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Differential Expression of Metastasis-Associated Genes in Papilla of Vater and Pancreatic Cancer Correlates With Disease Stage

From the Department of Visceral and Transplantation Surgery and Institute of Pathology, University of Bern, Inselspital, Bern, Switzerland, and Division of Endocrinology, Diabetes and Metabolism, and Departments of Medicine, Biological Chemistry, and Pharmacology, University of California, Irvine, CA.

Address reprint requests to Helmut Friess, MD, Department of Visceral and Transplantation Surgery, University of Bern, Inselspital, CH-3010 Bern, Switzerland; email: helmut.friess{at}insel.ch

	ABSTRACT

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PURPOSE: Papilla of Vater cancer has a much better prognosis than pancreatic cancer. It is not known whether this is the result of differences in the tumor biology of the two malignancies. Because metastasis formation is a critical step in tumor progression and a negative prognostic factor, we compared the expression of nm23-H1 and KAI1, two metastasis-suppressing genes, in papilla of Vater cancer and pancreatic cancer.

PATIENTS AND METHODS: Analysis was performed in nine normal human papilla of Vater samples, 27 papilla of Vater cancers, 16 normal pancreatic samples, and 29 pancreatic cancers. Expression of nm23-H1 and KAI1 was analyzed by Northern blot analysis and in situ hybridization. In addition, immunohistochemistry was performed to localize the respective proteins.

RESULTS: There was no difference in nm23-H1 and KAI1 mRNA expression levels in normal versus cancerous papilla of Vater samples. In contrast, nm23-H1 and KAI1 RNA expression was upregulated in early tumor stages of pancreatic cancer and reduced in advanced tumor stages. When expression of nm23-H1 and KAI1 RNA was analyzed by use of in situ hybridization, normal epithelial cells of the papilla of Vater exhibited mRNA staining intensity similar to that of papilla of Vater cancer cells. Similar levels of nm23-H1 and KAI1 immunoreactivity also were observed in these samples. In contrast, early stage pancreatic cancer samples exhibited stronger nm23-H1 and KAI1 immunoreactivity than normal controls. Furthermore, early pancreatic cancer stages exhibited higher KAI1 and nm23-H1 immunostaining than advanced tumor stages.

CONCLUSION: Differences in the expression patterns of the two tumor suppressor genes nm23-H1 and KAI1 may contribute to the different prognoses of papilla of Vater cancer and pancreatic cancer. Our findings support the hypothesis that biologic differences rather than earlier diagnosis influence the different outcomes of these two tumor entities.

	INTRODUCTION

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A CASCADE OF cellular, biochemical, and genetic events are known to contribute to the development and progression of tumors, which lead to malignancy and ultimately to metastasis. Metastatic status is an important clinical parameter that must be considered when the appropriate type of cancer therapy is chosen. Furthermore, the presence of metastases limits the surgical therapy of most malignant gastrointestinal tumors. Metastasis formation is a complex process that involves local invasion, intra- and extravasation of tumor cells, and decreased host immunologic responses.^1,2 In carcinoma of the papilla of Vater, lymph node metastases are present in 31% to 52% of patients at the time of diagnosis, and radical tumor resection results in 5-year survival rates between 21% and 61%.^3,4 In contrast to cancer of the papilla of Vater, pancreatic ductal adenocarcinoma has a dismal prognosis despite the proximity of these malignancies. At the time of diagnosis, most pancreatic cancers exhibit regional spread or distal metastasis, which limits therapy to nonresective palliative procedures. Therefore, the median survival time of most of patients with pancreatic cancer is 4 to 6 months. The aggressive growth behavior of pancreatic cancer results in a death/incidence ratio of approximately 0.99 in the United States and most European countries.^5,6 The reason pancreatic cancer has a completely different prognosis than cancer of the papilla of Vater is not known. It was postulated that jaundice, with a resultant earlier diagnosis, mainly accounts for the better prognosis of papilla of Vater cancer patients. However, it is not known whether differences in tumor cell biology also may contribute to this difference.

