RNA-seq from archival FFPE Breast Cancer samples: Molecular Pathway Fidelity and Novel Discovery
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PROVIDER: PRJNA539981 | ENA |
REPOSITORIES: ENA
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Project description:Archival Formalin-Fixed Paraffin Embedded (FFPE) tissue represents an abundant and more easily transferable source of patient tissue than live specimens. Furthermore cancer FFPE tissue deposited at major cancer research institutes is often paired with survival data increasing the value of biomarker and prediction based research from these samples. However the known fragility of RNA and artifacts introduced through the processes of tissue preservation and storage introduce the possibility of unfaithful RNA expression signatures from these sources. To evaluate this we established an RNA isolation, library prepration and data analysis pipeline at Oregon Health and Science University to evaluate the fidelity of RNA expression profiles from FFPE tissues utilizing gene and pathway expression comparisons between ER positive and ER negative samples in archival FFPE specimens compared to those obtained from public available data sets of fresh specimens.
2019-11-26 | GSE130397 | GEO
Project description:BackgroundFormalin-fixed, paraffin-embedded (FFPE) tissues for RNA-seq have advantages over fresh frozen tissue including abundance and availability, connection to rich clinical data, and association with patient outcomes. However, FFPE-derived RNA is highly degraded and chemically modified, which impacts its utility as a faithful source for biological inquiry.MethodsTrue archival FFPE breast cancer cases (n = 58), stored at room temperature for 2-23 years, were utilized to identify key steps in tissue selection, RNA isolation, and library choice. Gene expression fidelity was evaluated by comparing FFPE data to public data obtained from fresh tissues, and by employing single-gene, gene set and transcription network-based regulon analyses.ResultsWe report a single 10 μm section of breast tissue yields sufficient RNA for RNA-seq, and a relationship between RNA quality and block age that was not linear. We find single-gene analysis is limiting with FFPE tissues, while targeted gene set approaches effectively distinguish ER+ from ER- breast cancers. Novel utilization of regulon analysis identified the transcription factor KDM4B to associate with ER+ disease, with KDM4B regulon activity and gene expression having prognostic significance in an independent cohort of ER+ cases.ConclusionOur results, which outline a robust FFPE-RNA-seq pipeline for broad use, support utilizing FFPE tissues to address key questions in the breast cancer field, including the delineation between indolent and life-threatening disease, biological stratification and molecular mechanisms of treatment resistance.
| S-EPMC6924022 | biostudies-literature
Project description:Use of archival resources has been limited to date by inconsistent methods for genomic profiling of degraded RNA from formalin-fixed paraffin-embedded (FFPE) samples. RNA-seq offers a novel way to address this problem. In this study we evaluated transcriptomic dose responses using RNA-seq in paired FFPE and frozen (FROZ) samples from two archival studies in mice, one recent (<2 years old) and the other older (>20 years old). Experimental treatments included di(2-ethylhexyl)phthalate (DEHP) and dichloroacetic acid (DCA) for the <2 and >20 year-old studies, respectively. Total RNA was ribodepleted and sequenced using the Illumina HiSeq platform. In the recent study, FFPE samples showed high concordance in total reads (98% vs FROZ), fold-change values of differentially expressed genes (DEGs) (R2 = 0.99), highly enriched target pathways (90% overlap with FROZ), and benchmark dose estimates for preselected target genes (-2% overall vs FROZ). In contrast, RNA-seq data from older FFPE samples had lower total reads (70% vs FROZ) and poor concordance in global DEGs and pathways. Despite a 99% loss of counts, dose responses were still evident for target genes in FFPE samples and positively correlated with paired FROZ samples. These findings highlight potential variability in the quality of RNA-seq data from FFPE samples. More recent FFPE samples were highly similar to FROZ samples in sequencing quality metrics, DEG profiles, and dose-response parameters, while further methods development is needed for older or lower-quality FFPE samples. This work should help broaden the use of archival resources in both chemical safety and translational science.
