scholarly journals Leukemic transformation driven by an ASXL1 mutation after a JAK2V617F-positive primary myelofibrosis: clonal evolution and hierarchy revealed by next-generation sequencing

2013 ◽  
Vol 6 (1) ◽  
Author(s):  
Francisca Ferrer-Marín ◽  
Beatriz Bellosillo ◽  
Luz Martínez-Avilés ◽  
Gloria Soler ◽  
Pablo Carbonell ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Clonal Evolution ◽  
Primary Myelofibrosis ◽  
Next Generation ◽  
Leukemic Transformation ◽  
Generation Sequencing

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

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4641-4641
Author(s):  
Lan Zhang ◽  
Xingnong Ye ◽  
Shengjie Wang ◽  
Keyi Jin ◽  
Shuna Luo ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Comprehensive Evaluation ◽  
Myeloproliferative Neoplasms ◽  
Primary Myelofibrosis ◽  
Clinical Parameters ◽  
Next Generation ◽  
School Of Medicine ◽  
Medical Discipline ◽  
Generation Sequencing

Abstract 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. We conducted a retrospective study of MPN patients from October 2014 to June 2020 in the Fourth Affiliated Hospital of Zhejiang University. Patients who were diagnosed with ET, pre-PMF or overt-MF according to the 2016 WHO Classification were included. We reviewed the clinical parameters, haematologic information, and genetic mutations of patients using next-generation sequencing (NGS). Mutation screening was performed in 44 patients by next-generation sequencing techniques, 84 genes and 258 mutations were detected. JAK2 was the most frequently mutated gene (25/44, 56.82%), followed by TET2 (14/44, 31.82%), KMT2C (13/44, 29.55%), and ASXL1 (10/44, 23.73%) in MPN (Figure 1-A). The VAFs of all studied genes with mutation frequencies >10% are shown in Figure 1-B. Of the 20 patients with ET, 9 (45%) were positive for the JAK2 mutation, 5 (25%) carried FAT1, 5 (25%) carried KMT2C, and 4 (20%) carried CALR. Of the 5 patients with pre-PMF, 4 (80%) carried JAK2, 3 (60%) carried EP300, and 2 (40%) carried TET2. Of the 19 patients with overt PMF, 12 (63%) carried JAK2, 10 (53%) carried TET2, 7 (37%) carried ASXL1, and 6 (32%) carried KMT2C, as reported in Figure 2. The median follow-up was 36 months for ET, 42 months for pre-PMF, and 53 months for overt PMF. Overall survival between pre-PMF, overt PMF, and ET was significantly different (P<0.001), as shown in Figure 3. During the follow-up time, only one death of ET was registered, so we analysed the impact of clinical parameters and mutational status at diagnosis on outcome in PMF, including pre-PMF and overt PMF. We performed Kaplan-Meier curves to examine the relationships between the clinical parameters and patient survival. We found that male sex (P=0.0107), MPN10 symptoms (P=0.0354), anaemia (haemoglobin<120g/L, P=0.0239), and thrombocytopenia (platelet count <100 ×10 9/L, P=0.0002) were significantly related to inferior OS (Figure 4). 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 MPN10, anaemia, thrombocytopenia, and KMT2A and CUX1 mutations, indicated a poor prognosis for PMF patients. In conclusion, we identified differences in the clinical, haematologic, and molecular presentations of ET, pre-PMF, and overt PMF patients, indicating that comprehensive evaluation of not only BM features but also clinical, haematologic, and molecular profiles is needed for accurate diagnosis and treatment of these three disease entities. The molecular analysis revealed that pre-PMF might be relevant to EP300 mutation, demonstrating the value of molecular examination. 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. Acknowledgment:The research was supported by the Public Technology Application Research Program of Zhejiang, China (LGF21H080003), the Key Project of Jinhua Science and Technology Plan, China (2020XG-29 and 2020-3-011), the Academician Workstation of the Fourth Affiliated Hospital of the Zhejiang University School of Medicine (2019-2024), the Key Medical Discipline of Yiwu, China (Hematology, 2018-2020) and the Key Medical Discipline of Jinhua, China (Hematology, 2019-2021). Correspondence to: Dr Jian Huang, Department of Hematology, The Fourth Affiliated Hospital of Zhejiang University School of Medicine. N1 Shangcheng Road. Yiwu, Zhejiang, Peoples R China. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Next-Generation Sequencing and Real-Time Quantitative PCR for Minimal Residual Disease (MRD) Detection Using the Immunoglobulin Heavy Chain Variable Region: A Methodical Comparison in Acute Lymphoblastic Leukemia (ALL), Mantle Cell Lymphoma (MCL) and Multiple Myeloma (MM)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 788-788 ◽  
Author(s):  
Marco Ladetto ◽  
Monika Bruggemann ◽  
Simone Ferrero ◽  
Francois Pepin ◽  
Daniela Drandi ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Real Time ◽  
Heavy Chain ◽  
Clonal Evolution ◽  
Variable Region ◽  
Immunoglobulin Heavy Chain ◽  
Next Generation ◽  
Heavy Chain Variable Region ◽  
Generation Sequencing ◽  
Disease Specific

