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Molecular Marker Expression in Oral and Oropharyngeal Squamous Cell Carcinoma
Benjamin D. Smith, BA;
Grace L. Smith, MPH;
Darryl Carter, MD;
Michael P. DiGiovanna, MD, PhD;
Katherine M. Kasowitz, BS;
Clarence T. Sasaki, MD;
Bruce G. Haffty, MD
Arch Otolaryngol Head Neck Surg. 2001;127:780-785.
ABSTRACT
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Objective To determine the relative prognostic value of p53, cyclin D1, epidermal
growth factor receptor (EGFR), and vascular endothelial growth factor (VEGF)
expression in patients with oral and oropharyngeal squamous cell carcinoma.
Design Retrospective cohort study.
Patients Fifty-six patients with oral and oropharyngeal squamous cell carcinoma
referred to the Department of Therapeutic Radiology at Yale–New Haven
Hospital (Conn) between 1981 and 1992 who were treated with gross total surgical
resection and postoperative external beam radiotherapy.
Results Archival tumor tissue was stained with monoclonal antibodies directed
against these 4 oncoproteins and scored for staining intensity and percent
distribution by a pathologist blinded to the patients' clinical outcomes.
Frequency of marker expression was 48% for p53, 20% for cyclin D1, 64% for
EGFR, and 41% for VEGF. In multivariate analysis, EGFR positivity was protective
against locoregional relapse (relative risk [RR], 0.27; 95% confidence interval
[CI], 0.11-0.66; P = .002). In contrast, advanced
N stage predicted poor locoregional relapse-free survival (RR, 1.94; 95% CI,
1.03-3.66; P = .04). Predictors of poor overall survival
in multivariate analysis included VEGF positivity (RR, 3.53; 95% CI, 1.75-7.13; P<.001) and black race (RR, 2.48; 95% CI, 1.05-5.85; P = .04). Cyclin D1 and p53 expression were not significantly
associated with prognosis in this cohort.
Conclusions In oral and oropharyngeal squamous cell carcinoma treated with surgery
and postoperative radiotherapy, VEGF and EGFR expression may influence clinical
outcome. If confirmed, these results have potential implications for the determination
of patient prognosis and the development of biologically based pharmacotherapies.
INTRODUCTION
IN RECENT YEARS, a number of studies have attempted to determine the
prognostic relevance of certain molecular defects commonly found in head and
neck squamous cell carcinoma (HNSCC). Information gained from these studies
may ultimately supplement clinical staging, thereby enabling more accurate
determination of patient prognosis and more effective selection of therapeutic
modalities. In addition, identification of specific molecular defects that
influence tumor behavior may provide new avenues for the development of molecularly
based cancer pharmacotherapies.
Of the molecular markers studied in HNSCC, p53, cyclin D1, and epidermal
growth factor receptor (EGFR) have received particular attention in multiple
studies as possible factors influencing outcome.1 p53 is a tumor suppressor gene that protects the integrity
of the genome by initiating either apoptosis or cell cycle arrest following
DNA damage.2 Cyclin D1
is a proto-oncogene that responds to extracellular mitogens by promoting G1-phase
progression through the cell cycle.3 Similarly, EGFR is a proto-oncogene that, when activated at the cell
surface by transforming growth factor- , serves to promote cellular
proliferation.4 Inactivation of tumor suppressor
genes such as p53 and activation of proto-oncogenes
such as cyclin D1 and EGFR
results in deregulation of the cell cycle and thus promotes neoplasia. These
alterations are amenable to assessment via immunohistochemistry, as p53 gene mutations often confer stability to the p53 protein
resulting in elevated intracellular levels,5
and activation of cyclin D1 and EGFR is often associated with increased protein expression.
Another possible determinant of tumor aggressiveness is vascular endothelial
growth factor (VEGF), a protein that is induced under hypoxic conditions and
promotes vascular permeability and endothelial cell proliferation.6 We have recently published the first report to demonstrate
that VEGF protein levels are an independent risk factor for poor survival
in HNSCC.7 This study showed high levels of
VEGF expression to correlate with local recurrence, distant metastatic disease,
poor disease-free survival, and poor overall survival.
Given these intriguing results, the present study examined the same
cohort of patients to compare the prognostic utility of VEGF protein levels
with the prognostic value of p53, cyclin D1, and EGFR protein levels. Specifically,
this study used multivariate modeling to assess the prognostic significance
of VEGF, p53, cyclin D1, and EGFR protein levels with respect to the specific
end points of locoregional recurrence-free survival and overall survival.
