scholarly journals Analysis of genetic variants in myeloproliferative neoplasms using a 22-gene next-generation sequencing panel

2022 ◽  
Vol 15 (1) ◽  
Author(s):  
Jaymi Tan ◽  
Yock Ping Chow ◽  
Norziha Zainul Abidin ◽  
Kian Meng Chang ◽  
Veena Selvaratnam ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Myeloproliferative Neoplasms ◽  
Bone Marrow Failure ◽  
Biological Significance ◽  
Driver Mutations ◽  
Study Cohort ◽  
Polycythaemia Vera ◽  
Next Generation ◽  
Novel Variants ◽  
Generation Sequencing

Abstract Background The Philadelphia (Ph)-negative myeloproliferative neoplasms (MPNs), namely essential thrombocythaemia (ET), polycythaemia vera (PV) and primary myelofibrosis (PMF), are a group of chronic clonal haematopoietic disorders that have the propensity to advance into bone marrow failure or acute myeloid leukaemia; often resulting in fatality. Although driver mutations have been identified in these MPNs, subtype-specific markers of the disease have yet to be discovered. Next-generation sequencing (NGS) technology can potentially improve the clinical management of MPNs by allowing for the simultaneous screening of many disease-associated genes. Methods The performance of a custom, in-house designed 22-gene NGS panel was technically validated using reference standards across two independent replicate runs. The panel was subsequently used to screen a total of 10 clinical MPN samples (ET n = 3, PV n = 3, PMF n = 4). The resulting NGS data was then analysed via a bioinformatics pipeline. Results The custom NGS panel had a detection limit of 1% variant allele frequency (VAF). A total of 20 unique variants with VAFs above 5% (4 of which were putatively novel variants with potential biological significance) and one pathogenic variant with a VAF of between 1 and 5% were identified across all of the clinical MPN samples. All single nucleotide variants with VAFs ≥ 15% were confirmed via Sanger sequencing. Conclusions The high fidelity of the NGS analysis and the identification of known and novel variants in this study cohort support its potential clinical utility in the management of MPNs. However, further optimisation is needed to avoid false negatives in regions with low sequencing coverage, especially for the detection of driver mutations in MPL.

scholarly journals Analysis of Genetic Variants in Myeloproliferative Neoplasms Using a 22-Gene Next-Generation Sequencing Panel

2021 ◽  
Author(s):  
Jaymi Tan ◽  
Yock Ping Chow ◽  
Norziha Zainul Abidin ◽  
Kian Meng Chang ◽  
Veena Selvaratnam ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Myeloproliferative Neoplasms ◽  
Bone Marrow Failure ◽  
Biological Significance ◽  
Driver Mutations ◽  
Polycythaemia Vera ◽  
Next Generation ◽  
Single Nucleotide Variants ◽  
Novel Variants ◽  
Generation Sequencing

Abstract Background The Philadelphia (Ph)-negative myeloproliferative neoplasms (MPNs) (i.e. essential thrombocythaemia (ET), polycythaemia vera (PV), and primary myelofibrosis (PMF)) are a group of chronic clonal haematopoietic disorders that have the propensity to advance into bone marrow failure or acute myeloid leukaemia; often resulting in fatality. Although driver mutations have been identified in these Ph-negative MPNs, subtype-specific markers of the disease have yet to be discovered. Next-generation sequencing (NGS) technology can potentially improve the clinical management of Ph-negative MPNs by allowing for the simultaneous screening of many disease-associated genes. Methods The performance of a custom, in-house designed 22-gene NGS panel was technically validated using reference standards across two independent replicate runs. The panel was subsequently used to screen a total of 10 clinical MPN samples (ET n = 3, PV n = 3, PMF n = 4). The resulting NGS data was then analysed via a bioinformatics pipeline. Results The custom NGS panel had a detection limit of 1% variant allele frequency (VAF). A total of 44 variants with minor allele frequencies (MAFs) of ≥ 1%, and 20 variants with MAFs of < 1% were identified (4 of which were putatively novel variants with potential biological significance) across the 10 MPN samples. All single nucleotide variants with VAFs ≥ 15% were confirmed via Sanger sequencing. Conclusions The high fidelity of the NGS analysis and the identification of known and novel variants support its potential clinical utility in the management of Ph-negative MPNs.

