Immunohistochemical detection of aberrant p53 expression in hepatocellular carcinoma: Correlation with cell proliferative activity indices, including mitotic index and MIB-1 immunostaining

Immunohistochemical Detection of Aberrant p53 Expression in Hepatocellular Carcinoma: Correlation With Cell Proliferative Activity Indices, Including Mitotic Index and MIB-I Immunostaining TOSHITAKA NAGAO, MD, FUKUO KONDO, MD, TSUNENOBU SATO, MD, YOSHINOBU NAGATO, MD, AND YOICHIRO KONDO, MD We analyzed the p53 expression immunohistochemically in 50 specimens of hepatocellular carcinoma (HCC) using two m o n o c l o n a l antibodies (DO7 and PAbl801) and one polyclonal antibody (CM1), which recognize both wild and mutant type p53 proteins and can be used for paraffin-embedded sections. Fifteen of the 50 HCC specimens (30%) showed p53 expression localized at t u m o r nuclei, and this expression was significantly more frequent in HCCs with histologically lower differentiation. Except for serum titers of alpha-fetoprotein, the p53 expression had no statistically significant correlation with clinicopathological parameters, including hepatitis virus infection, tumor size, and background liver diseases. Conversely, the cell proliferative activities of tumor cells as assessed by mitotic index and immunostaining for MIB-1 were well correlated with the grade of histological differentiation. Moreover, MIB-1 immunostaining was shown to be useful in distinguishing well differentiated HCC f r o m

hepatocytes in chronic liver diseases. It also was shown that p53 expression was strongly associated with cell proliferative activity. Our results indicate that p53 expression takes place in the late stage of t u m o r progression and is related to the high malignant potential of HCCs. HUM PATHOL 26:326--333. Copyright © 1995 by W.B. Saunders Company Key words- p53, hepatocellniar carcinoma, MIB-1, paraffin section, mitotic index. Abbreviations: hsc, heat shock protein; HCC, hepatocellular carcinoma; PCNA, proliferating cell nuclear antigen; MAb, monoclonal antibody; PFA, paraformaldehyde; PBS, phosphate-buffered saline; AFP, alpha-fetoprotein; HPF, high-power field; Ig, immunoglobulin; APES, 3-aminopropyltriethoxylane; LI, labeing index; ANOVA, analysis of variance; LC, liver cirrhosis.

p53 is a t u m o r suppressor gene located on the short arm of c h r o m o s o m e 17.1 Wild type p53 protein is involved in negative regulation of cell growth by controlling cell entry into the S phase. 2 In general, wild type p53 protein has a short halfqife of only 20 minutes, whereas m u t a n t type p53, which forms stable complexes with heat shock protein (hsc) 70 and some other proteins, has a longer half-life of several h o u r s ? '4 accumulates within a nucleus, and thus is visible by immunohistochemistry. 5 Accumulation of the p53 protein has b e e n d e m o n s t r a t e d in several c o m m o n h u m a n cancers, such as those arising f r o m the lung, 6 breast, 7'8 colon, 9 esophagus, 1° and stomach. 11 Although the occurrence of p53 gene mutations has b e e n well recognized in hepatocellular carcinomas (HCCs) with the aid of molecular techniques, 12-16the precise patterns of aberrant p53 expression in t u m o r tissue have not yet b e e n fully clarified. It appears that such an alteration of a t u m o r suppressor gene is related to cell proliferation. ~ Several new methods for measuring t h e activity of cell proliferation have b e e n developed, such as b r o m o d e o x y u r i d i n e labeling, silver staining of the nucleolar organizer region, and immunostaining for Ki-67 and proliferating cell nuclear antigen (PCNA). 17 I m m u n o h i s t o c h e m i c a l in-

vestigations with Ki-67 m o n o c l o n a l antibody (MAb), which is expressed during the phases of G1, S, G2, and mitosis hut not during that of GO, require frozen material. TM Recently, however, a Ki-67 equivalent m u r i n e MAb (MIB-1) was g e n e r a t e d that can detect cell proliferative activity in formalin-fixed, paraffin-embedded tissue using a microwave oven heating procedure. 18'19 In the current i m m u n o h i s t o c h e m i c a l study we att e m p t e d to demonstrate accumulation of p53 protein in HCCs in paraffin-embedded sections by using two m o n o c l o n a l antibodies (DO7 and PAbl801) and one polyclonal antibody (CM1). We also applied two indices (ie, mitotic index and i m m u n o h i s t o c h e m i c a l staining for MIB-1) to correlate with the histological grade of differentiation and aberrant p53 expression.

