Reduced Expression of nm23 Protein is Related to Nodal Metastasis of Human Gastric Carcinoma

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The Seoul Journul of Mc~dic.ino V o l . 3 6 , No. 1 : Y - 1 4 , Murc.h l Y Y 5

Reduced Expression of nm23 Protein is Related to Nodal Metastasis of Human Gastric Carcinoma

Sang-Yong Song, and _Je G Chi1

= Abstract =

Reduced expression of nm23 gene or protein has been known to be related with nodal metastasis in a variety of malignant tumors of the breast, lung, liwr, prostate, ovary and stomach. To elucidate a possible prognostic factor, we studied 42 cases of gastric adenocarcinomas for the expression of nm23 protein using immunohistochemical methods and compared with the known prognostic parameters. The nm23 protein was intensely stained in the cytoplasm and/or the nucleus of carcinoma cells in 9 cases(21.4%). The nm23 protein expression of the non-metastatic group(46.7%) was higher than that of the nodal metastasis group(7.

4%). Perigastric lymph node metastases(p(0.001) were more frequently found in the nm23 protein negatiw group(75.8%) than in the nm23 positive group(22.2%). There was no significant correlation between nm23 protein expression and other para- meters such as patient age, sex, WHO grade, Lauren classification, depth of invasion, location of tumor and size. The results suggest that nm23 protein expr- ession plays a role in suppression of nodal metastasis in the gastric adenocarcinoma.

Key words : nn123, Nrrc~lcosi(ic~ (iip/lo.s/~ll(rtc' Kitllr.sc', Stotllrrc-11, Crr~lc.ot., Mtrr-

.sttr.si

INTRODUCTION

The nm23 gene has been reported to be a metastasls suppressor gene based on the experiment uslng murlne K-1735 melanoma cell lhnes w ~ t h low and h ~ g h metastatlc potentla1 (Steeg

et

^al.

1988a&b). The transfect~on of nm23 gene Into a h~ghly metastatlc murlne melanoma cell l ~ n e res- ulted In the reduction of metastatlc potentlal and primary tumor formation wlthout differences In In- trlnslc tumor cell growth (Leone

et

a / 1991) The

gene 1s located In the long arm of chromosome 17 (Varesco

et

a1 1992 Stephenson

et

^{a1 1993)}

and 1s known to be related wlth nucleoslde dtp- hosphate (NDP) klnase (Kimura

et

a / 1988231 990 Llotta & Steeg 1990 G~lles

et

a1 1991 Negat~ve correlations of nm23 expression wlth human cancers from the breast (Bevllacqua

et

al 1989 Bar- nes

et

a1 1991 Hlrayama

et

a1 1991 Royds

et

a1 1993 Yamashlta

et

a1 1993 Slmpson

et

a1 1993) ovary (Foulkes

et

a1 1993 Mandal

et

^{a1 1994)}

prostate ( K o n ~ s h ~

et

a1 1993 Brewster

et

a1 1994 lgawa

et

^a1 1994) pancreas(Nakamor1

et

^a1

1993) colon and rectum (Leone

et

a1 1991 Cohn

et

a1 1991 Myeroff

et

a1 1993 Yamaguch~

et

^{a /}

1993 Baflco

et

a1 1993) lung (Leone

et

a1 1991 Engel

et

a / 1993) thyroid (Aral

et

a1 1993 Zou

et

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a1 1993; Farley

et

a / . 1993) and stomach (Nak- ayama

et

a1 1993; Kodera

et

al. 1994) have been reported.

Because gastric cancer still remains the leadlng cause of cancer death in Korea, i t would be of great significance i f something other than the known cl~nical or histopathologic parameters could be predictable for the metastasis and prognosis of gastric cancer. We studied the nm23 protein expression of surgically resected gastric cancer t~ssues, with the hope that the ~mmunohistoc- hemical expression of nm23 protein might be one of such predlctlve factors.

MATERIAL AND METHOD

1. Material

Tissues were obtained from 42 surgically resected stomachs in the Department of Surgery.

Seoul National Univers~ty Hospital during the period from January 1990 to December 1992, with age and sex matching

2. Method

Samples were f~xed wlth 10% neutrally buffered formalin for 24 hours processed routinely, and embedded In paraffin Paraffin-embedded tlssues were cut Into 5 um-thlck sectlons and processed

~mmunohistochem~cally The tlssues were deparaf- flnlzed treated by 0 3 % H202 for 15 minutes and blocklng serum for 30 mlnutes Then, we used prlmary antlbody NCL-nm23 (Novocastra Laborat- ories Ltd 1 wlth the dllutlon o f 1 200 at room tem- perature for 2 hours Consequently, av~din-biottn peroxldase complex method was performed using an LSAB ktt (DAKO) Flnally, the tissues were stained with dlaminobenzid~ne and were counter-stained wtth hematoxyltn

For the stattstical analysts. we used F ~ s h e r s exact test or Chi-square test using the PC-SAS program 6 04 version

RESULTS

The nm23 proteln was intensely stalned In the cytoplasm and/or the nucleus of carcinoma cells In

