Expression of p53 and p21 Proteins in Oral Squamous Cell Carcinoma: Correlation with Lymph Node Metastasis and Response to Chemoradiotherapy

PATHOLOGY RESEARCH AND PRACTICE

© Gustav Fischer Verlag

Expression of p53 and p21 Proteins in Oral Squamous Cell Carcinoma: Correlation with Lymph Node Metastasis and Response to Chemoradiotherapy Yukihiro Tatemoto, Tokio Osaki, Kazunori Yoneda, Tetsuya Yamamoto, Eisaku Ueta and Tsuyoshi Kimura Department of Oral Surgery, Kochi Medical School, Kohasu, Oko-cho, Nankoku-city, Kochi 783-8505, Japan

Summary p53 protein, a product of the p53 cancer suppressor gene, and p21 protein, a cyclin-dependent kinase inhibitor, were immunohistochemically investigated in 150 oral squamous cell carcinomas (SCCs) and the relationship between their expression and clinicopathological findings were evaluated. The positivity for p53 and p21 proteins was not correlated with the T-stage, mode of tumor cell invasion or tumor cell differentiation. However, the expression of p53 and p21 proteins was correlated with lymph node metastasis. Of 62 SCCs with regional lymph node metastasis, 45 SCCs (72.6%) were positive for p53 while 45 (52.9%) of 88 SCCs without metastasis expressed p53 protein (p < 0.02). In addition, p21 protein was observed in 25 (38.5%) and 18 (21.2%) SCCs with and without metastasis, respectively (p < 0.05). Furthermore, p53 protein was inversely correlated with the histopathological effect of inductive chemoradiotherapy; the rate of chemoradiotherapy-induced lethal degeneration (56.7%) in p53-negative SCCs was significantly higher than that (28.9%) in p53-positive SCCs (p < 0.005). However, no clear difference in the effect was observed between p21-positive and p21-negative SCCs. Finally, the 5-year-survival rate was highest in p53(-)-p21(+) (80.0%) followed by 76.3% in p53(-)-p21(-), 65.9% in p53(+)-p21(+) and 65.4% in p53(+)-2Ip(-) SCCs. These results indicate that although the expression ofp21 protein is only weakly correlated with the clinico-histopathological findings, p53 protein is a useful prognostic marker and that inductive chemoradiotherapy can be successfully planned by immunohistochemical examination of p53 protein. Pathol. Res. Pract. 194: 821-830 (1998)

Key words: Oral squamous cell carcinoma - p53 - p21 - Metastasis - Chemoradiotherapy

Introduction p53 protein, a product of the p53 cancer suppressor gene, is a transcription factor that enhances the rate of transcription of genes exhibiting p53-dependent function in cells [26, 28]. The p53 gene and its products have been extensively studied since it became clear that more than 50% of human cancers contain mutations of this gene [20, 28]. When p53 is mutated, the gene product fails to induce its target genes such as p21/WAFlICipl [l2J, MDM2 [35J, GADD45 [24J, Bax [34J, Cyclin G [39J, IGF-BP3 [6J and GML [l3J, which are closely associated with G I and G2 arrest and apoptosis. The significance of p53 gene mutations as a prognostic marker for a variety of human cancers remains controversial. Several studies have described the importance of the p53 gene and its product as a prognostic marker [16, 17, 27, 37 J, while others have shown no significant correlation between p53 gene mutation and prognosis [1,5, 13, 14, 38]. Although mutations of the p53 gene have been reported in oral carcinomas [9, 18, 25, 41J, its correlation with lymph node metastasis and Address for correspondence: Prof. Tokio Osaki, DDS, DMD, Department of Oral Surgery, Kochi Medical School, Kohasu, Oko-cho, Nankoku-city, Kochi 783-8505, Japan. Tel.: 0888-80-2423, Fax: 0888-80-2424 0344-0338/98/0194-0821 $5.00/0

822 . Y. Tatemoto et al.

tumor response to chemoradiotherapy have not yet been satisfactorily investigated. A cyclin-dependent kinase (cdk) inhibitor, p21, inhibits the kinase activity of cdk2, cdk4 and cdk6 and arrests tumor cells at the Gl [15, 55J or G2 phase [51, 53J. The expression of p21 protein in tumor cells has been recognized as good prognostic evidence indicating suppression of cell proliferation and induction of apoptosis [8, 32, 50, 52]. However, an inverse relationship has recently been reported [3, 4, 7, 57]. The inversion makes the significance of p21 as a prognostic marker uncertain. In addition, large series concerning the expression of p21 protein have not yet been reported in oral squamous cell carcinomas (SCCs). In the present study, we examined the expression of p53 and p21 proteins in oral SCCs and sought the relationship between their expression and clinicopathological findings.

