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.

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 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.

scholarly journals The Application of Next-Generation Sequencing to Define Factors Related to Oral Cancer and Discover Novel Biomarkers

Life ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 228
Author(s):  
Soyeon Kim ◽  
Joo Won Lee ◽  
Young-Seok Park
Keyword(s):  
Next Generation Sequencing ◽  
Oral Cancer ◽  
Genetic Alterations ◽  
Eligibility Criterion ◽  
Next Generation ◽  
Novel Biomarkers ◽  
Targeted Gene Therapy ◽  
Next Generation Sequencing Ngs ◽  
Potential Use ◽  
Generation Sequencing

Despite the introduction of next-generation sequencing in the realm of DNA sequencing technology, it is not often used in the investigation of oral squamous cell carcinoma (OSCC). Oral cancer is one of the most frequently occurring malignancies in some parts of the world and has a high mortality rate. Patients with this malignancy are likely to have a poor prognosis and may suffer from severe facial deformity or mastication problems even after successful treatment. Therefore, a thorough understanding of this malignancy is essential to prevent and treat it. This review sought to highlight the contributions of next-generation sequencing (NGS) in unveiling the genetic alterations and differential expressions of miRNAs involved in OSCC progression. By applying an appropriate eligibility criterion, we selected relevant studies for review. Frequently identified mutations in genes such as TP53, NOTCH1, and PIK3CA are discussed. The findings of existing miRNAs (e.g., miR-21) as well as novel discoveries pertaining to OSCC are also covered. Lastly, we briefly mention the latest findings in targeted gene therapy and the potential use of miRNAs as biomarkers. Our goal is to encourage researchers to further adopt NGS in their studies and give an overview of the latest findings of OSCC treatment.

Performance of next-generation sequencing for detection of clinically actionable genetic variants in cancer.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 11099-11099
Author(s):  
Mohammed Omar Hussaini ◽  
Ian S. Hagemann ◽  
Teresa Mary Cox ◽  
Christina Lockwood ◽  
Karen Seibert ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Genetic Variants ◽  
Simultaneous Detection ◽  
Genetic Alterations ◽  
Sequence Variants ◽  
Tumor Type ◽  
Cancer Genes ◽  
Next Generation ◽  
Therapeutic Implications ◽  
Generation Sequencing

11099 Background: Next-generation sequencing (NGS) allows for simultaneous detection of numerous actionable somatic variants in cancer. We have implemented a clinical NGS panel to detect genetic alterations in 25 genes with established roles in cancer and report here the frequency of clinically actionable genetic variants in a variety of cancer types. Methods: NGS testing was performed in a CAP-certified, CLIA-licensed environment on DNA extracted from FFPE tissue in 209 cases spanning 41 histologic tumor types. DNA was enriched by hybrid capture and sequenced to >1,000x average coverage on Illumina sequencers with 2x101bp or 2x150bp reads. Variants were called using clinically validated parameters using the Genome Analysis Toolkit, Pindel, and the custom-written Clinical Genomicist Workstation. Results: Non-small cell lung cancer (45%), pancreatic cancer (10%), and colorectal cancer (8%) were the most common tumors sent for NGS analysis. An average of 3 (range 1- 16) non-synonymous, non-SNP sequence variants per case (SNVs and indels) were detected in the 130kb exonic target. Variants were most commonly seen in TP53, KRAS, and EGFR. 27% of cases (56/209) had one or more variants with therapeutic implications for the tumor type tested (e.g., EGFR mutation in NSCLC). 15% of cases (32/209) showed actionable variants not generally associated with the malignancy tested (e.g., detection of an activating KITvariant in thymic carcinoma). 10% of cases (21/209) had variants that were prognostically significant but not directly targetable. Some cases (9%) had variants that were prognostic/diagnostic and targetable. In 117 cases (56% of total), no therapeutically or prognostically significant variants were identified. Overall, in 92 cases (44%), NGS testing yielded information with therapeutic (majority), prognostic, or diagnostic ramifications. Conclusions: We found that 44% of unselected cancer cases have clinically relevant sequence variants in a set of 25 commonly mutated cancer genes. Our data suggest that clinical NGS testing may serve as an integral tool in realizing the potential of precision medicine in oncology.

