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Journal of Clinical Oncology, Vol 23, No 24 (August 20), 2005: pp. 5560-5567 © 2005 American Society of Clinical Oncology. DOI: 10.1200/JCO.2005.06.411 Epidermal Growth Factor Receptor Expression in Pretreatment Biopsies From Head and Neck Squamous Cell Carcinoma As a Predictive Factor for a Benefit From Accelerated Radiation Therapy in a Randomized Controlled TrialFrom the Gray Cancer Institute, The Cancer Centre, and Department of Pathology, Mount Vernon Hospital; Department of Oncology, University College London, London, United Kingdom; Department of Radiation Oncology, Marmara University School of Medicine, Istanbul, Turkey; and Barbara Ann Karmanos Cancer Institute, Detroit, MI Address reprint requests to Søren M. Bentzen, PhD, DSc, Department of Human Oncology, University of Wisconsin Medical School, K4/316 Clinical Sciences Center, 600 Highland Ave, Madison, WI 53792; e-mail: bentzen{at}humonc.wisc.edu
PURPOSE: Accelerated repopulation is a main reason for locoregional failure after fractionated radiotherapy for head and neck squamous cell carcinoma (HNSCC). Epidermal growth factor receptor (EGFR) is a key controller of cellular proliferation in HNSCC, which stimulated the current study to look for a direct link between EGFR status and a possible clinical advantage of accelerated radiotherapy. PATIENTS AND METHODS: Immunohistochemical staining for EGFR was performed in 304 patients with available pretreatment tumor biopsy material among 918 patients randomized to receive continuous hyperfractionated accelerated radiotherapy versus conventionally fractionated radiotherapy. The EGFR index was estimated as the proportion of tumor cells with EGFR membrane staining. RESULTS: Significant benefit in locoregional tumor control from continuous hyperfractionated accelerated radiotherapy was seen in patients with HNSCC with high EGFR expression (2P = .010) but not in those with low EGFR expression (2P = .85). EGFR status had no significant effect on survival or rate of distant metastases. The EGFR index was significantly associated with histologic grade and microvessel density. There was moderate support for an association between EGFR status and subsite within the head and neck region but no significant association with Ki-67 index, Ki-67 pattern, p53 index, p53 intensity, bcl-2 expression, or cyclin D1 index. CONCLUSION: This study indicates a key role for the EGFR receptor in determining the proliferative cellular response to fractionated radiotherapy in HNSCC. It also shows that we can select the dose-fractionation regime that has the greatest chance of benefiting the patient. These results also encourage further development of EGFR targeting combined with fractionated radiotherapy in HNSCC.
Accelerated repopulation of tumor cells during fractionated radiotherapy is one of the mechanisms by which human tumors may evade the cytotoxic effect of ionizing radiation.1-5 This is reflected clinically in an increasing tumor-control probability when a fixed total dose is delivered in a fixed number of fractions during a shorter overall treatment time, as recently demonstrated for head and neck squamous cell carcinoma (HNSCC) in a large randomized trial.6 Several randomized controlled trials have shown that increasing the rate of dose accumulation per week leads to an increased tumor-control probability in HNSCC (for a review, see Bernier and Bentzen7) as well as in non–small-cell lung cancer.8 Evidence for other tumor types is typically of lower strength, mainly being derived from nonrandomized studies. Traditionally, attempts to overcome the treatment resistance springing from accelerated repopulation of tumor cells have concentrated on altering the dose-time–fractionation pattern. However, the therapeutic gain from altered fractionation schedules observed in recent HNSCC trials may be close to the limit of what can be achieved by this means alone in unselected cases.5 Thus, further therapeutic progress is likely to require new strategies. One strategy is to develop predictive assays capable of selecting patients with a greater-than-average benefit from strongly accelerated radiotherapy. Promising approaches are gene-expression profiling9-11 