scholarly journals Next-Generation Sequencing (NGS) in Childhood Myeloproliferative Diseases (MPD)

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3049-3049
Author(s):  
Michelina Santopietro ◽  
Giovanna Palumbo ◽  
Maria Luisa Moleti ◽  
Anna Maria Testi ◽  
Luisa Cardarelli ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Children And Adolescents ◽  
Myeloproliferative Neoplasms ◽  
Primary Myelofibrosis ◽  
Advisory Board ◽  
Gene Panel ◽  
Driver Mutations ◽  
Next Generation ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Abstract Driver mutations of JAK2, CALR and MPL are found in >90% of adults with BCR-ABL1-negative myeloproliferative neoplasms (MPN). In children, the presence of clonal markers ranges between 22 and 40%, and inherited forms of MPD, such as familial erythrocytosis (FE) and hereditary thrombocytosis (HT), are common. Data on the mutational spectrum and biology of childhood MPD are limited. The aims of this study were: a) to evaluate the ability of a next-generation sequencing (NGS)-based 44-gene analysis to better characterize wild type (WT) MPD, and b) to identify non-canonical and/or non-driver mutations in children and adolescents with MPD. Eighty patients (pts) aged ≤20 years (yrs) at diagnosis of MPD, observed between June 1980 and September 2015, were first investigated with standardized methods for driver mutations of MPN (JAK, MPL, CALR), for genes involved in FE (HRE, EpoR, HIF2α, HIF1α, VHL, PHD1-3, STAT5, LNK, TET2) and HT (THPO, MPL, LNK and TET2). Then, a 44-gene panel providing diagnostic information in myeloid malignancies and in rare inherited erythrocytosis/thrombocytosis (JAK2, CALR, MPL, ASXL1, CBL, C-Kit, CSF3R, CUX1, DNMT3A, ETNK1, EZH2, IDH1, IDH2, IKZF1, KRAS, LNK, NFE2, NRAS, PTPN11, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53, U2AF1, ZRSR2, BPGM, EGLN1 (PHD2), EPAS1 (HIF2A), EPOR, GATA1, GELSOLIN, HBA1, HBA2, HBB, JAK2,MPL, RUNX1, SH2B3, SRC, THPO, VHL, WAS) was employed to better characterize these diseases. Sequencing analyses of DNA from mononuclear peripheral blood cells were performed in 57/80 pts. Eighty pts (M 41, F 39; median age at diagnosis: 149/12 yrs, range 3 months-1911/12 yrs), investigated by standardized methods, were retrospectively classified according to the WHO 2016 criteria as follows: 35 essential thrombocythemia (ET) (10 JAK2V617F, 2 CALR type1, 6 CALR type2, 1 CALR atypical, 16 WT), 9 polycythemia vera (PV) (4 JAK2V617F, 5 WT) and 3 primary myelofibrosis (PMF) (1 JAK2V617F, 2 WT). Twenty-three pts with MPLS505N or MPLV501A mutations and 10 pts with HIF mutations (3 pts) and/or anamnestic criteria of FE (7 WT) were considered HT and FE, respectively. The NGS-based 44-gene panel was applied to 57 MPD pts (11 JAK2V617F, 6 CALR, 12 MPLS505N, 2 MPLV501A, 3 HIF2α and 23 WT). According to the WHO 2016 criteria, 27 pts were ET, 14 HT, 8 FE, 7 PV and 1 PMF. By using the NGS panel, clonal markers were found in 12/23 (52%) pts with MPN WT: HBB and PDH2 in 2 FE, MPLW515_P518>KT in 1 ET pt and non-driver mutations in 9 pts (7 ET, 1 PF and 1 PV). Furthermore, two non-canonical driver mutations, MPLC322G and JAK2G301R were identified in 1 CALR type2 ET and in 1 JAK2V617FPV, respectively. An additional MPLV501M mutation was found in 1 MPLS505N HT. Taken together, among the 57 pts 18 (32%) had one (11/18=68%) or two (7/18=39%) non-driver mutations. Eight of the 34 pts (23.5%) with a clonal marker had additional non-driver mutations, that was single in 6 pts. Within the familial MPD, a single non-driver mutation was found in 3/8 FE pts (37.5%), while no mutations were detected in HT pts. Considering the functional classification of non-driver mutations, we found mutations in signaling (CBL, LNK/SH2B3, CSF3R, KIT, SETBP1) and splicing (U2AF1, ZRSR2) genes in ET and PMF pts, and mutations of epigenetic regulation genes (TET2, ASXL1, DNMT3A) in PV, FE and ET pts (Table 1). The co-occurrence of driver and non-driver mutations in the same individual is illustrated in the circos plot (Figure 1). The use of a NGS-based 44-gene panel in acquired and familial pediatric MPD enabled to identify driver and non-driver mutations, not otherwise detected by conventional methods, with a substantial proportion of MPD pts (81%) showing mutations in the genes analyzed. Interestingly, we found additional neoplastic mutations in some pts with FE. Although the utilized NGS-based panel proved useful to better characterize children and adolescents with MPD, 19% of our pts still remain without any identified clonal marker. Further targeted NGS and whole genome sequencing may enable to better define MPD children without molecular markers. Disclosures Malaspina: Sapienza University, Rome: Other: Resident in Hematology. Foà:ABBVIE: Other: ADVISORY BOARD, Speakers Bureau; CELGENE: Other: ADVISORY BOARD, Speakers Bureau; AMGEN: Other: ADVISORY BOARD; INCYTE: Other: ADVISORY BOARD; NOVARTIS: Speakers Bureau; ROCHE: Other: ADVISORY BOARD, Speakers Bureau; GILEAD: Speakers Bureau; JANSSEN: Other: ADVISORY BOARD, Speakers Bureau; CELTRION: Other: ADVISORY BOARD.

