scholarly journals Differential Expression of miR-101 and miR-744 in Nasopharyngeal Carcinoma in Pahang State of Malaysia

2018 ◽  
Vol 16 (5) ◽  
pp. 295-306
Author(s):  
Azmir AHMAD ◽  
Haziq Abdul WAHID ◽  
Wan Ishlah LEMAN ◽  
Noor Syamila OTHMAN ◽  
Azlina Abdul RAHMAN ◽  
...  
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Sample Size ◽  
Differential Expression ◽  
Control Group ◽  
Roc Curve Analysis ◽  
Diagnostic Biomarkers ◽  
Total Rna ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded

Previous study found that microRNA-101 (miR-101) and microRNA-744 (miR-744) were deregulated in head and neck cancers and were implicated in nasopharyngeal carcinoma (NPC) carcinogenesis. Thus, this study aimed to determine the expression of miR-101 and miR-744 in NPC and analyse the utility of these microRNAs (miRNAs) as diagnostic biomarkers. Total RNA was extracted from 31 NPC and 7 non-NPC control formalin-fixed paraffin-embedded (FFPE) samples. Complementary DNA (cDNA) was synthesized from the total RNA and proceeded with quantitative real-time polymerase chain reaction. Differential expression of miR-101 and miR-744 were calculated from quantification cycle (Cq) data using 2-ΔΔCq calculation. The performance of these miRNAs were calculated using receiver operating characteristic (ROC) curve analysis. The differential expression for miR-101 and miR-744 were -1.39 (p < 0.05) and 2.48 (p > 0.05), respectively, where the deregulations were consistent with the previous report. The area under curve for miR-101, miR-744 and combination of miR-101 and miR-744 were 0.654 (95 % CI: 0.465 - 0.844), 0.588 (95 % CI: 0.368 - 0.808) and 0.626 (95 % CI: 0.481 - 0.771), respectively. However, re-analysis using balanced sample size between NPC and non-NPC control group showed the value decreased to 0.653 (95 % CI: 0.347 - 0.959) for miR-101 but increased to 0.827 (95 % CI: 0.601 - 1.000) for miR-744 and 0.758 (95 % CI: 0.576 - 0.939) for the combination of miR-101 and miR-744, indicating the importance of having a balanced sample size. We have successfully determined the expression of miR-101 and miR-744 in NPC samples. We also demonstrated statistically the utility of these miRNAs as diagnostic biomarkers.

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 Time and tumor type (primary or metastatic) do not influence the detection of BRAF/NRAS mutations in formalin fixed paraffin embedded samples from melanomas

2016 ◽  
Vol 54 (11) ◽  
Author(s):  
Miriam Potrony ◽  
Celia Badenas ◽  
Bénédicte Naerhuyzen ◽  
Paula Aguilera ◽  
Joan Anton Puig-Butille ◽  
...  
Keyword(s):  
Melanoma Patient ◽  
Primary Melanoma ◽  
Sentinel Lymph Nodes ◽  
Tumor Type ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Complete Sequencing ◽  
Formalin Fixed ◽  
Tumor Area

AbstractBackground:Methods:DNA was obtained from 144 FFPE samples (62 primary melanoma, 43 sentinel lymph nodes [SLN] and 39 metastasis).Results:Complete sequencing results were obtained from 75% (108/144) of the samples, and at least one gene was sequenced in 89% (128/144) of them.Conclusions:Preserving sufficient tumor area in FFPE blocks is important. It is necessary to keep the FFPE blocks, no matter their age, as they are necessary to decide the best treatment for the melanoma patient.

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

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.

Results of first proficiency test for KRAS testing with formalin-fixed, paraffin-embedded cell lines in China

2014 ◽  
Vol 52 (12) ◽  
Author(s):  
Rui Zhang ◽  
Yanxi Han ◽  
Jie Huang ◽  
Liang Ma ◽  
Yulong Li ◽  
...  
Keyword(s):  
Cell Lines ◽  
Laboratory Testing ◽  
Proficiency Test ◽  
Cultured Cell ◽  
Proficiency Panel ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

AbstractLaboratory testing forArtificial FFPE samples were prepared from cultured cell lines to construct a proficiency panel of 10 samples covering eightThe percentages of mutant

scholarly journals Comparison of Whole Genome Amplification Methods for Analysis of DNA Extracted from Microdissected Early Breast Lesions in Formalin-Fixed Paraffin-Embedded Tissue