or more hypothesis-driven approaches such as molecular expression profiling using a panel of immunohistochemical markers involved in various aspects of cell proliferation or the response to radiation therapy.12 Another strategy is to improve radiotherapy outcome by targeting tumor cell repopulation by either the addition of cytotoxic chemotherapy or new targeted therapies developed from our improved understanding of the molecular mechanisms behind accelerated repopulation. Epidermal growth factor receptor (EGFR; also known as ErbB1 and HER 1), the first type I receptor tyrosine kinase identified,13 is a key regulator of cellular proliferation. Members of the ErbB family of receptors trigger a complex network of downstream signaling pathways that mediate a multitude of cellular responses including cell division, apoptosis, motility, invasion, adhesion, and DNA-damage repair.14 Of major importance in the present context is that ionizing radiation can activate EGFR and other receptor tyrosine kinases.15-17 This activation also induces a range of cytoprotective responses including increased cell proliferation, reduced apoptosis, and enhanced DNA repair. Overexpression of EGFR is a feature of many human cancers including head and neck, lung, breast, prostate, colon, and ovarian cancers,18,19 which makes this receptor an interesting potential target for cancer therapy. Indeed, a number of agents targeting EGFR are currently in clinical trials, and even more are in preclinical development.20 There is a strong rationale for combining these with radiation therapy because of the importance of tumor cell proliferation and accelerated repopulation as a clinical resistance mechanism. As a first proof of principle, we look here for a direct link between EGFR expression as assessed by immunohistochemistry on diagnostic biopsies and the effect of overall treatment time in HNSCC patients. The randomized controlled trial of strongly accelerated radiotherapy, continuous hyperfractionated accelerated radiotherapy (CHART), in patients with HNSCC is an ideal setting in which to test a possible interaction between high EGFR expression and a benefit from an increased rate of dose accumulation.
The CHART Head and Neck Trial The CHART head and neck phase III trial, sponsored by the Medical Research Council (United Kingdom), accrued 918 patients from March 1990 to April 1995. Patients were eligible if they were older than 18 years with HNSCC in the main subsites within the head and neck region except for T1N0 tumors. Exclusion criteria were evidence of distant metastasis or a WHO performance status of 2 or worse. Patients were centrally randomly assigned with a 3:2 allocation in favor of the CHART arm. Dische et al21 published details of the pretreatment investigations, the radiotherapy planning, and the radiotherapy quality-assurance program.
Briefly, radiotherapy was delivered by a shrinking-field technique. The large volume included the primary tumor, any involved lymph nodes, and the relevant area of lymphatic drainage. The small volume included the primary tumor and the known nodal involvement with a margin. Doses were prescribed at the intersection point of the central axes, and the range between the minimum and maximum tumor dose was required to be Conventional fractionation delivered 44 Gy in 22 fractions to the large volume followed by an additional 22 Gy in 11 fractions to the small volume. Thus, a total dose of 66 Gy in 2-Gy fractions was delivered with one fraction per day, 5 days per week, during a planned total overall treatment time of 45 days. CHART delivered 1.5 Gy per fraction, three fractions per day, with a strict 6-hour interval between fractions given in a day. Radiotherapy was delivered on 12 consecutive days, always starting on a Monday, treating on Saturday and Sunday, and finishing Friday of the second week. The large volume received 37.5 Gy in 25 fractions, and the small volume received an additional 16.5 Gy in 11 fractions, thus delivering a total dose of 54 Gy in 36 fractions in just 12 days to the gross tumor volume. For the present study, histologic slides of the primary tumor from 304 patients were available, and their clinicopathologic characteristics are listed in Table 1.