MATERIALS AND METHODS

Tissue Preparation

From the Department of Pathology and the First Department of Internal Medicine, School of Medicine, Chiba University, Chiba, Japan. Accepted for publication July 18, 1994. Address correspondence and reprint requests to Toshitaka Nagao, MD, Department of Pathology, School of Medicine, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba 260, Japan. Copyright © 1995 by W.B. Saunders Company 0046-8177/95/2603-001355.00/0

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Fifty Japanese patients with HCC were included in this study. Tumor samples were obtained at the time of surgery for 10 patients and by ultrasonography-guided needle biopsy for 40 patients at Chiba University Hospital and Chiba Cancer Center Hospital in 1992 and 1993. Surrounding nontumorous liver tissue also was sampled in 37 patients. For controls autopsy samples (within 5 hours postmortem) composed of five normal livers, one gastric carcinoma, and one normal tonsil were obtained at Asahi General Hospital. All materials were placed immediately in 4% paraformaldehyde (PFA) in phosphate-btfffered saline (PBS), at pH 7.4, fixed at 4°C for less than 15 hours, processed into paraffin blocks, and stored at 4°C. Nonfixed small tissue blocks of the normal tonsil were

p53 EXPRESSIONIN HCC (Nagao et al) frozen in optical cryopreserving tissue compound (Miles, Elkhart, IN) and stored at -80°C.

Clinicopathological Parameters We assessed the association of p53 expression with the age and sex of HCC patients, tumor size, background liver diseases, and hepatitis virus infection. Hepatitis B surface antigen and hepatitis C virus antibody in the serum were checked by radioimmunoassay and second-generation enzyme-linked immunosorbent assay, respectively. We also measured the titers of alpha-fetoprotein (AFP) and protein induced by vitamin K absence (P1VKA)-II in the serum.

Histological Grade of Differentiation and Mitotic Index Hematoxylin-eosin-stained sections were examined to evaluate the histological grade of tumor differentiation, and the HCCs were classified into well, moderately, and poorly differentiated subtypes as well as an undifferentiated subtype according to the General Rules for the Clinical and Pathological Study of Primary Liver Cancer of Japan, 2° which approximately correspond to Edmondson-Steiner's grades I, II, III, and IV,21 respectively. For the mitotic index the number of mitotic figures in five random high-power fields (HPFs, 10 × 40) was assessed in each case due to the limited number of samples obtained from biopsy specimens.

+ + , 10% to 50% of cells positive; + + + , more than 50% of cells positive. As a positive control we applied a specimen of gastric carcinoma that had been proven to be positive for p53 in more than 80% of tumor cells when tested with the three antibodies. MIB-1. Deparaffinized tissue sections were washed in distilled water, immersed in citrate buffer (pH 6.0) at 95°C for 15 minutes on a hot plate, and left for 1 hour at room temperature. Except for application of the primary monoclonal antibody, the remaining procedures were the same as for p53 immunostaining. The primary monoclonal antibody MIB-1 was diluted to 1:50. To check whether the MIB-1 immunostaining was reliable for showing cell proliferative activity in paraffin sections, we performed concurrent MIB-1 immunostaining for the normal tonsil in both frozen and paraffin sections. F o r the frozen sections 6-#m-thick specimens were put on 3-APES-coated slides, air dried for 30 minutes, and fixed in 4% PFA in PBS (pH 7.4) for 20 minutes. The remaining procedure was the same as for the paraffin-embedded sections. The percentage of positive cells was estimated by counting more than 500 cells in random HPFs and was recorded as the MIB-1 labeling index (LI).

