scholarly journals Analytical validation and performance characteristics of a 48-gene next-generation sequencing panel for detecting potentially actionable genomic alterations in myeloid neoplasms

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0243683
Author(s):  
Sun Hee Rosenthal ◽  
Anna Gerasimova ◽  
Charles Ma ◽  
Hai-Rong Li ◽  
Andrew Grupe ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Myeloproliferative Neoplasms ◽  
Simultaneous Detection ◽  
Molecular Testing ◽  
Next Generation ◽  
Analytical Validation ◽  
Bioinformatics Pipeline ◽  
Myeloid Neoplasms ◽  
Clinically Significant ◽  
Generation Sequencing

Identification of genomic mutations by molecular testing plays an important role in diagnosis, prognosis, and treatment of myeloid neoplasms. Next-generation sequencing (NGS) is an efficient method for simultaneous detection of clinically significant genomic mutations with high sensitivity. Various NGS based in-house developed and commercial myeloid neoplasm panels have been integrated into routine clinical practice. However, some genes frequently mutated in myeloid malignancies are particularly difficult to sequence with NGS panels (e.g., CEBPA, CARL, and FLT3). We report development and validation of a 48-gene NGS panel that includes genes that are technically challenging for molecular profiling of myeloid neoplasms including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and myeloproliferative neoplasms (MPN). Target regions were captured by hybridization with complementary biotinylated DNA baits, and NGS was performed on an Illumina NextSeq500 instrument. A bioinformatics pipeline that was developed in-house was used to detect single nucleotide variations (SNVs), insertions/deletions (indels), and FLT3 internal tandem duplications (FLT3-ITD). An analytical validation study was performed on 184 unique specimens for variants with allele frequencies ≥5%. Variants identified by the 48-gene panel were compared to those identified by a 35-gene hematologic neoplasms panel using an additional 137 unique specimens. The developed assay was applied to a large cohort (n = 2,053) of patients with suspected myeloid neoplasms. Analytical validation yielded 99.6% sensitivity (95% CI: 98.9–99.9%) and 100% specificity (95% CI: 100%). Concordance of variants detected by the 2 tested panels was 100%. Among patients with suspected myeloid neoplasms (n = 2,053), 54.5% patients harbored at least one clinically significant mutation: 77% in AML patients, 48% in MDS, and 45% in MPN. Together, these findings demonstrate that the assay can identify mutations associated with diagnosis, prognosis, and treatment options of myeloid neoplasms even in technically challenging genes.

scholarly journals Analytical validation and performance characteristics of a 48-gene next-generation sequencing panel for detecting potentially actionable genomic alterations in myeloid neoplasms

2020 ◽  
Author(s):  
Sun Hee Rosenthal ◽  
Anna Gerasimova ◽  
Charles Ma ◽  
Hai-Rong Li ◽  
Andrew Grupe ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Myeloproliferative Neoplasms ◽  
Simultaneous Detection ◽  
Molecular Testing ◽  
Next Generation ◽  
Analytical Validation ◽  
Bioinformatics Pipeline ◽  
Myeloid Neoplasms ◽  
Clinically Significant ◽  
Generation Sequencing

AbstractIdentification of genomic mutations by molecular testing plays an important role in diagnosis, prognosis, and treatment of myeloid neoplasms. Next-generation sequencing (NGS) is an efficient method for simultaneous detection of clinically significant genomic mutations with high sensitivity. However, due to lack of standard NGS protocols, the application of NGS for hematologic malignancies into clinical settings remains limited. We report development and validation of a 48-gene NGS panel for molecular profiling of myeloid neoplasms including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and myeloproliferative neoplasms (MPN). Target regions were captured by hybridization with complementary biotinylated DNA baits, and NGS was performed on an Illumina NextSeq500 instrument. A bioinformatics pipeline that was developed in-house was used to detect single nucleotide variations (SNVs), insertions/deletions (indels), and FLT3 internal tandem duplications (FLT3-ITD). An analytical validation study was performed on 184 unique specimens for variants with allele frequencies ≥5%. Variants identified by the 48-gene panel were compared to those identified by a 35-gene hematologic neoplasms panel using an additional 137 unique specimens. The developed assay was applied to a large cohort (n=2,053) of patients with suspected myeloid neoplasms. Analytical validation yielded 99.6% sensitivity (95% CI: 98.9-99.9%) and 100% specificity (95% CI: 100%). Concordance of variants detected by the 2 tested panels was 100%. Among patients with suspected myeloid neoplasms (n=2,053), 54.5% patients harbored at least one clinically significant mutation: 77% in AML patients, 48% in MDS, and 45% in MPN. Together, these findings demonstrate that the assay can identify mutations associated with diagnosis, prognosis, and treatment options of myeloid neoplasms.

