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 Next Generation Sequencing in Molecular Diagnosis of Lynch Syndrome – a Pilot Study Using New Stratification Criteria

2018 ◽  
Vol 61 (3) ◽  
pp. 98-102 ◽  
Author(s):  
Ivana Kašubová ◽  
Veronika Holubeková ◽  
Katarína Janíková ◽  
Barbora Váňová ◽  
Zuzana Sňahničanová ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Next Generation Sequencing ◽  
Pilot Study ◽  
Lynch Syndrome ◽  
New Technologies ◽  
Braf V600e ◽  
Sporadic Colorectal Cancer ◽  
Next Generation ◽  
Gene Panel Testing ◽  
Generation Sequencing

The development of the new technologies such as the next-generation sequencing (NGS) makes more accessible the diagnosis of genetically heterogeneous diseases such as Lynch syndrome (LS). LS is one of the most common hereditary form of colorectal cancer. This autosomal dominant inherited disorder is caused by deleterious germline mutations in one of the mismatch repair (MMR) genes – MLH1, MSH2, MSH6 or PMS2, or the deletion in the EPCAM gene. These mutations eventually result in microsatellite instability (MSI), which can be easily tested in tumor tissue. According to the actual recommendations, all patients with CRC that are suspect to have LS, should be offered the MSI testing. When the MSI is positive, these patients should be recommended to genetic counseling. Here we report a pilot study about the application of NGS in the LS diagnosis in patients considered to have sporadic colorectal cancer. The inclusion criteria for the NGS testing were MSI positivity, BRAF V600E and MHL1 methylation negativity. We have used 5 gene amplicon based massive parallel sequencing on MiSeq platform. In one patient, we have identified a new pathogenic mutation in the exon 4 of the MSH6 gene that was previously not described in ClinVar, Human Gene Mutation Database, Ensembl and InSight databases. This mutation was confirmed by the Sanger method. We have shown that the implementation of new criteria for colorectal patients screening are important in clinical praxis and the NGS gene panel testing is suitable for routine laboratory settings.

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.

A Novel Method for Microsatellite Instability Detection by Liquid Biopsy Based on Next- Generation Sequencing

2020 ◽  
Vol 15 ◽  
Author(s):  
Zheng Jiang ◽  
Hui Liu ◽  
Siwen Zhang ◽  
Jia Liu ◽  
Weitao Wang ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Microsatellite Instability ◽  
Liquid Biopsy ◽  
Tumor Tissue ◽  
High Sensitivity ◽  
Next Generation ◽  
Tissue Samples ◽  
Next Generation Sequencing Technology ◽  
Medication Selection ◽  
Generation Sequencing

Background: Microsatellite instability (MSI) is a prognostic biomarker used to guide medication selection in multiple cancers, such as colorectal cancer. Traditional PCR with capillary electrophoresis and next-generation sequencing using paired tumor tissue and leukocyte samples are the main approaches for MSI detection due to their high sensitivity and specificity. Currently, patient tissue samples are obtained through puncture or surgery, which causes injury and risk of concurrent disease, further illustrating the need for MSI detection by liquid biopsy. Methods: We propose an analytic method using paired plasma/leukocyte samples and MSI detection using next-generation sequencing technology. Based on the theoretical progress of oncogenesis, we hypothesized that the microsatellite site length in plasma equals the combination of the distribution of tumor tissue and leukocytes. Thus, we defined a window-judgement method to identify whether biomarkers were stable. Results: Compared to traditional PCR as the standard, we evaluated three methods in 20 samples (MSI-H:3/MSS:17): peak shifting method using tissue vs. leukocytes, peak shifting method using plasma vs. leukocytes, and our method using plasma vs. leukocytes. Compared to traditional PCR, we observed a sensitivity of 100%, 0%, and 100%, and a specificity of 100.00%, 94.12%, and 88.24%, respectively. Conclusion: Our method has the advantage of possibly detecting MSI in a liquid biopsy and provides a novel direction for future studies to increase the specificity of the method.

