Increased Epidermal Growth Factor Receptor Copy Number Research Articles

Cigarette Smoke Containing Acrolein Upregulates EGFR Signaling Contributing to Oral Tumorigenesis In Vitro and In Vivo.

Simple SummaryOral squamous cell carcinoma (OSCC) is one of the most common smoking-related cancer types in the world. Better understanding of the pathophysiology of OSCC would lead to the development of novel therapeutic options. The epidermal growth factor receptor (EGFR) pathway plays a crucial role in the development of OSCC, and aberrant EGFR expression levels have been associated with smoking. Cigarette smoke contains large amounts of aldehydes such as acrolein, which is a highly reactive environmental toxin. In this study, our results present that acrolein is important in oncogenic transformation through activating the EGFR signaling pathway, contributing to oral carcinogenesis. To the best of our knowledge, this is the first study to provide molecular evidence, showing that cigarette smoke containing acrolein contributes to EGFR amplification and activation of downstream signaling in OSCC. Thus, acrolein might be a novel target for early detection and prevention of oral cancer in the future.Oral squamous cell carcinoma (OSCC) accounts for 80–90% of all intraoral malignant neoplasms. The single greatest risk factor for oral cancer is tobacco use, including cigarettes, cigars, chewing tobacco, and snuff. Aberrations of the epidermal growth factor receptor (EGFR) pathway features prominently in oral tumorigenesis and progression. It was shown that cigarette smoking (CS) is associated with worse prognosis in OSCC patients and overexpression of EGFR in tumor tissue. However, the mechanism by which cigarette smoking induced EGFR pathway activation remains to be fully elucidated. Acrolein, an IARC group 2A carcinogen, is a highly reactive aldehyde found in CS. Here we report that acrolein is capable of inducing tumorigenic transformation in normal human oral keratinocytes (NOK). The acrolein-transformed NOK cells showed EGFR copy number amplification, increased EGFR expression, and activation of downstream ERK and AKT signaling pathway. No p53 mutations were observed in acrolein-transformed NOK cells. Inhibiting EGFR pathway using an anti-EGFR antibody, cetuximab, inhibits tumor growth. Furthermore, by examining tissue sample from patients, we found an increased EGFR copy number was positively associated with acrolein-induced DNA damages in OSCC patients. Taken together, our results indicate that acrolein is important in tumorigenic transformation through amplification of EGFR and activating the downstream signaling pathway, contributing to oral carcinogenesis. This is the first study to provide molecular evidence showing that CS containing acrolein contributes to EGFR amplification in OSCC.

Abstract 2571: An integral genomic/expression analysis reveals a cooperative role of APC and TP53 mutations in identifying cetuximab-sensitive colorectal cancer

Abstract The epidermal growth factor receptor (EGFR) is a major therapeutic target in metastatic colorectal cancer (CRC). Mutations in KRAS/NRAS have been identified as the resistance mechanisms to anti-EGFR antibody therapy (cetuximab/pantiumab). However, genomic understanding of the sensitivity to the EGFR-targeted therapy is lacking. A pre-specified, 203-gene cetuximab sensitivity (CTX-S) expression signature score was first validated on two previously published datasets: (1) 77 CTX-treated CRC tumors (progression free survival (PFS)>7 days), and (2) 147 in vitro CTX-treated CRC cell lines. The CTX-S signature was found to be consistently correlated with a newly developed 24-gene APC (truncating) mutations-specific Wnt/β-catenin score on four independent CRC datasets that also include Moffitt (n=468) and TCGA (n=624) CRC datasets. This promoted us to carry out a variety of analyses that revealed a cooperative role of mutant APC + mutant TP53 in identifying cetuximab sensitive subpopulations, which was further evidenced by additional validation analysis on two CRC PDX datasets (n=52 and n=98). While debating or inconclusive, other potential predictive markers of EGFRi therapies have been reported, including BRAF(V600E), PIK3CA mutations, loss of PTEN, and overexpression of HER2 as negative indicators, as well as increased EGFR copy number, and overexpression of EGFR ligands epiregulin and amphiregulin as positive indicators. Here, for the first time, we report that the mutant genes may cooperatively act as positive indicators of EGFRi response. Our study suggests that addition of the routine sequencing of APC and TP53 to extended RAS testing may refine the patient selection strategy to improve cetuximab/pantiumab response and potentially expand the therapeutic opportunity to previously-excluded mutant KRAS/NRAS patients who have “APC + TP53” mutations. Citation Format: Mingli Yang, Michael J. Schell, Andrey Loboda, Michael V. Nebozhyn, Jiannong Li, Jamie K. Teer, W. Jack Pledger, Timothy J. Yeatman. An integral genomic/expression analysis reveals a cooperative role of APC and TP53 mutations in identifying cetuximab-sensitive colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2571.

