Human Biospecimen Integrity in Breast Cancer Tissues Analytical Variance on Microarray-Based Pharmacogenomics Tests of Breast Cancer
Subcontractor: University of Texas — MD Anderson
Principal Investigator: W. Fraser Symmans, M.D.
Co-Principal Investigator: Christos Hatzis, Ph.D.
Dr. Symmans and his team investigated reliable stability and yield of RNA from breast cancer tissues that is important for biobanking, clinical trials, and diagnostic testing. The investigators also aimed to establish an annotated, publicly available source of genomic data that can be used to assess the specific contributions of preanalytical and analytical factors to the measurement of the expression of any single gene, or any multi-gene signatures for breast cancer. 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. Their team 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. Gne expression profiling was performed using Affymetrix Human Gene U133A microarrays. Their team found that sample preservation in RNA later 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 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 multi-gene signatures for breast cancer were largely unaffected by sample preservation method or cold ischemia. Dr. Symmans and his group concluded that 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.