In an effort to ensure homogeneity and enhance the validity of the study,
the patient population selected was limited to patients with squamous cell
carcinoma of the oral cavity and oropharynx who were treated with gross total
surgical resection and postoperative external beam radiation therapy. In addition,
we sought to compare the prognostic utility of these molecular markers with
standard clinicopathologic variables, including TNM stage, tumor site, tumor
grade, margin status, race, sex, and age at diagnosis.
PATIENTS, MATERIALS, AND METHODS
PATIENT SELECTION
Criteria for patient inclusion in this study were as follows: (1) presentation
to the Department of Therapeutic Radiology at Yale–New Haven Hospital
(Conn) between 1981 and 1992 with a histologically confirmed diagnosis of
squamous cell carcinoma in the oral cavity or oropharynx and (2) treatment
with primary surgical excision and postoperative external beam radiotherapy
with curative intent. Patients were excluded from this study if they had a
prior history of HNSCC, presented with metastatic disease, or failed to receive
a full course of radiation therapy. A review of radiation records identified
77 patients who met the entry criteria. Of these, 14 had incomplete follow-up
and 10 tissue samples were unavailable, leaving 56 patients for inclusion
in the study.
With the approval of the appropriate institutional review boards, paraffin-embedded
tissue samples were obtained from the hospital archives. Covariables including
demographic, staging, clinical, pathologic, and treatment parameters were
extracted from patient charts. Local recurrence was
defined as recurrence of disease at a site within the upper aerodigestive
tract anatomically contiguous with the primary tumor. Regional
recurrence was considered recurrence of disease within the cervical
lymphatic system. Distant recurrence was considered
recurrence of disease that did not meet the definitions of local or regional
recurrence and was not considered to represent a second primary tumor based
on its histological and/or clinical manifestations. Forty-one patients (73%)
were followed up until death, and the median follow-up time of the surviving
patients was 6.1 years with a minimum of 2.8 years.
All patients were treated with gross total surgical resection and postoperative
external beam radiotherapy to a median dose of 60 Gy. Final surgical margins
were negative in 38 patients (68%). Patients with evidence of tumor approaching
within 1 high-power field of final surgical margins were categorized as having
positive margins in accordance with the method of Beitler et al.8
Forty-nine patients (88%) received a radical or modified neck dissection.
Eleven patients (20%) received adjuvant chemotherapy with the hypoxic cytotoxins
mitomycin or porfiromycin as part of an institutional protocol.9-10
Five patients (9%) received intraoperative brachytherapy. All patients were
staged clinically according to the American Joint Committee on Cancer TNM
classification system.11 Clinically N0 patients
were restaged for the purposes of this analysis if pathologic examination
results of the neck dissection specimen were positive for nodal metastases.
IMMUNOHISTOCHEMISTRY
Immunohistochemical analysis was performed on 5-µm-thick tissue
sections prepared from formalin-fixed, paraffin-embedded archival tissue from
the resected primary tumor. Tissue sections were deparaffinized and then quenched
in 2% hydrogen peroxide–methanol solution. Samples stained for VEGF
were pretreated by microwaving at 50% power 3 times for 5 minutes in 10-mmol/L
sodium citrate (pH 6.0). Samples stained for EGFR were pretreated with 0.4%
pepsin in 0.1% hydrochloric acid for 15 minutes at 37°C. Samples stained
for p53 and cyclin D1 did not require an antigen retrieval step. Slides were
incubated overnight at 4°C with the following antibodies: (1) a mouse
monoclonal IgG1 reactive with the 34- to 50-kd isoforms of VEGF (1:200 dilution,
clone JH121; Oncogene Research Products, Cambridge, Mass); (2) a mouse monoclonal
IgG2b reactive with wild-type and mutant forms of p53 (1:50 dilution, clone
DO-7; DAKO, Carpinteria, Calif); (3) a mouse monoclonal IgG2b reactive with
human cyclin D1 (1:2500 dilution, clone A-12; Santa Cruz Biotechnology, Santa
Cruz, Calif); and (4) a mouse monoclonal IgG1 antibody specific for the protein
portion of the extracellular domain of EGFR (prediluted, clone E 30; BioGenex,
San Ramon, Calif). The next day, slides were incubated with horse antimouse
secondary antibody, labeled with avidin-biotin complex streptavidin-peroxidase
(Elite; Vector Laboratories, Inc, Burlingame, Calif), incubated with the chromogen
diaminobenzidine tetrahydrochloride, counterstained with hematoxylin, and
mounted.
