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Journal of Clinical Oncology, Vol 25, No 16 (June 1), 2007: pp. 2164-2170 © 2007 American Society of Clinical Oncology. DOI: 10.1200/JCO.2006.06.6605 Epidermal Growth Factor Receptor Copy Number Alterations Correlate With Poor Clinical Outcome in Patients With Head and Neck Squamous Cancer
From the Molecular Biology Laboratory and Departments of Thoracic/Head and Neck Medical Oncology, Leukemia, Biostatistics, and Pathology, The University of Texas M.D. Anderson Cancer Center; and the Cancer Biology Program, The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX Address reprint requests to Li Mao, MD, Department of Thoracic/Head and Neck Medical Oncology, Unit 432, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030; e-mail: lmao{at}mdanderson.org
Purpose Overexpression of epidermal growth factor receptor (EGFR) is common in head and neck squamous cell carcinoma (HNSCC). Recent studies showed that EGFR inhibitors are effective for patients with HNSCC. This study analyzed the genetic nature of EGFR gene in HNSCC and its clinical correlations. Patients and Methods The EGFR gene copy numbers in 134 HNSCC tumors were determined using quantitative real-time polymerase chain reaction. The status of EGFR gene copy numbers was analyzed with clinical parameters including clinical outcome. Mutation status of EGFR exons 18, 19, and 21 was determined in the HNSCC tumors. Results Aberrant EGFR copy numbers were found in 32 (24%) of 134 tumors, including 22 (17%) with increased copy number and 10 (7%) with decreased copy number. Patients whose tumors had EGFR copy number alterations (particularly patients with increased copy numbers) had significantly poorer overall, cancer-specific, and disease-free survivals compared with patients with normal copy numbers (P < .0001). At 5 years after initial diagnosis, 20 (91%) of the 22 patients with increased copy numbers died of disease compared with 30 (29%) of the 102 patients with normal copy number. No mutations on EGFR exons 18, 19, and 21 were detected in any of the tumors. Conclusion A subset of HNSCC manifests EGFR copy number alterations, and this is associated with a poor clinical outcome, suggesting a biologic role of the alterations. The rare mutation or small deletion at EGFR exons 18 to 21 indicates a minimal role of these events in HNSCC.
Epidermal growth factor receptor (EGFR), a member of the ErbB family of receptor tyrosine kinases, is overexpressed in a number of solid tumor types, including approximately 80% of head and neck squamous cell carcinomas (HNSCC). The overexpression occurs early in the head and neck tumorigenesis1 and is associated with advanced stages of the disease and a poor survival.2,3 Clinical successes using EGFR inhibitors in treating refractory colorectal and lung cancers underscore the potential use of this drug class. However, the objective tumor response to the agents is limited to a small number of patients. In patients with recurrent or metastatic HNSCC, the EGFR inhibitors have shown encouraging clinical activity in phase II studies.4,5 In a large, randomized, phase III study, the addition of cetuximab to high-dose radiation in patients with advanced HNSCC demonstrated a statistically significant prolongation in overall survival.6 Recently, somatic mutations in the kinase domain of EGFR have been identified in 10% to 15% of patients with non–small-cell lung cancer (NSCLC), particularly in females with adenocarcinoma histology, and have been correlated to response to gefitinib7-10 or erlotinib.11 In NSCLC, EGFR gene copy numbers have been associated with the protein expression levels, and EGFR gene amplification assessed by fluorescent in situ hybridization (FISH)12-14 or by quantitative real-time polymerase chain reaction (Q-PCR)10 was significantly associated with better clinical outcome in gefitinib-treated patients, suggesting that the copy number of the EGFR gene may be a useful predicting marker for patients treated with EGFR inhibitors. To elucidate the nature of EGFR gene alterations in HNSCC, we examined EGFR gene copy numbers in 134 primary HNSCC tumors using Q-PCR and examined mutations of the kinase domain using a sensitive PCR-restriction fragment length polymorphism (RFLP) assay and correlated the results with clinical parameters and patient outcome.
Study Population The 134 HNSCC patients include 41 patients (31%) treated at Institut Gustave-Roussy (Villejuif, France) and 93 patients (69%) treated at The University of Texas M.D. Anderson Cancer Center (Houston, TX) between 1985 and 2003. The institutional review boards approved the study, and informed consent was obtained from all patients. For the French group, all fresh frozen tumor biopsy specimens had a proportion of tumor cells greater than 70%. Tumor DNA was extracted using the QIAamp Tissue kit (Qiagen, Courtaboeuf, France). For the US group, DNA was extracted from formalin-fixed, paraffin-embedded tissue blocks. Microdissection was performed in specimens with less than 70% tumor cells. All of the tumors from the US group were graded as well-, moderately, or poorly differentiated tumors. Clinicopathologic and follow-up information was obtained by reviewing patient pathology reports and hospital charts.
