- Email: [email protected]
Early stage human colorectal cancer: prognostic value of nm23-H1 protein overexpression
CAMCEW (‘ancer
l,ettersI I I
( 1997) I-S
Early stage human colorectal cancer: prognostic value of nm23-H 1 protein overexpression
Kcceived
30 August
IYYh: accepted
IO September
lYY6
Abstract Nm23 gene codifies for a nucleoside diphosphate kinase allowing the intracellular transduction of the signals. In colorectal cancer nm2? protein expression seems related to the progression of the disease. By immunohistochemistry we have studied the intracytoplasmatic nm23 HI protein expression in 20 patients affected by colorectal cancer at initial stage. In 12 cases it resulted elevated and in four the disease recurred. The overexpression was not correlated with other prognostic factors. Nm23 t-1l-positive patients affected by colorectal cancer at initial stage could be considered at risk for disease recurrence and included in a more frequent follow-up protocol. 0 1907 Elscvier Science Ireland Ltd. All rights reserved Kc~~o~r/.v run23 gene: Colorcctal cancer; Recurrence; Prognostic factors _ ..-- _... _.-___---_____
I. Introduction Nm23 is a murine gene whose expression is associated with tumour metastatic potential. It was first identified by Steeg et al. by screening cDNA libraries from variants of murine melanoma cell lines in four different metastatic model systems with an inverse relationship between metastatic potential and nm23 RNA and/or protein levels [ 11. Therefore, nm23 has been proposed as a metastasis gene. Recently, two __--.. * Corresponding author. Tel./f’ax: ~mail: :c:~~cchione(~caspur.ir
+3Y 6 4441364:
G 1007 Elsevier 0~c~4-:ix.15!97is17.(x) P/l so?~tI-:x.~5(96)0447? 7_
Science lrcland
human nm23 CD&A clones, nm23 HI and H2, corresponding, respectively, to Nucleoside Diphosphate Kinase (NDPK) A and B, have been described [2,3 ]. They have an independent regulation of cellular proliferation which might play a role in the metastatic process of different tumours 131. Lacombe et al. reported an increase of NDPKinase expression and activity in malignant turnours, as compared with normal tissue 141.Several studies have reported the role of nm23 in breast, pancreas, lung, prostate and colon cancer and the relationship between its expression and the progression of disease [S--7]. In breast carcinoma reduced nm23-Hl expression has been associated with development of lymph node metastasis, reduced
Ltd. All rights reserved
M. Indinnimeo et al. I Cancer Letters 111 (1997) l-5
2 Table 1
Prognostic factors in colorectal cancer: our casuistry Case
T
ii
M
G
Diameter
% circumf.
Lymphargitis
1 2 3
2 2 2
0 0 0
0 0 0
1 0 1
>5 >.5 <5
>50 >50 <50
+ -
4 5 6 7
2 I 2 2
0 0 0 0
0 0 0 0
1 0 I 1
4 <5 >5 >5
50
+ -
8 9 10 11
I 2 2 2
0 0 0 0
0 0 0 0
1 1 2 1
>5 <5 >5 <5
>50 >50 >50
-
12 13 14 15 16 17 1x 19 20
2 1 2 I 2 3 2 7 2
0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0
1 1 1 1 I 2 I 2 1
cc5 15 15 c5 <5 <5 >S 4 <5
<50 2-50 <50 >50 <50 >50 >50 >50 <50
+ -
D/ocircumf., percentage involvement of the circumference.
disease-free survival and overall survival [5]. Genetic alterations as mutations in the coding sequence of nm23 Hl or as allelic deletions were detected in clinically advanced colon cancer, suggesting that nm23 gene might play a role in the development of metastasis in colon cancer [8]. In this preliminary study we have shown a casuistry of 20 patients with initial stage of disease (Tl-T2 NOMO). Immunohistochemistry evaluation was applied to observe the abnormal cytoplasmatic accumulation of NDPK nm23 Hl protein, its possible role of metastatic function and its correlation with the parietal invasion, the tumour’s diameter, the presence of neoplastic lymphangitis and the percentage involvement of the intestinal circumference, after a mean follow-up of 7 years.
