Impact of Epidermal Growth Factor Receptor Expression on Survival and Pattern of Relapse in Patients with Advanced Head and Neck Carcinoma

INTRODUCTION

HNSCCs3 are rather challenging to manage because of their heterogeneity in the natural history and the demand for not only better tumor control but also improved functional and cosmetic results. The data of large randomized trials addressing optimization of radiation fractionation regimen collectively show that biologically based modifications of the fractionation schedule have improved the LR control rate in the order of 10–15% but have had only a modest impact on the OS rate (1) . The results of scores of clinical trials testing combined modality therapy reveal that cytotoxic agents given before (induction or neoadjuvant chemotherapy) or after (adjuvant chemotherapy) surgery or radiation do not improve the therapeutic outcome appreciably relative to LR treatment alone. In contrast, administering chemotherapy concurrently with radiation has improved the OS rate by about 8% (2) ; unfortunately, it has done so at the expense of increased toxicity (3) . However, despite decades of intensive clinical investigations, the outcome of patients presenting with stage III−IV HNSCC is still poor, with 5-year actuarial survival rates fluctuating between 30% and 40% in most trials, depending on the patient eligibility criteria (2) . These findings underscore the need to develop novel strategies in the management of patients with advanced HNSCC.

Advances in the understanding of the molecular biology of HNSCC have opened many new research directions. Increasing effort has been directed toward developing molecular targeted therapies or searching for molecular markers that are useful either in predicting treatment outcome or in selecting patients for specific molecular targeted therapies based on particular tumor characteristics. Three recently published review articles (4, 5, 6) summarize the current status of the search for prognosis-predictive biomarkers. Although none of the studies has identified convincing data to warrant routine clinical application of any marker, encouraging leads have been generated for a number of molecules including p53, EGFR and one of its ligands, TGF-α, and cyclin D1 (6) .

We undertook a correlative biomarker study on a relatively large, prospective series of patients with advanced HNSCC enrolled in a Phase III trial of the RTOG and randomized to the standard therapy arm, i.e., conventionally fractionated radiotherapy (70 Gy in 7 weeks). The results revealed that EGFR expression was not correlated with disease stage at presentation or other known clinical prognostic variables. However, correlative analysis showed that EGFR expression was a strong prognostic indicator for OS and DFS and was highly predictive for the probability of LR relapse but not for distant metastasis. Our results suggest that quantitative EGFR IHC assay should be valuable for selecting patients for different currently available therapy modalities or enrollment into trials testing novel therapies targeting EGFR or its downstream signaling pathways.

MATERIALS AND METHODS

Study Population.

Between 1991 and 1998, RTOG completed accrual of 1113 patients to a large four-arm Phase III trial comparing the efficacy of three experimental radiation regimens with standard fractionated radiotherapy (7) . Patients with AJCC stage III−IV carcinoma of the oral cavity, oropharynx, and supraglottic larynx and stage II−IV carcinoma of the tongue base and hypopharynx were enrolled. Although 30 institutions participated in this study, 14 centers enrolled 80% of the patients. All patients had pretreatment tumor biopsy to establish histological diagnosis of HNSCC. Paraffin-embedded blocks or unstained slides were sent to the RTOG tissue repository center at the Department of Pathology, LDS Hospital, University of Utah School of Medicine for storage and preparation for biomarker studies.

Radiotherapy records of each patient, including simulation and verification portal films, total dose, number of fractions, and elapsed treatment days relative to the protocol prescription were verified by the two radiation oncology study co-chairs with the assistance of the RTOG staff. Follow-up assessment of LR control and (when indicated) systemic spread took place around 4 weeks after completion of treatment and then every 3 months for the first 1–1.5 years, every 4 months from 18 months through 3 years, biannually in years 3–5, and annually thereafter. The median follow-up for surviving patients was 41.2 months at the time of analysis.