Epidermal growth factor receptor protein overexpression and gene amplification are associated with aggressive biological behaviors of esophageal squamous cell carcinoma.

Alterations of the epidermal growth factor receptor (EGFR), including overexpression or gene mutations, contribute to the malignant transformation of human epithelial cells. The aim of this study was to assess EGFR overexpression or gene amplification in esophageal squamous cell carcinoma (ESCC) tissue samples and investigate their correlations with biological behaviors. Tissue specimens from 56 patients with surgically resected ESCC were obtained for immunohistochemical analysis of EGFR expression and fluorescence in situ hybridization analysis of EGFR amplification. The data were statistically analyzed to determine the associations with patient clinicopathological and survival data. EGFR was overexpressed in 30 of the 56 (53.6%) ESCC samples and was associated with poor tumor differentiation (P=0.047). EGFR amplification was detected in 13 cases (23.2%) and was associated with advanced pathological stage (P=0.042) and tumor lymph node metastasis (P=0.002). The univariate analysis identified no association between EGFR overexpression and the overall survival (OS) of the patients. By contrast, EGFR amplification predicted ESCC prognosis (P=0.031), while the multivariate analysis revealed a marginal statistical significance for the association between EGFR amplification and OS (P=0.056). EGFR overexpression and increased EGFR copy number were common events in ESCC and contributed to malignant biological behaviors, including tumor dedifferentiation and lymph node metastasis. EGFR amplification may therefore be useful in predicting OS in patients with ESCC.

Prognostic role of epidermal growth factor receptor in head and neck cancer: A meta-analysis

To evaluate the predicting value of the epidermal growth factor receptor (EGFR) for survival in patients with head and neck cancer (HNC). Data were collected from studies comparing overall survival (OS) or progression-free survival (PFS) in patients with higher or lower EGFR levels. Studies were pooled and combined hazard ratios (HRs) of EGFR for survival were calculated. A total of 68 studies involving 6,781 patients were included for meta-analysis. Either EGFR protein expression or gene copy number had prognostic value in HNC patients. EGFR overexpression could predict worse outcome, with HRs of 1.65 (95% CI: 1.45, 1.86) for OS and 1.27 (95% CI: 1.17, 1.37) for PFS. Increased EGFR copy number was also associated with reduced survival, with HRs of 1.5 (95% CI: 1.15, 1.96) for OS and 1.35 (95% CI: 1.14, 1.61) for PFS. Furthermore, EGFR overexpression could predict poorer OS in both eastern and western countries. Particularly, EGFR was considered a strong predictor in laryngeal squamous cell cancer (HR > 2). Elevated EGFR expression and gene copy number could predict poor survival in HNC patients.

Epidermal growth factor receptor protein expression and genomic alterations in renal cell carcinoma

Epidermal growth factor receptor (EGFR) is involved in the progression of many cancer types and represents an important therapeutic target. To determine the role of EGFR in renal cell carcinoma (RCC), the authors analyzed 1088 tumors in a tissue microarray format by using immunohistochemistry and fluorescence in situ hybridization (FISH). A subset of 63 cancers was sequenced for EGFR exon 18 through 21 mutations. EGFR expression was observed in 83.8% of clear cell carcinomas, in 68.2% of papillary carcinomas, in 75% of chromophobe carcinomas, and in 50% of oncocytomas. Within clear cell carcinomas, the expression level of EGFR was associated with high tumor grade (P < .0001), advanced pathologic tumor classification (P < .0001), and, to a lesser extent, lymph node status (P = .0326). FISH analysis revealed increased EGFR copy numbers (high polysomy) in 5.5% of tumors and amplification in 0.1% of tumors. EGFR copy number increases were associated with EGFR protein expression (P = .0015). Within clear cell carcinomas, EGFR copy number increases were associated with high tumor grade (P < .0001), advanced pathologic tumor classification (P = .0472), and lymph node status (P = .0065). No exon 18 through 21 mutations were identified in 63 sequenced tumors. The authors concluded that increased EGFR expression occurs in a fraction of patients who have RCC with an unfavorable histologic phenotype. EGFR copy number gain represents 1 possible cause for EGFR overexpression; however, many over expressing tumors have a normal genotype. High polysomy (which is suggested to be predictive of a response to tyrosine kinase inhibitors) occurs in 5.6% of RCCs. Thus, the potential utility of anti-EGFR medications may be worth further investigation in a small but significant subset of patients with RCC.

Relationship between epidermal growth factor receptor (EGFR) gene copy number, p16 status, and outcome in locally advanced squamous cell carcinoma of the head and neck (LASCCHN).

