High Epidermal Growth Factor Receptor Gene Copy Research Articles

EGFR gene copy number as a predictive/biomarker for patients with non-small-cell lung cancer receiving tyrosine kinase inhibitor treatment: a systematic review and meta-analysis

Epidermal growth factor receptor (EGFR) gene copy number has been proposed as a candidate biomarker for predicting treatment response to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in patients with advanced non-small-cell...

Assessment of epidermal growth factor receptor mutation/copy number and K-ras mutation in esophageal cancer.

The molecular status of epidermal growth factor receptor (EGFR) in esophageal cancer has not been well elucidated. The purpose of the study was to investigate the prevalence of EGFR and K-ras mutation, and EGFR gene copy number status as well as its association with clinicopathologic characteristics, and also to identify the prognostic value of EGFR gene copy number in esophageal cancer. EGFR mutation in exon 19/exon 21 and K-ras mutation in codon 12/codon 13 were detected by real-time PCR method, while EGFR gene copy number status was analyzed by fluorescent in situ hybridization (FISH). EGFR gene amplification and high polysomy were defined as high EGFR gene copy number status (FISH-positive), and all else were defined as low EGFR gene copy number status (FISH-negative). The relationship between EGFR gene copy number status and clinicpathologic characteristics was analyzed. Kaplan-Meier method and Cox proportional hazards regression model were employed to evaluate the effects of EGFR gene copy number status on the patients' survival. A total of 57 esophageal squamous cell carcinoma (ESCC) patients and 9 esophageal adenocarcinoma (EADC) patients were enrolled in the study. EGFR mutation was identified in one patient who was diagnosed as ESCC with stage IIIC disease. K-ras mutation was identified in one patient who was diagnosed as EADC. In all, 34 of 66 (51.5%) samples were detected as FISH-positive, which includes 30 ESCC and 4 EADC tumor samples. The correlation analysis showed that FISH-positive was significantly associated with the tumor stage (P=0.019) and lymph node metastasis (P=0.005) in esophageal cancer patients, and FISH-positive was also significantly associated with the tumor stage (P=0.007) and lymph node metastasis (P=0.008) in ESCC patients. Cox regression analysis showed that high EGFR gene copy number was not a significant predictor of a poor outcome for esophageal cancer patients (P=0.251) or for ESCC patients (P=0.092), but esophageal cancer patients or ESCC patients with low EGFR gene copy number may have longer survival than those with high EGFR gene copy number according to the survival curve trends. The results indicated that EGFR or K-ras mutation was rare in esophageal cancer, but high EGFR gene copy number is frequent, and correlated with advanced pathologic stage and more number of the metastatic regional lymph nodes, especially in ESCC. In addition, high EGFR gene copy number is likely to have a deleterious effect on prognosis of esophageal cancer patients or ESCC patients, although no statistical significance was reached in the study.

Epidermal growth factor receptor overexpression is common and not correlated to gene copy number in ependymoma.

The aim of this study was to investigate the epidermal growth factor receptor (EGFR) status in ependymoma specimens, as there is a need for new prognostic and druggable targets in this disease. Ependymomas (WHO grade II, n = 40; WHO grade III, n = 15) located spinal (n = 35), infratentorial (n = 14), and supratentorial (n = 6) of 53 patients with a median age of 40 (range, 2-79) years were analyzed for Ki-67, p53, and EGFR expression by immunohistochemistry using a tissue microarray and for EGFR gene copy number alterations/mutations. Results were correlated to clinical data. EGFR overexpression was found in 30/60% of ependymomas depending on the antibody used and was more pronounced in WHO grade III. High EGFR gene copy number gains were found in 6 (11%) ependymomas with half of them being amplifications. EGFR amplified ependymomas displayed an EGFR overexpression with both antibodies in two of three cases. A missense mutation in exon 20 of EGFR (S768I) was detected in one amplified case. EGFR is frequently overexpressed in ependymomas. Other mechanisms than amplification of the EGFR gene appear to contribute to EGFR overexpression in most cases. EGFR mutations may be present in a small subset of ependymomas.

