April 1998

class 1 2 3 4 5 < 45 y 45-55 y

(n=5) (n=4) (n=7) (n= 10) I (n=24) ] (n= 13) highMSI 3 (60%) 4 (100%) 1 (14%) 2 (20%) 4 (17%) 9 (70%) lowMSI 1 (20%) 0 0 0 0 0 IH*neg. 3 (60%) 3 (75%) 1 (14%) 2 (20%) 3 (13%) 9 (70%) Mutation (2) (1) (1) ? ? (1) IH = Immunohistochemistry

Although mutational analysis has currently been done in selected cases only (n=5), MSI status revealed low prevalence of HNPCC in class 3 and 4. In contrast, high MSI and loss of mismatch repair protein expression (IH neg) in class 1,2 and 5 indicate a high probability of HNPCC kindreds. Conclusions: The probability that a CRC is part of the HNPCC syndrome is not only related to the Amsterdam criteria. Patients with multiple HNPCC related primaries and those with CRC showing a distinct morphology are also at particular risk. Young age as a sole criterion is much less predictive as expected.

• (]12595 MICROSATELLITE INSTABILITY IN GASTRIC CARCINOMA: RELATION TO TUMOR TYPE, STAGE AND SITE IN THE STOMACH. F. Kullmann. V.D. Mohr °, S. Mehringer ° K.-W. Jauch °, J. SchiSlmerich, J. Riischoff*. Dpts. of Internal Medicine I, °Surgery and *Pathology, University of Regensburg, Germany.

Background: Microsatellites are normal regions of the genome and consist of short sequences of repeated DNA. Microsatellite instability in cancers results from length changes in these repeats due to deficiencies in repair systems for slipped-strand and perhaps base-base mismatches. Recently, we described a strong correlation between microsatellite instability (MSI) and a solid growth pattern in colonic adenocarcinoma, medullary type (Am J Pathol 1997;150: 1815-1825). Purnose: To determine the incidence of MSI from an European population and to relate these results to tumor type, especially growth pattern, stage and site in the stomach. Methods: 41 primary gastric adenocarcinoma (n=21 of solid/medullary type according to WHO 1990) from patients (19 women, 22 men, mean age 68,6 years) cared for at the University Hospital of Regensburg were selected. DNA was extracted by microdis-section from formalin-fixed, paraffin-embedded or fresh-frozen tissue. MSI was assessed at 4 selected loci (#5q:APC, #9p:D9S171, #10p:D10S89, #17p:TP53) in accordance to recently published recommendations (Cancer Res 1997; 57:4749-4756). MSI was correlated with clinical data of the patients and pathologic assessment of the cancers. Results" Nine of 41 cases (22%) demonstrated MSI at least at one locus. There was no loss of heterozygosity at any amplified loci. The tumors with solid growth pattern showed a higher frequency of MSI (7/21=33% vs. 2/20=10%, p=0.13). The following correlation between tumor site and MSI was found:

Tumor site Number of tumors (n) MSI (at least one locus) Cardia 13 3 (23%) Corpus 17 2 (12%) Antrum 11 4 (36%)

We observed no strong association between MSI and histology (Laur6n), TNM-classification, differentiation or mucin production of the tumor. Interestingly, the two "high" instable tumors (4 of 4 loci) were early stage (T1 and T2) tumors. Patients with MSI-tumors had a mean age of 71.8. However, the mean age decreased with the frequency of instable loci (MSI 1/4 loci: 76.3; 4/4 loci: 65.5). Conclusions: The incidence of MSI in gastric carcinoma is comparable with that of sporadic colorectal cancer. "High" MSI was associated with younger age of the patients, early tumor stage and occurrence at the proximal third of the stomach. Most interestingly, MSI tends to occur preferably in gastric tumors of solid/medullary type, as already observed in primary colonic adenocarcinoma with solid growth pattern.

G2596 DISTANT METASTASIS IN EARLY GASTRIC CANCER. K. Kumai 1,2, Y. Otani l, K. Aiura 1,2, Y. Iwao 2, Y. Sugino 2, T. Kubota I and M. Kitajima ],2. Department of Surgery l, Center for Diagnostic and Therapeutic Endoscopy 2, School of Medicine, Keio University, Tokyo, Japan

Distant metastasis of early gastric cancer is rarely observed, but it is very critical. Distant metastases were analyzed in 887 patients with early gastric cancer who underwent gastrectomy at our department since 1964. Four of 431 patients (0.9%) with mucosal gastric cancer died from gastric cancer associated with distant metastases to the lung, liver or bone. Among 456 patients with early gastric cancer invading the submucosa, 8 cases (1.7%) had distant metastasis and 9 cases (1.9%) had intra-abdominal recurrence. The mortality rate related to the disease type was 2.2% in Type I, 2.6% in Type IIc, 3.9% in Type IIa+llc and 3.2% in Type IIc+III. Superspreading type IIc

Gastrointestinal Oncology A631

with an ulcer scar in mucosal gastric cancer, and massive invasion of tumor into the submucosa in combined types of early gastric cancer (e.g., IIc+III, IIa+Ilc) were characteristic findings in the patients with lung metastasis. Liver metastases were observed in the patients with large Type I or superspreading type lie without ulcer scar. Two cases with bone metastases had severe lymph node metastases and massive infiltration of poorly differentiated adenocarcinoma into the submucosa. Superspreading type IIc and the combined types with a component of depression were characteristic of distant metastasis of early gastric cancer.