The current study population consisted of 155 of 268 (58%) patients (30% had stage III disease, and 66% had stage IV disease) randomized to the conventional radiotherapy (70 Gy in 7 weeks) arm and had sufficiently large pretreatment paraffin-embedded biopsy specimens for cutting additional slides for IHC assay. The distribution of patient and tumor characteristics and the treatment outcome of the study population were compared with those of the remaining 113 non-study group to detect the presence of potential systematic differences.

EGFR Immunohistochemical Assay.

The paraffin sections were dewaxed and hydrated in a graded series of alcohol, followed by blockage in 3% H₂O₂/methanol for 5 min and enzymatic digestion with 0.02% (w/v) protease type XXIV for 2 min at room temperature. The slides were incubated for 1 h with mouse monoclonal antibodies that react to the peptide backbone of the extracellular domain of the EGFR molecule (31G7; Zymed Laboratories, Inc.) diluted 1:50 in dilution buffer at room temperature. Subsequently, the slides were rinsed for 2 min in PBS (pH 7.6) for three times and incubated with Dako’s Mouse EnVision+ Peroxidase System for 30 min. The peroxidase-catalyzed product was visualized with the BioGenex DAB Chromogen Kit. The sections were finally lightly counterstained with Mayer’s hematoxylin (Sigma Chemical Co.) for 15 s, rinsed, intensified, dehydrated, and mounted for quantitative analysis.

Interassay consistency was monitored by inclusion of positive and negative controls with each batch of staining. These consisted of sections of a high EGFR-expressing lung cancer stained with primary antibody and isotype-matched irrelevant antibody, respectively. Each batch of 24 slides thus contained a positive control, a negative control, and slides of 22 cases. Measurements were normalized to these controls (negative control = 0, positive control = 100%).

Quantitation of EGFR Expression.

The magnitude of EGFR expression was measured by computerized quantitative image analysis using a SAMBA 4000 Cell Image Analysis System (SAMBA Technologies, Meylan, France) without knowledge of clinical data. The image analyzer is a PC-based integrated system for densitometric, morphometric, and colorimetric analysis of cells and tissues. It consists of a Zeiss microscope with ×10, ×20, and ×40 objectives and a Sony 960MD 3-chip charge-coupled device camera interfaced with a Power Spec computer (Micro Center Co., Houston, TX) equipped with a Matrox Meteor digitizer board (Matrox Electronic Systems Ltd., Dorval, Quebec, Canada).

Light and camera settings were standardized, resulting in average background values of 210 ± 5 (mean ± SD, scale 0–255 from black to white) for the red, green, and blue channels. Images were captured using ×20 objective. Background subtraction was automatically performed on every tissue after storing an empty field of the slide. Analysis was performed after transformation of the red-green-blue information to hue-saturation-intensity information. The SAMBA software (Immuno-Analysis Version 4.22) enables the operator, after evaluating several fields on control slides, to set an intensity threshold value for the best discrimination between tissue and background. Separation between the hematoxylin counterstain (blue) and the DAB immunostaining (brown) was achieved by setting a proper threshold on the hue value. The threshold for DAB-positive hues was obtained by evaluating several fields on positive and negative control slides for optimal separation between blue- and brown-stained areas. Stromal components were removed from the analysis by interactive cut and paste techniques.

Parameters measured were MOD (MOD = the mean of optical densities measured over the labeled areas within the structure, proportional to the mean stain concentration), SI (SI = the proportion of stained area relative to the total area of the structures), and QS (QS = MOD × SI/100). Averaging the quantitative computerized image analysis data obtained from nine fields of each section yielded these parameters. These parameters with or without subtraction of a paired negative control slide (isotype-matched irrelevant antibody) were correlated with clinical outcome data.

Data Analysis.

The parameters of EGFR expression were sent to the RTOG Statistical Unit for incorporation into the study data set for performing correlative analysis. To determined whether the study population is representative for the whole group, the known clinical prognostic profile and outcome end points of patients with successful EGFR assay were first compared with those of patients with no or insufficient specimens. The magnitude and distribution of pretreatment EGFR expression were then assessed using various descriptive statistics (mean, median, and so forth) along with their association with the known tumor- and patient-related prognostic variables and RPA (8) for survival and LR control. Finally, the magnitude and distribution of pretreatment EGFR expression were correlated with the OS, DFS, and pattern of relapse (LR recurrence and distant metastasis).