Quantification of EGFR Gene Copy Numbers
We quantified each gene (EGFR, ß-globin, and WNT9A) in separate reactions using a universal PCR protocol recommended by the manufacturer. Q-PCR reactions for each sample and each gene were performed in triplicate. The threshold amplification cycles (CT) at the normalized reporter signal minus the baseline signal level of 0.2 for each gene were determined, and their differences,
EGFR Mutation Analysis For PCR-RFLP, PCR products were digested using respective restriction enzymes before determining sizes of the PCR products. For determining sizes of DNA fragments, fluorescently labeled DNA was separated on 4% polyacrylamide gel on an ABI 377 DNA Sequencer (Applied Biosystems). Fluorescent bands were analyzed using GENESCAN 2.1 software (Applied Biosystems) to determine fragment length. H2255 cells with exon 21 mutation, HCC827 cells with a 15–base pair deletion in exon 19, and H406 cells with a 9–base pair deletion were used as positive controls in each experiment. Serial dilutions of normal DNA (H292 cells) and mutated DNA were used to determine the detection sensitivity.
Analyses of EGFR Gene Copy Number and Expression by FISH and Immunohistochemistry For EGFR expression, 4-µm paraffin-embedded tissue sections were deparaffinized followed by antigen retrieval. The sections were incubated for 1 hour with mouse monoclonal antibody (31G7; Zymed Laboratories, Inc, South San Francisco, CA) diluted 1:50 in dilution buffer at room temperature and then incubated with Dako's Mouse EnVision+ Peroxidase System for 30 minutes (Dako, Carpinteria, CA). The peroxidase-catalyzed product was visualized with the BioGenex DAB Chromogen Kit (BioGenex, San Ramon, CA). Finally, the sections were lightly counterstained with Mayer's hematoxylin (Sigma Chemical Co, St Louis, MO) and mounted for analysis. The expression was scored as weak, moderate, or strong based on the staining strength of at least 1,000 tumor cells measured.
Statistical Analysis
Patient Characteristics Forty-one patients were from France, and 93 were from the United States. The general characteristics of the patients are listed in Table 1. In the US group compared with the French group, there were significantly more female patients (47% v 17%, respectively; P = .0009), more oral cavity tumors (70% v 37%, respectively; P = .0008), and more T1-2 tumors (47% v 27%, respectively; P = .03; Table 1). The median follow-up time after surgery was 3.6 years for the overall population (3.9 years for the US group and 3.4 years for the French group). The 5-year overall survival rates were 48.6% for the US group, 19.1% for the French group, and 44.4% for the combined population.
EGFR Gene Copy Numbers Thirty-two (24%) of the 134 tumors exhibited abnormal EGFR gene copy numbers, including 22 tumors (17%) with an increased EGFR copy number ranging from three copies to 12 copies (median, 3.9 copies) and 10 tumors (7%) with a decreased EFGR copy number (< two copies). EGFR copy numbers in the remaining 102 tumors were in the normal range. There was no significant difference in the frequencies of the overall abnormal copy numbers between the US group and the French group (22% v 29%, respectively; P = .33). However, a higher percentage of tumors exhibited increased gene copy numbers in the French group (27%) than in the US group (12%; P = .06). The gene copy numbers were correlated with clinical parameters of the patients (Table 2). There was no significant association between the abnormal EGFR gene copies and age, sex, smoking status, tumor differentiation, tumor location, and tumor size. However, the aberrant gene copies were correlated significantly with increased lymph node metastasis (P = .045) and advanced pathologic stage (P = .03; Table 2).
EGFR Copy Number Measured by FISH and EGFR Expression Measured by Immunohistochemistry We performed FISH and immunohistochemistry (IHC) analyses in 16 of the tumors from the US cohort, including five with reduced EGFR copy number, five with normal EGFR copy number, and six with increased EGFR copy number based on the Q-PCR analysis described earlier (Fig 1). All of the six tumors with increased EGFR copy number determined by Q-PCR showed EGFR amplification by FISH analysis, whereas none of the 10 tumors with normal or reduced EGFR copy number was FISH positive (P = .0001), supporting the role of Q-PCR in measuring EGFR copy numbers in clinical specimens. EGFR expression was also measured in the 16 tumors by IHC. Although five of the six tumors with increased EGFR copy number showed strong EGFR expression, three of the 10 tumors without EGFR amplification also showed strong EGFR expression (P = .12). Examples of EGFR copy numbers and protein expression measured by FISH and IHC are presented in Figure 1.
