Characterization of gene mutations and copy number changes in acute myeloid leukemia using a rapid target enrichment protocol

Bolli, Niccolò; Manes, Nicla; McKerrell, Thomas; Chi, Jianxiang; Park, Naomi; Gundem, Gunes; Quail, Michael A.; Sathiaseelan, Vijitha; Herman, Bram; Crawley, Charles; Craig, Jenny I. O.; Conte, Nathalie; Grove, Carolyn; Papaemmanuil, Elli; Campbell, Peter J.; Varela, Ignacio; Costeas, Paul; Vassiliou, George S.

doi:10.3324/haematol.2014.113381

Cited by 43 publications

(37 citation statements)

References 34 publications

(41 reference statements)

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“…Moreover, increased discovery of clinically important mutations and structural variations not detectable by cytogenetics, FISH, or small gene panels (such as copynumber changes, amplifications, deletions, and gene fusions) begets the need for means to comprehensively evaluate molecular alterations of a variety of types in clinical practice. To this end, a number of targeted DNA-sequencing [174][175][176][177] and combined DNA/RNA-sequencing 178,179 panels evaluating recurrently altered genes across hematopoietic malignancies have been described, some of which are commercially available (reviewed recently by Kuo and Dong, 180 Meldrum et al, 181 and Kanagal-Shamanna et al 182 ) and allow use of formalin-fixed paraffinembedded specimens. Further improvements in next-generation sequencing technologies (reviewed by Sheikine et al 183 ) are expected to allow evaluation of mutations across the entire coding regions of hundreds to thousands of genes while also providing information on copy-number status and gene fusions in a clinically relevant timeframe.…”

Section: Discussionmentioning

confidence: 99%

Diagnosis and classification of hematologic malignancies on the basis of genetics

Taylor¹,

Xiao²,

Abdel-Wahab

2017

Blood

188

149

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Genomic analysis has greatly influenced the diagnosis and clinical management of patients affected by diverse forms of hematologic malignancies. Here, we review how genetic alterations define subclasses of patients with acute leukemias, myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPNs), non-Hodgkin lymphomas, and classical Hodgkin lymphoma. These include new subtypes of acute myeloid leukemia defined by mutations in RUNX1 or BCR-ABL1 translocations as well as a constellation of somatic structural DNA alterations in acute lymphoblastic leukemia. Among patients with MDS, detection of mutations in SF3B1 define a subgroup of patients with the ring sideroblast form of MDS and a favorable prognosis. For patients with MPNs, detection of the BCR-ABL1 fusion delineates chronic myeloid leukemia from classic BCR-ABL1− MPNs, which are largely defined by mutations in JAK2, CALR, or MPL. In the B-cell lymphomas, detection of characteristic rearrangements involving MYC in Burkitt lymphoma, BCL2 in follicular lymphoma, and MYC/BCL2/BCL6 in high-grade B-cell lymphomas are essential for diagnosis. In T-cell lymphomas, anaplastic large-cell lymphoma is defined by mutually exclusive rearrangements of ALK, DUSP22/IRF4, and TP63. Genetic alterations affecting TP53 and the mutational status of the immunoglobulin heavy-chain variable region are important in clinical management of chronic lymphocytic leukemia. Additionally, detection of BRAFV600E mutations is helpful in the diagnosis of classical hairy cell leukemia and a number of histiocytic neoplasms. Numerous additional examples provided here demonstrate how clinical evaluation of genomic alterations have refined classification of myeloid neoplasms and major forms of lymphomas arising from B, T, or natural killer cells.

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Section: Discussionmentioning

confidence: 99%

Diagnosis and classification of hematologic malignancies on the basis of genetics

Taylor¹,

Xiao²,

Abdel-Wahab

2017

Blood

188

149

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“…Similar findings were recently made for targeted re-sequencing in acute myeloid leukemia, and it was shown that increased coverage could improve the identification of copy number alterations in Haloplex assays. 27 Haloplex analyses should not be preferred over array comparative genomic hybridization or multiplex ligation probe amplification techniques for the detection of copy number variations, especially for the detection of duplications.…”

Section: Discussionmentioning

confidence: 99%

“…27 To this end, we normalized the coverage of all genes against the on-target mapped nucleotides across samples. First we looked at X chromosome genes (MTMR8, KDM6A, PHF6, MAGEC3, RPL10 and USP9X).…”

Section: Design a Design B (N=80 T-all Cases) (N=75 T-all Samples)mentioning

confidence: 99%

Targeted sequencing identifies associations between IL7R-JAK mutations and epigenetic modulators in T-cell acute lymphoblastic leukemia