Statistical Method.

Comparisons of baseline characteristics were done using the Pearson χ² test (9) , with the exception of age, which was performed with a t test. OS and DFS were estimated using the Kaplan-Meier method (10) and compared by the log-rank test (11) . Time to LR control and distant metastases were estimated by the method of cumulative incidence (12) and tested with Gray’s test (13) . Multivariate analysis was performed using the Cox proportional hazards model (14) .

RESULTS

Clinical Characteristics of the Study Population.

We compared the distribution of patient and tumor characteristics and treatment outcome between the study group and the remaining patients to assess the presence of potential imbalances in the known prognostic variables. Tables 1⇓ and 2⇓ show that there was no significant difference between the groups in the distribution of known clinical prognostic indicators of survival and LR control, i.e., KPS, age, T stage, N stage, combined stage grouping, and RPA classes. There was, however, some imbalance in the distribution by gender and primary tumor site, i.e., the study group had relatively more women and more patients with supraglottic cancer but fewer patients with oropharyngeal carcinoma. Table 3⇓ shows that there was no difference between the two groups in treatment outcome end points, i.e., OS, DFS, LR control, and distant metastasis rates.

Variation of EGFR Expression and Its Correlation with Known Prognostic Factors and Treatment Outcome.

Figs. 1⇓ and 2⇓ show that HNSCCs had a wide range of EGFR expression [i.e., the MOD varied from 0.2 to 66.0 (median, 24.0), SI varied from 0.3 to 97.0 (median, 80.7), and QS varied from 0.01 to 69.9 (median, 23.8)]. Of the 155 tumors examined, 148 (95%) had detectable EGFR expression. There was a relatively strong but nonlinear correlation between MOD and SI (correlation coefficient, 0.79).

Table 4⇓ reveals that there was no correlation between EGFR expression and the known clinical prognostic indicators, i.e., T stage, N stage, stage grouping, and RPA classes for survival and LR control. However, as shown in Fig. 3⇓ and Table 5⇓ , the level of EGFR expression, quantified either with or without subtraction of negative controls, had highly significant correlation with the OS, DFS, and LR control rates, but not with the incidence of distant metastasis. Patients with higher (>median MOD) EGFR-expressing HNSCCs had highly significantly poorer OS and DFS (Fig. 3, A and B)⇓ and lower LR control (Fig. 3C)⇓ than those with lower EGFR-expressing cancer. In contrast, EGFR expression had no influence on the incidence of distant metastasis (Fig. 3D)⇓ . Significant correlations, although slightly less robust than MOD, were also observed between SI and QS and the OS, DFS, and LR relapse rates (Table 5)⇓ . In multivariate analyses (Table 6)⇓ , EGFR MOD came out as a strong independent prognostic factor for OS and DFS and as the strongest predictor for LR control.

DISCUSSION

EGFR, a M_r 170,000 transmembrane glycoprotein, is a member of the large receptor tyrosine kinase family encoded by a gene located in human chromosome 7p12 (15) . EGFR activation by ligand binding leads to parallel signaling mainly through Ras, MAPK/MAPK-extracellular signal-regulated kinase kinase, phosphatidylinositol-3′ kinase/Akt, phospholipase-γ/protein kinase-α, and signal transducer and activator-3 pathways (16 , 17) . Activation of these pathways ultimately leads to transcription of other genes responsible for cell growth, differentiation, and death. However, how these downstream pathways regulate radiation sensitivity is not well understood and is the subject of investigation in many laboratories. A study using the DU145 prostate carcinoma cell line, for example, showed that after irradiation these cells become dependent on MAPK activation for cell cycle progression and that MAPK inhibition results in cell cycle arrest at the G₂-M-phase transition, which is accompanied by cell death when MAPK inhibition is sustained (18) . Another study revealed that EGFR blockade leads to redistribution of DNA-PK from the nucleus to the cytosol, resulting in reduced radiation-induced DNA damage repair and thus radiation sensitization, detectable through the classical split-dose experiment (19) .

