EGFR plasma levels in HNSCC patients compared with both control groups (P=0.001 and 0.005, respectively). Receiver operating characteristic curve analysis identified a sensitivity of 76.09%, a specificity of 67.27% and an area under curve of 0.727 for this comparison. No significant association was observed between EGFR plasma levels and tumor stage, tumor grade, lymph node or distant metastasis occurrence, smoking habit or hypertension. However, the presence of human papillomavirus infection and T2DM in HNSCC patients had borderline effect on the plasma EGFR levels. Survival analysis revealed no significant influence of plasmatic EGFR levels on the overall and disease-specific survival of HNSCC patients. In conclusion, EGFR plasma levels appear to be a relatively promising diagnostic, but poor prognostic, HNSCC marker.

Abstract 1303: In vivo analysis of EGFR family signalling as a bypass mechanism in prostate cancer

Abstract Background: Prostate cancers (PCa) rely on androgenic ligands and the androgen receptor (AR) for their growth and survival, making AR inhibition a predominant therapeutic strategy for these tumors. Some prostate tumors however fail this therapy due to ‘bypass’ mechanisms that emerge as a result of prolonged AR targeting. This in vivo study attempted to assess the expression and activation of the epidermal growth factor receptor (EGFR) family (whose role is well-documented in PCa) in response to androgen deprivation therapy (ADT). Methods: Nude mice were implanted (s.c.) with CWR22 tumors (human-patient-derived, androgen-dependent ‘AD’) and its castration-resistant (‘CR’) subline CWR22-Rv1 (relapsed CWR22). Androgen deprivation (i.e. AR inhibition) was achieved by surgical or ‘sham’ castration of mice. Tumors were analyzed (immunohistochemistry/immunoblot) for EGFR/ErbB2/ErbB3/AR proteins and proliferative/apoptotic markers. Results: Castration caused significant tumor regression in AD but not CR tumors. in vitro viability assays demonstrated that castration (mimicked by using charcoal-stripped serum, ‘css’) did not slow down CR cells to the same degree as it did AD cells. At baseline, intratumoral EGFR protein was unchanged in R22 tumors, ErbB2 levels decreased and ErbB3 protein increased in Rv1 tumors. Castration increased ErbB3 but not EGFR or ErbB2 proteins in CWR22 tumors. Phosphorylated forms of these receptors were generally difficult to detect but there was more phosphorylated ErbB3 protein in Rv1 tumors. Downstream of the EGFR family, there was less phosphorylated Erk but not Akt protein in CWR22-Rv1 tumors. Castration decreased Erk protein in AD tumors but increased it in CR tumors. Immunohistochemical quantification revealed that cytoplasmic EGFR and ErbB3 proteins were elevated in CR tumors but reduced in AD tumors. Castration greatly decreased Ki-67 staining in AD but not in CR tumors while the number of TUNEL-positive nuclei and intensity of PARP staining decreased in castrated CR but not in AD tumors. ErbB3 and AR proteins were significantly correlated with DNA damage and proliferation in CWR22 tumors but only nuclear AR levels and proliferation were significantly correlated in CR tumors. Conclusions: We conclude that androgen deprivation therapy may alter EGFR and ErbB3 protein levels and localization in androgen-dependent and castration-resistant tumors. The EGFR family is typically activated at the cell surface hence their presence and activity there, in response to castration, may initiate signalling pathways encouraging tumor cell proliferation and survival. Citation Format: Maitreyee K. Jathal, Thomas M. Steele, Salma Siddiqui, Benjamin A. Mooso, Leandro S. D’Abronzo, Christiana M. Drake, Paramita M. Ghosh. In vivo analysis of EGFR family signalling as a bypass mechanism in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1303.