Immunohistochemistry Formalin-fixed paraffin-embedded sections (3 to 4 µm) were dewaxed in xylene for 5 minutes before rehydration through graded alcohols (100%, 90%, and 70%) to water. Antigen retrieval was performed on the slides by placing them in a bath of 10 mmol/L citric acid (pH = 6) and boiling for 16 minutes using an 800-W microwave oven (2,450 MHz; Panasonic NN-6453BBPQ; John Lewis, Watford, UK). The volume of fluid was topped up, and the slides then were left to stand for 20 minutes at room temperature before being washed well in running tap water. Slides then were transferred to a DAKO (Carpinteria, CA) Autostainer machine containing peroxidase block (DAKO S2023), the detection reagent (Chemate Envision horseradish peroxidase, DAKO K5007), and antihuman EGFR antibody (NCL-L-EGFR; Novocastra Labs Ltd, Peterborough, UK) diluted 1:20 in antibody diluent (DAKO S2023). The Autostainer program included 5 minutes in peroxidase block, 1-hour incubation in primary antibody, 30-minute incubation in Envision polymer, and 5 minutes in diaminobenzidine substrate. When the program was complete, stained slides were removed from the machine and counterstained in Gills hematoxylin (01500E; Surgipath Europe Ltd, Peterborough, UK) for 30 seconds. Slides then were washed in tap water, dehydrated in graded alcohols (70%, 90%, and 100%), cleared in xylene, and mounted in DPX (08600E; Surgipath Europe Ltd). Each staining run incorporated a positive control slide of EGFR-positive squamous cell carcinoma of the cervix. A negative control was also incorporated and involved the substitution of the antihuman EGFR antibody for an isotypic control antibody at the same protein concentration. A series of markers was previously assessed in 290 of the present cases.12,22 The microvessel count identified by CD31 staining was classified in 10 high-power fields: 1, less than 35 microvessels; 2, 35 to 55 microvessels; and 3, more than 55 microvessels. Cyclin D1 was assessed by manual counting aided by an in-house image-acquisition system. The Ki-67 proliferative pattern was assessed as described previously23: 1, marginal (mostly organized); 2, intermediate (mainly organized); 3, mixed (> one pattern); and 4, random (diffuse, disorganized staining). Ki-67 slides were visually scanned and scored: 1, less than 20% positive cells; 2, 20% to 40% positive; and 3, more than 40% positivity. Bcl-2 was scored as negative if less than 5% of cells were stained and scored as positive otherwise.24 The p53 staining was scored as: 1, negative (< 5% positive cells); 2, sporadic (5% to 75% positive cells); and 3, all (> 75% cells). The staining intensity was classified as: 1, weak; 2, intermediate; and 3, strong. At least 10 high-power fields (x40 objective) were assessed for each specimen.
Evaluation of Slides The EGFR index was estimated as the percentage of tumor cells with membrane staining. All tumor present in the slide was evaluated. Slides were classified as negative, with an EGFR index of zero, if not a single tumor cell with stained membrane was observed.
Follow-Up, Clinical End Point, and Statistical Analysis The primary end point in the present analysis was locoregional treatment failure, defined as persistent viable tumor after primary radiotherapy or progression of disease in primary (T) or nodal (N) position within 3 years after the end of radiotherapy. The restriction of the time window was based on the observation of some late cases of locoregional progression or possibly new primaries at rather long follow-up times when relatively few patients were at risk. A second analysis used the whole range of available observation times. Box-and-whisker plots were used to summarize the distribution of EGFR index in subgroups of cases. The bottom and top of the box indicate the first and third quartile of the distribution, respectively, and the bold line indicates the median value. The whiskers indicate the lowest and highest values of the EGFR index that are not outliers in a statistical sense. Outliers are indicated as open circles and are defined as cases with values between one and a half and three times the interquartile range from the upper or lower edge of the box. Extreme values are indicated by an asterisk and are located more than three times the interquartile range from the upper or lower edge of the box.
Differences in baseline characteristics between patients in the CHART trial who were or were not included in the EGFR study were tested for statistical significance by using Pearson's
Patient and tumor characteristics were distributed similarly in the EGFR substudy population and in the remaining patients in the CHART trial. Also, the two groups had a similar outcome with respect to the main study end point: the 3-year locoregional control estimates (± 1 SE of the estimate, SEE) were 47.4% ± 3.0% and 51.4% ± 4.9% in patients who were and who were not included in the EGFR study, respectively (2P = .30).