Statistical Analysis The statistical significance of the individual findings and their associations was checked by the one-way analysis of variance (ANOVA), Student's t-test, Mann-Whimey Utest, the simple linear regTession test, and Fisher's exact probability test. Probability values less than .05 were considered to be significant.

Antibodies Staining for p53 protein was carried out using the following antibodies: 1) mouse monoclonal antibody DO7, isotype immunoglobulin (Ig)G2b (Novocastra Laboratories, Newcastle, UK) that recognizes a denaturation-resistant epitope at the N terminus o f p53; 22 2) mouse monoclonal antibody PAb1801, isotype IgG1 (Novocastra) that recognizes a denaturation-resistant epitope in human p53 located between amino acids 32 and 79; 23 and 3) rabbit polyclonal antibody CM1 (Novocastra Laboratories) that reacts with whole proteins expressed in Escherichia coli. 5 These three antibodies were capable of detecting both wild type and mutant type p53 proteins. Cell proliferative activity was determined using mouse monoclonal antibody MIB-1, isotype IgG1 (Immunotech SA, Marseille, France) that recognizes native Ki-67 antigen and recombinant fragments of the Ki-67 molecule. 18'm'24

Immunohistochemistry p53. Paraffin-embedded blocks were cut at 4-#m thickness and put on 3-aminopropyltriethoxylane (APES) (Sigma Chemical, St Louis, M O ) - c o a t e d slides. To block the endogenous peroxidase activity the slides were immersed in methanol with 0.3% hydrogen peroxide for 30 minutes. The sections were incubated with the primary antibody for 15 hours at 4°C. The optimal dilution for each antibody was 1:100 for DO7, 1:50 for PAbl801, and 1:1000 for CM1. The slides were incubated with biotinylated antimouse Ig for monoclonal antibodies or antirabbit Ig for the polyclonal antibody. They were subsequently incubated with streptavidin-biotin peroxidase complex (Dako, Glostrup, Denmark). 3,3'-diaminobenzidine was used as a chromogen and enhanced by 1% osmium in PBS. The sections were slightly counterstained with hematoxylin. Only nuclear staining was regarded as positive. The percentage of positive tumor cells was estimated by counting more than 500 cells and the samples containing more than 1% of cells positive for at least two of the three antibodies were determined to be positive. The tumors were semiquantitatively categorized into four groups as follows: , 0% to 1% of cells positive; +, 1% to 10% of cells positive;

327

RESULTS

p53 Expression Positive p53 i m m u n o s t a i n i n g was seen in nuclei o f t u m o r cells, b u t the intensity was quite variable f r o m cell to cell. N o i m m u n o s t a i n i n g was seen in t u m o r cells u n d e r g o i n g mitosis (Fig 1). W e a k cytoplasmic staining was sometimes seen, especially in the samples t r e a t e d with polyclonal a n t i b o d y (CM1). Exclusive cytoplasmic staining, however, was scored as negative. N o d e t e c t a b l e n u c l e a r staining was p r e s e n t in any o f the n o r m a l liver tissues. A s s o c i a t i o n o f p53 e x p r e s s i o n with h i s t o l o g i c a l g r a d e o f t u m o r d i f f e r e n t i a t i o n is p r e s e n t e d in T a b l e 1. F i f t e e n o f 50 H C C s a m p l e s (30%) were d e t e r m i n e d to b e positive, t h e i n c i d e n c e o f w h i c h was usually h i g h e r in H C C s with a l o w e r h i s t o l o g i c a l d i f f e r e n t i a t i o n . F o r instance, p53 e x p r e s s i o n was m o r e o f t e n s e e n in t h e m o d e r a t e l y a n d p o o r l y d i f f e r e n t i a t e d subtypes (including one sample of the undifferentiated subtype) than in t h e well d i f f e r e n t i a t e d H C C s ( P < .05, F i s h e r ' s e x a c t p r o b a b i l i t y test). W i t h r e g a r d to c h r o n i c liver diseases, o n e s a m p l e o f c h r o n i c active h e p a t i t i s s h o w e d p53 exp r e s s i o n with a s c a t t e r e d p a t t e r n (Fig 1). I n o n e positive s p e c i m e n o f well d i f f e r e n t i a t e d H C C , a similar s c a t t e r e d p a t t e r n also was o b s e r v e d . A s s o c i a t i o n o f p53 s t a i n i n g with c l i n i c o p a t h o l o g i cal p a r a m e t e r s is p r e s e n t e d in T a b l e 2. T h e r e was n o statistically significant c o r r e l a t i o n b e t w e e n p53 expression a n d several p a r a m e t e r s , such as t h e age a n d sex o f t h e p a t i e n t , t u m o r size, b a c k g r o u n d liver disease, h e p a t i t i s virus i n f e c t i o n , o r s e r u m titer o f PIVKA II. In contrast, t h e p a t i e n t s with a h i g h titer o f A F P ( m o r e t h a n 4,000 n g / m L ) s h o w e d significant p53 e x p r e s s i o n ( P < .04, c h i - s q u a r e d test).

