Assessment of p53 expression in nasopharyngeal carcinoma

Assessment of p53 Expression in Nasopharyngeal Carcinoma LAI-FA SHEU, MD, ANN CHEN, MD, PHD, HUI-HWA TSENG, MD, FUR-JIANG LEU, MD, PHD, JOHN K, LIN, MD, KOU-CHIEH HO, PHD, AND CHING-LIANG MENG, PHD We analyzed the expression of the p53 protein by immunohistochemical methods from 101 patients with nasophmTngeal carcinoma (NPC): 2 4 with NPC and dysplastic lesions adjacent to carcinoma and 14 with primary and metastatic specimens. Ninety-six of 101 lesions (95%) had detectable p53 protein in the nuclei of tumor cells, indicating that overexpression of the p53 protein might be closely associated with NPC. Among 24 patients who had NPC and dysplastic lesions adjacent to carcinoma, 19 of the dysplastic lesions (79.2%) and 22 of the carcinomas (91.7%) showed positive staining for the p53 protein. In dysplastic epithelia p53 antigenicity was generally in a basal location. The significant association of p53 expression in NPC and dysplastic lesions adjacent to carcinoma (P < .0001, Fisher's exact probability test) suggests that p53 overexpression seems to occur at an early stage in the development of NPC. p53 expression in NPC does not correlate with histological grading, degree of lymphocytic infiltration between tumor cells, clinical stage, sex, or age (P >

.05, chi-squared test). A comparison of p53 expression between primary and metastatic NPC was performed in 14 lesions. Although the p53 protein was consistently expressed in primary and metastatic tumor cells, there was no significant difference in p53 expression in both distinct but related lesions (P > .05, paired t-test). Our results suggest that the association of overexpression of the p53 protein in NPC may not be indicative of a mutant type p53 protein. HUM PATHOL 26:380-386. Copyright © 1995 by W.B. Saunders Company Key words: p53 protein, immunohistochemistry, nasopharyngeal carcinoma. Abbreviations: NPC, nasopharyngeal carcinoma; EBV, EpsteinBarr virus; SSCP, single-strand conformation polymorphism; HE, hematoxylin and eosin; SCC, squamons cell carcinoma; NKC, nonkerafinizing carcinoma; UC, undifferentiated carcinoma; TBS, Trisbuffered saline.

Nasopharyngeal carcinoma (NPC) is a common malignant neoplasm of the head and neck in the southeastern Asian area, particularly in the southeastern provinces of China, including Taiwan. 1'2 The etiologic factors of NPC include genetic susceptibility, EpsteinBarr virus (EBV) infection, 4 dietary factors,9 etc. Gene abnormalities are believed to underlie the development of human cancers. 6 Chromosomal analysis in several established NPC cell lines showed multiple genetic aberrations, including chromosome 17p13. 79 The human p53 gene is located at chromosome 17p13.1 and encodes a 375 amino acid nuclear phosphoprotein involved in the regulation of cell proliferation, a°'u p53 gene point mutations are common genetic alterations of human cancer. 12'13 A close correlation between the presence of point mutations in the evolutionary conserved region of the gene and overexpression of the mutant p53 protein has been demonstrated in tests of diverse tumors of epithelial origin) 4 The point mutation of the p53 gene was consistently demonstrated in the NPC cell lines by single-strand conformation polymorphism (SSCP) analysis but was only expressed infre-

quently (10%) in NPC biopsy specimens. 15-17Thus, the role of the p53 gene in carcinogenesis of NPC remains to be elucidated. Normal p53 protein has a very short half-life and is undetectable by immunohistochemical methods, whereas most mutant p53 proteins have a longer halflife and are easily detected by immunohistochemical methods. 18 The overexpressed p53 detected by the immunohistochemical method represents the mutant form of the protein and this effect is unrelated to upregulation of the wild type p53.19 Several methods are available to examine p53 gene expression in human tumors, and the detection of protein levels in neoplastic tissue is suggested as the most efficient for a large serial evaluation of p53 expression. 12Thus, the immunohistochemical method commonly is applied to demonstrate the overexpression of p53 protein in situ. In the past the structure of the p53 gene in only a few NPC samples was evaluated by SSCP analysis and few of them were demonstrated to have the mutated p53 gene) ~-17A large serial evaluation of p53 protein expression in NPC by immunohistochemical method has not been performed. We evaluated p53 protein expression in 101 NPC samples and found that the p53 protein was consistently overexpressed in the primary tumor, dysplastic lesions, and metastatic tumors. The correlations between p53 overexpression and primary tumor, dysplastic lesions, metastatic tumors, and clinical pathological features, such as histological grading, lymphocyte infiltration, clinical stage, age, and sex, were analyzed.

