A multisite blinded study for the detection of BRAF mutations in formalin-fixed, paraffin-embedded malignant melanoma.
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ABSTRACT:
SUBMITTER: Richter A
PROVIDER: S-EPMC3625889 | biostudies-other | 2013
REPOSITORIES: biostudies-other
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Project description:miRNA expression analysis was done on Uveal melanoma, metastatic and non - metastatic case in formalin fixed paraffin embedded sections, identified from case registry and confirmed by Chromosome insitu hybridization harboring monosomy 3 aberration in metastaic case and no aberration in non metastatic case. 19 miRNAs were found to be expressed only in class I tumors (choroidal melanoma) and not in class II (liver metastasis) and 11 miRNAs were found to be expressed only in class II and not in class I. The tumors were found to harbor oncomirs in both choroidal melanoma and metastasizing melanoma targeting tumor suppressor genes and metastatic suppressor genes. None of the differentially expressed miRNAs in either case were found to be located on the chromosomes which have been proved to carry chromosomal abnormalities. Rather it was found that genes targeted by the miRNAs were found to be present in chromosomal regions that are often found to be deleted 8p22, 13q and 17p. Organism used: Homo sapiens. * Slides: Human miRNA V2 8x15k Arrays AMADID: 19118. * Starting material: 18 slides with formalin fixed Paraffin embedded sections-20 μm. * RNA Samples used: 225-94-DUP, 225-94-E, 727-07-DUP, 727-07 -E. * Labeling kit: Agilent miRNA labeling reagent and Hybridization Kit. * Labeling Method: Ligation of one cyanine3-pCp molecule to the 3' end of RNA molecule with greater than 90% efficiency that generates fluorescent miRNA. * Total RNA and cRNA Purification Kit: Biorad MicroBio Spin 6 columns. * Hybridization Kit: Agilent miRNA Hybridization kit. Hybridization protocol See Agilent Technologies website for miRNA microarray hybridization and wash protocol: http://www.chem.agilent.com/scripts/LiteraturePDF.asp?iWHID=50500 Scan protocol Laser detection of Cyanine 3 and Cyanine 5 fluorescence is performed using a confocal scanning instrument containing two tuned lasers, which excite Cyanine dyes at the appropriate wavelengths. Description Microarrays were scanned on an Agilent scanner (G2505C) at 100% PMT with 10% for lower intensity (XDR Scanning).Data extraction was carried out with Agilent Feature Extraction software (version 9.1), and normalization was done using linear per array algorithm according to the manufacturer’s protocol Data processing Feature extracted data was analyzed using GeneSpring GX v 10.0.2 software from Agilent. Normalization of the data was done in GeneSpring GX using the recommended Percentile shift normalization (50th percentile) . Further quality control of normalized data was done using correlation based condition tree to eliminate bad experiments. High expressed miRNA's were clustered using gene tree to identify significant gene expression patterns.
| S-ECPF-GEOD-17037 | biostudies-other
Project description:Archived formalin-fixed paraffin-embedded (FFPE) samples are the global standard format for preservation of the majority of biopsies in both basic research and translational cancer studies, and profiling chromatin accessibility in the archived FFPE tissues is fundamental to understanding gene regulation. Accurate mapping of chromatin accessibility from FFPE specimens is challenging because of the high degree of DNA damage. Here, we first showed that standard ATAC-seq can be applied to purified FFPE nuclei but yields lower library complexity and a smaller proportion of long DNA fragments. We then present FFPE-ATAC, the first highly sensitive method for decoding chromatin accessibility in FFPE tissues that combines Tn5-mediated transposition and T7 in vitro transcription. The FFPE-ATAC generates high-quality chromatin accessibility profiles with 500 nuclei from a single FFPE tissue section, enables the dissection of chromatin profiles from the regions of interest with the aid of hematoxylin and eosin (H&E) staining, and reveals disease-associated chromatin regulation from the human colorectal cancer FFPE tissue archived for >10 yr. In summary, the approach allows decoding of the chromatin states that regulate gene expression in archival FFPE tissues, thereby permitting investigators to better understand epigenetic regulation in cancer and precision medicine.
| S-EPMC8744681 | biostudies-literature
Project description:Archived formalin-fixed, paraffin-embedded human tumors are widely available and represent a unique source of morphologically defined material. Formalin-fixed, paraffin-embedded tissue is known to contain a wealth of molecular information in the form of microRNAs (miRNAs), which could be correlated with clinical outcome for improved prognostication and/or treatment response. miRNAs are endogenous, noncoding RNAs ( approximately 22 nucleotides) and may function as tumor suppressors or oncogenes. A reliable, robust methodology is needed to take full advantage of archived human cancers, especially for those where fresh-frozen tumor banks are unavailable, for example, malignant melanoma. To this end, we applied a simple-to-use protocol for extracting total RNA from various formalin-fixed, paraffin-embedded specimens (colon, liver, prostate, thyroid, uterus, and skin), optimized for small RNA recovery. Using a "poison primer" strategy (ie, primer silencing), we blocked the amplification of ribosomal RNA, enabling the successful sequencing of 17 novel and 53 known miRNAs (including small RNAs) from 10-year-old archived normal skin, cutaneous scalp melanoma, and sentinel lymph nodes (both negative and positive for metastasis) excised from a 52-year-old man. The cloning incidence provided an estimation of the level of specific miRNA expression, which was confirmed by Northern analysis and quantitative real-time polymerase chain reaction. This methodology can therefore be used to facilitate miRNA discovery from archived human cancers.