Figure 1 shows the relative frequency distribution of EGFR indices in this study. Approximately 84% (56 of 304) of all tumor samples demonstrated EGFR expression. The 95% CI for this proportion is 80% to 88%. An EGFR index of
Correlation Between EGFR Status and Clinicopathologic Parameters The most prevalent primary anatomic subsites in the CHART trial were larynx, oropharynx, hypopharynx, and oral cavity, with only 4% of the tumors originating from other subsites (Table 1). Figure 2 shows the distribution of the EGFR index for various anatomic subsites. The variation among them was statistically significant (2P = .011, Kruskal-Wallis test). After the Bonferroni correction there was still moderate support for an association between subsite and EGFR index (2P = .13). Post hoc tests using Tamhane's procedure (for which equal variances are not assumed) showed that the significant differences arose between oral cavity on one side and larynx (2P = 5 x 10–7), oropharynx (2P = .005), and hypopharynx (2P = .03) on the other.
Histologic grade was available in 88% of the tumors in this study and was highly significantly associated with EGFR expression, with a higher EGFR index in more differentiated tumors (Fig 3). The two-tailed P value was highly significant also after the Bonferroni correction (Table 2).
Figure 4 illustrates the statistically significant association between the microvessel density and the EGFR index. The two-tailed P value was .0016 without and .02 with the Bonferroni correction.
There was no significant correlation between EGFR index and the Ki-67 index, Ki-67 pattern, P53 index, or P53 intensity (Table 2). Bcl-2-positive and -negative tumors did not differ significantly in their EGFR expression. The EGFR index showed no significant association with any of the clinicopathologic features, including T and N category or maximum tumor diameter (size). A weak correlation was seen between the EGFR and cyclin D1 indices (rS = 0.15; 2P = .018). However, this was not significant after the Bonferroni correction.
EGFR Status and Outcome of Radiotherapy
The one-sided hypothesis that high EGFR expression is predictive for a benefit from accelerated radiotherapy was tested as the interaction between randomization arm and EGFR status. Comparing the hazard ratios for a benefit from CHART relative to conventional fractionation in the two EGFR strata yielded a P value of .048. There was no significant effect of EGFR expression on overall survival (2P = .44). The estimated absolute 5-year survival advantage of a patient with a tumor with a low versus a high EGFR index was 4.5%, with 95% confidence limits of –7% and 16%. The distant-metastasis rate at 5 years was close to 18% irrespective of the EGFR status. The estimated absolute difference in 5-year metastasis rate, in favor of patients with low EGFR, was only 0.4% but had wide 95% confidence limits (–12%, 8%).
EGFR is a promising therapeutic target, partly because it is overexpressed in many solid cancers and partly because this receptor and other members of the ErbB family are actively involved in all the "hallmarks" of cancer.29 In relation to radiotherapy, the interest centers on the role of EGFR in regulating cellular proliferation. Here we present the first direct demonstration of the link between EGFR status and the time factor in fractionated radiotherapy in a clinical study with random allocation to 12 v 45 days of overall treatment time. Overall, there was no statistically significant difference between the locoregional control rate in the two arms of the CHART trial.21 The short overall time of CHART necessitates a reduction of the total dose to stay within the limit of mucosal tolerance.30 In the CHART arm, patients received 54 Gy in 36 fractions, and using the linear-quadratic model with an  /ß ratio of 10 Gy,31 this corresponds to an equivalent dose in 2-Gy fractions of 51.75 Gy. The identical locoregional tumor control in the two arms means that the 14.25-Gy higher dose in the conventional arm must have been offset by the effect of repopulation in the interval from 12 to 45 days. Figure 5 shows that in the low-EGFR stratum the trade-off between dose and time in the CHART relative to the conventional arm just balanced out. In high-EGFR tumors, CHART was more effective than conventional fractionation. The implications of this finding for optimal design of dose-fractionation schedules (with or