scholarly journals A Targeted Gene Panel for Circulating Tumor DNA Sequencing in Neuroblastoma

2020 ◽  
Vol 10 ◽  
Author(s):  
Flora Cimmino ◽  
Vito Alessandro Lasorsa ◽  
Simona Vetrella ◽  
Achille Iolascon ◽  
Mario Capasso
Keyword(s):  
Ad Hoc ◽  
Circulating Tumor Dna ◽  
Gene Panel ◽  
Liquid Biopsies ◽  
Targeted Next Generation Sequencing ◽  
Pathogenic Variation ◽  
Targeted Ngs ◽  
Molecular Landscape ◽  
Next Generation Sequencing Ngs ◽  
Tumor Biopsies

BackgroundLiquid biopsies do not reflect the complete mutation profile of the tumor but have the potential to identify actionable mutations when tumor biopsies are not available as well as variants with low allele frequency. Most retrospective studies conducted in small cohorts of pediatric cancers have illustrated that the technology yield substantial potential in neuroblastoma.AimThe molecular landscape of neuroblastoma harbors potentially actionable genomic alterations. We aimed to study the utility of liquid biopsy to characterize the mutational landscape of primary neuroblastoma using a custom gene panel for ctDNA targeted sequencing.MethodsTargeted next-generation sequencing (NGS) was performed on ctDNA of 11 patients with primary neuroblastoma stage 4. To avoid the detection of false variants, we used UMIs (unique molecular identifiers) for the library construction, increased the sequencing depth and developed ad hoc bioinformatic analyses including the hard filtering of the variant calls.ResultsWe identified 9/11 (81.8%) patients who carry at least one pathogenic variation. The most frequently mutated genes were KMT2C (five cases), NOTCH1/2 (four cases), CREBBP (three cases), ARID1A/B (three cases), ALK (two cases), FGFR1 (two cases), FAT4 (two cases) and CARD11 (two cases).ConclusionsWe developed a targeted NGS approach to identify tumor-specific alterations in ctDNA of neuroblastoma patients. Our results show the reliability of our approach to generate genomic information which can be integrated with clinical and pathological data at diagnosis.

scholarly journals Differential Mutation Detection Capability Through Capture-Based Targeted Sequencing in Plasma Samples in Hepatocellular Carcinoma

2021 ◽  
Vol 11 ◽  
Author(s):  
Jian Gao ◽  
Lei Xi ◽  
Rentao Yu ◽  
Huailong Xu ◽  
Min Wu ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Circulating Tumor Dna ◽  
Tumor Burden ◽  
Targeted Next Generation Sequencing ◽  
Targeted Ngs ◽  
Tumor Protein P53 ◽  
Next Generation Sequencing Ngs ◽  
Coding Variants ◽  
G Protein Coupled ◽  
Generation Sequencing

Circulating tumor DNA (ctDNA) is a promising biomarker for accurate monitoring and less invasive assessment of tumor burden and treatment response. Here, targeted next-generation sequencing (NGS) with a designed gene panel of 176 cancer-relevant genes was used to assess mutations in 90 ctDNA samples from 90 patients with multiple types of liver disease and 10 healthy donor samples for control. Using our ctDNA detection panel, we identified mutations in 98.89% (89/90) of patient plasma biopsy samples, and 19 coding variants located in 10 cancer-related genes [ACVR2A, PCLO, TBCK, adhesion G protein-coupled receptor (ADGRV1), COL1A1, GABBR1, MUC16, MAGEC1, FASLG, and JAK1] were identified in 96.7% of patients (87/90). The 10 top mutated genes were tumor protein p53 (TP53), ACVR2A, ADGRV1, MUC16, TBCK, PCLO, COL11A1, titin (TTN), DNAH9, and GABBR1. TTN and TP53 and TTN and DNAH9 mutations tended to occur together in hepatocellular carcinoma samples. Most importantly, we found that most of those variants were insertions (frameshift insertions) and deletions (frameshift deletions and in-frame deletions), such as insertion variants in ACVR2A, PCLO, and TBCK; such mutations were detected in almost 95% of patients. Our study demonstrated that the targeted NGS-based ctDNA mutation profiling was a useful tool for hepatocellular carcinoma (HCC) monitoring and could potentially be used to guide treatment decisions in HCC.

scholarly journals R-Score: A New Parameter to Assess the Quality of Variants’ Calls Assessed by NGS Using Liquid Biopsies