Nm23-H1 and KAI1, the so-called metastasis genes, influence the metastatic ability of a variety of gastrointestinal cancer cells.^7-9 Nm23-H1 mRNA expression was found to be increased in a nonmetastatic murine melanoma cell line compared with the low expression levels of a highly metastatic matched melanoma cell line.¹⁰ Furthermore, transfection of the nm23-H1 gene into highly metastatic murine melanoma cells that did not express nm23-H1 reduced the metastatic potential of these tumor cells, which indicates that nm23-H1 is important for tumor metastasis.¹¹ Although reduced nm23-H1 expression is associated with a higher metastatic potential of melanomas, breast cancers, pancreatic cancers, and hepatocellular cancers, increased nm23-H1 expression also has been associated with decreased disease-free survival of patients with squamous cell lung cancer, colorectal cancer, thyroid cancer, and neuroblastoma.^12,13

KAI1, a gene that was isolated originally in prostate cancer cells, also has been correlated with decreased metastatic potential of cancer cells. After transfer of the KAI1 gene into highly metastatic prostate cancer cells that exhibited low KAI1 mRNA expression, the cancer cells’ metastatic ability was suppressed, whereas their primary tumor growth behavior was not affected.⁸ It was reported recently that decreased KAI1 mRNA expression correlates with the formation of metastases in pancreatic cancer and hepatocellular cancer and with poorer survival in patients with non–small-cell lung cancer, urinary bladder cancer, and colorectal cancers.^14-18 In primary gastric and esophageal cancer, however, the expression of KAI1 seems to be of minor importance for metastasis. Taken together, these findings suggest that nm23-H1 and KAI1 are involved in metastasis formation in pancreatic cancer, and their loss seems to contribute to the progression of the tumor to a metastatic state.

We compared the expression and distribution patterns of nm23-H1 and KAI1 in papilla of Vater and pancreatic cancer patients to evaluate whether differences in these genes might contribute to the different clinical appearance and aggressiveness of these tumors.

	PATIENTS AND METHODS

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Patients
Sixteen normal human pancreatic tissue samples from six female and 10 male patients (median age, 41 years; range, 16 to 47 years) were obtained through a multiorgan donor program in which no recipient was available for pancreatic transplantation. In nine cases (four female, five male; median age, 39 years; range, 23 to 43 years), the pancreas was obtained with the duodenum and the papilla of Vater completely resected. Tissue samples of 27 patients with carcinoma of the papilla of Vater (12 female and 15 male; median age, 63 years; range, 40 to 81 years) were obtained after Whipple resection. The diagnosis of cancer of the papilla of Vater was confirmed by histopathologic analysis. According to the tumor-node-metastasis classification of the International Union Against Cancer,¹⁹ there were seven stage I, 11 stage II, seven stage III, and two stage IV papilla of Vater cancers. The organ donors were significantly younger than papilla of Vater cancer patients (P < .05).

Pancreatic cancer tissues were obtained from 29 patients (14 female and 15 male) who underwent surgery for pancreatic cancer. The median age of the pancreatic cancer patients was 67 years (range, 37 to 78 years). A partial duodenopancreatectomy (Whipple resection) was performed in 26 patients and a distal pancreatectomy was performed in three patients. In accordance with the tumor-node-metastasis classification of International Union Against Cancer, there were nine patients with stage I, four patients with stage II, 14 patients with stage III, and two patients with stage IV disease. Tumor differentiation was graded as follows: well in seven tumors, moderate in 17, and poor in five. The organ donors were significantly younger than the pancreatic cancer patients (P < .05).

Tissue Sampling
For RNA extraction and Northern blot analysis, normal and tumor specimens were frozen in liquid nitrogen immediately after surgical removal and stored at -80°C until use. In addition, freshly removed normal and cancerous tissue samples were fixed immediately in formaldehyde solution for 12 to 24 hours and paraffin-embedded for in situ hybridization and immunohistochemistry.

Northern Blot Analysis
Total RNA was extracted by the single-step guanidinium isothiocyanate method, size-fractionated on 1.2% agarose/1.8 mol/L formaldehyde gels, and stained with ethidium bromide for verification of RNA integrity and loading equivalency.²⁰ The RNA was electrotransferred onto nylon membranes (GeneScreen, Du Pont International, Regensdorf, Switzerland) and cross-linked by ultraviolet irradiation. For hybridization, a ³²P-labeled cRNA probe of nm23-H1, a digoxigenin (DIG)-labeled KAI cRNA probe, and a ³²P-labeled 7S cDNA probe were used.

