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Clinical Cancer Research Vol. 11, 6162-6170, September 1, 2005
© 2005 American Association for Cancer Research

Imaging, Diagnosis, Prognosis

Expression of Cdx2 and Hepatocyte Antigen in Gastric Carcinoma: Correlation with Histologic Type and Implications for Prognosis

Zhaoqing Fan1, Jiyou Li2, Bin Dong2 and Xinfu Huang1

Authors' Affiliations: Departments of 1 Surgery and 2 Pathology, Peking University School of Oncology and Beijing Cancer Hospital, Beijing, P.R. China

Requests for reprints: Jiyou Li, Peking University School of Oncology and Beijing Cancer Hospital, Haidian District, No. 52 Fucheng Road, Beijing 100036, P.R. China. Phone: 86-10-8812-2450; E-mail: lijiyou{at}263.net.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: This study was designed to (a) analyze the correlation between the expression of Cdx2 and Hep and the clinicopathologic features of patients with gastric carcinoma, and (b) determine the value of combined analysis of Cdx2 and Hep expression in distinguishing histologic types and prognoses of gastric carcinomas.

Experimental Design: The expression of Cdx2 and Hep were studied using immunohistochemistry of paraffin-embedded tumor specimens from 109 patients who underwent D2 resection for gastric adenocarcinoma from 1995 to 1998.

Results: Nuclear Cdx2 and Hep expression was detected in 36.7% (40 of 109) and 54.1% (59 of 109) of gastric carcinoma cases, respectively. Expression of Cdx2 and Hep was significantly higher in intestinal-type carcinomas than in diffuse-type carcinomas (P = 0.027 and P = 0.037, respectively). There was a clear negative correlation between Cdx2 expression and lymph node metastasis (P = 0.029), as well as between Hep expression and depth of wall invasion (P = 0.011). The patients with Cdx2-positive or Hep-positive expression shows higher survival rate than those with Cdx2-negative or Hep-negative expression (P = 0.0008 and P = 0.003, respectively). Multivariate analysis revealed that the expression of Cdx2 and Hep were independent prognostic indicators of gastric carcinoma. The combination of Cdx2 and Hep expression was significantly lower in diffuse-type carcinoma than in intestinal or mixed-type carcinoma. Multivariate analysis revealed that Cdx2 and Hep expression was an independent prognostic indicator of gastric carcinoma (P < 0.001).

Conclusions: These data suggest that combined analysis of Cdx2 and Hep has significant value in distinguishing histologic types and in predicting the prognosis of gastric carcinoma.

Gastric carcinoma is one of the most common malignant diseases and is the second most common cause of cancer-related death in China and in the world (1).

Gastric carcinoma is a markedly heterogeneous disease in histologic feature and biological characters, especially in the advanced stages. For pathologic diagnosis and classification of gastric carcinoma, two classification systems, the Lauren and WHO systems, are commonly used. The number of clinical documents showed that the biological behavior and prognosis could be significantly different among the patients with the same stage, histologic type, or differentiation grade. Therefore, searching for the biomarkers to indicate the biological characters, and predicting the outcome of patients with gastric carcinoma, is the major focus of research on gastric carcinoma.

Gastric carcinoma can be divided into two main types by the Lauren classification system (2): intestinal and diffuse-type carcinomas. Intestinal-type carcinoma is thought to develop from well-characterized sequential stages that include chronic gastritis, atrophy, intestinal metaplasia, and dysplasia (3). Recently, it was observed that intestinal-type gastric carcinoma developed from intestinal metaplasia in Cdx2-transgenic mice (4). This is the first direct evidence which shows that intestinal metaplasia is the precancerous lesion of gastric carcinoma.

