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FFPE sequencing

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https://www.readbyqxmd.com/read/29684684/targeted-data-independent-acquisition-for-mass-spectrometric-detection-of-ras-mutations-in-formalin-fixed-paraffin-embedded-tumor-biopsies
#1
Yeoun Jin Kim, Andrew G Chambers, Fabiola Cecchi, Todd Hembrough
Genomic testing for KRAS and NRAS mutations in clinical biopsies of various cancers is routinely performed to predict futility of anti-epidermal growth factor receptor (anti-EGFR) therapies. We hypothesized that RAS mutations could be detected and quantified at the protein level for diagnostic purposes using data-independent acquisition (DIA)-based mass spectrometry in formalin-fixed, paraffin-embedded (FFPE) tumor samples. We developed a targeted DIA assay that surveys the specific mass range of all possible peptides harboring activating mutations in KRAS exon 2...
April 20, 2018: Journal of Proteomics
https://www.readbyqxmd.com/read/29677173/what-does-this-mutation-mean-the-tools-and-pitfalls-of-variant-interpretation-in-lymphoid-malignancies
#2
REVIEW
Guillermin Yann, Lopez Jonathan, Chabane Kaddour, Hayette Sandrine, Bardel Claire, Salles Gilles, Sujobert Pierre, Huet Sarah
High throughput sequencing (HTS) is increasingly important in determining cancer diagnoses, with subsequent prognostic and therapeutic implications. The biology of cancer is becoming increasingly deciphered and it is clear that therapy needs to be individually tailored. Whilst translational research plays an important role in lymphoid malignancies, few guidelines exist to guide biologists and routine laboratories through this constantly evolving field. In this article, we review the challenges of interpreting HTS in lymphoid malignancies and provide a toolkit to interpret single nucleotide variants obtained from HTS...
April 20, 2018: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/29676606/awareness-of-mutational-artefacts-in-suboptimal-dna-samples-possible-risk-for-therapeutic-choices
#3
Sara Mariani, Luca Bertero, Vittoria Coppola, Giorgio Saracco, Alberto Arezzo, Paola Francia di Celle, Jasna Metovic, Caterina Marchiò, Paola Cassoni
BACKGROUND: Technical biases due to PCR artefacts could represent an insidious obstacle for mutational analysis and precision medicine. METHODS: The authors report a retrospective analysis by fast COLD-PCR and sequencing of 31 suboptimal tumor DNA samples obtained from FFPE tissues and liquid biopsies. RESULTS: In FFPE tumor tissues and plasma liquid biopsies of patients with lung and colorectal adenocarcinoma, we observed a significant rate of artefactual KRAS mutations, unveiled by repeated analysis following UDG pretreatment as well as by simple repetition without UDG pretreatment step, thus suggesting a DNA damage different from cytosine deamination...
April 20, 2018: Expert Review of Molecular Diagnostics
https://www.readbyqxmd.com/read/29630047/simple-and-rapid-method-to-obtain-high-quality-tumor-dna-from-clinical-pathological-specimens-using-touch-imprint-cytology
#4
Kenji Amemiya, Yosuke Hirotsu, Toshio Oyama, Masao Omata
It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissues are widely used for genetic testing. However, FFPE DNA is generally damaged and fragmented during the fixation process with formalin. Therefore, FFPE DNA is sometimes not adequate for genetic testing because of low quality and quantity of DNA. Here we present a method of touch imprint cytology (TIC) to obtain genomic DNA from cancer cells, which can be observed under a microscope...
