Stathmin Antibody Validation
Stathmin expression was evaluated in ten breast cancer cell lines and in a colorectal cancer cell line by western blot and tissue microarray. Western blots showed bands at the appropriate migration distances compared to molecular weight standards (). AQUA scores were collected for an overlapping series of cells lines that showed a range of expression with lowest expression observed in SKBR3 cells (AQUA score = 63) and highest in UACC 812 (AQUA score = 1,903). In addition, stathmin siRNA knockdown was performed in a BT20 cell line indicating decreased stathmin expression after 24 hours () and providing evidence of antibody specificity. Finally, two different lots of stathmin antibody were purchased and tested on breast TMAs cored from the same tissue block to confirm reproducibility of the antibody.
Stathmin Expression Pattern in Normal Breast Tissue and the Yale University Cohort
Stathmin was measured in normal epithelial ducts and lobules using a normal breast TMA (n= 110) with two-fold redundancy to determine the level of expression in normal breast tissue. Consistent cytoplasmic localization was observed and stathmin average expression scores in normal breast tissue showed mean and median AQUA scores of 25 and 18, respectively, and a score range between 13 and 127. A frequency distribution of these scores is shown (). Normal tissue TMA and an Index TMA were also used to determine the threshold of signal to noise. We found AQUA scores below 18 represent non-specific or background signal (images not shown). The threshold for detection was determined by measuring a control slide in which the primary antibody was omitted. All subsequent AQUA scores were normalized to this AQUA score range using an Index TMA.
Next we examined stathmin expression within the Yale University cohort (n= 651), a primary breast cancer cohort previously used to study many other biomarkers 33, 36, 37
. Stathmin showed cytoplasmic localization similar to our observations in normal breast tissue. A stathmin AQUA score was computed for each case by averaging observations from two TMA spots and normalizing using the Index TMA. The frequency distribution of stathmin expression scores in the Yale University cohort is shown along with the mean and median AQUA scores of 62 and 30, respectively, with a score range of 4 to 1,092 (). A total of 474 cases had sufficient tumor tissue for analysis. The mean stathmin expression score in normal breast tissue was used in all analyses to stratify patients as high expressers (AQUA score ≥ 25) versus low (AQUA < 25) with 41.8% classified as low stathmin expressers compared with 58.2% classified as high expressers in the Yale University cohort ()
Univariate analysis of tumor and clinical risk factors for overall survival in the Yale University cohort.
Premenopausal status, high nuclear grade, and ER negative status correlated most frequently with high stathmin expression compared to low expression (34.4% vs. 22.6%) (P = 0.005); (34.6% vs. 22.12%) (P = 0.0004), and (50.6% vs. 38.9%) (P = 0.0136). Stathmin expression did not correlate with tumor size, nodal status, PR or HER2 status (data not shown).
Stathmin Prognostic Value in the Yale University Cohort
The expression status of stathmin was evaluated for association with overall survival. Using Kaplan Meier analysis, patients with low stathmin expression (n=195) showed improved survival compared to those with high expression (n= 273) (66.6% vs 53.3 %, respectively; log-rank, P = 0.003; ). When stratified by ER status, stathmin retained prognostic value with ER positive/low stathmin expressers (n = 114) showing improved survival compared to ER negative/high stathmin expressers (n=136; 70.9% vs 48%; log-rank, P = 0.012; data not shown). Similarly, patients stratified by HER2 status indicated improved survival with HER2 negative/low stathmin expression (n= 156) compared to HER2 positive/high stathmin expression (n= 60) (66.4% vs 53.0% respectively; log-rank, P = 0.006).
Kaplan Meier survival analysis in the Yale University Cohort
In univariate analysis, high stathmin expression, postmenopausal status, nodal metastasis, ER negative and PR negative status were associated with worse overall survival (OS) (HR = 1.483, 1.434, 2.338, 1.402, 1.421, P = 0.0006, <0.001, 0.015, and 0.013, respectively) while small tumor size and low nuclear grade were associated with improved OS (HR = 0.493 and 0.614, P <0.001 and 0.024, respectively) (). Using the Cox PH model, multivariate analysis indicates that high stathmin expression has independent prognostic value with a hazard ratio of 1.566 (95% CI, 1.091 to 2.248, P = 0.015; ). Large tumor size, nodal metastasis, and ER negative status were also associated with worse OS.
Multivariate analysis of tumor and clinical risk factors for overall survival in the Yale University cohort.
Prognostic value for the ratio of MAP-tau to stathmin in the Yale University Cohort
We have shown that high MAP-tau is associated with favorable outcome (see ). Since MAP-tau and stathmin play opposite but complimentary roles in tubulin stabilization, we assessed the ratio of MAP-tau to stathmin expression levels. The frequency distribution of ratio scores for MAP-tau to stathmin expression in our cohort showed mean and median AQUA scores of 13 and 7, respectively, with a ratio score range of 0.098–184 and is shown in . The median ratio score of 7 was used as the cut-point to differentiate high MAP- tau/stathmin expressers from low MAP-tau/stathmin expressers (). Low MAP-tau/stathmin expression ratio correlated with premenopausal status, high nuclear grade, ER and PR negative status, and HER2 positive status.
Using Kaplan Meier survival analysis, patients with high MAP-tau/stathmin expression (n= 231) showed improved survival compared to those with low MAP-tau/stathmin expression (n= 237) (65.4% vs 52.5 %, respectively; log-rank, P = 0.0009; ). When stratified by ER status, in ER+ patients, the MAP-tau ratio has no prognostic value. But in ER- patients, high MAP-tau/stathmin expression showed improved survival compared to ER- and low MAP-tau/stathmin expression (n=139; 66.1% vs 45.5%; log-rank, P = 0.009). When patients were stratified by HER2 status, improved survival with high MAP-tau:low stathmin expression was seen in both HER2 positive and negative subgroups.
In univariate analysis, high MAP-tau/stathmin expression ratio was associated with improved OS (HR = 0.679; 95% CI, 0.517 to 0.891, P= 0.0053, see ). The ratio was also significant by multivariate analysis where patients with high MAP-tau/stathmin expression showed a hazard ratio of 0.609 (95% CI, 0.422 to 0.879, P = 0.008; ).
As a destabilizing protein, we hypothesized that stathmin would have an inverse relationship to MAP-tau. shows that in normal breast tissue that is not seen. However, in breast tumors, shown in 3b, there are many cases with high stathmin and low MAP-tau and visa versa and they define a subset of cases with the hypothesized inverse relationship. However, there are also many cases that are low for both proteins and no inverse relationship is seen. Since these cases predominate, no statistically significant inverse relationship is seen when assessing the whole cohort.
Correlation between stathmin expression and MAP-tau expression in normal versus Yale Cohort breast tissue