The human Cdx2 genes are the homologues of the caudal-related homeobox gene in the Drosophila. In normal adults, Cdx2 is expressed in mucosa from the duodenum to the distal colon but not in gastric or esophageal mucosa (5). As transcription factors, Cdx2 proteins are important for regulating the proliferation and differentiation of intestinal cells and maintaining the intestinal phenotype by binding the promoters of several intestine-specific genes and activating transcription processes. Cdx2 was reported to be involved in tumorigenesis of the colon (6) and is known to be a tumor suppressor (7). Studies have shown that Cdx2 expression is absent in normal gastric mucosa but is present in >85% of intestinal metaplasias (8–10), suggesting that Cdx2 might be associated with intestinal metaplasia formation. At the stage of chronic gastritis without intestinal metaplasia, expression of Cdx2 but not Cdx1, or other gene markers of intestinal metaplasia were detected. These findings suggest that the expression of Cdx2 precedes that of Cdx1 and other intestine-specific genes in the progression of intestinal metaplasia from chronic gastritis. Therefore, Cdx2 might be the triggering factor for intestinal metaplasia (10). Although expression of Cdx2 has been detected in some gastric carcinomas (8), few studies reported the relationship between Cdx2 expression and prognosis of gastric carcinoma (11, 12).

The epitope of hepatocyte antigen (Hep) is believed to be a component of the membrane of hepatocellular mitochondria (13). Due to the high sensitivity and specificity of Hep expression in normal and carcinomatous liver tissue, Hep is thought to be a useful marker for the diagnosis of hepatocellular carcinoma and for differential diagnosis of primary and metastatic hepatic malignancies (14, 15). Recently, Hep expression was found to be high in the intestinal epithelium (16, 17) and was found in 100% of intestinal metaplasia of gastric and esophageal mucosa (17). Fan et al. (18) reported that Hep expression was detected in 47% of gastric carcinomas. The clinicopathologic features and prognosis of gastric carcinoma in relation to Hep expression have not been reported.

In the present study, we examined the expression of Cdx2 and Hep in gastric carcinoma in order to analyze their correlation with histologic type, clinicopathologic features, and clinical outcomes of the patients in gastric carcinoma. We also investigated the efficacy of combined analysis of Cdx2 and Hep expression to distinguish histologic types and predict the prognosis of gastric carcinoma.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Patients and tissue samples. A consecutive series of 109 patients with gastric carcinoma were studied. All patients were treated by D2 resection in the Beijing Cancer Hospital between September 1995 and December 1998. There were 75 males and 34 females with a mean age of 59 years (range, 29-82 years). None of the patients had received chemotherapy or radiation therapy preoperatively. The age and gender of patients, maximum tumor size, depth of wall invasion, status of lymph node metastasis, vascular invasion, and histologic grade were obtained from histopathology reports. Stage of gastric carcinoma was described according to the 1997 tumor-node-metastasis (TNM) classification of malignant tumors by the International Union Against Carcinoma. All patients were given follow-ups until January 2004 with a minimum 5 years of follow-up. In addition, 6 samples of normal stomach tissue, 17 samples of normal small intestine tissue (duodenum and ileum), and 9 samples of colon tissue were selected from the surgical pathology files at the Beijing Cancer Hospital between 1996 and 1998. Nine cases of intestinal metaplasia were obtained from gastroscopic biopsies files at Beijing Cancer Hospital in 2003.

Immunohistochemistry. The tissue was fixed in 10% neutral formalin and embedded in paraffin. One paraffin-embedded block of tumorous tissue was selected from each case and was cut into 4 µm sections. The sections were put in the oven at 60°C for 4 hours, deparaffinized in xylene, rehydrated in a graded ethanol series, and treated with 3% hydrogen peroxide solution for 10 minutes. Antigen retrieval was done by microwaving tissue in 10 mmol/L citric acid buffer for 10 minutes, then cooling at room temperature for 2 hours. The sections were incubated with an anti-Cdx2 monoclonal antibody (1:100, BioGenex, San Ramon, CA) and an anti-Hep monoclonal antibody (1:100, Zymed, San Francisco, CA) overnight at 4°C. The primary antibodies were detected using the Powervision two-step histostaining reagent, with PV-6001, (Dako, Glostrop, Denmark) as the secondary antibody and were visualized with diaminobenzedine. Tissue was counterstained with hematoxylin, 1% hydrochloric acid of alcohol and eosin, dehydrated with graded ethanols, and mounted. Positive and negative immunohistochemistry controls were routinely used.

Assessment of staining. Two experienced pathologists independently examined nuclear Cdx2 and cytoplasmic Hep staining while blind to the clinicopathologic data and clinical outcomes of the patients. At least 10 high-power fields at 400x magnification were chosen randomly and >1,000 carcinoma cells were counted for each section. Cases with >5% positive tumor cells in a section were regarded as positive expression.

