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The Correlation of p53 and nm23-H1 Expression with Invasiveness and Metastasis in Esophageal Carcinoma L I U L i g a n g , P A N T i e c h e n g , LI J u n , Z H A N G N i , S O N G D i n g w e i , H U Min
Objective To study the relationship between expression of p53 and nm23-H1 and differentiation, invasiveness and metastasis in human esophageal carcinoma, and the correlation between expression of p53 and nm23-H1. Methods Expression of p53 and nm23-H1 in 50 patients with squamous cell carcinoma of esophagus was detected by using immunohistochemical S-P methods. Results 35 cases (70%) and 32 cases (64%) of esophageal squamous cell carcinoma were positive for nm23-H1 protein and p53 protein, respectively. The expression of nm23-H1 was related to lymphatic metastasis (P <0. 025), but not related to tumor differentiation, invasiveness, tumor location, tumor length, patient' s gender and age (P > 0.05). The lymphatic metastasis location positive group had a very lower expression of nm23-H1 and the negative rate was 70.8%, but the negative group had a higher expression and the positive rate was 65.4%. The expression of p53 was related to tumor differentiation and invasiveness ( P < 0.05 ), but not related to lymphatic metastasis, tumor location, tumor length, patient' s gender and age( P > 0.05 ). Amongthe three groups, the high differentiation group had the lowest expression of p53 and the positive rate was 29.2%, but the low differentiation group had the highest positive rate (71.4%). As for tumor invasiveness, the group of outer membrane of esophagus infiltrated had the highest p53 protein positive rate (56%), but in the group of mucous or submucous layer infiltrated p53 protein was not detectable. The low expression of nm23-H1 and the high expression of p53 were also correlated. The expression of nm23-H1 and p53 were both correlated with TNM stage of esophageal carcinoma ( P < 0.05 ). The better esophageal carcinomas differentiated, the lower nm23-Hl expressed and higher p53 expressed. Conclusion Low expression of nm23-H1 and high expression of p53 play an important role in the progression of squamous cell carcinoma of esophagus. Nm23-H1 might be a gene marker in the prophecy of patients' prognosis and benefit tumor treatment clinically. Key words esophageal carcinoma; p53 protein; nm23-H1 protein ecent researches have demonstrated that multiple
R genes changes result in tumor genesis and progression. In the complicated courses, many genes are involved, including activation of proto-oncogenes and inactivation of antioncogenes. In clinical tumor therapy, the major factor which influences patients' prognosis is the conditions of tumor invasiveness and metastasis, and tumor biological behavior is also a major element in determination of tumor therapy plan. Many tumor experts have always been working hard at the early diagnosis of tumor invasiveness and metastasis, in order to improve tumor prognosis and prolong patients' life as much as possible. Therefore, tumor markers which can provide information on the conditions of tumor invasiveness and metastasis have always been emphasized by many tumor experts and become a hot researching subject, among which are p53 and nm23. In clinical researches, it is demonstrated that nm23, of which nm23-H1 might play the most important role, has the function of inhibiting tumor metastasis. This function has also been demonstrated in many human malignant tumors ~2-51 . P53 (natural type) is also a kind of antioncogene. When it mutates, p53 will participate in tumor formation and progression. Esophageal carcinoma is a kind of common malignant tumors of the digestive system and many genes are also involved in its genesis and progression courses. In China, reports on the relationship between Thoracic and Cardiac Department, Tongji Hospital, Tongji Medical College, HuazhongUniversityof Scienceand Technology,Wuhan430030, China
nm23 gene and progression of esophageal carcinoma have still very fewer and their relationship still needs many more studies. This study was using immunohistochemical techniques to examine nm23-H1 and p53 protein expressed in tissues of esophageal carcinomas to analyze the correlation of nm23-H1 and p53 expression with invasiveness and metastasis of esophageal carcinoma.