without concurrent EGFR targeting) are the topic of work in progress. Three recent studies looked at the prognostic significance of EGFR in patients with HNSCC after radiotherapy.32-34 Ang et al32 performed an immunohistochemical analysis of pretreatment biopsy material from 155 of 268 patients randomly assigned to the conventional-fractionation arm of the Radiation Therapy Oncology Group (RTOG) 90.03 trial.35 They found a significantly higher locoregional relapse rate and significantly lower overall and disease-free survival rate in patients with high EGFR expression. Because the study included only patients from the conventional arm of the RTOG trial, there was no opportunity to look for a potential predictive value of EGFR in selecting candidates for accelerated radiotherapy. In the conventional arm of our study, patients with high EGFR expression tended to have a worse locoregional outcome than patients with low EGFR expression. However, after CHART, the high-EGFR patients did slightly better than the low-EGFR patients. Thus, when analyzing all patients together, there was no significant negative impact of high EGFR expression on locoregional tumor control (data not shown), which may also explain the significant effect of EGFR status on overall survival in the American trial but not in the present study. This would support a scenario in which high EGFR expression is associated with a poor prognosis, but most of this effect can be counteracted effectively by strongly accelerated radiotherapy. The upper limit of the 95% CI for the survival advantage of EGFR expression below rather than above the median in the present study was 16%. It is also encouraging from a therapeutic management perspective that the rate of distant metastasis was similar in the two EGFR strata, although the statistical power of this comparison was admittedly quite low. Eriksen et al33 analyzed pretreatment biopsies from 336 patients selected from three consecutive trials conducted by the Danish Head and Neck Cancer group. This design produced three groups of patients who received radiotherapy in 5.5, 6.5, or 9.5 weeks. A limitation of the study was the nonrandom assignment to the study groups, resulting in some marked differences in patient characteristics among them. For example, the proportion of patients with T3-T4 tumors was 64% in the 9.5-week group, 27% in the 6.5-week group, and only 9% in the 5.5-week group. Eriksen et al took this into consideration by looking at the prognostic value of EGFR expression within rather than across the treatment-time groups. High EGFR expression was associated with a significantly poorer 5-year local control rate only in the 9.5-week split-course radiotherapy group but not in the 5.5- and 6.5-week groups. This is in qualitative agreement with our findings that the negative impact of high EGFR expression might be counteracted by shortening the overall treatment time. Despite the nonrandom allocation of patients to the 5.5- and 6.5-week groups in the Danish study, the data presented agree with a larger benefit from accelerated fractionation in the high-EGFR patients, which again supports the observations in our study. Eriksen et al33 also found a highly significant correlation between EGFR expression and differentiation (P = .002), in agreement with our findings. However, the Danish group also observed a highly significant correlation between T category and EGFR expression, with higher expression in the T1-T2 tumors (P = .001). Because histologic differentiation is a strong predictor for an effect of overall treatment time in the Danish study, this is a confounding factor in the analysis of the EGFR effect. It is not clear whether EGFR expression could be the underlying cause of the effect of differentiation. The interpretation of the Danish Head and Neck Cancer study is complicated further by the fact that the proportion of tumors with high EGFR and moderately or well-differentiated tumors was significantly higher among the larynx cases than among the pharynx cases. Turning to the correlations between EGFR expression and clinicopathologic tumor characteristics in the present study, the most significant association was seen between histologic differentiation and EGFR status. However, the actual magnitude of the correlation was quite modest (rS = –0.27), and therefore histologic grade is not likely to be a useful surrogate for EGFR status.