Biology ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 954
Author(s):  
Roberto Serna-Blasco ◽  
Estela Sánchez-Herrero ◽  
María Berrocal Renedo ◽  
Silvia Calabuig-Fariñas ◽  
Miguel Ángel Molina-Vila ◽  
...  
Keyword(s):  
Egfr Mutation ◽  
False Negative ◽  
Digital Pcr ◽  
Liquid Biopsies ◽  
Molecular Alterations ◽  
Significant Linear Correlation ◽  
Molecular Landscape ◽  
Nsclc Patients ◽  
Next Generation Sequencing Ngs

Next-generation sequencing (NGS) has enabled a deeper knowledge of the molecular landscape in non-small cell lung cancer (NSCLC), identifying a growing number of targetable molecular alterations in key genes. However, NGS profiling of liquid biopsies risk for false positive and false negative calls and parameters assessing the quality of NGS calls remains lacking. In this study, we have evaluated the positive percent agreement (PPA) between NGS and digital PCR calls when assessing EGFR mutation status using 85 plasma samples from 82 EGFR-positive NSCLC patients. According to our data, variant allele fraction (VAF) was significantly lower in discordant calls and the median of the absolute values of all pairwise differences (MAPD) was significantly higher in discordant calls (p < 0.001 in both cases). Based on these results, we propose a new parameter that integrates both variables, named R-score. Next, we sought to evaluate the PPA for EGFR mutation calls between two independent NGS platforms using a subset of 40 samples from the same cohort. Remarkably, there was a significant linear correlation between the PPA and the R-score (r = 0.97; p < 0.001). Specifically, the PPA of samples with an R-score ≤ −1.25 was 95.83%, whereas PPA falls to 81.63% in samples with R-score ≤ 0.25. In conclusion, R-score significantly correlates with PPA and can assist laboratory medicine specialists and data scientists to select reliable variants detected by NGS.

scholarly journals The ICR96 exon CNV validation series: a resource for orthogonal assessment of exon CNV calling in NGS data

2017 ◽  
Vol 2 ◽  
pp. 35 ◽  
Author(s):  
Shazia Mahamdallie ◽  
Elise Ruark ◽  
Shawn Yost ◽  
Emma Ramsay ◽  
Imran Uddin ◽  
...  
Keyword(s):  
Sequencing Data ◽  
Targeted Next Generation Sequencing ◽  
Negative Results ◽  
Targeted Ngs ◽  
Predisposition Genes ◽  
Next Generation Sequencing Ngs ◽  
Ngs Data ◽  
Validation Series ◽  
Generation Sequencing ◽  
Dependent Probe

Detection of deletions and duplications of whole exons (exon CNVs) is a key requirement of genetic testing. Accurate detection of this variant type has proved very challenging in targeted next-generation sequencing (NGS) data, particularly if only a single exon is involved. Many different NGS exon CNV calling methods have been developed over the last five years. Such methods are usually evaluated using simulated and/or in-house data due to a lack of publicly-available datasets with orthogonally generated results. This hinders tool comparisons, transparency and reproducibility. To provide a community resource for assessment of exon CNV calling methods in targeted NGS data, we here present the ICR96 exon CNV validation series. The dataset includes high-quality sequencing data from a targeted NGS assay (the TruSight Cancer Panel) together with Multiplex Ligation-dependent Probe Amplification (MLPA) results for 96 independent samples. 66 samples contain at least one validated exon CNV and 30 samples have validated negative results for exon CNVs in 26 genes. The dataset includes 46 exon CNVs in BRCA1, BRCA2, TP53, MLH1, MSH2, MSH6, PMS2, EPCAM or PTEN, giving excellent representation of the cancer predisposition genes most frequently tested in clinical practice. Moreover, the validated exon CNVs include 25 single exon CNVs, the most difficult type of exon CNV to detect. The FASTQ files for the ICR96 exon CNV validation series can be accessed through the European-Genome phenome Archive (EGA) under the accession number EGAS00001002428.

scholarly journals Advancing quality-control for NGS measurement of actionable mutations in circulating tumor DNA