Material and Methods Patients and tumors

Oral squamous cell carcinomas (SCCs) (n = 150), consisting of tongue (48), gingiva (63), floor of the mouth (22), cheek mucosa (9) and palate (8) from the Department of Oral Surgery, Kochi Medical School from 1986 to 1996 were used in the present study (Table 1). Patients consisted of 92 men and 58 women ranging from 33 to 84 years old with a mean of 67.4 years old. The tumors were staged according to the VICC definition [48J except for gingival carcinomas, where T4 was limited to those tumors that had invaded into the level of the mandibular canal or intensively destroyed the maxillary sinus wall by radiograph. Even if the tumor invaded focally into the alveolar bone, it was classified as T2 or T3 depending on the maximum diameter of the tumor.

Table 1. Sites and stages of tumors examined Site

Tongue Maxillary gingiva Mandibular gingiva Floor of the mouth Cheek/Palate

Tumor stage II

III

IV

Total

19 11 14 5 8

16 5 6 7 5

13 4 23 10 4

48 20 43 22 17

------------------------------------------------

Total

57

39

54

150

day (2 Gy x 5/week). Concomitantly, 250 mg/day of 5-FU was intravenously injected and 5 mg of PLM was subcutaneously administered 3 times per week. This regimen of concomitant chemoradiotherapy was repeated 3 or 4 times depending on the patient's general and local condition. The mean PLM and 5-FU doses were 45.1 ± 12.2 mg and 3950 ± 1250 mg, respectively, and the mean radiation dose was 28.0 ± 8.4 Gy. After evaluating the clinical effects of inductive chemoradiotherapy, each tumor was extirpated with an appropriate surgical margin. Positive regional lymph nodes were dissected with the primary tumor and were processed for histological evaluation. Tumors that completely responded to chemoradiotherapy were periodically followed without surgical excision. Locally recurring tumors and late metastatic lymph nodes, that were included in the N-positive group, were extirpated when operable. Pathologic examination

Tumor materials biopsied before treatment and tumors extirpated after chemoradiotherapy were investigated histopathologically. The excised specimens were fixed with 10% buffered formalin and embedded in paraffin. Sections at 4 J.Im were stained with Hematoxylin and Eosin (H&E) and other sections were stained immunohistochemically. In sections obtained before treatment, tumor cell differentiation was categorized as well, moderate or poor, and the mode of tumor cell invasion was graded according to the YamamotolKohama classification [56]. In the tumor specimens obtained after chemoradiotherapy, histological degenerative effects of inductive chemoradiotherapy were determined by Shimosato/Oboshi's classification [43J. Immunohistochemical method

To retrieve the antigens, paraffin sections were heated in 10 mM citrate buffer solution (pH 6.0) for 5 minutes x 2 by a microwave oven [42J. Both p53 and p21 proteins were detected using the streptavidin-biotin peroxidase complex method using DO-7 (dilution 1:50, Dakopatts, Tokyo, Japan) and Ab1 (dilution 1:20, Calbiochem, Cambridge, USA), respectively, as primary antibodies. DO-7 recognizes an epitope in the Nterminal of the human p53 protein and reacts with both wild and mutant types of p53 protein. For counting total tumor cell numbers, the sections examined were counter-stained with veronal acetate-buffered 1% methyl green solution (pH 4.0). The degree of stainability was determined by observing 500 cells in 3 high magnification (x200) fields and the immunoreaction of the two proteins was classified into negative (-): less than 10% positive tumor cells, and positive (+): more than 10% positive tumor cells. Analysis

Treatments for oral SCCs

All patients underwent inductive chemoradiotherapy with peplomycin (PLM), 5-fluorouracil (5-FU) and cobalt 60 (60CO). Each tumor was radiated serially from Monday to Fri-

The difference in frequencies was evaluated by X2 test, and p-values less than 0.05 were considered significant. For multivariate survival analysis, Kaplan-Maier's curves were plotted for the p53/p21 stainability. Statistical significance of the survival difference was tested by generalized Wilcoxon method.

Expression of p53 and p21 in Oral Carcinomas . 823

Results The expression of p53 and p21 proteins was not correlated with tumor sites (Table 2) although p53 protein was strongly expressed in advanced (T3 and T4) carcinomas (Table 3). In the tongue, gingiva, floor of the mouth and cheek/palate SCCs, p53 protein was expressed in 54.2%, 66.7%, 59.1 % and 52.9%, respectively (Table 2 and Figs. lA, 2A and 3A). Likewise, there were no differences in the expression of p21 protein according to the tumor sites, although p21 expression was observed about one fourth less frequently than p53 (Fig. IB). In advanced carcinomas, p53 expression was

highly observed (62.5% in T3 and 62.1 % in T4) but p21 expression was infrequently observed (22.5% in T3 and 31.0% in T4, Table 3). However, there was no statistically significant difference between T1 + T2 and T3 + T4 carcinomas in expression of p53 and p21 proteins. Corresponding to the increase of the invasion grade, p53 expression increased; however, any statistically significant difference was not observed (Table 4). Of 37 carcinomas in grades 1 and 2 of the invasion mode, 20 tumors (54.1 %) were positive for p53 protein. While from 76 tumors in grade 3 and 37 tumors in grades 4C and 40, 43 (56.6%) and 27 (73.0%) were positive for p53 protein, respectively. No correlation was observed