For nm23-H1, the blots were prehybridized for 8 hours at 65°C in 50% formamide, 0.5% sodium dodecyl sulfate (SDS), 5x Denhardt’s solution (1x Denhardt’s solution = 0.02% Ficoll, 0.02% polyvinylpyrrolidone, and 0.02% bovine serum albumin), 250 µg/mL salmon sperm DNA, and 50 mmol/L sodium phosphate buffer, pH 6.5. The blots were then hybridized for 18 hours at 65°C in the presence of 1 x 10⁶ cpm/mL of the ³²P-labeled antisense nm23-H1 riboprobe, washed twice at 65°C in a solution that contained 2x standard saline citrate (SSC; 0.3 mol/L NaCl and 0.03 mol/L Na₃ citrate, pH 7) and 0.5% SDS, and washed three times for 15 minutes at 65°C in a solution that contained 0.1x SSC and 0.5% SDS.²¹

Prehybridization for KAI1 was performed for 2 hours at 65°C in a buffer that contained 50% formamide, 5x SSC, 2% blocking reagent (Roche Diagnostics, Rotkreuz, Switzerland), and 0.1% lauroylsarcosine. After the DIG-labeled KAI1 antisense probe was added, hybridization was performed at 65°C for 18 hours. The filters were washed afterward for 5 minutes in 2x SSC and for 5 minutes in 0.1% SDS at room temperature, followed by two washes at 68°C for 15 minutes each in 0.1x SSC and 0.1% SDS. Next, the filters were incubated in 20 mL of blocking buffer (1% blocking reagent in 100 mmol/L maleic acid, 150 mmol/L sodium chloride, and 175 mmol/L sodium hydroxide) that contained 1 µL of anti-DIG alkaline phosphatase antibodies (Roche Diagnostics) for 30 minutes, washed with blocking buffer for 15 minutes, and incubated with 4 µL of CDP-Star (25 mmol; Roche Diagnostics). The membranes were then exposed to x-ray films for 15 seconds at room temperature as reported previously.^20,21

To assess equivalent RNA loading, the membranes were rehybridized with the ³²P-labeled mouse 7S cDNA probe that cross-hybridizes with human 7S RNA to verify equivalent RNA loading.^20,21 The membranes were prehybridized for 4 to 8 hours at 42°C in a buffer that contained 50% formamide, 1% SDS, 0.75 mol/L NaCl, 5 mmol/L EDTA, 5x Denhardt’s solution, 100 µg/mL salmon sperm DNA, 10% dextran sulfate, and 50 mmol/L sodium phosphate, pH 7.4. The hybridization was performed at 42°C for 18 hours by addition of the labeled cDNA probe (1 x 10⁵ cpm/mL). The blots were rinsed twice in 2x SSC at room temperature and washed three times at 55°C in 0.2x SSC and 2% SDS.

The blots were then exposed at -80°C to Fuji x-ray films with intensifying screens for 24 to 48 hours. The intensity of the nm23-H1, KAI1, and 7S signals was quantified by video densitometry analysis with the BioRad 620 densitometer (BioRad, Hercules, CA) as reported previously.^20,21 The ratios between the nm23-H1 or KAI1 signals and the corresponding 7S signal were calculated for each sample.

In Situ Hybridization
In situ hybridization of nm23-H1 and KAI1 was performed by use of DIG-labeled cRNA probes as reported in detail previously.^14,22 Sections of normal and cancerous tissue samples were processed simultaneously. In addition, consecutive tissue slides were processed with one slide incubated with the sense probe and the other incubated simultaneously with the antisense probe. The prehybridization, hybridization, and wash conditions were the same for pancreatic and papilla of Vater tissue samples. Four-µm tissue sections were deparaffinized, rehydrated, and incubated in 0.2 mol/L HCl for 20 minutes. After they were washed with 2x SSC, the tissues were permeabilized with proteinase K at a concentration of 35 µg/mL for 15 minutes at 37°C. After postfixation with 4% paraformaldehyde in saline phosphate buffer (5 minutes) and a wash in 2x SSC, the sections were prehybridized for 1 hour at 60°C in a buffer that contained 50% formamide (vol/vol), 4x SSC, 2x Denhardt’s reagent, and 250 µg RNA/mL. Hybridization was performed overnight at the same temperature in 50% (vol/vol) formamide, 4x SSC, 2x Denhardt’s reagent, 500 µg RNA/mL, and 10% dextran sulfate (vol/vol). The final concentration of the DIG-labeled nm23-H1 and KAI1 probes (antisense or sense) was approximately 0.5 ng/µL. After hybridization, excess probe was removed by a wash in 2x SSC and by RNase treatment that consisted of 100 U/mL RNase T1 and 0.2 µg/mL RNase DNase-free (Roche Diagnostics) at 37°C for 30 minutes. Washes were performed at 60°C (nm23-H1) and 63°C (KAI1) in 2x SSC (10 minutes), and twice in 0.2x SSC (10 minutes each). Afterward the sections were incubated with an anti-DIG antibody conjugated with alkaline phosphatase (Roche Diagnostics). For the color reaction, 5-bromo-4-chloro-3-indolyl phosphatase and nitroblue tetrazolium (Sigma Chemical Co, Buchs, Switzerland) were used.