Abstract 3279: The effect of Erlotinib treatment on human angiogenesis in vitro

Abstract Background: Angiogenesis is a process by which new capillaries develop from previously formed venules. Epidermal growth factor receptor (EGFR) is a member of ErbB family of receptor tyrosine kinases (TKs). The EGFR signaling pathway is activated in proliferation, angiogenesis, tumor growth and progression. Overexpression of EGFR in many solid human tumors associates with poor prognosis. One of the effective strategies to target Erb receptors is use of small molecule inhibitors of the TK domain, such as Erlotinib. We hypothesized that inhibiting EGFR signaling by Erlotinib treatment will decrease physiologic and pathologic angiogenesis in an in vitro model system. Methods: Tissue was embedded in a three-dimensional fibrin-thrombin clot, and evaluated for neovessel growth as per our in vitro Human Angiogenesis Model (HAM). Physiologic angiogenesis was studied in two venous tissues, human placental vein (HPV) and inferior vena cava (IVC). Human liver neuroendocrine tumor (NET) tissue modeled pathologic angiogenesis. All tissues were treated with Erlotinib in a dose response manner (1 μM, 10 μM, 100 μM) and evaluated for three angiogenesis parameters: percent initiation (%I), angiogenic growth (AG), and overall angiogenic response (OAR). Concentrations of angiogenesis-relevant ligand-receptor pairs in supernatant were determined by Human Angiogenesis/Growth panel (Milliplex, EMD Millipore). Effect of Erlotinib treatment on EGF pathway genes expression in IVCs and liver NETs was analyzed by TaqMan EGF pathway array. Angiogenesis data was analyzed for significance using Z-test (Primer) and paired t-test (MedCalc). Results: Selected dose of Erlotinib [10 μM] consistently achieved statistically significant inhibition of both AG (p&amp;lt;0.0001) and OAR (p&amp;lt;0.0001) in all three tissue types compared to the control. Percent inhibition of%I was below 30% for HPV, IVC and liver NETs. Angiogenic growth was inhibited by 45.65% in HPV (p = 0.035), 52.14% in IVC (p&amp;lt;0.0001) and 47.59% in liver NETs (p = 0.0107) and OAR by 60% in HPV (p = 0.105*), 53.61% in IVC (p&amp;lt;0.0001) and 52.27% in liver NET (p = 0.0168). Preliminary ligand-receptor pair concentrations in IVCs and liver NETs show opposing response to Erlotinib treatment. Out of 92 EGF pathway-related genes, only twelve exhibited significant expression level changes [≥2-fold] in both IVC and liver NET Erlotinib-treated samples. Among them, only five genes demonstrated the same direction of change in both tissue types. The CAV2, GAB1, RHOD genes were down-regulated; MUC1 and PIK3c2b genes were upregulated. Conclusions: Erlotinib is an effective antiangiogenic kinase inhibitor in HPV, IVC and liver NETs in-vitro. It impedes physiologic and pathologic angiogenesis via regulation of neovessel growth rather than initiation of sprouts. Five EGF pathway related genes commonly affected in both physiologic and pathologic models by Erlotinib treatment may be essential modulators of human angiogenesis. Citation Format: Tanja Milosavljevic, Elise Juge, Russ Guidry, Eugene Woltering. The effect of Erlotinib treatment on human angiogenesis in vitro. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3279.

Abstract 1414: Treatment outcome under cetuximab-based therapy and individual ADCC capability in head and neck cancer

Abstract Introduction Cetuximab is a IgG1 monoclonal antibody against epidermal growth factor receptor (EGFR) used for head and neck squamous cell carcinoma (HNSCC) treatment. In addition to EGFR targeting, cetuximab is able to develop an antibody-dependent cell-mediated cytotoxicity (ADCC), triggered by Fc receptors (Fcγ-R) on Natural Killer cells, macrophages and polymorphonucleated leukocytes. This prospective study examined the possible prognostic value of cetuximab-mediated ADCC response in a cohort of HNSCC patients. Patients and Methods There were 63 patients treated with curative intent with chemo-radiotherapy (CT-RT), associated (N = 39) or not (N = 24) with cetuximab. Peripheral blood samples were collected at start of therapy. Intrinsic ADCC was evaluated from ex vivo NK-dependent activity measuring LDH release through the Cytotox 96® non radioactive cytotoxicity assay, previously standardized in our laboratory (Crit Rev Oncol Hematol, 2015). EGFR tumoral expression was analyzed by immunohistochemistry. Results Median ADCC response at treatment start for all patients was 56.1% (range 5-99%). Analysing the whole population, patients with ADCC above the median value (high) showed a better OS as compared to patients with ADCC below the median (low), HR = 0.3827 (95% CI, 0.1464 to 1.000), p = 0.05 (Long-rank Mantel-Cox Test). A sub-group analysis confirmed a possible link between high ADCC and a better OS (CT-RT group, p = 0.223 and CT-RT+cetuximab group, p = 0.244). Importantly, considering CT-RT+cetuximab treatment and EGFR tumoral expression (N = 21), patients with EGFR 3+ status and high ADCC showed an improved OS as compared to patients with ADCC below median value, HR = 0.09031 (95% CI, 0.009983 to 0.8170), p = 0.032 (Long-rank Mantel-Cox Test). Median OS values for patients with high EGFR expression were 34.8 months (range 9.1-53.7) and 13.1 months (range 2.1-39.5) for patients with respectively high ADCC and low ADCC. Conclusion ADCC is shown to be a strong driver of cetuximab-based therapy outcome in HNSCC. High tumoral EGFR expression and high ADCC confer a particularly long overall survival. These data suggest an ADCC-based and EGFR expression-based precision therapy under cetuximab in HNSCC and open perspectives for NK boosting. Citation Format: Cristiana Lo Nigro, Laura Lattanzio, Daniela Vivenza, Martino Monteverde, Federica Tonissi, Giuliana Strola, Marco Merlano, Gerard Milano. Treatment outcome under cetuximab-based therapy and individual ADCC capability in head and neck cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1414.

