Relationship between nm23-H1 gene product expression and lymphatic and blood vessel invasion in esophageal squamous cell carcinoma

Background: We assessed the relationship between nm23-H1 gene product expression and lymphatic and blood vessel invasion in patients with esophageal squamous cell carcinoma. Formalin-fixed and paraffin-embedded tissue sections from 45 patients who were treated surgically were used in this study. Pathologists graded lymphatic and blood vessel invasion in each of the tissue samples. Expression of nm23-H1 gene product was determined using a specific monoclonal antibody. Results: Expression of nm23-H1 gene product was present in 17 (37.8%) cases. We found an inverse correlation between nm23-H1 gene product expression and lymphatic vessel invasion although the data for lymphatic vessel invasion and lymph node metastasis did not reach statistical significance. There was no correlation between nm23-H1 gene product expression and blood vessel invasion. Conclusions: In patients with esophageal squamous cell carcinoma, there appears to be an inverse relationship between nm23-H1 gene product expression and lymphatic vessel invasion. Our data does not demonstrate any correlation between nm23-H1 gene product expression and blood vessel invasion. Introduction Expression of nm23-H1 gene product is thought to play a specific biological role in suppressing tumor metastasis [1,2]. A number of studies of human malignant tumors including breast [3–6], gastric [7,8], colorectal [9,10], hepatocellular [11], and ovarian [12] carcinomas have demonstrated a correlation between reduced expression of nm23 mRNA or its gene product and increased metastasis, resulting in poorer prognosis. Although very little attention has been given to the role of nm23-H1 gene product in esophageal squamous cell carcinoma, two studies report on the relationship between nm23-H1 expression and patient survival [13,14]. It is general knowledge that vessel invasion is the first and necessary step at the outset of the metastatic cascade. What remains to be seen, however, is whether nm23-H1 gene product expression is associated with lymphatic or blood vessel invasion in patients with esophageal squamous cell carcinoma. The present study examines this question. Materials and Methods Patients and Tissue Samples Forty-five randomly selected patients (39 men, 6 women) with primary esophageal squamous cell carcinoma who were treated surgically at Miyazaki Medical College were included in this study. TNM staging was accomplished in all patients [15]. Surgically resected tissue samples previously fixed in formalin and embedded in paraffin were used in this study. Following the Guidelines for the Clinical and Pathologic Studies on Carcinoma of the Esophagus (Japanese Society for Esophageal Diseases, 1999), pathologists graded lymphatic and blood vessel invasion in each of the tissue samples. Briefly, lymphatic vessel invasion was graded according to the following: ly0: none; ly1: mild; ly2: moderate; and ly3: severe. Blood vessel invasion was also graded as follows: v0: none; v1: mild; v2: moderate; and v3: severe. Immunohistochemical Studies The specific monoclonal antibody against nm23-H1 gene product (Novocastra Laboratories, Newcastle, UK) was used to detect gene product expression. Before staining, serial 4 μm-thick sections were deparaffinized in three changes of lemosol and rehydrated through a descending series of ethanol. These sections were then immersed in 0.6% H2O2 in methanol for 20 minutes at room temperature to block endogenous peroxidase activity. After blocking nonspecific protein bindings by an overnight incubation with Block Ace (Dainippon Inc., Osaka, Japan), the sections were incubated with primary antibodies (1:200) at 4oC overnight. Subsequently, sections were incubated with the secondary antiserum (1:500) for 1 hour, followed by an incubation with peroxidase antiperoxidase (PAP) complex for 30 minutes at room temperature. The sections were visualized using a Diaminobenzidine/Metal Concentration (10x) and Stable Peroxide Substrate Buffer (1x) system (Pierce, Rockford, IL, USA). The sections were then washed with water and counterstained with hematoxylin. Immunohistochemical results were assessed semiquantitatively by two authors, taking into account the percentage of nm23-H1 protein positive cancer cells within the maximum cut-surface specimen of the tumor tissue. Patients with greater than 50% of cancer cells staining more intensely than stromal cells were considered positive for nm23-H1 gene product expression. Statistical Analysis Data was analyzed using the X test with Yates’ correlation. A p value less than 0.05 was considered significant. Results Expression of nm23-H1 gene product was present in 17 (37.8%) cases and appeared diffusely throughout the cytoplasm of the cancer cells. Table 1 shows the relationship between nm23-H1 gene product expression and clinical characteristics. Expression of nm23-H1 gene product was not correlated with these clinical factors. Lymph node metastasis in positive nm23-H1 gene product cases was 9/17 (52.9%); in negative cases, 13/28 (46.4%). There was no significant correlation between nm23-H1 gene product expression and lymph node