5528 Background: Human papilloma virus infection (HPV) is a powerful prognostic biomarker in LASCCHN and can be indirectly identified by p16 immunohistochemistry (IHC). Increased EGFR gene copy number has been reported to be a negative predictor of outcome in LASCCHN. In this study the relationship of p16 to EGFR gene copy number in patients treated with chemoradiation was assessed. Methods: Formalin-fixed paraffin embedded tissue was obtained from 110 patients with LASCCHN treated on three clinical trials with conventional chemoradiation or with a tirapazamine containing regimen. EGFR gene copy number was assessed by Fluorescence in situ hybridization (FISH) using LSI EGFR SpectrumOrange/CEP 7 SpectrumGreen probes (Vysis/Abbott). Tumors with high polysomy or amplification of EGFR were scored as positive. p16 was assessed by IHC using a validated antibody (Neomarkers). Markers were correlated with outcome i.e., failure-free survival (FFS) and overall survival (OS) using Cox regression, adjusting for trial. Results: Results for both EGFR FISH and p16 IHC were obtained in 97 patients. EGFR FISH was positive in 21/97 (22%) tumors including 12/66 (18%) oropharyngeal tumors. p16 was positive in 40/97 (41%) tumors including 36/66 (55%) oropharyngeal tumours. Only 1/40 p16 positive tumors were EGFR FISH positive compared with 20/57 p16 negative tumors (p<0.001). EGFR FISH positivity was associated with inferior FFS (HR 2.48 (95%CI 1.14 to 5.38), p = 0.022) and OS (HR 2.33 (95%CI 0.98 to 5.57), p = 0.057). p16 positivity was associated with improved FFS (HR 0.19 (95%CI 0.07 to 0.56), p = 0.002) and OS (HR 0.18 (95%CI 0.05 to 0.62), p = 0.006). On multivariable Cox regression analysis only p16 was independently predictive of outcome (p = 0.007 and 0.0014 for FFS and OS respectively). Conclusions: EGFR FISH positivity and p16 expression were found to be almost mutually exclusive in LASCCHN with only 1 of 97 tumors positive for both markers suggesting that increased EGFR copy number is largely restricted to HPV-negative LASCCHN. EGFR FISH and p16 were predictive of outcome in univariate analyses, but only p16 remained predictive on multivariable analysis. No significant financial relationships to disclose.

Association of epidermal growth factor receptor (EGFR) gene mutations with EGFR amplification in advanced non‐small cell lung cancer

Somatic mutations in the epidermal growth factor receptor (EGFR) gene are associated with the response to EGFR tyrosine kinase inhibitors in patients with non-small cell lung cancer (NSCLC). Increased EGFR copy number has also been associated with sensitivity to these drugs. However, given that it is often difficult to obtain sufficient amounts of tumor tissue for genetic analysis from patients with advanced NSCLC, the relationship between these two types of EGFR alterations has remained unclear. We have now evaluated EGFR mutation status both by direct sequencing and with a high-sensitivity assay, the Scorpion-amplification-refractory mutation system, and have determined EGFR copy number by fluorescence in situ hybridization (FISH) analysis in paired tumor specimens obtained from 100 consecutive patients with advanced NSCLC treated with chemotherapy. EGFR mutations or FISH positivity (EGFR amplification or high polysomy) were apparent in 18% (18/100) and 32% (32/100) of patients, respectively. The Scorpion-amplification-refractory mutation system was more sensitive than direct sequencing for the detection of EGFR mutations. Furthermore, EGFR mutations were associated with EGFR amplification (P = 0.009) but not with FISH positivity (P = 0.266). Our results therefore suggest the existence of a significant association between EGFR mutation and EGFR amplification in patients with advanced NSCLC.

A multicenter stratified phase II study of cetuximab for the treatment of patients with recurrent high-grade glioma