Epidermal growth factor receptor and AKT1 gene copy numbers by multi‐gene fluorescence in situ hybridization impact on prognosis in breast cancer

The epidermal growth factor receptor (EGFR)/PI3K/AKT signaling pathway aberrations play significant roles in breast cancer occurrence and development. However, the status of EGFR and AKT1 gene copy numbers remains unclear. In this study, we showed that the rates of EGFR and AKT1 gene copy number alterations were associated with the prognosis of breast cancer. Among 205 patients, high EGFR and AKT1 gene copy numbers were observed in 34.6% and 27.8% of cases by multi-gene fluorescence in situ hybridization, respectively. Co-heightened EGFR/AKT1 gene copy numbers were identified in 11.7% cases. No changes were found in 49.3% of patients. Although changes in EGFR and AKT1 gene copy numbers had no correlation with patients' age, tumor stage, histological grade and the expression status of other molecular makers, high EGFR (P = 0.0002) but not AKT1 (P = 0.1177) gene copy numbers correlated with poor 5-year overall survival. The patients with co-heightened EGFR/AKT1 gene copy numbers displayed a poorer prognosis than those with tumors with only high EGFR gene copy numbers (P = 0.0383). Both Univariate (U) and COX multivariate (C) analyses revealed that high EGFR and AKT1 gene copy numbers (P = 0.000 [U], P = 0.0001 [C]), similar to histological grade (P = 0.001 [U], P = 0.012 [C]) and lymph node metastasis (P = 0.046 [U], P = 0.158 [C]), were independent prognostic indicators of 5-year overall survival. These results indicate that high EGFR and AKT1 gene copy numbers were relatively frequent in breast cancer. Co-heightened EGFR/AKT1 gene copy numbers had a worse outcome than those with only high EGFR gene copy numbers, suggesting that evaluation of these two genes together may be useful for selecting patients for anti-EGFR-targeted therapy or anti-EGFR/AKT1-targeted therapy and for predicting outcomes.

Prognostic and predictive values of EGFR overexpression and EGFR copy number alteration in HER2-positive breast cancer.

Background:Epidermal growth factor receptor (EGFR) is overexpressed in a subset of human epidermal growth factor receptor 2 (HER2)-positive breast cancers, and coexpression of HER2 and EGFR has been reported to be associated with poor clinical outcome. Moreover, interaction between HER2 and EGFR has been suggested to be a possible basis for trastuzumab resistance.Methods:We analysed the clinical significance of EGFR overexpression and EGFR gene copy number alterations in 242 HER2-positive primary breast cancers. In addition, we examined the correlations between EGFR overexpression, trastuzumab response and clinical outcome in 447 primary, and 112 metastatic HER2-positive breast cancer patients treated by trastuzumab.Results:Of the 242 primary cases, the level of EGFR overexpression was 2+ in 12.7% and 3+ in 11.8%. High EGFR gene copy number was detected in 10.3%. Epidermal growth factor receptor overexpression was associated with hormone receptor negativity and high Ki-67 proliferation index. In survival analyses, EGFR overexpression, but not high EGFR copy number, was associated with poor disease-free survival in all patients, and in the subgroup not receiving adjuvant trastuzumab. In 447 HER2-positive primary breast cancer patients treated with adjuvant trastuzumab, EGFR overexpression was also an independent poor prognostic factor. However, EGFR overexpression was not associated with trastuzumab response, progression-free survival or overall survival in the metastatic setting.Conclusions:Epidermal growth factor receptor overexpression, but not high EGFR copy number, is a poor prognostic factor in HER2-positive primary breast cancer. Epidermal growth factor receptor overexpression is a predictive factor for trastuzumab response in HER2-positive primary breast cancer, but not in metastatic breast cancer.

High EGFR gene copy number predicts poor outcome in triple-negative breast cancer