HUMAN PATHOLOGY

Volume 26, No. 3 (March 1995)

/

° ~

~n

_

B

FIGURE i . Detection of p53 expression using DO7 antibody. (A) Poorly differentiated HCC showing strong nuclear staining. No immunostaining is seen in a mitotic tumor cell (arrow). (B) Chronic active hepatitis. Scattered nuclear staining can be seen (arrows). (Immunoperoxidase technique, hematoxylin counterstain; original magnifications ×200.)

S e m i q u a n t i t a t i v e E v a l u a t i o n o f p53-Positive Tumor Cells

Distribution of p53-positive tumor cells assessed by the three anti-p53 antibodies (DO7, PAbl801, and CM1) is summarized in Table 3. The percentage of p53-positive tumor cells ranged from 0% to 76.6%, depending on the histological grade of differentiation; the positivity was higher in poorly and undifferentiated HCCs. As shown in Table 3, the staining pattern was basically similar with

all three anti-p53 antibodies used (Fig 2), and 11 of 15 samples were positively stained for all antibodies. One specimen that showed positive staining for only one antibody was judged to be negative. Cell Proliferative A c t i v i t y I n d i c e s

We used two indices for the examination of cell proliferative activities, including mitotic index and MIB1 immunostaining. In HCCs the mitotic index ranged

~~

~

FIGURE 2. Detection of p53 expression using three anti-p53 antibodies on serial sections of HCC: (A) DO7, (B) PAb1801, and (C) CM1. Similar staining paffern is seen with all three antibodies. (Immunoperoxidase technique, hematoxylin counterstain; original magnifications x 100.)

328

p53 EXPRESSION IN HCC (Nagao et al)

Association of p53 Expression With Histological Grade (n : 92)

Semiquantitative Evaluation of p53 Expression of HCC Using Three Anti-p53 Antibodies (n = 50)

TABLE 1.

Histological Grade

No. of Samples

p53Positive Samples (%)

15

1(7)

26

9(35) ] /

TABLE 3.

p53Negative Samples (%)

Well differentiated HCC Moderately differentiated HCC Poorly differentiated HCC Undifferentiated HCC Total

8 1 50

15 (30)

35 (70)

Chronic liver diseases Normal liver

37 5

1 (3)

36 (97)

0 (0)

].14(93)

Histological Subtype of HCC

1

17(65)]/*

5 (100)

Abbreviations: HCC, hepatocellular carcinoma. *Fisher's exact probability test (P < .05).

from 0 to 18/5 HPFs and was intimately associated with the histological grade of differentiation (F = 15.373; P < .0001, one-way ANOVA) as shown in Fig 3. In fact, mitotic figures could rarely be found in well differentiated HCCs. In both frozen and paraffin sections of normal tonsil tissues, MIB-1 was similarly visualized with a strong nuclear pattern in the germinal center, a site of active

