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https://www.readbyqxmd.com/read/28228113/validation-of-an-ngs-mutation-detection-panel-for-melanoma
#1
Anne Reiman, Hugh Kikuchi, Daniela Scocchia, Peter Smith, Yee Wah Tsang, David Snead, Ian A Cree
BACKGROUND: Knowledge of the genotype of melanoma is important to guide patient management. Identification of mutations in BRAF and c-KIT lead directly to targeted treatment, but it is also helpful to know if there are driver oncogene mutations in NRAS, GNAQ or GNA11 as these patients may benefit from alternative strategies such as immunotherapy. METHODS: While polymerase chain reaction (PCR) methods are often used to detect BRAF mutations, next generation sequencing (NGS) is able to determine all of the necessary information on several genes at once, with potential advantages in turnaround time...
February 22, 2017: BMC Cancer
https://www.readbyqxmd.com/read/28201998/clinical-performance-evaluation-of-a-sensitive-rapid-low-throughput-test-for-kras-mutation-analysis-using-formalin-fixed-paraffin-embedded-tissue-samples
#2
Christine Weyn, Sofie Van Raemdonck, Robina Dendooven, Vincent Maes, Karen Zwaenepoel, Suzan Lambin, Patrick Pauwels
BACKGROUND: Testing for KRAS mutations in metastatic colorectal cancer (mCRC) on formalin-fixed, paraffin embedded (FFPE) tumor tissue has become standard of care. Different molecular methods exist to determine hotspot KRAS mutations in exon 2, 3 and 4, but testing is often limited by the sensitivity and the speed of analysis. The aim of this retrospective study was to establish the clinical performance of the Idylla™ KRAS Mutation Test on FFPE tumor samples of patients with mCRC. METHODS: KRAS mutation analysis was performed using the therascreen KRAS on the RotorGene Q platform (CE-IVD; Qiagen) and results were subsequently compared to the Idylla™ KRAS Mutation Test...
February 16, 2017: BMC Cancer
https://www.readbyqxmd.com/read/28196074/clinical-applicability-and-cost-of-a-46-gene-panel-for-genomic-analysis-of-solid-tumours-retrospective-validation-and-prospective-audit-in-the-uk-national-health-service
#3
Angela Hamblin, Sarah Wordsworth, Jilles M Fermont, Suzanne Page, Kulvinder Kaur, Carme Camps, Pamela Kaisaki, Avinash Gupta, Denis Talbot, Mark Middleton, Shirley Henderson, Anthony Cutts, Dimitrios V Vavoulis, Nick Housby, Ian Tomlinson, Jenny C Taylor, Anna Schuh
BACKGROUND: Single gene tests to predict whether cancers respond to specific targeted therapies are performed increasingly often. Advances in sequencing technology, collectively referred to as next generation sequencing (NGS), mean the entire cancer genome or parts of it can now be sequenced at speed with increased depth and sensitivity. However, translation of NGS into routine cancer care has been slow. Healthcare stakeholders are unclear about the clinical utility of NGS and are concerned it could be an expensive addition to cancer diagnostics, rather than an affordable alternative to single gene testing...
February 2017: PLoS Medicine
https://www.readbyqxmd.com/read/28192086/variant-call-concordance-between-two-laboratory-developed-solid-tumor-targeted-genomic-profiling-assays-using-distinct-workflows-and-sequencing-instruments
#4
Ken J Hampel, Francine B de Abreu, Nikoletta Sidiropoulos, Jason D Peterson, Gregory J Tsongalis
Targeted genomic profiling (TGP) using massively parallel DNA sequencing is becoming the standard methodology in clinical laboratories for detecting somatic variants in solid tumors. The variety of methodologies and sequencing platforms in the marketplace for TGP has resulted in a variety of clinical TGP laboratory developed tests (LDT). The variability of LDTs is a challenge for test-to-test and laboratory-to-laboratory reliability. At the University of Vermont Medical Center (UVMMC), we validated a TGP assay for solid tumors which utilizes DNA hybridization capture and complete exon and selected intron sequencing of 29 clinically actionable genes...
