Results and lessons from dual extraction of DNA and RNA from formalin-fixed paraffin-embedded breast tumor tissues for a large Cancer epidemiologic study.

BACKGROUND: The use of archived formalin-fixed paraffin-embedded (FFPE) tumor tissues has become a common practice in clinical and epidemiologic genetic research. Simultaneous extraction of DNA and RNA from FFPE tissues is appealing but can be practically challenging. Here we report our results and lessons learned from processing FFPE breast tumor tissues for a large epidemiologic study. METHODS: Qiagen AllPrep DNA/RNA FFPE kit was adapted for dual extraction using tissue punches or sections from breast tumor tissues. The yield was quantified using Qubit and fragmentation analysis by Agilent Bioanalyzer. A subset of the DNA samples were used for genome-wide DNA methylation assays and RNA samples for sequencing. The QC metrices and performance of the assays were analyzed with pre-analytical variables. RESULTS: A total of 1859 FFPE breast tumor tissues were processed. We found it critical to adjust proteinase K digestion time based on tissue volume to achieve balanced yields of DNA and RNA. Tissue punches taken from tumor-enriched regions provided the most reliable output. A median of 1475 ng DNA and 1786 ng RNA per sample was generated. The median DNA integrity number (DIN) was 3.8 and median DV200 for RNA was 33.2. Of 1294 DNA samples used in DNA methylation assays, 97% passed quality check by qPCR and 92% generated data deemed high quality. Of the 130 RNA samples with DV200 ≥ 20% used in RNA-sequencing, all but 5 generated usable transcriptomic data with a mapping rate ≥ 60%. CONCLUSIONS: Dual DNA/RNA purification using Qiagen AllPrep FFPE extraction protocol is feasible for clinical and epidemiologic studies. We recommend tissue punches as a reliable source material and fine tuning of proteinase K digestion time based on tissue volume. IMPACT: Our protocol and recommendations may be adapted by future studies for successful extraction of archived tumor tissues.

Evaluation of microRNA expression profiles and their associations with risk alleles in lymphoblastoid cell lines of familial ovarian cancer.

Interindividual variations of microRNA expression are likely to influence the expression of microRNA target genes and, therefore, contribute to phenotypic differences in humans, including cancer susceptibility. Whether microRNA expression variation has any role in ovarian cancer development is still unknown. Here, we evaluated microRNA expression profiles in lymphoblastoid cell lines from 74 women with familial ovarian cancer and 47 unrelated controls matched on gender and race. We found that the cases and unrelated controls can be clustered using 95 differentially expressed microRNAs with 91% accuracy. To assess the potential implications of microRNAs in ovarian cancer, we investigated the associations between microRNA expression and seven ovarian cancer risk variants discovered from genome-wide association studies (GWAS), namely, rs3814113 on 9p22.2, rs2072590 on 2q31, rs2665390 on 3q25, rs10088218, rs1516982, rs10098821 on 8q24.21 and rs2363956 on 19p13. We observed 130 significant associations at a permutation level of 0.01. Compared with other risk variants, rs3814113 and rs2072590 had the greatest number of significant associations (68 and 37, respectively). Interestingly, 14 microRNAs that were associated with ovarian cancer risk alleles belong to five microRNA clusters. The most notable cluster is the tumorigenic miR-17-92 cluster with five microRNAs, all of which are significantly associated with rs3814113. Using pathway analysis, several key biological pathways were significantly overrepresented, such as cellular response to stress (P = 2.87 × 10(-06)), etc. Further characterization of significant associations between microRNAs and risk alleles could facilitate the understanding of the functions of these GWAS discovered risk alleles in the genetic etiology of ovarian cancer.

Differences in oocyte development and estradiol sensitivity among mouse strains.