Epidermal growth factor receptor (EGFR) is frequently overexpressed in triple-negative breast cancer and is emerging as a therapeutic target. EGFR gene copy number alteration and mutation are highly variable and scientists have been challenged to define their prognostic significance in triple-negative breast cancer. We examined EGFR protein expression, EGFR gene copy number alteration and mutation of exon 18 to 21 in 151 cases of triple-negative breast cancer and correlated these findings with clinical outcomes. In addition, intratumoral agreement of EGFR protein overexpression and gene copy number alteration was evaluated. EGFR overexpression was found in 97 of 151 cases (64%) and high EGFR gene copy number was detected in 50 cases (33%), including 3 gene amplification (2%) and 47 high polysomy (31%). Five EGFR mutations were detected in 4 of 151 cases (3%) and included G719A in exon 18 (n=1), V786M in exon 20 (n=1), and L858R in exon 21 (n=3). One case had two mutations (G719A and L858R). High EGFR copy number, but not EGFR mutation, correlated with EGFR protein overexpression. Intratumoral heterogeneity of EGFR protein overexpression and EGFR copy number alteration was not significant. In survival analyses, high EGFR copy number was found to be an independent prognostic factor for poor disease-free survival in patients with triple-negative breast cancer. Our findings showed that EGFR mutation was a rare event, but high EGFR copy number was relatively frequent and correlated with EGFR overexpression in triple-negative breast cancer. Moreover, high EGFR copy number was associated with poor clinical outcome in triple-negative breast cancer, suggesting that evaluation of EGFR copy number may be useful for predicting outcomes in patients with triple-negative breast cancer and for selecting patients for anti-EGFR-targeted therapy.

Synergistic Antitumor Activity of Cetuximab and Namitecan in Human Squamous Cell Carcinoma Models Relies on Cooperative Inhibition of EGFR Expression and Depends on High EGFR Gene Copy Number

Despite the frequent overexpression of epidermal growth factor receptor (EGFR) in squamous cell carcinoma (SCC), the efficacy of cetuximab alone is limited. Given the marked activity of namitecan, a hydrophilic camptothecin, against SCC models, the present study was performed to explore the efficacy of the cetuximab-namitecan combination in a panel of SCC models. We examined the antiproliferative and antitumor activities of the cetuximab-namitecan combination in four SCC models characterized by a different EGFR gene copy number/EGFR protein level. We also assessed the effects of the combination on EGFR expression at both mRNA and protein levels and investigated the molecular basis of the interaction between the two agents. Cetuximab and namitecan exhibited synergistic effects, resulting in potentiation of cell growth inhibition and, most importantly, enhanced therapeutic efficacy, with high cure rates in three SCC models characterized by high EGFR gene copy number, without increasing toxicity. The synergistic antitumor effect was also observed with the cetuximab-irinotecan combination. At the molecular level, the two agents produced a cooperative effect resulting in complete downregulation of EGFR. Interestingly, when singly administered, the camptothecin was able to strongly decrease EGFR expression mainly by transcriptional inhibition. Our results (i) demonstrate a marked efficacy of the cetuximab-namitecan combination, which reflects a complete abrogation of EGFR expression as a critical determinant of the therapeutic improvement, in SCC preclinical models, and (ii) suggest EGFR gene copy number as a possible marker to be used for patient selection in the clinical setting.

Highlights of This Issue

The epidermal growth factor receptor (EGFR) is a validated therapeutic target in many human cancers. In spite of the frequent EGFR overexpression in squamous cell carcinoma (SCC), the anti-EGFR antibody, cetuximab, as a single agent exhibits marginal efficacy. Our study shows an impressive efficacy of cetuximab/namitecan combination, which reflects a complete abrogation of EGFR expression, in SCC preclinical models characterized by high EGFR gene copy number. The study provides a molecular basis for a rational combination to be exploited for possible therapeutic applications in the clinical setting and suggests EGFR gene copy number as a possible marker for patient selection.Shortened constitutional telomere length is a well-established risk factor for the development of primary malignancies, related to genomic instability that may result from excessive telomere shortening. However, the relationship between telomere length and risk for second malignancies has not been explored. Gramatges and colleagues performed a matched case-control analysis of telomere length between survivors with and without second malignancies within the Childhood Cancer Survivor Study. A relationship was observed with shortened telomeres, driven by a highly significant association with thyroid second cancers. These results suggest cancer predisposition related to genetic or therapy-related telomere shortening, or other factors affecting telomere homeostasis.Both protein kinase C (PKC) and Twist1 play an important role in cancer pathology, including epithelial-mesenchymal transition as well as treatment resistance. Shiota and colleagues have explored the signaling pathway evoked by androgen-deprivation therapy (ADT) for prostate cancer. ADT induced PKC activation and Twist1 induction. On the other hand, an inhibition of PKC, especially PKCβ and PKCϵ, suppressed Twist1 expression, indicating the novel relationship between PKC and Twist1. Accordingly, PKC inhibition combined with ADT such as novel antiandrogen enzalutamide exerted excellent synergistic anticancer effects, suggesting that PKC inhibition as an alternative of Twist1 inhibition might be a promising therapeutic strategy.The uptake transporter SLC22A1 (OCT1) has been suggested to contribute to imatinib resistance; however, its role for imatinib transport into cells is controversial. Nies and colleagues applied an array of in vitro and in vivo model systems to systematically elucidate the contribution of OCT1 to imatinib transport. Using various OCT1-overexpressing cells, leukemic cells being devoid of OCT1 mRNA and protein, and Oct1-deficient mice the authors demonstrate that cellular uptake of imatinib is independent of OCT1. Therefore, OCT1 is apparently not a valid biomarker for imatinib resistance.