2016-08-30 | GSE78962 | GEO
Project description:Formalin-fixed paraffin-embedded (FFPE) samples are the only remaining biological archive for many toxicological and clinical studies, yet their use in genomics has been limited due to variability in RNA quality from formalin fixation. Older FFPE samples, which often have highly degraded RNA, pose a particularly difficult technical challenge. Recent developments in probe-based targeted sequencing technologies show promise in addressing this issue, while also being more cost effective than traditional RNA-sequencing (RNA-seq) methods. In the current study, we evaluated dose-dependent transcriptional changes from paired frozen and archival FFPE liver samples stored for over 20 years using targeted resequencing (TempO-Seq) and whole-genome RNA-seq methods. Samples were originally collected from male mice exposed to a reference chemical (dichloroacetic acid, DCA) at 0, 198, 313 and 427 mg/kg-day, (n=6/dose) by drinking water for 6 days. TempO-Seq showed high overlap in differentially expressed genes (DEGs) between matched FFPE and flash-frozen (FROZ) samples and concordance in fold-changes across the highest two dose levels of DCA vs. control (R2 ≥ 0.94). Similarly, high concordance in fold-changes was observed between TempO-Seq FFPE and RNA-seq FROZ results (R2 ≥ 0.92). Only 28 DEGs or fewer were identified in RNA-seq FFPE samples. Modeling of DCA-dependent changes in gene sets identified benchmark doses from TempO-Seq FROZ and FFPE samples that were within 1.4-fold of RNA-seq FROZ samples (93.9 mg/kg-d), whereas RNA-seq FFPE samples were 3.3-fold higher (310.3 mg/kg-d). This work demonstrates that targeted sequencing may provide a more robust method for quantifying gene expression profiles from aged archival FFPE samples.
2022-10-10 | GSE186113 | GEO
Project description:The study described a comprehensive high-fidelity method to successfully amplify low starting amounts of RNA extracted from FFPE tissue obtained from bronchial biopsies. Both amplified and unamplified RNA resulted in comparable gene expression for lung cancer and asthma samples.
2023-03-30 | GSE228217 | GEO
Project description:Refractory/relapsed diffuse large B-cell lymphoma (DLBCL) has a poor prognosis. Novel drugs targeting the constitutively activated NF- B pathway characteristic of ABC-DLBCL are promising, but evaluation depends on accurate activated B cell-like (ABC)/germinal center B cell-like (GCB) molecular classification. This is traditionally performed on gene microarray expression profiles of fresh biopsies, which are not routinely collected, or by immunohistochemistry on formalin-fixed, paraffin-embedded (FFPE) tissue, which lacks reproducibility and classification accuracy. We explored the possibility of using routine archival FFPE tissue for gene microarray applications. We examined Affymetrix HG U133 Plus 2.0 gene expression profiles from paired archival FFPE and fresh-frozen tissues of 40 ABC/GCB-classified DLBCL cases to compare classification accuracy and test the potential for this approach to aid the discovery of therapeutic targets and disease classifiers in DLBCL. Unsupervised hierarchical clustering of unselected present probe sets distinguished ABC/GCB in FFPE with remarkable accuracy, and a Bayesian classifier correctly assigned 32 of 36 cases with >90% probability. Enrichment for NF- B genes was appropriately seen in ABC-DLBCL FFPE tissues. The top discriminatory genes expressed in FFPE separated cases with high statistical significance and contained novel biology with potential therapeutic insights, warranting further investigation. These results support a growing understanding that archival FFPE tissues can be used in microarray experiments aimed at molecular classification, prognostic biomarker discovery, and molecular exploration of rare diseases.
2014-12-29 | GSE64555 | GEO
Project description:BACKGROUND: Array Comparative Genomic Hybridization (aCGH) is a rapidly evolving technology that still lacks complete standardization. Yet, it is of great importance to obtain robust and reproducible data to enable meaningful multiple hybridization comparisons. Special difficulties arise when aCGH is performed on archival formalin-fixed, paraffin-embedded (FFPE) tissue due to its variable DNA quality. Recently, we have developed an effective DNA quality test that predicts suitability of archival samples for BAC aCGH. METHODS: In this report, we first used DNA from a cancer cell-line (SKBR3) to optimize the aCGH protocol for automated hybridization, and subsequently optimized and validated the procedure for FFPE breast cancer samples. We aimed for highest throughput, accuracy, and reproducibility applicable to FFPE samples, which can also be important in future diagnostic use. RESULTS: Our protocol of automated array-CGH on archival FFPE ULS-labeled DNA showed very similar results compared with published data and our previous manual hybridization method. CONCLUSION: This report combines automated aCGH on unamplified archival FFPE DNA using non-enzymatic ULS labeling, and describes an optimized protocol for this combination resulting in improved quality and reproducibility.
