Multivariate analysis of CPCC or MAI for correlation with RFS using Cox proportional hazard regression model at two institutions.

<p>The co-variables of age at diagnosis (groups 1–5), T stage (T1, T2 or T3), N stage (N0, N1, N2 or N3), HER2 status (negative or positive), ER status (negative or positive), PR status (negative or positive), institution (KCCH or IIPH), and year of surgical resection (2000, 2001, 2002, 2003, 2005, or 2006) were used in the multivariate analysis, treating each co-variable as a categorical variable.</p>†<p><i>P</i> for linear trend.</p><p>KCCH  =  Korean Cancer Center Hospital; IIPH  =  Ilsan Inje Paik Hospital; CI  =  confidence interval.</p

Univariate analysis of clinicopathological factors for correlation with RFS.

<p>* KCCH  =  Korean Cancer Center Hospital; IIPH  =  Ilsan Inje Paik Hospital; CI  =  confidence interval; HER2  =  human epidermal growth factor receptor 2; ER  =  estrogen receptor; PR, progesterone receptor; NOS, not otherwise specified.</p><p>–: not analyzed due to limited number of cases.</p

Prognostic significance of proliferation activity in breast cancer subgroups.

<p>Shown are Kaplan-Meier CPCC plots for the luminal A (A), HER2-positive (B), and TN (C) subgroup cases, as well as MAI plots for the luminal A (D), HER2-positive (E), and TN (F) subgroup cases. The <i>P</i> values were determined by log-rank test, and the hazard ratios (HRs) and their 95% confidence interval (CI) by the Cox proportional hazard regression model according to the co-variables of age, T stage, N stage, institution, and year of surgical resection. The X-axis is RFS in months, and the Y-axis, RFS probability. CPCC  =  chromatin CKAP2-positive cell count; MAI  =  mitotic activity index.</p

CKAP2 immunohistochemical staining in breast cancer tissues.

<p>CKAP2-positive cells are rare in normal breast ductal cells (A), but present variably in breast cancer tissues (B). The chromatin CKAP2-positive cell numbers were variable: low as in (C), or high as in (D). One hundred µl rulers are shown. The arrows indicate chromatin CKAP2 staining.</p

Clinicopathological characteristics of breast cancer patients.

<p>^*Luminal A subgroup: cases with hormone receptor (HR)-positive and HER2-negative status.</p><p>^**HER2-positive subgroup: cases with HER2-positive status with or without HR positivity.</p><p>^***Triple-negative subgroup (TN): HR-negative and HER2-negative status.</p><p>IIPH  =  Ilsan Inje Paik Hospital; KCCH  =  Korean Cancer Center Hospital; ER  =  estrogen receptor; PR  =  progesterone receptor; HR  =  hormone receptor; HER2  =  human epidermal growth factor receptor 2; CPCC  =  chromatin CKAP2-positive cell count; MAI  =  mitotic activity index.</p

Correlations of CPCC and MAI with RFS.

<p>Shown are a Kaplan-Meier CPCC plot for total, KCCH, or IIPH cases (A-C) and an MAI plot for total, KCCH, or IIPH cases (D-F). The <i>P</i> values were determined by log-rank test, and the hazard ratios (HRs) and their 95% confidence interval (CI) by the Cox proportional hazard regression model according to the co-variables of age, T stage, N stage, HER2 status, estrogen receptor status, progesterone receptor status, institution, and year of surgical resection. The X-axis is RFS in months, and the Y-axis, RFS probability. CPCC  =  chromatin CKAP2-positive cell count; MAI  =  mitotic activity index.</p

Inter-observer correlations of CPCC or MAI and correlation between CPCC and MAI.

<p>A. Inter-observer correlation of CPCC among 100 cases. B. Inter-observer correlation of MAI among 100 cases. C. Correlation between CPCC and MAI in total 375 cases. Two data points are outside the axis limits in C. The slope and Y intercept are shown. The correlations were calculated by two-sided Spearman test. CPCC  =  chromatin CKAP2-positive cell count; MAI  =  mitotic activity index.</p

Multivariate analysis of CPCC or MAI for correlation with RFS in breast cancer subgroups by Cox proportional hazard regression model.

<p>The co-variables of age at diagnosis (groups 1–5), T stage (T1, T2 or T3), N stage (N0, N1, N2 or N3), institution (KCCH or IIPH), and year of surgical resection (2000, 2001, 2002, 2003, 2005 or 2006) were used in the multivariate analysis, treating each co-variable as a categorical variable.</p><p>*Luminal A subgroup: cases with hormone receptor (HR)-positive and HER2-negative status.</p><p>**HER2-positive subgroup: cases with HER2-positive status with or without HR positivity.</p><p>***Triple-negative subgroup (TN): HR-negative and HER2-negative status.</p>†<p><i>P</i> for linear trend.</p

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