Use of FFPE-derived DNA in next generation sequencing: DNA extraction methods.

Archival tissues represent a rich resource for clinical genomic studies, particularly when coupled with comprehensive medical records. Use of these in next generation sequencing (NGS) is a priority. Nine formalin-fixed paraffin-embedded (FFPE) DNA extraction methods were evaluated using twelve FFPE samples of varying tissue types. Quality assessment included total yield, percent dsDNA, fragment analysis and multiplex PCR. After assessment, three tissue types from four FFPE DNA methods were selected for NGS downstream evaluation, targeted and whole exome sequencing. In addition, two low input library protocols were evaluated for WES. Analysis revealed average coverage across the target regions for WES was ~20-30X for all four FFPE DNA extraction methods. For the targeted panels, the highest molecular tag coverage was obtained with the Kingfisher FFPE extraction method. The genotype concordance was 99% for the commonly called variant positions between all four extraction methods with the targeted PCR NGS panel and 96% with WES. Assessing quality of extracted DNA aids in selecting the optimal NGS approach, and the choice of both DNA extraction and library preparation approaches can impact the performance of archival tissue in NGS.

Multi-platform analysis of microRNA expression measurements in RNA from fresh frozen and FFPE tissues.

MicroRNAs play a role in regulating diverse biological processes and have considerable utility as molecular markers for diagnosis and monitoring of human disease. Several technologies are available commercially for measuring microRNA expression. However, cross-platform comparisons do not necessarily correlate well, making it difficult to determine which platform most closely represents the true microRNA expression level in a tissue. To address this issue, we have analyzed RNA derived from cell lines, as well as fresh frozen and formalin-fixed paraffin embedded tissues, using Affymetrix, Agilent, and Illumina microRNA arrays, NanoString counting, and Illumina Next Generation Sequencing. We compared the performance within- and between the different platforms, and then verified these results with those of quantitative PCR data. Our results demonstrate that the within-platform reproducibility for each method is consistently high and although the gene expression profiles from each platform show unique traits, comparison of genes that were commonly detectable showed that detection of microRNA transcripts was similar across multiple platforms.

Quantitative miRNA expression analysis using fluidigm microfluidics dynamic arrays.

BACKGROUND: MicroRNAs (miRNAs) represent a growing class of small non-coding RNAs that are important regulators of gene expression in both plants and animals. Studies have shown that miRNAs play a critical role in human cancer and they can influence the level of cell proliferation and apoptosis by modulating gene expression. Currently, methods for the detection and measurement of miRNA expression include small and moderate-throughput technologies, such as standard quantitative PCR and microarray based analysis. However, these methods have several limitations when used in large clinical studies where a high-throughput and highly quantitative technology needed for the efficient characterization of a large number of miRNA transcripts in clinical samples. Furthermore, archival formalin fixed, paraffin embedded (FFPE) samples are increasingly becoming the primary resource for gene expression studies because fresh frozen (FF) samples are often difficult to obtain and requires special storage conditions. In this study, we evaluated the miRNA expression levels in FFPE and FF samples as well as several lung cancer cell lines employing a high throughput qPCR-based microfluidic technology. The results were compared to standard qPCR and hybridization-based microarray platforms using the same samples. RESULTS: We demonstrated highly correlated Ct values between multiplex and singleplex RT reactions in standard qPCR assays for miRNA expression using total RNA from A549 (R = 0.98; p < 0.0001) and H1299 (R = 0.95; p < 0.0001) lung cancer cell lines. The Ct values generated by the microfluidic technology (Fluidigm 48.48 dynamic array systems) resulted in a left-shift toward lower Ct values compared to those observed by ABI 7900 HT (mean difference, 3.79), suggesting that the microfluidic technology exhibited a greater sensitivity. In addition, we show that as little as 10 ng total RNA can be used to reliably detect all 48 or 96 tested miRNAs using a 96-multiplexing RT reaction in both FFPE and FF samples. Finally, we compared miRNA expression measurements in both FFPE and FF samples by qPCR using the 96.96 dynamic array and Affymetrix microarrays. Fold change comparisons for comparable genes between the two platforms indicated that the overall correlation was R = 0.60. The maximum fold change detected by the Affymetrix microarray was 3.5 compared to 13 by the 96.96 dynamic array. CONCLUSION: The qPCR-array based microfluidic dynamic array platform can be used in conjunction with multiplexed RT reactions for miRNA gene expression profiling. We showed that this approach is highly reproducible and the results correlate closely with the existing singleplex qPCR platform at a throughput that is 5 to 20 times higher and a sample and reagent usage that was approximately 50-100 times lower than conventional assays. We established optimal conditions for using the Fluidigm microfluidic technology for rapid, cost effective, and customizable arrays for miRNA expression profiling and validation.