2. Materials and methods 2.1. Clinical samples From January 1987 to March 1991 we evaluated 20 patients with initial stage colorectal cancer submitted to radical surgery. Nine of them were females (45%)
and 11 males (550/o),mean age 61 years (range 39-84 years). In all patients histological examination demonstrated the presence of initial stage adenocarcinoma (Tl-T2 NOMO), localized in one case in the high rectum, in four cases in the middle rectum, in seven patients in the low rectum, in eight at the level of the sigmoid colon, and in one in the descending colon. In these patients a postoperative staging according to UICC TNM classification resulted in 17 cases T2 and in three cases Tl. In the same patients we evaluated the percentage involvement of the circumference, the tumour’s diameter, the presence or absence of neoplastic lymphangitis and the differentiation degree (G) (Table 1). 2.2. Immunohistochemistry As adjunctive prognostic factor we evaluated by means of immunohistochemistry the cytoplasmatic expression of the nm23 HI tumour suppressor gene. The anti nm23-NDPK (37.6) protein is a murine monoclonal antibody purified from human erythrocytes and purchased from Novocastra diagnostic. Step sections, cut at 5 pm, were dewaxed in xylene. hydrated through graded ethanols and washed in PBS (pH 7.4). Endogenous peroxidase activities were quenched in 0.3% (v/v) hydrogen peroxidase in absolute methanol for 15 min. Immunoperoxidase staining was performed using a streptavidin biotin system kit for primary mouse antibodies (Zymed, San Francisco. CA), according to the manufacturer’s specifications. The primary monoclonal antibody was used at the dilution of 1: 125 and incubated for 30 min at room temperature. In the experiment a section of breast carcinoma with high nm23 expression was included as a positive control. Expression of nm23 Hl protein was primarily cytoplasmatic and was scored for distribution and evaluation of the percentage of stained cancer cells and staining intensity. The degree of staining intensity was evaluated by counting DAB, staining tumor cells at a minimum 200 cells in five microscopic fields at 400 x magnification, and was considered positive when at least >lO% of the tumor cells were immunostained. All the patients were followed up for a mean of 7 years (range 5-9 years) with evaluation of the tumour markers every 4 months, hepatic echography and thorax X-ray every 6 months and CT total body every year.
4. Discussion
Table 2
Nm23 protein expression correlated with other prognostic factors Case
nm2.3 7
N
bl
Diameter
“i circumf.
ci
Scurc: t. overexpression -50%: -. negative. Cc cirwmi.. percentage involvement of the circumference.
3. Resuits The cytoplasmatic expression of nm23 NDPK resulted positive in 12 out of the 20 studied patients (60%). In five cases (25%). three T2NOMO and two TINOMO, we observed a marking intensity greater than 50%; in seven cases (35%), six T2NOMO and one TlNOMO, it was less than SO%; in eight cases (40%). all T2NOM0, nm23 resulted negative. No correlation was observed either with the diameter or with the involvement percentage of the circumference or with the differentiation degree of the neoplasia (Table 2). Only in four patients with overexpression of nm23 protein greater than 50% we observed the disease recurrence: in two cases after 3 years, in one after 1 year and in one after 11 months; at postoperative stadiation they resulted in three cases T2NOMO and in one case TlNOMO. The correlation with the other prognostic factors is summarized in Table 3. In one patient the disease recurrence was local, in one patient hepatic and in the others spread.