ISRN Oncology ◽  
2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Nona Arneson ◽  
Juan Moreno ◽  
Vladimir Iakovlev ◽  
Arezou Ghazani ◽  
Keisha Warren ◽  
...  
Keyword(s):  
Comparative Genomic Hybridization ◽  
Whole Genome Amplification ◽  
Comparative Genomic ◽  
Whole Genome ◽  
Genomic Hybridization ◽  
Genome Amplification ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

To understand cancer progression, it is desirable to study the earliest stages of its development, which are often microscopic lesions. Array comparative genomic hybridization (aCGH) is a valuable high-throughput molecular approach for discovering DNA copy number changes; however, it requires a relatively large amount of DNA, which is difficult to obtain from microdissected lesions. Whole genome amplification (WGA) methods were developed to increase DNA quantity; however their reproducibility, fidelity, and suitability for formalin-fixed paraffin-embedded (FFPE) samples are questioned. Using aCGH analysis, we compared two widely used approaches for WGA: single cell comparative genomic hybridization protocol (SCOMP) and degenerate oligonucleotide primed PCR (DOP-PCR). Cancer cell line and microdissected FFPE breast cancer DNA samples were amplified by the two WGA methods and subjected to aCGH. The genomic profiles of amplified DNA were compared with those of non-amplified controls by four analytic methods and validated by quantitative PCR (Q-PCR). We found that SCOMP-amplified samples had close similarity to non-amplified controls with concordance rates close to those of reference tests, while DOP-amplified samples had a statistically significant amount of changes. SCOMP is able to amplify small amounts of DNA extracted from FFPE samples and provides quality of aCGH data similar to non-amplified samples.

scholarly journals Characterizations of Gene Alterations in Melanoma Patients from Chinese Population

2020 ◽  
Vol 2020 ◽  
pp. 1-8
Author(s):  
Yi Luo ◽  
Zhenzhen Zhang ◽  
Jianfan Liu ◽  
Linqing Li ◽  
Xuezheng Xu ◽  
...  
Keyword(s):  
Braf Mutations ◽  
Driver Genes ◽  
Cancer Driver ◽  
Formalin Fixed Paraffin ◽  
Significant Difference ◽  
The Status ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Comprehensive Genomic Profiling ◽  
Gene Alterations

Melanoma is a human skin malignant tumor with high invasion and poor prognosis. The limited understanding of genomic alterations in melanomas in China impedes the diagnosis and therapeutic strategy selection. We conducted comprehensive genomic profiling of melanomas from 39 primary and metastatic formalin-fixed paraffin-embedded (FFPE) samples from 27 patients in China based on an NGS panel of 223 genes. No significant difference in gene alterations was found between primary and metastasis melanomas. The status of germline mutation, CNV, and somatic mutation in our cohort was quite different from that reported in Western populations. We further delineated the mutation patterns of 4 molecular subgroups (BRAF, RAS, NF1, and Triple-WT) of melanoma in our cohort. BRAF mutations were more frequently identified in melanomas without chromic sun-induced damage (non-CSD), while RAS mutations were more likely observed in acral melanomas. NF1 and Triple-WT subgroups were unbiased between melanomas arising in non-CSD and acral skin. BRAF, RAS, and NF1 mutations were significantly associated with lymph node metastasis or presence of ulceration, implying that these cancer driver genes were independent prognostic factors. In summary, our results suggest that mutational profiles of malignant melanomas in China are significantly different from Western countries, and both gene mutation and amplification play an important role in the development and progression of melanomas.

scholarly journals A novel use of random priming-based single-strand library preparation for whole genome sequencing of formalin-fixed paraffin-embedded tissue samples

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Emily A Saunderson ◽  
Ann-Marie Baker ◽  
Marc Williams ◽  
Kit Curtius ◽  
J Louise Jones ◽  
...  
Keyword(s):  
Genome Sequencing ◽  
Preparation Method ◽  
Degraded Dna ◽  
Whole Genome ◽  
Library Preparation ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed ◽  
Library Preparation Method