An experienced pathologist (D.C.), blinded to the clinical outcomes,
examined multiple microscopic fields to score the tissue sections for tumor
staining intensity (0, none; 1, faint and focal; 2, moderate; 3, strong; 4,
intense) and percent distribution. Positivity for cyclin D1 staining was defined
as 5% or more staining distribution in accordance with the method of Pignataro
et al.12 Positivity for p53 staining was defined
as 10% or greater staining, a cut point used in previous studies.13-14 To maximize the power of statistical
comparisons using the VEGF status variable, VEGF status was determined by
dichotomizing the VEGF staining intensity variable with the cut point at the
median between moderate and strong staining. The EGFR status was also determined
by dichotomizing the EGFR staining intensity variable with the cut point at
the median between faint and moderate staining. The EGFR status was not divided
into tertiles as described by Grandis et al15
due to the smaller sample size in this study.
STATISTICAL ANALYSIS
Molecular marker status and relevant covariables were assembled in a
database and analyzed using statistical software.16
All tests of statistical significance were 2-sided. Follow-up time and time
to recurrence were calculated from the date of surgery to the date of the
relevant outcome. To enhance the power of statistical comparisons, the following
categories were collapsed: T stages 1 and 2, T stages 3 and 4, N stages 2
and 3, and tumor grades poorly and poorly to moderately differentiated.
Bivariate analyses for the associations between predictor variables
and the main outcome variables (locoregional relapse-free survival and overall
survival) were conducted using the Kaplan-Meier log-rank test. Variables violating
the linearity assumption entered into the analysis as categorical variables,
with cut points determined by quartiles. Unadjusted relative risks were calculated
using a Cox proportional hazards model.
In the multivariate analysis, Cox proportional hazards regression determined
significant predictors of locoregional recurrence-free survival and overall
survival at an = .05 level in the final model. Molecular marker status
and all clinicopathologic covariables were initially entered into a forward
parsimonious selection. Variables with P .10 were
retained for the final model.
RESULTS
DESCRIPTIVE STATISTICS
A total of 77 patients were eligible for the analysis, but 21 were excluded
due to incomplete follow-up data and/or insufficient archival tissue, leaving
56 patients (73%) in the analysis. Frequency statistics for clinicopathologic
characteristics of the patient cohort are presented in Table 1. Fifty-one patients (91%) presented with stage III or IV
disease, with the remaining 5 patients presenting with stage II. A total of
14 patients (25%) experienced local recurrence, 10 had regional recurrence
(18%), and 14 had distant recurrence (25%). Forty-one patients (73%) died
during the follow-up period. Five-year survival for this cohort was 40% (22
patients).
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Table 1. Clinicopathologic Variables and Outcome*
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Positivity rates for molecular marker expression were as follows: 41%
for VEGF, 48% for p53, 20% for cyclin D1, and 64% for EGFR. Examples of tumors
positive for each of these molecular markers are presented in Figure 1. The VEGF-positive tumors demonstrated cytoplasmic granular
staining, p53 and cyclin D1–positive tumors displayed nuclear staining,
and EGFR-positive tumors showed membranous staining.
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Immunohistochemical staining of formalin-fixed, paraffin-embedded
tumor tissue with monoclonal antibodies directed against vascular endothelial
growth factor (A), p53 (B), cyclin D1 (C), and epidermal growth factor receptor
(D) (original magnification x100, scale bar = 200 µm).
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BIVARIATE ANALYSIS
The relationships between clinicopathologic covariables and the main
outcome measures in this study (locoregional relapse-free survival and overall
survival) are presented in Table 1.
None of the clinicopathologic variables were significantly predictive of locoregional
relapse-free survival in log-rank statistics at = .05. N stage, however,
was significantly predictive of locoregional relapse-free survival in an unadjusted
Cox proportional hazards model (relative risk [RR], 1.97; 95% confidence interval
[CI], 1.03-3.80). Several clinicopathologic covariables were significant risk
factors for poor overall survival, including advanced T stage (RR, 2.09; 95%
CI, 1.01-4.32; P = .04), advanced N stage (RR, 1.91;
95% CI, 1.20-3.05; P = .02), and black race (RR,
2.57; 95% CI, 1.19-5.57; P = .01).
The relationships between molecular marker positivity and the main outcome
variables are presented in Table 2.