EGFR Gene Copy Numbers and Survival Patients whose tumors had abnormal EGFR copy numbers experienced statistically significant poorer survival compared with patients whose tumors had normal EGFR copy numbers (P < .001 for overall, cancer-specific, and disease-free survivals; Fig 2). Because the biologic consequences are likely different for tumors with increased EGFR and decreased copy number, we analyzed the survival by considering this factor. Among the three groups (102 tumors with normal copy number, 22 with increased copy number, and 10 with decreased copy number), a striking difference was observed between the patients whose tumors exhibited normal EGFR copy number and patients whose tumors had increased EGFR copy numbers (Fig 3). At 5 years, 20 (91%) of the 22 patients with increased copy numbers died of the disease compared with 30 (29%) of the 102 patients with normal copy numbers (Fig 3B). Patients whose tumors had decreased EGFR copy numbers also had poorer cancer-specific and disease-free survivals (Figs 3B and 3C), although the differences were less striking compared with patients with increased copy numbers.
Multicovariate analysis was used to determine whether the gene copy number abnormality is an independent factor for patients' survival. Because differences in certain clinic parameters and patterns of EGFR abnormalities were observed between the US patients and the French patients, we stratified the data by patient country of origin. Table 3 lists the results of the multicovariate analysis. As expected, age, tumor size, and lymph node metastasis were independent prognostic factors for overall survival, whereas tumor size and lymph node metastasis were also independent prognostic factors for cancer-specific and disease-free survival. Interestingly, the aberrant EGFR copy number was a strong prognostic factor independent of all the other known factors in overall, cancer-specific, and disease-free survival.
EGFR Gene Mutation Serial mixed dilutions of normal DNA and mutated DNA from control cell lines showed that the sensitivity of our mutation detection method for exon 18 and 21 mutations and exon 19 in-frame deletions was high (detected approximately 1% of mutated or deleted copies among the normal background; data not shown). We used positive controls for mutations and deletions in each experiment. Among the 134 tumors, five samples showed PCR fragments suspicious for mutations or deletions. However, direct sequencing of these specimens failed to identify any mutation or deletion in the corresponding genomic regions.
The identification of mutations in EGFR kinase domain and the correlation between the mutations and tumor response to EGFR inhibitors have spiked extensive search for these mutations in various epithelial tumor types. In NSCLC, these EGFR mutations centralize in exons 18 to 21, with more than 90% located in the following three hotspots: in-frame deletions in exon 19, L858R (2573T>G) point mutation in exon 21, and G719S (2155G>A) point mutation in exon 18.7-11 These mutations are more frequent in nonsmokers, women with adenocarcinoma, and patients from Asian nations.10,16,17 In this study, we failed to detect any mutation in these hotspots in 134 patients with primary HNSCC, suggesting that the mutations are rare in HNSCC patients in the US and French populations. It should be noted that only the three mutation hotspots were examined in this study. Although we failed to confirm mutation or deletion in the five tumors with suspicious alterations, it may be a result of the low sensitivity of direct sequencing analysis in the confirmative analysis, as suggested in a recent study.18 Nevertheless, our finding is consistent with recent reports in which mutation or deletion was rarely identified in HNSCC specimens from whites.19,20 In another study, Lee et al21 detected three EGFR-mutated tumors (exon 19 deletion) among 41 HNSCC specimens from a Korean population, suggesting that mutations may be more frequent in Asian populations with HNSCC. EGFR copy number in tumors is another factor important for EGFR expression level and response to EGFR inhibitors. Using FISH, increased copy numbers of EGFR have been observed in 10% to 13% of the HNSCC samples.22,23 In this study, we showed that patients whose tumors had aberrant copy numbers were more advanced and had poorer clinical outcomes independent of other clinical parameters. In a recent study, Morrison et al24 analyzed 59 laryngeal cancers and found similar frequencies of EGFR amplification (n = 14, 24%) and deletion (n = 6, 10%). These authors found that patients whose tumors had either EGFR amplification