The third recently published study of EGFR expression in HNSCC is a rather small series of 54 patients with advanced-stage nasopharyngeal carcinoma.34 These patients received induction chemotherapy with two to three cycles of 60 mg/m2 cisplatin and 110 mg/m2 epirubicin every 3 weeks followed by radiotherapy and therefore may not be readily comparable with patients receiving definitive radiotherapy. There was no attempt to link EGFR status to the overall treatment time, but patients with a high EGFR index had a significantly poorer relapse-free survival and a significantly lower locoregional control probability. This study from the University of Hong Kong used the Novocastra NCL-EGFR-384 (clone EGFR.25) antibody raised to the cytoplasmic domain of the EGFR molecule, whereas we used the NCL-L-EGFR (clone EGFR.113) raised to the extracellular domain. Both can be used to assess the EGFR status of tissues and tumors. Chau et al34 found EGFR expression in 89% (48 of 54) of their cases, in good agreement with the 84% reported here (Fisher's exact test, 2P = .53). Also, the proportion of patients with an EGFR index of In our study, tumors with a high EGFR expression had a significantly higher microvessel count, supporting a possible mechanistic relationship mediated by the proangiogenic property of EGFR. It is interesting to note that there was no direct relationship between EGFR overexpression and increased proliferative index as measured by Ki-67. However, there was a suggestion that overexpression of cyclin D1 was associated with the high-EGFR-status tumors, although this did not reach significance after correction for multiple comparisons. This latter relationship might be expected because cyclin D1 is directly involved in the signal transduction pathway linking EGF stimulation to cell-cycle activation.36 There was also some variation in EGFR expression among the anatomic subsites of the head and neck, with a lower EGFR expression in oral cavity tumors and also possibly nasopharyngeal tumors, although there were only nine such tumors in the present study. Whether these findings have any real biologic significance should be tested in an independent study. In conclusion, there was a significant benefit from strongly accelerated CHART in patients with HNSCC who had high EGFR expression and no benefit in patients with an EGFR index below the median. These findings encourage further developments of EGFR targeting in combination with fractionated radiotherapy in this disease. The present study supports the hypothesis that the EGFR receptor, possibly in concert with the other members of the ErbB receptor family, may be the master switch for the proliferative cellular response to fractionated radiotherapy in HNSCC.
The authors indicated no potential conflicts of interest.
We acknowledge the great efforts of all members of the continuous hyperfractionated accelerated radiotherapy steering committee: A. Barrett (Chairman), B. Cottier, D. Coyle, A.M. Crellin, P. Dawes, S. Dische, M.F. Drummond, C. Gaffney, D. Gibson, A. Harvey, J.M. Henk, T. Herrmann, B. Littbrand, J. Littler, F. Macbeth, D.A. L. Morgan, H. Newman, M. K.B. Parmar, A. G. Robertson, M. Robinson, R.I. Rothwell, M. I. Saunders, R.P. Symonds, J.S. Tobias, M.J. Whipp, and H. Yosef.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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32. Ang KK, Berkey BA, Tu X, et al: Impact of epidermal growth factor receptor expression on survival and pattern of relapse in patients with advanced head and neck carcinoma. Cancer Res 62:7350-7356, 2002 33. Eriksen JG, Steiniche T, Askaa J, et al: The prognostic value of epidermal growth factor receptor is related to tumor differentiation and the overall treatment time of radiotherapy in squamous cell carcinomas of the head and neck. Int J Radiat Oncol Biol Phys 58:561-566, 2004[Medline] 34. Chua DT, Nicholls JM, Sham JS, et al: Prognostic value of epidermal growth factor receptor expression in patients with advanced stage nasopharyngeal carcinoma treated with induction chemotherapy and radiotherapy. Int J Radiat Oncol Biol Phys 59:11-20, 2004 35. Fu KK, Pajak TF, Trotti A, et al: A Radiation Therapy Oncology Group (RTOG) phase III randomized study to compare hyperfractionation and two variants of accelerated fractionation to standard fractionation radiotherapy for head and neck squamous cell carcinomas: First report of RTOG 9003. Int J Radiat Oncol Biol Phys 48:7-16, 2000[CrossRef][Medline] 36. Lukas J, Bartkova J, Bartek J: Convergence of mitogenic signalling cascades from diverse classes of receptors at the cyclin D-cyclin-dependent kinase-pRb-controlled G1 checkpoint. Mol Cell Biol 16:6917-6925, 1996[Abstract] Submitted November 18, 2004; accepted February 17, 2005.
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Copyright © 2005 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
10%. 
2 test for independence except in the case of patient age, for which the Mann-Whitney test was used. Associations between EGFR index and other immunohistochemical or clinicopathologic characteristics were quantified by using the nonparametric Spearman's rank-correlation coefficient, rS, with the P value for rejecting the hypothesis rS = 0. The only exception was the association between EGFR index and subsite within the head and neck region, for which the nonparametric Kruskal-Wallis test was used. A Bonferroni correction was applied to adjust for multiple comparisons when estimating the P value for these associations. Time-of-occurrence data were analyzed by using the Kaplan-Meier method
25% was seen in 63% of all samples with a 95% CI of 58% to 69%. 