Association between epidermal growth factor receptor gene copy number and ERCC1, BRCA1 protein expression in Chinese patients with non-small cell lung cancer

Mutation in epidermal growth factor receptor (EGFR) gene may predict response to chemotherapy in non-small cell lung cancer (NSCLC). However, the correlation between EGFR gene copy status and protein levels of drug-resistant genes, such as excision repair cross-complementing 1 (ERCC1) and breast cancer 1 (BRCA1), remains unclear. We retrospectively analyzed formalin-fixed, paraffin-embedded tumor tissues from 109 Chinese patients with NSCLC. EGFR gene copy number was evaluated by fluorescence in situ hybridization (FISH), and protein levels of platinum-resistance-associated genes, including ERCC1 and BRCA1, were determined by immunohistochemical staining. High EGFR gene copy (EGFR FISH-positive) was found in 21.1% of the patients (amplification in 7.3% and high polysomy in 13.8%, respectively). Immunohistochemical analysis revealed that ERCC1 protein expression was not associated with clinicopathological factors, whereas a significantly higher BRCA1 positive rate was found in poorly differentiated tumors (P=0.02). Further association studies demonstrated that EGFR gene copy number status was not correlated with protein levels of ERCC1 or BRCA1; however, expression of ERCC1 was significantly associated with that of BRCA1 in this set of Chinese patients with NSCLC (P<0.001, r=0.484). Our study demonstrated that EGFR gene copy number status was not correlated with ERCC1 or BRCA1 protein expression, but ERCC1 protein levels were significantly correlated to BRCA1 protein expression levels in tumor tissues from Chinese patients with NSCLC.

Abstract 2396: High EGFR gene copy number predicts poor outcome in triple-negative breast cancer.

Abstract INTRODUCTION: Epidermal growth factor receptor (EGFR) is frequently expressed in triple-negative breast cancer and is emerging as a new therapeutic target. However, the rate of EGFR gene copy number alteration and mutation has been reported to be quite variable and their prognostic significance is poorly defined in triple-negative breast cancer. METHOD: We examined EGFR protein expression, gene copy number alteration and mutation in 150 cases of tripe-negative breast cancer and correlated their findings with clinical outcome of the patients. EGFR protein expression was evaluated by immunohistochemistry using whole tissue sections and EGFR copy number alteration was assessed by dual-color fluorescence in situ hybridization using tissue microarrays. EGFR mutation analysis was performed by polymerase chain reaction of exon 18 to 21 of EGFR gene and direct sequencing of PCR products. RESULT: EGFR protein was expressed in 117 of 150 cases (78.0%). High EGFR gene copy number was found in 33.3% of the cases (amplification in 2.0% and high polysomy in 31.3%). Five EGFR mutations were detected in 4 of 150 cases (2.7%) with G719A in exon 18 (n=1), V786M in exon 20 (n=1) and L858R in exon 21 (n=3). One case had two mutations (G719A and L858R). High EGFR copy number, but not EGFR mutation, was correlated with EGFR protein over-expression. In survival analyses, high EGFR copy number gain was found to be a poor prognostic factor for relapse-free survival (p=0.046), especially for metastasis-free survival (p=0.016). CONCLUSION: EGFR mutation is found to be a rare event in triple-negative breast cancer. However, high EGFR copy number is relatively frequent and is associated with poor clinical outcome in triple-negative breast cancer, suggesting that evaluation of EGFR gene copy number is beneficial for the prediction of outcome in patients with triple-negative breast cancer and selection of the patients for anti-EGFR targeted therapy. Citation Format: Heae Surng Park, Min Hye Jang, Eun Joo Kim, Hyun Jeong Kim, Hee Jin Lee, So Yeon Park. High EGFR gene copy number predicts poor outcome in triple-negative breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2396. doi:10.1158/1538-7445.AM2013-2396