2021 ◽  
Author(s):  
James C. Willey ◽  
Tom Morrison ◽  
Brad Austermiller ◽  
Erin L. Crawford ◽  
Daniel J. Craig ◽  
...  
Keyword(s):  
Quality Control ◽  
Limit Of Detection ◽  
Internal Standard ◽  
Circulating Tumor Dna ◽  
Primary Objective ◽  
Nucleotide Position ◽  
Targeted Next Generation Sequencing ◽  
Next Generation Sequencing Ngs ◽  
Rate Limit ◽  
Tumor Dna

SUMMARYThe primary objective of the FDA-led Sequencing and Quality Control Phase 2 (SEQC2) project is to develop standard analysis protocols and quality control metrics for use in DNA testing to enhance scientific research and precision medicine. This study reports a targeted next generation sequencing (NGS) method that enables more accurate detection of actionable mutations in circulating tumor DNA (ctDNA) clinical specimens. This advancement was enabled by designing a synthetic internal standard spike-in for each actionable mutation target, suitable for use in NGS following hybrid-capture enrichment and unique molecular index (UMI) or non-UMI library preparation. When mixed with contrived ctDNA reference samples, internal standards enabled calculation of technical error rate, limit of blank, and limit of detection for each variant at each nucleotide position, in each sample. True positive mutations with variant allele fraction too low for detection by current practice were detected with this method, thereby increasing sensitivity.

scholarly journals Characterizing the pharmacogenome using molecular inversion probes for targeted next-generation sequencing

2019 ◽  
Vol 20 (14) ◽  
pp. 1005-1020 ◽  
Author(s):  
Oscar Suzuki ◽  
Olivia M Dong ◽  
Rachel M Howard ◽  
Tim Wiltshire
Keyword(s):  
Next Generation Sequencing ◽  
Control Cell ◽  
Next Generation ◽  
Targeted Next Generation Sequencing ◽  
Technical Performance ◽  
Targeted Ngs ◽  
Molecular Inversion Probes ◽  
Next Generation Sequencing Ngs ◽  
Molecular Inversion Probe ◽  
Generation Sequencing

Aim: This study assesses the technical performance and cost of a targeted next-generation sequencing (NGS) multigene pharmacogenetic (PGx) test. Materials & methods: A genetic test was developed for 21 PGx genes using molecular inversion probes to generate library fragments for NGS. Performance of this test was assessed using 53 unique reference control cell lines from the Genetic Testing Reference Materials Coordination Program (GeT-RM). Results: 93.7% of variants were successfully called and the repeatability rate was 99.9%. Reference calls were available for 78.4% of diplotype calls resulting from PGx testing, and concordance for the test was 85.7%. Cost per sample was $32–$56. Conclusion: A targeted NGS assay using molecular inversion probe technology is able to characterize the pharmacogenome efficiently.

scholarly journals Synchronous Pulmonary Adenocarcinomas

2020 ◽  
Vol 154 (1) ◽  
pp. 57-69
Author(s):  
Carlos A Pagan ◽  
Catherine A Shu ◽  
John P Crapanzano ◽  
Galina G Lagos ◽  
Mark B Stoopler ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Gene Panel ◽  
Driver Mutations ◽  
Molecular Testing ◽  
Next Generation ◽  
Synchronous Tumors ◽  
Targeted Next Generation Sequencing ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Abstract Objectives To determine concordance/discordance between morphology and molecular testing (MT) among synchronous pulmonary carcinomas using targeted next generation sequencing (NGS), with and without comprehensive molecular review (CMR), vs analyses of multiple singe genes (non-NGS). Methods Results of morphologic and MT assessment were classified as concordant, discordant, or indeterminate. For discordant cases, comprehensive histologic assessment (CHA) was performed. Results Forty-seven cases with 108 synchronous tumors were identified and underwent MT (NGS, n = 23 and non-NGS, n = 24). Histology and MT were concordant, discordant, and indeterminate in 53% (25/47), 21% (10/47), and 26% (12/47) of cases, respectively. CHA of the 10 discordant cases revised results of three cases. Conclusions There is discordance between histology and MT in a subset of cases and MT provides an objective surrogate for staging synchronous tumors. A limited gene panel is sufficient for objectively assessing a relationship if the driver mutations are distinct. Relatedness of mutations require CMR with a larger NGS panel (eg, 50 genes).

Molecular profiling of early-stage colorectal cancer (CRC) using targeted next generation sequencing (NGS) to predict signatures of recurrence.