From the Graduate Institute of Medical Science, National Defense Medical Center, Taipei; the Departments of Pathology, Otolaryngology, and Dentistry, Tri,Service General Hospital, National Defense Medical Centre, Taipei; the Department of Pathology, Veterans General Hospital-Kaohsiung, Kaohsiung; and the Department of Botany, National Taiwan University, Taipei, Taiwan, Republic of China. Accepted for publication July 25, 1994. Supported by grants from the National Science Council (NSC 80-0412-B-016-57 and NSC 81-0412-B-075-43), Taiwan, Republic of China. Address correspondence and reprint requests to Lai-Fa Sheu, MD, Department of Pathology, Tri-Service General Hospital, No. 8, Sec 3, Ting-Chow Road, Taipei, Taiwan 10750, Republic of China. Copyright © 1995 by W.B. Saunders Company 0046-8177/95/2604-000455.00/0

380

MATERIALS AND METHODS

Patients and Tissue Preparation T h e tissue s p e c i m e n s were collected f r o m 101 p a t i e n t s

with NPC from August 1988 to April 1993. Among these pa-

p53 EXPRESSIONIN NASOPHARYNGEALCARCINOMA(Sheuet al) tients, 73 were male and 28 were female (male-to-female ratio, 2.6:1). The ages ranged from 19 to 89 years (mean, 48.5 years). The diagnosis of all patients was verified by nasopharyngeal biopsy in the Department of Pathology of the TriService General Hospital. Fourteen patients with primary tumor specimens (nasopharyngeal biopsy) and metastatic specimens (such as cervical lymph node, liver, kidney, and bone marrow) were collected simultaneously for comparative examination. Twenty-four nasopharyngeal biopsies specimens had NPC and dysplastic lesions adjacent to carcinoma. All tissue specimens were routinely processed, formalin fixed, and paraffin embedded. Paraffin sections 4-#m thick were stained with hematoxylin and eosin (HE) for histological examination and classification.

Histological Classification All sections were routinely stained with HE. For differential diagnosis the special stains for mucin and reticulin fiber as well as immunohistochemical stains for common leukocyte antigen and cytokeratin were performed in selected sections. The sample were classified into three main groups: keratinizing squamous cell carcinoma (SCC), nonkeratinizing carcinoma (NKC), and undifferentiated carcinoma (UC), according to their predominant pattern under optical microscopy as proposed by the World Health Organization histological classification. 2° The cases also were classified independently of histological type on the basis of degree of lymphocytic infiltration among the tumor cells as insignificant, moderate, or marked. 21

Clinical Staging For clinical staging all patients were evaluated by fiberoptic endoscopy, plain radiography, polytomography, computed tomography of the nasopharynx, sonography of the abdomen, and whole-body bone scan. Except for 21 patients lost to clinical follow-up, the clinical staging of patients was performed according to the classification system of the Union Internationale Contre le Cancer 1987. 22

Immunohistochemical Staining for the p53 Protein Sections 4-#m thick were cut from the formalin-fixed, paraffin-embedded specimens and placed on gelatin-coated slides. After heating at 65°C for 60 minutes, the tissue sections were deparaffinized in xylene for 5 minutes, three times each. The sections were rehydrated in graded alcohol and rinsed in Tris-buffered saline (TBS; 145 m m o l / L NaC1 and 20 m m o l / L Tris at pH 7.6). Normal rabbit serum (diluted as 1:5 in TBS) was used as a blocking reagent. The sections were incubated with monoclonal antibody (1:50 dilution) specific for human p53 protein (DO-7; Dako, Carpinteria, CA), which reacts with both the wild and mutant types of p53 protein, for 90 minutes, followed by biotin-labeled rabbit immunoglobulin antimouse (Dako) and streptavidin-biotin complex linked to alkaline phosphatase (Dako). Slides were washed in TBS three times for 5 minutes each time. The color was developed in naphthol phosphate-new fuchsin solution (Dako) after which the slides were slightly counterstained with hematoxylin and mounted.

