scholarly journals Next-Generation Sequencing in Adult B Cell Acute Lymphoblastic Leukemia Patients

2017 ◽  
Vol 23 (4) ◽  
pp. 691-696 ◽  
Author(s):  
Olga Sala Torra ◽  
Megan Othus ◽  
David W. Williamson ◽  
Brent Wood ◽  
Ilan Kirsch ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Lymphoblastic Leukemia ◽  
Next Generation ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Generation Sequencing

Next Generation Sequencing Identifies Novel Oncogene Alterations In CRLF2-rearranged Precursor B-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 472-472 ◽  
Author(s):  
Akinori Yoda ◽  
Terry Haley ◽  
Oliver B. Weigert ◽  
Jeffery Kutok ◽  
Adalis Maisonet ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Exome Sequencing ◽  
Rna Editing ◽  
Lymphoblastic Leukemia ◽  
Genetic Alterations ◽  
Next Generation ◽  
High Confidence ◽  
Generation Sequencing

Abstract Abstract 472 We and others recently identified locus rearrangements involving the type I cytokine receptor subunit CRLF2 (also known as thymic stromal lymphopoietin receptor (TSLPR)) in 5–7% of all adult and pediatric B-cell precursor acute lymphoblastic leukemia (B-ALL). CRLF2 rearrangement places full-length CRLF2 under alternate transcriptional control, and can either result from an intrachromosomal CRLF2-P2RY8 deletion or from a CRLF2-IGH translocation. Prognosis for CRLF2-rearranged B-ALL is particularly poor, suggesting that a sizable fraction of relapsed and ultimately fatal B-ALL harbor CRLF2 rearrangements. Approximately 50% of CRLF2-rearranged B-ALL harbor mutations in JAK2 (and rarely JAK1) that cluster around JAK2 Arg683. A separate 15% have a CRLF2 F232C mutation that promotes constitutive homodimerization and signaling. In the remaining cases, the driver of CRLF2 signaling is not known. To identify additional genetic alterations that contribute to CRLF2-mediated leukemogenesis, we performed next-generation sequencing on 3 CRLF2-rearranged B-ALL specimens, including one with CRLF2 F232C (#536). Exome libraries were assembled from bone marrow specimens with greater than 90% B-ALL involvement and from paired remission bone marrows. Transcriptome sequencing was also performed on cDNA isolated from the involved marrow of patient #536 to: 1) confirm the findings from exome sequencing, 2) focus on transcribed genes and 3) identify possible RNA editing events. Sequencing utilized the SOLiD (Sequencing by Oligonucleotide Ligation and Detection; Applied BioSystems) platform, with a target recovery of greater than 40 million (transcriptome) and 25 million (exome) uniquely mapping reads per sample. Mutations were considered high confidence if a minimum of 3 individual reads identified the same mutation within the involved specimen and the mutation was not identified in any reads from the remission specimen (minimum 10 reads at that base-pair). Paired exome sequencing of germline and involved marrow specimens identified 129, 297 and 630 high-confidence, non-synonymous, somatic mutations in the three samples. Of the total 1056 mutations, 1023 (96.9%) were missense and the remaining 33 (3.1%) were nonsense mutations. The expected mutation in CRLF2 F232C (#536) was recovered, as was a novel JAK2 mutation from a sample previously thought to be JAK2 wild-type (#002). Comparison between transcriptome and exome sequence markedly reduced the number of potential “driver” alterations, i.e., nonsynonymous coding mutations expressed in the tumor but not present in the remission exome. For example, from the 129 somatic alterations in the involved exome from #536, only 15 were recovered from the involved transcriptome. Sequence alterations affected genes known to be involved in histone modification, oxygen metabolism, and steroid responsiveness. To our knowledge, none of the identified mutations have previously been described. One mutation in a gene involved in E2F transcription was observed in two of three cases. In addition, 2,428 coding sequence mutations were identified from the involved marrow transcriptome of #536 that were not present in the involved marrow exome, possibly indicating a high rate of RNA editing. Additional sequence analysis and molecular epidemiology of the novel sequence alterations in CRLF2-rearranged and —unrearranged B-ALL is underway. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Exome Sequencing Identifies Highly Recurrent Somatic GATA2 and CEBPA Mutations in Acute Erythroid Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1394-1394
Author(s):  
Suning Chen ◽  
Nana Ping ◽  
Jia Yin ◽  
Wenxiu Cheng ◽  
Qinrong Wang ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Acute Lymphoblastic Leukemia ◽  
Exome Sequencing ◽  
Myeloid Leukemia ◽  
Transcriptional Activity ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Next Generation ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Generation Sequencing