February 10, 2017: Experimental and Molecular Pathology
https://www.readbyqxmd.com/read/28147344/detection-and-localization-of-viral-infection-in-the-pancreas-of-patients-with-type-1-diabetes-using-short-fluorescently-labelled-oligonucleotide-probes
#5
Niels Busse, Federico Paroni, Sarah J Richardson, Jutta E Laiho, Maarit Oikarinen, Gun Frisk, Heikki Hyöty, Eelco de Koning, Noel G Morgan, Kathrin Maedler
Enteroviruses, specifically of the Coxsackie B virus family, have been implicated in triggering islet autoimmunity and type 1 diabetes, but their presence in pancreata of patients with diabetes has not been fully confirmed.To detect the presence of very low copies of the virus genome in tissue samples from T1D patients, we designed a panel of fluorescently labeled oligonucleotide probes, each of 17-22 nucleotides in length with a unique sequence to specifically bind to the enteroviral genome of the picornaviridae family...
29, 2017: Oncotarget
https://www.readbyqxmd.com/read/28137276/towards-standardization-of-next-generation-sequencing-of-ffpe-samples-for-clinical-oncology-intrinsic-obstacles-and-possible-solutions
#6
Maxim Ivanov, Konstantin Laktionov, Valery Breder, Polina Chernenko, Ekaterina Novikova, Ekaterina Telysheva, Sergey Musienko, Ancha Baranova, Vladislav Mileyko
BACKGROUND: Next generation sequencing has a potential to revolutionize the management of cancer patients within the framework of precision oncology. Nevertheless, lack of standardization decelerated entering of the technology into the clinical testing space. Here we dissected a number of common problems of NGS diagnostics in oncology and introduced ways they can be resolved. METHODS: DNA was extracted from 26 formalin fixed paraffin embedded (FFPE) specimens and processed with the TrueSeq Amplicon Cancer Panel (Illumina Inc, San Diego, California) targeting 48 cancer-related genes and sequenced in single run...
January 31, 2017: Journal of Translational Medicine
https://www.readbyqxmd.com/read/28127742/performance-comparison-of-nextseq-and-ion-proton-platforms-for-molecular-diagnosis-of-clinical-oncology
#7
Fei Cao, Lianju Gao, Longgang Wei, Zhanni Chen, Yan Wang, Xia Ran, Xuehong Meng, Ji Tao
PURPOSE: Next-generation sequencing is a powerful approach to detect genetic mutations with which cancer diagnosis and treatment can be tailored to the individual patient in the era of personalized and precision medicine. Ion Torrent Systems Ion Proton and Illumina NextSeq are 2 major targeted sequencing platforms; however, not much work has been done to compare these platforms' performance for mutation detection in formalin-fixed paraffin-embedded (FFPE) materials. METHODS: We benchmarked the performance by using a collection of FFPE samples from 23 patients with different cancers for NextSeq and Ion Proton platforms...
January 25, 2017: Tumori
https://www.readbyqxmd.com/read/28125707/digital-pcr-improves-mutation-analysis-in-pancreas-fine-needle-aspiration-biopsy-specimens
#8
Shonan Sho, Colin M Court, Stephen Kim, David R Braxton, Shuang Hou, V Raman Muthusamy, Rabindra R Watson, Alireza Sedarat, Hsian-Rong Tseng, James S Tomlinson
Applications of precision oncology strategies rely on accurate tumor genotyping from clinically available specimens. Fine needle aspirations (FNA) are frequently obtained in cancer management and often represent the only source of tumor tissues for patients with metastatic or locally advanced diseases. However, FNAs obtained from pancreas ductal adenocarcinoma (PDAC) are often limited in cellularity and/or tumor cell purity, precluding accurate tumor genotyping in many cases. Digital PCR (dPCR) is a technology with exceptional sensitivity and low DNA template requirement, characteristics that are necessary for analyzing PDAC FNA samples...