EGFR status and EGFR ligand expression influence the treatment response of head and neck cancer cell lines

Combination treatment (chemoradiotherapy) is the standard treatment for locally advanced head and neck squamous cell carcinoma (HNSCC); however, treatment resistance and local recurrence are significant problems. A high level of epidermal growth factor receptor (EGFR) has been associated with a more aggressive phenotype as well as decreased responsiveness to radio- or chemotherapy. We examined the role of EGFR status and EGFR ligand expression for the treatment response. Intrinsic sensitivity to radiotherapy, cisplatin, and cetuximab treatments was investigated in 25 HNSCC cell lines. EGFR gene copy number, mRNA and protein expression, EGFR and Akt phosphorylation status, and mRNA expression of the EGFR ligands were analyzed using quantitative PCR and ELISA and assessed for their impact on treatment sensitivity. Different treatment modalities yielded great diversity in outcome; of note, cetuximab treatment stimulated growth in one cell line. When treatments were combined primarily additive effects were observed. While radioresistance tended to be associated with a high level of phosphorylated EGFR (pEGFR; P = 0.09), cetuximab-resistant cells had low levels of pEGFR (P = 0.13). The three most cetuximab-sensitive cell lines had high EGFR gene copy numbers. Furthermore, cetuximab treatment response was significantly correlated with epiregulin mRNA expression (r = -0.408, P = 0.043). Cisplatin-resistant tumor cells expressed significantly lower levels of EGFR protein (P = 0.04) compared to cisplatin-sensitive cells and tended to have lower levels of phosphorylated Akt (pAkt; P = 0.13) and lower expression levels of amphiregulin (P = 0.18). Epidermal growth factor receptor status and ligand expression influence the treatment sensitivity of HNSCC cells and may be useful as predictive markers.

Biomarker Analyses and Final Overall Survival Results From a Phase III, Randomized, Open-Label, First-Line Study of Gefitinib Versus Carboplatin/Paclitaxel in Clinically Selected Patients With Advanced Non–Small-Cell Lung Cancer in Asia (IPASS)

The results of the Iressa Pan-Asia Study (IPASS), which compared gefitinib and carboplatin/paclitaxel in previously untreated never-smokers and light ex-smokers with advanced pulmonary adenocarcinoma were published previously. This report presents overall survival (OS) and efficacy according to epidermal growth factor receptor (EGFR) biomarker status. In all, 1,217 patients were randomly assigned. Biomarkers analyzed were EGFR mutation (amplification mutation refractory system; 437 patients evaluable), EGFR gene copy number (fluorescent in situ hybridization; 406 patients evaluable), and EGFR protein expression (immunohistochemistry; 365 patients evaluable). OS analysis was performed at 78% maturity. A Cox proportional hazards model was used to assess biomarker status by randomly assigned treatment interactions for progression-free survival (PFS) and OS. OS (954 deaths) was similar for gefitinib and carboplatin/paclitaxel with no significant difference between treatments overall (hazard ratio [HR], 0.90; 95% CI, 0.79 to 1.02; P = .109) or in EGFR mutation-positive (HR, 1.00; 95% CI, 0.76 to 1.33; P = .990) or EGFR mutation-negative (HR, 1.18; 95% CI, 0.86 to 1.63; P = .309; treatment by EGFR mutation interaction P = .480) subgroups. A high proportion (64.3%) of EGFR mutation-positive patients randomly assigned to carboplatin/paclitaxel received subsequent EGFR tyrosine kinase inhibitors. PFS was significantly longer with gefitinib for patients whose tumors had both high EGFR gene copy number and EGFR mutation (HR, 0.48; 95% CI, 0.34 to 0.67) but significantly shorter when high EGFR gene copy number was not accompanied by EGFR mutation (HR, 3.85; 95% CI, 2.09 to 7.09). EGFR mutations are the strongest predictive biomarker for PFS and tumor response to first-line gefitinib versus carboplatin/paclitaxel. The predictive value of EGFR gene copy number was driven by coexisting EGFR mutation (post hoc analysis). Treatment-related differences observed for PFS in the EGFR mutation-positive subgroup were not apparent for OS. OS results were likely confounded by the high proportion of patients crossing over to the alternative treatment.