Abstract Acute erythroid leukemia (AEL) is a distinct subtype of acute myeloid leukemia (AML) characterized by predominant erythropoiesis. Currently, only few studies using next-generation sequencing were reported in AEL. To decipher the somatic mutation spectrum and discover disease-driving genes responsible for the pathogenesis of AEL, we performed whole exome-sequencing (WES) in 6 AEL and validating using targeted next generation sequencing (NGS) and Sanger sequencing in 58 AEL. From August 2003 to October 2014, a total of 158 patients fulfilling the WHO criteria for AEL were identified, comprising 91 males and 67 females. Median age was 50 years. These patients were further subclassified into 3 groups: 37 AEL after MDS, 108 de novo AEL, and 13 AML with myelodysplasia-related changes. In total, we identified 52 genes with somatic mutations in at least 2 patients, including CEBPA in 4 patients and GATA2 in 2 patients. We identified high frequencies of mutations in CEBPA (40.0%, 22/55; about 1/4 are biallelic mutations), GATA2 (22.4%, 13/58), NPM1 (15.5%, 9/58), SETBP1 (12.1%, 7/58), and U2AF1 (12.1%, 7/58), followed by TP53 (5.2%, 3/58), RUNX1 (3.5%, 2/58), TET2 (3.5%,2/58), ASXL1 (3.5%, 2/58), DNMT3A (3.5%, 2/58), SRSF2 (1.7%, 1/58) and FLT3 (1.7%, 1/58). We did not detect alterations of some of commonly mutated genes associated with AML, including IDH1, IDH2 and RAS. The results are summarized in Figure 1. To further identify the prevalence of GATA2 mutations in hematologic malignancies, we amplified and sequenced the entire coding region of GATA2 gene in 253 non-AEL AML, 40 chronic myeloid leukemia in blast crisis (CML-BC), 55 B-cell acute lymphoblastic leukemia (B-ALL), and 38 T-cell acute lymphoblastic leukemia (T-ALL). We detected GATA2 mutations in 5.5% of non-AEL AML, 15% of CML-BC, and none of B-ALL or T-ALL. The GATA2 mutations in AEL are mainly localized in ZF1 domain (P304H, D309E, A318V/T, G320S/D, L321P, and R330X) and TAD domain (Q20H). To find out the implications of GATA2 mutations in the leukemogenesis of AEL, we overexpressed the mutants of GATA2 (P304H, L321P, and R330X) in 293T cells and demonstrate that GATA2 mutant led to reduced transcriptional activity. Whereas overexpression of GATA2 mutants in mouse myeloid progenitor cell line, 32D, has no effect on the proliferative or colony formation abilities, it caused increased expression of erythroid-related antigen Ter-119 (Figure 2), b-globin and bh1-globin. Furthermore, 32D cells transfected with GATA2 mutants showed increased positivity than control cells by Benzidine staining. Taken together, our findings demonstrate that the mutatome of AEL is different from other types of AML. AEL is associated with a high frequency of mutations in GATA2 and CEBPA. GATA2 mutations resulted in a decrease of transcriptional activity and erythroid development of mouse myeloid progenitors, suggest an important role of GATA2 mutations in AEL. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

scholarly journals Next-Generation Sequencing in Acute Lymphoblastic Leukemia

2019 ◽  
Vol 20 (12) ◽  
pp. 2929 ◽  
Author(s):  
Nicoletta Coccaro ◽  
Luisa Anelli ◽  
Antonella Zagaria ◽  
Giorgina Specchia ◽  
Francesco Albano
Keyword(s):  
Next Generation Sequencing ◽  
Acute Lymphoblastic Leukemia ◽  
Residual Disease ◽  
Age Of Onset ◽  
Lymphoblastic Leukemia ◽  
Genomic Analysis ◽  
Next Generation ◽  
Acute Leukemias ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer and accounts for about a quarter of adult acute leukemias, and features different outcomes depending on the age of onset. Improvements in ALL genomic analysis achieved thanks to the implementation of next-generation sequencing (NGS) have led to the recent discovery of several novel molecular entities and to a deeper understanding of the existing ones. The purpose of our review is to report the most recent discoveries obtained by NGS studies for ALL diagnosis, risk stratification, and treatment planning. We also report the first efforts at NGS use for minimal residual disease (MRD) assessment, and early studies on the application of third generation sequencing in cancer research. Lastly, we consider the need for the integration of NGS analyses in clinical practice for genomic patients profiling from the personalized medicine perspective.