Pretreatment of the slides with RNase abolished the hybridization signals, and hybridization with the nm23-H1 or KAI1 sense probes that corresponded to the antisense probes failed to produce an in situ hybridization signal.

The in situ hybridization signals were evaluated semiquantitatively by two independent observers blinded to patient status, followed by resolution of any differences by joint review and consultation with a third observer. The in situ hybridization results were scored as described previously^23,24: -, no detectable signal; +, weak detectable signal; ++, moderate detectable signal; and +++, strong detectable signal.

Preparation of nm23-H1 and KAI1 Antisense and Sense cRNA Probes
To prepare radiolabeled nm23-H1 and DIG-labeled nm23-H1 and KAI1 cRNA probes for Northern blot analysis and in situ hybridization, a 168-bp nm23-H1 sample and a 500-bp fragment of KAI1 cDNA were subcloned into the pCR-II vector (Invitrogen, San Diego, CA), which contains promoters for DNA-dependent SP6 and T7 RNA polymerases. After linearization of the plasmid, the antisense nm23-H1 and KAI probes were transcribed by use of SP6 polymerase and the Ribomax system (Promega Biotechnology, Madison, WI). For nm23-H1, a ³²P-labeled cRNA probe was prepared for Northern blot analysis and a DIG-labeled cRNA was prepared for in situ hybridization. A DIG-labeled KAI1 cRNA probe was used for Northern blot analysis and in situ hybridization. To evaluate the specificity of the in situ hybridization reaction, DIG-labeled sense probes of nm23-H1 and KAI1 were generated after linearization of the plasmid with BamHI and HindIII, respectively, and after in vitro transcription with T7 polymerase and the Ribomax system.^14,25 For the in situ hybridization experiments, the KAI1 antisense and sense probes were shortened to a length of approximately 150 bases as described previously.^14,25

Preparation of 7S cDNA Probe
To verify equivalent RNA loading and transfer on Northern blot membranes, all filters were rehybridized with a 190-bp fragment of 7S cDNA.^20,21 The 7S cDNA probe was radiolabeled with [-³²P]dCTP using a random primer labeling system (Pharmacia Biotech AG, Dubendorf, Switzerland).^20,21

Immunohistochemistry
From each normal and cancer tissue sample, three tissue sections were evaluated. After deparaffinization and hydration, 3-µm tissue sections were submerged for 15 minutes in Tris-buffered saline (10 mmol/L Tris-HCl and 0.85% NaCl, pH 7.4) that contained 0.1% (vol/vol) Triton X-100, and they were rinsed briefly three times for 1 to 2 minutes in Tris-buffered saline solution.^26,27 After incubation in methanol-containing 0.6% hydrogen peroxide for 30 minutes to block endogenous peroxidase activity, the slides were covered with 10% normal goat serum at 23°C for 30 minutes before overnight incubation with mouse monoclonal antihuman KAl1 antibody (antibody C33, kindly supplied by J.C. Barrett, PhD, Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC) or a 1:100 dilution of mouse monoclonal antihuman nm23-H1 antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA). After they were washed with Tris-buffered saline, biotinylated goat antimouse immunoglobulin and a streptavidin-peroxidase complex (Kirkegaard and Perry Laboratories, Gaithersburg, MD) were added at 23°C for 45 and 30 minutes, respectively, followed by incubation with a 3,3'-diaminobenzidine tetrahydrochloride and hydrogen peroxide mixture.²⁸ The samples were counterstained with Mayer’s hematoxylin.