Nm23 gene is considered an antimetastatic tumour suppressor gene, codifying for a protein with nucleoside diphosphate kinase activity, enzyme which allows the intracellular transduction of the signals [9]. A decrease in the intracytoplasmatic expression of NDPK has been observed in the hepatic carcinoma with metastatic potentiality [lo] .Other authors report that some solid tumours such as hepatocarcinoma, malignant melanoma, gastric cancer, cancer of the thyroid and pancreas, are not characterized by an inverse relationship between nm23 protein expression and the metastatic potential 141.On the other hand no correlation was identified between the nm23 protein expression and the metastatic activity of the other tumours (7). It is probable that many other mechanisms such as a transcriptional or postranscriptional check regulate the function of this protein, which probably is not the only one to contribute to the metastatic process. The cytoplasmatic accumulation of this protein might be due to a molecular alteration or to a deficit of the mechanisms of nuclear reentry. Cohn et al. suggested a possible antimetastatic role, observing an allelic deletion of nm23 Hl in the colorectal cancer with distant metastases [I 11. Ham et al., on the other hand, did not find any correlation between the expression of nm23 and the metastatic potential [ 121. Royds et al. analyzed, using a purified polyclonal antibody (Ab 1l), 46 patients submitted to surgical resection for cancer of the colorectum, followed up for a mean period of 24 months, and correlated the presence of nm23 protein with survival, age, sex, vascular invasion and Dukes stage. They demonstrated the expression of nm23 protein both in the normal epithelium and in the neoplastic one, where it was anyway more represented. In the normal epithelium nm23 was found mainly at the border with the proliferative zones at the basis of the crypts. By correlation of the nm23 protein with survival, the authors observed that only ten patients with overexpression of the protein were alive after 5 years, and 36 were dead (29 for disseminated illness and seven for causes not depending on the tumour). The risk of death for each positivity increment of nm23 showed an increase of 0.573. No significant association was demonstrated between the nm23 overexpression and a more advanced Dukes’ stage (P = 0.404). Furthermore, the study
4
M. Lndinnimeo et al. / Cancer Lxtters I II (1997) I-5
Table 3 Nm23 protein overexpression and other prognostic factors correlated with recurrence of disease Case
nm23 protein
T
N
M
Diameter
% circumf.
G
Recurrence
1 2 3 4 5 6 I 8 9 10 11 12
+ + ++ ++ +t + f f +t + +T +
2 2 2 2 1 2 2 I 1 2 2 2
0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0
>5 >5 <5 <5 -3 >5 >5 >5 <5 <5 >5 <5
>SO% >50% <50% (50% <50% >50% <50% >50% <50% >50% >50% <5070
1 0 I 1 0 1 1 1 1 1 2 1
Local Spread Hepatic Spread -
Score: +, overexpression <5OW; + +, overexpression >50%; -, negative. ‘7~circumf., percentage involvement of the circumference.
did not show a correlation among nm23 and vascular invasion, lymphnodal involvement, age and sex [13]. Ham et al. compared the nm23 expression with the malignancy degree of the colorectal lesions, analyzing normal mucosa, adenomas, primary non-metastatic and metastatic tumours. In the metastatic tumours the protein is expressed at a very high level, but without difference between high and low metastatic potential neoplasias [12]. Yamaguchi et al. studied 36 colorectal cancers and the normal adjacent mucosa. Lymph node metastases were present in 23 patients (63.9%) and hepatic metastases in nine patients (25%). In all examined cases nm23 Hl protein showed higher expression in the neoplastic tissue compared to the normal mucosa, where it was anyway present. The authors noticed that the increase of nm23 Hl protein did not correlate in a statistically significant way with other prognostic factors such as histological type of the tumor, lymphatic invasion, venous invasion or lymph node metastasis. The expression of nm23 Hl protein resulted significantly lower in the tumours with hepatic metastases compared to those without metastases (P < 0.05) [ 141. Lacombe and Sastre-Garau suggested that the overexpression of nm23 H 1 gene might be correlated to the proliferation of malignant cells [ 151. Keim recently demonstrated that peripheral lymphocytes stimulated with phytohemoagglutinin show high levels of expression of nm23 Hl protein, probably according to the percentage of S