2007-02-23 | GSE7122 | GEO
Project description:Advantages of RNA-Seq over array based platforms are quantitative gene expression and discovery of expressed single nucleotide variants (eSNVs) and fusion transcripts from a single platform, but the sensitivity for each of these characteristics is unknown. We measured gene expression in a set of manually degraded RNAs, nine pairs of matched fresh-frozen, and FFPE RNA isolated from breast tumor with the hybridization based, NanoString nCounter, (226 gene panel) and with whole transcriptome RNA-Seq using RiboZeroGold ScriptSeq V2 library preparation kits. We performed correlation analyses of gene expression between samples and across platforms. We then specifically assessed whole transcriptome expression of lincRNA and discovery of eSNVs and fusion transcripts in the FFPE RNA-Seq data. For gene expression in the manually degraded samples, we observed Pearson correlation of >0.94 and >0.80 with NanoString and ScriptSeq protocols respectively. Gene expression data for matched fresh-frozen and FFPE samples yielded mean Pearson correlations of 0.874 and 0.783 for NanoString (226 genes) and ScriptSeq whole transcriptome protocols respectively. Specifically for lincRNAs, we observed superb Pearson correlation (0.988) between matched fresh-frozen and FFPE pairs. FFPE samples across NanoString and RNA-Seq platforms gave a mean Pearson correlation of 0.838. In FFPE libraries, we detected 53.4% of high confidence SNVs and 24% of high confidence fusion transcripts. Sensitivity of fusion transcript detection was not overcome by an increase in depth of sequencing up to 3-fold (increase from ~56 to ~159 million reads). Both NanoString and ScriptSeq RNA-Seq technologies yield reliable gene expression data for degraded and FFPE material. The high degree of correlation between NanoString and RNA-Seq platforms suggests discovery based whole transciptome studies from FFPE material will produce reliable expression data. The RiboZeroGold ScriptSeq protocol performed particularly well for lincRNA expression from FFPE libraries but detection of eSNV and fusion transcripts was less sensitive.
2013-12-06 | GSE51124 | GEO
Project description:Archival formalin-fixed paraffin-embedded (FFPE) tissue samples hold a wealth of transcriptomic information; however, little is known about potential artifacts. Previously, we identified a consistent shift in global RNA-sequencing profiles between matching frozen and FFPE samples. We hypothesized that this shift was from fixing fresh tissue in formalin. To test this idea, RNA-sequencing was performed on liver samples collected from male mice treated with 600 ppm of a reference chemical (phenobarbital, 600 ppm phenobarbital) or vehicle control for 7 days. Samples were divided into: (1) fresh-frozen (FR); (2) directly fixed in 10% buffered formalin for 18 hours followed by paraffin embedding (FFPE); (3) frozen then fixed as FFPE (FR>FFPE); or (4) frozen then fixed in 70% ethanol followed by paraffin embedding (FR>OH) (n=6/group/condition). Direct fixation resulted in 2946 differentially expressed genes (DEGs), 98% of which were down-regulated. Freezing prior to fixation resulted in ≥95% fewer DEGs vs. FR, indicating that most formalin-derived transcriptional effects occurred with fixation. This was supported by follow-up studies, which identified consistent enrichment in oxidative stress, mitochondrial dysfunction, and transcription elongation pathways with formalin fixation. Notably, formalin fixation in the parent study did not significantly impact chemical response profiles, which were consistent with CAR/PXR activation and 600 ppm phenobarbital exposure. Our results demonstrate distinct transcriptional effects of formalin fixation that could impact gene expression studies using FFPE samples.
2020-09-08 | GSE148174 | GEO
Project description:Over 20 million archival tissue samples are stored annually in the United States as formalin-fixed, paraffin-embedded (FFPE) tissue blocks, but only recently has whole-genome expression profiling from these samples become technically feasible. Here, we introduce novel general methods for assessing, summarizing, and visualizing expression data quality from archival samples. We validated these methods in technical study of 144 clinical breast cancer and autopsy samples and in an overview of all current publicly available FFPE whole-genome expression data. We additionally performed a case study incorporating over 1,000 colorectal cancer (CRC) samples collected from patients across the United States over a period of more than 25 years, integrating clinicopathological information, tumor molecular data, and archival tissue gene expression on an unparalleled scale. Both large-scale clinical studies presented a much greater range of data quality than previous smaller studies, emphasizing the need for rigorous quality control in translational applications of archival tissue gene expression profiling. This series includes 1,003 FFPE-preserved CRC tumors, assayed by Illumina HumanRef-v3 WG-DASL microarray.
2012-10-02 | E-GEOD-32651 | biostudies-arrayexpress