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Increased Epidermal Growth Factor Receptor Gene Copy Number Is Associated With Poor Prognosis in Head and Neck Squamous Cell Carcinomas

Christine H. Chung, Kim Ely, Loris McGavran, Marileila Varella-Garcia, Joel Parker, Natalie Parker, Carolyn Jarrett, Jesse Carter, Barbara A. Murphy, James Netterville, Brian B. Burkey, Robert Sinard, Anthony Cmelak, Shawn Levy, Wendell G. Yarbrough, Robbert J.C. Slebos, Fred R. Hirsch

From the Division of Hematology/Oncology, Department of Medicine, Department of Cancer Biology, Department of Pathology, Department of Otolaryngology, Department of Radiation Oncology, Department of Biomedical Informatics, Vanderbilt-Ingram Comprehensive Cancer Center, Vanderbilt University School of Medicine, Nashville, TN; Department of Pathology, Colorado Genetics Laboratory, Department of Medicine, University of Colorado Cancer Center, University of Colorado Health Sciences Center, Aurora, CO; and Constella Health Sciences, Durham, NC

Address reprint requests to Christine H. Chung, MD, Division of Hematology/Oncology, Department of Medicine, Vanderbilt University School of Medicine, 777 Preston Research Building, Nashville, TN 37232-6307; e-mail: Christine.Chung{at}Vanderbilt.edu

	ABSTRACT

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Purpose: High epidermal growth factor receptor (EGFR) gene copy number is associated with poor prognosis in lung cancer, but such findings have not been reported for HNSCC. A better understanding of the EGFR pathway may improve the use of EGFR inhibitors in HNSCC.

Patients and Methods: EGFR status was analyzed in 86 tumor samples from 82 HNSCC patients by fluorescent in situ hybridization (FISH) to determine EGFR gene copy number, by polymerase chain reaction and direct sequencing for activating mutations, and by DNA microarray and immunohistochemistry for RNA and protein expression. The results were associated with patient characteristics and clinical end points.

Results: Forty-three (58%) of 75 samples with FISH results demonstrated EGFR high polysomy and/or gene amplification (FISH positive). The FISH-positive group did not differ from the FISH-negative group with respect to age, sex, race, tumor grade, subsites and stage, or EGFR expression by analyses of RNA or protein. No activating EGFR mutations were found. However, the FISH-positive group was associated with worse progression-free and overall survival (P < .05 and P < .01, respectively; log-rank test). When microarray data were interrogated using the FISH results as a supervising parameter, ECop (which is known to coamplify with EGFR and regulate nuclear factor-kappa B transcriptional activity) had higher expression in FISH-positive tumors.

Conclusion: High EGFR gene copy number by FISH is frequent in HNSCC and is a poor prognostic indicator. Additional investigation is indicated to determine the biologic significance and implications for EGFR inhibitor therapies in HNSCC.

	INTRODUCTION

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Head and neck squamous cell carcinoma (HNSCC) is the fifth most common cancer in the United States and comprises approximately 80% to 90% of cancers arising from head and neck region.^1,2 The role of epidermal growth factor receptor (EGFR) expression as a prognostic marker in HNSCC was first described by Grandis et al.^3,4 They demonstrated that high EGFR protein and its ligand (transforming growth factor alpha [TGF-]), expression by immunohistochemical staining (IHC) was associated with poor survival. However, the correlation between the EGFR gene copy number and its RNA or protein expression has not been determined clearly, and association of gene copy number with prognosis is not known. Freier et al⁵ evaluated EGFR gene copy numbers by fluorescence in situ hybridization (FISH) on a tissue microarray containing 609 HNSCCs and found that 12.7% were amplified, but no correlation with survival was seen. In a smaller study by Mrhalova et al,⁶ seven of 23 tumors (30%) showed increased EGFR gene copy number by FISH, but FISH status did not correlate with protein expression by IHC. In addition, Gebhart et al⁷ examined 35 oral squamous cell carcinomas by comparative genomic hybridization, and 19 tumors (54%) showed a gain of chromosome 7p including band p12. The patients with the 7p genomic gain had a higher rate of relapse and worse survival.⁷