TABLE 2, Association of p53 Expression With Clinicopathological Parameters No. of Samples Age (yrs) (Mean _+ SD) Sex (%) Male Female Tumor size (%) _--<20mm 21-49 mm 50 mm =< Background liver diseases

50

p53Positive Samples

p53Negative Samples

59.4 ± 9.1 60.9 ± 7.5

P NS*

42 8

13 (31) 2 (25)

29 (69) 6 (75)

NS t

17 24 9

5 (29) 6 (25) 4 (44)

12 (71) 18 (75) 5 (56)

NS++

17 33

3 (18) 12 (36)

14 (82) 21 (64)

NS t

8 42

2 (25) 13 (31)

6 (75) 29 (69)

NS t

43 7

14 (33) 1 (14)

29 (67) 6 (86)

NSJ-

14 29 7

3 (21) 7 (24) 5 (71)

11 (79) 22 (76) 2 (29)

<.04++

11 33

5 (45) 9 (27)

6 (55) 24 (73)

NSt

Total No. of p53Positive Samples

1 1 1

3 3 6

1 1 1

5 3 3

1

8 7 9

1 1 1

3 3 3

4 4 4

1 1 1

1 1 1

Abbreviation: HCC, hepatocellular carcinoma.

lymphocyte proliferation (Fig 4). The availability of MIB-1 immunostaining for suitably fixed paraffin sections thus was confirmed. MIB-l-stained hepatocytes were scattered t h r o u g h o u t the tissue in chronic liver diseases, whereas in HCCs the MIB-l-stained tumor cells tended to be distributed around sinusoidal spaces forming cell clusters (Fig 5). Mitotic figures also were stained strongly (Fig 4). The MIB-1 LI ranged from 0% to 84.7%. T h e r e was a good correlation between mitotic index and the MIB-1 LI in HCCs (y = 3.875x + 3.342; r = 0.88; P < .0001; n = 50) (Fig 3). The MIB-1 LI was intimately associated with the histological grade of liver disease ( F = 35.784, P < .0001; one-wayANOVA) (Fig 3); MIB-1 LI expression in well differentiated HCC was the lowest among the HCC group. Yet, the MIB-1 LI of well differentiated HCC was significantly higher than that of liver cirrhosis (LC) (P < .05, Student's t-test).

DISCUSSION

p53 Expression

PWKA II (%) _-<0.06 A U / m L 0.06 A U / m L <

+++

Hepatocellular carcinomas with positive p53 staining had a significantly higher MIB-1 LI than those without (P < .003; the Mann-Whitney Utest). Furthermore, the positivity also was significantly related to mitotic index (P < .01; the Mann-Whitney Utest) (Table 4).

AFP (%) <20 n g / m L 204000 n g / m L 4000 n g / m L <

++

Comparison Between p53 Expression and Cell Proliferative Activity Indices

(%)

Noncirrhotic Cirrhotic HBsAg (%) (+) (-) HCVAb (%) (+) (-)

Well differentiated DO7 PAb1801 CM1 Moderately differentiated DO7 PAbl801 CM1 Poorly differentiated DO7 PAb1801 CM1 Undifferentiated DO7 PAbl801 CM1

+

Abbreviations: HBsAb, hepatitis B surface antigen; HCVAb, hepatitis C antibody; AFP, alpha-fetoprotein; NS, not significant. * Student's t test. t Fisher's exact probability test. .+ Chi-squared test.

329

Several studies have shown that the prevalence of p53 mutations in HCCs was strikingly related to geographic factors, as demonstrated by a close association with dietary aflatoxin B1.12'13'15 In the current study we showed that aberrant p53 expression could be detected immunohistochemically in 30% of Japanese patients with HCC in an aflatoxin low exposure area. Laurent-

HUMAN PATHOLOGY

Volume 26, No. 3 (March 1995)

20i

F

I

1816" 14" 12..

10-

6 4 ooo

2

;;'::.

