Negative control was obtained by omitting the primary antibody. Whole tissue sections of 5 randomly selected cases were stained in parallel as validation of the TMA results. c. EPZ-5676 leukemia Assessment of tumor budding Tumor budding was defined as detached single cells or clusters of 5 cells. Cases were evaluated Inhibitors,Modulators,Libraries for tumor budding using a 10 in 10 approach. Briefly, whole tissue sections of each case underwent immunohisto chemistry for AE1AE3 staining. The 10 densest hot spots of tumor budding were evaluated at high magnification and counted. The average number of Inhibitors,Modulators,Libraries buds per case was obtained. Although tumor budding is described to occur mostly at the invasive front of cancers, in our PDAC series we frequently observed the presence of buds within the main tumor body as well.
Using a receiver operat ing characteristic curve approach, a cut off score of 10 buds on average was identified as most discriminatory for survival. Cases with an average of 10 buds were classified as high grade budders. those with 10 buds were assigned as low grade budders. d. Assessment of RKIP staining Immunohistochemistry was evaluated by Inhibitors,Modulators,Libraries estimating visually the percentage of positive cells per tissue microarray punch in 5% intervals. In the case of multiple tumor punches per localization, the average protein expression was calculated across all punches from the same localization. The end result was that each patient had a final protein expression score for the main tumor body, the tumor buds, the matched lymph node metastases, the precursor lesions and the normal pancreatic tissue.
Evaluation was performed blinded Inhibitors,Modulators,Libraries to clinical endpoints. e. Statistical analysis In order to determine a valid cut off score for RKIP expression, receiver operating characteristic curve analysis was performed, using the end point of tumor budding. A threshold value of 10% was identified. Association of RKIP expression with categorical clinicopathological features was performed using the Chi Square test with continuity correction and the Fishers Exact tests. for continuous variables such as age and tumor size, the non parametric Wilcoxons Rank Sum test was used. For matched analyses, the Wilcoxons Signed Rank test for pairs and the Cochran Mantel Haenszel test for three of more groups were used. Logistic regression analysis was used to determine the odds ratio and 95%CI for loss of RKIP expression with certain clinicopathological features.
Missing data were few and were assumed to be missing Inhibitors,Modulators,Libraries at random. No imputation for missing values was performed. Univariate survival time analysis was performed using the log rank test and differences plotted using Kaplan Meier curves. P values Calcitriol proliferation 0. 05 were considered statistically significant. Correction for multiple hypothesis testing was not carried out. Analyses were carried out using SAS. Results Patient characteristics and RKIP expression One hundred and twenty patients with PDAC were included in this study.