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FFPE sequencing

Fiona Taylor, James Bradford, Penella J Woll, Dawn Teare, Angela Cox
Molecular profiling using low coverage whole genome sequencing of cell free DNA (cfDNA) represents a non-targeted approach to identify multiple somatic copy number alterations (SCNA) across different lung cancer subtypes. We aim to establish that SCNA can be detected in cfDNA of lung cancer cases.Standard protocols were followed to process matched cfDNA, formalin-fixed paraffin embedded (FFPE) tumour and lymphocyte DNA. Copy number profiles for cfDNA or FFPE DNA were normalised to profiles from matched lymphocyte DNA with the software CNAnorm...
2016: Advances in Experimental Medicine and Biology
Lori D Racsa, Marlene DeLeon-Carnes, Matthew Hiskey, Jeannette Guarner
16S sequencing on formalin-fixed paraffin-embedded (FFPE) material has been used to identify bacteria when culture-based phenotyping techniques have not worked. The objective of this study was to determine how frequently 16S sequencing used in FFPE material was helpful to clinicians in the diagnosis and treatment of infectious diseases. Requests for testing occurred upon consultation between an infectious disease pathologist and a surgical pathologist or an infectious disease physician. A selected paraffin block from each case was referred for 16S sequencing...
October 4, 2016: Human Pathology
Christopher S Hughes, Melissa K McConechy, Dawn R Cochrane, Tayyebeh Nazeran, Anthony N Karnezis, David G Huntsman, Gregg B Morin
Although re-sequencing of gene panels and mRNA expression profiling are now firmly established in clinical laboratories, in-depth proteome analysis has remained a niche technology, better suited for studying model systems rather than challenging materials such as clinical trial samples. To address this limitation, we have developed a novel and optimized platform called SP3-Clinical Tissue Proteomics (SP3-CTP) for in-depth proteome profiling of practical quantities of tumour tissues, including formalin fixed and paraffin embedded (FFPE)...
October 7, 2016: Scientific Reports
Jerome Ambroise, Jamal Badir, Louise Nienhaus, Annie Robert, Anne-France Dekairelle, Jean-Luc Gala
BACKGROUND: Pyrosequencing Allele Quantification (AQ) is a cost-effective DNA sequencing method that can be used for detecting somatic mutations in formalin-fixed paraffin-embedded (FFPE) samples. The method displays a low turnaround time and a high sensitivity. Pyrosequencing suffers however from two main drawbacks including (i) low specificity and (ii) difficult signal interpretation when multiple mutations are reported in a hotspot genomic region. RESULTS: Using a constraint-based regression method, the new AdvISER-PYRO-SMQ algorithm was developed in the current study and implemented into an R package...
2016: Algorithms for Molecular Biology: AMB
Yu Kakimoto, Masayuki Tanaka, Hiroshi Kamiguchi, Eriko Ochiai, Motoki Osawa
MicroRNAs (miRNAs) are small non-coding RNAs responsible for fine-tuning of gene expression at post-transcriptional level. The alterations in miRNA expression levels profoundly affect human health and often lead to the development of severe diseases. Currently, high throughput analyses, such as microarray and deep sequencing, are performed in order to identify miRNA biomarkers, using archival patient tissue samples. MiRNAs are more robust than longer RNAs, and resistant to extreme temperatures, pH, and formalin-fixed paraffin-embedding (FFPE) process...
2016: PloS One
Nadezda P Velizheva, Markus P Rechsteiner, Christine E Wong, Qing Zhong, Matthias Rössle, Beata Bode, Holger Moch, Alex Soltermann, Peter J Wild, Verena Tischler
BACKGROUND: Molecular testing of lung adenocarcinomas (ADCs) is crucial for therapy stratification of patients. Because of the often limited diagnostic material, the authors aimed to explore the suitability of cytology smears for next-generation sequencing (NGS) and compared the results with concurrent histological specimens or cell blocks. METHODS: A total of 16 formalin-fixed paraffin-embedded (FFPE) ADCs with known genetic alterations were used as the first cohort for targeted DNA and RNA sequencing...