The EGFR positivity was strongly protective against locoregional recurrence
(RR, 0.27; 95% CI, 0.11-0.66, P = .002). Borderline
predictors of locoregional recurrence included VEGF status (RR, 2.09; 95%
CI, 0.87-5.02; P = .09) and p53 status (RR, 0.45;
95% CI, 0.18-1.17; P = .09). With respect to overall
survival, the only significant predictor was VEGF status (RR, 3.21; 95% CI,
1.63-6.32; P<.001).
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Table 2. Molecular Marker Expression and Outcome*
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Finally, the relationships between molecular marker expression and distant
recurrence were examined (Table 2).
Significant predictors included VEGF status (RR, 4.62; 95% CI, 1.41-15.10; P = .006), p53 status (RR, 3.02; 95% CI, 0.95-9.64; P = .05), and cyclin D1 status (RR, 2.88; 95% CI, 0.96-8.65; P = .05).
MULTIVARIATE ANALYSIS
Both Cox proportional hazards models included all 56 patients. The VEGF,
p53, cyclin D1, and EGFR status, along with the aforementioned covariables,
were entered into the model. For locoregional relapse-free survival, the variables
N stage and EGFR status were retained at a level of P
= .10 (Table 3). With a final
= .05 criterion, both variables were predictive of locoregional relapse-free
survival. The most significant predictor, EGFR positivity, was protective
against locoregional recurrence (RR, 0.27; 95% CI, 0.11-0.66; P = .002). Thus, EGFR-negative tumors recurred locally or regionally
more than 3 times as often as EGFR-positive tumors. Expression of VEGF, cyclin
D1, and p53 was not significantly associated with locoregional relapse-free
survival in this multivariate model. N stage, the only clinicopathologic variable
included in the final model, was positively correlated with locoregional recurrence
(RR, 1.94; 95% CI, 1.03-3.66; P = .04).
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Table 3. Multivariate Models for Locoregional Relapse-Free Survival
and Overall Survival*
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For overall survival, age, race, and VEGF status were retained at a
level of P = .10 (Table 3). Because age was entered as a 3-level dummy variable, all
age categories were forced into the final model. The most significant predictor
of poor survival in this cohort was VEGF status, with death more than 3 times
as likely in VEGF-positive patients (RR, 3.53; 95% CI, 1.75-7.13; P<.001). Despite entry of cyclin D1, EGFR, and p53 status into the
initial model, none of these markers significantly predicted overall survival
when controlling for other relevant variables. Black race, the only demographic
variable included in the final model, was associated with increased mortality
(RR, 2.48; 95% CI, 1.05-5.85; P = .04). T stage and
N stage were not predictive of survival in multivariate modeling.
COMMENT
This study, conducted on patients with oral cavity and oropharyngeal
primary tumors treated with surgical excision and postoperative radiotherapy,
demonstrates the potential prognostic utility of molecular marker analysis.
Specifically, low levels of EGFR expression were predictive of poor locoregional
recurrence-free survival in multivariate analysis. In addition, high levels
of VEGF expression were highly predictive of poor overall survival in multivariate
analysis. Thus, in this cohort of patients with relatively advanced disease
and poor prognosis, EGFR and VEGF status proved to be better indicators of
outcome than traditional staging parameters, such as T stage and N stage.
In addition, EGFR and VEGF status were more important predictors of outcome
than p53 and cyclin D1 expression.
This study was limited to a relatively homogeneous cohort to control
for variations in tumor behavior imposed by different primary tumor sites
and therapeutic modalities. These criteria enhanced the internal validity
of the study, while limiting its external validity and power. Larger studies
will be needed to verify the results of this study and determine their applicability
to other sites within the head and neck. Furthermore, the scoring systems
used in this study to define VEGF and EGFR positivity have not been used in
previous studies and will need to be prospectively verified in future work.
This study was also limited to the qualitative immunohistochemical assessment
of oncoprotein expression. This technique offers important advantages because
it is relatively inexpensive and readily available in most pathology departments.
Hence, molecular markers that can be assessed via immunohistochemical methods
may be more easily integrated into clinical practice than markers dependent
on various techniques of molecular biology. However, qualitative immunohistochemistry
is subject to interobserver variability and, at least in the case of p53,
does not always correlate with the specific genotypic changes shown to be
associated with poor prognosis.5 As a result,
a study that uses immunohistochemistry alone can never rule out the possibility
that a specific genetic marker affects prognosis.