2020 ◽  
Vol 38 (4_suppl) ◽  
pp. 242-242
Author(s):  
Michael C. Burns ◽  
Kristen Carroll ◽  
Ryan Jones ◽  
Masha Kocherginsky ◽  
Kirsten Bell Burdett ◽  
...  
Keyword(s):  
Adjuvant Chemotherapy ◽  
Early Stage ◽  
Late Recurrence ◽  
Stage I ◽  
Recurrence Free Survival ◽  
Primary Tumors ◽  
Free Survival ◽  
Targeted Next Generation Sequencing ◽  
Targeted Ngs ◽  
Next Generation Sequencing Ngs

242 Background: While the 5-year recurrence rate in early stage CRC is low (12%) and there is currently limited role of adjuvant chemotherapy in such cases, a unique subset of patients (pts) will have late recurrences. To identify molecular signatures predictive of late recurrence after pts undergo intended curative resection, we employed a 22 targeted gene NGS panel in pts with early CRC. Association between mutation status and recurrence free survival (RFS) was analyzed. Methods: Pts with stage I-II CRC had their tumor prospectively sequenced between 09/2015-12/2018 by an ion torrent targeted 22 gene hotspot NGS panel, including KRAS, EGFR, BRAF, PIK3CA, AKT1, ERBB2/4, PTEN, NRAS, STK11, MAP2K1, ALK, DDR2, CTNNB1, MET, TP53, SMAD4, FBX7, NOTCH1, and FGFR1/2/3. Associations were analyzed with unadjusted p-values (p) and Benjamini & Hochberg adjusted (BHp) shown. Results: Clinical and pathologic data from 180 pts were analyzed: median age 66 (range 24-86), male (47%), stage I (41%), stage II (69%), left (54%) vs right (36%) sided primary tumors, and microsatellite stable (85%). 35 (19%) pts had adjuvant therapy (n = 21 rectal, n = 14 colon). Pathological mutations were found in 160 (89%) of pts, including TP53 (56%), KRAS (44%), PIK3CA (22%), BRAF (12%), SMAD4 (8%), MET (6%) and NRAS (3%). There was only 1 case of ERBB2 mutation. 33 pts (18%) had evidence of recurrence. 36 month RFS was 82%. Common sites of recurrence included liver (13 pts, 39%), lung (10 pts, 30%), and bone (2 pts, 6%). Alterations in MET cDNA and protein were associated with recurrence-free survival (RFS) (HR = 4.1; p =0.0026, BHp= 0.057). Interestingly, while TP53 mutations are typically associated with worse prognosis in metastatic colorectal cancers, it was not associated with RFS (HR = 0.8; p = 0.55, BHp= 0.98). There was also no association between the number of gene alterations and RFS (p = 0.45). Conclusions: These data highlight that targeted NGS tumor profiling of early stage CRC, including sequencing MET among other genes, may be utilized alongside known prognostic pathological factors to predict pts with a higher risk of recurrence and may facilitate tailored adjuvant chemotherapy to mitigate this risk.

scholarly journals NovelGUCY2DGene Mutations in Japanese Male Twins with Leber Congenital Amaurosis

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Katsuhiro Hosono ◽  
Yuko Harada ◽  
Kentaro Kurata ◽  
Akiko Hikoya ◽  
Miho Sato ◽  
...  
Keyword(s):  
Clinical Features ◽  
Illumina Miseq ◽  
Fundus Photography ◽  
Compound Heterozygous ◽  
Leber Congenital Amaurosis ◽  
Targeted Next Generation Sequencing ◽  
Retinal Dystrophies ◽  
Japanese Male ◽  
Targeted Ngs ◽  
Next Generation Sequencing Ngs

Purpose. Leber congenital amaurosis (LCA), a genetically and clinically heterogeneous disease, is the earliest onset retinitis pigmentosa (RP) and is the most severe of hereditary retinal dystrophies. This study was conducted to investigate genetic and clinical features of LCA in a set of Japanese male twins with LCA.Methods. To identify causative mutations, 74 genes known to cause RP or LCA were examined by targeted-next generation sequencing (NGS). Targeted-NGS was performed using a custom designed Agilent HaloPlex target enrichment kit with Illumina Miseq sequencer. Identified potential pathogenic mutations were confirmed using Sanger sequencing. Clinical analyses were based on ophthalmic examination, fundus photography, and electroretinography (ERG).Results. Compound heterozygousGUCY2Dmutations of novel splicing mutation c.2113+2_2113+3insT and novel missense mutation p.L905P were detected in both twins. Their father and mother were heterozygous for c.2113+2_2113+3insT and p.L905P, respectively. The twins had phenotypic features similar to those previously reported in patients withGUCY2Dmutations. This included early childhood onset of visual loss, nystagmus, unrecordable ERG, photophobia, and hyperopia.Conclusions. To the best of our knowledge, this is the first report of genetic and clinical features of Japanese LCA twins withGUCY2Dmutation, which were detected using targeted-NGS.

