Affiliations of authors: D. M. Shin, L. Mao, V. M. Papadimitrakopoulou, J. S. Lee, W. K. Hong (Department of Thoracic/Head and Neck Medical Oncology), G. Clayman, A. Gillenwater, J. Myers (Department of Head and Neck Surgery), A. El-Naggar, H. J. C. Shin (Department of Pathology), J. J. Lee (Department of Biostatistics), S. M. Lippman (Department of Clinical Cancer Prevention), W. N. Hittelman (Department of Clinical Investigation). The University of Texas M. D. Anderson Cancer Center, Houston.
Correspondence to: Dong M. Shin, M.D., Department of Thoracic/Head and Neck Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Box 80, 1515 Holcombe Blvd., Houston, TX 77030.
Oral premalignant lesions are a useful model for developing
chemoprevention trials involving lesions throughout the upper
aerodigestive tract (1). These lesions most often develop in
association with exposure to carcinogens, such as tobacco and alcohol,
and frequently precede the development of invasive carcinoma
(2,3). Although early oral premalignant lesions (hyperplasia
and mild dysplasia) are particularly responsive to single-agent
retinoid therapy (4-8), advanced oral premalignant lesions
(i.e., moderate to severe dysplasia) are known to be resistant to
single-agent retinoid therapy (9,10). Since the combination of
retinoids and interferons (IFNs) has been shown to have an enhancing
effect on the induction of cell differentiation and on the suppression
of cell proliferation as seen in preclinical (10-14) and
clinical (15-17) studies, we designed a biochemoprevention
study for testing a combination of isotretinoin
(13-cis-retinoic acid), IFN-, and
-tocopherol for
advanced premalignant lesions in the head and neck. The mutations in
the p53 (also known as TP53) gene and alterations in the p53 protein
resulting in its accumulation in cells may play a critical role in
tumorigenesis (18-20), and both are common in human cancers,
including premalignant lesions and cancers of the head and neck region
(21-23). We conducted a prospective, nonrandomized phase II
trial to assess the effect of biochemopreventive therapy on the
responsiveness of lesions in the head and neck area, particularly
consisting of mild to severe dysplasia in a laryngeal site or moderate
to severe dysplasia in the oral cavity, and to compare laboratory
findings involving p53 alterations.
All eligible patients received high-dose isotretinoin (80 mg/m2 per day),
IFN- (3 million U/m2 twice a week), and
-tocopherol (1200 IU/day) for
12 months, unless the lesions showed progressive disease. The patients were evaluated for clinical
and pathologic responses at 6 and 12 months after beginning of therapy. All analyses were based
on the outcome observed at 6 months unless otherwise specified. All participants had follow-up
examinations without therapy for the following 6 months and then had a final biopsy at 18
months. These tissue samples were analyzed for pathologic response, p53 protein expression, and
p53 gene mutations. Pathologic changes in tissue in response to the therapy were consistently
evaluated according to the criteria previously described (24) by one
pathologist (A. El-Naggar). All pathology slides were coded by numbers, and the pathologist was
blinded to the time the biopsies were performed. Immunohistochemical staining was performed on
tissue sections by use of monoclonal anti-p53 antibody (clone D07; BioGenex, Inc., San Ramon,
CA) (22). The epithelial layer of each lesion was microdissected in three
or four serial sections from each biopsy sample, as described previously (25). The p53 positivity was stratified as low or high, based on two different sets of cut
points, i.e., labeling index (L.I.) of less than 0.2 as low and greater than or equal to 0.2 as high or
L.I. of less than 0.1 as low and greater than or equal to 0.1 as high (see Fig. 1
legend). P values were determined with the use of both sets of cut points.
Three separate fragments of the p53 gene encompassing exons 5-9 were amplified from the
extracted DNA from these microdissections and directly sequenced, as described previously (26). The optimal two-stage design by Simon (27)
was applied, and a 6-month pathologic response was used as the major end point. The pathologic
complete response was described as the complete disappearance of dysplastic cells from the
epithelium, and the pathologic partial response was described as a reduction in the degree of
dysplasia by at least one degree (i.e., severe to moderate dysplasia). Absence of any response (no
response) was described as no pathologic change or a minor change involving focal improvement
within the same degree of dysplasia. Progressive disease indicated an increase in the severity of
dysplasia by at least one degree (i.e., moderate dysplasia to severe dysplasia) or the development
of invasive carcinoma. The standard and target response rates were chosen to be 10% and
30% of all patients, respectively. Both the type I and type II error rates were set at
10%. Associations between categorical variables were determined either by the
2 test or Fisher's exact test. Differences between the two groups with respect
to continuous variables, such as the differences in p53 protein expression in relation to pathologic
progression in laryngeal lesions and in oral cavity lesions, were compared with the use of the
Wilcoxon rank sum test. All statistical tests were two-sided; P values of <.05 were
considered to be statistically significant.