scholarly journals Simultaneous Detection of Pathogens and Tumors in Patients With Suspected Infections by Next-Generation Sequencing

2021 ◽  
Author(s):  
Jiachun Su ◽  
Xu Han ◽  
Xiaogang Xu ◽  
Wenchao Ding ◽  
Ming Li ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Chromosomal Abnormalities ◽  
Malignant Tumors ◽  
Simultaneous Detection ◽  
Prior History ◽  
Next Generation ◽  
Clinical Value ◽  
Microbiological Testing ◽  
Number Variation ◽  
Generation Sequencing

Abstract Background: Differential diagnosis of patients with suspected infections is particularly difficult, but necessary for prompt diagnosis and rational use of antibiotics. A substantial proportion of these patients have non-infectious diseases that include malignant tumors. Metagenomic next-generation sequencing (mNGS) technologies are used with increasing frequency to aid clinical diagnosis of patients with suspected infections. Methods: Based upon mNGS technologies and chromosomal copy number variation (CNV) analysis on abundant human genome, a new workflow named Onco-mNGS was established to simultaneously detect pathogens and malignant tumors in patients with suspected infections. Results: Of 140 patients screened by Onco-mNGS testing at four hospitals in Shanghai, 115 patients were diagnosed with infections; 17 had obvious abnormal CNV signals indicating malignant tumors that were confirmed clinically. The sensitivity and specificity of mNGS testing for diagnosis of a clinically relevant infection was 53.0% (61/115) and 60% (15/25), respectively, vs 20.9% (24/115) and 96.0% (24/25), respectively, for conventional microbiological testing (both P<0.01). Klebsiella pneumoniae was the most common pathogen detected by mNGS, followed by E. coli and viruses. The chromosomal abnormalities of the 17 cases included genome-wide variations and local variations of a certain chromosome. Five of 17 patients had a final confirmed with malignant tumors, including three lung adenocarcinomas and two hematological tumors; one patient was highly suspected to have lymphoma; and 11 patients had a prior history of malignant tumor.Conclusions: This preliminary study demonstrates the feasibility and clinical value of using Onco-mNGS to simultaneously search for potential pathogens and malignant tumors in patients with suspected infections.

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 Next-Generation Sequencing Improves Diagnosis, Prognosis and Clinical Management of Myeloid Neoplasms

Cancers ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 1364 ◽  
Author(s):  
Diego Carbonell ◽  
Julia Suárez-González ◽  
María Chicano ◽  
Cristina Andrés-Zayas ◽  
Juan Carlos Triviño ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Copy Number Variants ◽  
Genetic Alterations ◽  
Next Generation ◽  
Pathogenic Variants ◽  
Myeloid Neoplasms ◽  
Diagnostic Samples ◽  
Gene Panels ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Molecular diagnosis of myeloid neoplasms (MN) is based on the detection of multiple genetic alterations using various techniques. Next-generation sequencing (NGS) has been proved as a useful method for analyzing many genes simultaneously. In this context, we analyzed diagnostic samples from 121 patients affected by MN and ten relapse samples from a subset of acute myeloid leukemia patients using two enrichment-capture NGS gene panels. Pathogenicity classification of variants was enhanced by the development and application of a custom onco-hematology score. A total of 278 pathogenic variants were detected in 84% of patients. For structural alterations, 82% of those identified by cytogenetics were detected by NGS, 25 of 31 copy number variants and three out of three translocations. The detection of variants using NGS changed the diagnosis of seven patients and the prognosis of 15 patients and enabled us to identify 44 suitable candidates for clinical trials. Regarding AML, six of the ten relapsed patients lost or gained variants, comparing with diagnostic samples. In conclusion, the use of NGS panels in MN improves genetic characterization of the disease compared with conventional methods, thus demonstrating its potential clinical utility in routine clinical testing. This approach leads to better-adjusted treatments for each patient.

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