2017 Background: An increased copy number and mutation of the epidermal growth factor receptor (EGFR) gene are frequently found in high-grade gliomas (HGG). We investigated the activity of the EGFR-blocking monoclonal antibody cetuximab for the treatment of patients (pts) with recurrent HGG following surgery, radiotherapy, and chemotherapy. Methods: Eligible adult pts were treated with cetuximab (400 mg/m2 2 h IV on d1 and weekly 250 mg/m2 1h IV thereafter). Pts were stratified in 2 treatment arms according to the amplification status of the EGFR gene of their HGG (determined by fluorescence in situ hybridization on archival tumor material). Results: Between May 2005 and December 2007 a total of 55 pts with performance status 0–2 initiated treatment with cetuximab (28 pts with and 27 pts without an increased EGFR copy number, 17F/38M, median age 53 years [range 32–73]). Cetuximab was generally well tolerated. During a total of 689 treatment weeks the most frequent treatment-related adverse events were: skin toxicity (grade 2, n=12; grade 3, n=4), thrombocytopenia (grade 2, n=1; grade 3, n=2), confusion/diminished consciousness (grade 3, n=4 pt), lymphopenia (grade 4, n=1), infusion related allergic reaction (grade 2, n=1), intratumoral hemorrhage (grade 2, n=1), diarrhea (grade 2, n=1), and fatigue (grade 2, n=3). A dose reduction of cetuximab (to 200 mg/m² weekly) was necessary in 2 pts. After a median follow-up of 15 months, 11 pts are alive (5 are still receiving cetuximab) and 44 pts have died. The disease control rate was 35% (3 pts (5.6%) had a PR and 16 pts (29.6%) had SD) and 35 pts (64.8%) had PD. The median PFS was 1.9 months, median OS was 5.0 months. The 6-month PFS and OS rates were 10% and 40%, respectively. Whereas PFS was less than 5 months in the majority (n=49) of pts, a subgroup of 5 pts (9.2%) have a PFS on cetuximab of more than 9 months (range 9.5 to >16.5). No significant correlation was found between response, survival and EGFR copy number. Conclusions: Cetuximab as a single agent was safe and well tolerated in this population of pretreated patients with recurrent high-grade glioma. Durable disease control was observed in a small subgroup of patients. Mature results of this study will be available for presentation at the meeting. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Amgen, Merck, sanofi-aventis, Schering-Plough, Wyeth Merck

EGFR Mutations in Lung Adenocarcinomas : Clinical Testing Experience and Relationship to EGFR Gene Copy Number and Immunohistochemical Expression

Lung adenocarcinomas responsive to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors possess EGFR mutations and often increased EGFR copy number. We prospectively studied 334 clinical cases using polymerase chain reaction-based assays to detect deletions within exon 19 and the L858R mutation in exon 21, which together account for approximately 90% of EGFR mutations. Seventy-eight (23%) of these tumors had an EGFR mutation, with 55 (71%) exon 19 deletions and 23 (29%) exon 21 L858R mutations. We were able to compare mutant and normal EGFR alleles and found a preferential amplification of the mutant allele. The association of mutations with EGFR amplification (determined by chromogenic in situ hybridization) and EGFR expression (determined by immunohistochemistry) was further examined in a subset of 60 tumors. EGFR amplification (> or =5 EGFR signals per nucleus) was seen in 15 of 29 (52%) EGFR-mutated tumors but in only five of 31 (6%) non-mutated tumors (P = 0.006). EGFR overexpression was strongly associated with amplification but was statistically independent of EGFR mutation. Most patients with EGFR mutations (17 of 29, 59%) never smoked compared with 13% (four of 31) of patients lacking such mutations (P = 0.0003). The association of amplification with smoking status was marginal and was nonexistent with EGFR expression. Thus, these results indicate that EGFR amplification, preferentially of the mutant allele, often accompanies EGFR mutation, whereas EGFR immunohistochemical staining associates with amplification but cannot predict EGFR mutation status.

Increased epidermal growth factor receptor ( EGFR) gene copy number is strongly associated with EGFR mutations and adenocarcinoma in non-small cell lung cancers: A chromogenic in situ hybridization study of 182 patients

To evaluate the association of epidermal growth factor receptor (EGFR) gene copy number with EGFR and k-ras mutation status and tyrosine kinase inhibitor (TKI) sensitivity in non-small cell lung cancer (NSCLC), EGFR gene copy number of 182 NSCLC tumor specimens were analyzed by chromogenic in situ hybridization (CISH). EGFR and k-ras mutation analyses were also performed for, respectively, 176 and 157 of the 182 patients. Additionally, 36 patients in this study had received TKI monotherapy. The tumor was considered to be CISH positive if the gene copy number was >or=5 signals per nucleus in >or=40% of tumor cells. CISH-positive tumors were strongly associated with adenocarcinoma (56.8%) compared with squamous cell carcinoma (15.9%) (p<0.0001). The CISH-positive tumors were also strongly associated with EGFR mutations (78%) compared with wild type (20.2%) (p<0.0001). Only six tumors had k-ras mutations. None had EGFR mutation and only one was CISH positive. In the patients treated with TKI, EGFR mutation was strongly associated with TKI responsiveness (22/25 responders) (p<0.0001), but the CISH-positive tumors were only marginally significant (18/25 responders) (p=0.0665). Patients with EGFR mutations or CISH-positive tumors were all associated with longer median survival, although not statistically significant. Our results suggest Increased EGFR copy number was highly correlated with EGFR mutation in adenocarcinoma. Although it is less correlated with TKI responsiveness when compared with EGFR mutations, it still could be a good alternative molecular predictive marker for TKI responsiveness, since CISH can be done on paraffin section and is much quicker than DNA sequencing.