Statistical analyses. All data were analyzed using SPSS 10.0 software. The association of Cdx2 and Hep expression with various clinicopathologic features was analyzed using the {chi}2 test. Cumulative survival was estimated by the Kaplan-Meier method and differences between survival curves were analyzed by the log-rank test. The influence of each variable on survival was analyzed by the multivariate analysis of Cox proportional hazard model (backward, stepwise). Differences at P < 0.05 were considered to be statistically significant.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Cdx2 and Hep expression in normal gastric, intestinal tissue, and colon tissue, and intestinal metaplasia. Cdx2 expression was detected in the nuclei of most of the normal small intestinal mucosa (14 of 17, 82.4%), normal colon mucosa (8 of 9, 88.9%), and intestinal metaplasia (7 of 9, 77.8%), but not in any of the six cases of normal gastric mucosa (0 of 6, 0%; Fig. 1). Hep expression was detected in the cytoplasm of one case of normal gastric mucosa (1 of 6, 16.7%), most cases of normal small intestinal mucosa (15 of 17, 88.2%), one case of normal colon mucosa (1 of 9, 11.1%), and in all cases of intestinal metaplasia (9 of 9, 100%; Fig. 2).



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  		Fig. 1. Immunohistochemical staining for Cdx2 in normal gastrointestinal mucosa and intestinal metaplasia (x200). A, Cdx2 staining is negative-in normal gastric mucosa; B, Cdx2 staining is observed in the nuclei of ileum epithelial cells; C, Cdx2 staining is observed in the nuclei of colon epithelial cells; D, Cdx2 staining is observed in intestinal metaplasia of the stomach.

 


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  		Fig. 2. Immunohistochemical staining for Hep in normal gastrointestinal mucosa and intestinal metaplasia. A, Hep staining is negative-in normal gastric mucosa (x40); B, Hep staining is observed in the cytoplasm of ileum epithelial cells (x40); C, Hep staining is negative-in colon mucosa (x40); D, Hep staining is observed in intestinal metaplasia of the stomach (x100).

 
Cdx2 and Hep expression in gastric carcinoma. Cdx2 was expressed in the nuclei of gastric carcinoma cells in 36.7% (40 of 109) of cases, whereas it was expressed granularly in the cytoplasm of gastric carcinoma cells in 33.9% (37 of 109) of cases. Cdx2 was expressed in both nucleus and cytoplasm in six cases. Hep was expressed in the cytoplasm of gastric carcinoma cells in 54.1% (59 of 109) of cases and exhibited focal positivity (Fig. 3).



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  		Fig. 3. Immunohistochemical staining for Cdx2 and Hep in gastric carcinoma (x200). A, Cdx2 staining is observed in the nuclei of carcinoma cells; B, granular positive staining of Cdx2 is observed in the cytoplasm of carcinoma cells; C, Hep staining is observed in the cytoplasm of carcinoma cells but is absent from adjacent normal tissue.

 
Cdx2 and Hep expression in relation to clinicopathologic features. The expression of Cdx2 in the nucleus was significantly higher in males than in females (P = 0.019), in stages I and II than that in stages III and IV (P = 0.018), and in intestinal-type carcinoma (46.7%) than diffuse-type gastric carcinoma (23.1%, P = 0.027). There was a negative correlation between nuclear Cdx2 expression and lymph node metastasis (P = 0.029). The expression of nuclear Cdx2 was not found to correlate with age, maximum tumor diameter, depth of wall invasion, distant metastasis, vascular invasion, or histologic grade. No significant association was observed between cytoplasmic Cdx2 expression and any of these clinicopathologic features (Table 1).


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  		Table 1. Correlation between Cdx2 nuclear or cytoplasmic expression and clinicopathologic features

 
There was a negative correlation between Hep expression and depth of wall invasion (P = 0.011). There was significantly less Hep expression in diffuse-type carcinoma (46.2%) than in intestinal-type carcinoma (70.0%, P = 0.037) but expression did not correlate with other clinicopathologic features (Table 2).