scholarly journals Next Generation Sequencing in MPNs. Lessons from the Past and Prospects for Use as Predictors of Prognosis and Treatment Responses

Cancers ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 2194
Author(s):  
Vibe Skov
Keyword(s):  
Next Generation Sequencing ◽  
Clinical Decision Making ◽  
Myeloproliferative Neoplasms ◽  
Clonal Evolution ◽  
Scoring Systems ◽  
Treatment Modalities ◽  
Driver Mutations ◽  
Next Generation ◽  
The Past ◽  
Generation Sequencing

The myeloproliferative neoplasms (MPNs) are acquired hematological stem cell neoplasms characterized by driver mutations in JAK2, CALR, or MPL. Additive mutations may appear in predominantly epigenetic regulator, RNA splicing and signaling pathway genes. These molecular mutations are a hallmark of diagnostic, prognostic, and therapeutic assessment in patients with MPNs. Over the past decade, next generation sequencing (NGS) has identified multiple somatic mutations in MPNs and has contributed substantially to our understanding of the disease pathogenesis highlighting the role of clonal evolution in disease progression. In addition, disease prognostication has expanded from encompassing only clinical decision making to include genomics in prognostic scoring systems. Taking into account the decreasing costs and increasing speed and availability of high throughput technologies, the integration of NGS into a diagnostic, prognostic and therapeutic pipeline is within reach. In this review, these aspects will be discussed highlighting their role regarding disease outcome and treatment modalities in patients with MPNs.