Despite these limitations, the results of this study provide novel information
that may prove clinically relevant. Because a high level of EGFR was the most
important predictor of locoregional control, EGFR may serve as a marker of
radiosensitivity in this cohort. Indeed, at least 3 studies conducted on cell
lines derived from human squamous cell carcinomas have shown that in vitro
activation of EGFR enhances radiosensitivity.17-19
However, in a different cell line derived from a human HNSCC, Huang et al20 found that inhibition of EGFR with a specific monoclonal
antibody enhanced radiosensitivity. Hence, the role of EGFR in mediating radiosensitization
may depend on the downstream signaling components present in a specific clonal
population of neoplastic cells. This study suggests that, in neoplasms derived
from the oral cavity and oropharynx, high levels of EGFR expression may correlate
with increased radiosensitivity.
In contradistinction to the results of this study, at least 3 excellent
clinical studies have shown a relationship between elevated levels of EGFR
and poor prognosis.15, 21-22
These conflicting results could be attributed to differences in the anatomical
subsites of HNSCC included in the specific studies and differences in therapeutic
modalities used. Indeed, none of these studies were limited to oral cavity
and oropharyngeal primary tumors, and only 1 study included a significant
number of patients (62%) treated with radiotherapy.15
In contrast to these 3 studies, Wen et al23
found that EGFR expression was not predictive of recurrent disease or overall
survival in a cohort of 68 patients with early stage laryngeal cancer treated
exclusively with radiotherapy. Hence, although elevated EGFR expression may
generally portend aggressive disease, the specific contribution of EGFR expression
to radiosensitivity has yet to be consistently determined in clinical series.
Additional clinical information regarding the role of EGFR in mediating response
to radiotherapy will be forthcoming, as a phase 3 clinical trial using C225,
a monoclonal antibody targeted against EGFR, recently began accrual for patients
with advanced head and neck tumors.24
In this cohort, VEGF was the only molecular marker that significantly
predicted overall survival in multivariate analysis. Hence, VEGF may prove
to be a more important determinant of prognosis in patients with head and
neck cancer than EGFR, p53, and cyclin D1. In addition, VEGF expression was
a more significant predictor of overall survival than clinicopathologic variables
such as T stage and N stage. If this relationship is verified in further studies,
quantitation of tumor VEGF expression may provide useful information in staging
for patients with head and neck cancer.
Vascular endothelial growth factor may mediate poor prognosis through
several potential mechanisms. For example, because VEGF is induced under hypoxic
conditions, and hypoxic tumors tend to display increased radioresistance,
VEGF protein expression may serve as a surrogate marker of hypoxic radioresistance.
This hypothesis is consistent with the borderline correlation between VEGF
levels and locoregional relapse observed in this study. In addition, VEGF
expression likely promotes distant metastatic disease by at least 2 mechanisms.
First, angiogenesis may promote metastatic disease by exposing tumors to a
greater endothelial surface area, thus increasing the likelihood of hematogenous
dissemination.25 In addition, once a tumor
has formed a distant micrometastasis, it must recruit a vascular supply to
proliferate to a clinically significant size. These hypotheses are consistent
with the strong relationship between VEGF expression and distant metastatic
disease observed in this study.
Ultimately, the growing literature on molecular markers in head and
neck cancer should contribute to the management of patients with HNSCC by
enabling more precise staging and more elegant selection of therapeutic modalities.
For example, certain markers may enable prediction of the tumor response to
radiation therapy, with obvious clinical implications. In addition, certain
molecular defects are amenable to pharmacological modification or possibly
replacement via gene therapy. Continued efforts to evaluate the influence
of molecular lesions on tumor behavior will prove important in the identification
of certain pathways for which intervention will result in clinically significant
advances in the treatment of patients with HNSCC.
AUTHOR INFORMATION
Accepted for publication November 14, 2000.
Presented at the annual meeting of the American Head and Neck Society,
Fifth International Conference on Head and Neck Cancer, San Francisco, Calif,
August 1, 2000.
Corresponding author and reprints: Bruce G. Haffty, Department of
Therapeutic Radiology, Yale University School of Medicine, PO Box 208040,
New Haven, CT 06520-8040 (e-mail: bruce.haffty{at}yale.edu).
From the Departments of Therapeutic Radiology (Mr B. D. Smith, Ms G.
L. Smith, and Dr Haffty), Pathology (Dr Carter), Internal Medicine, Section
of Medical Oncology (Dr DiGiovanna and Ms Kasowitz), and Surgery, Section
of Otolaryngology (Dr Sasaki), Yale University School of Medicine, New Haven,
Conn.
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