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We observed mutations in p53 gene in eight of 48 baseline tissue samples or in the baseline samples of seven of 34 patients: three with laryngeal lesions and four with oral cavity lesions. Of 27 patients, six had no follow-up data. Of the remaining 21 patients whose baseline tissue samples expressed wild-type p53 gene, at 6 months, 14 patients maintained the wild-type gene in post-treatment samples, while seven subsequently showed p53 gene mutations in the tissues following treatment. Five of these 14 patients (all with laryngeal lesions), whose baseline tissue samples expressed wild-type p53 gene and whose post-treatment tissue samples maintained wild-type during therapy, achieved complete response by pathologic evaluation. In contrast, among 12 patients (five with oral lesions and seven with laryngeal lesions) whose baseline tissue samples expressed mutant p53 gene or whose post-treatment tissue samples showed an outgrowth into a mutant form of p53 gene during the follow-up period between 6 months after therapy to 18 months after therapy, only one patient expressed wild-type p53 after treatment and then showed complete response by pathologic evaluation (P = .17; Fisher's exact test). Four of eight baseline tissue samples showing mutations in the p53 gene had high p53 protein expression, and the remaining four had low p53 protein expression. Among 39 of 40 baseline samples (one sample did not have p53 sequencing data) that had wild-type p53 gene expression, 18 (46%) had high p53 protein expression, and 21 (54%) exhibited low p53 protein expression. Therefore, we did not find in this study any direct association between p53 protein expression and p53 gene mutations.
This translational study of biochemoprevention of advanced premalignant lesions in the head
and neck region was a biomarker-driven clinical trial involving multiple biopsy specimens of
lesions in an attempt to better understand the biologic consequences of response to treatment and
resistance to this therapy. Combined treatment with isotretinoin, IFN-, and
-tocopherol achieved a substantial rate of pathologic responses, particularly in patients with
laryngeal lesions. This finding is striking because 88.6% (31 of 35 patients) of the
patients' lesions monitored in this study were pathologically advanced premalignant lesions
(moderate or severe dysplasia). Therefore, it appears that IFN and retinoids have enhancing
effects in this setting, as suggested by preclinical and clinical studies (10-17). It is well established that moderately or severely dysplastic lesions have a
significantly higher risk of transformation to invasive carcinoma than do early premalignant lesions
(28,29). In our previous studies using retinoids alone (3,23), only a minority of the patients studied presented with moderate or severe dysplasia,
and response was predominantly observed in less advanced lesions (1,3).
The degree of responsiveness to treatment in this study, however, depended markedly on disease
site. Complete pathologic response occurred exclusively in laryngeal lesions, whereas oral cavity
lesions were found to be more resistant to this regimen. This differential responsiveness associated
with different sites in the head and neck area may be an important clue for better understanding
the underlying pathobiology of tumorigenesis, tumor heterogeneity, and organ specificity of
response to therapy, although the exact mechanism should be explored further.
In this study, we document the reappearance of the wild-type p53 gene in post-treatment biopsies from seven patients whose pretreatment tissue samples expressed mutant p53 gene. To the best of our knowledge, these results represent the first demonstration of elimination of clonal cells expressing mutant p53 gene by effective biochemopreventive therapy, which not only produced histologic improvement but also allowed the reappearance of the functional or wild-type p53 gene. On the other hand, new mutations of the p53 gene emerged in the lesions of seven other patients during treatment, and no patients in this group achieved a pathologic complete response. This observation suggests that a subset of clonal cells with p53 mutations emerged during the therapy and may have been resistant and survived this therapy.
Patients with low p53 protein expression in baseline biopsy samples achieved higher level
pathologic responses (seven [58%] of 12 patients) than those whose baseline
biopsy samples had high p53 expression (two [14%] of 14 patients). By p53
sequence analyses, patients whose pretreatment and post-treatment tissue samples maintained
wild-type p53 gene also achieved a high rate of pathologic responses. Retention of p53 function
appears to be a critical parameter in cancer treatment (30). Tumor cell
killing by radiation therapy or chemotherapeutic agents has been reported to be an active process
of apoptotic cell death (31,32). Since apoptotic cell death is mediated by a
functional p53 gene in many situations, the effectiveness of a given therapy can hinge on the p53
status (32). In certain circumstances, the p53 effects may be rendered in
indirect ways. Since IFN- has an antiangiogenic activity and its combination with retinoids
has an enhancing effect on antiangiogenesis (11,12), this combined
regimen may shut down vascularization in tumorigenesis and possibly push the dysplastic cells
into the apoptotic pathway. Those lesions with p53 functional defects, however, may not undergo
apoptotic cell death (30) and may then become resistant to this therapy.
In conclusion, biochemopreventive therapy appears to be highly active in advanced premalignant lesions, particularly those of the larynx. Our study extends the valuable record of translational chemopreventive studies (33,34) that have incorporated biomarkers, such as p53 and other genetic markers. Taken together, these results demonstrate the critical importance of this translational approach for advancing our understanding of the pathobiologic basis of tumorigenesis and therapeutic outcome and for identifying relevant targets to overcome resistance to an effective biochemopreventive regimen. The results of this single-arm, phase II trial should be confirmed in randomized, placebo-controlled phase III trials in the future.
NOTES
Supported in part by Public Health Service grants U01CA68089 (to W. K. Hong), CA69025 (to D. M. Shin), and CA16672 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services. W. K. Hong is an American Cancer Society Clinical Research Professor.
We thank the patients who participated in this study and their families; K. Lawhorn and L. Martinez for assisting with patient care; A. Shellshear and J. Starr for the preparation of the manuscript; S. Cweren, C. Corrales, H. Ibarguen, E. Yoo, and Y.-H. Fan for their technical excellence in the studies relating p53 expression and mutations; and R. Yu and D. Liu for assistance with statistical analysis.
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Manuscript received May 6, 1999; revised August 27, 1999; accepted November 8, 1999.
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