Abstract Abstract 788 Background. Real-Time Quantitative (RQ) PCR-based MRD detection using tumor-specific primers derived from the immunoglobulin heavy chain variable region (IGH) is an established disease monitoring tool with high predictive value in ALL, MCL and MM. It is highly sensitive and has been standardized in the context of international cooperative groups such as the European Scientific Foundation for Laboratory Hematooncology (ESLHO). However it has some limitations, including marker identification failure, particularly in hypermutated tumors and false negatives due to clonal evolution, particularly in ALL. IGH-based next-generation sequencing (NGS) might overcome some of these limitations and further increase sensitivity, specificity, accuracy and reproducibility. We have thus performed a head to head comparison of the two methods on diagnostic (DG) and post-treatment follow-up (FU) samples on a panel of 55 patients. Patients and Methods. Overall, 381 samples (215 bone marrow, 166 peripheral blood; 62 DG, 319 FU) were collected from 55 patients (15 ALL, 30 MCL, 10 MM) in which RQ-PCR based MRD analysis had been performed in the context of prospective clinical trials. IGH-based RQ-PCR was carried out as previously described [Ladetto et al, BBMT 2000; Brüggemann et al, Blood 2006], according to the criteria of the European Study Group on MRD detection [van der Velden et al, Leukemia 2007], at two experienced MRD laboratories (Kiel, DE, 238 samples; Torino, IT, 143 samples). NGS was performed at the Sequenta facilities in San Francisco, CA, USA. Using universal primer sets, we amplified IGH variable, diversity, and joining gene segments from genomic DNA. Amplified products were sequenced to obtain a high degree of coverage (14 reads per each IGH molecule) and analyzed using standardized algorithms for clonotype determination. Tumor-specific clonotypes were identified for each patient based on their high-frequency in DG sample and then quantitated in FU samples. A quantitative and standardized measure of clone level among all leukocytes was determined using internal reference DNA. NGS analysis was performed independently and data were blinded until comparison. Comparability of MRD results by RQ-PCR and NGS was assessed by means of bivariate correlations between methods analysis (software R 2.15.1 package irr). Discordances were classified as follows: a positive/negative discordance was defined as major when the positive result was >1 E-05 and minor when ≤1 E-05; a quantitative discordance was defined as the presence of two positive results with a quantitative discrepancy >1 log. Results. 51 pts (93%) were evaluable with at least one tool (RQ-PCR 45, NGS 49), 43 (78%) with both and 4 (7%) with none. Disease-specific success rates are shown in Tab. 1. Overall, 333 samples (87%) were evaluated with at least one tool (RQ-PCR 282, NGS 319) and 268 (70%) with both. The latter group was thus comparable in terms of MRD output. A correlation analysis between RQ-PCR and NGS results is shown in Fig. 1. Overall a significant concordance was observed (p<0.001, R=0.791). 210/268 (78%) samples had an optimal level of concordance. Of these 99 were MRD negative and 111 MRD positive. Major discordances were 17 (6,3%) with NGS overestimating compared to RQ-PCR in 11 samples and underestimating in 6; minor discordances were 23 (8,6%) with NGS overestimating in 5 samples and underestimating in 18; quantitative discordances were 18 (6,7%) with NGS overestimating in 14 cases and underdestimating in 4. In 2 ALL clonal evolution hampered straightforward MRD assessment in one of both methods. In one case IGH RQ-PCR underestimated MRD while a second RQ-PCR marker (TCRD) led to results comparable to NGS. In a second case NGS did not detect the tumor clone identified at diagnosis due to loss of the complete IGHV at relapse whereas the preceeding IGHDJ was preserved and detected by RQ-PCR targeting the preserved DJ region. Conclusions. NGS is a feasible tool for IGH-based MRD monitoring in ALL, MCL and MM, in selected cases reaching similar sensitivities compared to standardized RQ-PCR. Good concordance was seen in the vast majority of cases. However, disease-specific pitfalls (clonal evolution, somatic hypermutations, frequency of complete IGH rearrangements) have to be considered for both methods and prospective comparative analysis of unselected cases is required to verify the clinical impact of NGS-based MRD assessment. Disclosures: No relevant conflicts of interest to declare.

