scholarly journals An optimised protocol for isolation of RNA from small sections of laser-capture microdissected FFPE tissue amenable for next-generation sequencing

2017 ◽  
Vol 18 (1) ◽  
Author(s):  
Parisa Amini ◽  
Julia Ettlin ◽  
Lennart Opitz ◽  
Elena Clementi ◽  
Alexandra Malbon ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Ffpe Tissue ◽  
Next Generation ◽  
Laser Capture ◽  
Laser Capture Microdissected ◽  
Generation Sequencing

Rapid Somatic Mutation Testing in Colorectal Cancer by Use of a Fully Automated System and Single-Use Cartridge: A Comparison with Next-Generation Sequencing

2018 ◽  
Vol 3 (2) ◽  
pp. 178-184 ◽  
Author(s):  
M Rabie Al-Turkmani ◽  
Kelley N Godwin ◽  
Jason D Peterson ◽  
Gregory J Tsongalis
Keyword(s):  
Colorectal Cancer ◽  
Next Generation Sequencing ◽  
Ffpe Tissue ◽  
Braf V600e ◽  
Automated System ◽  
Next Generation ◽  
Tissue Samples ◽  
Tissue Sections ◽  
Single Use ◽  
Generation Sequencing

AbstractBackgroundMolecular tests have been increasingly used in the management of various cancers as more targeted therapies are becoming available as treatment options. The Idylla™ system is a fully integrated, cartridge-based platform that provides automated sample processing (deparaffinization, tissue digestion, and DNA extraction) and real-time PCR-based mutation detection with all reagents included in a single-use cartridge. This retrospective study aimed at evaluating both the Idylla KRAS and NRAS-BRAF-EGFR492 Mutation Assay cartridges (research use only) against next-generation sequencing (NGS) by using colorectal cancer (CRC) tissue samples.MethodsForty-four archived formalin-fixed paraffin-embedded (FFPE) CRC tissue samples previously analyzed by targeted NGS were tested on the Idylla system. Among these samples, 17 had a mutation in KRAS proto-oncogene, GTPase (KRAS), 5 in NRAS proto-oncogene, GTPase (NRAS), and 12 in B-Raf proto-oncogene, serine/threonine kinase (BRAF) as determined using the Ion AmpliSeq 50-gene Cancer Hotspot Panel v2. The remaining 10 samples were wild-type for KRAS, NRAS, and BRAF. Two 10-μm FFPE tissue sections were used for each Idylla run, 1 for the KRAS cartridge, and 1 for the NRAS-BRAF-EGFR492 cartridge. All cases met the Idylla minimum tumor content requirement for KRAS, NRAS, and BRAF (≥10%). Assay reproducibility was evaluated by testing commercial controls derived from human cell lines, which had an allelic frequency of 50% and were run in triplicate.ResultsThe Idylla system successfully detected all mutations previously identified by NGS in KRAS (G12C, G12D, G12V, G13D, Q61K, Q61R, A146T), NRAS (G12V, G13R, Q61H), and BRAF (V600E). Compared with NGS, Idylla had a sensitivity of 100%. Analysis of the mutated commercial controls demonstrated agreement with the expected result for all samples and 100% reproducibility. The Idylla system produced results quickly with a turnaround time of approximately 2 h.ConclusionThe Idylla system offers reliable and sensitive testing of clinically actionable mutations in KRAS, NRAS, and BRAF directly from FFPE tissue sections.

scholarly journals Targeted Next-Generation Sequencing of Cancer-Related Genes in a Norwegian Patient Cohort With Head and Neck Squamous Cell Carcinoma Reveals Novel Actionable Mutations and Correlations With Pathological Parameters

2021 ◽  
Vol 11 ◽  
Author(s):  
Harsh N. Dongre ◽  
Hilde Haave ◽  
Siren Fromreide ◽  
Fredrik A. Erland ◽  
Svein Erik Emblem Moe ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Head And Neck ◽  
Squamous Cell ◽  
Ffpe Tissue ◽  
Next Generation ◽  
Targeted Next Generation Sequencing ◽  
Mutational Burden ◽  
Custom Made ◽  
Targeted Ngs ◽  
Generation Sequencing

BackgroundTargeted next-generation sequencing (NGS) is increasingly applied in clinical oncology to advance personalized treatment. Despite success in many other tumour types, use of targeted NGS panels for assisting diagnosis and treatment of head and neck squamous cell carcinomas (HNSCC) is still limited.AimThe focus of this study was to establish a robust NGS panel targeting most frequent cancer mutations in long-term preserved formalin-fixed paraffin-embedded (FFPE) tissue samples of HNSCC from routine diagnostics.Materials and MethodsTumour DNA obtained from archival FFPE tissue blocks of HNSCC patients treated at Haukeland University Hospital between 2003-2016 (n=111) was subjected to mutational analysis using a custom made AmpliSeq Library PLUS panel targeting 31 genes (Illumina). Associations between mutational burden and clinical and pathological parameters were investigated. Mutation and corresponding clinicopathological data from HNSCC were extracted for selected genes from the Cancer Genome Atlas (TCGA) and used for Chi-square and Kaplan-Meier analysis.ResultsThe threshold for sufficient number of reads was attained in 104 (93.7%) cases. Although the specific number of PCR amplified reads detected decreased, the number of NGS-annotated mutations did not significantly change with increased tissue preservation time. In HPV-negative carcinomas, mutations were detected mainly in TP53 (73.3%), FAT1 (26.7%) and FLG (16.7%) whereas in HPV-positive, the common mutations were in FLG (24.3%) FAT1 (17%) and FGFR3 (14.6%) genes. Other less common pathogenic mutations, including well reported SNPs were reproducibly identified. Presence of at least one cancer-specific mutations was found to be positively associated with an extensive desmoplastic stroma (p=0.019), and an aggressive type of invasive front (p=0.035), and negatively associated with the degree of differentiation (p=0.041). Analysis of TCGA data corroborated the association between cancer-specific mutations and tumour differentiation and survival analysis showed that tumours with at least one mutation had shorter disease-free and overall survival (p=0.005).ConclusionsA custom made targeted NGS panel could reliably detect several specific mutations in archival samples of HNSCCs preserved up to 17 years. Using this method novel associations between mutational burden and clinical and pathological parameters were detected and actionable mutations in HPV-positive HNSCC were discovered.

