First generation prognostic gene signatures for breast cancer predict both survival and chemotherapy sensitivity and identify overlapping patient populations.

The aims of this study were to compare the performance of six different genomic prognostic markers to predict long-term survival and chemotherapy response on the same patient cohort and assess if clinicopathological variables carry independent prognostic and predictive values. We examined seven clinical variables and six previously described prognostic signatures on 228 tumors from patients who received homogeneous preoperative chemotherapy and had long-term follow-up information for survival. We used the area under the receiver operator characteristic curve (AUC) to compare predictors and also performed univariate and multivariate analyses including the genomic and clinical variables and plotted Kaplan-Meir survival curves. All genomic prognostic markers had statistically similar AUCs and sensitivity to predict 5-year progression-free survival (PFS, sensitivities ranged from 0.591 to 0.773, and AUCs: 0.599-0.673), overall survival (OS, sensitivities: 0.590-0.769, AUCs: 0.596-0.684) and pathologic complete response (pCR, sensitivities: 0.596-0.851, AUCs: 0.614-0.805). In multivariate analysis, the genomic markers were not independent from one another; however, estrogen receptor (Odds Ratio [OR] 7.63, P < 0.001) and HER2 status (OR: 0.37, P = 0.021) showed significant independent predictive values for pCR. Nodal status remained an independent prognostic, but not predictive, variable (OR for PFS: 2.77, P = 0.021, OR for OS: 3.62, P = 0.01). There was moderate to good agreement between different prediction results in pair-wise comparisons. First-generation prognostic-gene signatures predict both chemotherapy response and long-term survival. When multiple predictors are applied to the same case discordant risk prediction frequently occurs.

PIK3CA-activating mutations and chemotherapy sensitivity in stage II-III breast cancer.

INTRODUCTION: In vitro evidence suggests that PIK3CA (phosphatidylinositol 3-kinase, catalytic, alpha polypeptide) activation may be associated with altered chemotherapy sensitivity in cancer. METHODS: Tumor DNA from 140 patients with stage II-III breast cancer undergoing neoadjuvant chemotherapy was sequenced for PIK3CA mutations on exons 1, 9, and 20. Mutation status was correlated with clinical/pathological parameters and chemotherapy response as (a) pathological complete response (pCR) versus residual cancer or (b) quantitative residual cancer burden (RCB) scores, including stratification for estrogen receptor (ER) expression status, type of chemotherapy, and by exons. RESULTS: Twenty-three patients (16.4%) harbored a PIK3CA mutation, with 12, 11, and 0 mutations located in exons 9, 20, and 1, respectively. PIK3CA exon 9 mutations were more frequent among node-negative (52% versus 25%; P = 0.012) than node-positive tumors, particularly among ER-positive tumors. pCR rates and RCB scores were similar among patients with the wild-type and mutant PIK3CA genes, even after stratification by ER status, chemotherapy regimen (anthracycline versus anthracycline plus paclitaxel), or exon. CONCLUSION: PIK3CA mutations are not associated with altered sensitivity to preoperative anthracycline-based or taxane-based chemotherapies in ER-positive and ER-negative breast tumors. In this study, PIK3CA mutation was associated with a decreased rate of node-positive disease, particularly among ER-positive tumors.

Effects of tissue handling on RNA integrity and microarray measurements from resected breast cancers.

BACKGROUND: Reliable stability and yield of RNA from breast cancer tissues are important for biobanking, clinical trials, and diagnostic testing. METHODS: Aliquots of fresh primary tumor tissue from 17 surgically resected invasive breast cancers were placed into RNAlater at room temperature after tumor removal (baseline) and up to 3 hours thereafter or were snap frozen at baseline and 40 minutes thereafter. Samples were stored at -80°C until gene expression profiling with Affymetrix Human Gene U133A microarrays. We evaluated the effects of cold ischemic time (the time from tumor specimen removal to sample preservation) and sample preservation method on RNA yield, Bioanalyzer-based RNA integrity number, microarray-based 3'/5' expression ratios for assessing transcript integrity, and microarray-based measurement of single-gene and multigene expression signatures. The statistical significance of the effects was assessed using linear mixed effects regression models. All statistical tests were two-sided. RESULTS: Sample preservation in RNAlater statistically significantly improved RNA integrity compared with snap freezing as assessed by the RNA integrity number, which increased from 7.31 to 8.13 units (difference = 0.82 units, 95% confidence interval [CI] = 0.53 to 1.11 units, P < .001), and RNA yield, which increased threefold from 8.9 to 28.6 µg (difference = 19.7 µg, 95% CI = 14.1 to 25.3 µg, P < .001). Prolonged cold ischemic delay at room temperature before sample stabilization decreased the RNA integrity number by 0.12 units/h (95% CI = 0.02 to 0.23 units/h) compared with a projected average RNA integrity number of 7.39 if no delays were present (P = .008) and decreased the RNA yield by 1.5 µg/h (95% CI = 0 to 4 µg/h) from a baseline mean RNA yield of 33.5 µg if no delays were present (P = .019). Prolonged cold ischemia statistically significantly increased the 3'/5' ratio of control gene transcripts, particularly of STAT1 (P < .001). Snap freezing statistically significantly increased the 3'/5' ratio of three control transcripts (ACTB, GAPDH, and 18S rRNA). Expression levels of single genes and multigene signatures for breast cancer were largely unaffected by sample preservation method or cold ischemia. CONCLUSIONS: Sample preservation in RNAlater improves RNA yield and quality, whereas cold ischemia increases RNA fragmentation as measured by the 3'/5' expression ratio of control genes. However, expression levels of single genes and multigene signatures that are of diagnostic relevance in breast cancer were mostly unaffected by sample preservation method or prolonged cold ischemic duration.