Table 2. Staining results of p53 and p21 proteins in each tumor site

Table 3. Relationship between T-stage and expression of p53 and p21 proteins T-stage

Stainability of p53 and p21 proteins p53(+) n=90

p53(-) n=60

p21(+) n=43

p21(-) n= 107

Tl n=2

1 (50.0%) ,1<

1 (50.0%)

1 (50.0%)

1 (50.0%)

0

0

T2 n=79

46 (58.2%)

33 (41.8%)

24 (30.4%)

55 (69.6%)

28

T3 n=40

25 (62.5%)

15 (37.5%)

9 (22.5%)

31 (77.5%)

19

T4 n=29

18 (62.1%)

11 (37.9%)

9 (31.0%)

20 (69.0%)

10

.

::~.

".~

positive cases positive + negative cases

x 100

p53(+)p21(-) n =57

p53(-)p21(+) n= 10

p53(+)p21(+) n= 33

p53(-)p21(-) n=50

6

18

27

3

6

12

8

10

824 . Y. Tatemoto et al.

Fig. I. p53 and p21 immunostaining in a well differentiated tongue carcinoma exhibiting grade 3 of invasion mode. A: p53 immunostaining; most tumor cells reveal prominent nuclei positive for p53 (xI20). B: p21 immunostaining; many tumor cells having p21-positive nucleus are seen (x 120).

Fig. 2. Poorly-differentiated carcinoma of the lower gingiva. A: p53 immunostaining; about half of the tumor cells are positive for p53 protein (x40). B: H&E staining; a histological view of specimens prepared from the tumor resected after chemoradiotherapy. The degree of histological effect is grade II B (x40).

Table 4. Correlation between mode of invasion and stainability ofp53 and p21 proteins Mode of invasion

Stainability of p53 and p21 proteins p53(+) n=90

p53(-) n=60

p21(+) n=43

p21(-) n= 107

p53(+)p21(-) n=57

2 (66.7%)

I (33.3%)

2 (66.7%)

0

0

p53(-)p21(+) n= 10

p53(+)p21(+) n = 33

P53(-)p21(-) n=50 2

grade I n=3

I (33.3%)

grade 2 n=34

19 (55.9%)

15 (44.1%)

10 (29.4%)

24 (70.6%)

II

2

8

13

grade 3 n=76

43 (56.6%)

33 (43.4%)

17 (22.4%)

59 (77.6%)

32

6

II

27

grade4C+4D 27 (73.0%) n=37

10 (27.0%)

15 (40.5%)

22 (59.5%)

14

2

13

8

~;~:

.,*

positive cases positive + negative cases

x 100

Expression of p53 and p21 in Oral Carcinomas . 825

between the p21 stainability and the invasion mode, however tumors with grades 4C and 4D expressed p21 protein more frequently than other tumors. In 57 tumors of p53(+)-p21(-), 46 exhibited an invasion mode of grade 3 or grades 4C and 4D. In 35 of 50 tumors with p53(-)-p21(-), the invasion mode was grade 3 or grade 4. Furthermore, invasion modes of grade 3 and 4 were observed in 24 of 33 tumors with p53(+)-p21(+). The degree of cell differentiation was not correlated with the expression of p53 or p21 proteins; p53 was positively stained in about 58%, 56% and 73% of well-, moderately- and poorly-differentiated carcinomas, respectively. Positive p21 staining was observed in about 30% without large differences according to differentiation (Table 5). Among the p53(+)-p21(-), p53(-)-p21(+), p53(+)p21(+) and p53(-)-p21(-) groups, no clear difference of tumor cell differentiation was observed. The histopathological effects of chemoradiotherapy were inversely correlated with the expression of p53 protein (Figs. 2B and 3B), although they were not correlated with p21 protein expression (Table 6). Of 90 tumors exhibiting non-lethal degeneration of grade II A (14 tumors) and grade II B (76 tumors), 64 tumors (71.1 %) were positive for p53. Amongst 60 tumors exhibiting lethal degeneration of grade III and grade IV, 26 (43.3%) expressed p53 protein (p < 0.005). In 90 tumors with non-lethal degeneration and 60 tumors with lethal degeneration, positive p21 staining was observed in 28 (31.1 %) and 15 tumors (25.0%), respectively. In the 4 groups, the p53(+)-p21(+)" group revealed the lowest lethal degeneration rate (27.3%), being followed by the p53(+)-p21(-) group (29.8%). The lethal degeneration rates in the remaing 2 groups, p53(-)-p21(+) and p53(- )-p21 (-), were statistically significantly higher than those in the p53(+) groups (60.0% in the p53(-)-p21(+) and 56.0% in the p53(-)-p21(-) group) (p < 0.02).