Clinical Significance of Epidermal Growth Factor Receptor Protein Overexpression and Gene Copy Number Gains in Prostate Cancer

The epidermal growth factor receptor (EGFR) is a protein involved in the tumor progression of many cancer types and is an important therapeutic target. To determine its role in prostate cancer, we analyzed 2,497 prostate cancers on the DNA and protein level. Tissue samples from each tumor were brought into a tissue microarray and analyzed by immunohistochemistry and fluorescence in situ hybridization. A subset of cancers was also sequenced for EGFR exon 18 to 21 mutations. Detectable EGFR expression was found in 18% of cancers and was significantly associated with high grade, advanced stage, and high risk for prostate-specific antigen recurrence in univariate analysis (P < 0.0001, each). Fluorescence in situ hybridization analysis with a dual-labeling probe for centromere 7 and EGFR showed increased EGFR copy number in 3.3% of cases. EGFR copy number gains were mostly due to an overrepresentation of the entire chromosome and were associated with EGFR protein expression (P < 0.0001), high grade (P < 0.0001), and advanced stage (P = 0.0056). Only one cancer had a high-level amplification (>20 EGFR gene copies per cell). This amplification was heterogeneous, involving only approximately 30% of the cancer volume. EGFR mutations were not found in 35 of the cases analyzed. Increased EGFR expression is often seen in prostate cancer and is associated with poor prognosis. The significant association of EGFR copy number gains with protein expression argues for the significant role of minimal gene copy number changes for protein expression. Although EGFR expression was not an independent prognostic variable, the potential utility of anti-EGFR medications might be worth further investigation in EGFR-expressing prostate cancer.

Gene Expression Profiles as Markers of Aggressive Disease—EGFR as a Factor

We previously reported that 43 (58%) of 75 head and neck squamous cell carcinoma (HNSCC) tumors harbor increased epidermal growth factor receptor (EGFR) gene copy numbers as determined by fluorescent in situ hybridization. In this study, an increased EGFR copy number was associated with decreased progression-free survival and overall survival of HNSCC patients. However, activated EGFR protein levels are difficult to quantify by immunohistochemistry and are subject to dynamic regulation, specifically receptor downregulation on ligand binding. Therefore, we generated an activated EGFR gene expression signature in an in vitro HaCaT keratinocyte model system to further study genes involved in the EGFR signaling pathway in HNSCC. The results from this model system have suggested that the activated EGFR signature might reflect the activated state of the EGFR pathway in human HNSCC tumors and that it is associated with the increased EGFR gene copy number by fluorescent in situ hybridization. Furthermore, the activated EGFR signature has provided additional leads, because they are related to co-regulated molecular pathways and associated gene products on activation of EGFR. These could be exploited to refine and optimize combination therapies to be used in conjunction with available EGFR inhibitors in individual HNSCC patients.

KRASMutation Is an Important Predictor of Resistance to Therapy with Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Non–Small-Cell Lung Cancer

EGFR gene mutations and increased EGFR copy number have been associated with favorable response to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKI) in patients with non-small-cell lung cancer (NSCLC). In contrast, KRAS mutation has been shown to predict poor response to such therapy. We tested the utility of combinations of these three markers in predicting response and survival in patients with NSCLC treated with EGFR-TKIs. Patients with advanced NSCLC treated with EGFR-TKI with available archival tissue specimens were included. EGFR and KRAS mutations were analyzed using PCR-based sequencing. EGFR copy number was analyzed using fluorescence in situ hybridization. The study included 73 patients, 59 of whom had all three potential markers successfully analyzed. EGFR mutation was detected in 7 of 71 patients (9.8%), increased EGFR copy number in 32 of 59 (54.2%), and KRAS mutation in 16 of 70 (22.8%). EGFR mutation (P<0.0001) but not increased EGFR copy number (P=0.48) correlated with favorable response. No survival benefit was detected in patients with either of these features. KRAS mutation correlated with progressive disease (P=0.04) and shorter median time to progression (P=0.0025) but not with survival. Patients with both EGFR mutation and increased EGFR copy number had a >99.7% chance of objective response, whereas patients with KRAS mutation with or without increased EGFR copy number had a >96.5% chance of disease progression. KRAS mutation should be included as indicator of resistance in the panel of markers used to predict response to EGFR-TKIs in NSCLC.