• G2597

ACTIVATION OF HER2 PATHWAY INDUCES EXPRESSION OF CYCLOOXYGENASE-2, RELEASE OF PROSTAGLANDIN E2, AND MITOGENESIS IN HUMAN COLORECTAL CANCER CELLS. Rakesh Kumar, Mahitosh Mandal, Liana Adam, Debdutta Bandyopadhyay, Gideon Steinbach, John Mendelsohn, Ratna Vadlamudi. Cell Growth Regulation Laboratory, The University of Texas MD Anderson Cancer Center, 1515 Holc0mbe Blvd., Houston, TX 77030.

Growth factors and their receptors play an essential role in the regulation of the proliferation of colonic epithelial ceils. Cyclooxygenase (COX-2), an enzyme responsible for the conversion of arachidonic acid to prostaglandins (PGs), is implicated in colorectal tumorigenesis. Here, we report that human colorectal cancer cell lines express abundant levels of HER3 and HER2 receptors, and are growth-stimulated by recombinant neu-differentiation factor-beta 1 (NDF). NDF-treatment of colorectal cancer cells was accompanied by increased tyrosine phosphorylation and heterodimerization of HER3 with HER2. In addition, we demonstrated that HER3 and HER2 receptors in colorectal cancer cells are constitutively phosphorylated on tyrosine residues and form heterodimeric complexes in the absence of exogenous NDF. Inhibition of HER3 signaling by an anti-HER3 mAb against the ligand binding site resulted in a decrease in the levels of constitutively activated HER3/HER2 heterodimers, and the unexpected reduction of COX-2 expression. Activation of the HER3/HER2 pathway by NDF induced the expression of COX-2 mRNA, COX-2 protein and accumulation of prostaglandin E2 in the culture medium. Finally, we demonstrated that NDF promotes the ability of colorectal cancer ceils to survive in an extracellular matrix milieu, such as Matrigel, and also to invade through a 0.8 lam porous membrane. These biological activities of NDF and its stimulation of cell proliferation are blocked by a specific inhibitor of COX-2. Taken together, our findings provide the first biochemical evidence of a possible role of the COX-2 pathway in the mitogenic action of NDF in colorectal cancer cells where it may be constitutively upregulated due to the autocrine/paracrine activation of HER3/HER2 heterodimers.

• G2598

BCL-2 PREVENTS ACTIVATION OF Caspase-3 THAT CLEAVES DNA-DEPENDENT PROTEIN KINASE IN THE NUCLEUS DURING SHORT-CHAIN FATTY ACID-INDUCED APOPTOSIS. Rakesh Kumar and Mahitosh Mandal, Cell Growth Regulation Laboratory, The University of Texas MD Anderson Cancer Center, Houston, TX 77030.

The colonic epithelial cells near the top of crypt and in the lumen have been shown to undergo apoptosis. Since butyric acid is the major short-chain fatty acid produced by fermentation of dietary fiber in the large bowel, it has been proposed that it could act an important regulator of apoptosis in colorectal cancer. We have earlier shown that upregulation of Bcl-2 in colorectal carcinoma cells confers resistance to butyric acidinduced apoptosis (Mandal, M., Wu, X. and Kumar, R. Carcinogenesis 18, 229-232, 1997). Here we report that the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) was preferentially cleaved in colorectal carcinoma cells treated with butyric acid, and resulted in a significant inhibition of DNA-PK kinase activity. In cells undergoing apoptosis, the cleavage of DNA-PKcs was paralleled or preceded by the induction of processing of pro-Caspase-3 into active Caspase-3 fragments. Deregulation of Bcl-2 completely inhibited butyric acidinduced activation of Caspase-3 as well as cleavage of DNA-PKcs. We also demonstrate that DNA-PKcs was proteolytically cleaved by active Caspase-3 in the nucleus and cleaved DNA-PKcs fragments were released in the cytosolic compartment. The observed processing of pro-Caspase-3 into active Caspase-3, nuclear cleavage of DNA-PKcs, and release of cleaved DNA-PKcs fragments in the cytosolic compartment were inhibited by a specific caspase-3 inhibitor, the tetrapeptide DEVDCHO. Finally, we demonstrated that butyric acid induces the expression of Bax protein in cells with or without Bcl-2 deregulation, implying that Bcl-2 overexpression may alter the ratio of Bax to Bcl-2 in favor of cell survival. Induction of Bax by butyric acid was not associated with increased p53 expression. However, butyric acid treatment triggered an uniform peri-nuclear accumulation of p53 against the nuclear membrane in Bcl-2 overexpressing cells. These findings suggest that short- chain fatty acid may utilize pathways both down-stream and up-stream of Bcl-2, to trigger apoptosis in colorectal carcinoma cells.