CD133, CD166, CD44, CD24, CD90, EpCAM, and ABCG5 were evaluated for both membrane and cytoplasmic staining; ALDH1 was exclusively evaluated in the cytoplasm of tumor buds. The number of CD166, CD44s, http://www.selleckchem.com/products/PF-2341066.html EpCAM, ALDH1, CD133, CD24, CD90, and ABCG5 positive tumor buds was then evaluated in the area of densest tumor budding as determined by CK22 staining. Statistical analysis Univariate survival analysis was carried out using the Kaplan-Meier method and log rank test. Two multivariable Cox regression analyses were performed. First, to test the independent prognostic value of tumor budding, the effects of pT stage, pN stage, tumor grade, and vascular invasion were adjusted for.
Subsequently, because of the small number of positive cases, only 2 variables could be entered into the multivariable Cox regression analysis along with positive expression of the protein in tumor buds, hence pT classification and pN classification were selected. The assumption of proportional hazards was verified prior to this analysis. Hazard ratios (HR) and 95% CI were obtained to determine the prognostic effect of positive cases adjusting for pT and pN. Kendall��s correlation coefficient (r) was obtained for correlation analysis of markers. P < 0.05 was considered statistically significant. RESULTS Prognostic value of tumor budding In order to confirm the prognostic value of tumor budding in our series, cases were divided into 3 groups based on the distribution of number of tumor buds: those with < 40 buds, between 41-60 buds, and finally those with > 60 buds per 20 �� field.
The greater the number of tumor buds the more unfavorable was the prognosis both in univariate (P < 0.001) and multivariable analysis with pT, pN, tumor grade, and vascular invasion (HR: 1.6, 95% CI: 1.2-2.1). Expression of putative stem cell markers within tumor buds CK22 staining was used to identify regions of densest tumor budding with epithelial cells exclusively immunoreactive for the protein. Staining for ABCG5, ALDH1, CD133, CD166, CD24, and CD44s could be observed in both tumor cells and inflammatory or stromal cells. EpCAM staining was predominantly limited to expression in tumor cells whereas CD90 was almost always expressed by stromal cells and only in 3 cases in the tumor itself. Marker expression was then evaluated in the area of densest budding. Representative immunostains for all markers are shown in Figure Figure2.
2. Only one case (1.03%) was positive for CD90, while 5 (5.1%) and 6 (6.1%) cases were positive for CD44s and CD133, respectively. On the other hand, a considerably larger number of positive cases was found to express ALDH1 (16/97, 16.5%), CD24 (16/99, 16.2%) and CD166 (34/100, 34%). Finally, ABCG5 and EpCAM staining Batimastat were frequent events with 39/97 (40.2%) and 69/100 (69%) positive cases, respectively (Figure (Figure33).
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