Expression profiling of formalin-fixed paraffin-embedded primary breast tumors using cancer-specific and whole genome gene panels on the DASL® platform.

BACKGROUND: The cDNA-mediated Annealing, extension, Selection and Ligation (DASL) assay has become a suitable gene expression profiling system for degraded RNA from paraffin-embedded tissue. We examined assay characteristics and the performance of the DASL 502-gene Cancer Panel v1 (1.5K) and 24,526-gene panel (24K) platforms at differentiating nine human epidermal growth factor receptor 2- positive (HER2+) and 11 HER2-negative (HER2-) paraffin-embedded breast tumors. METHODS: Bland-Altman plots and Spearman correlations evaluated intra/inter-panel agreement of normalized expression values. Unequal-variance t-statistics tested for differences in expression levels between HER2 + and HER2 - tumors. Regulatory network analysis was performed using Metacore (GeneGo Inc., St. Joseph, MI). RESULTS: Technical replicate correlations ranged between 0.815-0.956 and 0.986-0.997 for the 1.5K and 24K panels, respectively. Inter-panel correlations of expression values for the common 498 genes across the two panels ranged between 0.485-0.573. Inter-panel correlations of expression values of 17 probes with base-pair sequence matches between the 1.5K and 24K panels ranged between 0.652-0.899. In both panels, erythroblastic leukemia viral oncogene homolog 2 (ERBB2) was the most differentially expressed gene between the HER2 + and HER2 - tumors and seven additional genes had p-values < 0.05 and log2 -fold changes > |0.5| in expression between HER2 + and HER2 - tumors: topoisomerase II alpha (TOP2A), cyclin a2 (CCNA2), v-fos fbj murine osteosarcoma viral oncogene homolog (FOS), wingless-type mmtv integration site family, member 5a (WNT5A), growth factor receptor-bound protein 7 (GRB7), cell division cycle 2 (CDC2), and baculoviral iap repeat-containing protein 5 (BIRC5). The top 52 discriminating probes from the 24K panel are enriched with genes belonging to the regulatory networks centered around v-myc avian myelocytomatosis viral oncogene homolog (MYC), tumor protein p53 (TP53), and estrogen receptor α (ESR1). Network analysis with a two-step extension also showed that the eight discriminating genes common to the 1.5K and 24K panels are functionally linked together through MYC, TP53, and ESR1. CONCLUSIONS: The relative RNA abundance obtained from two highly differing density gene panels are correlated with eight common genes differentiating HER2 + and HER2 - breast tumors. Network analyses demonstrated biological consistency between the 1.5K and 24K gene panels.

Follicle luteinization in hyperandrogenic follicles of polycystic ovary syndrome patients undergoing gonadotropin therapy for in vitro fertilization.