Control Staining As a positive control for p53 immunostaining, a p53-positive infiltrating ductal carcinoma of the breast was used. Negative controls for all immunostaining were performed by substituting the primary antibody with TBS or normal rabbit serum.

381

TABLE 1, p53 Protein Expression in NPC p53-Positive (%) p53-Negative

<10

10-50

>50

5 5

30 29.7

32 31.7

34 33.7

No. of specimens (N = 101) Percentage of specimens

95

Analysis of p53 Reactivity The results were evaluated visually and divided into four groups (negative, < 10% of cells positive, 10% to 50% of cells positive, and >50% of cells positive) according to the estimated number of positive nuclei of either dysplasia or tumor cells.

Statistical Analysis Differences in the degree of p53 expression in formalinfixed specimens with diverse clinicopathological features were evaluated by chi-squared tests with Yates' correction. Probability values less than .05 were considered to be statistically significant. The significant association of p53 expression in NPC and dysplastic lesions adjacent to carcinomas was determined using Fisher's exact probability test. Probability values less than .05 were considered to be significant, p53 expression in primary and metastatic NPC was verified according to the paired t-test.

RESULTS

Expression of the p53 Protein in NPC T h e results o f i m m u n o s t a i n i n g o f the p53 p r o t e i n are s u m m a r i z e d in Table 1. A m o n g 101 specimens examined, 96 (95%) showed positive staining for the p53 p r o t e i n a n d five (5%) s h o w e d n o detectable p53 protein in t u m o r cells. T h e positive specimens, i n c l u d i n g each histological subtype, showed overexpression o f the p53 p r o t e i n c o n f i n e d to nuclei o f t u m o r cells; n o cytoplasmic staining was observed (Fig 1). N o r m a l n a s o p h a ryngeal epithelia, stromal cells, a n d infiltrating lymphocytes d e m o n s t r a t e d n o i m m u n o r e a c t i v i t y for p53 staining. Positive staining within the t u m o r nuclei was h o m o g e n o u s a n d evenly distributed, a l t h o u g h it sometimes s h o w e d a c c e n t u a t i o n a r o u n d the n u c l e a r b o r d e r . C o m p a r i s o n between p53 staining patterns in three histological subtypes o f N P C s h o w e d n o significant differences. T h e intensity o f i m m u n o s t a i n i n g varied a m o n g t u m o r nuclei; some were strong, some were weak, a n d others showed a p r e d o m i n a n t l y n u c l e a r counterstain.

Correlation Between p53 Expression and Clinicopathological Features T h e correlations b e t w e e n p53 expression a n d histological type, d e g r e e o f l y m p h o c y t e infiltration, clinical stage, age, a n d sex distribution were tested a n d are s u m m a r i z e d in Table 2. After statistical analysis acc o r d i n g to the chi-squared test a n d Yates' correction, n o significant difference was d e m o n s t r a t e d (P > .05). Thus, p53 p r o t e i n overexpression in N P C does n o t cor-

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FIGURE 1. p53 protein expression in NPC. (A) Keratinizing SCC. (Original magnification ×400.) (B) Nonkeratinizing carcinoma. (Original magnification ×200.) (C) Undifferentiated carcinoma. (Original magnification ×200.) (D) Scanty expression in a case of UC (Original magnification ×400). Immunohistochemical staining with light hematoxylin counterstain, paraffin-embedded material.

relate with histological grading, degree of lymphocyte infiltration, clinical stage, sex, or age. Coexistence of p53 Expression in NPC a n d Dysplastic Lesions A d j a c e n t to C a r c i n o m a s

Among 24 patients who had NPC and dysplastic lesions adjacent to carcinomas, 19 NPC and dysplastic lesions showed coexpression of p53 protein (Table 3). 382