2017: PloS One
https://www.readbyqxmd.com/read/28122052/a-comparison-of-rna-seq-results-from-paired-formalin-fixed-paraffin-embedded-and-fresh-frozen-glioblastoma-tissue-samples
#9
Anna Esteve-Codina, Oriol Arpi, Maria Martinez-García, Estela Pineda, Mar Mallo, Marta Gut, Cristina Carrato, Anna Rovira, Raquel Lopez, Avelina Tortosa, Marc Dabad, Sonia Del Barco, Simon Heath, Silvia Bagué, Teresa Ribalta, Francesc Alameda, Nuria de la Iglesia, Carmen Balaña
The molecular classification of glioblastoma (GBM) based on gene expression might better explain outcome and response to treatment than clinical factors. Whole transcriptome sequencing using next-generation sequencing platforms is rapidly becoming accepted as a tool for measuring gene expression for both research and clinical use. Fresh frozen (FF) tissue specimens of GBM are difficult to obtain since tumor tissue obtained at surgery is often scarce and necrotic and diagnosis is prioritized over freezing. After diagnosis, leftover tissue is usually stored as formalin-fixed paraffin-embedded (FFPE) tissue...
2017: PloS One
https://www.readbyqxmd.com/read/28118988/evaluation-of-positive-rift-valley-fever-virus-formalin-fixed-paraffin-embedded-samples-as-a-source-of-sequence-data-for-retrospective-phylogenetic-analysis
#10
B Mubemba, P N Thompson, L Odendaal, P Coetzee, E H Venter
Rift Valley fever (RVF), caused by an arthropod borne Phlebovirus in the family Bunyaviridae, is a haemorrhagic disease that affects ruminants and humans. Due to the zoonotic nature of the virus, a biosafety level 3 laboratory is required for isolation of the virus. Fresh and frozen samples are the preferred sample type for isolation and acquisition of sequence data. However, these samples are scarce in addition to posing a health risk to laboratory personnel. Archived formalin-fixed, paraffin-embedded (FFPE) tissue samples are safe and readily available, however FFPE derived RNA is in most cases degraded and cross-linked in peptide bonds and it is unknown whether the sample type would be suitable as reference material for retrospective phylogenetic studies...
January 22, 2017: Journal of Virological Methods
https://www.readbyqxmd.com/read/28116522/molecular-characterization-of-cd44-cd24-ck-cd45-cells-in-benign-and-malignant-breast-lesions
#11
Arnaud Da Cruz Paula, Catarina Leitão, Oriana Marques, Ana Margarida Rosa, Ana Helena Santos, Alexandra Rêma, Maria de Fátima Faria, Ana Rocha, José Luís Costa, Margarida Lima, Carlos Lopes
Breast cancer epithelial cells with the CD44(+)/CD24(-/low) phenotype possess tumor-initiating cells and epithelial-mesenchymal transition (EMT) capacity. Massive parallel sequencing can be an interesting approach to deepen the molecular characterization of these cells. We characterized CD44(+)/CD24(-)/cytokeratin(Ck)(+)/CD45(-) cells isolated through flow cytometry from 43 biopsy and 6 mastectomy samples harboring different benign and malignant breast lesions. The Ion Torrent Ampliseq Cancer Hotspot panel v2 (CHPv2) was used for the identification of somatic mutations in the DNA extracted from isolated CD44(+)/CD24(-)/Ck(+)/CD45(-) cells...
January 23, 2017: Virchows Archiv: An International Journal of Pathology
https://www.readbyqxmd.com/read/28106345/blood-based-detection-of-ras-mutations-to-guide-anti-egfr-therapy-in-colorectal-cancer-patients-concordance-of-results-from-circulating-tumor-dna-and-tissue-based-ras-testing
#12
Wolff Schmiegel, Rodney J Scott, Susan Dooley, Wendy Lewis, Cliff J Meldrum, Peter Pockney, Brian Draganic, Steve Smith, Chelsee Hewitt, Hazel Philimore, Amanda Lucas, Elva Shi, Kateh Namdarian, Timmy Chan, Danilo Acosta, Su Ping-Chang, Andrea Tannapfel, Anke Reinacher-Schick, Waldemar Uhl, Christian Teschendorf, Heiner Wolters, Josef Stern, Richard Viebahn, Helmut Friess, Klaus-Peter Janssen, Ulrich Nitsche, Julia Slotta-Huspenina, Michael Pohl, Deepak Vangala, Alexander Baraniskin, Barbara Dockhorn-Dworniczak, Susanne Hegewisch-Becker, Philippe Ronga, Daniel L Edelstein, Frederick S Jones, Stephan Hahn, Stephen B Fox
An accurate blood-based RAS mutation assay to determine eligibility of metastatic colorectal cancer (mCRC) patients for anti-EGFR therapy would benefit clinical practice by better informing decisions to administer treatment independent of tissue availability. The objective of this study was to determine the level of concordance between plasma and tissue RAS mutation status in patients with mCRC to gauge whether blood-based RAS mutation testing is a viable alternative to standard-of-care RAS tumor testing. RAS testing was performed on plasma samples from newly diagnosed metastatic patients, or from recurrent mCRC patients using the highly sensitive digital PCR technology, BEAMing (beads, emulsions, amplification, and magnetics), and compared with DNA sequencing data of respective FFPE (formalin-fixed paraffin-embedded) tumor samples...
