Contrasted Frequencies of p53 Accumulation in the Two Age Groups of North African Nasopharyngeal Carcinomas¹

NPCs³ are rare in most countries but occur with a high incidence in Southeast Asia and to a lesser extent in North Africa and Artic regions (1, 2). Although NPC is not as frequent in North Africa as it is in Southeast Asia, it remains the most frequent head and neck cancer in Tunisia, Algeria, and Morocco (2, 3). Asian and African NPCs share a number of common clinical and biological characteristics. In both parts of the world, >90% of the cases are EBV-associated tumors with the WHO type III (undifferentiated) or II (nonkeratinizing)histological type (4, 5, 6). In both places, they display a characteristic high degree of lymphocytic infiltration on tissue sections and are, on average, more sensitive to radiotherapy and chemotherapy than other head and neck carcinomas (7). However, one remarkable difference between Asian and African NPCs is their age distribution. Several epidemiological studies have revealed a bimodal pattern for North African NPCs. Whereas in Southeast Asia there is only one single peak of incidence about the age of 50, in North Africa an additional minor peak of incidence occurs between the ages of 10 and 20, including about 15% of all NPC patients (3, 6, 8). A similar form of juvenile NPC is also observed among African American teenagers, although with a much lower incidence than in North Africa (2, 8). Thus far, there has been only limited data on biological characteristics that could differentiate tumors from both age groups (6).

p53 and Bcl2, two key proteins for apoptosis control and oncogenesis,have been previously investigated by immunostaining of tissue sections in a number of Asian NPC series (9, 10, 11, 12). Both proteins were consistently detected in malignant cells of a large majority of tumors. The selective detection of p53 in tumor cells has been interpreted as indicative of p53 abnormal stabilization, as is often the case in human malignancies (10, 13). Thus far, there has been no report concerning p53 and Bcl2 expression in North African NPCs. Therefore, we started to investigate both molecules in a series of 90 NPC biopsies from Tunisia. Investigations on CD95 (or Fas-receptor) were also included in this study. CD95 is a member of the tumor necrosis factor receptor superfamily, containing an intracytoplasmic death domain. It is a potent mediator of apoptosis in a wide range of nonmalignant cells (14, 15). Its expression is often decreased or its function impaired in various types of human malignancies (16, 17).

Here, we report that p53 and Bcl2 were detectable in a majority of tumors and CD95 in all our NPC specimens. However, there was significantly much less p53-positive tumors in the group of young patients (under 30 years of age) than in the main age group (above 30 years of age).

Primary NPC biopsies were collected from 90 patients, before any treatment, in the Sfax University Hospital (Sfax, Tunisia) from January 1993 to December 1998. The male:female ratio was 257:1, with 64 men and 26 women. The ages ranged from 9–77 years (mean age, 43.3). The clinical stage of the disease was determined according to the tumor,node, metastasis (TNM) classification of the American Joint Committee on Cancer/Union International Contre le Cancer (1997). Six(6.67%) patients were stage II, 25 (27.77%) patients were stage III,and 59 (65.55%) were stage IV. All patients were treated by irradiation of the nasopharynx and/or cervical lymph nodes, except eight patients with early distant metastases who only received chemotherapy. The effect of radiotherapy was evaluated for 71 patients(the follow-up was insufficient for 4 patients). The extent of tumor response was graded as: complete disappearance (95%), partial response(1.3%), and no response (3.8%).

All tissue specimens were fixed in Bouin’s fixative (75% saturated picric acid, 25% formalin, and 5% glacial acetic acid) and paraffin-embedded for light microscopy and immunohistochemistry. The diagnosis was based on morphological examination after H&E staining. It was further assisted by immunostaining of leukocyte common antigen and cytokeratin in 20 cases, to facilitate the differential diagnosis with a malignant lymphoma or a sarcoma. NPC specimens were classified according to the WHO Histological Classification into three main subtypes: keratinizing SCC (1 of 90), NKC (69 of 90), and UC (20 of 90). Several patterns of differentiation were often mixed in the same specimen, but only the predominant pattern was taken in account.