Three specimens showed negative staining of p53 in dysplastic lesions but were positive in carcinomas. The remaining two specimens showed negative p53 protein in both lesions simultaneously. In dysplastic lesions positive p53 antigenicity was located generally in the basal part of the epithelium and more commonly in a high grade of dysplasia (Fig 2). There was a significant association between p53 expression in NPC and dysplastic lesions adjacent to carcinoma (P < .0001, Fisher's exact

p53 EXPRESSIONIN NASOPHARYNGEALCARCINOMA (Sheu et al) TABLE 2. p53 Protein Expression in NPC: Correlations With Histological Type, Degree of Lymphocyte Infiltration, Stage, Age, and Sex I m m u n o h i s t o c h e m i c a l Staining of p53 Protein Correlative Data Histological type SCC NKC UC Lymphocyte infiltration Insignificant Moderate Marked Stage (UICC 1987) I II III IV U n known Age (yr) <30 30-60 >60 Sex Male Female

No. of Specimens

Negative

< 10%

10%-50%

>50%

4 24 73

0 2 3

1 6 23

1 7 24

2 9 23

31 23 47

2 0 3

7 5 18

10 8 14

12 10 12

0 2 9 69 21

0 1 0 2 2

0 0 1 21 8

0 1 6 23 2

0 0 2 23 9

14 65 22

0 5 0

7 19 4

3 21 8

4 20 10

73 28

4 1

24 6

21 11

24 10

P Value .71

.49

.24

.08

.85

* Chi-squared test with Yates' correction. If P > .05, the difference is n o t significant.

probability test), p53 overexpression seems to occur from an early stage in the development of NPC. p53 Expression in Primary and Metastatic NPC A comparative examination of p53 expression in primary and metastatic NPC was performed in lesions from 14 patients. The results are summarized in Table 4. Concurrent p53 protein expression in primary and metastatic NPC was demonstrated in 13 of 14 specimens. The other specimen expressed no p53 protein in primary and metastatic NPC. However, the fraction of tumor cells with p53 overexpression in primary and metastatic tumor showed no significant difference (P > .05, paired t-test), p53 protein is expressed concurrently in primary and metastatic tumor cells but does not correlate with metastatic activity. DISCUSSION We used the monoclonal antibody DO-7, which is specific to human beings and reacts with the wild and TABLE 3. Coexistence of p53 Expression in NPC and Dysplastic Lesions Adjacent to Carcinoma Nasopharyngeal Carcinoma (No. of Specimens) Dysplasia

p53-Positive

p53-Negative

Total

p53-Positive p53-Negative Total

19 3 22

0 2 2

19 5 24

NOTE. Fisher's exact probability test: P < .0001, significant association.

383

mutant types of the p53 protein, to detect p53 protein expression in NPC. We analyzed the p53 protein immunohistochemically in 101 patients with NPC, of whom 24 had carcinoma and dysplastic lesions adjacent to carcinoma and 14 had primary and metastatic specimens for comparison. We demonstrated overexpression of the p53 protein in 96 of 101 NPC specimens (95%). The data indicate that p53 protein overexpression may be closely associated with NPC. It was reported that point mutation of the p53 gene was not a common genetic abnormality in NPC biopsy specimens with less than 10% of specimens being demonstrated by SSCP analysis.15!7 A similar incompatible result between p53 overexpression and point mutation in NPC biopsies was reported by Niedobitek et al. 23 They demonstrated that p53 overexpression was detected in five of nine EBVnegative squamous cell NPCs and in nine of 13 EBVpositive undifferentiated NPCs and suggested that there is no obvious correlation of p53 overexpression with EBV infection, p53 overexpression in NPC seems less correlated with the point mutation of the p53 gene. It has been shown that the p53 protein can bind to cellular proteins, such as the mdm-2 oncogene product 24 and heat-shock protein 7 0 , 25 and to several DNA tumor virus proteins, including SV40 T antigen 26 and Elb protein from adenovirus type 5, 27 leading to its stabilized accumulation and inactivation. Although overexpression of the p53 protein detected by immunohistochemical methods does not necessarily indicate an underlying gene mutation, it may be a useful indicator of an altered wild type p53 function. Besides the point mutation, there are several potential explanations for p53 overexpression in NPC, such as inactivation of an enzymatic pathway responsible for p53 degradation 2s or stabilization by complex formation with unknown activated cel-

