Trial Information

The Expression and Effect of Cyr61 in Urinary Tract Transitional Cell Carcinoma

1. Patients: as shown in inclusion criteria

2. Surgical samples Surgical specimens from included patients with TCC who underwent
surgery will be collected. The samples will be examined histologically for the presence
of tumor cells. Independent pathologists who are blinded to the records of the patients
will perform histological examinations. Tumor grade is determined on the basis of
cytological features. There are three grades of TCC, as proposed by Mostofi et al. and
adopted by the American Bladder Tumor Registry of the World Health Organization. These
are based on degree of anaplasia: Grade 1 tumors show mild cytological atypia and rare
mitosis; Grade 2 tumors show moderate cytological atypical and the presence of mitotic
figures; and Grade 3 tumors show severe cytological atypia and frequent mitotic
figures. Lymph node metastasis, lymphatic invasion, and venous invasion will be
evaluated by pathology reports and image study. Clinical staging of TCC is based on the
American Joint Committee on Cancer staging (AJCC) TNM system.

3. Immunohistochemical stain Paraffin-embedded, formalin-fixed surgical specimens were
collected for Cyr61 immunohistochemical staining. Heat-induced epitope retrieval will
be performed with a pressure cooker and TRIS buffer (pH 9.0) for 2 min. They will be
allowed to cool for 15 min, rinsed in distilled water twice and in PBS for 5 min. The
sections will then be treated with 0.5% hydrogen peroxidase/PBS for 20 min at room
temperature to block the endogenous peroxidase. They will subsequently be blocked with
10% normal goat serum for 30 min at room temperature, and then incubated with
polyclonal anti-Cyr61 antibody (Santa Cruz, CA) at 4°C overnight. The specific
antibodies will be omitted in sections as negative controls. The sections will be
washed three times in PBS/0.2% Triton X-100 for 10 min and incubated with
biotin-conjugated secondary antibodies (DAKO, Carpinteria, CA) for 1 hour at room
temperature the following day. They will then be incubated with the
avidin-biotin-peroxidase reagent (DAKO) for another 1 hour at room temperature. After
three washes in PBS/0.2% Triton X-100 for 10 min each, the reactions on sections will
be detected with peroxidase substrate containing diaminobenzidine chromagen (DAKO). The
slides will be counterstained with hematoxylin. If >50% of the tumor cells are
positively stained, the specimen will be grouped as "positive". All other staining
results will be regarded as negative.

4. Statistical analysis For statistical analysis, P values are based on two-sided,
parametric Student's t tests. A P value of <0.05 on the basis of at least three
independent sets of experiments is considered to be statistically significant. In the
clinical sets of project, Chi-squire test and Student's t test will be used to study
the association of Cyr61 expression with single clinical factors (age, gender,
pathology, and grade). Kaplan-Meier survival curves for patients with positive versus
negative Cyr61 expression will be plotted and log-rank test will be used for comparing
the equality of the two survival curves. Cox proportional hazard model will also be
developed to correlate the clinical characteristics, survival, and the expression of
Cyr61.