Identifying large indels in targeted next generation sequencing assays for myeloid neoplasms: a cautionary tale of the ZRSR1 pseudogene

2017 ◽  
Vol 70 (12) ◽  
pp. 1069-1073 ◽  
Author(s):  
Isaac KS Ng ◽  
Christopher Ng ◽  
Jia Jin Low ◽  
Lily Chiu ◽  
Elaine Seah ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Clinical Diagnostics ◽  
Great Promise ◽  
Next Generation ◽  
Bioinformatics Pipeline ◽  
Targeted Next Generation Sequencing ◽  
Molecular Features ◽  
Myeloid Neoplasms ◽  
Large Indels ◽  
Generation Sequencing

Targeted next generation sequencing platforms have been increasingly utilised for identification of novel mutations in myeloid neoplasms, such as acute myeloid leukaemia (AML), and hold great promise for use in routine clinical diagnostics. In this study, we evaluated the utility of an open source variant caller in detecting large indels in a targeted sequencing of AML samples. While we found that this bioinformatics pipeline has the potential to accurately capture large indels (>20 bp) in patient samples, we highlighted the pitfall of a confounding ZRSR1 pseudogene that led to an erroneous ZRSR2 variant call. We further discuss possible clinical implications of the ZRSR1 pseudogene in myeloid neoplasms based on its molecular features. Knowledge of the confounding ZRSR1 pseudogene in ZRSR2 sequencing assays could be particularly important in AML diagnostics because the detection of ZRSR2 in AML patients is highly specific for an s-AML diagnosis.

scholarly journals Next Generation Sequencing in Cytopathology: Focus on Non-Small Cell Lung Cancer

2021 ◽  
Vol 8 ◽  
Author(s):  
Pasquale Pisapia ◽  
Francesco Pepe ◽  
Antonino Iaccarino ◽  
Roberta Sgariglia ◽  
Mariantonia Nacchio ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Next Generation Sequencing ◽  
Small Cell Lung Cancer ◽  
Cell Lung Cancer ◽  
Simultaneous Detection ◽  
Small Cell ◽  
Molecular Testing ◽  
Next Generation ◽  
Small Cell Lung ◽  
Generation Sequencing

Molecular cytopathology is a rapidly evolving field embracing both conventional microscopy and molecular pathology. Its growing popularity stems from the fact that in many types of advanced cancers, including non small cell lung cancer (NSCLC), cytological samples often constitute the only available specimens for morphomolecular analysis. Indeed, non formalin fixed and paraffin embedded (FFPE) cytological samples feature a higher quality of extracted nucleic acids than histological specimens. However, because of the growing complexity of molecular testing, several efforts should be made to validate the analytical performance of the wide array of currently available molecular technologies, including next generation sequencing (NGS). This technology has the terrific advantage of allowing simultaneous detection of scores of predictive biomarkers even in low-input DNA/RNA specimens. Here, we briefly review the role of the modern cytopathologist in the morphomolecular diagnosing of advanced stage NSCLC and the adoption of NGS in conventional cytopreparations (cell blocks, direct smears, and liquid-based cytology) and supernatants.

scholarly journals A Single-Run Next-Generation Sequencing (NGS) Assay for the Simultaneous Detection of Both Gene Mutations and Large Chromosomal Abnormalities in Patients with Myelodysplastic Syndromes (MDS) and Related Myeloid Neoplasms

Cancers ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1947
Author(s):  
Alessandro Liquori ◽  
Iván Lesende ◽  
Laura Palomo ◽  
Gayane Avetisyan ◽  
Mariam Ibáñez ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Myelodysplastic Syndromes ◽  
Chromosomal Abnormalities ◽  
Myeloproliferative Neoplasms ◽  
Simultaneous Detection ◽  
Genetic Alterations ◽  
Next Generation ◽  
Snp Arrays ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Myelodysplastic syndromes (MDS) and myelodysplastic/myeloproliferative neoplasms are clonal disorders that share most of their cytogenetic and molecular alterations. Despite the increased knowledge of the prognostic importance of genetics in these malignancies, next-generation sequencing (NGS) has not been incorporated into clinical practice in a validated manner, and the conventional karyotype remains mandatory in the evaluation of suspected cases. However, non-informative cytogenetics might lead to an inadequate estimation of the prognostic risk. Here, we present a novel targeted NGS-based assay for the simultaneous detection of all the clinically relevant genetic alterations associated with these disorders. We validated this platform in a large cohort of patients by performing a one-to-one comparison with the lesions from karyotype and single-nucleotide polymorphism (SNP) arrays. Our strategy demonstrated an approximately 97% concordance with standard clinical assays, showing sensitivity at least equivalent to that of SNP arrays and higher than that of conventional cytogenetics. In addition, this NGS assay was able to identify both copy-neutral loss of heterozygosity events distributed genome-wide and copy number alterations, as well as somatic mutations within significant driver genes. In summary, we show a novel NGS platform that represents a significant improvement to current strategies in defining diagnosis and risk stratification of patients with MDS and myeloid-related disorders.