phase cells [ 161. In the patients affected by initial stage colorectal cancer and considered in this study cytoplasmatic nm23 Hl protein resulted expressed in 12 cases (60%). Nm23 Hl protein was expressed in the tumours with invasion of the mucosa and submucosa tunica, in the neoplasias with diameter <5 cm and with involvement of the circumference <50%. Moreover, it resulted positive in ten cases, which had no neoplastic lymphangitis, and in ten patients with well differentiated tumour (GO-G]). Therefore, this proteic mutation appeared in a very initial stage. In four patients who showed an overexpression of the nm23 Hl protein, we detected a local or distant recurrence (33.3%). In one case there were two factors that we consider unfavourable (tumour diameter >5 cm, circumference involvement >50%). In the other three patients all anatomopathologic and clinical factors were prognostically favourable. Though the experience is modest as to the number of examined patients, in our opinion the research of the mutation of nm23 Hl protein in initial stage is justified. If the results we have achieved are confirmed, we will be able to consider the initial stage nm23 HI protein-positive patients at risk for disease recurrence and, therefore, include them in a more intensive follow-up protocol or even in pre- and postoperative radio-chemotherapeutic schemes. In our experience in three of the four cases that showed neoplastic recurrence, the disease was not resectable at the time of diagnosis because of
the less frequent follow-up by which these patients were observed, not showing negative prognostic factors. References [ t 1 Steeg, P.S.. Bevilacqua, G.. Kopper, L., Thorgeirsson, U.P.. Taimadge. J.B., Liotta, L.A. and Sobel, M.E. (1988) Evidence for a novel gene associated with low tumour metastatic potentral. J. Natl. Cancer Inst., 80, 200-204. 121 Rosengard, A.. Krutzsch, H., Sheam. A., Bigga, J., Barker, E. and Margulies. I. (1989) Reduced nm23/AWD protein in human metastasis and aberrant Drosophilrr development. Nature, 342, 177-180. /?I Staht. J.. Leone, A.. Rosengard, A., Porter. L., King. C. and Steeg, P. (1991) Identification of a second human nm23 gene, om23-J-J?. Cancer Res.. 5 I, 445-449. (41 Lacombe. M.L.. Sastre-Garau, X.. Lascu. I., Wallet, V., Thiery, J.P. and Veron. M. (1991) Overexpression of nucleoside diphosphate kinase nm23 in human solid turnours, Eur. J. Cancer, 27. 1322137. [5/ Bevitacyua. G., Sobet, ME., Lance, A., Liotta, L.A. and Steeg, P.S. (1989) Association of low nm23 RNA levels in human primary breast carcinoma with lymphnode involvement and other histopathoiogical indicators of high metastatic potential, Cancer Res.. 49. 18% 190. 161 Hennersey. C. and May, B. (1991) Expression of the antimetastattc gene nm23 in human breast cancer: an association with good prognosis, .I. Natl. Cancer Inst., 83, 281-285. 17) Barnes. R., Shala, M. and Barker. E. (1991) Low nm23 protein expression in infiltrating ductal breast carcinoma correlates with reduced patient survival, Am. J. Pathol., 139, 245 250. [Xl Wang, L. and Patel. U. (1993) Mutation in the nm23 gene i\