•

o

i

i

W-HCC n= 15

i

M-HCC n=26

A

i

P-HCC n=8

U-HCC n= 1

90807060~50 40-

_=

30-

°to 10

E

0

90 ¸

I

'

z~

'

6

'

8

'

10

'

12

'

14

'

16

'

18

' 2(]

Mitotic Index i

I

i

80 70

~60'v 5040.

t

30. 2010-

°

0

M e a n (%) ± SD

CH

LC

n=14

n=23

1 . 6 ± 1.1

2.2±1.4

W-HCC

M-HCC

P-HCC

U-HCC

n=15

n=26

n=8

n=l

12.1±t1.1

28.4±16.6

--

3.4±2.1

FIGURE 3. Cell proliferative activity indices in liver diseases. (A) Association of mitotic index with histological g r a d e of HCC. *, o n e - w a y ANOVA (F = 15.373; P < .0001). (B) Correlation b e t w e e n mitotic index a n d MIB-1 LI (y = 3.875x + 3.342; r = 0.88; P < .0001; n = 50). (C) Association of MIB-1 LI with histological g r a d e of liver disease. *, o n e - w a y ANOVA (F = 35.784; P < .0001); **, Student's t-test (P < .05). CH, chronic hepatitis; LC, liver cirrhosis; W-HCC, well differentiated HCC; M-HCC, m o d e r a t e l y differentiated HCC; P-HCC, poorly differentiated HCC; U-HCC, undifferentiated HCC.

Puig et a125 observed p53 expression in 16% of white patients with HCC using immunohistochemical techniques. This discrepancy could be accounted for by differences in criteria regarding the classification of positive and negative cases. Alterations of the p53 gene may

330

not always lead to an accumulation of the p53 protein and thus cannot be detected by immunohistochemistry. 26 Nevertheless, our results were in general accord with those obtained by molecular techniques. 14'16 In our observations the prevalence of p53 expression was significantly higher in HCCs with histologically lower differentiation. Oda et a116 proposed a genetic model of multistage hepatocarcinogenesis, hypothesizing that alterations of the p53 gene occur at a relatively late stage of tumor progression and may not be involved in carcinogenesis in HCC.16 In our study p53 expression was seen in HCCs with a somewhat lower prevalence than other h u m a n cancers. 7'9'1°'11 This fact suggests that p53 expression is a later event taking place during tumor progression in HCCs. T h e r e also may be a possibility that some of the HCC cases do not involve p53 gene alterations during tumor progression. Although many investigators consider that hepatitis B virus and hepatitis C virus are certainly involved in hepatocarcinogenesis, the possible mechanisms of cellular transformation induced by these viruses are still unknown. Feitelson e t a127 suggest the existence of HB × antigen that is capable of binding to p53 protein and, through this association, inducing the development of HCC. In our study, except for the serum titer of AFP, p53 expression was not significantly correlated with clinicopathological parameters, including hepatitis virus infection. The reason for the association between the high titer of AFP secretion of tumor cells and p53 expression remains unknown. In a tumor positive for p53 immunostaining the intensity was quite variable from cell to cell, possibly reflecting variation in the p53 protein level during the cell cycle, as suggested in wild type p53. 3 The negligible immunoreactivity for p53 in mitotic cells may correspond to a p r o m i n e n t reduction of the p53 level at the M phase. The percentage of p53-positive tumor cells was higher in poorly and undifferentiated HCCs. It has been demonstrated that mutation sites of the p53 gene in HCCs differ according to the histological grade of differentiation. 16 Such a difference also may be related to variabilities in the p53 protein level and cell kinetics. 4,28 Unexpectedly, one sample of chronic liver disease showed p53 expression when assessed by three anti-p53 antibodies that recognize both wild and mutant type p53 proteins. One possible explanation for this unusual finding may be reduced p53 protein breakdown, resulting in an accumulation of the wild type p53 protein. Another possibility is that other unknown gene products somehow modulate the p53 gene to enhance p53 protein synthesis to a level that can be detected by immunohistochemistry. The three anti-p53 antibodies (DO7, PAbl801, and CM1) used in this study, which can be used to demonstrate p53 expression on paraffin sections, recognize different epitopes, 5'2z'2~'29 accounting for some differences in the results obtained by the individual antibodies. The epitope structure also may be modified by the fact that mutant type p53 forms a complex with h s c 7 0 4

p53 EXPRESSION IN HCC (Nagao et al)