Targeting of Epidermal Growth Factor Receptor in Patients Affected by Biliary Tract Carcinoma

TO THE EDITOR: A study by Philip et al published in the July 1, 2006, issue of the Journal of Clinical Oncology shows the therapeutic benefit of epidermal growth factor receptor (EGFR) blockade with erlotinib in patients with biliary tract carcinoma. The results are interesting because in a cohort composed of 24 chemotherapy-refractory patients and 18 chemotherapy-naive patients, erlotinib as a single agent can achieve disease control in 50% of patients with a median duration of disease control of 5.1 months. However, the authors conclude that the activity of erlotinib is modest because only 17% of the patients achieved the primary end point of the study, which was progression-free survival at 24 weeks. Although gefitinib and erlotinib are active in unselected patients with non–small-cell lung cancer (NSCLC), clinical characteristics and tumor molecular markers associated with enhanced benefit have been identified. Notably, mutations in the tyrosine kinase domain of EGFR were subsequently found to be associated with sensitivity to small molecule inhibitors in NSCLC. Emerging data also suggest that increased EGFR copy number is a predictor of response to gefitinib therapy, regardless of EGFR kinase domain mutational status. Furthermore, in contrast to wild-type EGFR, the EGFR mutations are mutually exclusive of K-Ras mutations, which are found in biliary tract carcinomas with a particularly high incidence in some geographical areas. Mutations of the EGFR kinase domain have recently been described by our group in 15% of biliary tract carcinoma specimens. One of these mutations was a T790M substitution, which is known to correlate with an acquired resistance to small molecule inhibitor treatment in NSCLC. Whereas 28% of the EGFR-positive patients were progression free at 24 weeks, the study by Philip et al, based on the lack of correlation between EGFR expression level and treatment outcome in other tumor types, includes six undetermined and seven EGFRnegative patients. This patient number is too small to provide evidence of any correlation. Thus, it would be of interest to know if other molecular analyses, such as sequencing of EGFR tyrosine kinase or of downstream members of the signaling pathway, like K-Ras, B-Raf or PTEN, or EGFR gene amplification, have been planned in this group of biliary tract cancer patients treated with erlotinib. This finding will have extraordinary implications and will serve as a critical step toward individualized patient-specific treatment plans based on the molecular constitution of the tumor of each individual.

Impact of epidermal growth factor receptor (EGFR) kinase mutations, EGFR gene amplifications, and KRAS mutations on survival of pancreatic adenocarcinoma

Erlotinib, in combination with gemcitabine, has shown clinical benefits in pancreatic adenocarcinoma patients. The presence of EGFR mutations and increased EGFR copy numbers in pancreatic adenocarcinoma was explored. Sixty-six pancreatic cancer patients were included in the analysis. The EGFR mutation was analyzed by DNA sequencing of exons 18-21 in the tyrosine kinase domain. KRAS mutation was analyzed by sequencing codons 12, 13, and 61. Quantitative real-time polymerase chain reaction was performed to analyze the copy number of EGFR. In the current study the EGFR mutation was harbored in only 1 (1.5%) of the 66 inoperable or metastatic pancreatic adenocarcinoma patients. Amino acid substitution was detected in exon 20 of the EGFR gene. Increased EGFR copy numbers (> or =3.0 per cell) were detected in 26 (41%) patients. There was only 1 patient, who had a highly increased EGFR copy number (> or =6.0 per cell), who died, 2.1 months from the date of diagnosis. The EGFR amplification did not significantly influence survival in pancreatic adenocarcinoma patients (P = .935). Thirty-two (49%) of the 65 pancreatic adenocarcinomas examined harbored a point mutation in codons 12 (n = 31) and 61 (n = 1) of the KRAS gene. The presence of a point mutation in codon 12 adversely influenced survival of pancreatic cancer patients (P = .030). The incidence of somatic mutations in the tyrosine kinase domains of EGFR was very low and the increased gene copy number of EGFR did not significantly influence survival.

EGFRvIII mutation in lung cancer correlates with increased EGFR copy number

Overexpression of the epidermal growth factor receptor (EGFR) is caused by EGFR gene amplification and is sometimes associated with expression of a variant EGFR (deletion exon 2-7 or EGFRvIII). EGFRvIII mutation has oncogenic potential and is investigated as a potential therapeutic target. We genotyped the EGFRvIII mutation status in 252 surgically treated lung cancer cases. The presence or absence of EGFRvIII mutation was analyzed by real-time quantitative polymerase chain reaction (PCR) with mutation specific sensor and anchor probes. EGFR copy number was evaluated with PCR-based assay. EGFR mutation status at kinase domain has been examined and reported. EGFRvIII mutation was found on 8 of 252 patients. All patients were male, smokers, and 7 had squamous cell carcinoma. The mutation status was significantly correlated with pathological subtypes (squamous cell carcinoma vs. adenocarcinoma, p=0.0114). Sixty EGFR mutations at kinase domain exclusively existed with EGFRvIII mutations. EGFR gene copy number was significantly higher in EGFRvIII mutant (4.711+/-4.968) than in non-EGFRvIII mutant (2.284+/-1.224) (p=0.0001). EGFRvIII gene mutation might be one of the mechanisms of increased EGFR copy number. Further studies are needed to confirm the mechanisms of EGFRvIII mutations for possible anti-EGFR therapy for lung cancer.