associated with metastasis in colorectal cancer, Cancer Res., 53. 717-720. [91 Chen, H.C., Wang, L. and Banerjee, S. (1994) Isolation and characterization of the promoter region of human nm23-HI, a metastatic suppressor gene, Oncogene, 9, 2905-29 12. 1101 Campo. E. and Miquet, R. (1994) Prognostic significance of the loss of heterozigosity of nm23-Hl and pS3 genes in human colorectal carcinomas, Cancer, 73, 2913-2921. 1111 Cohn,K.H.,Wang,F.,Solomon,W.B.andSteeg,P.S.(1991) Association of nm23 allelic deletions with distant metastases in colorectal carcinoma, Lancet. 338, 722-723. 1121 Haut. M., Steeg, P.. Willson. J.K.W. and Markowitz, S. (1991) Induction of nm23 gene expression in human colonic neoplasm and equal expression in colon tumors of high and low metastatic potential, J. Natl. Cancer Inst., 83, 712-716. [I31 Royds, J.A., Cross. S.S., Silcocks, P.B.. Scholetied. J.H., Recs, R.C. and Stephenson, T.J. (1994) Nm23 antimetastatic gene product expression in colorectal carcinoma, J. Pathol., 72, 26 l-266. 1141 Yamaguchi, A.. Urano, T., Fushida, S., Furukawa, K., Nishimura, G., Yonemura, Y., Miyazaki, I., Nakagavara, G. and Shiku, H. (1993) Inverse association of nm23-HI expression by colorectal cancer with liver metastasis, Cancer, X8. 1020-t 024. [ 151 Heilat, N., Kein, D.R., Melhem, RF., Zhu, X.X., Eckerskom, C., Brodeur, G.R., Reynolds, C.P., Seeger, R.C., Lottspeich, F.. Strahler, J.R. and Hanash, S.M. (1991) High levels of p19/ nm23 protein in neuroblastoma are associated with advanced stage disease and with N-Myc gene amplification, J. Clin. Invest., 88, 34 I-345. 116) Keim, D., Heilat, N., Melhem, R., zhu. X.X., Lascu, l., Veron, M. and Strahler. J. (1992) Proliferation-related expression of p19/nm23 nucleoside diphosphate kinase, J. Ctin. Invest.. 89. 9 19-924.
l,ettersI I I
( 1997) I-S
Early stage human colorectal cancer: prognostic value of nm23-H 1 protein overexpression
Kcceived
30 August
IYYh: accepted
IO September
lYY6
Abstract Nm23 gene codifies for a nucleoside diphosphate kinase allowing the intracellular transduction of the signals. In colorectal cancer nm2? protein expression seems related to the progression of the disease. By immunohistochemistry we have studied the intracytoplasmatic nm23 HI protein expression in 20 patients affected by colorectal cancer at initial stage. In 12 cases it resulted elevated and in four the disease recurred. The overexpression was not correlated with other prognostic factors. Nm23 t-1l-positive patients affected by colorectal cancer at initial stage could be considered at risk for disease recurrence and included in a more frequent follow-up protocol. 0 1907 Elscvier Science Ireland Ltd. All rights reserved Kc~~o~r/.v run23 gene: Colorcctal cancer; Recurrence; Prognostic factors _ ..-- _... _.-___---_____
I. Introduction Nm23 is a murine gene whose expression is associated with tumour metastatic potential. It was first identified by Steeg et al. by screening cDNA libraries from variants of murine melanoma cell lines in four different metastatic model systems with an inverse relationship between metastatic potential and nm23 RNA and/or protein levels [ 11. Therefore, nm23 has been proposed as a metastasis gene. Recently, two __--.. * Corresponding author. Tel./f’ax: ~mail: :c:~~cchione(~caspur.ir
+3Y 6 4441364:
G 1007 Elsevier 0~c~4-:ix.15!97is17.(x) P/l so?~tI-:x.~5(96)0447? 7_
Science lrcland
human nm23 CD&A clones, nm23 HI and H2, corresponding, respectively, to Nucleoside Diphosphate Kinase (NDPK) A and B, have been described [2,3 ]. They have an independent regulation of cellular proliferation which might play a role in the metastatic process of different tumours 131. Lacombe et al. reported an increase of NDPKinase expression and activity in malignant turnours, as compared with normal tissue 141.Several studies have reported the role of nm23 in breast, pancreas, lung, prostate and colon cancer and the relationship between its expression and the progression of disease [S--7]. In breast carcinoma reduced nm23-Hl expression has been associated with development of lymph node metastasis, reduced
Ltd. All rights reserved
M. Indinnimeo et al. I Cancer Letters 111 (1997) l-5
2 Table 1
Prognostic factors in colorectal cancer: our casuistry Case
T
ii
M
G
Diameter
% circumf.