Materials and m e t h o d s Materials
Sample Sources Paraffin-embedded tissues of esophageal carcinoma from 50 randomly selected patients who were treated surgically from 1996 to 1997 at thoracic and cardiac department of Tongji Hospital attached to Tongji Medical College of Huazhong University of Science and Technology. Before operation, they had not received radiotherapy and (or) chemotherapy. Among these patients, 38 patients were male and 12 female. They were aged from 40 to 69 years and mean age was 54 years. According to tumor position, these samples were categorized as follows: carcinoma at the upper part of esophagus 8 cases, carcinoma at the middle part of esophagus 26 cases, carcinoma at the lower part of esophagus 16 cases, including 5 cases at both of the middle and lower parts. They were all squamous cell carcinoma. And tumor length ranged from 2 cm to 8.5 cm, and the mean length was 5 . 2 cm. According to tumor gross appearance, 8 cases were fungating, 13 cases were ulcerative, 12 cases were stenosing and 17 cases were
The Chinese-GermanJournal of Clinical Oncology, December2002, Vol.1, No.4 medullary. Of 50 patients, there were 24 cases with paraesophageal lymphatic metastasis or paraaortic lymphatic metastasis. Reagents Mouse-anti-human monoclonal antibody against nin23-H1 protein, mouse-anti-human monoclonal antibody against p53 protein, S-P reagent kit (Zhongshan biological and technical company, Peking, China). Methods Immunohistochemical S-P techniques were applied. All the sections were routinely deparaffinized and rehydrated, then immersed in 3% H202 to block the endogenous enzymes, subsequently were digested by 0 . 4 % gastric protein enzyme, and the rest operations were performed according to the introduction of the reagent kit. The sections of breast carcinoma tissues known nm23-H1 protein positive were used as positive controls and normal mouse serum and PBS substituting the first antibody were used as negative controls. These sections were stained with DAB and counterstained with hematoxylin, and the results were examined under microscopy. Evaluation of results P53 protein was mainly expressed in the nucleus of tumor ceils and under microscopy it was stained like brown and yellow granules or masses. According to the percentage of the p53 protein positive cancer cells within the maximum cut-surface specimen of the tumor tissue, the percentage of the p53 protein positive cancer cells was categorized into four groups: negative (-), <25% (+), > 2 5 % to 7 5 % ( q - + ) , > 7 5 % to 100% (}}}). Nm23-H1 protein was mainly expressed in the cytoplasm of tumor cells and under microscopy it was stained like light brown to deep brown granules or masses. According to the same principles as p53 above, the percentage of the nm23-H1 protein positive cancer cells was categorized into four groups: negative ( - ) , < 25% (+), >25% to 75% ( + + ) , >75% to 100% (+++). Statistical analysis Data was analyzed by using the X2 test. A P value less than 0.05 was considered signifiTable 1
195 cant.
Results Among all the stained sections of esophageal carcinoma tissues, nm23-H1 protein expressed ( + - + + ) could be detected in 35 cases ( 7 0 % ) , and 15 cases ( 3 0 % ) were negative. As for expression of p53 protein, 32 cases ( 6 4 % ) could be detected ( + -}H-) and 18 cases were negative. Under microscopy nm23-H1 protein expression was shown that there were many light brown to deep brown granules or masses in the cytoplasm of carcinoma cells (Fig. 1 ). And p53 protein was expressed in the nucleus of carcinoma cells stained like brown and yellow granules or masses under microscopy (Fig. 2 ) . In order to analyze easily, The (++) or ( }+Jr) expression of nm23-H1 and p53 protein was categorized into the high expression group ( H E ) , and the ( - ) or ( + ) expression was categorized into the low expression group (LE).
The relationship between expression of p53 and nm23HI protein and differentiation of squamous esophageal carcinoma The expression of nm23-H1 protein was not related to the differentiation of squamous esophageal carcinomas ( P > 0.05, Table 1 ). But on the contrary, the expression of p53 was related ( P < 0 . 0 5 , Table 1 ). The worse squamous cell carcimomas differentiated, the higher p53 protein was expressed.
The relationship between expression of nm23-H1 and p53 and lymphatic metastasis in squamous cell carcinoma of esophagus The tissues of esophageal squamous cell carcinoma with lymphatic metastasis had lower expression of nm23-H1 protein apparently. The low expression of nm23H1 was associated with lymphatic metastasis closely ( P < 0.025, Table 2 ) , but expression of p53 was not ( P > 0.05, Table 2).