PA-MSHA in combination with EGFR tyrosine kinase inhibitor: A new strategy to overcome the drug resistance of non-small cell lung cancer cells

The inhibition of epidermal growth factor receptor (EGFR) signaling by Gefitinib provides a promising treatment strategy for non-small cell lung cancer (NSCLC); however, drug resistance to Gefitinib and other tyrosine kinase inhibitors presents a major issue. Using NSCLC cell lines with differential EGFR status, we examined the potency of PA-MSHA (Pseudomonas aeruginosa-mannose-sensitive hemagglutinin) in combination with Gefitinib on proliferation, apoptosis, cell cycle arrest, EGFR signaling and tumor growth. PC-9, A549, and NCI-H1975 cells were treated with PA-MSHA, Gefetinib, or PA-MSHA plus Gefetinib at different concentrations and times. The effects of the drugs on proliferation, cell cycle distribution and apoptosis were evaluated. The activation of EGFR and apoptotic signaling-related molecules was evaluated by Western blotting in the presence or absence of EGFR siRNA. Tumor growth and pathway signaling activation was assessed by xenografts in nude mice. A time-dependent and concentration-dependent cytotoxic effect of PA-MSHA was observed in all NSCLC cells tested. The combination of PA-MSHA plus Gefitinib enhanced the growth inhibition, sub-G1 content and apoptosis over that observed with either agent alone. Furthermore, the combination of PA-MSHA plus Gefitinib resulted in caspase-3/caspase-9 cleavage and increased inhibition of EGFR-dependent activation of AKT and ERK phosphorylation. Combination treatment was more effective in reducing tumor size and EGFR activation than either agent alone. These data suggest that PA-MSHA and Gefitinib function additively to suppress the proliferative effects of NSCLC cells of differential EGFR status. The combination of PA-MSHA and Gefitinib provides a potential new strategy to conquer drug resistance for anti-EGFR-targeted therapy of NSCLC.

Relationship between serum carcinoembryonic antigen level and epidermal growth factor receptor mutations with the influence on the prognosis of non-small-cell lung cancer patients

ObjectiveTo investigate the relationship between serum carcinoembryonic antigen (CEA) level and epidermal growth factor receptor (EGFR) gene mutations in non-small-cell lung cancer (NSCLC) patients and to analyze the influence of CEA level on postoperative survival time in lung cancer patients.MethodsA total of 296 patients who were treated in Thoracic Surgery Department of Henan Provincial Chest Hospital from September 2011 to September 2013 were recruited. The level of tumor markers, such as CEA, was determined before the surgery, and EGFR gene mutations were detected after surgery. Thereby, the relationship between tumor makers, including CEA, and EGFR mutation and its influence on prognosis could be investigated.ResultsAmong 296 patients, the positive rate of EGFR gene mutation was 37.84% (112/296); the mutation occurred more frequently in nonsmokers, adenocarcinoma patients, women, and patients aged <60 years (P<0.05). Both tumor markers and chemosensitivity indicators were related to the profile of EGFR mutations. Elevated squamous cell carcinoma and Cyfra21-1 as well as positively expressed ERCC1 were more common in patients with wild-type EGFR (P<0.05), whereas increased CEA level was observed more frequently in patients with EGFR gene mutation (P=0.012). The positive rate of EGFR gene mutations was higher as the serum CEA level increased, that is, the positive rate in patients with serum CEA level <5, 5–20, and >20 μg/L was 39.81%, 45.32%, and 65.47%, respectively (P=0.004). Logistic regression analysis showed that CEA level was an independent factor in predicting EGFR gene mutations, and serum CEA level was also an independent factor in affecting the prognosis of NSCLC patients, as the overall 2-year survival rate was 73.86% in elevated CEA group and 86.43% in normal group (P<0.01).ConclusionThe prognosis of NSCLC patients receiving resection can be predicted according to serum CEA level, which is associated with EGFR mutations in NSCLC patients and provides a preliminarily guidance for EGFR mutations.