scholarly journals Profiling miRNAs in nasopharyngeal carcinoma FFPE tissue by microarray and Next Generation Sequencing

Genomics Data ◽  
2014 ◽  
Vol 2 ◽  
pp. 285-289 ◽  
Author(s):  
Jin Peng ◽  
Yanjun Feng ◽  
Gabriel Rinaldi ◽  
Paul Levine ◽  
Samantha Easley ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Nasopharyngeal Carcinoma ◽  
Ffpe Tissue ◽  
Next Generation ◽  
Generation Sequencing

scholarly journals Using Targeted RNA Next Generation Sequencing and Differential Expression to Compare Primary Central Nervous System Lymphoma with CD+ DLBCL, Nodal DLBCL and EBV+ DLBCL

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 22-22
Author(s):  
Haifaa Abdulhaq ◽  
Maher Albitar
Keyword(s):  
Next Generation Sequencing ◽  
Differential Expression ◽  
Central Nervous System Lymphoma ◽  
Ffpe Tissue ◽  
Next Generation ◽  
Advisory Committees ◽  
Tissue Samples ◽  
T Score ◽  
Significant Difference ◽  
Generation Sequencing

Introduction:Primary central nervous system lymphoma (PCNSL) is a diffuse large B-cell lymphoma (DLBCL) with a tropism for the CNS microenvironment. Limited studies suggested differential expression of multiple extracellular matrix (ECM) and adhesions-related genes in PCNSL as compared to nodal DLBCL. We used next generation sequencing to evaluate expression profiles of 1408 genes and and performed differential expression profiling to compare PCNSL with nodal DLBCL, CD5+ DLBCL and EBV+ DLBCL. CD5+ DLBCL was selected because it is frequently ABC subtype. We also selected EBV+ DLBCL due to its poor outcome, similar to PCNSL. Methods:RNA was extracted from 30 formalin-fixed paraffin-embedded (FFPE) tissue samples from PCNSL patients and 23 FFPE tissue samples from cases with lymph node DLBCL-NOS, 8 samples CD5+DLBCL, 14 samples EBV+ DLBCL.. We sequenced the RNA using a 1408 gene panel (Illumina). Next Generation Sequencing was based on hybrid capture methodology. Total number of reads per sample must exceed 5 million to be accepted. RNA levels were quantified using FPKM. Levels of expression of each of the 1408 genes were compared between groups. The T-score is used to show differences. Considering the number of analyzed samples and to correct for multiple testing, only T-score less or greater than 3 was considered clinically significant. Results:PCNSL showed significant overexpression in 25 genes as compared with CD5+ DLBCL, while only 5 genes were highly expressed in PCNSL as compared with nodal DLBCL and 8 when compared with EBV+ DLBCL (Table below). Four genes were significantly less expressed in PCNSL as compared with CD5+ DLBCL, and only one gene when compared to nodal and EBV+ DLBCL. There was no significant difference in RNA expression of MYD88, CD79B, CARD11, KMT2D or SPP1 between PCNSL and DLBC-NOS, CD5+ DLBCL and EBV+ DLBCL . There was no significant difference between PCNSL and the other sub types in MYC, Ki67, BCL2, CD44, or CD274 (PD-L1) expression. Several genes emerged as highly expressed in PCNSL as compared with other subtypes of DLBCL (negative T-score). However, CLTC (Clathrin Heavy Chain) gene is uniquely highly overexpressed in PCNSL and not in any of the other 3 types of lymphoma. The CLTC gene is involved in anaplastic lymphoma as a partner gene for ALK (CLTC-ALK) in t(2,17) translocation. The SEPT2 (SEPTIN2) gene is uniquely highly expressed in PCNSL as compared with nodal and EBV DLBC lymphoma. SEPT2 gene plays significant role in Hodgkin lymphoma and in maintaining Reed-Stenberg cells . In contrast, VEGFC was significantly lower in PCNSL compared with nodal DLBCL. Conclusions:RNA molecular profiling suggests significant difference between PCNSL and CD5+ DLBCL and less difference when compared with nodal and EBV+ DLBCL. The strikingly high expression of CLTC gene in PCNSL suggests possible targeting of this gene might present a specific therapeutic approach for PCNSL. Figure Disclosures Abdulhaq: Novartis:Consultancy, Honoraria, Speakers Bureau;Astellas:Honoraria, Speakers Bureau;Amgen:Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau;Morphosys:Membership on an entity's Board of Directors or advisory committees, Research Funding;Oncopeptide:Research Funding.

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