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  		Table 2. Correlation between Hep expression and clinicopathologic features

 
Prognostic implications of Cdx2 or Hep expression in gastric carcinoma. Cdx2-nucleus-positive patients had significantly higher cumulative postoperative 5-year overall survival rates (57.5%) than Cdx2-nucleus-negative patients (23.2%, P = 0.0009; Fig. 4A). However, Kaplan-Meier survival curves showed no statistically significant correlation between cytoplasmic positive (21.6%) and negative cases (43.1%, P = 0.1601; Fig. 4B). Cases were divided using the Lauren classification and grade of differentiation, and the survival rates of groups with different Cdx2 nuclear expression status were analyzed. In intestinal-type carcinoma, the 5-year survival rate of Cdx2-positive patients tended to be higher than that of Cdx2-negative patients (60.0% versus 33.3%). However, statistical significance was not reached (P = 0.083). In diffuse or mixed-type carcinoma, the Cdx2-positive group (58.3%, 54.6%, respectively) showed a significantly higher 5-year survival rate than the Cdx2-negative group (22.5%, P = 0.0279; 10.0%, P = 0.0162, respectively). In moderately or poorly differentiated carcinoma, the Cdx2-positive groups (56.3% and 59.1%, respectively) had significantly higher 5-year survival rate than that of negative group (15.8%, P = 0.0141; 24.4%, P = 0.0039, respectively). There were too few cases of well-differentiated carcinoma to be analyzed by statistical analysis (Table 3).



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  		Fig. 4. Kaplan-Meier survival curves for overall survival of 109 patients with D2 resected gastric adenocarcinoma. A, categorized by nuclear Cdx2 expression, survival was significantly better for patients with positive Cdx2 nuclear expression than those with negative expression (P = 0.0008); B, Categorized by cytoplasmic Cdx2 expression, no significant difference can be observed between the two groups of patients (P = 0.1064); C, categorized by Hep expression, survival was significantly better for patients with positive Hep expression than those with negative expression (P = 0.003).

 

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  		Table 3. Postoperative survival and Cdx2 nuclear expression in each histologic type and grade

 
The Hep-positive group had significantly higher 5-year survival rates (44.1%) than the Hep-negative group (26.0%, P = 0.003; Fig. 4C). Using a Cox regression analysis for the 109 patients, positive Cdx2 nuclear expression (P = 0.001), Hep expression (P = 0.029), TNM stage (P < 0.001), and vascular invasion (P = 0.015) seemed to be independent prognostic indicators (Table 4).


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  		Table 4. Multivariate analysis of the prognostic factors in 109 cases by Cox proportional hazard model

 
Combined analysis of Cdx2 and Hep expression in distinguishing histologictype. No significant association was observed between Cdx2 and Hep expression by {chi}2 test. When the two markers were combined to evaluate (Cdx2 and Hep expression), positivity was defined as Cdx2 and/or Hep expression. Table 5 shows the correlation between the expression profiles of Cdx2 and Hep expression and Lauren classification. Cdx2 and Hep expression positivity in diffuse gastric carcinoma (50%) was significantly less than that in intestinal-type (93.3%, P < 0.001) or mixed-type carcinoma (76.2%, P = 0.04).


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  		Table 5. Correlation between expression of Cdx2 and Hep combined (Cdx2 and Hep expression) and Lauren classification

 
Survival analysis of combined evaluation of Cdx2 and Hep. On the basis of the expression profiles of Cdx2 and Hep, the 109 patients were categorized as (a) Cdx2+/Hep+, (b) Cdx2+/Hep–, (c) Cdx2–/Hep+, and (d) Cdx2–/Hep–. We analyzed the postoperative survival (log-rank test) and found significant differences between groups a and b (P = 0.0165) and between groups c and d (P = 0.0436). However, there was no significant differences between groups b and c (P = 0.7411), and thus they were combined: (a) Cdx2+/Hep+ (n = 21), (b) Cdx2+/Hep– or Cdx2–/Hep+ (n = 57), (c) Cdx2–/Hep– (n = 31). There were significant differences in survival rates among the three groups (P < 0.0001; Fig. 5). The survival rate of group a was significantly higher than that of group b (P = 0.0039), and the survival rate of group b was significantly higher than that of group c (P = 0.0106) and the survival rate of group a was significantly higher than that of group c (P < 0.0001). Using Cox regression analysis of the 109 patients, Cdx2 and Hep expression (P < 0.001), TNM stage (P < 0.001), and vascular invasion (P = 0.001) seemed to be independent prognostic indicators (Table 6).