scholarly journals Sensitive Monitoring of BCR-ABL1 Kinase Domain Mutations By Next Generation Sequencing for Optimizing Clinical Decisions in Philadelphia-Positive Acute Lymphoblastic Leukemia in the Graaph-2014 Trial

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1295-1295 ◽  
Author(s):  
Jean-Michel Cayuela ◽  
Francois Lay ◽  
Yves Chalandon ◽  
Philippe Rousselot ◽  
Xavier Thomas ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Acute Lymphoblastic Leukemia ◽  
Limit Of Detection ◽  
Lymphoblastic Leukemia ◽  
Intensive Therapy ◽  
Kinase Domain ◽  
Rapid Expansion ◽  
Next Generation ◽  
Low Level ◽  
Generation Sequencing

A high proportion of Ph-positive Acute Lymphoblastic Leukemia (Ph+ ALL) patients still undergo relapses despite the use of Tyrosine Kinase Inhibitors (TKIs) in addition to chemotherapy as frontline therapy. In this leukemia subtype, the role of BCR-ABL1 kinase domain (KD) mutations as a driver of resistance to TKIs has already been documented by previous studies and such mutations have been reported in up to 80% of the patients at relapse. Next-generation sequencing (NGS) has been proposed to characterize these mutations with a higher sensitivity than Sanger. We report here a prospective study aiming at detecting potentially resistant cell populations by NGS in Ph+ ALL patients enrolled in the GRAAPH 2014-trial. Between March 2016 and February 2019, 156 patients aged 18 to 59 years with newly diagnosed Ph+ and/or BCR-ABL1 positive ALL have been included in the GRAAPH 2014 trial (NCT02619630). BCR-ABL1 isoforms were E1A2 69%, B2A2/B3A2 29%, atypical 2%. After a prephase of steroid, treatment consisted of 4 blocks of chemotherapy + nilotinib. 118 patients (76%) underwent allogeneic or autologous stem cell transplantation (SCT). 22 medullary relapses were recorded within a median time of 9 months (range, 2 - 35). Blood and marrow samples harvested at diagnosis, after each treatment block, before and 3 months after SCT, and at relapse, were sequenced if BCR-ABL1/ABL1 ratio were above 0.001. Mutated BCR-ABL1 transcripts were detected by sequencing the KD of BCR-ABL1 transcripts by NGS with a limit of detection (LOD) of 0.03. T315I allele specific oligonucleotide (ASO) droplet digital RT-PCR (ddRT-PCR) with a LOD of 0.0005 was also performed at diagnosis on a subset of 63 patients, including 5 who have subsequently developed a T315I clone. NGS. At diagnosis, no KD mutation was found by NGS in pretreatment samples of 137 patients. During follow-up (FU), only 12 mutations were found by NGS in 7 out of the 88 patients tested (81, 45, 30, 20, 19, 9 after block 1, 2, 3, 4, before and 3 months after SCT, respectively). Mutations were T315I (N=6), Y253H (N=1), E255K (N=2), E255V (N=1), Q252H (N=1), Y253F (N=1). At relapse, 16 mutations were identified by NGS in 12 patients out of the 17 tested (71%). Mutations were T315I (N=7), Y253H (N=n=3), F359V (N=2), E255K (N=1), E255V (N=1), Q252H (N=1), Y253F (N=1). More than 1 mutated clone were present in 2 patients (E255V+T315I+F359V and Y253H+F359V), and a compound mutation was found in 1 patient (Q252H/Y253F). Out of the 7 patients found mutated during FU, 5 have relapsed with a rapid expansion (1 to 3 months) of the mutated clone. One patient harboring a sub-clonal (10%) E255K at MRD1 has relapsed 9 months later without any detectable mutation. One patient identified with 3 mutated clones (E255K 10%, E255V 10%, T315I 80%) underwent SCT and has not relapsed so far. We failed to anticipate expansion of any mutated clone in the 7 remaining patients found mutated at relapse. T315I ASO ddRT-PCR on diagnostic samples. Low-level T315I mutated BCR-ABL1 transcripts (0.00051 to 0.0013) were detected in 14 out of 63 patients (24%) tested. Only one has expanded a T315I clone later on. In the context of the GRAAPH 2014 trial, 71% of the 17 relapses tested so far were associated with BCR-ABL1 KD mutations. Expansion of the mutated clone could have been characterized before the onset of hematological relapses in only 5 out of 12 patients (42%). Unfortunately in these cases, lags between first detection and relapse were very short (1 to 3 months). On the contrary, occurrences of relapses associated with expansion of KD-mutated clones could not have been anticipated in 58%. All mutations identified, including T315I, F359V, E255K/V and Y253F/H, Q252H/Y253F are known for conferring resistance to nilotinib. NGS is a valuable method for KD mutation detection in Ph+ ALL. It allows a quantitative characterization of KD mutations at relapse. However in our hands and in the context of an intensive therapy combining chemotherapy, nilotinib and SCT, its enhanced sensitivity as compared to Sanger (3% vs 20%) does not translate into the capacity of anticipating expansion of KD-mutated clones. Moreover, in this study, NGS did not detect any mutation in pre-therapeutic samples while T315I mutated BCR-ABL1 transcripts were found at low-level in 24% of these samples by ddRT-PCR. However it should be emphasized that when detected, low-level T315I mutated sub-clones present at diagnosis failed to expand in most instances. Disclosures Cayuela: Incyte: Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau. Chalandon:Pfizer: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Incyte Biosciences: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Rousselot:Pfizer: Research Funding. Thomas:PFIZER: Honoraria; ABBVIE: Honoraria; INCYTE: Honoraria; DAICHI: Honoraria. Huguet:Amgen: Honoraria; Servier: Honoraria; Jazz Pharmaceuticals: Honoraria; BMS: Honoraria; Pfizer: Honoraria; Incyte Biosciences: Honoraria; Novartis: Honoraria. Chevallier:Incyte: Consultancy, Honoraria; Jazz Pharmaceuticals: Honoraria; Daiichi Sankyo: Honoraria. Boissel:NOVARTIS: Consultancy. Vey:Novartis: Consultancy, Honoraria; Janssen: Honoraria. Berthon:PFIZER: Other: DISCLOSURE BOARD; JAZZPHARMACEUTICAL: Other: DISCLOSURE BOARD; CELGEN: Other: DISCLOSURE BOARD.

