Gene expression profile comparison of malignant melanoma and benign melanocytic nevi from formalin-fixed paraffin-embedded specimens

2006 ◽  
Vol 16 (Supplement 1) ◽  
pp. S56-S57
Author(s):  
M. Moore ◽  
M. Opel ◽  
P. Duck ◽  
M. Reyes ◽  
K. Yau ◽  
...  
2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 15039-15039
Author(s):  
M. G. Ghi ◽  
A. Paccagnella ◽  
G. Stanta ◽  
B. Murer ◽  
F. Petrera ◽  
...  

15039 Background: Patients with chemoresponsive tumors are more likely to have a survival advantage, consequently a great interest is being placed on the identification of predictive markers. Currently, the improving in the extraction techniques allow the detection of gene profile at the mRNA level from FFPE materials. The purpose of the study was to analyse the m-RNA level of specific genes from FFPE (Stanta et al, BioTechniques 1998), both primary tumor (T) and locoregional lymphnodes (N) in CRC patients treated with chemotherapy (CT), and to relate it with chemoresponsiveness. Material and Methods: RNA was extract from FFPE tumor specimen both in T and N. RNA was reversing transcribed to cDNA. From the cDNA sample, BRCA1, ERCC1, CES2 and TS gene transcripts were specifically amplified by PCR. ERCC1 and BRCA1 are involved in platinum-compound resistance; TS is involved in responses to 5Fluorouracil (5FU) and CES2 level expression was recently related to Irinotecan pro-drug activation. Eligible patients included metastatic CRC patients treated from March 2000 to December 2003 as first line CT with Oxaliplatin/5FU or Irinotecan/5FU or 5FU alone. Results: Forty-five consecutive patients were retrospectively analysed. 15 of them received Oxaliplatin/5FU, 15 Irinotecan/5FU and the other 15 5FU alone. Median age was 64 (range 46–75). 13 patients (28%) had received adjuvant CT. 32 patients (72%) had metastatic disease at the time of surgery. Global Response Rate was 44%. All 45 patients received 5FU and they were analysed for the level of TS expression. With Multiple Regression Analysis, no statistical significant relation between TS level expression and response to 5FU was observed (P=0.36). A strong relation was observed between ERCC1 and response to Oxaliplatin (P=0.006) and a possible correlation of BRCA1-exon11 level expression and response to Irinotecan (P=0.06). The analyses of CES2 and the relation between gene expression and survival are ongoing. Conclusions: The analyses of mRNA gene expression profile from FFPE could be use to predicting response to CT in CRC patients. To test this hypothesis, a randomized phase II-III prospective study of tailored therapy in metastatic CRC is planned. No significant financial relationships to disclose.


BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Michal Marczyk ◽  
Chunxiao Fu ◽  
Rosanna Lau ◽  
Lili Du ◽  
Alexander J. Trevarton ◽  
...  

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.


2016 ◽  
Vol 70 (1) ◽  
pp. 25-32 ◽  
Author(s):  
Carla Thomas ◽  
Cleo Robinson ◽  
Ben Dessauvagie ◽  
Benjamin Wood ◽  
Greg Sterrett ◽  
...  

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.


2019 ◽  
Author(s):  
Christopher A. Hilker ◽  
Aditya V. Bhagwate ◽  
Jin Sung Jang ◽  
Jeffrey G Meyer ◽  
Asha A. Nair ◽  
...  

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.


2021 ◽  
Vol 4 (1) ◽  
pp. 61-65
Author(s):  
Elahe Esmaeili ◽  
◽  
Sara Ghaffarpour ◽  
Alireza Sadeghipour ◽  
Tooba Ghazanfari ◽  
...  

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.


2017 ◽  
Vol 19 (1) ◽  
pp. 65-71 ◽  
Author(s):  
Assunta De Rienzo ◽  
Robert W. Cook ◽  
Jeff Wilkinson ◽  
Corinne E. Gustafson ◽  
Waqas Amin ◽  
...  

2009 ◽  
Vol 15 (1) ◽  
pp. 80-85 ◽  
Author(s):  
Sean T. Glenn ◽  
Karen L. Head ◽  
Bin T. Teh ◽  
Kenneth W. Gross ◽  
Hyung L. Kim

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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