Staining of p53 and Bcl2 proteins was performed on 90 NPC specimens, of which 88 were also stained with the anti-CD95. Five-micrometer sections attached on silanized slides were dewaxed in xylene, rehydrated in graded ethanol, and heated in a microwave oven in 10 mmcitrate buffer (pH 6) twice for 10 min at 500 W. They were then incubated for 15–30 min with the primary antibodies against p53, Bcl2,or CD95, all three being mouse monoclonal antibodies from DAKO (clones DO7, 124, and DX2, respectively). Immunobinding of anti-p53 and Bcl2 was visualized with biotin-labeled secondary antibodies and streptavidine-peroxidase complexes with use of diaminobenzidine as a chromogenic substrate (LSAB system; DAKO). CD95-staining was visualized with the Catalyzed Amplification System (DAKO), which includes an amplification step with biotinyl tyramide (18). Biopsies were recorded as positive when >5% of tumor cells were stained,taking in account five consecutive microscope fields. In addition, CD95 staining was scored with regard to the approximate percentage of positive tumor cells and relative immunostaining intensity, as previously reported (19). The percentage of positive tumor cells was graded as: 0, none; 1, 1–25%; 2, 26–50%; 3, 51–75%; 4,76–100%. Immunostaining intensity was rated as: 0, none; 1, weak; 2,moderate; 3, intense. When tumors were heterogeneous in staining intensity, each component of the tumor was scored independently and the results were summed. For example, when a specimen contained 50% of the tumor cells with moderate intensity (2 × 2 = 4), 25% of tumor cells with intense immunostaining (1 × 3 = 3), and 25% of cells with weak intensity (1 × 1 = 1), the score was 4 + 3 + 1 = 8. The maximal possible score was 12.

The χ² test with the Yates correction was used to evaluate differences in p53 and Bcl2 staining, which were analyzed as qualitative variables. The Student’s t test was used to assess the variations of the CD95 staining scores according to various clinical parameters.

Sixty-four (71%) NPC biopsies were positively stained for p53 (1 SCC, 48 NKCs, and 15 UCs; Table 1). As reported previously for NPC, the p53 staining was restricted to the nuclei of tumor cells (Refs. 10 and 20; Fig. 1). Immunohistochemical detection of p53 was not correlated with patient sex, disease stage, or tumor histological type. In contrast, there was a very strong correlation with patient age (Table 1). Only 38% of specimens were p53 positive for patients <30 years of age compared with 81% for older patients(P = 0.00013). The impact of patient age on p53 detection is clearly shown in Fig. 2. Among NPC patients recruited in our hospital, there was a relative excess of patients in the 2nd decade of their life, as is consistently the case for North African NPC series (3, 6). In contrast,there were only few patients in the 3rd decade. The number of patients started, again, to increase after the age of 30, with a maximum between 41 and 60. For patients in the 2nd decade, a large majority of biopsies were p53 negative. In contrast, most biopsies from patients over 30 years of age were p53 positive. Even more, in the case of patients between 51 and 60 years of age, all biopsies (17 of 17) were p53 positive, without a single exception.

Concerning Bcl2, the staining was cytoplasmic, sometimes with a granular appearance. Sixty-nine (77%) biopsies were positive (1 SCC,56 NKCs, and 12 UCs; Table 1 and Fig. 1). In most cases, Bcl2 and p53 were both detected or both absent. This association was statistically significant (P = 0.0066; Table 2). Like p53, the presence of Bcl2 was not correlated with patient sex, disease stage, or tumor histological type (Table 1). Again, there was a trend toward a correlation with patient age, with more Bcl2-negative biopsies for patients under 30 years of age, but it was not statistically significant (Table 1).