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  		Fig. 5. Kaplan-Meier survival curves for three phenotypes of Cdx2 and Hep expression in gastric carcinoma. Patients with Cdx2–/Hep– expression profile had the worst outcome for overall survival among the three phenotypes (P < 0.0001).

 

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  		Table 6. Prognostic value of Cdx2 and Hep expression in multivariate analysis by Cox proportional hazard model

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
In this study, we found that Cdx2 and Hep, especially combined analysis of these two markers are very sensitive and specific for identifying intestinal-type carcinoma, as well as being significant for understanding carcinogenesis and biological behavior, and predicting outcome of patients with gastric carcinoma.

Extensive epidemiologic investigation revealed that intestinal metaplasia increased the risk for development of gastric carcinoma, especially intestinal-type carcinoma. Intestinal metaplasia is an important precancerous lesion in the multisteps of gastric carcinoma proposed by Correa (3). Several studies to elucidate the mechanism underlying Cdx2 expression and its role have been done. The study by Silberg et al. (19) showed that gastric expression of Cdx2 alone was sufficient to induce intestinal metaplasia in mice. A recent study in Cdx2-transgenic mouse by Mutoh et al. (4) provided direct evidence that intestinal-type carcinoma could develop from intestinal metaplasia. Therefore, intestinal metaplasia and intestinal-type carcinoma may partly share similar expression of some markers, such as Cdx2 and Hep.

Cdx2 is one member of the caudal-related homeobox gene family and is an intestine-specific transcription factor. In normal adults, Cdx2 is expressed in mucosa from the duodenum to the distal colon but not in gastric or esophageal mucosa (5). As a transcription factor, Cdx2 has a key role for regulating the proliferation and differentiation of intestinal cells and maintaining intestinal phenotypes (20–24). Thus, Cdx2 is thought to be a marker for intestinal epithelium. Others reported that expression of Cdx2 was also observed in non–gastrointestinal cancers with intestinal phenotype, including pancreatic carcinomas and carcinomas of ampulla of Vater (25, 26). In our study, expression of Cdx2 was detected in the majority of cases of intestinal metaplasia and was higher in intestinal-type than diffuse-type gastric carcinomas. Marchetti et al. (27) reported that chronic acid exposure induced Cdx2 expression in murine esophageal epithelium cells. Ectopic expression of Cdx2 in the gastric mucosa could cause transdifferentiation of the epithelium to an intestinal type (19). Lorentz et al. (24) has shown that Cdx2 plays a key role in the extracellular matrix-mediated intestinal cells differentiation. Liver intestine–cadherin is an intestine-specific cell adhesion molecule and expression in intestinal metaplasia and intestinal-type gastric carcinoma. Hinoi et al.'s study showed that liver intestine–cadherin expression was strongly induced by Cdx2 and was suggested to mediate Cdx2 function in intestinal cell morphogenesis and differentiation (28).

Hep is initially used as a specific marker for diagnosis of hepatocellular carcinoma. Later studies found that Hep was also expressed in other types of cancer. Interestingly, Hep was expressed specifically in intestinal metaplasia (17). Our data are consistent with this result. A study by Lee et al. (29) showed that gastric carcinoma with Hep expression was significantly associated with early stage and well differentiation. The Hep antigen was thought to be a component of the membrane of hepatocellular mitochondria (13). Hep-positive expression in gastric and intestinal mucosa can be explained by common embryologic derivation of liver, stomach, and small intestine, which raises the possibility of expression of common antigens in cells from these organs. However, the mechanism of Hep expression in intestinal metaplasia and gastric carcinoma remains to be investigated.

Taken together, this and other studies have provided evidence that Cdx2 and Hep were special and sensitive markers for intestinal metaplasia and intestinal-type gastric carcinoma. Moreover, our results showed that combined analysis of Cdx2 and Hep could offer a biological variable to identify intestinal-type carcinoma.

