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The 31 pairs of FF (fresh frozen) andFFPE (Formalin-fixed paraffin-embedded) data for the application of 95GC to FFPE in 2019.

ABSTRACT: The study aimed to apply 95GC, originally developed using fresh‑frozen (FF) tissues, to formalin‑fixed paraffin‑embedded (FFPE) tissues, because FFPE tissues are routinely prepared and are readily available. Although we previously reported the applicability of 72GC to FFPE tissues (DOI: 10.1016/j.clbc.2013.11.006), the present study aimed to improve the accuracy of 95GC for FFPE tissues using the reference robust multiarray average (refRMA) method, optimized for FFPE tissues. Therefore, a 95GC RS (Recurrence Score) was first developed and then the accuracy of the newly developed 95GC algorithm for FFPE tissues was evaluated using the 95GC RS. These 31 pairs of FF and FFPE data are included in the concordance analysis of 95GC high/low results between FF and FFPE (Figure 3B). A long time passed, and now it is unclear how these 31 cases were distributed among the analysis. *Note: This old data has been updated multiple times by the other members. Then, there are some differences from the original paper and unclear points still remain. Therefore, do not use it for formal analysis aimed at public insurance coverage etc. This is for research purposes only. Please cite this paper when writing a new paper. PMID: 31638234 DOI: 10.3892/or.2019.7358

ORGANISM(S): Homo sapiens

PROVIDER: GSE234686 | GEO | 2023/06/16

REPOSITORIES: GEO

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Project description:This study was performed to evaluated RNA extraction and gene expression analysis of FFPE specimen stored for more than 20 years. Using long time stored FFPE material; large retrospective studies correlating molecular features with therapeutic response and clinical outcome, can be performed. Quantitative PCR (qPCR) was used to evaluate RNA extraction methods and to compare gene expression profiles of FFPE and fresh frozen (FF) tissue. Extracted RNA was subsequently subjected to microarray analysis and compared to qPCR data. The Ambion RecoverAll kit appears to be particularly suited for RNA extraction of long time stored FFPE tissues. Gene expression analysis using Affymetrix platform displayed a high degree of correlation for endogenous control genes comparing FF and FFPE tissues. We conclude that high quality gene expression signatures can be recognized using Affymetrix gene expression platform on FFPE tissue stored for more than 20 years. However, a general interpretation must be done with caution as different FFPE procedures have varying effects on RNA quality. Three RNA extraction methods from Roche (Basel, Switzerland), Ambion (Austin, TX, USA) and Qiagen (Hilden, Germany), designed for FFPE material was used. The methods were compared using qPCR. For the qPCR analysis, two different concentrations of input cDNA were used (20ng and 100ng) and 32 human endogenous control genes were examined. RNA from the Ambion FFPE kit was further analyzed by using microarray. Amplification of RNA prior to microarray analysis was performed using Nugen technologies (San Carlos, CA, USA). Nugen has developed amplification kits both for FFPE and FF materials: WT- ovation FFPE RNA amplification System V2 (Nugen FFPE), Ovation FF RNA Amplification System V2 (Nugen V2 FF) and Ovation FF Pico RNA Amplification System (Nugen PICO FF). The Nugen FFPE kit, designed for FFPE material was only used for FFPE tissue. The Nugen Pico FF kit, designed to target small amounts of FF RNA (>500 pg) and the Nugen V2 FF kit designed to target total FF RNA, were only used for FF tissue. Affymetrix standard amplification protocol (Affy FF) designed for FF RNA was also included as the standard method for amplification.

Project description:This experiment contains the RNA-Seq samples only. Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable resource for clinical research. However, nucleic acids extracted from FFPE tissues are fragmented and chemically modified making them challenging to use in molecular studies. We analysed 23 fresh-frozen (FF), 35 FFPE and 38 paired FF/FFPE specimens, representing six different human tissue types (bladder, prostate and colon carcinoma; liver and colon normal tissue; reactive tonsil) in order to examine the potential use of FFPE samples in next-generation sequencing (NGS) based retrospective and prospective clinical studies. Two methods for DNA and three methods for RNA extraction from FFPE tissues were compared and were found to affect nucleic acid quantity and quality. DNA and RNA from selected FFPE and paired FF/FFPE specimens were used for exome and transcriptome analysis. Preparations of DNA Exome-Seq libraries was more challenging (29.5 % success) than that of RNA-Seq libraries, presumably because of modifications to FFPE tissue-derived DNA. Libraries could still be prepared from RNA isolated from two-decade old FFPE tissues. Data were analysed using the CLC Bio Genomics Workbench and revealed systematic differences between FF and FFPE tissue-derived nucleic acid libraries. In spite of this, pairwise analysis of DNA Exome-Seq data showed concordance for 70-80 % of variants in FF and FFPE samples stored for fewer than three years. RNA-Seq data showed high correlation of expression profiles in FF/FFPE pairs (Pearson Correlations of 0.90 +/- 0.05), irrespective of storage time (up to 244 months) and tissue type. A common set of 1,494 genes was identified with expression profiles that were significantly different between paired FF and FFPE samples irrespective of tissue type. Our results are promising and suggest that NGS can be used to study FFPE specimens in both prospective and retrospective archive-based studies in which FF specimens are not available.

Project description:As proteins represent cell function, proteomic analysis of prostate tissues may elucidate the mechanisms of prostate cancer (PCa) progression. To that end, formalin-fixed paraffin-embedded (FFPE) tissues are valuable resources, but associated with large analytical difficulties due to protein cross-linking. Our aim is to characterize tissue protein changes associated with castration resistant PCa. This study focused at the development of a reliable protocol for the proteomic analysis of FFPE tissues which was followed by a pilot study using FFPE PCa clinical samples to investigate whether the optimized protocol can provide biologically relevant data in the context of PCa. Archival FFPE mouse tissues were processed using seven protein extraction protocols including combinations of homogenization means (beads, sonication, boiling) and buffers (Tris-HCl and Urea-Thiourea). The proteomics output was evaluated by SDS electrophoresis and high resolution LC-MS/MS analysis. FFPE tissues (3 sections, 15��m each per sample) from 10 patients with PCa corresponding to tumor (GS=6 or GS��8) and adjacent benign regions were processed with the optimized protocol. Extracted proteins were analyzed by GeLC-MS/MS followed by statistical and bioinformatics analysis using Cytsoscape and Open Targets tool. A combination of cell lysis by Tris-HCl, SDS and DTE with bead homogenization, sonication and boiling provided most efficient protein extraction from FFPE tissues based on protein identifications and reproducibility. Comparison between the FFPE and matched FF tissues showed a substantial overlap in protein identifications (502 common out of 1,216 IDs commonly identified in all FF samples and 530 IDs commonly identified in all FFPE samples) with a strong correlation in relative abundances (rs=0.846, p<.001). Proteins significantly deregulated between PCa GS��8 and PCa GS=6 represented extracellular matrix organisation, phosphorylation and gluconeogenesis pathways, while proteins deregulated between cancerous tissues and benign counterparts, reflected increased translation, peptide synthesis and protein metabolism in the former, as expected. Further disease association analysis using the Open Targets platform showed that 60% of the differentially expressed proteins in cancer versus normal adjacent were significantly associated with PCa. These results support the relevance of the proteomic findings in the context of PCa and the reliability of the optimized protocol for proteomics analysis of FFPE material. A large scale study including 160 clinical FFPE prostate cancer and benign tissues to define protein changes associated with aggressive PCa, is planned.

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The 31 pairs of FF (fresh frozen) andFFPE (Formalin-fixed paraffin-embedded) data for the application of 95GC to FFPE in 2019.

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