Epidermal Growth Factor Receptor Inhibitor Gefitinib Added to Chemoradiotherapy in Locally Advanced Head and Neck Cancer

Assess efficacy and toxicity of gefitinib, an epidermal growth factor receptor (EGFR) inhibitor, added to, and in maintenance after, concurrent chemoradiotherapy (CCRT) in locally advanced head and neck cancer (LA-HNC) and correlate outcomes with EGFR gene copy number alterations. Patients with stage III to IV LA-HNC received two cycles of carboplatin/paclitaxel induction chemotherapy (IC) followed by split-course CCRT with fluorouracil, hydroxyurea, twice daily radiotherapy (FHX), and gefitinib (250 mg daily) followed by continued gefitinib for 2 years total. The primary end point was complete response (CR) rate after CCRT. EGFR gene copy number was assessed by fluorescent in situ hybridization. Sixty-nine patients (66 with stage IV disease, 37 with oropharynx primary tumors, and 67 with performance status 0 to 1) were enrolled with a median age of 55 years. Predominant grade 3 or 4 toxicities during IC and CCRT were neutropenia (n = 20) and in-field mucositis (n = 59) and dermatitis (n = 23), respectively. CR rate after CCRT was 90%. After median follow-up of 3.5 years, 4-year overall, progression-free, and disease-specific survival rates were 74%, 72%, and 89%, respectively. To date, one patient has developed a second primary tumor in the aerodigestive tract. In 31 patients with available tissue, high EGFR gene copy number was associated with worse overall survival (P = .02). Gefitinib can be administered with FHX and as maintenance therapy for at least 2 years, demonstrating CR and survival rates that compare favorably with prior experience. High EGFR gene copy number may be associated with poor outcome in patients with LA-HNC treated with this regimen.

Comparison of FISH, PCR, and Immunohistochemistry in Assessing EGFR Status in Lung Adenocarcinoma and Correlation With Clinicopathologic Features

The purpose of this study is to investigate the relationship between mutation status, copy number, and protein expression of epidermal growth factor receptor (EGFR) in lung adenocarcinoma, and to correlate the genetic status and clinicopathologic features. Forty-nine adenocarcinomas were tested by polymerase chain reaction (PCR) for EGFR gene mutations, by fluorescence in situ hybridization (FISH) for increased EGFR gene copy number, and by immunohistochemistry (IHC) for EGFR protein expression. Terminal respiratory unit (TRU) morphology, tumor grade, mitotic count, and pleural and lymphovascular invasion were also examined. Five of 49 tumors (10%) had EGFR mutation by PCR. Eighteen (36%) had high EGFR gene copy number by FISH, all of which were polysomies without gene amplification. Fifteen (31%) had EGFR protein overexpression by IHC, 13 (87%) of which were also positive by FISH. Of the PCR-positive cases, 4/5 (80%) were positive by FISH and 3/5 (60%) were positive by IHC. TRU morphology was observed in all PCR-positive cases and in about 50% of FISH-positive and IHC-positive cases. Pleural invasion was significantly more common in IHC and FISH-positive cases, and lymphovascular invasion was more often present in FISH-positive cases. No statistically significant differences in mitotic count, age, sex, smoking status, degree of differentiation, or stage were seen. Our findings suggest that a staged approach, with FISH testing as the first step, followed by PCR for the FISH-negative cases would be most effective in the identification of patients with EGFR gene alterations.

Protein overexpression and gene amplification of epidermal growth factor receptor in nonsmall cell lung carcinomas: Comparison of four commercially available antibodies by immunohistochemistry and fluorescence in situ hybridization study