or deletion had a poorer survival.24 Considering that the majority of the patients had oral or pharyngeal cancers in our study, these data together suggest that the alteration rates of EGFR copy numbers are similar at the different anatomic locations of HNSCC. It is possible that tumor cells with increased EGFR copy numbers possess a greater capability to invade under stress conditions because they might produce a higher peak level of receptors than cells with normal copy number. It should be noted that the prognosis and the rate of abnormal EGFR copy numbers were different between the US and French patients in the study, probably as a result of the more oropharyngeal tumors, more advanced disease stage, and more males in the French population. We postulate that both genetic background and environmental etiology might contribute to the development of EGFR gene deletion or amplification. It is possible that the deletion occurs as a cellular response to reduce overexpressed EGFR and that the cells survive through alternative pathways. We observed some tumor cell clusters with reduced EGFR signal in the tumors with reduced EGFR copy number measured by Q-PCR in our FISH analysis (Fig 1A), which suggests that a subpopulation of the tumors might have contributed to the Q-PCR result. Whether the tumor cells with reduced EGFR capability developed other surviving mechanisms remains to be determined. Tsao et al11 analyzed 197 tumors for EGFR mutations and 221 tumors for EGFR gene copy numbers from patients with NSCLC who participated in a phase III clinical trial to determine efficacy of erlotinib. Although increased gene copy number was associated with longer survival in patients treated with erlotinib in a univariate analysis, such association was not statistically significant in multivariate analysis,11 suggesting that gene amplification might be associated with other confounding factors in the study population. A recent study showed a lack of EGFR amplification in HNSCC tumors that responded to gefitinib treatment.20 Major differences between NSCLC and HNSCC include the heterogeneous histology and the higher EGFR mutation rate in lung cancers. Nevertheless, patients with HNSCC seem to benefit from treatment with EGFR inhibitors. A recent phase III clinical trial using cetuximab in combination with high-dose radiation for patients with locally advanced HNSCC demonstrated an improved locoregional disease control and survival.6 Because approximately 10% improvement was achieved at 2 years using this treatment strategy compared with radiotherapy alone, it is reasonable to hypothesize that patients whose tumors contain increased EGFR copy number might be the ones who benefited from the addition of the EGFR inhibitor. Analyzing tumor samples from these clinical trials or incorporating the genetic analysis into future clinical trials may answer this important question. Although FISH is commonly used to determine gene copy numbers, Q-PCR can obtain reliable gene copy number and is less expensive. In FISH analysis, only a small part of tumors is analyzed, typically using the mean copy number of 60 cells, whereas real-time Q-PCR may be used to analyze a large area of tumors to minimizing the issue of tumor heterogeneity. The determination of gene copy numbers based on real-time Q-PCR is objective and, therefore, makes data interpretation more reliable.
The authors indicated no potential conflicts of interest.
Conception and design: Stephane Temam, Jean-Pierre Issa, Li Mao Financial support: Li Mao Provision of study materials or patients: Stephane Temam, Adel K. El-Naggar Collection and assembly of data: Stephane Temam, Hidetoshi Kawaguchi, Jaroslav Jelinek, Hongli Tang, Wenhua Lang Data analysis and interpretation: Stephane Temam, Hidetoshi Kawaguchi, Adel K. El-Naggar, Jaroslav Jelinek, Diane D. Liu, J. Jack Lee, Li Mao Manuscript writing: Stephane Temam, Li Mao Final approval of manuscript: Hidetoshi Kawaguchi, Adel K. El-Naggar, Li Mao
Supported by National Cancer Institute Grants No. CA106451 and CA16672 and Department of Defense Grant No. W81XWH-05-2-0027. S.T. and H.K. contributed equally to the work. Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
