scholarly journals PATH-26. RNA SEQUENCING OF FORMALIN-FIXED PARAFFIN-EMBEDDED SPECIMENS IN DIAGNOSTIC ROUTINE IDENTIFIES CLINICALLY RELEVANT GENE FUSIONS

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii429-iii430
Author(s):  
Damian Stichel ◽  
Daniel Schrimpf ◽  
Jochen Meyer ◽  
Annika Wefers ◽  
Philipp Sievers ◽  
...  
Keyword(s):  
Brain Tumor ◽  
Rna Sequencing ◽  
Fusion Gene ◽  
Detection Accuracy ◽  
Gene Fusions ◽  
Sequencing Data ◽  
Methylation Array ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

Abstract Pediatric brain tumor entities harbor a variety of gene fusions. Whilst other molecular parameters like somatic mutations and copy number alterations have become pivotal for brain tumor diagnostics, gene fusions are only less well covered by routinely applied methylation arrays or targeted next-generation sequencing of DNA. In a routine diagnostic setting we established and optimized a workflow for investigation of gene fusions in formalin-fixed paraffin-embedded (FFPE) tumor tissues by using RNA sequencing. Assessing different tools for calling fusions from raw data, we found relevant fusions in 66 out of 101 (65%) analyzed cases in a prospective cohort collected over 26 months. In 43 (43%) cases the fusions were of decisive diagnostic relevance and in 40 (40%) cases the fusion genes rendered a druggable target. Besides the relevance of pathognomonic fusions for diagnostics, especially the detection of druggable gene fusions yields direct benefit to the patients. This approach allows for an unbiased search for fusion events in the tested samples. Besides rare variants of established fusions which were not detected by prior targeted analyses, we identified previously unreported fusion events. Exemplified on KIAA1549:BRAF fusion, we in addition provide an overview of the detection accuracy of different methods, including breakpoint detection in DNA methylation array data and fusion gene detection in DNA panel sequencing data. Our data show that RNA sequencing has great diagnostic as well as therapeutic value by clinically detecting relevant alterations.

scholarly journals The impact of RNA extraction method on accurate RNA sequencing from formalin-fixed paraffin-embedded tissues

BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Michal Marczyk ◽  
Chunxiao Fu ◽  
Rosanna Lau ◽  
Lili Du ◽  
Alexander J. Trevarton ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Rna Sequencing ◽  
Rna Extraction ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
The Impact ◽  
Formalin Fixed

Abstract Background Utilization of RNA sequencing methods to measure gene expression from archival formalin-fixed paraffin-embedded (FFPE) tumor samples in translational research and clinical trials requires reliable interpretation of the impact of pre-analytical variables on the data obtained, particularly the methods used to preserve samples and to purify RNA. Methods Matched tissue samples from 12 breast cancers were fresh frozen (FF) and preserved in RNAlater or fixed in formalin and processed as FFPE tissue. Total RNA was extracted and purified from FF samples using the Qiagen RNeasy kit, and in duplicate from FFPE tissue sections using three different kits (Norgen, Qiagen and Roche). All RNA samples underwent whole transcriptome RNA sequencing (wtRNAseq) and targeted RNA sequencing for 31 transcripts included in a signature of sensitivity to endocrine therapy. We assessed the effect of RNA extraction kit on the reliability of gene expression levels using linear mixed-effects model analysis, concordance correlation coefficient (CCC) and differential analysis. All protein-coding genes in the wtRNAseq and three gene expression signatures for breast cancer were assessed for concordance. Results Despite variable quality of the RNA extracted from FFPE samples by different kits, all had similar concordance of overall gene expression from wtRNAseq between matched FF and FFPE samples (median CCC 0.63–0.66) and between technical replicates (median expression difference 0.13–0.22). More than half of genes were differentially expressed between FF and FFPE, but with low fold change (median |LFC| 0.31–0.34). Two out of three breast cancer signatures studied were highly robust in all samples using any kit, whereas the third signature was similarly discordant irrespective of the kit used. The targeted RNAseq assay was concordant between FFPE and FF samples using any of the kits (CCC 0.91–0.96). Conclusions The selection of kit to purify RNA from FFPE did not influence the overall quality of results from wtRNAseq, thus variable reproducibility of gene signatures probably relates to the reliability of individual gene selected and possibly to the algorithm. Targeted RNAseq showed promising performance for clinical deployment of quantitative assays in breast cancer from FFPE samples, although numerical scores were not identical to those from wtRNAseq and would require calibration.

scholarly journals Impact of RNA Extraction and Target Capture Methods on RNA Sequencing Using Formalin-Fixed, Paraffin Embedded Tissues

2019 ◽  
Author(s):  
Christopher A. Hilker ◽  
Aditya V. Bhagwate ◽  
Jin Sung Jang ◽  
Jeffrey G Meyer ◽  
Asha A. Nair ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fusion Gene ◽  
Rna Extraction ◽  
Extraction Methods ◽  
Rna Seq ◽  
Gene Detection ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Whole Transcriptome ◽  
Formalin Fixed

AbstractFormalin fixed paraffin embedded (FFPE) tissues are commonly used biospecimen for clinical diagnosis. However, RNA degradation is extensive when isolated from FFPE blocks making it challenging for whole transcriptome profiling (RNA-seq). Here, we examined RNA isolation methods, quality metrics, and the performance of RNA-seq using different approaches with RNA isolated from FFPE and fresh frozen (FF) tissues. We evaluated FFPE RNA extraction methods using six different tissues and five different methods. The reproducibility and quality of the prepared libraries from these RNAs were assessed by RNA-seq. We next examined the performance and reproducibility of RNA-seq for gene expression profiling with FFPE and FF samples using targeted (Kinome capture) and whole transcriptome capture based sequencing. Finally, we assessed Agilent SureSelect All-Exon V6+UTR capture and the Illumina TruSeq RNA Access protocols for their ability to detect known gene fusions in FFPE RNA samples. Although the overall yield of RNA varied among extraction methods, gene expression profiles generated by RNA-seq were highly correlated (>90%) when the input RNA was of sufficient quality (≥DV200 30%) and quantity (≥ 100 ng). Using gene capture, we observed a linear relationship between gene expression levels for shared genes that were captured using either All-Exon or Kinome kits. Gene expression correlations between the two capture-based approaches were similar using RNA from FFPE and FF samples. However, TruSeq RNA Access protocol provided significantly higher exon and junction reads when compared to the SureSelect All-Exon capture kit and was more sensitive for fusion gene detection. Our study established pre and post library construction QC parameters that are essential to reproducible RNA-seq profiling using FFPE samples. We show that gene capture based NGS sequencing is an efficient and highly reproducible strategy for gene expression measurements as well as fusion gene detection.

Next-generation RNA Sequencing of Archival Formalin-fixed Paraffin-embedded Urothelial Bladder Cancer

2014 ◽  
Vol 66 (6) ◽  
pp. 982-986 ◽  
Author(s):  
Yu Liu ◽  
Aidan P. Noon ◽  
Eduardo Aguiar Cabeza ◽  
Jess Shen ◽  
Cynthia Kuk ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Rna Sequencing ◽  
Next Generation ◽  
Urothelial Bladder Cancer ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Urothelial Bladder ◽  
Formalin Fixed
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