880, 0.863, 0.729, 0.699, and 0.799 respectively, and all these comparisons were statistically

significant at p ≤ 0.0001 (Figure 4A–E). Figure 3 Representative example of human breast cancer specimens from TMA3 that expressed either low (left panel) or high (right panel) eIF4E. Matching specimens from the same patient are shown for c-Myc, cyclin D1, ODC, TLK1B, and VEGF (200 × magnification). Figure 4 CP673451 Correlation of immunohistochemical expression of eIF4E vs c-Myc [A], cyclin D1 [B], ODC [C], TLK1B [D], VEGF [E] from TMA3. Figures represent the integrated optical density (IOD) of immunohistochemical staining intensity normalized to cytokeratin. Protein expression of eIF4E and TLK1B were also find more compared by western blot analysis [F], in which values represent expression of eIF4E and TLK1B as fold- over benign. All comparisons were done using Spearman’s rank correlation. Rho- and p- values for each comparison are displayed in each panel. Western blot analysis: Correlation of eIF4E with TLK1B We have previously shown by western blot analysis that the expression of eIF4E correlated with that of TLK1B [23]. As further validation of our TMA results, we also compared eIF4E with TLK1B using the corresponding fresh-frozen specimens from the same tumors as those used for TMA3 (Figure 4F). Due to limited

amounts of fresh-frozen specimens, the other proteins were not analyzed. Protein expressions of eIF4E to TLK1B were positively correlated (rho value 0.485, p

value 0.0054). Non-correlation to independent markers We have previously demonstrated that western blot analysis MGCD0103 mw of eIF4E did not correlate with node status, ER, PR, or HER-2/neu [18, 19]. In the current study, expression of eIF4E (by both TMA-IHC and western blot) was also compared to ER, PR, and HER-2/neu expression. There was no correlation of eIF4E on TMA3 with any of these independent markers by either TMA-IHC or western blot analysis of eIF4E (Table 2). Table 2 Lack of correlation of ER, PR, or HER-2/neu with eIF4E     95% Confidence Interval       Rho Value Lower Upper n P TMA expression of eIF4E a eIF4E and ER -0.137 -0.469 0.228 31 0.452 eIF4E and PR -0.069 -0.413 0.293 31 0.707 eIF4E and HER-2/neu -0.013 -0.406 0.384 25 0.949 Western blot expression of eIF4E b eIF4E and ER -0.192 -0.479 0.132 39 0.237 eIF4E and PR -0.295 -0.558 0.023 39 0.069 eIF4E and Dimethyl sulfoxide HER-2/neu -0.143 -0.469 0.216 32 0.425 a For the first three rows, comparisons were made of immunohistochemical staining of each protein normalized to cytokeratin to ER, PR, and HER-2/neu.bLast three rows, comparison of protein expression of eIF4E assayed by western blot (fold- over benign) to ER, PR, and HER-2/neu. All comparisons were done using Spearman’s Rank Correlation. Discussion In the current study, we have analyzed the expression of eIF4E along with 5 of its downstream effector proteins in human breast carcinoma specimens using immunohistochemical analysis of TMAs.