Integrated differential DNA methylation and gene expression of formalin-fixed paraffin-embedded uveal melanoma specimens identifies genes associated with early metastasis and poor prognosis

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.

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Expression of proliferation genes in formalin-fixed paraffin-embedded (FFPE) tissue from breast carcinomas. Feasibility and relevance for a routine histopathology laboratory

Journal of Clinical Pathology ◽

10.1136/jclinpath-2016-203786 ◽

2016 ◽

Vol 70 (1) ◽

pp. 25-32 ◽

Cited By ~ 1

Author(s):

Carla Thomas ◽

Cleo Robinson ◽

Ben Dessauvagie ◽

Benjamin Wood ◽

Greg Sterrett ◽

...

Keyword(s):

Gene Expression ◽

Breast Carcinoma ◽

Expression Profiling ◽

Proliferative Activity ◽

Breast Cancers ◽

Breast Carcinomas ◽

Ki 67 ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed

AimBreast carcinoma proliferative activity, histological grade and commercial molecular tests are all important in prognostication and treatment. There is a particular need for improved, standardised techniques for subclassification of grade 2 breast cancers into low-risk and high-risk prognostic groups. In this study we investigated whether gene expression profiling of five proliferation genes was feasible using breast cancer tissue in a clinical setting and whether these profiles could enhance pathological assessment.MethodsExpression of five proliferation gene mRNAs; Ki-67, STK 15, CCNB1, CCND1 and MYBL2, was quantified in 27 breast carcinomas and compared with Ki-67 proliferation index (PI) and Nottingham mitotic score.ResultsExpression of Ki-67, STK15 and MYBL2 mRNA showed moderate Spearman's correlation with Ki-67 PI (p<0.01), but CCND1 and CCNB1 showed weak, non-significant correlation. Individual gene expression did not associate with mitotic score but combined mRNA expression correlated with both Ki-67 PI (p=0.018) and mitotic score (p=0.03; 0.007).ConclusionsThis study confirms mRNA analysis in breast carcinoma formalin-fixed, paraffin-embedded samples is feasible and suggests gene expression profiling, using a small set of five proliferation genes, has potential in aiding histological grading or assessment of proliferative activity of breast cancers. To fully evaluate the clinical applicability of this approach, a larger cohort study with long-term follow-up data is required.

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

10.1101/656736 ◽

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.

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The Gene Expression of MMP-2 and TIMP-1 in Non-pathological Paratumoral and Autopsy Lung Tissues

Immunoregulation ◽

10.32598/immunoregulation.4.1.7 ◽

2021 ◽

Vol 4 (1) ◽

pp. 61-65

Author(s):

Elahe Esmaeili ◽

◽

Sara Ghaffarpour ◽

Alireza Sadeghipour ◽

Tooba Ghazanfari ◽

...

Keyword(s):

Gene Expression ◽

Lung Tissue ◽

Matrix Metalloproteinase 2 ◽

Control Group ◽

Extracellular Matrix Remodeling ◽

Matrix Remodeling ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed ◽

Gene Expression Levels

Background: Finding a sample of healthy tissue is a critical challenge in research studies. Non-pathological Tissue adjacent to the tumor (NAT) specimens is usually used as the control in several studies. However, little is known about the similarity of NAT to healthy tissues. Here, we compared the expression of Matrix Metalloproteinase 2 (MMP-2) and its inhibitor, Tissue Inhibitors of MMP (TIMP)-1 as extracellular matrix remodeling factors in NAT and autopsy lung tissue. Materials and Methods: RNA of 7 NAT and 6 Formalin-Fixed Paraffin-Embedded (FFPE) lung autopsies from healthy people as the control group was extracted, and cDNA was synthesized. The gene expression levels of MMP-2 and TIMP-1 were evaluated by real-time PCR. Results: There were no significant differences in the expression of MMP-2, TIMP-1, or their ratio between the two groups. Conclusion: The results showed that NAT could be used as healthy controls in lung tissue studies for MMP-2 and TIMP-1.

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Validation of a Gene Expression Test for Mesothelioma Prognosis in Formalin-Fixed Paraffin-Embedded Tissues

Journal of Molecular Diagnostics ◽

10.1016/j.jmoldx.2016.07.011 ◽

2017 ◽

Vol 19 (1) ◽

pp. 65-71 ◽

Cited By ~ 6

Author(s):

Assunta De Rienzo ◽

Robert W. Cook ◽

Jeff Wilkinson ◽

Corinne E. Gustafson ◽

Waqas Amin ◽

...

Keyword(s):

Gene Expression ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed

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Formalin Fixed Paraffin Embedded Tissue-Based Gene Expression and Immune Cell Profiling as a Tool for Monitoring Immune Response in Kidney Biopsies with Low Grade Inflammation

Transplantation Journal ◽

10.1097/01.tp.0000543529.71659.0b ◽

2018 ◽

Vol 102 ◽

pp. S624

Author(s):

Henrik Junger ◽

Dejan Dobi ◽

Tara Sigdel ◽

Adeline Chen ◽

Zoltan Laszik

Keyword(s):

Gene Expression ◽

Immune Response ◽

Immune Cell ◽

Low Grade ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Low Grade Inflammation ◽

Formalin Fixed

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Maximizing RNA Yield from Archival Renal Tumors and Optimizing Gene Expression Analysis

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057109355059 ◽

2009 ◽

Vol 15 (1) ◽

pp. 80-85 ◽

Cited By ~ 10

Author(s):

Sean T. Glenn ◽

Karen L. Head ◽

Bin T. Teh ◽

Kenneth W. Gross ◽

Hyung L. Kim

Keyword(s):

Gene Expression ◽

Expression Analysis ◽

Reverse Transcription ◽

Gene Expression Analysis ◽

Rna Isolation ◽

Proteinase K ◽

Specific Primers ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed

Formalin-fixed, paraffin-embedded tissues are widely available for gene expression analysis using TaqMan ® PCR. Five methods, including 4 commercial kits, for recovering RNA from paraffin-embedded renal tumor tissue were compared. The MasterPure™ kit from Epicentre produced the highest RNA yield. However, the difference in RNA yield between the kit from Epicenter and Invitrogen’s TRIzol method was not significant. Using the top 3 RNA isolation methods, the manufacturers’ protocols were modified to include an overnight Proteinase K digestion. Overnight protein digestion resulted in a significant increase in RNA yield. To optimize the reverse transcription reaction, conventional reverse transcription with random oligonucleotide primers was compared to reverse transcription using primers specific for genes of interest. Reverse transcription using gene-specific primers significantly increased the quantity of cDNA detectable by TaqMan ® PCR. Therefore, expression profiling of formalin-fixed, paraffin-embedded tissue using TaqMan® qPCR can be optimized by using the MasterPure™ RNA isolation kit modified to include an overnight Proteinase K digestion and gene-specific primers during the reverse transcription.

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