| To explore the expression and clinical significance of Tiam - 1 protein in NSCLC.MaterialsPatients and SpecimenWe study 86 patients with primary NSCLC who had definitive pulmonary resection with complete ipsilateral mediastinal lymph nodes resection at the department of thoracic surgery of the First affiliated clinical hospital of China Medical University between April 1999 and June 2000. All patients underwent potentially curative surgery without pre-operative adjuvant therapy. There were 61 men and 25 women, with a mean age of 58.19 years (range 36 ~76). The histological typing was performed according to the revised WHO classification (1998). 38 patients were SQC and 47 were AC. All of the specimens, fixed in 4% formaldehyde, were buried in paraffin. Among them,47 cases have follow - up data more than 3 years.Antibodies: Rabbit anti - human Tiam - 1 antibody was offeredby Santa Crutz Ltd. USA, working dilution 1:100.MethodsImmunohistochemistiy :Mounting of slide; All specimens of paraffin - embedded blocks were mounted to 4um section, and fixed with acetone for 10 min, air -dried.Staining; All subsequent steps were performed at room temperature. The sections were incubated with methanol containing 0.3% hydrogen peroxide for 20 minutes to block endogenous peroxidase activity. After treatment with PBS containing 5% normal serum for 20 mins, the sections were incubated with monoclonal Tiam -1 antibody at a dilution of 1:100 for 2 hours in a moist chamber. After treated with biotinylatede rabbit anti - mouse immunoglobulin dilution of 1: 100 for 30 minutes, the sections were incubated with S ?P for 20 mins, and followed by DAB and hydrogen peroxide mixture. The sections were counterstained with hematoxylin, dehydrated in graded eth-anol, cleared in xylene and mounted. Negative controls were established by replacing the primary antibody (Tiam -1) with PBS.Evaluation of the specimens; The comprehensive criteria of im-munostaining positive degree for Tiam ? 1 expression in cancer cells were based on staining intensity and positive cell percentage. The staining intensity can be classified to: Lack of color (0 point) , light yellow (1 point) , brown (2 points). The percentage of Tiam -1 positive cancer cells; negative (0 point) , < =10% (1 point) , 10% ~ 50% (2 points) , 50% -75% (3 points), and >75% (4 points). The Points of both types should be multiplied. And if the results aremore than 3, it could be regard as positive cases ( + ) , the rest was defined as negative cases ( - ). The final slides were underwent double - blind evaluation by two pathologic doctors using light microscopy.Statistical analysis the x2 test was used to compare the association between Tiam -1 expression level and clinic pathologic factors. P values < 0.05 were considered significant. Cox regression was employed to analyze prognosis.ResultIn normal alveolar epithelium and lymph tissue, Tiam -1 expression defect was found; In normal epithelial cell of bronchiole it shows slight positive. Expression defect could be found in mucoid and transparent cell carcinoma, and weak expression in alveolar cell carcinoma. In most of adenocarcinoma and squamous carcinoma ideal positive expression often be found in cell plasm and distribute evenly, but no clear expression in membrane. Tiaml in metastastic cell in lymph node show strong expression. The result shows that there are significant different between normal pulmonary tissue group and tumor tissue group (P<0.01) .When study the relationship between Tiam - 1 expression and clinical pathologic factors, we found that the overexpression of Tiaml has not significant correlation with age, sex, histology type, diameter of tumor and clinical staging( P > 0.05 ). But it has strong relationship with the state of tumor metastasis and differentiation level of cancer cell. Among 86 NSCLC cases, 55 high and moderate differentiated cases have 40% positive percentage and in 31 undifferentiated cases,the positive incidence hold 67.74%. The positive rate of Tiaml... |
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