March 21, 2018: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29621323/performance-comparison-of-three-dna-extraction-kits-on-human-whole-exome-data-from-formalin-fixed-paraffin-embedded-normal-and-tumor-samples
#5
Eric Bonnet, Marie-Laure Moutet, Céline Baulard, Delphine Bacq-Daian, Florian Sandron, Lilia Mesrob, Bertrand Fin, Marc Delépine, Marie-Ange Palomares, Claire Jubin, Hélène Blanché, Vincent Meyer, Anne Boland, Robert Olaso, Jean-François Deleuze
Next-generation sequencing (NGS) studies are becoming routinely used for the detection of novel and clinically actionable DNA variants at a pangenomic scale. Such analyses are now used in the clinical practice to enable precision medicine. Formalin-fixed paraffin-embedded (FFPE) tissues are still one of the most abundant source of cancer clinical specimen, unfortunately this method of preparation is known to degrade DNA and therefore compromise subsequent analysis. Some studies have reported that variant detection can be performed on FFPE samples sequenced with NGS techniques, but few or none have done an in-depth coverage analysis and compared the influence of different state-of-the-art FFPE DNA extraction kits on the quality of the variant calling...
2018: PloS One
https://www.readbyqxmd.com/read/29615338/use-of-the-ion-ampliseq-cancer-hotspot-panel-in-clinical-molecular-pathology-laboratories-for-analysis-of-solid-tumours-with-emphasis-on-validation-with-relevant-single-molecular-pathology-tests-and-the-oncomine-focus-assay
#6
Ahwon Lee, Sung-Hak Lee, Chan Kwon Jung, Gyungsin Park, Kyo Young Lee, Hyun Joo Choi, Ki Ouk Min, Tae Jung Kim, Eun Jung Lee, Youn Soo Lee
Targeted application of next-generation sequencing (NGS) technology allows detection of specific mutations that can provide treatment opportunities for cancer patients. We evaluated the applicability of the Ion AmpliSeq Cancer Hotspot Panel V2 (CHV2) using formalin-fixed, paraffin-embedded (FFPE) tissue of clinical specimens. Thirty-five FFPE tumour samples with known mutational status were collected from four different hospitals and sequenced with CHV2 using an Ion Chef System and Ion S5 XL system. Out of 35 cases, seven were sequenced with Oncomine focus Assay Panel for comparison...
March 29, 2018: Pathology, Research and Practice
https://www.readbyqxmd.com/read/29608913/shallow-whole-genome-sequencing-for-robust-copy-number-profiling-of-formalin-fixed-paraffin-embedded-breast-cancers
#7
Suet-Feung Chin, Angela Santonja, Marta Grzelak, Soomin Ahn, Stephen-John Sammut, Harry Clifford, Oscar M Rueda, Michelle Pugh, Mae A Goldgraben, Helen A Bardwell, Eun Yoon Cho, Elena Provenzano, Federico Rojo, Emilio Alba, Carlos Caldas
Pathology archives with linked clinical data are an invaluable resource for translational research, with the limitation that most cancer samples are formalin-fixed paraffin-embedded (FFPE) tissues. Therefore, FFPE tissues are an important resource for genomic profiling studies but are under-utilised due to the low amount and quality of extracted nucleic acids. We profiled the copy number landscape of 356 breast cancer patients using DNA extracted FFPE tissues by shallow whole genome sequencing. We generated a total of 491 sequencing libraries from 2 kits and obtained data from 98...
March 30, 2018: Experimental and Molecular Pathology
https://www.readbyqxmd.com/read/29602637/importin-11-overexpression-promotes-the-migration-invasion-and-progression-of-bladder-cancer-associated-with-the-deregulation-of-cdkn1a-and-thbs1
#8
Junjie Zhao, Lei Shi, Shuxiong Zeng, Chong Ma, Weidong Xu, Zhensheng Zhang, Qingzuo Liu, Peng Zhang, Yinghao Sun, Chuanliang Xu
OBJECTIVES: We recently determined that a novel oncogene, IPO11 from 5q12, participates in bladder cancer (BCa) progression. However, the biological function of IPO11 and the molecular mechanisms through which it contributes to BCa progression remain unclear. The aim of this study was to investigate the role of IPO11 in BCa aggressiveness and elucidate the molecular mechanisms underlying its effects in BCa. MATERIALS AND METHODS: The mRNA expression levels of IPO11 in BIU-87, RT4, UMUC3, EJ, 5637, T24, J82, and HT-1376 cell lines were determined using quantitative real-time polymerase chain reaction...