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FIGURE 2. p53 protein expression in a dysplastic lesion adjacent to NPC. (A) p53 expression in the basal location of a severe dysplastic epithelium (adjacent neoplastic island not shown here). (Original magnification × 100.) (B) p53 expression in the basal location of a hyperplastic squamous epithelium with dysplastic change and invasive carcinoma. (Original magnification x200.) (C) p53 expression in a ciliated, pseudostratified epithelium with dysplastic change and carcinoma in situ (adjacent neoplastic island not shown here). (Original magnification x40.) Immunohistochemical staining with light hematoxylin counterstain, paraffin-embedded material.

lular proteins or with a protein f r o m a virus infection. The last m e c h a n i s m is of particular interest because EBV is consistently associated with NPC. 4 C o n c u r r e n t expression of the p53 protein in carcin o m a and dysplasfic lesions has b e e n r e p o r t e d in lungs, 29 oral mucosa, ~° and colorectum 31'32 by i m m u n o histochemical methods. O u r results also d e m o n s t r a t e d that p53 was overexpressed c o m m o n l y and simultaneously in NPC and dysplasfic lesions adjacent to carcinoma, which suggests that p53 overexpression seems to occur from an early stage in the d e v e l o p m e n t of NPC. Correlation tests between p53 overexpression and

384

clinicopathological features also were performed. Overexpression of the p53 protein in NPG does not correlate with histological subtype, degree of lymphocyte infiltration, clinical stage, sex, or age (P > .05). T h e p53 protein was preferentially expressed in a high histological grade of carcinoma of breast a n d n e u r o e n d o c r i n e tumors of lungs 34 but showed no such effect in carcinoma of the head and neck. a5 Data f r o m o u r tests confirm the latter observation. T h e regulation of p53 protein expression of malignant neoplasms m i g h t vary a m o n g neoplasms. Davidoff et al ~6 showed a significant association be-

p53 EXPRESSION IN NASOPHARYNGEAL CARCINOMA (Sheu et al)

TABLE 4.

p53 Expression in Primary and Metastatic NPC p53 Expression in NPC (%)

Specimen No.

Histological Subtype

Metastatic Site

Primary

Metastasis

1 2 3 4 5 6 7 8 9 10 11 12 13 14

SCC NKC UC UC UC NKC UC NKC NKC UC UC UC NKC UC

Lymph node Lymph node Lymph node Lymph node Lymph node Lymph node Lymph node Lymph node Lymph node Liver Liver Bone marrow Bone marrow Kidney

20 15 45 60 0 5 10 75 45 55 5 10 15 25

20 10 40 70 0 1 10 65 60 50 5 0 15 30

27.5% -+ 0.239% 0.064

26.8% +_ 0.255% 0.068

Mean _+ SD SE NOTE. Paired Student's t-test: P ~> .05, difference not significant.

tween a high level of expression of the p53 protein and a late stage of disease and metastatic tumor spread. The presence of elevated levels of mutant p53 may of itself be a prognostic factor in h u m a n breast cancer and important in the ability of a tumor to metastasize. In contrast, no similar results were observed in small cell carcinoma of the l u n g Y adenocarcinoma of the coltrectum, 31 or our studies. Although p53 overexpression was shown to be associated with the transition from dysplastic lesions to carcinoma in NPC, it did not uniquely determine metastatic ability or modulate tumor progression. In conclusion, we characterized p53 expression in NPC. Expression of the p53 protein is associated consistently with NPC. C o n c u r r e n t p53 expression in NPC and dysplastic lesions adjacent to carcinomas indicates that p53 overexpression seems to occur in an early stage in the development of NPC. T h e r e is no significant correlation between p53 protein expression and clinical pathological features, including histological grading, lymphocyte infiltration, clinical stage, age, and sex. The p53 protein is expressed consistently in primary and metastatic NPC, but its expression does not correlate with the potential of tumor cell metastasis, p53 overexpression in NPC possibly plays a role during malignant transformation but does not account for tumor progression and metastasis. The mechanisms of p53 protein overexpression in NPC seem to be less correlated with the point mutation of the p53 gene, but other factors, such as activated cellularity or EBV infection, remain to be elucidated. The possibility that cellularity or EBV proteins alter the biological functions of the p53 protein in NPC is being investigated currently in our laboratory.

Acknowledgment, We t h a n k Ying-Sau C h a n g a n d FuG o n g Lin for t h e i r e x c e l l e n t technical work a n d statistical analysis, respectively. 385

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