In the present study, the Cdx2-positive patients show better outcome than the Cdx2-negative patients. Multivariate analysis revealed that Cdx2 represents an independent prognostic indicator. The results are consistent with reports from Seno and Mizoshita (11, 12). Our data show a negative correlation between Cdx2 expression and tumor stage or lymph node metastatic status, which suggests that Cdx2 may play a suppressive role in the progression of gastric carcinoma. Previous studies suggested that Cdx2 is a tumor suppressor of colon carcinoma. Cdx2 can promote differentiation, inhibit proliferation, and increase sensitivity to apoptosis of intestinal epithelium cells and colon cancer–derived cells (21, 30). Cdx2 is involved in the signaling pathways of some tumor suppressors. For example, Cdx2 can transactivate the transcription of the cyclin-dependent kinase inhibitor p21/WAF1/CIP1 via binding to the promoter of p21 (31), which induces tumor cell growth suppression. Cdx2 also contributes to APC's tumor suppressive effects (32). Cdx2 may have a similar function in gastric carcinoma. Seno et al. reported that Cdx2-positive cases showed a lower Ki-67 index (11), which suggested that Cdx2-positive carcinoma might have low rate of cell proliferation. Recently, Cdx2 was reported to have a close relation to the intestinal-like tumor as well as being a prognostic factor of pancreatic invasive ductal carcinomas and carcinomas of ampulla of Vater (25, 26). Consistent with our viewpoint, Cdx2 is regarded as an intestinal phenotypic marker that implicates better biological behavior and outcome.

As a marker of intestinal epithelium, Cdx2 expression in gastric carcinoma suggests that not only intestinal-type but that a part of diffuse-type carcinomas also have biological characteristics of intestinal-type carcinoma. The prognosis of Cdx2-positive cases is better than that of Cdx2-negative cases in diffuse-type gastric carcinoma. This indicates that although the former did not have morphologic characteristics of intestinal epithelium, it was similar in biological behavior of intestinal-type carcinoma. Cdx2 expression was significantly correlated with prognosis in spite of the same histologic type or differentiation grade. These results suggest that expression of Cdx2 may reflect the biological behavior and prognosis of gastric carcinoma more accurately than histologic classification.

Our data show that Hep-positive expression has a negative correlation with the depth of wall invasion, suggesting that Hep may inhibit the progression of cancer. In this study, Hep expression was associated with good prognosis and was an independent prognostic indicator of gastric carcinoma, which has not yet been previously reported. However, the mechanism of Hep expression in normal or neoplastic tissues is, thus far, still unclear.

Given that Cdx2 and Hep are both intestinal markers and independent prognostic factors, we studied the significance of Cdx2 and Hep coexpression of the prognosis for gastric carcinoma. The patients could be categorized into three groups according to Cdx2 and Hep coexpression profiles: (a) Cdx2+/Hep+, (b) Cdx2+/Hep– or Cdx2–/Hep+, (c) Cdx2–/Hep–. Significant differences of prognosis were found among these groups. Patients with both positive expression of Cdx2 and Hep have the best outcome among the three groups. In contrast, patients with both negative expression of Cdx2 and Hep have the worst outcome among the three groups (relative risk, 3.087; 95% confidence interval, 1.946-7.444). Moreover, the survival rate of group b is significantly different from groups a and c, and indicates that combined evaluation of Cdx2 and Hep predicts the prognosis more accurately than using a single marker of each. Multivariate analysis revealed that the Cdx2 and Hep coexpression profile was an independent prognostic indicator of gastric carcinoma. These results suggest that Cdx2 and Hep coexpression may be a new and powerful indicator for predicting the prognosis of gastric carcinoma. This may be helpful for the individualized therapy of patients with gastric carcinoma.

In conclusion, our findings indicate that the prognosis of gastric carcinoma patients with Cdx2- or Hep-positive expression is significantly better than that with Cdx2- or Hep-negative expression. We revealed for the first time that combined analysis of Cdx2 and Hep may provide additional evidence of biological behavior to histologic type and might be useful to predict the prognosis for gastric carcinoma.


   Footnotes
 
Grant support: Key Technologies Research and Development Program 2002BA711A06 and National Basic Research Priorities Program 973 Project 1998051203 from the Ministry of Science and Technology of China, and grant H020920030390 from the Beijing Science and Technology Commission.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 2/ 7/05; revised 5/10/05; accepted 6/ 7/05.


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 Materials and Methods
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