Statistical Analysis
Results are expressed as median and upper and lower quartile or median and range. For statistical analysis the Mann-Whitney U test, the ² test, and the Spearman correlation test were used. Significance was defined as P < .05.

	RESULTS

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Nm23-H1/KAI1 mRNA Expression by Northern Blot Analysis
Papilla of Vater samples. In 44% and 67% of the normal papilla of Vater tissue samples, nm23-H1 and KAI1 mRNA signals were detectable, respectively. In papilla of Vater cancer samples, slightly higher nm23-H1 and KAI1 mRNA expression levels were present in 50% and 46% of the samples, respectively. In the remaining cancer samples, expression of both genes was comparable with the normal controls. Densitometric analysis of the expression signals revealed a 23% increase (P = .67) and a 11% decrease (P = .59) in nm23-H1 and KAI1 mRNA levels, respectively, in papillary cancer compared with the normal controls when all cancerous tissue samples were included ( Fig 1A). When only cancer samples with increased nm23-H1 and KAI1 mRNA expression levels were analyzed statistically, the increases were 1.7-fold (not significant) for nm23-H1 and 1.6-fold for KAI1 (not significant) ( Fig 2; lanes 1 to 9 in normal samples and lanes 10 to 20 in papilla of Vater cancer). Comparison of primary papilla of Vater cancer samples in which lymph node metastases were present at the time of tumor resection (stage III/IV) with those in which no lymph node metastases (stage I/II) were present revealed no differences in nm23-H1 or KAI1 mRNA levels (Fig 1A). In papilla of Vater cancer tissue samples, but not in normal samples, there was a positive relationship between nm23-H1 and KAI1 mRNA levels (normal, r = .25, P = .51; cancer, r = .57, P < .002).

View larger version (36K): [in this window] [in a new window]   Fig 1. (A) nm23-H1 and KAI1 mRNA expression in normal versus cancerous papilla of Vater tissue. (B) nm23-H1 and KAI1 mRNA expression in normal pancreas versus pancreatic cancer tissue. Stage I/II, without metastases; stage III/IV, with metastases; median, , lower quartile; , upper quartile. Abbreviation: n.s., not significant.

View larger version (50K): [in this window] [in a new window]   Fig 2. Northern blot analysis of nm23-H1 and KAI1 mRNA expression in normal papilla of Vater tissue and in papilla of Vater cancer. No difference exists between the normal and cancerous samples.

View larger version (57K): [in this window] [in a new window]   Fig 3. Northern blot analysis of nm23-H1 and KAI1 mRNA expression in normal pancreas and pancreatic cancer. Nm23-H1 and KAI1 mRNA are upregulated in selected cases of pancreatic cancer compared with the normal pancreas. Lanes: 12, 14, 18 to 22, nm23-H1; 12, 14, 16, 18 to 20, KAI1.

View larger version (51K): [in this window] [in a new window]   Fig 4. Comparative analysis of nm23-H1 and KAI1 mRNA expression in papilla of Vater cancer and pancreatic cancer samples. Levels of nm23-H1 and KAI1 mRNA are higher in papilla of Vater cancer compared with pancreatic cancer.

Papilla. In the normal papilla of Vater, moderate nm23-H1 ( Fig 5A) and strong KAI1 mRNA (Fig 5G) signals were present in the cytoplasm of most epithelial cells. Lymphocytes in the submucosal areas of the normal papilla of Vater exhibited weak or moderate expression of nm23-H1 (Fig 5A, arrows) and KAI1 (Fig 5G, arrows) mRNA. When normal tissue adjacent to the tumor was compared with the normal tissue of organ donors, no difference in nm23-H1 and KAI1 mRNA expression was observed. In the papilla of Vater cancer samples, the signal intensity for nm23-H1 (Fig 5B) and KAI1 (Fig 5H) mRNA in the cytoplasm of the cancer cells was comparable to the signal observed in the normal controls. Furthermore, there was no difference in the intensity of the nm23-H1 (Fig 5B) and KAI1 (Fig 5H) mRNA signals in primary papilla of Vater cancer samples without metastases (Fig 5B and 5H) and with metastases (Fig 5C and 5I).