Recently, a randomized trial showed that a combination of cetuximab and radiation therapy increased clinical response and survival compared with radiation alone for locally advanced HNSCC patients.⁸ It is well known that activation of the EGFR pathway is associated with radiation resistance, and that EGFR blockade with cetuximab can modulate the radiation effect in head and neck cancer.^9,10 In addition, several EGFR inhibitors have been studied in recurrent or metastatic HNSCC patients and found to have modest response rates in the range of 4.3% to 16.5% as single agents.^11-13 Combination regimens including EGFR inhibitors were studied mostly with cetuximab and either cisplatin or carboplatin, and the response rates were in the range of 10% to 26% in platinum-refractory patients.^14-16 Therefore, there is a clear clinical benefit of EGFR inhibition in a small subset of HNSCC patients. Consequently, it is critical to identify biomarkers to predict treatment response after EGFR inhibitor therapy, which can be applied for patient selection.

In non–small-cell lung cancer (NSCLC), activating mutations in EGFR were identified and associated with a better response to the EGFR tyrosine kinase (TK) inhibitors.^17,18 However, the EGFR mutations found in NSCLC are rare in HNSCC. One study found a mutation rate of 7.3% (three of 41 patients) in HNSCC with the in-frame deletion mutation in exon 19 (E746_A750del) in a Korean population.¹⁹ Unlike the NSCLC data in which mutations were more prevalent in Asian nonsmoking women, all three HNSCC patients with mutations were males who currently smoked.¹⁹ Cohen et al²⁰ examined HNSCC patients who were responders and nonresponders to EGFR TK inhibitors, as well as 65 unselected HNSCC patients for activating mutations within the TK domain of EGFR (exons 18 to 24), and found no mutations. Furthermore, EGFR gene copy number analysis by FISH in NSCLC was shown to be associated with poor prognosis and with improved survival on treatment with the EGFR TK inhibitors, whereas such associations are unknown in HNSCC.^21-24 In this study, we assessed the EGFR gene copy number, EGFR gene mutation, and EGFR RNA and protein expression, and the results were correlated with clinical outcome to gain insight into the biologic and clinical significance of the EGFR pathway in HNSCC.

	PATIENTS AND METHODS

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Patients and Tissue Samples
Eighty-six formalin-fixed, paraffin-embedded tumor samples were collected from 82 patients undergoing surgery or biopsy at the University of North Carolina at Chapel Hill (Chapel Hill, NY; 50 patients) and Vanderbilt University Medical Center (Nashville, TN; 32 patients) between 1994 and 2005. All patients consented to participate in the tissue collection for research purposes only under protocols approved by the institutional review boards at the two institutions.

EGFR FISH Analyses
The FISH assays were performed as described previously using the LSI EGFR SpectrumOrange/CEP 7 SpectrumGreen probe (Vysis/Abbott Molecular, Des Plaines, IL).²¹ FISH patterns were defined and described in previous studies.^22,24 Briefly, the samples were grouped as normal disomy, two gene copies in more than 90% of cells; trisomy, three gene copies in more than 10% of cells; low polysomy, four gene copies in more than 10% but less than 40% of cells; high polysomy, four gene copies in 40% cells; and gene amplification, ratio gene/chromosome more than two or 15 gene copies in 10% of cells. Tumors showing high polysomy and gene amplification were considered to be FISH positive.

DNA Sequence Analyses and DNA Deletion Assay
DNA from 52 selected frozen tumors with tumor cellularity of more than 70% was isolated using Qiagen DNA isolation kit (Qiagen, Valencia, CA), and polymerase chain reaction (PCR) amplified and sequenced for EGFR exons 18, 19, and 21 using previously published amplimer sequences.¹⁷ To examine the deletion in EGFR exon 19 described by Lee et al¹⁹ using a more sensitive method (lower detection limit of 5%), a DNA deletion assay was performed using a fluorescently labeled primer set: EGFR19AS-T7 5'-TAA TAC GAC TCA CTA TAG GGT GAG GTT CAG AGC CAT GGA C-3' and EGFR19S-FAM 5'-TCT GGA TCC CAG AAG GTG AG-3'. The length of the PCR products was determined by capillary electrophoresis on an ABI 3100-Avant (Applied Biosystems, Foster City, CA).