March 27, 2018: Urologic Oncology
https://www.readbyqxmd.com/read/29600293/-usp8-mutations-in-pituitary-cushing-adenomas-targeted-analysis-by-next-generation-sequencing
#9
Cora Ballmann, Anne Thiel, Hannah E Korah, Anna-Carinna Reis, Wolfgang Saeger, Stefanie Stepanow, Karl Köhrer, Guido Reifenberger, Christiane B Knobbe-Thomsen, Ulrich J Knappe, Ute I Scholl
Gain-of-function somatic mutations in the ubiquitin specific protease 8 ( USP8 ) gene have recently been reported as a cause of pituitary adenomas in Cushing disease. Molecular diagnostic testing of tumor tissue may aid in the diagnosis of specimens obtained through therapeutic transsphenoidal surgery; however, for small tumors, availability of fresh tissue is limited, and contamination with normal tissue is frequent. We performed molecular testing of DNA isolated from single formalin-fixed and paraffin-embedded (FFPE) tissue sections of 42 pituitary adenomas from patients with Cushing disease (27 female patients and 15 male patients; mean age at surgery, 42...
March 1, 2018: Journal of the Endocrine Society
https://www.readbyqxmd.com/read/29596374/dissecting-the-mutational-landscape-of-cutaneous-melanoma-an-omic-analysis-based-on-patients-from-greece
#10
Georgia Kontogianni, Georgia Piroti, Ilias Maglogiannis, Aristotelis Chatziioannou, Olga Papadodima
Melanoma is a lethal type of skin cancer, unless it is diagnosed early. Formalin-fixed, paraffin-embedded (FFPE) tissue is a valuable source for molecular assays after diagnostic examination, but isolated nucleic acids often suffer from degradation. Here, for the first time, we examine primary melanomas from Greek patients, using whole exome sequencing, so as to derive their mutational profile. Application of a bioinformatic framework revealed a total of 10,030 somatic mutations. Regarding the genes containing putative protein-altering mutations, 73 were common in at least three patients...
March 29, 2018: Cancers
https://www.readbyqxmd.com/read/29581779/idh1-2-mutations-predict-shorter-survival-in-chondrosarcoma
#11
Iwona Lugowska, Pawel Teterycz, Michal Mikula, Maria Kulecka, Anna Kluska, Aneta Balabas, Magdalena Piatkowska, Michal Wagrodzki, Andrzej Pienkowski, Piotr Rutkowski, Jerzy Ostrowski
Background . Recent studies have shown that isocitrate dehydrogenase 1/2 ( IDH1/2 )- activating mutations occur in a variety of cancers, including acute myeloid leukaemia, gliomas, and chondrosarcomas (CHS)s. The effect of IDH1/2 mutation on overall survival (OS) has not been reported in CHS. The aim of our study was to assess the prevalence of known cancer-related gene mutations in CHS, as well as their prognostic role in patient survival. Methods . DNA from FFPE samples of 80 patients (F:M- 1:1.3; mean age: 58 years; range 27-86) with histologically confirmed CHS (G1:29; G2:34; G3:17) was subjected to library preparation with the Ion AmpliSeq Cancer Hotspot Panel v2 and sequenced on the PGM Ion Torrent...
2018: Journal of Cancer
https://www.readbyqxmd.com/read/29573606/repair-of-dna-damage-caused-by-cytosine-deamination-in-mitochondrial-dna-of-forensic-case-samples
#12
Erin M Gorden, Kimberly Sturk-Andreaggi, Charla Marshall
DNA sequence damage from cytosine deamination is well documented in degraded samples, such as those from ancient and forensic contexts. This study examined the effect of a DNA repair treatment on mitochondrial DNA (mtDNA) from aged and degraded skeletal samples. DNA extracts from 21 non-probative, degraded skeletal samples (aged 50-70 years) were utilized for the analysis. A portion of each sample extract was subjected to DNA repair using a commercial repair kit, the New England BioLabs' NEBNext FFPE DNA Repair Kit (Ipswich, MA)...