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Baselga J, Trigo JM, Bourhis J, et al: Phase II multicenter study of the antiepidermal growth factor receptor monoclonal antibody cetuximab in combination with platinum-based chemotherapy in patients with platinum-refractory metastatic and/or recurrent squamous cell carcinoma of the head and neck. J Clin Oncol 23:5568-5577, 2005 6. Bonner JA, Harari PM, Giralt J, et al: Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and neck. N Engl J Med 354:567-578, 2006 7. Lynch TJ, Bell DW, Sordella R, et al: Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 350:2129-2139, 2004 8. Paez JG, Janne PA, Lee JC, et al: EGFR mutations in lung cancer: Correlation with clinical response to gefitinib therapy. Science 304:1497-1500, 2004 9. Pao W, Miller V, Zakowski M, et al: EGF receptor gene mutations are common in lung cancers from "never smokers" and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad Sci U S A 101:13306-13311, 2004 10. Takano T, Ohe Y, Sakamoto H, et al: Epidermal growth factor receptor gene mutations and increased copy numbers predict gefitinib sensitivity in patients with recurrent non–small-cell lung cancer. J Clin Oncol 23:6829-6837, 2005 11. Tsao MS, Sakurada A, Cutz JC, et al: Erlotinib in lung cancer: Molecular and clinical predictors of outcome. N Engl J Med 353:133-144, 2005 12. Hirsch FR, Varella-Garcia M, Bunn PA Jr, et al: Epidermal growth factor receptor in non-small-cell lung carcinomas: Correlation between gene copy number and protein expression and impact on prognosis. J Clin Oncol 21:3798-3807, 2003 13. Hirsch FR, Varella-Garcia M, McCoy J, et al: Increased epidermal growth factor receptor gene copy number detected by fluorescence in situ hybridization associates with increased sensitivity to gefitinib in patients with bronchioloalveolar carcinoma subtypes: A Southwest Oncology Group study. J Clin Oncol 23:6838-6845, 2005 14. Cappuzzo F, Hirsch FR, Rossi E, et al: Epidermal growth factor receptor gene and protein and gefitinib sensitivity in non-small-cell lung cancer. J Natl Cancer Inst 97:643-655, 2005 15. Amann J, Kalyankrishna S, Massion PP, et al: Aberrant epidermal growth factor receptor signaling and enhanced sensitivity to EGFR inhibitors in lung cancer. Cancer Res 65:226-235, 2005 16. Riely GJ, Pao W, Pham D, et al: Clinical course of patients with non-small cell lung cancer and epidermal growth factor receptor exon 19 and exon 21 mutations treated with gefitinib or erlotinib. Clin Cancer Res 12:839-844, 2006 17. Shigematsu H, Lin L, Takahashi T, et al: Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers. J Natl Cancer Inst 97:339-346, 2005 18. Janne PA, Borras AM, Kuang Y, et al: A rapid and sensitive enzymatic method for epidermal growth factor receptor mutation screening. Clin Cancer Res 12:751-758, 2006 19. Loeffler-Ragg J, Witsch-Baumgartner M, Tzankov A, et al: Low incidence of mutations in EGFR kinase domain in Caucasian patients with head and neck squamous cell carcinoma. Eur J Cancer 42:109-111, 2006[CrossRef][Medline] 20. Cohen EW, Lingen MW, Martin LE, et al: Response of some head and neck cancers to epidermal growth factor receptor tyrosine kinase inhibitors may be linked to mutation of ERBB2 rather than EGFR. Clin Cancer Res 11:8105-8108, 2005 21. Lee JW, Soung YH, Kim SY, et al: Somatic mutations of EGFR gene in squamous cell carcinoma of the head and neck. Clin Cancer Res 11:2879-2882, 2005 22. Koynova DK, Tsenova VS, Jankova RS, et al: Tissue microarray analysis of EGFR and HER2 oncogene copy number alterations in squamous cell carcinoma of the larynx. J Cancer Res Clin Oncol 131:199-203, 2005[CrossRef][Medline] 23. Freier K, Joos S, Flechtenmacher C, et al: Tissue microarray analysis reveals site-specific prevalence of oncogene amplifications in head and neck squamous cell carcinoma. Cancer Res 63:1179-1182, 2003 24. Morrison LE, Jacobson KK, Friedman M, et al: Aberrant EGFR and chromosome 7 associate with outcome in laryngeal cancer. Laryngoscope 115:1212-1218, 2005[CrossRef][Medline] Submitted March 17, 2006; accepted October 6, 2006.
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Copyright © 2007 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
CT1 (globin – EGFR) and 
2.1; normal if the value was more than 1.5 but less than 2.1, and decreased if the value was 
1.5. Four NSCLC cell lines with known EGFR gene amplification
2 and Fisher's exact tests were used to test equal proportion between groups in two-way contingency tables. Survival probability as a function of time was computed using the Kaplan-Meier method. The log-rank test was used to compare patient survival times between groups. Overall, cancer-specific survival (ie, patients who died of HNSCC-related causes) and disease-free survival (ie, patients who developed recurrence and/or metastasis) were analyzed. Multicovariate Cox regression was used to model the risks of abnormal EGFR gene copy numbers on survival time, with adjustment for clinical and histopathologic parameters. All statistical tests are two sided, and a P 