scholarly journals Frequency detection of BRAF V600E mutation in a cohort of pediatric langerhans cell histiocytosis patients by next-generation sequencing

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Shunqiao Feng ◽  
Lin Han ◽  
Mei Yue ◽  
Dixiao Zhong ◽  
Jing Cao ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Langerhans Cell Histiocytosis ◽  
Mutation Screening ◽  
Langerhans Cell ◽  
Braf V600e ◽  
P Value ◽  
Type I ◽  
Next Generation ◽  
Mutation Status ◽  
Generation Sequencing

Abstract Background Langerhans cell histiocytosis (LCH) is a rare neoplastic disease that occurs in both children and adults, and BRAF V600E is detected in up to 64% of the patients. Several studies have discussed the associations between BRAF V600E mutation and clinicopathological manifestations, but no clear conclusions have been drawn regarding the clinical significance of the mutation in pediatric patients. Results We retrieved the clinical information for 148 pediatric LCH patients and investigated the BRAF V600E mutation using next-generation sequencing alone or with droplet digital PCR. The overall positive rate of BRAF V600E was 60/148 (41%). The type of sample (peripheral blood and formalin-fixed paraffin-embedded tissue) used for testing was significantly associated with the BRAF V600E mutation status (p-value = 0.000 and 0.000). The risk of recurrence declined in patients who received targeted therapy (p-value = 0.006; hazard ratio 0.164, 95%CI: 0.046 to 0.583). However, no correlation was found between the BRAF V600E status and gender, age, stage, specific organ affected, TP53 mutation status, masses close to the lesion or recurrence. Conclusions This is the largest pediatric LCH study conducted with a Chinese population to date. BRAF V600E in LCH may occur less in East Asian populations than in other ethnic groups, regardless of age. Biopsy tissue is a more sensitive sample for BRAF mutation screening because not all of circulating DNA is tumoral. Approaches with low limit of detection or high sensitivity are recommended for mutation screening to avoid type I and II errors.

scholarly journals Panel of serum miRNAs as potential non-invasive biomarkers for pancreatic ductal adenocarcinoma

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Imteyaz Ahmad Khan ◽  
Safoora Rashid ◽  
Nidhi Singh ◽  
Sumaira Rashid ◽  
Vishwajeet Singh ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Pancreatic Ductal Adenocarcinoma ◽  
Early Stage ◽  
Ductal Adenocarcinoma ◽  
Next Generation ◽  
Serum Samples ◽  
Tissue Samples ◽  
Non Invasive ◽  
Specific Symptoms ◽  
Generation Sequencing

AbstractEarly-stage diagnosis of pancreatic ductal adenocarcinoma (PDAC) is difficult due to non-specific symptoms. Circulating miRNAs in body fluids have been emerging as potential non-invasive biomarkers for diagnosis of many cancers. Thus, this study aimed to assess a panel of miRNAs for their ability to differentiate PDAC from chronic pancreatitis (CP), a benign inflammatory condition of the pancreas. Next-generation sequencing was performed to identify miRNAs present in 60 FFPE tissue samples (27 PDAC, 23 CP and 10 normal pancreatic tissues). Four up-regulated miRNAs (miR-215-5p, miR-122-5p, miR-192-5p, and miR-181a-2-3p) and four down-regulated miRNAs (miR-30b-5p, miR-216b-5p, miR-320b, and miR-214-5p) in PDAC compared to CP were selected based on next-generation sequencing results. The levels of these 8 differentially expressed miRNAs were measured by qRT-PCR in 125 serum samples (50 PDAC, 50 CP, and 25 healthy controls (HC)). The results showed significant upregulation of miR-215-5p, miR-122-5p, and miR-192-5p in PDAC serum samples. In contrast, levels of miR-30b-5p and miR-320b were significantly lower in PDAC as compared to CP and HC. ROC analysis showed that these 5 miRNAs can distinguish PDAC from both CP and HC. Hence, this panel can serve as a non-invasive biomarker for the early detection of PDAC.

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