Practical guide for the comparison of two next-generation sequencing systems for solid tumour analysis in a universal healthcare system

2018 ◽  
Vol 72 (3) ◽  
pp. 225-231 ◽  
Author(s):  
Perry Maxwell ◽  
Seán O Hynes ◽  
Marc Fuchs ◽  
Stephanie Craig ◽  
Claire McGready ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Cost Effective ◽  
Standard Of Care ◽  
Personal Genome ◽  
Molecular Testing ◽  
Next Generation ◽  
Bioinformatics Pipeline ◽  
Universal Healthcare ◽  
Gene Panels ◽  
Generation Sequencing

AimsAlthough there have been excellent reports in the literature of validating next-generation sequencing, comparisons between two systems are not often published due to cost and time. We set out to establish that targetable mutations could be reliably detected with different gene panels and different chemistries using a common bioinformatics pipeline for meaningful comparisons to be made.MethodsAfter running selected formalin-fixed, paraffin-embedded samples through QPCR, Sanger sequencing and the 50 gene hotspot v2 panel from Life Technologies to determine standard-of-care variants, we compared the Oncomine panel from Life Technologies performed on a Personal Genome Machine (PGM) and the eight-gene actionable panel from Qiagen performed on a MiSeq platform. We used a common bioinformatics program following the creation of respective VCF files.ResultsBoth panels were accurate to above 90%, the actionable panel workflow was easier to perform but the lowest effective starting DNA load was obtained on the Oncomine workflow at 4 ng. Such minimal DNA can help with samples where there is limited material such as those for lung cancer molecular studies. We also discuss gene panel content and propose that increasing the gene profile of a panel will not benefit clinical laboratories where standard-of-care testing is all that is required.ConclusionsOnce recognised, it may be cost-effective for such laboratories to begin validation with an appropriate bioinformatics pipeline for targeted multigene hotspot molecular testing.

scholarly journals The genetic analysis of Chinese patients with clonal cytopenias using targeted next-generation sequencing

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Lijuan Zhang ◽  
YuYe Shi ◽  
Yue Chen ◽  
Shandong Tao ◽  
Wenting Shi ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Myelodysplastic Syndromes ◽  
Rna Splicing ◽  
Myeloproliferative Neoplasms ◽  
Chinese Patients ◽  
Newly Diagnosed ◽  
Next Generation ◽  
Targeted Next Generation Sequencing ◽  
Significant Difference ◽  
Generation Sequencing

Abstract Background Clonal hematopoiesis (CH) can be found in various myeloid neoplasms (MN), such as myelodysplastic syndromes (MDS), myelodysplastic syndromes/myeloproliferative neoplasms (MDS/MPN), also in pre-MDS conditions. Methods Cytogenetics is an independent prognostic factor in MDS, and fluorescence in-situ hybridization (FISH) can be used as an adjunct to karyotype analysis. In the past 5 years, only 35 of 100 newly diagnosed MDS and MDS/MPN patients were identified abnormalities, who underwent the FISH panel. In addition, we examined a cohort of 51 cytopenic patients suspected MDS or MDS/MPN with a 20-gene next generation sequencing (NGS), including 35 newly diagnosed MN patients and 16 clonal cytopenias of undetermined significance (CCUS) patients. Results Compared with the CCUS group, the MN group had higher male ratio (22/13 vs 10/6), cytogenetics abnormalities rate (41.4% vs 21.4%) and frequency of a series of mutations, such as ASXL1 (28.6% vs 25%), U2AF1 (25.7% vs 25%), RUNX1 (20% vs 0.0%); also, higher adverse mutations proportion (75% vs 85.2%), and double or multiple mutations (54.3% vs 43.75%). There were 7 MN patients and 4 CCUS patients who experienced cardio-cerebrovascular embolism events demonstrated a significant difference between the two groups (25% vs 20%). Ten of the 11 patients had somatic mutations, half had DNA methylation, while the other half had RNA splicing. Additionally, six patients had disease transformation, and four patients had mutated U2AF1, including two CCUS cases and two MDS-EB cases. Following up to January 2021, there was no significant difference in over survival between the CCUS and MN groups. Conclusion NGS facilitates the diagnosis of unexplained cytopenias. The monitoring and management of CCUS is necessary, also cardio-cerebrovascular embolism events in patients with CH need attention in the clinical practice.