scholarly journals Capture-Based Next-Generation Sequencing Improves the Identification of Immunoglobulin/T-Cell Receptor Clonal Markers and Gene Mutations in Adult Acute Lymphoblastic Leukemia Patients Lacking Molecular Probes

Cancers ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 1505
Author(s):  
Roberta Cavagna ◽  
Marie L. Guinea Montalvo ◽  
Manuela Tosi ◽  
Michela Paris ◽  
Chiara Pavoni ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
T Cell Receptor ◽  
Lymphoblastic Leukemia ◽  
Cell Receptor ◽  
Molecular Probes ◽  
Next Generation ◽  
Mutational Status ◽  
Generation Sequencing

The monitoring of minimal residual disease (MRD) in Philadelphia-negative acute lymphoblastic leukemia (ALL) requires the identification at diagnosis of immunoglobulin/T-cell receptor (Ig/TCR) rearrangements as clonality markers. Aiming to simplify and possibly improve the patients’ initial screening, we designed a capture-based next-generation sequencing (NGS) panel combining the Ig/TCR rearrangement detection with the profiling of relevant leukemia-related genes. The validation of the assay on well-characterized samples allowed us to identify all the known Ig/TCR rearrangements as well as additional clonalities, including rare rearrangements characterized by uncommon combinations of variable, diversity, and joining (V-D-J) gene segments, oligoclonal rearrangements, and low represented clones. Upon validation, the capture NGS approach allowed us to identify Ig/TCR clonal markers in 87% of a retrospective cohort (MRD-unknown within the Northern Italy Leukemia Group (NILG)-ALL 09/00 clinical trial) and in 83% of newly-diagnosed ALL cases in which conventional method failed, thus proving its prospective applicability. Finally, we identified gene variants in 94.7% of patients analyzed for mutational status with the same implemented capture assay. The prospective application of this technology could simplify clonality assessment and improve standard assay development for leukemia monitoring, as well as provide information about the mutational status of selected leukemia-related genes, potentially representing new prognostic elements, MRD markers, and targets for specific therapies.

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