The relationship between expression of p53 and nm23-H1 and differentiation of squamous cell carcinoma of esophagus
Differentiation of esophageal carcinoma
LE
High 9 Moderate 7 Low 10 Total * X2 =3.226, P>0.05; A • =6.44, Table 2 Lymphatic metastasis
(37.5%) (58.3%) (71.4%) 26 P<0.05
Nm23-H1 * HE 15 (62.5%) 5 (41.7%) 4 (28.6%) 24
Total 24 12 14 50
LE 17 (70.8%) 7 (58.3%) 4 (71.4%) 28
P53 ~ HE 7 (29.2%) 5 (41.7%) 10 (28.6%) 22
The relationship between expression of nm23-H1 and p53 and lymphatic metastasis in squamous cell carcinoma of esophagus Nm23-H1 * P53 ~ LE HE Total LE HE
Negative 9 (34.6%) Positive 17(70.8%) Total 26 9 X2 =6.559, P<0.025; zxX2 =0. 101, P>0.05
17 (65.4%) 7 (29.2%) 24
26 24 50
14 (53.8%) 14 (58.3%) 28
12 (46.2%) l0 (41.7%) 22
Total 24 12 14 50
Total 26 24 50
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Table 3
The relationship between expression of nm23-H1 and p53 and depth of invasiveness in squamous cell carcinoma of esophagus
Depth of invasiveness
LE
Mucous or submucous layer Muscular layer Shallowlayer** Deep layer Outer membrane Total * X2 = 1. 332, P > 0.05 ; z~• mucous or submucous layer.
Nm23-H1 * HE
1(25 % ) 4(50% ) 6(46.2% ) 15(60% ) 26
Total
3 ( 75 % ) 4(50% ) 7(53.8% ) 10(40% ) 24
LE
4 8 13 25 50
4 ( 100% ) 7(87.5% ) 6(46.2% ) 11 (44%) 28
P53 A HE
Total
0 1( 12.5% ) 7(63.8% ) 14(56% ) 22
4 8 13 25 50
= 8. 168, P < 0.05 ; * * In order to analyze easily, the group of shallow muscular layer was included into the group of
Table 4 The correlation of expression of nm23-H1 and p53 with TNM stage of esophageal carcinoma (according to the international classification principle of esophageal carcinoma modified by UICC in 1987) Nm23-H1 P53 Stage TNM Number LE HE LE HE 0
Tis NO, M0
I
T1 NO, MO
Ha
3"2 NO, T1 N1, T2 N1, T3 NO, T3 N1, T4 any N MO T4 any N M1
llb III IV
MO MO MO MO MO
0 4 15
1(25%) 3(20%)
6 3 16 6
4(66.7% 2(66.7% 12(75% ) 4(66.7%
3(75% ) 12(70%) 2(33.3%) 1(33.3% ) 4(25% ) 2(33.3%)
4(100% ) 10(66.7%)
0 5(33.3%
3(50%) 2(66.7% 7(43.8% 2(33.3%
3(50% ) 1(33.3% 9(56.2% 4(66.7%
0
Note: Analyzing the groups of IIa, Ilb, III, 1V got the P value, P < 0.05
The relationship between expression of nm23-H1 and p53 and depth of invasiveness in squamous cell carcinoma of esophagus The expression of nm23-H1 protein was not related to the depth of invasiveness ( P > 0 . 0 5 , Table 3 ) , but the expression of p53 protein was related ( P < 0 . 0 5 , Table 3 ) . The deeper esophageal carcinoma infiltrated, the higher p53 expressed. The correlation of expression of nm23-H1 and p53 with
patients' age, gender, tumor location and tumor length The expressions of nm23-H1 and p53 were not correlated with patients' age, gender, tumor location and tumor length, respectively ( P > 0.05 ). The correlation of expression of nm23-H1 and p53 with TNM stage of esophageal carcinoma The expression of nm23-H1 and p53 was correlated with TNM stage of esophageal carcinoma ( P < 0.05 ). From stage 0 to 1W, the expression of nrn23-H1 had a higher negative rate (Table 4 ) , namely, the more advanced esophageal carcinomas were, the lower nm23-H1 expressed. P53 was also related, but different from nm23-H1, the more advanced esophageal carcinomas were, the higher p53 expressed (Table 4 ) . The relationship between p53 expression and nm23-H1 expression in esophageal carcinoma In this experiment, it was demonstrated that there was a correlation between p53 expression and nm23-H1 expression. From the Table 1, it was shown that there were 24 eases with nm23-H1 protein high expression and 26 eases low expression. As for p53 protein expression, 22 cases had high expression and 28 eases low expression. Under microscopy, the same sections with high expression of p53 and nrn23-H1 were very fewer,
but it was interesting that the sections with low expression of nm23-H1 and high expression of p53 at the same time were more, both of which had a close correlation P < 0.01, Table 5). Table 5
The relationship between p53 protein expression and nm23-H1 expression in esophageal carcinoma Nm23-H1 Factor Total HE LE P53
HE LE
Total
5 19 24
17 9 26
22 28 50
Xz =10.052, P < 0 . 0 1