scholarly journals Signed in Blood: Circulating Tumor DNA in Cancer Diagnosis, Treatment and Screening

Cancers ◽  
2021 ◽  
Vol 13 (14) ◽  
pp. 3600
Author(s):  
Jacob J. Adashek ◽  
Filip Janku ◽  
Razelle Kurzrock
Keyword(s):  
Point Mutations ◽  
Circulating Tumor Dna ◽  
Molecular Testing ◽  
Dynamic Changes ◽  
Tissue Acquisition ◽  
Polymerase Chain ◽  
Molecular Landscape ◽  
Metastatic Sites ◽  
Next Generation Sequencing Ngs ◽  
Tumor Dna

With the addition of molecular testing to the oncologist’s diagnostic toolbox, patients have benefitted from the successes of gene- and immune-directed therapies. These therapies are often most effective when administered to the subset of malignancies harboring the target identified by molecular testing. An important advance in the application of molecular testing is the liquid biopsy, wherein circulating tumor DNA (ctDNA) is analyzed for point mutations, copy number alterations, and amplifications by polymerase chain reaction (PCR) and/or next-generation sequencing (NGS). The advantages of evaluating ctDNA over tissue DNA include (i) ctDNA requires only a tube of blood, rather than an invasive biopsy, (ii) ctDNA can plausibly reflect DNA shedding from multiple metastatic sites while tissue DNA reflects only the piece of tissue biopsied, and (iii) dynamic changes in ctDNA during therapy can be easily followed with repeat blood draws. Tissue biopsies allow comprehensive assessment of DNA, RNA, and protein expression in the tumor and its microenvironment as well as functional assays; however, tumor tissue acquisition is costly with a risk of complications. Herein, we review the ways in which ctDNA assessment can be leveraged to understand the dynamic changes of molecular landscape in cancers.

scholarly journals Intermediate between Idiopathic Hypereosinophilia and Chronic Eosinophilic Leukemia: A Report of Two Hypereosinophilic Cases with Possible Novel Molecular Mutations

2021 ◽  
Vol 2021 ◽  
pp. 1-6
Author(s):  
Jui Choudhuri ◽  
Mohammad Eskandari ◽  
Yang Shi ◽  
Yanhua Wang
Keyword(s):  
Steroid Therapy ◽  
Young Patients ◽  
Disease Process ◽  
Organ Damage ◽  
Targeted Next Generation Sequencing ◽  
Chronic Eosinophilic Leukemia ◽  
Molecular Abnormalities ◽  
Targeted Ngs ◽  
Normal Cytogenetics ◽  
Next Generation Sequencing Ngs

To distinguish a reactive eosinophilia from its malignant counterpart is challenging. Establishing clonality of the eosinophils is crucial and considered the determining factor for establishing a diagnosis. Cases of hypereosinophilia without clear reactive etiologies, no evidence of end-organ damage, normal cytogenetics, and no molecular mutations are termed as “Idiopathic Hypereosinophilia (IHE).” For cases which lie between the spectrum of chronic eosinophilic leukemia (CEL) and IHE, identification of underlying molecular abnormalities might be helpful in better understanding the disease process and prognosis. Here, we report two cases of hypereosinophilia in which five possible novel molecular mutations were identified by targeted next-generation sequencing (NGS) analysis. They were FBXW7, KM2A, TCF3, ERBB4, and MET. With multiple genetic mutations, these cases could be classified as chronic eosinophilic leukemia. Both these young patients responded well to steroid therapy. While targeted NGS is a useful tool in identifying new molecular mutation associated with hypereosinophilia, our cases raise the question of further investigating this entity and if there is a possibility of an intermediate category lying between the spectrum of CEL and IHE. Defining hypereosinophilia with clonal molecular abnormality as a malignant process may need to be revisited. Even though attempts are being made to identify mutations in IHE, it might be more significant clinically to differentiate them based on response to steroid therapy and prognosis.

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