Does histological assessment accurately distinguish separate primary lung adenocarcinomas from intrapulmonary metastases? A study of paired resected lung nodules in 32 patients using a routine next-generation sequencing panel for driver mutations

2021 ◽  
pp. jclinpath-2021-207421
Author(s):  
Frido K Bruehl ◽  
Erika E Doxtader ◽  
Yu-Wei Cheng ◽  
Daniel H Farkas ◽  
Carol Farver ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Molecular Analysis ◽  
Low Frequency ◽  
Driver Mutations ◽  
Lung Nodules ◽  
Driver Gene ◽  
Next Generation ◽  
Histological Assessment ◽  
Lung Adenocarcinomas ◽  
Generation Sequencing

AimVarious approaches have been reported for distinguishing separate primary lung adenocarcinomas from intrapulmonary metastases in patients with two lung nodules. The aim of this study was to determine whether histological assessment is reliable and accurate in distinguishing separate primary lung adenocarcinomas from intrapulmonary metastases using routine molecular findings as an adjunct.MethodsWe studied resected tumour pairs from 32 patients with lung adenocarcinomas in different lobes. In 15 of 32 tumour pairs, next-generation sequencing (NGS) for common driver mutations was performed on both nodules. The remainder of tumour pairs underwent limited NGS, or EGFR genotyping. Tumour pairs with different drivers (or one driver/one wild-type) were classified as molecularly unrelated, while those with identical low-frequency drivers were classified as related. Three pathologists independently and blinded to the molecular results categorised tumour pairs as related or unrelated based on histological assessment.ResultsOf 32 pairs, 15 were classified as related by histological assessment, and 17 as unrelated. Of 15 classified as related by histology, 6 were classified as related by molecular analysis, 4 were unrelated and 5 were indeterminate. Of 17 classified as unrelated by histology, 14 were classified as unrelated by molecular analysis, none was related and 3 were indeterminate. Histological assessment of relatedness was inaccurate in 4/32 (12.5%) tumour pairs.ConclusionsA small but significant subset of two-nodule adenocarcinoma pairs is inaccurately judged as related by histological assessment, and can be proven to be unrelated by molecular analysis (driver gene mutations), leading to significant downstaging.

Mutational profiling of acute myeloid leukemia with normal cytogenetics in Brazilian patients: the value of next-generation sequencing for genomic classification

2017 ◽  
Vol 65 (8) ◽  
pp. 1155-1158 ◽  
Author(s):  
Thiago Rodrigo de Noronha ◽  
Miguel Mitne-Neto ◽  
Maria de Lourdes Chauffaille
Keyword(s):  
Acute Myeloid Leukemia ◽  
Next Generation Sequencing ◽  
Myeloid Leukemia ◽  
Regulation Of Gene Expression ◽  
Uniparental Disomy ◽  
Driver Mutations ◽  
Next Generation ◽  
Normal Cytogenetics ◽  
Generation Sequencing ◽  
Acute Myeloid

Karyotype (KT) aberrations are important prognostic factors for acute myeloid leukemia (AML); however, around 50% of cases present normal results. Single nucleotide polymorphism array can detect chromosomal gains, losses or uniparental disomy that are invisible to KT, thus improving patients’ risk assessment. However, when both tests are normal, important driver mutations can be detected by the use of next-generation sequencing (NGS). Fourteen adult patients with AML with normal cytogenetics were investigated by NGS for 19 AML-related genes. Every patient presented at least one mutation:DNMT3Ain nine patients;IDH2in six;IDH1in three;NRASandNPM1in two; andTET2,ASXL1,PTPN11, andRUNX1in one patient. No mutations were found inFLT3,KIT,JAK2,CEBPA,GATA2,TP53,BRAF,CBL,KRAS,andWT1genes. Twelve patients (86%) had at least one mutation in genes related with DNA methylation (DNMT3A,IDH1,IDH2,andTET2), which is involved in regulation of gene expression and genomic stability. All patients could be reclassified based on genomic status and nine had their prognosis modified. In summary, NGS offers insights into the molecular pathogenesis and biology of cytogenetically normal AML in Brazilian patients, indicating that the prognosis could be further stratified by different mutation combinations. This study shows a different frequency of mutations in Brazilian population that should be confirmed.

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.

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