Discussion The nm23 gene Ell was detected by Steeg and his colleagues in the year of 1988. From then on, many researches were carried out and demonstrated that level of nm23 mRNA in cancer cells was correlated with metastatic ability of cancer cells and nm23 gene had the function of metastasis inhibition ~2-51 . Level of its mRNA or gene products were highly associated with tumor metastasis. Lately, further studies demonstrated that nrn23 is also a kind of normal gene in human body. At present, it has already known that there arc six members in the gene family, namely nm23-H1 ( 1 9 8 8 - 1990), nm23-H2 ( 1 9 9 1 ) , nm23-H3 (1995), nm23-H4 ( 1 9 9 7 ) , nm23-H5 (1998) and nm23H6 (1999). Because nrn23-H1 has a close relationship with tumor metastasis, it has always been the focus of
The Chinese-GermanJournal of Clinical Oncology, December2002, Vol. 1, No. 4
Fig. 1 Positivestaining for nm23-H1 protein in esophageal squamous cell carcinoma ( original magnificationx 200) researching. As researches has shown, nm23-H1 gene can regulate several processes in ceils, such as cell message transmission, cell differentiation etc. by performing the functions of nucleoside diphosphate kinase ( N D P K ) , then further inhibit tumor metastasis. Therefore, normal expression of nm23-H1 is an important mechnism of tumor metastasis inhibition in human body. On the contrary, if nm23-H1 mutates, defects or takes place other changes, it might lose the function of inhibiting tumor metastasis. In many human malignant tumors, such as breast carcinomas I31 , gastric carcinomas I41 , colorectal carcinomas ESI etc. it has been demonstrated that low expression of nm23-H1 gene products is correlated with increased tumor metastatic potential. Therefore, many tumor experts believe that nm23-H1 mRNA or its gene products might be looked on as prognostic markers of cancer metastasis and might benefit tumor treatment clinically. From the results of this experiment, it could be learn that the positive rate of nm23-H1 protein expression in these esophageal carcinomas was 70%. And low expression of nm23-H1 was not related to tumor differentiation, tumor location, depth of invasiveness, tumor length, patients' gender and age ( P > 0.05 ) , but related to lymphatic metastasis, significantly. Those without lymphatic metastasis had higher nm23-H1 positive rate than the rest with lymphatic metastasis ( 6 5 . 4 % vs 2 9 . 2 % ) , and the difference between the two group was significant ( P < 0.025 ). The expression of nm23-H1 was also correlated with TNM stage of esophageal carcinoma ( P < O. 05 ) , the more advanced esophageal carcinomas were, the lower nm23-H1 ex-
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Fig. 2 Positioe staining for p53 protein in esophageal squamous cell carcinoma (original magnification x200)
pressed. Therefore, in this experiment it could be concluded that nm23-H1 gene negatively regulated the processes of metastasis in esophageal carcinoma, and this conclusion was in accordance with those some foreign tumor experts, such as Masaki E61, Sarbia ETIetc. , drew from their experiments. P53 gene is a kind of antioncogene, and is also an important regulating gene in cytorecycling. Its inactivation plays a n important role in tumor formation and its biological functions act as "molecular police" examining the damages of DNA and supervising the integrity of gene groups during G1 stage, If there is some gene damaged, it can make p53 overexpress to inhibit DNA duplication in order to provide enough time for damaged gene to be repaired. But if the damaged gene fails to be repaired, then p53 will induce the cell to develop programmatic death, so as to block the formation of carcinomatous cell colony. P53 gene might take place mutation-namely, becoming a mutant type and the mutant type can result in cytorecycling uncontrollable, even it will activate some genes, such as Rus gene, which would make growth of cells uncontrollable and increase tumor infiltrating ability, so the risk of tumor lymphatic metastasis is much increased. Proteins of p53 natural type have a short half-life time and are difficult to examine. On the contrary, proteins of p53 mutant type have a longer half-life time and have a higher stability in cells, because their configurations are changed. Thus they can be detected by using immunohistochemical methods. In addition, the mutation of p53 gene is in accordance with the expression of p53 protein in carcinomatous tissues Esj 9 In this experiment, it is demonstrated that expression