View larger version (145K): [in this window] [in a new window]   Fig 5. In situ hybridization of nm23-H1 (A through F) and KAI1 (G through L). (A, G) Normal papilla; (B, H) papilla of Vater cancer without metastases; (C, I) papilla of Vater cancer with metastases; (D, J) normal pancreas; (E, K) pancreatic cancer without metastases; (F, L) pancreatic cancer with metastases (arrows show positive lymphocytes).

Immunohistochemistry of nm23-H1 and KAI1
Papilla. In the normal papilla of Vater samples, moderate nm23-H1 ( Fig 6A) and strong KAI1 (Fig 6G) immunoreactivity was present in the cytoplasm of epithelial cells. In addition, moderate to strong membranous immunostaining was found for KAI1 in the normal papilla of Vater samples (Fig 6G). No differences in nm23-H1 and KAI1 immunoreactivity were found between normal tissue adjacent to the tumor and normal tissue of organ donors. Papilla of Vater cancer cells exhibited an nm23-H1 (Fig 6B) and KAI1 (Fig 6H) staining pattern and intensity similar to that of the corresponding normal samples. However, only a few cancer cells exhibited membranous KAI1 immunoreactivity (Fig 6H). No difference in the pattern and in the signal intensity of nm23-H1 (Fig 6B) and KAI1 (Fig 6H) immunoreactivity was found in primary cancer cells of tumors without metastases (Fig 6B and 6H) and with metastases (Fig 6C and 6I).

View larger version (145K): [in this window] [in a new window]   Fig 6. Immunohistochemistry of nm23-H1 (A through F) and KAI1 (G through L). (A, G) Normal papilla; (B, H) papilla of Vater cancer without metastases; (C, I) papilla of Vater cancer with metastases; D, J: normal pancreas; E, K: pancreatic cancer without metastases; F, L: pancreatic cancer with metastases.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Patients with carcinoma of the papilla of Vater have the best prognosis of all patients with periampullary carcinomas,^4,5 whereas patients with pancreatic head carcinomas have the worst prognosis.^4,5,29 The reason papilla of Vater cancer has a much better long-term prognosis than pancreatic cancer is not known. It has been hypothesized that papilla of Vater cancers become symptomatic in early tumor stages, whereas pancreatic malignancies become symptomatic and detectable in advanced tumor stages if a larger tumor mass already exists. Therefore, it has been postulated that one of the reasons for the better prognosis of patients with papilla of Vater cancer might be that they undergo surgery during earlier tumor stages. However, recent data suggest that molecular differences exist in these tumor entities that might influence their growth behavior.³⁰

In past years, a variety of genetic alterations have been reported in pancreatic adenocarcinomas, including overexpression of a variety of growth factors and their receptors, disturbances in growth inhibitory pathways, and mutations in tumor suppressor genes.^31-34 Thus, accumulation of these molecular alterations in pancreatic cancer cells might give the cells a major growth advantage and contribute to the dismal prognosis of pancreatic cancer patients. Whether papilla of Vater cancers exhibit a comparable spectrum of molecular alterations, or whether major molecular differences between these two disorders exist that might account subsequently for the different prognoses is not yet known. Furthermore, the reason pancreatic cancer cells have a greater propensity to metastasize is not known. Therefore, in the present study we compared the expression patterns of nm23-H1 and KAI1, two genes that influence the metastatic potential of various cancers, in papilla of Vater and pancreatic cancer tissues.

By Northern blot analysis, there were no major differences in the nm23-H1 and KAI1 mRNA levels of normal and cancerous papilla of Vater samples. Furthermore, primary papilla of Vater cancer tissues with and without lymph node metastases exhibited similar nm23-H1 and KAI1 mRNA expression levels. The Northern blot data were confirmed by in situ hybridization and immunohistochemistry. In contrast, in pancreatic tissues there were clear differences in the expression of nm23-H1 and KAI1 between the normal pancreas and pancreatic cancer. Therefore, the persistent expression of two motility- and metastasis-suppressing genes in papilla of Vater cancers might contribute to the less aggressive growth and metastatic potential of these tumors compared with pancreatic cancer. In this study, patients with papilla of Vater or pancreatic cancer were significantly older than normal controls. However, in situ hybridization and immunohistochemistry revealed no differences in nm23-H1 and KAI1 signals between the organ donor tissues, and normal tissues adjacent to cancer lesions in both types of cancer. Therefore, differences in age between the organ donors and the cancer patients do not seem to influence nm23-H1 and KAI1 expression levels.