February 2017: Molecular Oncology
https://www.readbyqxmd.com/read/28075488/generating-exome-enriched-sequencing-libraries-from-formalin-fixed-paraffin-embedded-tissue-dna-for-next-generation-sequencing
#13
Beth A Marosy, Brian D Craig, Kurt N Hetrick, P Dane Witmer, Hua Ling, Sean M Griffith, Benjamin Myers, Elaine A Ostrander, Janet L Stanford, Lawrence C Brody, Kimberly F Doheny
This unit describes a technique for generating exome-enriched sequencing libraries using DNA extracted from formalin-fixed paraffin-embedded (FFPE) samples. Utilizing commercially available kits, we present a low-input FFPE workflow starting with 50 ng of DNA. This procedure includes a repair step to address damage caused by FFPE preservation that improves sequence quality. Subsequently, libraries undergo an in-solution-targeted selection for exons, followed by sequencing using the Illumina next-generation short-read sequencing platform...
January 11, 2017: Current Protocols in Human Genetics
https://www.readbyqxmd.com/read/28074285/t-cell-receptor-repertoire-usage-in-cancer-as-a-surrogate-marker-for-immune-responses
#14
REVIEW
David Schrama, Cathrin Ritter, Jürgen C Becker
Characterizing the interaction of cancer cells with the host adaptive immune system is critical for understanding tumor immunology and the modus operandi of immunotherapeutic interventions to treat cancer. As the key cellular effectors of adaptive immunity, T cells are endowed with specialized receptors (the T cell receptor; TCR), to recognize and to eliminate cancer cells. The diversity of the TCR repertoire results from specialized genetic diversification mechanisms that generate an incredible variability allowing recognizing extensive collections of antigens...
January 10, 2017: Seminars in Immunopathology
https://www.readbyqxmd.com/read/28049581/validation-of-a-next-generation-sequencing-panel-for-detection-of-hotspot-cancer-mutations-in-a-clinical-laboratory
#15
Reza Shahsiah, Jenefer DeKoning, Saeed Samie, Seyed Ziaeddin Latifzadeh, Zahra Mehdizadeh Kashi
Recent advances in sequencing technologies have enabled us to scrutinize the versatile underlying mechanisms of cancer more precisely. However, adopting these new sophisticated technologies is challenging for clinical labs as it involves complex workflows, and requires validation for diagnostic purposes. The aim of this work is towards the analytical validation of a next generation sequencing (NGS) panel for cancer hotspot mutation analysis. Characterized formalin-fixed paraffin-embedded (FFPE) samples including biopsy specimens and cell-lines were examined by NGS methods utilizing the Ion Torrent™ Oncomine™ Focus DNA Assay and the PGM™ platform...
December 16, 2016: Pathology, Research and Practice
https://www.readbyqxmd.com/read/27995571/evaluating-the-feasibility-of-dna-methylation-analyses-using-long-term-archived-brain-formalin-fixed-paraffin-embedded-samples
#16
Stine T Bak, Nicklas H Staunstrup, Anna Starnawska, Tina F Daugaard, Jens R Nyengaard, Mette Nyegaard, Anders Børglum, Ole Mors, Karl-Anton Dorph-Petersen, Anders L Nielsen
We here characterize the usability of archival formalin-fixed paraffin-embedded (FFPE) brain tissue as a resource for genetic and DNA methylation analyses with potential relevance for brain-manifested diseases. We analyzed FFPE samples from The Brain Collection, Aarhus University Hospital Risskov, Denmark (AUBC), constituting 9479 formalin-fixated brains making it one of the largest collections worldwide. DNA extracted from brain FFPE tissue blocks was interrogated for quality and usability in genetic and DNA methylation analyses by different molecular techniques...