Anticipating High-Risk Chromosome Clonal Evolution in Chronic Lymphocytic Leukemia: A Next Generation Sequencing and Longitudinal FISH Combined Study

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3305-3305
Author(s):  
Ana Maria Hurtado ◽  
Chahinez Hamedi ◽  
Bartlomiej P Przychodzen ◽  
Ana I. Anton ◽  
Maria Dolores Garcia-Malo ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
High Risk ◽  
Chronic Lymphocytic Leukemia ◽  
Clonal Evolution ◽  
Lymphocytic Leukemia ◽  
Next Generation ◽  
Mutational Hotspots ◽  
Generation Sequencing ◽  
11Q Deletion

Abstract Background: Chronic lymphocytic leukemia (CLL), the most common leukemia of adults in Western countries, is clinically characterized by a variable clinical outcome, ranging from indolent to agressive cases. The acquisition of new 17p and/or 11q chromosomal lesions during the disease course (high-risk chromosomal clonal evolution), detected either by FISH or conventional cytogenetics has been shown to confer an adverse prognosis. In a high proportion of these patients, a concomitant p53 and ATM mutation can be found in the remaining allele. Next Generation Sequencing (NGS) of tumors is now an affordable, rapid and comprehensive technology for detecting somatic coding mutations and its depth-sensitivity enables reliable detection of subclonal mutations not detectable by classic methods. The aim of this study was to assesed whether the presence of a detectable p53 or ATM mutation by NGS might anticipate a high risk chromosomal clonal evolution during the follow up. Methods: To this end, we performed targeted NGS sequencing of blood samples from 168 CLL patients at diagnosis who did not present a 17p and/or 11q deletion by FISH and did not meet criteria for active treatment. We designed a TruSeq Custom Amplicon panel (TSCA, Illumina) targeting 12 genes recurrently mutated in CLL, including p53 and ATM. In genes with well-defined mutational hotspots only these regions were targeted; otherwise the entire coding sequence of the gene was sequenced. The panel covers a total of 46605 base pairs with 305 amplicons. Libraries prepared from 250 ng DNA were subjected to 250 bp paired-end sequencing. A second FISH was perfomed in the course of the disease if progression data fullfilling criteria for starting therapy was observed or during of the third year of disease follow up otherwise. Results: With a median age of 71 y.o. (range, 43-95) and a slight male predominance (56%), the median follow up time of our cohort was 43 months (24-104). Median absolute lymphocyte count at baseline was 17660/uL (interquartile range, 7300-25250), with a 49% and 33% of ZAP70 and CD38 positive cases, respectively. At baseline, 52% of patients presented a 13q deletion and 13% a trisomy 12.Twenty-eight percent of patients presented, at least, a panel mutation, being NOTCH1 the gene most frequenly mutated (n=14).Thirteen patients (9%) developed a high risk chromosomal clonal evolution during the follow up: 8 patients acquired a 17p deletion and 5 cases a 11q deletion. In eight out of these cases, a p53/ATM mutation could be found in the baseline simple, with a clonal size ranging from 4% to 50% (median=9%). The presence of a p53/ATM mutation was associated with de development od a high-risk chromosomal clonal evolution (p=0.02) Conclusions: Our study shows, in a clinical setting, that the use of targeted next-generation sequencing technology can anticípate the high-risk chromosomal clonal evolution during the follow up in a subset of patients with chronic lymphocytic leukemia, Disclosures No relevant conflicts of interest to declare.