Alterations in nm23-H1 and KAI1 mRNA expression are associated with metastasis formation in various gastrointestinal and nongastrointestinal cancers.^9-11,35,36 Nm23-H1 upregulation is associated with lymph node metastasis and poor prognosis in human pancreatic cancer samples,¹³ whereas downregulation of KAI1 in the cancer tissues contributes to tumor invasion and metastasis formation.^14,17,18,25 In gastric cancer, reduced expression of nm23-H1 mRNA is associated with the formation of tumor metastases.³⁵ In contrast, KAI1 mRNA expression levels demonstrate no association with the metastatic status of gastric and esophageal cancers.³⁷ These findings suggest that the effects of nm23-H1 and/or KAI1 up- and downregulation on formation of metastases may depend on the underlying tumor type.

The exact functions of nm23-H1 and KAI1 in tumor metastasis are not well understood. Original results of nm23-H1 cloning by differential hybridization of melanoma cell lines with high and low metastatic potential suggested that this gene might function as a potent metastasis suppressor. Although the exact function of nm23-H1 remains unclear, its gene product is identical to human nucleotide diphosphatase kinase, which modulates intracellular signal transduction through phosphorylation of GTP-binding proteins.³⁸ Analysis of nm23-H1 in a variety of gastrointestinal cancers revealed that it does not exert a uniform effect on cancer cell spread. Thus, in colorectal cancer, nm23-H1 does not apparently influence metastasis,³⁹ whereas in hepatocellular cancers, loss of nm23-H1 mRNA expression is associated with the presence of metastases.^40,41 The role of nm23-H1 in pancreatic cancer remains controversial, because positive as well as negative effects on local tumor progression, tumor spread, and prognosis have been described.^13,42,43

In contrast to nm23-H1, most studies have demonstrated that KAI1 functions as a metastasis-suppressor gene.^8,14,18,28 The KAI1 gene product is identical to CD82, a glycoprotein on the cell membrane of leukocytes that belongs to the transmembrane 4 super family (TM4SF).⁴⁴ In mammalian cells, more than 14 members of the TM4 superfamily have been identified, and although their exact functions are not well understood, many studies suggest that these molecules are involved in cell migration, proliferation, and adhesion. Therefore, disturbances in these cell surface glycoproteins may influence the ability of cancer cells to form tumor metastases. Transfection of KAI1 into highly metastatic prostate cancer cells was demonstrated to reduce their metastatic ability when tumor cells were transplanted onto nude mice.⁸ Similar effects could be observed in cultured colon cancer cells, in which KAI1 upregulation by transfection suppressed cancer cell motility and in vitro invasiveness and increased tumor cell aggregatability.^17,45 Analysis of KAI1 in human prostate, pancreatic, hepatic, urinary bladder, lung, and breast cancer tissues revealed that loss of KAI1 expression is associated with formation of metastases. In contrast to these malignancies, KAI1 mRNA expression is comparable in normal and cancerous papilla of Vater tissue samples. Furthermore, no difference in mRNA levels of KAI1 exists between nonmetastatic and metastatic primary tumors. These findings suggest that papilla of Vater tissues do not lose KAI1 gene expression during malignant transformation and tumor progression. Therefore, these tumors do not gain a higher metastatic potential that could reduce the chance of cure and enhance the risk of tumor recurrence and metastases after radical local tumor resection. In conjunction with nm23-H1, which also demonstrates little alteration in papilla of Vater cancer samples compared with normal controls, our study results demonstrate a distinct difference in expression of metastasis-influencing genes in pancreatic and papilla of Vater cancers. Together, these findings suggest that papilla of Vater and pancreatic cancers exhibit molecular differences that might account in part for the differences in prognosis experienced by patients with these cancers.

	ACKNOWLEDGMENTS

 
Supported by grant no. 32-049494.96 from the Swiss National Foundation.

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TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
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Submitted April 3, 2000; accepted January 25, 2001.

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