Assessing the quality of RNA isolated from human breast tissue after ambient room temperature exposure.

High quality human tissue is essential for molecular research, but pre-analytical conditions encountered during tissue collection could degrade tissue RNA. We evaluated how prolonged exposure of non-diseased breast tissue to ambient room temperature (22±1°C) impacted RNA quality. Breast tissue received between 70 to 190 minutes after excision was immediately flash frozen (FF) or embedded in Optimal Cutting Temperature (OCT) compound upon receipt (T0). Additional breast tissue pieces were further exposed to increments of 60 (T1 = T0+60 mins), 120 (T2 = T0+120 mins) and 180 (T3 = T0+180 mins) minutes of ambient room temperature before processing into FF and OCT. Total exposure, T3 (T0+180 mins) ranged from 250 minutes to 370 minutes. All samples (FF and OCT) were stored at -80°C before RNA isolation. The RNA quality assessment based on RNA Integrity Number (RIN) showed RINs for both FF and OCT samples were within the generally acceptable range (mean 7.88±0.90 to 8.52±0.66). No significant difference was observed when RIN at T0 was compared to RIN at T1, T2 and T3 (FF samples, p = 0.43, 0.56, 0.44; OCT samples, p = 0.25, 0.82, 1.0), or when RIN was compared between T1, T2 and T3. RNA quality assessed by quantitative real-time PCR (qRT-PCR) analysis of beta-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclophilin A (CYPA), and porphobilinogen deaminase (PBGD) transcripts showed threshold values (Ct) that indicate abundant and intact target nucleic acid in all samples (mean ranging from 14.1 to 25.3). The study shows that higher RIN values were obtained for non-diseased breast tissue up to 190 minutes after resection and prior to stabilization. Further experimental exposure up to 180 minutes had no significant effect on RIN values. This study strengthens the rationale for assessing RIN and specific gene transcript levels as an objective method for determining how suitable RNA will be for a specific research purpose ("fit-for purpose").

Molecular Heterogeneity in Primary Breast Carcinomas and Axillary Lymph Node Metastases Assessed by Genomic Fingerprinting Analysis.

Molecular heterogeneity within primary breast carcinomas and among axillary lymph node (LN) metastases may impact diagnosis and confound treatment. In this study, we used short tandem repeated sequences to assess genomic heterogeneity and to determine hereditary relationships among primary tumor areas and regional metastases from 30 breast cancer patients. We found that primary carcinomas were genetically heterogeneous and sampling multiple areas was necessary to adequately assess genomic variability. LN metastases appeared to originate at different time periods during disease progression from different sites of the primary tumor and the extent of genomic divergence among regional metastases was associated with a less favorable patient outcome (P = 0.009). In conclusion, metastasis is a complex process influenced by primary tumor heterogeneity and variability in the timing of dissemination. Genomic variation in primary breast tumors and regional metastases may negatively impact clinical diagnostics and contribute to therapeutic resistance.

Importance of substantial weight loss for altering gene expression during cardiovascular lifestyle modification.

OBJECTIVE: To examine relationships between weight loss through changes in lifestyle and peripheral blood gene expression profiles. METHODS: A prospective nonrandomized trial was conducted over 1 year in participants undergoing intensive lifestyle modification to reverse or stabilize progression of coronary artery disease. Cardiovascular risk factors, inflammatory biomarkers, and gene expression as a function of weight loss were assessed in 89 lifestyle participants and 71 retrospectively matched controls undergoing usual care. RESULTS: Substantial weight loss (-15.2 ± 3.8%) in lifestyle participants (n = 33) was associated with improvement in selected cardiovascular risk factors and significant changes in peripheral blood gene expression from pre- to post-intervention: 132 unique genes showed significant expression changes (false discovery rate corrected P-value <0.05 and fold-change ≥1.4). Altered molecular pathways were related to immune function and inflammatory responses involving endothelial activation. In contrast, participants losing minimal weight (-3.1 ± 2.5%, n = 32) showed only minor changes in cardiovascular risk factors and markers of inflammation and no changes in gene expression compared to non intervention controls after 1 year. CONCLUSIONS: Weight loss (≥10%) during lifestyle modification is associated with down-regulation of genetic pathways governing interactions between circulating immune cells and the vascular endothelium and may be required to successfully reduce CVD risk.

Abundance and distribution of polychlorinated biphenyls (PCBs) in breast tissue.

Many environmental chemicals accumulate in human tissues and may contribute to cancer risk. Polychlorinated biphenyls (PCBs) are associated with adverse health effects, but relationships between PCB exposure and breast cancer are unclear. In this study, we sought to determine whether bioaccumulation of PCBs differs within regions of the human breast and whether PCB levels are associated with clinical and pathological characteristics in breast cancer patients. Tissue sections (n=245) were collected from breast quadrants from 51 women with a diagnosis ranging from disease-free to metastatic breast cancer. Ninety-seven PCB congeners were assayed by high resolution gas chromatography. ANOVA was used to examine PCB distribution within the breast and relationships with clinical/pathological variables. Pearson product-moment correlations assessed relationships between age at mastectomy and PCB levels. PCBs were abundant in breast tissues with a median concentration of 293.4ng/g lipid (range 15.4-1636.3ng/g). PCB levels in breast tissue were significantly different (p<0.001) among functional groupings of congeners defined by structure-activity properties: Group I (28.2ng/g), Group II (96.6ng/g), Group III (166.0ng/g). Total PCB concentration was highly correlated with age at mastectomy, but the distribution of PCBs did not differ by breast quadrant. PCB levels were not associated with patient status or tumor characteristics. In conclusion, PCB congeners with carcinogenic potential were present at high levels in the human breast, but were not associated with clinical or pathological characteristics in breast cancer patients.

Intensive cardiovascular risk reduction induces sustainable changes in expression of genes and pathways important to vascular function.

BACKGROUND: Healthy lifestyle changes are thought to mediate cardiovascular disease risk through pathways affecting endothelial function and progression of atherosclerosis; however, the extent, persistence, and clinical significance of molecular change during lifestyle modification are not well known. We examined the effect of a rigorous cardiovascular disease risk reduction program on peripheral blood gene expression profiles in 63 participants and 63 matched controls to characterize molecular responses and identify regulatory pathways important to cardiovascular health. METHODS AND RESULTS: Dramatic changes in dietary fat intake (-61%; P<0.001 versus controls) and physical fitness (+34%; P<0.001) led to significant improvements in cardiovascular disease risk factors. Analysis of variance with false discovery rate correction for multiple testing (P<0.05) identified 26 genes after 12 weeks and 143 genes after 52 weeks that were differentially expressed from baseline in participants. Controls showed little change in cardiovascular disease risk factors or gene expression. Quantitative reverse transcription polymerase chain reaction validated differential expression for selected transcripts. Lifestyle modification effectively reduced expression of proinflammatory genes associated with neutrophil activation and molecular pathways important to vascular function, including cytokine production, carbohydrate metabolism, and steroid hormones. Prescription medications did not significantly affect changes in gene expression. CONCLUSIONS: Successful and sustained modulation of gene expression through lifestyle changes may have beneficial effects on the vascular system not apparent from traditional risk factors. Healthy lifestyles may restore homeostasis to the leukocyte transcriptome by downregulating lactoferrin and other genes important in the pathogenesis of atherosclerosis. Clinical Trial Registration- URL: www.clinicaltrials.gov. Unique identifier: NCT01805492.