February 19, 2018: Forensic Science International. Genetics
https://www.readbyqxmd.com/read/29556922/idh-mutation-analysis-in-glioma-patients-by-cadma-compared-with-snapshot-assay-and-two-immunohistochemical-methods
#13
Irena Urbanovska, Magdalena Houdova Megova, Zachary Dwight, Ondrej Kalita, Magdalena Uvirova, Jarmila Simova, Lucie Tuckova, Petr Buzrla, Tomas Palecek, Marian Hajduch, Jana Dvorackova, Jiri Drabek
Mutations in IDH1/2 genes are a marker of good prognosis for glioma patients, associated with low grade gliomas and secondary glioblastomas. Immunohistochemistry and Sanger sequencing are current standards for IDH1/2 genotyping while many other methods exist. The aim of this study was to validate Competitive amplification of differentially melting amplicons (CADMA) PCR for IDH genotyping by comparison with SNaPshot assay and two immunohistochemical methods. In our study, 87 glioma patients (46 from Olomouc and 41 from Ostrava) were analyzed...
March 19, 2018: Pathology Oncology Research: POR
https://www.readbyqxmd.com/read/29529386/cell-type-specific-quantification-of-telomere-length-and-dna-double-strand-breaks-in-individual-lung-cells-by-fluorescence-in-situ-hybridization-and-fluorescent-immunohistochemistry
#14
Aernoud A van Batenburg, Karin M Kazemier, Ton Peeters, Matthijs F M van Oosterhout, Joanne J van der Vis, Jan C Grutters, Roel Goldschmeding, Coline H M van Moorsel
Telomeres are small repetitive DNA sequences at the ends of chromosomes which act as a buffer in age-dependent DNA shortening. Insufficient telomere repeats will be recognized as double-strand breaks. Presently, it is becoming more evident that telomere attrition, whether or not caused by mutations in telomere maintenance genes, plays an important role in many inflammatory and age-associated diseases. In this report, a method to (semi)quantitatively assess telomere length and DNA double-strand breaks in formalin-fixed paraffin-embedded (FFPE) tissue is described...
March 1, 2018: Journal of Histochemistry and Cytochemistry: Official Journal of the Histochemistry Society
https://www.readbyqxmd.com/read/29525983/identification-of-novel-mutations-in-ffpe-lung-adenocarcinomas-using-deparray-sorting-technology-and-next-generation-sequencing
#15
Ji Won Lee, Jong-Yeon Shin, Jeong-Sun Seo
Formalin-fixed paraffin-embedded (FFPE) tissues are utilized as the standard diagnostic method in pathology laboratories. However, admixture of unwanted tissues and shortage of normal samples, which can be used to detect somatic mutation, are considered critical factors to accurately diagnose cancer. To explore these challenges, we sorted the pure tumor cells from 22 FFPE lung adenocarcinoma tissues via Di-Electro-Phoretic Array (DEPArray) technology, a new cell sorting technology, and analyzed the variants with next-generation sequencing (NGS) for the most accurate analysis...
March 10, 2018: Journal of Applied Genetics
https://www.readbyqxmd.com/read/29523855/a-simple-and-robust-real-time-qpcr-method-for-the-detection-of-pik3ca-mutations
#16
Virginia Alvarez-Garcia, Clare Bartos, Ieva Keraite, Urmi Trivedi, Paul M Brennan, Maïwenn Kersaudy-Kerhoas, Karim Gharbi, Olga Oikonomidou, Nicholas R Leslie
PIK3CA mutations are seemingly the most common driver mutations in breast cancer with H1047R and E545K being the most common of these, accounting together for around 60% of all PIK3CA mutations and have promising therapeutic implications. Given the low sensitivity and the high cost of current genotyping methods we sought to develop fast, simple and inexpensive assays for PIK3CA H1047R and E545K mutation screening in clinical material. The methods we describe are based on a real-time PCR including a mutation specific primer combined with a non-productive oligonucleotide which inhibits wild-type amplification and a parallel internal control reaction...