9

(21 4'0) out of 42 cases(Ftg 1 ) Although

some cases showed weak stain~ng, we discarded the cases that showed stalnabillty either weaker than the adjacent non-cancerous mucosa or heterogeneous statnlng Some cases showed more intense staining In the well differentiated area and weaker staining In the poorly differentiated area Furthermore, the deeper p o r t ~ o n o f the tumor showed the weaker staining in cases The nm23 protein expression was definite in 7 of 15 cases (46 7 % ) of the non-metastatic group, while it was definite in 2 of 27 cases (7 4 % ) of the nodal metastasis group (Fig 2) Thus the positive ratio was much higher in the non-metastatic group, and ~t was statistically significant by a p value less than 001 We tried to compare this finding to other data related to the prognosis There was n o slg- nlflcant correlation between nm23 protein expression and other parameters such as patient age sex, WHO grade, Lauren classification, depth of invasion, l o c a t ~ o n of tumor and size (Table 1 ) although the Lauren's diffuse type and larger tum- o r ( > 5 c m ) were more frequently found In the nm23 negative group than in the nm23 positive group (Fig 3) Only perigastrlc lymph node met- asis was significantly associated with nm23 protein expression (p< 0 001 )

DISCUSSION

The actual mechanism by whlch nm23 gene or product affects invasive or metastatic potential still remains unknown Because of the homology between the nucleoside diphosphate (NDP) kinase and the role of NDP kinase in cytoskeleton status and cell to cell commun~cation (Nickerson & Wells 1984 Gilles

et

a1 1991 ), ~t could be pred~cted that the nm23 takes part In cell adhesion or migration, namely invasion or metastas~s (Kodera et a1 1994) However, controversies are still present, and additional studies are mandatory

The expression of nm23 gene has been known to be Inversely correlated with metastatic potential in various human cancers as well as gastrlc cancer In gastric cancer, it has been reported that nm23 gene expression was higher in primary cancer t~ssue than in matched mucosa (Nakayama et a1 1993) Furthermore, it has been suggested that

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Fig. 1. Photomicrographs of the nm23 p o s ~ t ~ v e ( A ) and negat~ve ( B ) gastr~c cancer cells

N M 2 3 ( + ) NM23(-)

.metastasis C d i f f u s e .size>5cm

Fig. 3. The rates of lymph node metastas~s. Laur- en's diffuse type and large tumor size (>5 cm) a c c o r d ~ n g to nm23 protein expression -. The nm23 protein expression rate accord-

ing to lymph node metastasis

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Table 1. Relationship between nm23 protein expression and clinicopathologic parameters in gastric adenocarcinoma

Parameters

- . . . -

Samples(n ⁼42) Nodal metastasis

absent present Tumor size

5 c m

<

5 c m

>

Lauren classification intestinal

dlffuse Sex

male female

T classificat~on T2

T3

WHO grade well

moderately poorly

--

NS not s~gnif~cant

nm23 reactivity p value

negative positive

the down-regulation of nm23 gene mlght have a role In metastas~s and lnvaslon In gastrlc cancer.

poss~bly leadlng to a poor prognosls(Kodera

et

a1 1994) W ~ t h regard to the loss of heterozygos-

~ t y ~t was not only rare or hardly found but was also not related wlth cltnlco-pathologic features (Nakayama

et

a1 1993 Kim et a1 1993) The cur- rent study demonstrates that reduced expresslon of nm23 proteln In gastrlc adenocarclnoma would be related w ~ t h lymph node metastasls Some factors such as Lauren s dlffuse type and large tum- or size were also related wlth the down-regu- latlon of nm23 proteln expression although there was no stat~st~cal s~gnlf~cance There has been controversy as to whether the dlfferentlat~on of gastrlc adenocarc~noma ^IS associated w ~ t h nm23 expresslon (Kodera

et

a1 1993 Nakayarr~a

et

a1 1994) The actlon mechanrsm of nm23 In cancer

might be explained by a bimodal pattern. First, it might act as a suppressor of invasion in mam- mary ductal carcinoma In situ (Simpson et a/.

1994). Second, it mlght act as a well-known suppressor of lymph node metastasis. Gastric carcin- ogenesis has been explained by two pathways, de novo or by way of colonic metaplasia. In that sen- se, we presume that the genetic events of gastric carcinomas with reference to Lauren's classification would show discrepancy with each other.

Our study has the limitations of a small number of cases and short follow-up period. Therefore, we are preparing more reliable data with a larger number of cases with longer follow-up period.

In Korea, studies related to the metastatic potentlal of human cancer and nm23 expression have only been performed in breast carcinomas, especially using the immunoh~stochemical method

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(Song et a / . submitted). Although the loss of het- erozygosity has been studied in gastric adenocarcinoma, the result was not contributory(Kim et a/.

1993). We hope that additional extensive studies elucidating the role of nm23 in the metastasis of various human cancers will be performed In the near future.

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Reduced Expression of nm23 Protein is Related to Nodal Metastasis of Human Gastric Carcinoma