RNA Analyses by DNA Microarray and RT-PCR
DNA microarray data using Affymetrix Human 133U 2.0 Plus GeneChips and the array data validation using five genes (TAF7L, SYCP1, RFC4, CDKN2A, and NAP1L2) by reverse transcriptase (RT) -PCR were published in Slebos et al.²⁵ The raw microarray data from this earlier study were deposited in the Nation Institutes of Health Gene Expression Omnibus database under accession No. GSE3292.²⁵ For the array experiments, total RNA was purified from frozen tumors analyzed, as reported previously.²⁵ Total RNA from 13 tumors were also analyzed for EGFR expression levels by RT-PCR analysis using the endogenous genes 18S, PPIA, and GUSB as controls. Each sample was analyzed in triplicate on an Applied Biosystems 7900HT instrument (Applied Biosystems). RT-PCR data were analyzed by the 2^-CT method as described previously.²⁶ Expression differences were tested between the FISH-positive and FISH-negative samples using Student's t test.

IHC Staining
Fifty tumors were immunostained for EGFR protein (DAKO EGFR PharmDx kit, K1494; Carpinteria, CA), and the method and initial results were published in Chung et al.²⁷ In the present study, the samples were rescored using a semiquantitative method described and published, which considers the intensity of the staining (1, negative or trace; 2, weak; 3, moderate; and 4, strong) and the percentage of positive tumor cells (0% to 100%), thus generating an overall score ranging from 0 to 400 points.²¹

Determination of the Differentially Expressed Genes Between the EGFR FISH-Positive and FISH-Negative Tumors
Differential expression of genes mapping at 7p12, the location of the EGFR FISH probe, and on the entire chromosomal arm of 7p were examined using Student's t test. Genes with P values less than .05 between the two groups were determined to be significantly different. Furthermore, the samples with EGFR gene amplification and high polysomy were compared separately with FISH-negative samples because the two genetic abnormalities may have different biologic effects.

Statistical Analyses for Survival
Progression-free survival (PFS) was defined as the time from diagnosis to recurrence or death as a result of all causes. Overall survival (OS) was defined as the time from the diagnosis to death as a result of all causes. Univariate analyses for PFS and OS were determined using log-rank test using the SAS/STAT software package (SAS Institute, Cary, NC) and plotted as Kaplan-Meier curves. Multivariate analyses were performed using a Cox model that included age, stage, major subsites, and FISH status.

	RESULTS

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Patient Characteristics
Total of 86 HNSCC tumor blocks were obtained from 82 patients; 72 samples were from primary tumors before any treatment and 14 samples were from recurrent tumors. There were three paired samples for primary and recurrent tumors from same patients and one paired sample from two different parts of the same tumor. All four major subsites of HNSCC were represented, including oral cavity, oropharynx, hypopharynx, and larynx. The detailed patient characteristics are described in Table 1 . The median follow-up was 24.5 months. Although the patients had various clinical treatments, 73 of 75 patients with the assessable FISH results received the standard of care with curative intent at the time of disease presentation and these patients were included in the survival analyses. Major primary treatments were surgery in 51 patients (70%) and nonsurgical approaches such as chemoradiotherapy in 21 patients (30%). No statistically significant difference in PFS and OS was apparent based on the treatment approaches. Four patients received single-agent cetuximab as a second-line systemic treatment for recurrent and/or metastatic disease. Two of these four patients had sustained partial response over 6 months. Two patients had stable disease for 6 and 11 weeks, respectively, and then experienced progression.

View larger version (59K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 1. Epidermal growth factor receptor (EGFR) gene copy number evaluation by fluorescent in situ hybridization. (A) gene amplification, (B) cluster gene amplification, (C) low polysomy, (D) trisomy, (E) normal disomy, and (F) high polysomy (EGFR, SpectrumOrange, Centromere 7 SpectrumGreen).

View larger version (13K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 2. Bar graph of the assessable fluorescent in situ hybridization results from 75 head and neck squamous cell carcinomas.

View larger version (8K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 3. Kaplan-Meier plots for (A) progression-free survival, and (B) overall survival based on EGFR gene copy number determined by fluorescent in situ hybridization.