CONTEXT: Polycystic ovary syndrome (PCOS) is a reproductive disorder of ovarian hyperandrogenism and insulin resistance characterized by abnormal luteinization of small follicles. After exposure to GnRH analog/FSH stimulation for in vitro fertilization (IVF), however, it is unclear whether such PCOS follicles remain abnormally luteinized during the resumption of oocyte maturation in vivo. OBJECTIVE: The aim of this study was to determine whether PCOS follicles exposed to GnRH analog/FSH stimulation for IVF show abnormal luteinization. DESIGN: This study was a prospective cohort. SETTING: The setting was an institutional practice. PATIENTS: Eleven PCOS and 30 normoandrogenic ovulatory women were included. INTERVENTION(S): All subjects received GnRH analog/FSH therapy after basal serum hormone determinations. MAIN OUTCOME MEASURE(S): Follicle fluid aspirated at oocyte retrieval from the first follicle of each ovary was assayed for gonadotropins, steroids, insulin, and glucose. LH receptor mRNA expression was determined in granulosa cells of the same follicle. RESULTS: In PCOS patients with basal hyperandrogenemia and hyperinsulinemia, total oocyte number was increased and follicle diameter was decreased, despite normal maximal serum estradiol levels. Within PCOS follicles, progesterone levels were reduced (P < 0.01), despite comparable bioactive LH and insulin levels and granulosa cell LH receptor mRNA expression; estradiol levels were normal, despite diminished FSH availability (P < 0.004). Elevated androstenedione (P < 0.01), testosterone (P < 0.001), and glucose (P < 0.01) levels also occurred. In PCOS follicles containing mature oocytes, however, elevated androgen levels were accompanied by both normal progesterone concentrations and a normal inverse relationship between glucose depletion and lactate accumulation. CONCLUSION: Hyperandrogenic follicles with mature oocytes from PCOS women receiving GnRH analog/recombinant human FSH therapy for IVF show sufficient glucose utilization for normal luteinization.

Insulin and messenger ribonucleic acid expression of insulin receptor isoforms in ovarian follicles from nonhirsute ovulatory women and polycystic ovary syndrome patients.

Insulin action is mediated by two insulin receptor (IR) isoforms, differing in mitogenic and metabolic function. IR isoform expression might occur in human granulosa cells and could be altered in polycystic ovary syndrome (PCOS) from hyperinsulinemia. To determine the relationship between granulosa cell IR isoform expression and follicular fluid insulin concentration in individual follicles, 18 normal women and seven PCOS patients receiving gonadotropins for in vitro fertilization were studied. Glucose tolerance testing was performed before pituitary desensitization, and fasting serum insulin was measured at oocyte retrieval. Granulosa cells and fluid aspirated from the first follicle were used to determine IR isoform mRNA expression and insulin concentration, respectively. IR isoform A mRNA expression was greater than that of IR isoform B expression in normal mural granulosa and cumulus cells, without a cell type effect. Intrafollicular insulin levels increased with adiposity and serum insulin levels at oocyte-retrieval but did not predict IR mRNA expression. Total IR mRNA expression, but not intrafollicular insulin levels, was elevated in PCOS patients, whereas intrafollicular insulin levels were increased in women with impaired glucose tolerance. Granulosa cell IR heterogeneity, together with adiposity-dependent intrafollicular insulin availability, introduces a novel mechanism by which insulin may affect granulosa cell function within the follicle.

Functional insulin receptors on human epithelial ovarian carcinoma cells: implications for IGF-II mitogenic signaling.

The insulin receptor mediates a proliferative response in certain transformed cells, but little is known about its function in ovarian cancer. We used human epithelial ovarian carcinoma cell lines and lifespan-extended normal ovarian surface epithelial (OSE) cells to examine (125)I-insulin binding and mitogenic responses to insulin. All cancer cell and OSE cultures specifically bound (125)I-insulin. Except for OV202, the carcinoma lines had elevated insulin binding compared with OSE cells. All carcinoma lines except OV202 expressed insulin receptor as detected by flow cytometry and increased (3)H-thymidine incorporation or cell number in response to 0.1-10 nM insulin. Interestingly, similar concentrations of IGF-II also induced proliferation of the insulin-responsive cancer cell lines and displaced (125)I-insulin binding. Direct binding of (125)I-IGF-II to the insulin receptor was visualized by cross-linking and immunoprecipitation. Binding of IGF-II to the insulin receptor and a proliferative effect of insulin suggest the presence of insulin receptor isoform A. Real-time PCR analyses confirm that insulin receptor isoform A expression predominates over isoform B expression in the ovarian carcinoma cell lines. This report suggests that the insulin receptor may play a role in the regulation of ovarian cancer cell growth.