March 9, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29523759/landscape-of-tumor-mutation-load-mismatch-repair-deficiency-and-pd-l1-expression-in-a-large-patient-cohort-of-gastrointestinal-cancers
#17
Mohamed E Salem, Alberto Puccini, Axel Grothey, Derek Raghavan, Richard M Goldberg, Joanne Xiu, W Michael Korn, Benjamin A Weinberg, Jimmy J Hwang, Anthony F Shields, John L Marshall, Philip A Philip, Heinz-Josef Lenz
The efficacy of immunotherapy varies widely among different gastrointestinal cancers. Response to immune checkpoint inhibitors is shown to correlate with tumor mutation load (TML), mismatch repair deficiency (dMMR) status, and programmed cell death-ligand 1 (PD-L1) expression. Herein, we quantify TML, dMMR, and PD-L1 expression and determine their interrelationship in gastrointestinal cancers. Here, a total of 4125 tumors from 14 different gastrointestinal cancer sites were studied using validated assays. Next-generation sequencing (NGS) was performed on genomic DNA isolated from formalin-fixed paraffin-embedded (FFPE) tumor specimens using the NextSeq platform...
March 9, 2018: Molecular Cancer Research: MCR
https://www.readbyqxmd.com/read/29484115/transcriptomes-define-distinct-subgroups-of-salivary-gland-adenoid-cystic-carcinoma-with-different-driver-mutations-and-outcomes
#18
Candace A Frerich, Kathryn J Brayer, Brandon M Painter, Huining Kang, Yoshitsugu Mitani, Adel K El-Naggar, Scott A Ness
The relative rarity of salivary gland adenoid cystic carcinoma (ACC) and its slow growing yet aggressive nature has complicated the development of molecular markers for patient stratification. To analyze molecular differences linked to the protracted disease course of ACC and metastases that form 5 or more years after diagnosis, detailed RNA-sequencing (RNA-seq) analysis was performed on 68 ACC tumor samples, starting with archived, formalin-fixed paraffin-embedded (FFPE) samples up to 25 years old, so that clinical outcomes were available...
January 26, 2018: Oncotarget
https://www.readbyqxmd.com/read/29476606/integrated-multi-omic-analyses-support-distinguishing-secretory-carcinoma-of-the-breast-from-basal-like-triple-negative-breast-cancer
#19
Min-Sun Jin, Hyebin Lee, Jongmin Woo, Seongmin Choi, Mi Sol Do, Kwangsoo Kim, Min Ji Song, Youngsoo Kim, In Ae Park, Dohyun Han, Han Suk Ryu
PURPOSE: Secretory carcinoma (SC) of the breast is defined as an indolent tumor but is still categorized into a basal-like triple-negative breast cancer (BL-TNBC) subgroup that generally shows aggressive behaviour according to the current classification. Despite the unique clinical behaviour of SC, molecular characteristics that reflect biological behaviours of SC remain largely unknown. EXPERIMENTAL DESIGN: We employed a combinatorial approach of whole-exome sequencing and mass spectrometry-based in-depth quantitative proteomics to determine the entire molecular landscape of SC using three SC formalin-fixed paraffin-embedded (FFPE) tissues...
February 24, 2018: Proteomics. Clinical Applications
https://www.readbyqxmd.com/read/29471422/phylogenetic-analysis-of-histoplasma-capsulatum-var-duboisii-in-baboons-from-archived-formalin-fixed-paraffin-embedded-tissues
#20
M Hensel, A Rodrigues Hoffmann, M Gonzales, M A Owston, E J Dick
Histoplasma capsulatum var. duboisii (Hcd) infections have been well documented to cause chronic granulomatous disease, mainly involving the skin of baboons and humans in African countries primarily. This retrospective study classified the subspecies of Histoplasma and developed a phylogenetic tree utilizing DNA sequences extracted from formalin-fixed, paraffin embedded (FFPE) tissues from 9 baboons from a research colony in Texas histologically diagnosed with Hcd. Based on sequence analysis of ITS-2, Tub-1, and ARF, Hcd isolated from the archived samples closely aligns with the African clade and has 88% sequence homology with a sample isolated from an individual in Senegal...
February 16, 2018: Medical Mycology: Official Publication of the International Society for Human and Animal Mycology
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