Gene Expression Analyses by EGFR FISH Status
Given that there was no correlation between EGFR gene copy numbers and RNA or protein expression levels, the RNA expression of genes located on chromosome 7p12 was examined to identify candidates whose expression correlates with EGFR gene status. None of the genes expressed differentially with statistical significance. Given that the interphase FISH data do not provide information on the exact content of each amplicon, all genes on chromosome 7p were examined. In this analysis, hypothetical protein DKFZp564K0822, also known as EGFR-coamplified and overexpression protein (ECop; accession No. AK126848) or glioblastoma amplified secreted protein (GASP, accession No. AF395824), on 7p11.2 was identified as having higher expression in FISH-positive tumors (FISH positive v FISH negative, P = .059; high polysomy v FISH negative, P = .046; gene amplification v FISH negative, P = .224, Student's t test).

	DISCUSSION

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This is the first study to show that increased EGFR gene copy number as measured by FISH is common and is associated with worse PFS and OS in HNSCC. Prognostic markers, which predict outcome in unselected patient populations, may assist in risk stratification of patients; however, predictive markers of a given therapy can only be determined by studies of uniformly treated patient populations, and allow tailored therapies to the individual patients. One limitation of this study is that the samples were obtained from a heterogeneous cohort of patients who received both surgical and nonsurgical (ie, concurrent chemoradiotherapy) therapies as their primary treatments. However, all patients in the survival analyses were treated with curative intent using the standard of care. Because of the availability of the FISH assay in current clinical laboratories, EGFR gene copy number evaluation could be integrated rapidly into the risk stratification of HNSCC patients as a prognostic marker.

The association between poor prognosis and high expression of EGFR protein and its ligand, TGF-, determined by IHC has been known for many years.⁴ Therefore, we expected to detect an increase in RNA or protein expression levels in FISH-positive tumors, but such a correlation was not found. There are several potential explanations for the observed lack of correlation. One is that the majority of EGFR gene copies may be inactive through epigenetic mechanisms such as promoter methylation. In this scenario, gene copy number may have increased due to an early genetic pressure but became transcriptionally silent at a later stage of tumorigenesis. It is well known that cell lines with high EGFR expression, such as A431 and MDA-468, undergo apoptosis on stimulation with growth factors and that excessive ligand binding deregulates growth signaling, causing growth arrest.^28-30 The most prominent characteristic of the high-risk tumors in our previous study was increased expression of TGF- and amphiregulin.²⁷ A second explanation could be that all EGFR gene copies are transcribed but that these transcripts are aberrantly spliced so that they can not be detected by the EGFR probes in the DNA microarray and RT-PCR tests. Several EGFR mRNA species exist under normal circumstances, each with different lengths including 10, 5.6, and 2.9 kb.^31,32 The lack of correlation at the protein level could be from the limitation of EGFR protein quantification by IHC. Multiple EGFR-specific antibodies are available commercially and recognize different epitopes of the protein with variable affinities.³³ The breadth of the detection in differential expression is narrow using conventional scoring of 0 to 3+ intensity and percent-positive cells. Because it is done on formalin-fixed paraffin-embedded tissue, antigen retrieval can be an issue as well as the possibility of staining the cross-reactive proteins rather than EGFR. In addition, the scoring can be subjected to intra- and interexaminer in consistency, although it can be alleviated by automated tools such as automated quantitative analysis.^34-36

Alternatively, the prognostic association of EGFR FISH status may not be related to EGFR alone, but it is either a surrogate marker of generalized genetic instability in the tumors that are FISH positive or a marker for additional genes that are coamplified with EGFR. In support of this theory, genomic gain at 7p was detected in 32% to 54% of HNSCC by comparative genomic hybridization.^7,37 Therefore, we examined the DNA microarray data for changes on the short arm of chromosome 7. We found a novel gene, ECop, on 7p11.2 to be differentially expressed based on EGFR FISH status with greater association in high polysomy than in gene amplification. As the name implies, ECop is coamplified and expressed with EGFR in 35% of glioblastomas.³⁸ Recently, ECop was shown to regulate nuclear factor kappa B (NF-B) transcriptional activity by modulating inhibitor of kappa light polypeptide gene enhancer in B cells, alpha (IB) degradation and induce resistance to apoptosis.³⁹ Increased NF-B signaling in HNSCC is known to be associated with poor prognosis.^40-43 In our previous study, the tumors with high-risk of recurrence showed the gene expression pattern of a deregulated NF-B signaling pathway.⁴⁴