Molecular Mechanisms of Epidermal Growth Factor Receptor (EGFR) Activation and Response to Gefitinib and Other EGFR-Targeting Drugs

The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, including EGFR, HER2/erbB2, and HER3/erbB3, is an attractive target for antitumor strategies. Aberrant EGFR signaling is correlated with progression of various malignancies, and somatic tyrosine kinase domain mutations in the EGFR gene have been discovered in patients with non-small cell lung cancer responding to EGFR-targeting small molecular agents, such as gefitinib and erlotinib. EGFR overexpression is thought to be the principal mechanism of activation in various malignant tumors. Moreover, an increased EGFR copy number is associated with improved survival in non-small cell lung cancer patients, suggesting that increased expression of mutant and/or wild-type EGFR molecules could be molecular determinants of responses to gefitinib. However, as EGFR mutations and/or gene gains are not observed in all patients who respond partially to treatment, alternative mechanisms might confer sensitivity to EGFR-targeting agents. Preclinical studies showed that sensitivity to EGFR tyrosine kinase inhibitors depends on how closely cell survival and growth signalings are coupled with EGFR, and also with HER2 and HER3, in each cancer. This review also describes a possible association between EGFR phosphorylation and drug sensitivity in cancer cells, as well as discussing the antiangiogenic effect of gefitinib in association with EGFR activation and phosphatidylinositol 3-kinase/Akt activation in vascular endothelial cells.

Epidermal growth factor receptor mutations and gene amplification in Japanese non-small cell lung cancer patients treated with gefitinib

17063 Background: To evaluate relationship between epidermal growth factor receptor (EGFR) gene status and clinical outcome in patients with non-small-cell lung cancer (NSCLC) treated with gefitinib. Also, to examine an involvement of human papilloma virus (HPV) in EGFR status of these patients. Methods: Twenty seven patients with NSCLC who had relapsed after surgery and received gefitinib were included. Genomic DNA was extracted from the 12 paraffin and the 15 frozen surgical specimens. PCR and sequencing for genotyping were done for EGFR (exon 18–21) and ERBB2 (exon 19–22) and KRAS (exon 1). Gene amplification for EGFR was analyzed by quantitative real-time PCR. HPV was detected by PCR and in-situ PCR. Results: Nine patients (33%) had EGFR mutations; seven patients had deletion mutations in exon 19, one patient had missense mutations (L858R) in exon 21. Two patients had missense mutations (G719S, exon 18 and L838P, exon 21) No mutations were identified in ERBB2 and KRAS. EGFR copy number in the tumor cells ranged from 1.1 to 9.7, and increased EGFR copy numbers (≥3) were observed in six patients ( 22.2%). Two patients (7%) had HPV, and had no mutation and no amplification. Response rate (67% {six of nine patients} v 18% {three of 17 patients}; p = 0.012) was significantly better in patients with EGFR mutations than in patients with wild-type EGFR. Overall survival (median 17 v 9 months) was better in patients with EGFR mutations than in patients with wild-type EGFR. EGFR copy number and presence of HPV were not associated with clinical outcome. Conclusions: EGFR mutations were significantly associated with better clinical outcome in gefitinib treated NSCLC patients. No significant financial relationships to disclose.

High gene copy number of epidermal growth factor receptor by fluorescence in situ hybridization is frequent in head and neck squamous cell carcinomas and associates with worse recurrence-free survival

5502 Background: Epidermal growth factor receptor (EGFR) is expressed in 90–95% of head and neck squamous cell carcinoma (HNSCC). High EGFR gene copy number and activating mutations correlate with response and survival in lung cancer upon the treatment with EGFR tyrosine kinase inhibitors, but such findings have not been reported for HNSCC. A better understanding of the EGFR pathway may improve the use of EGFR inhibitors and provide a rationale for combination therapy. Methods: Tumor samples were obtained from 47 patients and analyzed for increased EGFR copy numbers by fluorescence in situ hybridization (FISH) (Vysis/Abbot) (N = 47), by immunohistochemistry (IHC) using antibodies against EGFR, phospho-EGFR (Tyr1173) and phospho-AKT (Ser473) (N = 31), for activating mutations in EGFR exons 18, 19 and 21 (N = 29), and for gene expression using Affymetrix Human 133U Plus 2.0 GeneChip (N = 18). Results from these assessments were tested for associations with patient characteristics and clinical endpoints. Univariate analysis was performed to test for differences in recurrence-free survival (RFS) and overall survival. Results: Twenty-six (63%) of 41 samples with FISH result demonstrated EGFR high polysomy and/or gene amplification (FISH+). The FISH(+) group did not differ from the FISH(−) group with regard to age, sex, race, tumor grade and site, disease stage, total EGFR, phospho-EGFR, or phospho-AKT, although the total EGFR protein score was close to significance (p = 0.10, Chi2-test for trend). The FISH(+) were associated with worse RFS, although this association did not reach significance (p = 0.057, Wilcoxon test). No activating EGFR point mutations were found. Using the FISH results as a supervising parameter, microarray data was interrogated and expression of genes involved in wnt signalling, cell motility, G-protein regulation and epithelial-mesenchymal transition pathways were found to be enhanced in the FISH(+) group (FDR &lt;25%). Conclusions: High gene copy number of EGFR by FISH is frequent in HNSCC and may be a poor prognosticator. This data suggests that FISH(+) may be a marker of clinical benefit upon EGFR inhibition in HNSCC and further investigation is indicated. [Table: see text]