Epidermal growth factor receptor (EGFR) overexpression in nonsmall cell lung carcinomas (NSCLC) is variable ranging from 19% to 89% and its prognostic value remains controversial. We tried to investigate (1) EGFR protein expression using four different antibodies, (2) the correlation between protein overexpression and EGFR gene amplification, and (3) the correlation between EGFR genetic status and clinicopathologic features in NSCLC. We examined EGFR protein expression using four different antibodies including Zymed EGFR kit (Clone 31G7), Dako EGFR pharmDx kit (Clone 2-18C9), Dako (Clone H11) and Novocastra (Clone EGFR 113) by immunohistochemical analysis. The protein overexpression was compared to gene amplification status by fluorescence in situ hybridization (FISH). EGFR protein overexpression was observed in 56% of tumors with Zymed EGFR kit, in 51% with Dako EGFR pharmDx kit, in 5% with Dako and in 18% with Novocastra ( p = 0.010). Both Zymed and Dako pharmDx kit were more sensitive than the Dako test (clone H11) and Novocastra clone EGFR 113. EGFR overexpression was more prominent in squamous cell carcinomas (SCC) than adenocarcinomas (ADC) (71% vs. 48%, p = 0.001 with Zymed, 61% vs. 45%, p = 0.011 with Dako pharmDx kit; respectively). EGFR FISH-positivity as represented by high polysomy and gene amplification was observed in 45% of the NSCLC patients. Protein expression levels significantly correlated with the gene copy number per tumor cell ( p < 0.001). Our data showed a higher percentage of positive cells detected by Zymed and Dako pharmDx tests. The EGFR protein overexpression rate varied from 4% to 72% according to different antibody clones and histologic types. EGFR protein expression detected by Zymed and Dako pharmDx was significantly associated with a high EGFR gene copy number.

EGFR protein overexpression and gene copy number increases in oral tongue squamous cell carcinoma

New promising therapeutic agents targeting epidermal growth factor receptor (EGFR) have been developed although clinical information concerning EGFR status in oral tongue squamous cell carcinoma (OTSCC) is limited. We investigated EGFR protein expression and gene copy numbers in 78 pretreatment OTSCC paraffin samples. EGFR protein expression was found in all 78 tumours, of which 72% showed an intense staining. Fifty-four percent of the tumours had high (⩾four gene copies) EGFR gene copy numbers. EGFR gene copy number was significantly associated with EGFR protein expression ( P = 0.002). Pretreatment EGFR staining intensity tended to be associated with non-pathological complete remission after preoperative radiotherapy for Stage II OTSCC. No correlation was found between EGFR status and survival. EGFR FISH results were significantly ( P = 0.003) higher in more advanced tumours (Stages II, III and IV) than in the tumours in Stage I. Non-smokers exhibited a significantly higher EGFR gene copy number and protein overexpression in Stages I and II OTSCC than smokers ( P = 0.001, P = 0.009). In conclusion, EGFR was found to be overexpressed in all OTSCCs making this cancer type interesting for exploring new therapeutic agents targeting the EGFR receptor.

Epidermal growth factor receptor gene amplification in surgical resected Japanese lung cancer

To evaluate the epidermal growth factor receptor (EGFR) protein expression and increased copy number as predictors of clinical outcome in patients with non-small-cell lung cancer (NSCLC), we have performed fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). We investigated the EGFR increased copy number and EGFR protein expression statuses in 109 surgically treated NSCLC cases. The presence or absence of EGFR mutations of kinase domains was analyzed by genotyping analysis and sequences, and already reported. EGFR increased copy number was defined as Cappuzzo et al. criteria. FISH positive was found from 36/109 (33.0%) lung cancer patients, including 30 high polysomy cases and 6 gene amplification cases. FISH-positive cases were significantly correlated with worse prognosis (log-rank test p = 0.0097). Within EGFR-mutant patients ( n = 55), FISH-positive cases were also correlated with poor prognosis ( p = 0.0255). FISH-negative tumors were found to be more frequently well-differentiated histology. Smoking status (never smoker vs. smoker, p = 0.1510), and gender ( p = 0.5248) did not correlated with FISH positive. EGFR IHC results were correlated with FISH results ( p = 0.004), but not correlated with prognosis ( p = 0.2815). Although EGFR FISH-positive rate did not correlated with EGFR mutation ( p = 0.1973), EGFR polysomy or amplification cases were correlated with EGFR mutations ( p = 0.0023). In conclusion, the EGFR FISH-positive rate in Japanese patients with NSCLC was similar to rates in Western populations, unlike the higher frequencies of EGFR mutation in East Asians. A high EGFR gene copy number might have shorter survival in NSCLC.