December 19, 2016: Molecular Neurobiology
https://www.readbyqxmd.com/read/27939411/identification-of-novel-mutations-in-japanese-ovarian-clear-cell-carcinoma-patients-using-optimized-targeted-ngs-for-clinical-diagnosis
#17
Yoshiaki Maru, Naotake Tanaka, Miki Ohira, Makiko Itami, Yoshitaka Hippo, Hiroki Nagase
OBJECTIVE: Ovarian clear cell carcinoma (OCCC) is an aggressive ovarian cancer with a higher frequency in Japan and often becomes chemorefractory disease. Reliable genetic diagnosis is essential to affirm the success of precision medicine for OCCC treatment. The aim of this study is, therefore, to identify novel mutations in OCCCs and develop a feasible clinical next generation sequencing (NGS) approach using formalin-fixed paraffin-embedded (FFPE) rather than preferable but not always available fresh frozen (FF) samples...
December 8, 2016: Gynecologic Oncology
https://www.readbyqxmd.com/read/27930669/development-of-rna-fish-assay-for-detection-of-oncogenic-fgfr3-tacc3-fusion-genes-in-ffpe-samples
#18
Masahiro Kurobe, Takahiro Kojima, Kouichi Nishimura, Shuya Kandori, Takashi Kawahara, Takayuki Yoshino, Satoshi Ueno, Yuichi Iizumi, Koji Mitsuzuka, Yoichi Arai, Hiroshi Tsuruta, Tomonori Habuchi, Takashi Kobayashi, Yoshiyuki Matsui, Osamu Ogawa, Mikio Sugimoto, Yoshiyuki Kakehi, Yoshiyuki Nagumo, Masakazu Tsutsumi, Takehiro Oikawa, Koji Kikuchi, Hiroyuki Nishiyama
INTRODUCTION AND OBJECTIVES: Oncogenic FGFR3-TACC3 fusions and FGFR3 mutations are target candidates for small molecule inhibitors in bladder cancer (BC). Because FGFR3 and TACC3 genes are located very closely on chromosome 4p16.3, detection of the fusion by DNA-FISH (fluorescent in situ hybridization) is not a feasible option. In this study, we developed a novel RNA-FISH assay using branched DNA probe to detect FGFR3-TACC3 fusions in formaldehyde-fixed paraffin-embedded (FFPE) human BC samples...
2016: PloS One
https://www.readbyqxmd.com/read/27911273/robust-detection-of-immune-transcripts-in-ffpe-samples-using-targeted-rna-sequencing
#19
Benjamin E Paluch, Sean T Glenn, Jeffrey M Conroy, Antonios Papanicolau-Sengos, Wiam Bshara, Angela R Omilian, Elizabeth Brese, Mary Nesline, Blake Burgher, Jonathan Andreas, Kunle Odunsi, Kevin Eng, Ji He, Maochun Qin, Mark Gardner, Lorenzo Galluzzi, Carl D Morrison
Current criteria for identifying cancer patients suitable for immunotherapy with immune checkpoint blockers (ICBs) are subjective and prone to misinterpretation, as they mainly rely on the visual assessment of CD274 (best known as PD-L1) expression levels by immunohistochemistry (IHC). To address this issue, we developed a RNA sequencing (RNAseq)-based approach that specifically measures the abundance of immune transcripts in formalin-fixed paraffin embedded (FFPE) specimens. Besides exhibiting superior sensitivity as compared to whole transcriptome RNAseq, our assay requires little starting material, implying that it is compatible with RNA degradation normally caused by formalin...
January 10, 2017: Oncotarget
https://www.readbyqxmd.com/read/27910019/fluorescence-in-situ-hybridization-on-tissue-sections
#20
Alvin S T Lim, Tse Hui Lim
Formalin-fixed paraffin-embedded (FFPE) tissues are typically the specimens available for FISH analysis of solid tissues, particularly of tumor specimens. Occasionally, tissue cores constructed as tissue microarrays from several patients are presented for simultaneous evaluation. FFPE sections can also be prepared from cell blocks derived from cell suspensions. The interphase fluorescence in situ hybridization assay employs specific nucleic acid sequences (probes) that target complementary sequences of interest to detect gains or losses of genes/gene loci or a fusion gene within the tissue...
2017: Methods in Molecular Biology
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