| S-EPMC2729839 | biostudies-literature
Project description:Detection and characterization of novel viruses is hampered frequently by the lack of properly stored materials. Especially for the retrospective identification of viruses responsible for past disease outbreaks, often only formalin-fixed paraffin-embedded (FFPE) tissue samples are available. Although FFPE tissues can be used to detect known viral sequences, the application of FFPE tissues for detection of novel viruses is currently unclear. In the present study it was shown that sequence-independent amplification in combination with next-generation sequencing can be used to detect sequences of known and unknown viruses, although with relatively low sensitivity. These findings indicate that this technique could be useful for detecting novel viral sequences in FFPE tissues collected from humans and animals with disease of unknown origin, when other samples are not available. In addition, application of this method to FFPE tissues allows to correlate with the presence of histopathological changes in the corresponding tissue sections.
| S-EPMC7119673 | biostudies-literature
Project description:Formalin fixed paraffin embedded (FFPE) tissues are a vast resource of annotated clinical samples. As such, they represent highly desirable and informative materials for the application of high definition genomics for improved patient management and to advance the development of personalized therapeutics. However, a limitation of FFPE tissues is the variable quality of DNA extracted for analyses. Furthermore, admixtures of non-tumor and polyclonal neoplastic cell populations limit the number of biopsies that can be studied and make it difficult to define cancer genomes in patient samples. To exploit these valuable tissues we applied flow cytometry-based methods to isolate pure populations of tumor cell nuclei from FFPE tissues and developed a methodology compatible with oligonucleotide array CGH and whole exome sequencing analyses. These were used to profile a variety of tumors (breast, brain, bladder, ovarian and pancreas) including the genomes and exomes of matching fresh frozen and FFPE pancreatic adenocarcinoma samples.
| S-EPMC3511535 | biostudies-literature
Project description:BackgroundInvestigating the expression of candidate genes in tissue samples usually involves either immunohistochemical labelling of formalin-fixed paraffin-embedded (FFPE) sections or immunofluorescence labelling of cryosections. Although both of these methods provide essential data, both have important limitations as research tools. Consequently, there is a demand in the research community to be able to perform routine, high quality immunofluorescence labelling of FFPE tissues.ResultsWe present here a robust optimised method for high resolution immunofluorescence labelling of FFPE tissues, which involves the combination of antigen retrieval, indirect immunofluorescence and confocal laser scanning microscopy. We demonstrate the utility of this method with examples of immunofluorescence labelling of human kidney, human breast and a tissue microarray of invasive human breast cancers. Finally, we demonstrate that stained slides can be stored in the short term at 4 degrees C or in the longer term at -20 degrees C prior to images being collected. This approach has the potential to unlock a large in vivo database for immunofluorescence investigations and has the major advantages over immunohistochemistry in that it provides higher resolution imaging of antigen localization and the ability to label multiple antigens simultaneously.ConclusionThis method provides a link between the cell biology and pathology communities. For the cell biologist, it will enable them to utilise the vast archive of pathology specimens to advance their in vitro data into in vivo samples, in particular archival material and tissue microarrays. For the pathologist, it will enable them to utilise multiple antibodies on a single section to characterise particular cell populations or to test multiple biomarkers in limited samples and define with greater accuracy cellular heterogeneity in tissue samples.
| S-EPMC2288605 | biostudies-literature
Project description:BackgroundMolecular characterization of breast and other cancers by gene expression profiling has corroborated existing classifications and revealed novel subtypes. Most profiling studies are based on fresh frozen (FF) tumor material which is available only for a limited number of samples while thousands of tumor samples exist as formalin-fixed, paraffin-embedded (FFPE) blocks. Unfortunately, RNA derived of FFPE material is fragmented and chemically modified impairing expression measurements by standard procedures. Robust protocols for isolation of RNA from FFPE material suitable for stable and reproducible measurement of gene expression (e.g. by quantitative reverse transcriptase PCR, QPCR) remain a major challenge.ResultsWe present a simple procedure for RNA isolation from FFPE material of diagnostic samples. The RNA is suitable for expression measurement by QPCR when used in combination with an optimized cDNA synthesis protocol and TaqMan assays specific for short amplicons. The FFPE derived RNA was compared to intact RNA isolated from the same tumors. Preliminary scores were computed from genes related to the ER response, HER2 signaling and proliferation. Correlation coefficients between intact and partially fragmented RNA from FFPE material were 0.83 to 0.97.ConclusionWe developed a simple and robust method for isolating RNA from FFPE material. The RNA can be used for gene expression profiling. Expression measurements from several genes can be combined to robust scores representing the hormonal or the proliferation status of the tumor.