September 16, 2016: Cancer Cytopathology
Mohammad Mahdi Forghanifard, Elham Emami Vahid, Ezzat Dadkhah, Mehran Gholamin, Samaneh Broumand Noghabi, Martha Ghahraman, Mehdi Farzadnia, Mohammad Reza Abbaszadegan
OBJECTIVES: Variation in microsatellite sequences that are dispersed in the genome has been linked to a deficiency in cellular mismatch repair system and defects in several genes of this system are involved in carcinogenesis. Our aim in this study was to illustrate microsatellite DNA alteration in esophageal cancer. MATERIALS AND METHODS: DNA was extracted from formalin fixed paraffin embedded (FFPE) tissues from surgical and matched margin-normal samples. Microsatellite instability (MSI) and loss of heterozygosity (LOH) were studied in 50 cases of esophageal squamous cell carcinoma (ESCC) by amplifying six microsatellite markers: D13S260 (13q12...
July 2016: Iranian Journal of Basic Medical Sciences
Andrea Orue, Manuel Rieber
UNLABELLED: Establishing the KRAS mutational status of tumor samples is essential to manage patients with colorectal or lung cancer, since these mutations preclude treatment with monoclonal anti-epidermal growth factor receptor (EGFR) antibodies. We report an inexpensive, rapid multiplex allele-specific qPCR method detecting the 7 most clinically relevant KRAS somatic mutations with concomitant amplification of non-mutated KRAS in tumor cells and tissues from CRC patients. Positive samples evidenced in the multiplex assay were further subjected to individual allele-specific analysis, to define the specific mutation...
2016: PloS One
Pradyot Prakash, Shashikant C U Patne, Ashish Kumar Singh, Mohan Kumar, Mukti Nath Mishra, Anil Kumar Gulati
AIMS: To devise nested multiplex polymerase chain reaction (NMPCR) protocol for detection of mucosal human papilloma viruses (HPVs) and typing of HPV-16 and -18 in formalin-fixed, paraffin-embedded (FFPE) tissues of carcinoma cervix (CaCx). SETTINGS AND DESIGN: Cross-sectional observational study. MATERIALS AND METHODS: NMPCR was done for simultaneous detection of HPV, targeting 134 bp L1 capsid gene employing GP+/mGP+ primers and typing of genotypes-16 and -18, targeting E6/E7 gene from 34 FFPE tissue blocks of CaCx and cervical intraepithelial neoplasia (CIN)...
July 2016: Journal of Global Infectious Diseases
M Bujko, P Kober, N Rusetska, M Wakuła, K Goryca, E Grecka, E Matyja, J Neska, T Mandat, W Bonicki, J A Siedlecki
DLC1 encodes GTPase-activating protein with a well-documented tumor suppressor activity. This gene is downregulated in various tumors through aberrant promoter hypermethylation. Five different DLC1 isoforms can be transcribed from alternative promoters. Tumor-related DNA methylation of the DLC1 isoform 1 alternative promoter was identified as being hypermethylated in meningiomas in genome-wide DNA methylation profiling. We determined the methylation pattern of this region in 50 meningioma FFPE samples and sections of 6 normal meninges, with targeted bisulfite sequencing...
September 10, 2016: Journal of Neuro-oncology
Malachia Hoover, Yvess Adamian, Mark Brown, Ali Maawy, Alexander Chang, Jacqueline Lee, Armen Gharibi, Matthew H Katz, Jason Fleming, Robert M Hoffman, Michael Bouvet, Robert Doebler, Jonathan A Kelber
Next-generation sequencing (NGS) can identify and validate new biomarkers of cancer onset, progression and therapy resistance. Substantial archives of formalin-fixed, paraffin-embedded (FFPE) cancer samples from patients represent a rich resource for linking molecular signatures to clinical data. However, performing NGS on FFPE samples is limited by poor RNA purification methods. To address this hurdle, we developed an improved methodology for extracting high-quality RNA from FFPE samples. By briefly integrating a newly-designed micro-homogenizing (mH) tool with commercially available FFPE RNA extraction protocols, RNA recovery is increased by approximately 3-fold while maintaining standard A260/A280 ratios and RNA quality index (RQI) values...