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of p53 was correlated with differentiation of squamous cell carcinoma and infiltrating depth ( P < 0.05 ). And the worse squamous cell carcinoma of esophagus differentiated and the deeper tumors infiltrated, the higher p53 protein expressed in tumor tissues. But in this experiment it could not be concluded that expression of p53 was related to lymphatic metastasis, for data collected did not support this point, and still needs further studies. But the expression of p53 was also related to TNM stage of esophageal carcinoma ( P < 0.05). Different from nrn23-Hl, the more advanced esophageal carcinomas were, the higher p53 expressed (Table 4). In addition, expression of p53 was also not associated with tumor location, tumor length, patients' gender and age ( P > 0.05 ) , which was the same with nm23H1. Therefore, from this study it could be concluded that p53 gene participates in the course of invasiveness and accelerates tumor progression in esophageal carcinoma. In addition, in this experiment it was also demonstrated that low expression of nm23-H1 was associated with high expression of p53 in the tissues of esophageal squamous cell carcinomas. The tissues with higher expression of p53 and lower expression of nrn23-H1 had a higher rate of lymphatic metastasis than other groups. On the other hand, the sections with lower expression of nm23-H1 and deeper depth of infiltration had higher expression of p53 than those with higher expression of nm23-H1. And the differences between the two groups were significant. Therefore, low expression of nm23-H1 and high expression of p53 might perform the synergic action in the processes of tumor invasiveness and metastasis in squamous cell carcinoma of esophagus. And both of the two factors might accelerate the progression of squamous cell carcinoma. Certainly, both of them might perform its own functions separately and use
different mechanisms to accelerate the progression of squamous cell carcinoma. At present, it still needs further investigation. But anyway, examining expression of p53 and nm23-H1 proteins will be beneficial to make the best therapy plan, in order to improve patients' prognosis and prolong their 5-year survival as much as possible, which has already been supported by many researches at home and abroad, including this experiment. References 1 Steeg PS, Bevi]acqua G, Kooper L, et al. Evidence for a novel gene associated with low tumor metastatic potential. J Natl Cancer [nst, 1988, 80: 200. 2 Leone A, Flatow U, King CR, e t a [ . Reduced tumor incidence, metastatic potential and cytokine responsiveness of nm23-transfectod melanoma cell. Cell, 1991,65: 25. 3 [rayama R, Sawal S, Takagi Y, et al. Positive relationship between expression of anti-metastatic factor ( nm23 gene product or nncleotide diphosphate kinase) and good prognosis in human breast cancer. J Nail Cancer Inst, 1991, 83: 1249. Miiller W, Schneidem A, Hommel G, et al. Expression of nm23 in gastric carcinoma: association with tumor progression and poor prognosis. Cancer, 1998, 83: 2481. Tannapfel A, Katalinic A, Kockerling F, et al. The prediction of lymph node metastases in colorectal cancer by expression of the nucleoside diphosphate kinase/nm23-Hl and histopathologieal variables. Am J Gastroenterol, 1997, 92: 1182. Masaki T, Takanori A, Yasunori M, et al. Expression of nm23-H1 gene product in esophageal squamous cell carcinoma and its association with vessel invasion and survival. BMC Cancer, 2001, 1 : 3. Sarbia M, Porschen R, Borchard F, et al. Incidence and prognostic significance of vascular and neural invasion in squamous cell carcinoma of the esophagus. Int Cancer, 1995, 61 : 333. Zhou Q, Yang GH. Some developments of p53 researches. Chin J Patho, 1995, 24: 267.
( Received 2002 - 06 - 12)