The CD95 molecule was detected in all specimens without any exception,however, with some variations in both the percentage of positive malignant cells and the intensity of immunostaining (Table 3 and Fig. 1). To assess possible variations related to patient age or tumor stage, CD95 expression was graded with a scoring system, taking in account the approximate percentage of positive cells and the staining intensity. The observed scores were comprised between 4 and 12, with an average of 8.42. As shown in Table 3, there was a modest, but statistically significant,increase in the level of CD95 expression for patients at stage IV compared with patients at stages II and III. In contrast, CD95 expression was not influenced by the age of the patients or the histopathological type.

According to most reports, p53 and Bcl2 can be visualized in malignant cells of about 90% and 80% Asian NPCs, respectively (9, 10, 12, 21). The Chinese series reported by Harn et al. (11) is inconsistent with regard to Bcl2, which was found in only 33% of biopsies, but not with regard to p53, which was detected in 90% of the cases. In the present series of North African NPCs, p53 and Bcl2 were seen in 71% and 77% of biopsies, respectively, suggesting that the overall frequency of p53 overexpression is relatively smaller than in Asian NPCs. There was a strong correlation of p53 and Bcl2 expression as reported for the Chinese series of Sheu et al. (12). Concerning CD95, we have previously reported that it was strongly expressed in two NPC xenografted tumor lines and in a small series of frozen NPC biopsies (22). In addition, in the same report, we have shown that CD95 was still capable to mediate apoptosis in both NPC tumor lines, including one derived from a metastatic lesion. Here, we confirm that CD95 is constantly expressed by NPC cells, generally at a high level, even at late stages of tumor evolution (stage IV). These data contrast with previous studies on other types of human carcinomas—especially lung and breast carcinomas—that have shown a dramatic decrease in CD95 expression for advanced tumors (16, 17).

The main finding of this study was the relatively low frequency of p53 accumulation for tumors from patients under 30 years of age. This observation might be important for a better understanding of NPC oncogenesis, which is known to result from the combination of viral,genetic, and environmental factors (1). These factors might, in fact, combine differently for Asian and North African patients, and among North African patients, for patients of the two age groups. Interestingly, distinct characteristics related to virus-tumor interactions have already been reported for the two groups of North African NPCs. Most patients <30 years of age have only one or two copies of the EBV genome per malignant cell and no detectable serum IgA against EBV antigens. In contrast, patients over 30 consistently have an average copy number of 10 EBV genomes per malignant cells and high titers of anti-EA and -VCA IgA (6). Presently, we can only speculate on possible relationships between the EBV load in tumor cells and the level of p53. But at least, these data suggest that in both age groups malignant NPC cells tend to use distinct pathways to escape apoptosis, retain the EBV genome, and complete malignant transformation.

It is highly probable that p53 is in some way inactivated in NPC but it is not yet clear how this is achieved. Several studies based on PCR-single-strand conformational polymorphism and DNA sequence have found a very low frequency of p53 mutations in Asian and North African NPCs, generally below 10% of the tumors (23, 24, 25). Although there has been one recent study with a higher estimate, it does not go beyond 25% of the cases (26). This suggests that NPC p53 is often inactivated, and in many cases stabilized, by epigenetic mechanisms. Because MDM2 is a major cellular effector of p53 inactivation, several groups have investigated its possible role in NPC (27, 28). Detection of MDM2 by immunohistochemistry has been reported in about 30% tumors in a mixed series of Chinese and European NPC biopsies (28). Surprisingly, an accumulation of p53 was observed in the MDM2-positive specimens of this series, a finding that seems inconsistent with the known stimulation of p53 degradation by MDM2 (29). Alternative mechanisms might account for p53 inactivation in NPC, for example a loss of expression of the p33 protein. p33, the product of the ING1 tumor suppressor gene, binds p53; this interaction is required for p53 induction of p21 (30). Interestingly, loss of p33 expression in association with a nonmutated p53 has been recently reported in a subset of gastric carcinomas (31). Our aim,in future studies, will be to compare the status of MDM2, p33, and p53 in both age groups of North African NPC.