September 1, 2016: Oncotarget
Silvia Bonfiglio, Irene Vanni, Valeria Rossella, Anna Truini, Dejan Lazarevic, Maria Giovanna Dal Bello, Angela Alama, Marco Mora, Erika Rijavec, Carlo Genova, Davide Cittaro, Francesco Grossi, Simona Coco
BACKGROUND: Next Generation Sequencing (NGS) has become a valuable tool for molecular landscape characterization of cancer genomes, leading to a better understanding of tumor onset and progression, and opening new avenues in translational oncology. Formalin-fixed paraffin-embedded (FFPE) tissue is the method of choice for storage of clinical samples, however low quality of FFPE genomic DNA (gDNA) can limit its use for downstream applications. METHODS: To investigate the FFPE specimen suitability for NGS analysis and to establish the performance of two solution-based exome capture technologies, we compared the whole-exome sequencing (WES) data of gDNA extracted from 5 fresh frozen (FF) and 5 matched FFPE lung adenocarcinoma tissues using: SeqCap EZ Human Exome v...
2016: BMC Cancer
Susan D Hester, Virunya Bhat, Brian N Chorley, Gleta Carswell, Wendell Jones, Leah C Wehmas, Charles E Wood
Use of archival resources has been limited to date by inconsistent methods for genomic profiling of degraded RNA from formalin-fixed paraffin-embedded (FFPE) samples. RNA-sequencing offers a promising way to address this problem. Here we evaluated transcriptomic dose responses using RNA-sequencing in paired FFPE and frozen (FROZ) samples from two archival studies in mice, one <2 years old and the other >20 years old. Experimental treatments included 3 different doses of di(2-ethylhexyl)phthalate or dichloroacetic acid for the recently archived and older studies, respectively...
August 25, 2016: Toxicological Sciences: An Official Journal of the Society of Toxicology
Hassan Ashktorab, Hamed Azimi, Michael L Nickerson, Sara Bass, Sudhir Varma, Hassan Brim
BACKGROUND AND AIM: Next generation sequencing (NGS) has quickly the tool of choice for genome and exome data generation. The multitude of sequencing platforms as well as the variabilities within each platform need to be assessed. In this paper we used two platforms (ION TORRENT AND ILLUMINA) to assess single nucleotides variants in colorectal cancer (CRC) specimens. METHODS: CRC specimens (n = 13) collected from 6 CRC (cancer and matched normal) patients were used to establish the mutational profile using ION TORRENT AND ILLUMINA sequencing platforms...
June 2016: Next Generation, Sequencing & Applications
Márton Zs Enyedi, Gábor Jaksa, Lajos Pintér, Farkas Sükösd, Zoltán Gyuris, Adrienn Hajdu, Erika Határvölgyi, Katalin Priskin, Lajos Haracska
The development of breast and ovarian cancer is strongly connected to the inactivation of the BRCA1 and BRCA2 genes by different germline and somatic alterations, and their diagnosis has great significance in targeted tumor therapy, since recently approved PARP inhibitors show high efficiency in the treatment of BRCA-deficient tumors. This raises the need for new diagnostic methods that are capable of performing an integrative mutation analysis of the BRCA genes not only from germline DNA but also from formalin-fixed and paraffin-embedded (FFPE) tumor samples...