| S-EPMC2359756 | biostudies-literature
Project description:Lack of routine fresh or frozen tissue is a barrier to widespread transcriptomic analysis of adrenal cortical tumors and an impediment to translational research in endocrinology and endocrine oncology. Our group has previously pioneered the use of targeted amplicon-based next-generation sequencing for archival formalin-fixed paraffin-embedded (FFPE) adrenal tissue specimens to characterize the spectrum of somatic mutations in various forms of primary aldosteronism. Herein, we developed and validated a novel 194-amplicon targeted next-generation RNA sequencing (RNAseq) assay for transcriptomic analysis of adrenal tumors using clinical-grade FFPE specimens. Targeted RNAseq-derived expression values for 27 adrenal cortical tumors, including aldosterone-producing adenomas (APA; n=8), cortisol-producing adenomas (CPA; n=11), and adrenal cortical carcinomas (ACC; n=8), highlighted known differentially-expressed genes (DEGs; i. e., CYP11B2, IGF2, etc.) and tumor type-specific transcriptional modules (i. e., high cell cycle/proliferation transcript expression in ACC, etc.), and a subset of DEGs was validated orthogonally using quantitative reverse transcription PCR (qRT-PCR). Finally, unsupervised hierarchical clustering using a subset of high-confidence DEGs revealed three discrete clusters representing APA, CPA, and ACC tumors with corresponding unique gene expression signatures, suggesting potential clinical utility for a transcriptomic-based approach to tumor classification. Overall, these data support the use of targeted amplicon-based RNAseq for comprehensive transcriptomic profiling of archival FFPE adrenal tumor material and indicate that this approach may facilitate important translational research opportunities for the study of these tumors.
| S-EPMC7880170 | biostudies-literature
Project description:Metabolite profiling has significantly contributed to a deeper understanding of the biochemical metabolic networks and pathways in cancer cells. Metabolomics-based biomarker discovery would greatly benefit from the ability to interrogate retrospective annotated clinical specimens archived as formalin-fixed, paraffin-embedded (FFPE) material. Mass spectrometry-based metabolomic analysis was performed in matched frozen and FFPE human prostate cancers as well as isogenic prostate cancer cell lines. A total of 352 and 460 metabolites were profiled in human tissues and cell lines, respectively. Classes and physical-chemical characteristics of the metabolites preserved in FFPE material were characterized and related to their preservation or loss following fixation and embedding. Metabolite classes were differentially preserved in archival FFPE tissues, regardless of the age of the block, compared with matched frozen specimen, ranging from maximal preservation of fatty acids (78%) to loss of the majority of peptides and steroids. Generally, FFPE samples showed a decrease of metabolites with functional groups, such as carboxamide. As an adjunct technique, metabolic profiles were also obtained in situ from FFPE tissue sections where metabolites were extracted in a manner that preserves tissue architecture. Despite the fact that selected metabolites were not retained after processing, global metabolic profiles obtained from FFPE can be used to predict biologic states and study biologic pathways. These results pave the way for metabolomics-based biomarker discovery/validation utilizing retrospective and clinically annotated FFPE collections.Implications: Metabolic profiles can be performed in archival tissue and may be used to complement other profiling methods such as gene expression for biomarker discovery or pathway analysis in the assessment of biologic states. Mol Cancer Res; 15(4); 439-47. ©2017 AACR.
| S-EPMC5552883 | biostudies-literature
Project description:MotivationRecent studies have shown that RNA-sequencing (RNA-seq) can be used to measure mRNA of sufficient quality extracted from formalin-fixed paraffin-embedded (FFPE) tissues to provide whole-genome transcriptome analysis. However, little attention has been given to the normalization of FFPE RNA-seq data, a key step that adjusts for unwanted biological and technical effects that can bias the signal of interest. Existing methods, developed based on fresh-frozen or similar-type samples, may cause suboptimal performance.ResultsWe proposed a new normalization method, labeled MIXnorm, for FFPE RNA-seq data. MIXnorm relies on a two-component mixture model, which models non-expressed genes by zero-inflated Poisson distributions and models expressed genes by truncated normal distributions. To obtain maximum likelihood estimates, we developed a nested EM algorithm, in which closed-form updates are available in each iteration. By eliminating the need for numerical optimization in the M-step, the algorithm is easy to implement and computationally efficient. We evaluated MIXnorm through simulations and cancer studies. MIXnorm makes a significant improvement over commonly used methods for RNA-seq expression data.Availability and implementationR code available at https://github.com/S-YIN/MIXnorm.Contactswang@smu.edu.Supplementary informationSupplementary data are available at Bioinformatics online.
| S-EPMC7267832 | biostudies-literature