Fig. 3. Well-differentiated carcinoma of the floor of the mouth. A: p53 immunostaining; the tumor cells do not express p53 protein (x40). B: H&E staining; a histological view of specimens prepared from the tumor resected after chemoradiotherapy. No viable tumor cells are visible and there are many giant cells phagocytizing keratin fragments (x40).

Table 5. Correlation between tumor cell differentiation and stainability of p53 and p21 proteins Differentiation Stainability of p53 and p21 proteins of tumor cells p53(+) p53(-) p21(+) p21(-) n=90 n=60 n=43 n= 107 well n= 101

59 (58.4%)

moderate n=27 poor n=22 ~T~:

p53(+)p21(-) n= 57

p53(-)p21(+) n= 10

p53(+)p21(+) p53(-)p21(-) n=33 n=50

42 (41.6%)

27 (26.7%)

74 (73.3%)

38

6

21

36

15 (55.6%)

12 (44.4%)

8 (29.6%)

19 (70.4%)

9

2

6

10

16 (72.7%)

6 (27.3%)

8 (36.4%)

14 (63.6%)

10

2

6

4

*

positive cases positive + negative cases

x 100

826 . Y. Tatemoto et al. Table 6. Correlation between histological effect of chemoradiotherapy and stainability of p53 and p21 proteins Histological effect in the tumor cells

Stainability of p53 and p21 proteins p53(+) n=90

p53(-) n=60

p21(+) n=43

Non-lethal degeneration n=90

64

26

28

grade IIA n= 14

10

4

grade II B n=76

54

Lethal degeneration n=60

p21(-) n= 107

p53(+)p21 (-) n=57

p53(-)p21(+) n= 10

p53(+)p21(+) n= 33

p53(-)p21 (-) n=50

62

40

4

24

22

4

10

6

0

4

4

22

24

52

34

4

20

18

26

34

15

45

17

6

9

28

grade III n= 13

8

5

3

10

6

2

4

grade IV n=47

18

29

12

35

11

5

7

24

Lethalffotal

26/90 (28.9%)

34/60 (56.7%)

15/43 (34.9%)

17/57 (29.8%)

6/10 (60.0%)

45/107 (43.9%)

~

,

p<0.005

9/33 (27.3%)

p<0.02

28/50 (56.0%) ,

v

p<0.02

Table 7. Relationship between the imrnunostainability and lymph node metastasis Metastasis

p53(+) n=90

p53(-) n=60

p21(+) n=43

p21(-) n = 107

p53(+)p21(-) n = 57

p53(-)p21(+) p53(+)p21(+) n = 10 n = 33

(+) n=62

45 (72.6%)

17

24 (38.7%)

38

27 (47.2%)

6

'-----v--------

p<0.02

(-) n=88

18 (54.5%)

43

11 (22.0%)

~

p<0.05

~

45 (52.9%)

(60.0%)

p53(-)p21(-) n=50

P <0.01

~

19 (21.6%)

69

30 (52.6%)

4 (40.0%)

15 (45.5%)

39 (78.0%)

positive cases .,!<-: - - - - - - - - x 100 positive + negative cases

Lymph node metastasis was correlated with the expression of both proteins. In 62 tumors with lymph node metastasis, positive expression of p53 and p21 protein was observed in 45 (72.6%) and 24 tumors (38.7%), respectively (p < 0.02, P < 0.05) (Table 7). Of 88 tumors without lymph node metastasis, 45 (52.9%) and 19 tumors (21.6%) were positive for p53 and p21 protein, respectively. The expression of p53 but not p21 protein was correlated with the survival rate. The 5-year-survival rate of patients with p53-positive see (67.8%) was lower than that (75.3%) of patients with p53-negative see, while the 5-year-survival rate in p21-positive and p21-nega-

tive patients as 69.8% and 70.4%, respectively. When both proteins were evaluated, the 5-year-survival rate was the lowest in p53(+)-p21(-) patients (65.4%) and the highest in p53(-)-p21(+) patients (80.0%), although a statistically significant difference was not observed (Fig. 4).