Gefitinib for Epidermal Growth Factor Receptor Mutant Lung Cancers: Searching for a Weapon of Mass Destruction

In the spring of 2004, the association between somatic mutations in the epidermal growth factor receptor (EGFR) in non– small-cell lung cancer (NSCLC) and sensitivity to the EGFR tyrosine kinase inhibitor gefitinib was first described. Since that time, numerous retrospective studies have confirmed these original observations. Patients with EGFR mutations treated with gefitinib have a 60% to 90% chance of achieving a radiographic partial response and a median time to progression of approximately 12 months (Table 1). However, these were all retrospective observations that were limited by multiple potential biases including patient selection based on the availability of tumor material, differences in prior treatments, and the methods of EGFR mutation detection. In addition, other biomarkers associated with gefitinib’s clinical benefit, including increased EGFR copy number and EGFR expression as detected by immunohistochemistry, have also emerged over the last 2 years. The contribution and role of EGFR mutations remain to be definitively determined from ongoing prospective studies. In this issue, Inoue et al report the first prospective clinical trial in which chemotherapy-naive advanced NSCLC patients with somatic EGFR mutations were treated with gefitinib as their first systemic therapy. They started the study in June 2004, performed mutational analyses in 99 patients (75 patients were assessable), and found EGFR mutations in 25 of the patients (33%). The mutation frequency in this study is consistent with the previously published somatic EGFR mutation frequency in Japanese lung cancer patients. The 16 patients with EGFR mutations had a response rate of 75% and a median progression-free survival time of 9.7 months, which is consistent with the retrospective observations (Table 1). Inoue et al also found the same types of EGFR mutations as previously identified, with in-frame deletion of exon 19 being the most common type of mutation (Table 1). Despite the small sample size, the study met its primary protocol-defined end point of a 70% response rate, and this observation in a prospective trial confirms the previous retrospective observations associating EGFR mutations and the efficacy of gefitinib. What are the implications of this study? The findings of Inoue et al leave little doubt about the predictive value of EGFR somatic mutations (exon 19 deletions and L858R) for radiographic response after treatment with gefitinib. However, their study does not address whether these patients will survive longer when treated with gefitinib compared with chemotherapy. This will require a phase III trial comparing the outcome of patients with EGFR somatic mutations treated with chemotherapy with the outcome of patients treated with gefitinib. In addition, the role of adding systemic chemotherapy to EGFR tyrosine kinase inhibitors for this patient population remains to be determined and is being evaluated in ongoing studies. Inoue et al also demonstrate the feasibility of EGFR mutation detection in the routine clinical care of chemotherapy-naive patients with advanced NSCLC. The Japanese population is more likely to have a somatic EGFR mutation than the white population (30% in Japanese patients compared with 10% to 15% in white patients). This increased mutation frequency, combined with the rapid turn around time (median, 7 days) for EGFR sequencing, clearly helped this study. In the United States or Western Europe, approximately 300 patients would need to be screened (rather than the 99 patients screened in the study by Inoue et al) to find patients with sufficient tumor material for EGFR sequencing and, ultimately, patients with EGFR mutations to obtain a similar number of assessable patients. Nevertheless, these findings suggest that this may be a worthwhile effort in Western populations as well. Inoue et al found the most common types of EGFR mutations (exon 19 deletions and L858R) in their patient population. To date, more than 20 different EGFR mutations have been described. The predictive value for radiographic response to gefitinib treatment of EGFR mutations, other than exon 19 deletions or L858R, still needs to be determined from ongoing clinical studies. In the study by Inoue et al, there were four patients with NSCLC and EGFR mutations who either had stable (n 2) or progressive disease (n 2) after treatment with gefitinib. What is different about these patients compared with the patients with EGFR mutations who respond to treatment with gefitinib? There are no obvious differences in the types of EGFR mutations among these patients compared with the other 12 patients who achieved a partial response. In addition, the DNA from the tumors of the patients with stable or progressive disease did not contain the previously described EGFR T790M mutation associated with gefitinib resistance. Several recent studies have demonstrated that, both in EGFR mutant NSCLC cell lines and in patients with JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 24 NUMBER 21 JULY 2