CKAP2 (cytoskeleton-associated protein2) is a new prognostic marker in HER2-negative luminal type breast cancer

<div><p>Background</p><p>Recently, we reported cytoskeleton-associated protein2 (CKAP2) as a possible new prognostic breast cancer marker. However, it has not yet been applied in clinic. Therefore, clinical significance of CKAP2 was evaluated in comparison with that of Ki-67 in a cohort of breast cancer patients, and the expression difference was analyzed in cell cycle-arrested cancer and fibroblast cells.</p><p>Methods</p><p>A total of 579 early breast cancer patients who underwent surgery at the National Cancer Center Hospital in Korea between 2001 and 2005 were accrued. CKAP2-positive cell count (CPCC) and Ki-67 labeling index (Ki-67LI) were evaluated by immunohistochemcal staining. The immunocytochemical staining patterns of CKAP2 and Ki-67 were analyzed in HeLa and human fibroblast cells after synchronization by double thymidine block.</p><p>Results</p><p>Although there was a significant correlation (R = 0.754, <i>P</i> < 0.001) between CPCC and Ki-67LI, only CPCC was correlated with DFS in overall population (HR, 2.029; 95% CI, 1.012–4.068; <i>P</i> = 0.046) and HER2-negative luminal subgroup (HR, 3.984; 95% CI, 1.350–11.762; <i>P</i> = 0.012) by multivariate analysis. In immunocytochemical staining, more than 50% of serum-starved or non-mitotic cell phase HeLa cells were positive for Ki-67, in comparison to the low CKAP2-positivity, which might explain the prognostic difference between CPCC and Ki-67LI.</p><p>Conclusions</p><p>The current study showed that CPCC but not Ki-67LI is an independent prognostic indicator in early breast cancer, more specifically in HER2-negative luminal breast cancer. The difference between two markers may be related to the lower background expression of CKAP2 in cancer cells.</p></div

Ki-67-positive or CKAP2-positive cell ratios in human fibroblast and HeLa cells after cell cycle synchronization.

<p>Peak chromatin CKAP2 positive cell rates were observed in 7–8 hours after thymidine double block for HeLa cell (A), and in 8–10 hours for human fibroblast cell (B). In contrast, peak Ki-67-positive cell rate was not clear in human fibroblast (C) and HeLa cells (C). In serum starved HeLa cells for 48 (48 SFM), 72 (72 SFM or serum free media) hours, Ki-67-positive cell rate was about 50%, in contrast to low CKAP2-positive rate. The CKAP2 or Ki-67 positive ratios were determined by calculating the positive cells out of total cells, and counting positive cells were performed three times. Cell cycle synchrony was shown in human fibroblast cells (E) and HeLa cells (F). Expression patterns of CKAP2 and cell cycle dependent proteins such as phospho-S10-histone 3 (p-H3), Rb, phospho-Rb-S807, S811 (p-Rb), cyclin D1, cyclin E, cyclin A, cyclin B1, and GAPDH in various cell phases in human fibroblast (G) and HeLa cell (H) was analyzed by Western blot. Cell cycle dependent proteins for Western blot analysis were indicated on the right side of each strip. x-axis in A-D, the release time after the double thymidine block or incubation time in serum free media for 24, 48, or 72 h; y-axis in A-D, chromatin CKAP2-positive or Ki-67-positive cell ratios. For E and F, the number of cells was plotted against DNA content at the indicated release time points after the double thymidine block. Serum-starved samples for 48 h (48) and 72 h (72), and samples cultured for indicated hours (0, 2, 4, 6, 7, 8, and 10) after thymidine double block were indicated at the top of G and H.</p

Multivariate analysis of CPCC or Ki-67LI for correlation with DFS in breast cancer subgroups by Cox proportional hazard regression model.

<p>Multivariate analysis of CPCC or Ki-67LI for correlation with DFS in breast cancer subgroups by Cox proportional hazard regression model.</p

Kaplan-Meier plot for CPCC and KI-67LI in breast cancer.

<p>CPCC showed significant correlations with poor DFS in total cases (A) or HER2-negative luminal cases (B), but neither in HER2-positive (C), nor in triple-negative cases (D). Ki-67LI showed a significant correlation with poor DFS in total cases (E), but not in any subgroups such as HER2-negative luminal cases (F), HER2-positive (G), and triple-negative cases (H). The <i>P</i> value was determined by log-rank test. The X-axis is DFS in months, and the Y-axis, DFS probability. CPCC = chromatin CKAP2-positive cell count; Ki-67LI = Ki-67 labeling index.</p

Immunohistochemical staining for CKAP2 and Ki-67 in breast cancer tissues.

<p>A case with higher CKAP2-positive cells (A) showed higher Ki-67-positive cells (B). A case with lower CKAP2-positive cells (C) showed lower Ki-67-positive cells (D). The black arrow heads indicate cells with positive chromatin CKAP2 staining. The red ones indicate post-cytokinetic cells. Higher magnification for CKAP2 staining was shown in the box for A. One hundred μm rulers are shown.</p

Clinicopathologic characteristics.

<p>Clinicopathologic characteristics.</p