scholarly journals Next generation sequencing of RB1gene for the molecular diagnosis of ethnic minority with retinoblastoma in Yunnan

2020 ◽  
Author(s):  
Huaiyu Gu ◽  
Zhen Zhang ◽  
Yi-shuang Xiao ◽  
Ru Shen ◽  
Hong-chao Jiang ◽  
...  
Keyword(s):  
Genetic Testing ◽  
Next Generation Sequencing ◽  
Low Income ◽  
Eastern China ◽  
Low Income Countries ◽  
Next Generation ◽  
Single Nucleotide Variants ◽  
Bioinformatics Pipeline ◽  
Generation Sequencing

Abstract Background: Retinoblastoma is a rare intraocular malignancy and typically initiated by inactivating biallelic mutations of RB1 gene. Each year, ~8,000 children worldwide are diagnosed for retinoblastoma. In high-income countries, patient survival is over 95% while low-income countries is ~30%.If disease is diagnosed early and treated in centers specializing in retinoblastoma, the survival might exceed 95% and many eyes could be safely treated and support a lifetime of good vision. In China, approximate 1,100 newly diagnosed cases are expected annually and 28 hospitals covering 25 provinces established centers classified by expertise and resources for better treatment options and follow-up. Comparing with other province of eastern China, Yunnan province is remote geographically. This might result that healthcare staff have low awareness of the role of genetic testing in management and screening in families.Methods: The patients with retinoblastoma were selected in Yunnan. DNA from blood was used for targeted gene sequencing. Then, an in-house bioinformatics pipeline was done to detect both single nucleotide variants and small insertions/deletions. The pathogenic mutations were identified and further confirmed by conventional methods and cosegregation in families.Results: Using our approach, targeted next generation sequencing was used to detect the mutation of these 12 probands. Bioinformatic predictions showed that nine mutations were found in our study and four were novel pathogenic variants in these nine mutations.Conclusions: It’s the first report to describe RB1 mutations in Yunnan children with retinoblastoma. This study would improve role of genetic testing for management and family screening.

scholarly journals Next-Generation Sequencing Mutational Landscape and Clinical Features of Chinese Adults with Myeloproliferative Neoplasms

2021 ◽  
Author(s):  
Shuna Luo ◽  
Zanzan Wang ◽  
Xiaofei Xu ◽  
Lan Zhang ◽  
Shengjie Wang ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Comprehensive Evaluation ◽  
Myeloproliferative Neoplasms ◽  
Primary Myelofibrosis ◽  
Next Generation ◽  
Chinese Adults ◽  
Leukocyte Counts ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Abstract Background: Myeloproliferative neoplasms (MPNs) include three classical subtypes: polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Since prefibrotic primary myelofibrosis (pre-PMF) was recognized as a separate entity in the 2016 revised classification of MPN, it has been a subject of debate among experts due to its indefinite diagnosis. However, pre-PMF usually has a distinct outcome compared with either ET or overt PMF. In this study, we examined the clinical, haematologic, genetic, and prognostic differences among pre-PMF, ET, and overt PMF.Methods: We retrospectively reviewed the clinical parameters, haematologic information, and genetic mutations of patients who were diagnosed with pre-PMF, ET, and overt PMF according to the WHO 2016 criteria using next-generation sequencing (NGS).Results: Pre-PMF patients exhibited higher leukocyte counts, higher LDH values, a higher frequency of splenomegaly, and a higher incidence of hypertension than ET patients. On the other hand, pre-PMF patients had higher platelet counts and haemoglobin levels than overt PMF patients. Molecular analysis revealed that the frequency of EP300 mutations was significantly increased in pre-PMF patients compared with ET and overt PMF patients. In terms of outcome, male sex, along with symptoms including MPN-10, anaemia, thrombocytopenia, and KMT2A and CUX1 mutations, indicated a poor prognosis for PMF patients.Conclusion: The results of this study indicated that comprehensive evaluation of BM features, clinical phenotypes, haematologic parameters, and molecular profiles is needed for the accurate diagnosis and treatment of ET, pre-PMF, and overt PMF patients.

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