Lymphargitis
1 2 3
2 2 2
0 0 0
0 0 0
1 0 1
>5 >.5 <5
>50 >50 <50
+ -
4 5 6 7
2 I 2 2
0 0 0 0
0 0 0 0
1 0 I 1
4 <5 >5 >5
+ -
8 9 10 11
I 2 2 2
0 0 0 0
0 0 0 0
1 1 2 1
>5 <5 >5 <5
>50 >50 >50
-
12 13 14 15 16 17 1x 19 20
2 1 2 I 2 3 2 7 2
0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0
1 1 1 1 I 2 I 2 1
cc5 15 15 c5 <5 <5 >S 4 <5
<50 2-50 <50 >50 <50 >50 >50 >50 <50
+ -
D/ocircumf., percentage involvement of the circumference.
disease-free survival and overall survival [5]. Genetic alterations as mutations in the coding sequence of nm23 Hl or as allelic deletions were detected in clinically advanced colon cancer, suggesting that nm23 gene might play a role in the development of metastasis in colon cancer [8]. In this preliminary study we have shown a casuistry of 20 patients with initial stage of disease (Tl-T2 NOMO). Immunohistochemistry evaluation was applied to observe the abnormal cytoplasmatic accumulation of NDPK nm23 Hl protein, its possible role of metastatic function and its correlation with the parietal invasion, the tumour’s diameter, the presence of neoplastic lymphangitis and the percentage involvement of the intestinal circumference, after a mean follow-up of 7 years.
2. Materials and methods 2.1. Clinical samples From January 1987 to March 1991 we evaluated 20 patients with initial stage colorectal cancer submitted to radical surgery. Nine of them were females (45%)
and 11 males (550/o),mean age 61 years (range 39-84 years). In all patients histological examination demonstrated the presence of initial stage adenocarcinoma (Tl-T2 NOMO), localized in one case in the high rectum, in four cases in the middle rectum, in seven patients in the low rectum, in eight at the level of the sigmoid colon, and in one in the descending colon. In these patients a postoperative staging according to UICC TNM classification resulted in 17 cases T2 and in three cases Tl. In the same patients we evaluated the percentage involvement of the circumference, the tumour’s diameter, the presence or absence of neoplastic lymphangitis and the differentiation degree (G) (Table 1). 2.2. Immunohistochemistry As adjunctive prognostic factor we evaluated by means of immunohistochemistry the cytoplasmatic expression of the nm23 HI tumour suppressor gene. The anti nm23-NDPK (37.6) protein is a murine monoclonal antibody purified from human erythrocytes and purchased from Novocastra diagnostic. Step sections, cut at 5 pm, were dewaxed in xylene. hydrated through graded ethanols and washed in PBS (pH 7.4). Endogenous peroxidase activities were quenched in 0.3% (v/v) hydrogen peroxidase in absolute methanol for 15 min. Immunoperoxidase staining was performed using a streptavidin biotin system kit for primary mouse antibodies (Zymed, San Francisco. CA), according to the manufacturer’s specifications. The primary monoclonal antibody was used at the dilution of 1: 125 and incubated for 30 min at room temperature. In the experiment a section of breast carcinoma with high nm23 expression was included as a positive control. Expression of nm23 Hl protein was primarily cytoplasmatic and was scored for distribution and evaluation of the percentage of stained cancer cells and staining intensity. The degree of staining intensity was evaluated by counting DAB, staining tumor cells at a minimum 200 cells in five microscopic fields at 400 x magnification, and was considered positive when at least >lO% of the tumor cells were immunostained. All the patients were followed up for a mean of 7 years (range 5-9 years) with evaluation of the tumour markers every 4 months, hepatic echography and thorax X-ray every 6 months and CT total body every year.