Discussion It has been recently reported that the anti-tumor effect of anticancer drugs and radiation depends not only on necrosis but also on differentiation and apoptosis of

Expression ofp53 and p2l in Oral Carcinomas· 827 ~ pS3(+}p21(+}

pS3(+) ...... pS3(-) __ p21(+}

~

100

100....---.,.------...,

...... pS3(+) p21 (-) __ pS3(-}p21(+) ....... pS3(-} p21 (-)

....... p21(-}

-e- total

-e- total

90

90

~

.. III

Ql

i! 80 co> .~

:> III

70

70

Fig. 4. Comparison of Kaplan-Meier's cumulative survival rates in each p53/ p21 stainability.

60

'--.....,...-..,.....--,--r----. lY

2Y

the neoplastic cells [23, 46, 54J - the induction of tumor cell differentiation having been recognized as one of the important steps for tumor control [21, 49]. However, differentiation of cells is limited to the G1 phase [24, 58J, differentiation of solid tumor cells is difficult to induce in general, and, apoptosis is inducible in any cell phase. Therefore, induction of apoptosis appears easier than induction of differentiation. However, induction of apoptosis sufficient for controlling malignant tumors has not yet been established. p53 protein regulates the cell cycle by promoting the function of target genes associated with apoptosis and differentiation [6, 12, 13, 22, 26, 28, 34, 35, 37, 39]. The mutant p53 protein, however, induces neither G 1 arrest nor differentiation or apoptosis. Mutation of the p53 gene and its product has been intensively investigated in a variety of tumors, and poor prognosis of tumors with mutated p53 protein has been reported in carcinomas including oral SCCs [9, 16-18, 25, 37, 41]. The present study clearly revealed a correlation between p53 expression and lymph node metastasis, as well as poor response to chemoradiotherapy, and a lower 5-year-survival rate in p53-positive SCCs than in p53-negative SCCs. These findings suggest that the expression of p53 protein is regarded as a poor prognosispredicting factor in oral SCCs. Although anti-p53 antibody used in the present study reacts with both mutant and wild type p53 proteins, the detected p53 protein appeared to be mutated because wild type p53 protein is very labile and easily destroyed. However, there are some papers describing the fact that overexpression of p53 protein assessed with DO-7 immunohistochemistry was higher than the incidence of p53 gene mutations in some kinds of cancers, ranging from about 10% to 30% depending on the kinds of carcinomas [2, 47]. In these investigations, mutations of the p53 gene were exam-

3Y

4Y

SY

60

L.--..,._...,....._..--..,._..., lY

2Y

3Y

4Y

SY

ined in exon 2 to 9, and exon 5 to 9. Therefore, undetected mutations might exist in the remaining exons although there is the possibility that p53 protein detected in the present study and other reports was a wild type in some tumors [1-3, 25, 44, 47]. It is generally recognized that the mutated p53 gene product does not induce p21. However, p21 protein expression has been reported in many types of tumors expressing mutated p53 protein [3, 4, 7, 32, 50, 52, 57]. These reports indicate that p21 protein is induced by a p53-independent manner [4, 11, 33, 36]. In addition to these studies, our results revealed p53-independent p21 expression in oral SCCs. p21 protein inhibits cdk activity and down regulates the cell cycle by arresting cells in the Gl phase [15, 55J as well as the G2 phase [15, 53J; some favorable prognosis of adenocarcinomas expressing p21 in colorectal and breast carcinomas have been reported [8, 50, 52]. In the present study, the 5-year-survival rate in patients with p21-positive SCC was similar to that in patients with p21-negative SCC, although the expression ofp21 protein was correlated with lymph node metastasis. In 10 of 24 SCCs which possessed positive lymph nodes and expressed p21 protein, the N stage was N 1a which was fully controlled surgically. In addition, the degenerative effect summed up of grades II B, III and IV in p21-positive SCCs was similar to that in p21-negative SCCs although lethal degeneration of p21 protein-expressing tumor cells was rather mild compared with that of p21 protein-negative tumor cells. The expression of p21 did not positively indicate good prognosis. Many investigators reported that mutation of the p21 gene, however rarely, occurred in malignant tumor cells and that products of the mutated p21 do not work in the regulation of the cell cycle [29,30,40]. Therefore, there is a possibility that the p21-protein detected in this study was mutated. Further study is essential to clarify this

828 . Y. Tatemoto et al.

matter, while the biological role of p21 protein expressed in SCCs should be further explored. The expression of the p53 protein was intensively associated with lymph node metastasis. This relationship, however, has not previously been reported in oral SCCs, and suggests that p53 gene-mutated tumor cells have a tendency to metastasize to the lymph nodes. Further detailed investigation concerning the relationship between p53 gene mutation and metastasis appears to offer useful information for cancer treatment. 5-FU and y-radiation have tumor cell differentiationand apoptosis-inducing activities [10, 19, 45J. Wild type p53 protein plays a critical role in the induction of differentiation and apoptosis [12, 24, 31, 57 J, so that the induction of differentiation and apoptosis in tumor cells possessing mutated p53 gene is probably less likely than in tumor cells without p53 gene mutation. The poor histopathological degeneration after chemoradiotherapy in the SCCs positive for p53 protein may have resulted from this mechanism. The present results reveal that although further study is required to clarify the meaning of p21 protein expression, p53 is a useful prognostic marker in oral SCCs and that immunohistochemical examination for detection of the protein before treatment is advantageous for predicting the response of oral SCCs to chemoradiotherapy.