August 12, 2016: Oncotarget
Caroline Razafinjatovo, Svenja Bihr, Axel Mischo, Ursula Vogl, Manuela Schmidinger, Holger Moch, Peter Schraml
BACKGROUND: The VHL protein (pVHL) is a multiadaptor protein that interacts with more than 30 different binding partners involved in many oncogenic processes. About 70 % of clear cell renal cell carcinoma (ccRCC) have VHL mutations with varying impact on pVHL function. Loss of pVHL function leads to the accumulation of Hypoxia Inducible Factor (HIF), which is targeted by current targeted treatments. In contrast to nonsense and frameshift mutations that highly likely nullify pVHL multipurpose functions, missense mutations may rather specifically influence the binding capability of pVHL to its partners...
2016: BMC Cancer
Doreen Nguyen, Diana Taheri, Simeon Springer, Morgan Cowan, Gunes Guner, Maria Angelica Mendoza Rodriguez, Yuxuan Wang, Isaac Kinde, Christopher J VandenBussche, Matthew T Olson, Bernardo F P Ricardo, Isabela Cunha, Kazutoshi Fujita, Dilek Ertoy, Kenneth W Kinzler, Trinity J Bivalacqua, Nickolas Papadopoulos, Bert Vogelstein, George J Netto
Somatic activating mutations in the promoter of the telomerase reverse transcriptase (TERT) gene are the most common genetic alterations in urothelial carcinoma (UC) of the bladder and upper urinary tract. Little is known, however, about TERT-mutation status in the relatively uncommon but clinically aggressive micropapillary (MPC) variant. We evaluated the presence of TERT promoter mutations in MPC of the bladder and upper urinary tract. A retrospective search of our archives for MPC and UC with micropapillary features (2005-2014) was performed...
October 2016: Virchows Archiv: An International Journal of Pathology
Jennifer Dang, Pedro Mendez, Sharon Lee, James W Kim, Jun-Hee Yoon, Thomas W Kim, Charles J Sailey, David M Jablons, Il-Jin Kim
Next-generation sequencing (NGS) is becoming a standard for genetic analyses of clinical samples. DNAs retrieved from formalin-fixed, paraffin-embedded (FFPE) tissue specimens are commonly degraded, and specimens such as core biopsies are sometimes too small to obtain enough DNA for NGS applications. Thus, it is important to measure both the DNA quantity and quality accurately from clinical samples. However, there is no standard method for DNA quantity and quality analyses for NGS library preparation. We tested four different methods (PicoGreen, Qubit® fluorometry, TaqMan and SYBR-Green-based qPCR assay) and compared each to RNase P TaqMan as a reference control...
October 2016: International Journal of Oncology
George Jour, Lu Wang, Sumit Middha, Ahmet Zehir, Wen Chen, Justyna Sadowska, John Healey, Narasimhan P Agaram, Lisa Choi, Khedoudja Nafa, Meera Hameed
Extraskeletal osteosarcoma (ESOSA) is a rare soft tissue neoplasm representing <5% of osteosarcomas and <1% of all soft-tissue sarcomas. Herein, we investigate the clinicopathological and molecular features of ESOSA and explore potential parameters that may affect outcome. Thirty-two cases were retrieved and histomorphology was reviewed. Clinical history and follow-up were obtained through electronic record review. DNA from formalin-fixed paraffin-embedded (FFPE) tissue was extracted and processed from 27 cases...
January 2016: Journal of Pathology. Clinical Research
Mathias Stiller, Antje Sucker, Klaus Griewank, Daniela Aust, Gustavo Bruno Baretton, Dirk Schadendorf, Susanne Horn
DNA derived from formalin-fixed and paraffin-embedded (FFPE) tissue has been a challenge to large-scale genomic sequencing, due to its low quality and quantities. Improved techniques enabling the genome-wide analysis of FFPE material would be of great value, both from a research and clinical perspective.Comparing a single-strand DNA library preparation method originally developed for ancient DNA to conventional protocols using double-stranded DNA derived from FFPE material we obtain on average 900-fold more library molecules and improved sequence complexity from as little as 5 ng input DNA...
July 24, 2016: Oncotarget
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