Mouse oocytes develop in clusters of interconnected cells called germline cysts. Shortly after birth, the majority of cysts break apart and primordial follicles form, consisting of one oocyte surrounded by granulosa cells. Concurrently, oocyte number is reduced by two-thirds. Exposure of neonatal females to estrogenic compounds causes multiple oocyte follicles that are likely germline cysts that did not break down. Supporting this idea, estrogen disrupts cyst breakdown and may regulate normal oocyte development. Previously, the CD-1 strain was used to study cyst breakdown and oocyte survival, but it is unknown if there are differences in these processes in other mouse strains. It is also unknown if there are variations in estrogen sensitivity during oocyte development. Here, we examined neonatal oocyte development in FVB, C57BL/6, and F2 hybrid (Oct4-GFP) strains, and compared them with the CD-1 strain. We found variability in oocyte development among the four strains. We also investigated estrogen sensitivity differences, and found that C57BL/6 ovaries are more sensitive to estradiol than CD-1, FVB, or Oct4-GFP ovaries. Insight into differences in oocyte development will facilitate comparison of mice generated on different genetic backgrounds. Understanding variations in estrogen sensitivity will lead to better understanding of the risks of environmental estrogen exposure in humans.

Length heteroplasmies in human mitochondrial DNA control regions and breast cancer risk.

It has been proposed that the presence of heteroplasmy in the hypervariable (HV) regions of the mitochondrial DNA (mtDNA) may be an indicator of mitochondrial genome instability, mtDNA dysfunction, and, thus, may be associated with increased cancer risk. However, whether heteroplasmy in the HV regions of mtDNA could be a risk predictor of oxidative stress-related human cancers, such as breast cancer, remains to be determined. To explore the role of heteroplasmy in the HV regions of mtDNA in breast cancer etiology, we analyzed heteroplasmy in the HV regions of mtDNA in whole blood from 103 patients with breast cancer and 103 matched control subjects. Both cases and controls displayed heteroplasmies in both of the HV1 and HV2 regions. Closer examination of the prevalence of length heteroplasmy indicated that the prevalence of heteroplasmies in both of the HV1 and HV2 regions was much higher in the cases than in the controls (HV1: 68% vs 49%, P=0.007; HV2: 46% vs 25%, P=0.002). The presence of length heteroplasmies in both of the HV1 and HV2 regions was associated with 2.18- and 2.49-folds increased risk of breast cancer, respectively, (HV1: OR=2.18, 95% CI: 1.19 - 4.00; HV2: OR=2.49, 95% CI: 1.32 - 4.69). Interestingly, we observed that the controls with length heteroplasmies in both HV1 and HV2 had statistically significantly lower copy number of mtDNA than the ones without heteroplasmies. These results suggest that the length heteroplasmy in the HV regions of mtDNA could be associated with a risk of breast cancer, perhaps through affecting the copy number of mtDNA.

A pilot study of circulating miRNAs as potential biomarkers of early stage breast cancer.

BACKGROUND: To date, there are no highly sensitive and specific minimally invasive biomarkers for detection of breast cancer at an early stage. The occurrence of circulating microRNAs (miRNAs) in blood components (including serum and plasma) has been repeatedly observed in cancer patients as well as healthy controls. Because of the significance of miRNA in carcinogenesis, circulating miRNAs in blood may be unique biomarkers for early and minimally invasive diagnosis of human cancers. The objective of this pilot study was to discover a panel of circulating miRNAs as potential novel breast cancer biomarkers. METHODOLOGY/PRINCIPAL FINDINGS: Using microarray-based expression profiling followed by Real-Time quantitative Polymerase Cycle Reaction (RT-qPCR) validation, we compared the levels of circulating miRNAs in plasma samples from 20 women with early stage breast cancer (10 Caucasian American (CA) and 10 African American (AA)) and 20 matched healthy controls (10 CAs and 10 AAs). Using the significance level of p<0.05 constrained by at least two-fold expression change as selection criteria, we found that 31 miRNAs were differentially expressed in CA study subjects (17 up and 14 down) and 18 miRNAs were differentially expressed in AA study subjects (9 up and 9 down). Interestingly, only 2 differentially expressed miRNAs overlapped between CA and AA study subjects. Using receiver operational curve (ROC) analysis, we show that not only up-regulated but also down-regulated miRNAs can discriminate patients with breast cancer from healthy controls with reasonable sensitivity and specificity. To further explore the potential roles of these circulating miRNAs in breast carcinogenesis, we applied pathway-based bioinformatics exploratory analysis and predicted a number of significantly enriched pathways which are predicted to be regulated by these circulating miRNAs, most of which are involved in critical cell functions, cancer development and progression. CONCLUSIONS: Our observations from this pilot study suggest that the altered levels of circulating miRNAs might have great potential to serve as novel, noninvasive biomarkers for early detection of breast cancer.