Abstract The desire to analyse limited amounts of biological material, historic samples and rare cell populations has collectively driven the need for efficient methods for whole genome sequencing (WGS) of limited amounts of poor quality DNA. Most protocols are designed to recover double-stranded DNA (dsDNA) by ligating sequencing adaptors to dsDNA with or without subsequent polymerase chain reaction amplification of the library. While this is sufficient for many applications, limited DNA requires a method that can recover both single-stranded DNA (ssDNA) and dsDNA. Here, we present a WGS library preparation method, called ‘degraded DNA adaptor tagging’ (DDAT), adapted from a protocol designed for whole genome bisulfite sequencing. This method uses two rounds of random primer extension to recover both ssDNA and dsDNA. We show that by using DDAT we can generate WGS data from formalin-fixed paraffin-embedded (FFPE) samples using as little as 2 ng of highly degraded DNA input. Furthermore, DDAT WGS data quality was higher for all FFPE samples tested compared to data produced using a standard WGS library preparation method. Therefore, the DDAT method has potential to unlock WGS data from DNA previously considered impossible to sequence, broadening opportunities to understand the role of genetics in health and disease.

Comparison between two different next generation sequencing platforms for clinical relevant gene mutation test in solid tumours

2020 ◽  
Vol 73 (9) ◽  
pp. 602-604
Author(s):  
Silvia Bessi ◽  
Francesco Pepe ◽  
Marco Ottaviantonio ◽  
Pasquale Pisapia ◽  
Umberto Malapelle ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
High Performance ◽  
Solid Tumours ◽  
Next Generation ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Thermo Fisher Scientific ◽  
Formalin Fixed Paraffin Embedded ◽  
Sequencing Platforms ◽  
Generation Sequencing

In the present study, we analysed 44 formalin fixed paraffin embedded (FFPE) from different solid tumours by adopting two different next generation sequencing platforms: GeneReader (QIAGEN, Hilden, Germany) and Ion Torrent (Thermo Fisher Scientific, Waltham, Massachusetts, USA). We highlighted a 100% concordance between the platforms. In addition, focusing on variant detection, we evaluated a very good agreement between the two tests (Cohen’s kappa=0.84) and, when taking into account variant allele fraction value for each variant, a very high concordance was obtained (Pearson’s r=0.94). Our results underlined the high performance rate of GeneReader on FFPE samples and its suitability in routine molecular predictive practice.

PDL-1 and immunohistochemistry markers in esophageal adenocarcinoma.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e15567-e15567
Author(s):  
Dawn E. Jaroszewski ◽  
Joanne Xiu ◽  
Zoran Gatalica ◽  
Staci Beamer ◽  
Melissa Stanton ◽  
...  
Keyword(s):  
Clinical Outcomes ◽  
Esophageal Adenocarcinoma ◽  
Neoadjuvant Treatment ◽  
Formalin Fixed Paraffin ◽  
Ffpe Tumor ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

e15567 Background: Esophageal adenocarcinoma (EAC) prognosis is poor and there is a need to identify patients that benefit most from neoadjuvant therapy. To examine the association of various biomarkers with clinical outcomes in neoadjuvant treatment of EAC, we retrospectively evaluated the biomarker expression (TS, ERCC1, TOPO1, PD-L1, PD-1) in patient matched formalin-fixed paraffin-embedded (FFPE) tumor samples. Methods: Immunohistochemistry of TS (TS106/4H4B1) , ERCC1 (Ab. 8F1), TOPO1 (1D6), PD-L1 (both 22c3 and SP142), PD-1 (NAT105), and chromogenic in-situ hybridization (CISH) of Her2 were performed on FFPE samples from 35 patients across 2 institutions at time of EAC diagnosis and after treatment when available. Retrospective clinical data and survival (5/2006-1/2016) was analyzed with a mean follow up of 110 months (range 22-306). Results: Overexpression (pre/post-treatment) of TS (60%/54%), ERCC1 (69%/16%), TOPO1 (74%/50%), PD-1 (54%/63%), PD-L1 (SP142) (2.9%/4%), PD-L1 (22c3) (0%/4%) and amplification of Her2 (18%/23%) were observed. Pretreatment observed PD-L1 levels were lower in our study (3%) when compared to other studies in EAC specimens (35%). Immunohistochemistry and changes observed after chemoradiation are reviewed in Table. No markers had significant correlation with prognosis however TS negative expression showed a non-significant (p=0.15) trend towards improved survival. Conclusions: Analyzing biomarkers in our neoadjuvant EAC cohort demonstrated a lower than expected PD-L1 positivity. In the largest cohort, to our knowledge, of patient matched FFPE tumor samples, we did not observe a statistically significant association between TS, ERCC1, TOPO1, PD-L1, or PD-1 with improved clinical outcomes. [Table: see text]

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