4. Discussion
Table 2
Nm23 protein expression correlated with other prognostic factors Case
nm2.3 7
N
bl
Diameter
“i circumf.
ci
Scurc: t. overexpression -
3. Resuits The cytoplasmatic expression of nm23 NDPK resulted positive in 12 out of the 20 studied patients (60%). In five cases (25%). three T2NOMO and two TINOMO, we observed a marking intensity greater than 50%; in seven cases (35%), six T2NOMO and one TlNOMO, it was less than SO%; in eight cases (40%). all T2NOM0, nm23 resulted negative. No correlation was observed either with the diameter or with the involvement percentage of the circumference or with the differentiation degree of the neoplasia (Table 2). Only in four patients with overexpression of nm23 protein greater than 50% we observed the disease recurrence: in two cases after 3 years, in one after 1 year and in one after 11 months; at postoperative stadiation they resulted in three cases T2NOMO and in one case TlNOMO. The correlation with the other prognostic factors is summarized in Table 3. In one patient the disease recurrence was local, in one patient hepatic and in the others spread.
Nm23 gene is considered an antimetastatic tumour suppressor gene, codifying for a protein with nucleoside diphosphate kinase activity, enzyme which allows the intracellular transduction of the signals [9]. A decrease in the intracytoplasmatic expression of NDPK has been observed in the hepatic carcinoma with metastatic potentiality [lo] .Other authors report that some solid tumours such as hepatocarcinoma, malignant melanoma, gastric cancer, cancer of the thyroid and pancreas, are not characterized by an inverse relationship between nm23 protein expression and the metastatic potential 141.On the other hand no correlation was identified between the nm23 protein expression and the metastatic activity of the other tumours (7). It is probable that many other mechanisms such as a transcriptional or postranscriptional check regulate the function of this protein, which probably is not the only one to contribute to the metastatic process. The cytoplasmatic accumulation of this protein might be due to a molecular alteration or to a deficit of the mechanisms of nuclear reentry. Cohn et al. suggested a possible antimetastatic role, observing an allelic deletion of nm23 Hl in the colorectal cancer with distant metastases [I 11. Ham et al., on the other hand, did not find any correlation between the expression of nm23 and the metastatic potential [ 121. Royds et al. analyzed, using a purified polyclonal antibody (Ab 1l), 46 patients submitted to surgical resection for cancer of the colorectum, followed up for a mean period of 24 months, and correlated the presence of nm23 protein with survival, age, sex, vascular invasion and Dukes stage. They demonstrated the expression of nm23 protein both in the normal epithelium and in the neoplastic one, where it was anyway more represented. In the normal epithelium nm23 was found mainly at the border with the proliferative zones at the basis of the crypts. By correlation of the nm23 protein with survival, the authors observed that only ten patients with overexpression of the protein were alive after 5 years, and 36 were dead (29 for disseminated illness and seven for causes not depending on the tumour). The risk of death for each positivity increment of nm23 showed an increase of 0.573. No significant association was demonstrated between the nm23 overexpression and a more advanced Dukes’ stage (P = 0.404). Furthermore, the study
4
M. Lndinnimeo et al. / Cancer Lxtters I II (1997) I-5
Table 3 Nm23 protein overexpression and other prognostic factors correlated with recurrence of disease Case
nm23 protein
T
N
M
Diameter
% circumf.
G
Recurrence
1 2 3 4 5 6 I 8 9 10 11 12
+ + ++ ++ +t + f f +t + +T +
2 2 2 2 1 2 2 I 1 2 2 2
0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0
>5 >5 <5 <5 -3 >5 >5 >5 <5 <5 >5 <5
>SO% >50% <50% (50% <50% >50% <50% >50% <50% >50% >50% <5070
1 0 I 1 0 1 1 1 1 1 2 1
Local Spread Hepatic Spread -
Score: +, overexpression <5OW; + +, overexpression >50%; -, negative. ‘7~circumf., percentage involvement of the circumference.