References 1. Ahomadegbe JC, Barrois M, Fogel S, Le Bihan ML, Douc-Rasy S, Duvillard P, Armand JP, Riou G (1995) High incidence of p53 alteration (mutation, deletion, overexpression) in head and neck primary tumors and metastasis; absence of correlation with clinical outcome. Frequent protein overexpression in normal epithelium and in early non-invasive lesions. Oncogene 10: 1217-1227 2. Baas 10, Mulder J-WR, Offerhaus JA, Vogelstein B, Hamilton SR (1994) An evaluation of six antibodies for immunohistochemistry of mutant p53 gene product in archival colorectal neoplasms. J Pathol172: 5-12 3. Barbareschi M, Caffo 0, Doglioni C, Fina P, Marchetti A, Buttitta F, Leek R, Morelli L, Leonardi E, Bevilacqa G, Dalla Palma P, Harris AL (1996) p21 WAFI immunohistochemical expression in breast carcinoma: correlations with e1inico-pathological data, oestrogen receptor status, MIB expression, p53 gene and protein alterations and relapse-free survival. Br J Cancer 74: 208-215 4. Barboule N, Mazars P, Baldin V, Vidal S, Jozan S, Martel P, Valette A (1995) Expression of p21 WAF I is heterogenous and unrelated to proliferation index in human ovarian carcinoma. Int J Cancer 63: 611-615 5. Bourhis J, Bosq J, Wilson GD, Bressac B, Talbot M, Leridant AM, Dendale R, Janin N, Armand JP, Luboinski B, Malaise EP, Wibault P, Eschwege F (1994) Correlation between p53 gene expression and tumor-cell proliferation in oropharyngeal cancer. Int J Cancer 57: 458-462

6. Buckbinder L, Talbott R, Valasco-Miguel S, Takenaka I, Faha B, Seizinger BR, Kley N (1995) Induction of the growth inhibitor IGF-binding protein 3 by p53-target genes. Nature (Lond.) 377: 646-649 7. Byrne RL, Wilson Home CH, Robinson MC, Autzen P, Apakama I, Bishop RI, Neal DE, Hamdy FC (1997) The expression of waf-I, p53 and bel-2 in prostatic adenocarcinoma. Br J Uro179: 190-195 8. Chen YQ, Sherry CC, Arenkiel JM, Miller FR (1995) Tumor suppression by p21 wAFI • Cancer Res 55: 4536-4539 9. De Araujo VC, Loyola AM, Santos Pinto Jr DD, Borra RC, De Araujo NS (1997) p53 in biopsies of oral squamous cell carcinoma. A comparative study with a malignancy grading system. Oral Oncol 33: 5-9 10. Dewey WC, Ling CC, Meyn RE (1995) Radiation-induced apoptosis: relevance to radiotherapy. Int J Radiation Oncol BioI Phys 33: 781-796 II. DiGiuseppe JA, Redston MS, Yeo CJ, Kern SE, Hruban RH (1995) p53-independent expression of the cyelin-dependent kinase inhibitor p21 in pancreatic carcinoma. Am J Pathol147: 884-888 12. EI-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, Lin D, Mercer WE, Kinzler KW, Vogelstein B (1993) WAFI, a potential mediator of p53 tumor suppression. Cell 75: 817-825 13. Furuhara T, Tokino T, Urano T, Nakamura Y (1996) Isolation of a novel GPI-anchored gene specifically regulated by p53; correlation between its expression and anti-cancer drug sensitivity. Oncogene 13: 1965-1970 14. Hall PA, Lane DP (1994) p53 in tumour pathology: can we trust immunohistochemistry ? -revisited ! J Pathol 172: 1-4 15. Harper JW, Adami GR, Wei N, Keyomarsi K, Elledge SJ (1993) The p21 Cdk-interacting protein CIPI is a potent inhibitor of G 1 cyelin-dependent kinases. Cell 75: 805-816 16. Harris CC, Hollstein M (1993) Clinical implication of p53 tumor-suppressor gene. N Eng J Med 329: 1318-1327 17. Hayashi N, Ito I, Yanagisawa A, Kato Y, Nakamori S, Imaoka S, Watanabe H, Ogawa M, Nakamura Y (1995) Genetic diagnosis of lymph-node metastasis in colorectal canceL Lancet 345: 1257-1259 18. Heinzel PA, Balaram P, Bernard H-U (1996) Mutations and polymorphisms in the p53, p21, and p16 genes in oral carcinomas of indian betelquid chewers. Int J Cancer 68: 420-423 19. Hickman JA (1992) Apoptosis induced by anticancer drugs. Cancer Metastasis Rev 11: 121-139 20. Hollstein M, Shomer B, Greenblatt M, Soussi T, Hovig E, Montesano R, Harris CC (1996) Somatic point mutations in the p53 gene of human tumors and cell lines: updated complication. Nuc Acids Res 24: 141-146 21. Hozumi M (1994) Fundamentals of chemo-differentiation therapy of myeloid leukemia. Anticancer Res 14: 1177-1192 22. Kastan MB, Zhan Q, EI-Deiry WS, Carrier F, Jacks T, Walsh WV, Plunkett BS, Vogelstein B, Fornace AJ Jr (1992) A mammalian cell cyele checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia. Cell 71: 587-592