FIGURE 4. Immunostaining for MIB-1. (A) Frozen section of normal tonsil tissue. (B) Paraformaldehyde-fixed, paraffin-embedded section of normal tonsil tissue, (C) Paraformaldehyde-fixed, paraffin-embedded section of HCC, Note strong chromosomal staining of mitotic cells (arrows), (Hematoxylin counterstain; original magnifications ×200.)

and some other proteins 3 or by point mutation of the p53 gene in individual cases.

heating. We d e m o n s t r a t e d that MIB-1 also could be used for heating processed paraffin sections without using the microwave oven. Several reports have assessed the proliferative activity of hepatocytes using PCNA or Ki-67 immunostaining in various liver diseases. 31'32However, it has now b e c o m e clear that PCNA detected by

Cell P r o l i f e r a t i v e A c t i v i t y i n d i c e s

S h i e t al 3° first described antigen retrieval for immunohistochemical staining using microwave oven

FIGURE 5. Comparison between chronic liver diseases and HCC in MIB-1 immunostaining, (A) Liver cirrhosis. Positive cells are scattered, (B) Hepatocellular carcinoma. Positive cells are distributed around sinusoidal spaces, (Hematoxylin counterstain; original magnifications × 100.)

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Volume 26, No, 3 (March 1995)

Association of p53 Expression With Cell Proliferative Activity Indices of HCC (n = 50)

TABLE 4.

MIB-1 LI (%) (mean ± SEM) Mitotic index (mean ± SEM)

p53-Positive Samples (n = 15)

p53-Negative Samples (n = 35)

p*

23.3 ± 6.0

9.4 ± 1.8

<.003

4.7 ± 1.2

1.8 ± 0.5

<.01

Abbreviations: HCC, hepatocellular carcinoma; SEM, standard error of the mean. * Mann-Whitney U test.

PC10 antibody is expressed in noncycling cells and that the reactivity of PCNA is greatly modified by fixation procedures. In contrast, the immunoreactivity of MIB1 is not so sensitive to fixation modification and is not expressed in noncycling cells. 18 Significant inverse correlations between high numbers of Ki-67-positive cells and survival rate have been shown in many tumors. 33'34According to previous data as well as ours, MIB-1 immunostaining seems to satisfactorily assess the malignant potential of HCCs. T h e r e were some differences in MIB-1 immunostaining pattern between chronic liver diseases and HCCs. The MIB-l-stained hepatocytes were scattered t h r o u g h o u t the tissue in chronic liver diseases, whereas in HCCs the MIB-l-stained tumor cells tended to be clustered. Because there was a significant difference in terms of MIB-1 LI between well differentiated HCC and LC, this index could be useful in distinguishing well differentiated HCC from LC..

Association Between p53 Expression and Cell Proliferative Activity Indices When the wild type p53 gene is introduced into transformed cells they are arrested in the G1 phase of the cell cycle. ~5 In contrast, mutant forms of p53 can immortalize cells in c u l t u r e Y Several reports have suggested that p53 expression is associated with increased cell proliferation in tumors arising from the breast 7'8 and b l a d d e r F According to our observation p53 expression was associated with high cell proliferative activity. Unfortunately, we had no appropriate benign test controls, such as actively regenerating liver tissue with a high mitotic index. The issue regarding whether this association is exclusive to malignant hepatic tissue thus remains to be settled in future studies. Because wild type p53 protein is capable of suppressing cell proliferat i o n , - t h e current results suggest that mutant type p53 interferes with the normal cell cycle to enhance the proliferative activity of HCC cells. In conclusion, p53 expression, which may largely result from accumulation of mutant type p53 protein, could be a sign of high malignant potential as far as HCCs are concerned. REFERENCES 1. Isobe M, Emanuel BS, Givol D, et al: Localization of gene for human p53 tumor antigen to band 17p13. Nature 320:84-85, 1986

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