Clonal Evolution in Relapsed or Refractory Diffuse Large B Cell Lymphoma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 77-77
Author(s):  
Thomas Melchardt ◽  
Clemens Hufnagl ◽  
Oliver Weigert ◽  
David M. Weinstock ◽  
Nadja Kopp ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Primary Tumor ◽  
Clonal Evolution ◽  
B Cell Lymphoma ◽  
Next Generation ◽  
Synonymous Mutations ◽  
Risk Patients ◽  
Relapsed Disease ◽  
Generation Sequencing ◽  
Formalin Fixed

Abstract Background: Despite modern chemoimmunotherapy more than a third of patients with diffuse large B cell lymphoma (DLBCL) will experience relapse or refractory disease. Multiple mutations associated with the biology of DLBCL have recently been identified by next-generation sequencing in primary tumor samples, but little is known about their prognostic role. Furthermore, histological comparison of the primary tumor and relapsed disease is often not available in clinical practice due to the lack of centralized assessment and complicated by the difficulties to perform exome-wide sequencing in formalin fixed tissues. Therefore, the role of certain mutations and their mechanisms in clonal evolution during relapse is unknown and the rising of chemo-resistant DLBCL subclones has not been described in the literature so far. Methods: We identified all patients with available histologically confirmed relapsed or refractory DLBCL in our single center cohort of 346 patients with aggressive lymphoma treated at our tertiary cancer center in Salzburg, Austria. Primary formalin fixed paraffin embedded tumor sample, sample of refractory or relapsed disease and matched germ line were available for targeted next generation sequencing in 27 patients. A targeted exon capture and next-generation sequencing of all coding exons of 104 selected genes known to be frequently mutated in lymphoma were performed on a HiSeq 2500. Results: Sequencing was successful in 96.8% of all samples resulting in 25 patients with sequencing of the primary tumor and 24 patients with available pairs of primary lymphoma and histologically confirmed relapse. In these 24 patients two relapse samples were available in 10 patients and three relapse samples in one patient. Non-synonymous mutations were present in 74 of the 104 genes tested. Individual tumor samples showed between 0 and 29 non-synonymous mutations (median: 10). Less than six non-synonymous mutations in the primary tumor were associated with a better median OS than more mutations (28 versus 15 months p=0.031). We also compared the frequency of mutated genes in our cohort consisting of high risk patients defined by actual relapse with the literature containing patients with extensive sequencing but only little clinical data and clinical follow-up. Common mutated genes such as CARD11, CD58, CD79B, CREBBP, EZH2, BTG1 or B2M showed no difference in frequency to our patient cohort indicating no or only small driver function in resistant or refractory disease. Nevertheless, mutations previously reported to be at low frequency in DLBCL were significantly more often observed in our primary samples (NOTCH1, MYC, RB1, FAT2, ATM, SMARCA4, BCL7A) and relapsed samples (TP53, MCL1, ATM, FAT2, MYC, RB1, SMARCA4) of high risk patients when compared to the literature. We also observed the gain and the loss of several mutations between first diagnosis and histologically confirmed relapse. Overall, we observed an increase of the amount of non-synonymous mutations at first relapse in 12, no change in 6 and a decrease in 6 paired cases. A completely stable pattern of non-synonymous mutations was detected in 4 cases, but in the majority of cases relevant dynamic was observed: e.g.: gain of non-synonymous mutations in the p53 gene was seen in 3 patients (5 mutations), in the TNFRSF-14 gene in 2 patients (3 mutations), in the RB1 gene in 1 patient (1 mutation), in the NOTCH2 gene in 3 patients (4 mutations) and in the MYD88 gene in 1 patient (1 mutation) or loss of non-synonymous mutations in the CREBBP gene in 3 patients (3 mutations) or in the CRAD11 gene in 2 patients (2 mutations). Monitoring of subclones during disease was also possible using the allelic fraction over time e.g.: showing an increase of the NOTCH1 mutation burden in 2 biopsies after first diagnosis. Discussion: To the best of our knowledge clonal evolution detected by next generation sequencing has not been reported in DLBCL so far. We demonstrate the feasibility of such an approach from fixed tissue samples and using a curated set of target genes. In analogy to other lymphoid malignancies we can show the increase of allelic burden of certain mutations over time and the loss or gain of several others. While this approach is limited by the bias introduced by the selection of genes in the gene set, we feel that deep sequencing of selected mutations will provide further insights into subclone dynamics, which may be responsible for clonal evolution. Disclosures No relevant conflicts of interest to declare.