The EGFR pathways attracted the attention of head and neck cancer investigators because the majority of HNSCCs have elevated expression of EGFR and its ligand, TGF-α. For example, Grandis and Tweardy (20) found a mean 69-fold (range, 0.3–690) EGFR mRNA increase and a 5-fold (range, 0.1–19.9) TGF-α mRNA elevation in 91% and 87.5%, respectively, of 24 HNSCCs assessed relative to mRNA levels in normal mucosa. The corresponding magnitudes of increase in EGFR and TGF-α proteins, determined by IHC (MOD), were 1.7-fold (P = 0.005) and 1.9-fold (0.006), respectively (21) . The nature of the protein overexpression is thought to result from enhanced transcription with no apparent change in mRNA stability (20) , although gene amplification has also been observed (22) .

Three teams of investigators explored the potential prognostic significance of EGFR expression in patients with laryngeal carcinomas treated with radiotherapy (23 , 24) or surgery (25) . The studies of Miyaguchi et al. (23) and Wen et al. (24) yielded conflicting results in relatively small series of patients with early laryngeal carcinomas (T_1–2) receiving various radiation doses. The lower radiation dose range administered (24) may be considered substandard by current norm and, therefore may have accounted for the rather poor overall outcome. Maurizi et al. (25) determined the EGFR expression by radioligand receptor assay on frozen tumor samples in 140 patients with T_1–4 laryngeal carcinomas with or without palpable nodes who underwent conservative (n = 67) or radical (n = 73) laryngectomy alone. The median EGFR value in this series was 8.4 fmol mg⁻¹ protein but ranged from 0 to 169.9 fmol mg⁻¹ protein. Multivariate analysis revealed that T stage and EGFR level were the most important independent prognostic factors for relapse-free survival and OS. Unfortunately, the pattern of failure was not addressed in this study.

More recently, Grandis et al. (26) determined the expression of EGFR and TGF-α proteins by IHC in 91 patients with various stages (T₁4N_0–1) and sites of HNSCC all treated with surgical resection and followed by radiotherapy in 56 patients and chemotherapy in 16 patients. Analysis of IHC data and treatment outcome using a proportional hazard model revealed that the combination of EGFR or TGF-α level with lymph node stage was the strongest predictor of cause-specific survival. The combination of EGFR level with lymph node stage was found to be as strong a predictor as the triple combination of EGFR level plus TGF-α level plus lymph node stage. The exclusion of the EGFR level from the model resulted in a statistically significant reduction in the predictive power. This study also did not address the effects of protein expression on the pattern of relapse of the index cancer.

We performed a quantitative IHC study using antibodies predominantly recognizing the membrane-associated extracellular domain of the EGFR molecule (see Fig. 1⇓ ) in a larger series of representative patients enrolled in a prospective clinical trial and treated with a consistent radiation regimen alone. The results showed that advanced, predominantly stage III−IV HNSCC had a wide range of EGFR expression, which did not correlate with the tumor or nodal stage, other established prognostic combined stage groupings (AJCC or RPA), or patient performance status. Correlative analysis showed that EGFR expression was a strong, independent prognostic indicator of OS and DFS.

Analysis of the patterns of relapse in this series revealed a new, clinically relevant finding that EGFR expression was a robust predictor for LR relapse but not for distant metastasis. This observation provides compelling clinical evidence in support of experimental data showing that EGFR expression is the strongest and most consistent indicator of cellular radioresistance in vitro (27) and that there is a close relationship between EGFR overexpression and tumor radioresistance in vivo (28 , 29) . Multivariate analysis (Table 6)⇓ of the current data revealed that EGFR expression was even a stronger predictor of LR control than T stage. Furthermore, Table 7⇓ shows that its addition to T stage improves the predictive power for LR control. Because the incidence of LR relapse is much higher than that of distant metastasis, this finding suggests that patients with EGFR-overexpressing tumor would benefit more from the development of more effective LR therapy. Work aimed at assessing the impact of EGFR expression on the relapse pattern of patients treated with novel radiation regimens or with radiation plus concurrent chemotherapy is in progress.