Expression ofp53 and p21 in Oral Carcinomas· 829 23. Kerr IFR, Wyllie AH, Currie AR (1972) Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. Br 1 Cancer 26: 239-257 24. Kuerbitz S, Plunkett B, Walsh W, Kastan M (1992) Wildtype p53 is a cell cycle checkpoint determinant following radiation. Nature (Lond.) 89: 7491-7495 25. Kusama K, Okutsu S, Takeda K, Himiya T, Kojima A, Kidokoro Y, Chu L, Iwanari S, Kodo I, Moro I (1996) p53 gene alteration and p53 protein in oral epithelial dysplasia and squamous cell carcinoma. 1 Pathol178: 415-421 26. Lane DP (1992) p53, guardian of the genome. Nature (Lond.) 358: 15-16 27. Levesque MA, Katsaros D, Yu H, Giai M, Genta F, Roagna R, Ponzone R, Massobrio M, Sismondi P, Diamandis EP (1998) Immunofluorometrically determined p53 accumulation as a prognostic indicator in italian breast cancer patients. Int 1 Cancer (Pred Oncol) 79: 147-152 28. Levine Al (1997) p53, the cellular gatekeeper for growth and division. Cell 88: 323-331 29. Lukas 1, Groshen S, Saffari B, Niu N, Reles A, Wen W-H, Felix 1, 10nes LA, Hall FL, Press MF (1997) WAFl/Cipl gene polymorphism and expression in carcinomas of the breast, ovary, and endometrium. Am 1 Pathol 150: 167-175 30. Lu Y, Yamagishi N, Yagi T, Takebe H (1998) Mutated p21 (WAFl/Cipl/SDIl) lacking CDK-inhibitory activity fails to prevent apoptosis in human colorectal carcinoma cells. Oncogene 16: 705-712 31. Macleod KF, Sherry N, Hannon G, Beach D, Tokino T, Kinzler K, Vogelstein B, lacks T (1995) p53-dependent and independent expression of p21 during cell growth, differentiation, and DNA damage. Genes & Dev 15: 935-944 32. Mrelandsmo GM, Holm R, Fodstad 0, Kerbal RS, Fll/lrens VA (1996) Cyclin kinase inhibitor p21 WAF IICIP I in malignant melanoma. Reduced expression in metastatic lesions. Am 1 Pathol149: 1813-1822 33. Marchetti A, Doglioni C, Barbareschi M, Buttitta F, Pellegrini S, Bertacca G, Chella A, Merlo G, Angeletti CA, Palma PD, Bevilacqua G (1996) p21 RNA and protein expression in non-small cell lung carcinomas: evidence of p53-independent expression and association with tumoral differentiation. Oncogene 12: 1319-1324 34. Miyashita T, Reed lC (1995) Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell 80: 293-299 35. Momand 1, Zambetti GP, Olson DC, George DL, Levine Al (1992) The MDM oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation. Cell 69: 1237-1245 36. Nadal A, lares P, Cazorla M, Fernandes PL, Sanjuan X, Hernandez L, Pinyol M, Aldea M, Mallofre 1, Muntane 1, Traserra 1, Campo E, Cardesa A (1997) p21 WAFl/Cipl expression is associated with cell differentiation but not with p53 mutations in squamous cell carcinomas of the larynx. 1 Pathol183: 156-163 37. Nigro 1M, Baker Sl, Preisinger AC, lessup 1M, Hostetter R, Cleary K, Bigner SH, Davidson N, Baylin S, Devilee P, Glover T, Collins FC, Weston A, Modali R, Harris CC, Vogelsteip B (1989) Mutations of p53 gene occur in diverse human tumour types. Nature (Lond.) 342: 705-708