Comprehensive targeted next-generation sequencing to reveal limited clonal evolution after concurrent chemoradiation in patients with squamous cell carcinoma of the head and neck.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. 6059-6059 ◽  
Author(s):  
Inge Tinhofer ◽  
Theresa Eder ◽  
Robert Konschak ◽  
Franziska Niehr ◽  
Korinna Jöhrens ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Disease Progression ◽  
Clonal Selection ◽  
Clonal Evolution ◽  
Intratumoral Heterogeneity ◽  
Concurrent Chemoradiation ◽  
Tumor Evolution ◽  
Next Generation ◽  
Generation Sequencing ◽  
Selection Of

6059 Background: Recent next-generation sequencing (NGS) studies revealed a wide mutational spectrum in SCCHN. However, little is known about spatial intratumoral heterogeneity and temporal clonal evolution. Precise understanding of the genomic architecture of primary and recurrent/metastatic (R/M) tumors will be crucial for the development of personalized treatment and molecular biomarkers. Methods: In this pilot study, paired tumor samples (primary, R/M lesions) from 10 patients with locally advanced SCCHN who progressed after concurrent chemoradiation (CTRX) were included. Mutational profiling was performed by NGS targeting the exonic regions of 327 genes. Only somatic mutant variants with a difference of ≥0.15 in allele frequency (AF) between primary and R/M tumors were considered for further analysis. Results: Median time to progression was 6.2 months (range: 2.5-30.2). Overall, the difference in mutational patterns of primary and R/M tissue was very small. On average, one mutant variant (range: 0-2) was selectively detected in only one of the paired samples or differed in AF for ≥0.15. Nonetheless, clonal selection of mutant variants previously linked to disease progression was observed in 8 of 10 cases. In line with their gain of function, an increase in AF of TP53 missense mutations (R175H, R248Q) in the recurrent tumor - suggestive of the selection of treatment-resistant mutant TP53 subclones - was observed in two patients. Further variants with increased mutant AF in recurrent tumors were found in ADCY2, CDKN2A, FGFR3, MET, NOTCH1, PIK3CA and TGFBR2. Conclusions: We here provide first evidence that treatment-induced clonal selection after CRTX frequently occurs in SCCHN but is limited to only few gene alterations associated with an aggressive phenotype. This result is surprising given CRTX being a DNA-damaging regimen with inherent risk of de-novo mutagenesis and abundant time for clonal tumor evolution. Further investigations of spatial intratumoral heterogeneity and clonal evolution in larger patient cohorts are required for improving our understanding of treatment resistance and disease progression in SCCHN.

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