In view of our finding of lack of correlation between EGFR expression and the propensity for distant metastasis, the interpretation of the data of the surgical series of Maurizi et al. (25) is more complicated because the details of pattern of failure were not presented. It appears reasonable to postulate that the poorer survival of patients with EGFR-overexpressing tumor might have been the consequence of a higher LR relapse rate after surgical resection. If this is the case, then EGFR overexpression may be a marker for more extensive contiguous local spread and would call for more extensive resection or combination of surgery and adjunctive postoperative radiotherapy or combined therapy to yield better LR control. Resolution of this matter will thus have an important therapeutic implication.

Therapeutic approaches targeting EGFR signaling pathway either alone or in combination with radiation or cytotoxic agents are being intensively investigated. Strategies that are in various stages of development include blockade of the extracellular receptor domain (19 , 30, 31, 32, 33, 34, 35) , inhibition of the intracellular tyrosine kinase activity [reviewed by Fry (36) and Noonberg and Benz (37)] , inhibition of receptor production by antisense approaches (32 , 38) , expression of a truncated dominant-negative EGFR mutant (39) , and so on. For example, anti-EGFR antibody, C225, in combination with chemotherapy or radiation, is being addressed in Phase III clinical trials, and many small-molecule tyrosine kinase inhibitors are in Phase I−II clinical testing. Data presented in Fig. 3C⇓ suggest that tumor radiation sensitization through inhibition of EGFR signaling, when successful in humans, could yield a therapeutic gain by increasing the LR control rate in patients with EGFR-overexpressing HNSCC. The nonlinear correlation between MOD (a measure of EGFR density of individual malignant cells) and SI (reflecting the proportion of malignant cells expressing EGFR) raised a question regarding how to define EGFR overexpression. Data presented in Fig. 2⇓ show that the natural break points might be around the median values for both MOD and SI because below the median MOD, there is a wide variation in SI, and above the median, >80% of malignant cells express EGFR in most tumors. Fig. 4⇓ shows that additional cutoff points below or above the median value did not improve the prediction of the risk of LR relapse. The ongoing studies in other cohorts of patients will hopefully refine the cutoff values for use in future trials. Nevertheless, because many types of antibodies are available and some variability in assay techniques exists, an individual laboratory needs to perform assay calibration until a validated standardized kit becomes available. Meanwhile, using SI of 80% as a cutoff point would be a reasonable strategy.

Should ongoing preclinical studies consistently show that EGFR antagonists predominantly affect the radiation sensitivity of tumors with greater than median EGFR expression, our results suggest that the anticipated improvement in the LR control rate would be in the order of 20%, which is less than that shown by the data of Grandis et al. (26) . The relatively simple quantitative EGFR IHC can serve as an important eligibility criterion for selecting patients for enrollment in clinical trials testing these targeted therapies. Such an approach will reduce the required sample size for detecting therapeutic efficacy.

The following RTOG institutions and their affiliates enrolled the vast majority of patients into the randomized trial and contributed pretreatment tumor samples: University of South Florida-H. Lee Moffitt Cancer Center; Radiological Associates of Sacramento; University of Alabama Birmingham Medical Center; University of California San Francisco; University of Texas M. D. Anderson Cancer Center; Fox Chase Cancer Center; New York University Hospital; McGill University; Medical College of Wisconsin; Washington University; Montefiore Medical Center; University of Western Ontario; Akron City Hospital; State University of New York Health Science Center Brooklyn; Wayne State University; University of Pennsylvania Medical Center; Dartmouth Hitchcock Medical Center; Albert Einstein Medical Center; University of Puerto Rico; Emory University Affiliated Hospitals; University of Alberta; Thomas Jefferson University Hospital; University of Rochester; and LDS Hospital.