38. Nylander K, Stenling R, Gustafsson H, Zackrisson B, Roos G (1995) p53 expression and cell proliferation in squamous cell carcinomas of the head and neck. Cancer 75:87-93 39. Okamoto K, Bearch D (1994) Cyclin G is a transcriptional target of the p53 tumor suppressor protein. EMBO 1 13:4816-4822 40. Papandreou CN, Bogenrieder T, Loganzo F, Albino AP, Nanus DM (1997) Expression and sequence analysis of the p21 (WAFl/Cipl) gene in renal cancers. Urology 49: 481-486 41. Sakai E, Tsuchida N (1992) Most human squamous cell carcinomas in the oral cavity contain mutated p53 tumorsuppressor genes. Oncogene 7: 927-933 42. Shi SR, Key ME, Kalra KL (1991) Antigen retrieval in formalin-fixed, paraffin-embedded tissues; an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. 1 Histochem Cytochem 39: 741-748 43. Shimosato T, Oboshi S, Baba K (1971) Histological evaluation of effects of radiotherapy and chemotherapy of carcinomas. Ipn 1 Clin Oncoll: 19-35 44. Soong R, Robbins PD, Dix BR, Grieu F, Lim B, Knowles S, Williams KE, Turbett GR, House AK, Iacopetta Bl (1996) Concordance between p53 protein overexpression and gene mutation in a large series of common human carcinomas. Hum Pathol27: 1050-1055 45. Sugamura K, Makino M, Shirai H, Kimura A, Maeta M, Itoh H, Kaibara N (1997) Enhanced induction of apoptosis of human gastric carcinoma cells after preoperative treatment with 5-fluorouracil. Cancer 79: 12-17 46. Thompson CB (1995) Apoptosis in the pathogenesis and treatment of disease. Science 267: 1456-1462 47. Trivedy C, Wamakulasuriya KAAS, Tabassoli M, Steingrimsdottir H, Penhallow 1, Maher R, 10hnson NW (1998) p53 oberrations in oral submucous fibrosis and oral squamous cell carcinoma detected by immunocytochemistry and PCR-SSCP. 1 Oral Pathol Med 27: 72-77 48. VICC (1987) TNM classification of malignant tumours, 4th ed., pp 16-18. Springer-Verlag, Berlin 49. Vaux DL (1993) Toward a understanding of the molecular mechanisms of physiological cell death. Proc Nat! Acad Sci USA 90: 786-789 50. Wakasugi E, Kobayashi T, Tamaki Y, Ito Y, Miyashiro I, Komoike Y, Takeda T, Shin E, Takatsuka Y, Kikkawa N, Monden T, Monden M (1997) p21 (Wafl/Cipl) and 53 protein expression in breast cancer. Am 1 Clin Patholl 07: 684-691 51. Waldman T, Lengauer C, Kinzler KW, Vogelstein B (1996) Uncoupling of S phase and mitosis induced by anticancer agents in cells lacking p21. Nature 381: 713-716 52. Wang A, Yoshimi N, Ino N, Tanaka T, Mori H (1997) WAFt expression and p53 mutations in human colorectal cancers. 1 Cancer Res Clin Oncol23: 118-123 53. Weinberg RA (1995) The retinoblastoma protein and cell cycle control. Cell 81: 323-330 54. Wyllie AH (1992) Apoptosis and the regulation of cell numbers in normal and neoplastic tissues: an overview. Cancer Metastasis Rev 11: 95-103 55. Xiong Y, Hannon G, Zhang H, Casso D, Kobayashi R, Bearch D (1993) p21 is an universal inhibitor or cyclin kinases. Nature (Lond.) 366: 701-704

830 . Y. Tatemoto et al. 56. Yamamoto E, Kohama G, Sunakawa H, Iwai M, Hiratsuka H (1983) Mode of invasion: bleomycin sensitivity, and clinical course in squamous cell carcinoma of the oral cavity. Cancer 50: 2175-2180 57. Yasui W, Akama Y, Kuniyasu H, Yokozaki H, Semba S, Shimamoto F, Tahara E (1996) Expression of cyclin-dependent kinase inhibitor p21 WAFI/CIPI in non-neoplastic mucosa and neoplasia of the stomach: Relationship with p53 status and proliferative activity. J Pathol180: 122-128

58. Yonish-Arouach E, Resnitzky D, Lotem J, Sachs L, Kimchi A, Oren M (1991) Wild-type p53 induces apoptosis of myeloid leukaemaic cells that is inhibit by interleukin-6. Nature (Lond.) 352: 345-347

Received: June 9, 1998 Accepted in revised form: August 3, 1998