Inverse relationship between the length of the EGFR CA repeat polymorphism in lung carcinoma and protein expression of EGFR in the carcinoma

There is a CA dinucleotide repeat polymorphism in the first intron of the epidermal growth factor receptor (EGFR) gene. Our aim was to examine the relationship between the CA repeat length in lung carcinoma and EGFR mutation, EGFR gene copy number, and EGFR protein expression level in the carcinoma. We examined 168 lung carcinomas for the length of the CA repeat polymorphism by PCR-polyacrylamide gel electrophoresis analysis, for EGFR mutations by sequencing analysis, for EGFR gene copy number by fluorescence in situ hybridization analysis, and for EGFR protein expression by immunohistochemical analysis. When the carcinomas were divided into two groups according to the length of their CA repeat polymorphism, the EGFR protein expression level was found to be significantly higher in the shorter allele group than in the longer allele group (P = 0.0116), but its length was not associated with EGFR somatic mutations, high EGFR gene copy numbers, or clinicopathological factors, such as histological type or stage. The results suggested that the length of the EGFR CA repeat polymorphism in lung carcinoma is inversely related with level of EGFR protein expression in the carcinoma.

Role of KRAS and EGFR As Biomarkers of Response to Erlotinib in National Cancer Institute of Canada Clinical Trials Group Study BR.21

To evaluate the effect of KRAS and epidermal growth factor receptor (EGFR) genotype on the response to erlotinib treatment in the BR.21, placebo-controlled trial. We analyzed 206 tumors for KRAS mutation, 204 tumors for EGFR mutation, and 159 tumors for EGFR gene copy by fluorescent in situ hybridization (FISH). We reanalyzed EGFR deletion/mutation using two highly sensitive techniques that detect abnormalities in samples with 5% to 10% tumor cellularity. KRAS mutation was analyzed by direct sequencing. Thirty patients (15%) had KRAS mutations, 34 (17%) had EGFR exon 19 deletion or exon 21 L858R mutations, and 61 (38%) had high EGFR gene copy (FISH positive). Response rates were 10% for wild-type and 5% for mutant KRAS (P = .69), 7% for wild-type and 27% for mutant EGFR (P = .03), and 5% for EGFR FISH-negative and 21% for FISH-positive patients (P = .02). Significant survival benefit from erlotinib therapy was observed for patients with wild-type KRAS (hazard ratio [HR] = 0.69, P = .03) and EGFR FISH positivity (HR = 0.43, P = .004) but not for patients with mutant KRAS (HR = 1.67, P = .31), wild-type EGFR (HR = 0.74, P = .09), mutant EGFR (HR = 0.55, P = .12), and EGFR FISH negativity (HR = 0.80, P = .35). In multivariate analysis, only EGFR FISH-positive status was prognostic for poorer survival (P = .025) and predictive of differential survival benefit from erlotinib (P = .005). EGFR mutations and high copy number are predictive of response to erlotinib. EGFR FISH is the strongest prognostic marker and a significant predictive marker of differential survival benefit from erlotinib.

A Phase II Pharmacodynamic Study of Erlotinib in Patients with Advanced Non–Small Cell Lung Cancer Previously Treated with Platinum-Based Chemotherapy

To examine potential markers of clinical benefit and the effects of erlotinib on the epidermal growth factor receptor (EGFR) signaling pathway in advanced non-small cell lung cancer patients refractory to platinum-based chemotherapy. Patients were given erlotinib (150 mg/d). Tumor biopsies were done immediately before treatment and in a subgroup of patients after 6 weeks' treatment. Of 73 evaluable patients, 7 (10%) had partial response and 28 (38%) had stable disease. In 53 patients with baseline tumor samples, no relationship was observed between pretreatment levels of EGFR, phosphorylated (p)-EGFR, p-AKT, p-mitogen-activated protein kinase (MAPK), or p27 and clinical benefit (i.e., response, or stable disease >/=12 weeks). Tumors from 15 of 57 patients had high EGFR gene copy number, assessed using fluorescence in situ hybridization (FISH positive), 10 of whom had clinical benefit, compared with 5 of 42 FISH-negative patients. FISH-positive patients had longer median progression-free [137 versus 43 days, P = 0.002; hazard ratio (HR), 0.37] and overall (226 versus 106 days, P = 0.267; HR, 0.70) survival than FISH-negative patients. In paired biopsy samples from 14 patients, p-EGFR (P = 0.002), p-MAPK (P = 0.001), and Ki-67 (P = 0.025) levels were significantly reduced after 6 weeks' treatment. Apoptosis was significantly increased in patients with clinical benefit (P = 0.029), and may be a marker of clinical benefit. In this study, EGFR FISH-positive status was associated with improved outcome after erlotinib therapy. Erlotinib led to reduced levels of p-EGFR, p-MAPK, and Ki-67, and stimulated apoptosis in tumor samples from patients with clinical benefit.