In NSCLC, EGFR FISH-positive status is associated with increased RNA and protein expression, and with improved survival after treatment with the EGFR TK inhibitors, gefitinib and erlotinib.^21-24,45 However, in HNSCC, the gene copy numbers determined by quantitative PCR in responders to EGFR TK inhibitors did not show high EGFR copy number, whereas one of the nonresponders had low-level amplification.²⁰ In a recent randomized trial of cisplatin with and without cetuximab in a recurrent or metastatic HNSCC patient population, lower EGFR scores by IHC were associated with better cetuximab response.^14,46 Likewise, the four patients who were treated with cetuximab in our study did not have increased EGFR gene copy number. Two patients with partial response of more than 6 months had normal disomy copy number and two patients with stable disease had trisomy. Conclusions cannot be drawn from these four anecdotal reports, and there are no data regarding the association between cetuximab response and EGFR FISH status in NSCLC; however, caution should be exercised in extrapolating data from the NSCLC experience to HNSCC.

In conclusion, increased EGFR gene copy number determined by FISH is a prognostic marker of PFS and OS; however, the underlying molecular mechanisms are not clear. FISH-positive status may be a surrogate marker of genetic instability or of genes that coamplify with EGFR such as ECop. Additional studies are required to determine the biology involving the EGFR pathway and to gain insight regarding the clinical application of the EGFR FISH assay as a prognostic marker in clinical trials using EGFR inhibitors in HNSCC.

	Authors' Disclosures of Potential Conflicts of Interest

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Although all authors completed the disclosure declaration, the following authors or their immediate family members indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. For a detailed description of the disclosure categories, or for more information about ASCO’s conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.

Authors Employment Leadership Consultant Stock Honoraria Research Funds Testimony Other Christine H. Chung Astra-Zeneca (C) Fred R. Hirsch AstraZeneca (A); Lilly Oncology (A); OSI Pharmaceuticals (A); Ligand Pharmaceuticals (A); Genmab (A); Genentech (A) AstraZeneca (C); OSI Pharmaceuticals (B); Genentech (C)

Dollar Amount Codes (A) < $10,000 (B) $10,000-$99,900 (C) $100,000 (N/R) Not Required

	Author Contributions

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Conception and design: Christine H. Chung, Fred R. Hirsch Financial support: Christine H. Chung, Wendell G. Yarbrough Administrative support: Christine H. Chung Provision of study materials or patients: Christine H. Chung, Kim Ely, Barbara A. Murphy, James Netterville, Brian B. Burkey, Robert Sinard, Anthony Cmelak, Wendell G. Yarbrough Collection and assembly of data: Christine H. Chung, Kim Ely, Loris McGavran, Marileila Varella-Garcia, Joel Parker, Natalie Parker, Carolyn Jarrett, Jesse Carter, Robbert J.C. Slebos, Fred R. Hirsch Data analysis and interpretation: Christine H. Chung, Kim Ely, Loris McGavran, Marileila Varella-Garcia, Joel Parker, Natalie Parker, Carolyn Jarrett, Jesse Carter, Shawn Levy, Robbert J.C. Slebos, Fred R. Hirsch Manuscript writing: Christine H. Chung, Marileila Varella-Garcia, Wendell G. Yarbrough, Robbert J.C. Slebos, Fred R. Hirsch Final approval of manuscript: Christine H. Chung, Fred R. Hirsch

 

	ACKNOWLEDGMENTS

 
We thank David P. Carbone, MD, PhD, for critical review of this article.

	NOTES

 
Supported by Vanderbilt Physician-Scientist Development Award (C.H.C.), the Damon Runyon Clinical Investigator Award (CI-28-05, C.H.C.), the Robert J. Kleberg Jr, and Helen C. Kleberg Foundation (C.H.C. and W.G.Y.), the Barry Baker Laboratory for Head and Neck Oncology (W.G.Y.), and the Vanderbilt-Ingram Cancer Center.

Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.

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Submitted May 1, 2006; accepted July 10, 2006.

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