Relapse-free survival of statistically standardized continuous RT-PCR estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2): NCIC CTG MA.14.

Recent ASCO/CAP guidelines focus on decision making associated with the presence/absence of continuous breast biomarkers. Statistical standardization (SS) is demonstrated as a method to evaluate the effects of continuous RT-PCR biomarker expression levels on breast cancer outcomes. MA.14 allocated 667 postmenopausal patients to tamoxifen based on locally determined ER/PR. Of 299 available patient tumor samples, 292 passed internal quality control. All tumors were centrally assessed by RT-PCR ER/PR/HER2 with each biomarker's z-scores categorized: ≥1.0 standard deviation (SD) below mean; <1.0 SD below mean; ≤1.0 SD above mean; >1.0 SD above mean. Log-rank statistics tested univariate differences in breast cancer relapse-free survival (RFS). Continuous SS-ER/PR/HER2 were assessed in multivariate Cox step-wise forward regression, adding a factor if p ≤ 0.05. Sensitivity analyses examined an external HER2+ cut-point of 1.32. Patients whose tumors were tested were representative of the MA.14 population (p values = 0.18-0.90). At 9.8 years median follow-up, SS-ER did not univariately impact RFS (p = 0.31). SS-PR values above the mean (z ≥ 0.0) had the best univariate RFS (p = 0.03). SS-HER2 also univariately impacted RFS (p = 0.004) with lowest (z-scores ≤ -1.0) and highest (z-scores > 1.0) having shortest RFS. Multivariate stratified/unstratified Cox models indicated patients with T1 tumors (p = 0.02/p = 0.0002) and higher SS-PR (p = 0.02/p = 0.01) had longer RFS; node-negative patients had better RFS (in unstratified analysis, p < 0.0001). Local ER/PR status did not impact RFS (p > 0.05). Patients with SS HER2+ ≥ 1.32 had worse RFS (univariate, p = 0.05; multivariate, p = 0.06). We demonstrated that higher SS-PR, and SS HER2 levels, measured by RT-PCR impacted breast cancer RFS outcomes. Evaluation in other trials may provide support for this methodology.

Assessment of the prognostic and predictive utility of the Breast Cancer Index (BCI): an NCIC CTG MA.14 study.

BACKGROUND: Biomarkers that can be used to accurately assess the residual risk of disease recurrence in women with hormone receptor-positive breast cancer are clinically valuable. We evaluated the prognostic value of the Breast Cancer Index (BCI), a continuous risk index based on a combination of HOXB13:IL17BR and molecular grade index, in women with early breast cancer treated with either tamoxifen alone or tamoxifen plus octreotide in the NCIC MA.14 phase III clinical trial (ClinicalTrials.gov Identifier NCT00002864; registered 1 November 1999). METHODS: Gene expression analysis of BCI by real-time polymerase chain reaction was performed blinded to outcome on RNA extracted from archived formalin-fixed, paraffin-embedded tumor samples of 299 patients with both lymph node-negative (LN-) and lymph node-positive (LN+) disease enrolled in the MA.14 trial. Our primary objective was to determine the prognostic performance of BCI based on relapse-free survival (RFS). MA.14 patients experienced similar RFS on both treatment arms. Association of gene expression data with RFS was evaluated in univariate analysis with a stratified log-rank test statistic, depicted with a Kaplan-Meier plot and an adjusted Cox survivor plot. In the multivariate assessment, we used stratified Cox regression. The prognostic performance of an emerging, optimized linear BCI model was also assessed in a post hoc analysis. RESULTS: Of 299 samples, 292 were assessed successfully for BCI for 146 patients accrued in each MA.14 treatment arm. BCI risk groups had a significant univariate association with RFS (stratified log-rank p = 0.005, unstratified log-rank p = 0.007). Adjusted 10-year RFS in BCI low-, intermediate-, and high-risk groups was 87.5 %, 83.9 %, and 74.7 %, respectively. BCI had a significant prognostic effect [hazard ratio (HR) 2.34, 95 % confidence interval (CI) 1.33-4.11; p = 0.004], although not a predictive effect, on RFS in stratified multivariate analysis, adjusted for pathological tumor stage (HR 2.22, 95 % CI 1.22-4.07; p = 0.01). In the post hoc multivariate analysis, higher linear BCI was associated with shorter RFS (p = 0.002). CONCLUSIONS: BCI had a strong prognostic effect on RFS in patients with early-stage breast cancer treated with tamoxifen alone or with tamoxifen and octreotide. BCI was prognostic in both LN- and LN+ patients. This retrospective study is an independent validation of the prognostic performance of BCI in a prospective trial.

Preanalytical variables and phosphoepitope expression in FFPE tissue: quantitative epitope assessment after variable cold ischemic time.

Individualized targeted therapies for cancer patients require accurate and reproducible assessment of biomarkers to be able to plan treatment accordingly. Recent studies have shown highly variable effects of preanalytical variables on gene expression profiling and protein levels of different tissue types. Several publications have described protein degradation of tissue samples as a direct result of delay of formalin fixation of the tissue. Phosphorylated proteins are more labile and epitope degradation can happen within 30 min of cold ischemic time. To address this issue, we evaluated the change in antigenicity of a series of phosphoproteins in paraffin-embedded samples from breast tumors as a function of time to formalin fixation. A tissue microarray consisting of 93 breast cancer specimens with documented time-to-fixation was used to evaluate changes in antigenicity of 12 phosphoepitopes frequently used in research settings as a function of cold ischemic time. Analysis was performed in a quantitative manner using the AQUA technology for quantitative immunofluorescence. For each marker, least squares univariate linear regression was performed and confidence intervals were computed using bootstrapping. The majority of the epitopes tested revealed changes in expression levels with increasing time to formalin fixation. Some phosphorylated proteins, such as phospho-HSP27 and phospho-S6 RP, involved in post-translational modification and stress response pathways increased in expression or phosphorylation levels. Others (like phospho-AKT, phosphor-ERK1/2, phospho-Tyrosine, phospho-MET, and others) are quite labile and loss of antigenicity can be reported within 1-2 h of cold ischemic time. Therefore specimen collection should be closely monitored and subjected to quality control measures to ensure accurate measurement of these epitopes. However, a few phosphoepitopes (like phospho-JAK2 and phospho-ER) are sufficiently robust for routine usage in companion diagnostic testing.