did not show a correlation among nm23 and vascular invasion, lymphnodal involvement, age and sex [13]. Ham et al. compared the nm23 expression with the malignancy degree of the colorectal lesions, analyzing normal mucosa, adenomas, primary non-metastatic and metastatic tumours. In the metastatic tumours the protein is expressed at a very high level, but without difference between high and low metastatic potential neoplasias [12]. Yamaguchi et al. studied 36 colorectal cancers and the normal adjacent mucosa. Lymph node metastases were present in 23 patients (63.9%) and hepatic metastases in nine patients (25%). In all examined cases nm23 Hl protein showed higher expression in the neoplastic tissue compared to the normal mucosa, where it was anyway present. The authors noticed that the increase of nm23 Hl protein did not correlate in a statistically significant way with other prognostic factors such as histological type of the tumor, lymphatic invasion, venous invasion or lymph node metastasis. The expression of nm23 Hl protein resulted significantly lower in the tumours with hepatic metastases compared to those without metastases (P < 0.05) [ 141. Lacombe and Sastre-Garau suggested that the overexpression of nm23 H 1 gene might be correlated to the proliferation of malignant cells [ 151. Keim recently demonstrated that peripheral lymphocytes stimulated with phytohemoagglutinin show high levels of expression of nm23 Hl protein, probably according to the percentage of S
phase cells [ 161. In the patients affected by initial stage colorectal cancer and considered in this study cytoplasmatic nm23 Hl protein resulted expressed in 12 cases (60%). Nm23 Hl protein was expressed in the tumours with invasion of the mucosa and submucosa tunica, in the neoplasias with diameter <5 cm and with involvement of the circumference <50%. Moreover, it resulted positive in ten cases, which had no neoplastic lymphangitis, and in ten patients with well differentiated tumour (GO-G]). Therefore, this proteic mutation appeared in a very initial stage. In four patients who showed an overexpression of the nm23 Hl protein, we detected a local or distant recurrence (33.3%). In one case there were two factors that we consider unfavourable (tumour diameter >5 cm, circumference involvement >50%). In the other three patients all anatomopathologic and clinical factors were prognostically favourable. Though the experience is modest as to the number of examined patients, in our opinion the research of the mutation of nm23 Hl protein in initial stage is justified. If the results we have achieved are confirmed, we will be able to consider the initial stage nm23 HI protein-positive patients at risk for disease recurrence and, therefore, include them in a more intensive follow-up protocol or even in pre- and postoperative radio-chemotherapeutic schemes. In our experience in three of the four cases that showed neoplastic recurrence, the disease was not resectable at the time of diagnosis because of
the less frequent follow-up by which these patients were observed, not showing negative prognostic factors. References [ t 1 Steeg, P.S.. Bevilacqua, G.. Kopper, L., Thorgeirsson, U.P.. Taimadge. J.B., Liotta, L.A. and Sobel, M.E. (1988) Evidence for a novel gene associated with low tumour metastatic potentral. J. Natl. Cancer Inst., 80, 200-204. 121 Rosengard, A.. Krutzsch, H., Sheam. A., Bigga, J., Barker, E. and Margulies. I. (1989) Reduced nm23/AWD protein in human metastasis and aberrant Drosophilrr development. Nature, 342, 177-180. /?I Staht. J.. Leone, A.. Rosengard, A., Porter. L., King. C. and Steeg, P. (1991) Identification of a second human nm23 gene, om23-J-J?. Cancer Res.. 5 I, 445-449. (41 Lacombe. M.L.. Sastre-Garau, X.. Lascu. I., Wallet, V., Thiery, J.P. and Veron. M. (1991) Overexpression of nucleoside diphosphate kinase nm23 in human solid turnours, Eur. J. Cancer, 27. 1322137. [5/ Bevitacyua. G., Sobet, ME., Lance, A., Liotta, L.A. and Steeg, P.S. (1989) Association of low nm23 RNA levels in human primary breast carcinoma with lymphnode involvement and other histopathoiogical indicators of high metastatic potential, Cancer Res.. 49. 18% 190. 161 Hennersey. C. and May, B. (1991) Expression of the antimetastattc gene nm23 in human breast cancer: an association with good prognosis, .I. Natl. Cancer Inst., 83, 281-285. 17) Barnes. R., Shala, M. and Barker. E. (1991) Low nm23 protein expression in infiltrating ductal breast carcinoma correlates with reduced patient survival, Am. J. Pathol., 139, 245 250. [Xl Wang, L. and Patel. U. (1993) Mutation in the nm23 gene i\
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