et al. 2015

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JAK signaling pathway members in 27.7% of T-ALL samples screened; an observation with therapeutic potential. Statistically significant pairwise associations were found between different mutations, indicating the presence of functional interactions among different pathways in T-ALL pathogenesis. Of significance, we found a mutually exclusive relationship between IL7R-JAK mutations and the presence of TAL1/LMO2 rearrangements. We also identified positive correlations among IL7R-JAK mutations and mutations/deletions in PHF6 and members of the PRC2 complex. Our findings begin to unravel the diversity of genetic lesions that are implicated in the development of T-ALL. Methods DNA samplesT-ALL samples from patients (n=155: 111 children, 44 adults) were collected from various institutions (Online Supplementary Table S1). The diagnosis of T-ALL was based on morphology, cytochemistry and immunophenotyping according to World Health Organization criteria. Genomic DNA was isolated from bone marrow (either fixed or fresh bone marrow cells). 5,21,22 To investigate the prognostic relevance of IL7R, JAK1 and JAK3 mutations, Sanger sequencing was used to screen for mutations in these three genes in an independent cohort of 78 T-ALL patients. Those patients were all enrolled into the United Kingdom (UK) Children's Cancer and Leukaemia Group (CCLG) ALL2003 trial. 23This study was approved by the ethics committees of the institutes involved and informed consent was obtained from the participants. Samples and clinical data were stored in accordance with the declaration of Helsinki. Capture designSureDesign software was used to design two slightly different Haloplex capture assays (Table 1). The total amplicon number for design A was 23,127 with a region size of 472.006 kbp and a predicted target coverage of >99%. For design B the total amplicon number was 19,694 with a region size of 418.373 kbp and a predicted target coverage of >99%. For this study, 80 samples were processed with design A and 75 samples with design B. In both assays, the coding exons of selected genes (based on RefSeq, CCDS and VEGA databases) were targeted with an extra ten bases upstream and downstream. Targeted regions comprised the coding sequence of genes that were either recently identified as recurrently mutated in ALL or other hematologic malignancies (known driver genes) or were similar to known oncogenes (candidate driver genes) to be sequenced. 18,19,24,25 For statistical analyses we only considered the 115 genes that were sequenced in both Haloplex designs (Online Supplementary Table S2). Library preparation and sequencing were performed as described in the Online Supplementary Material. Data analysesIn NextGENe software (v2.2.1, Softgenetics, State College, PA, USA), we performed the following steps: (i) the fastQ output file was converted into a FASTA file to eliminate reads that were not "paired" and that did not meet the criteria of the default settings; (ii) reads from the converted unique FASTA file were aligned to the reference genome (...

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“…[20][21][22] but difficult to identify reliably using conventional short-read NGS data. 1,2,5 To address this, we developed F-TAFI, a novel bio-informatic tool that uses a de novo graph-based assembly-like approach to identify sequence "loops" within FLT3 exons 14 and 15, and this detected all 12 cases of FLT3-ITDs in our samples without false positives among 171 FLT3-ITD-negative samples (Figure 2; supplemental Methods). MLL-PTD is also associated with an adverse prognosis [23][24][25] and cannot be detected by standard mutational callers, because it does not change the exonic nt sequence.…”

Section: Sequencing (Hiseq -75bp Pe)mentioning

confidence: 99%

“…[1][2][3][4] However, traditional diagnostic approaches such as karyotyping and fluorescence in situ hybridization (FISH), also remain critical to the complete characterization of AML and a number of important mutations such as internal tandem duplications (ITD) of Fms-like tyrosine kinase 3 (FLT3) (FLT3-ITD) and partial tandem duplications (PTD) of mixed-lineage leukemia (MLL) (MLL-PTD) genes are difficult to detect using conventional next-generation sequencing (NGS)-based approaches. 1,5 Furthermore, copy neutral loss-of-heterozygosity (CN-LOH) events, a frequent and prognostically significant class of mutations in AML, [6][7][8] are not detectable by mainstream diagnostic platforms.…”

Section: Introductionmentioning

confidence: 99%

Development and validation of a comprehensive genomic diagnostic tool for myeloid malignancies

McKerrell

Moreno

Ponstingl

et al. 2016

Blood

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Key Points• We develop and validate Karyogene, a comprehensive one-stop diagnostic platform for the genomic analysis of myeloid malignancies.• Karyogene simultaneously detects substitutions, insertions/deletions, translocations, copy number and zygosity changes in a single assay.The diagnosis of hematologic malignancies relies on multidisciplinary workflows involving morphology, flow cytometry, cytogenetic, and molecular genetic analyses. Advances in cancer genomics have identified numerous recurrent mutations with clear prognostic and/or therapeutic significance to different cancers. In myeloid malignancies, there is a clinical imperative to test for such mutations in mainstream diagnosis; however, progress toward this has been slow and piecemeal. Here we describe Karyogene, an integrated targeted resequencing/analytical platform that detects nucleotide substitutions, insertions/deletions, chromosomal translocations, copy number abnormalities, and zygosity changes in a single assay. We validate the approach against 62 acute myeloid leukemia, 50 myelodysplastic syndrome, and 40 blood DNA samples from individuals without evidence of clonal blood disorders. We demonstrate robust detection of sequence changes in 49 genes, including difficult-to-detect mutations such as FLT3 internal-tandem and mixed-lineage leukemia (MLL) partial-tandem duplications, and clinically significant chromosomal rearrangements including MLL translocations to known and unknown partners, identifying the novel fusion gene MLL-DIAPH2 in the process. Additionally, we identify most significant chromosomal gains and losses, and several copy neutral loss-of-heterozygosity mutations at a genome-wide level, including previously unreported changes such as homozygosity for DNMT3A R882 mutations. Karyogene represents a dependable genomic diagnosis platform for translational research and for the clinical management of myeloid malignancies, which can be readily adapted for use in other cancers. (Blood. 2016;128(1):e1-e9)

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Characterization of gene mutations and copy number changes in acute myeloid leukemia using a rapid target enrichment protocol

Cited by 43 publications

References 34 publications

Diagnosis and classification of hematologic malignancies on the basis of genetics

Diagnosis and classification of hematologic malignancies on the basis of genetics

Targeted sequencing identifies associations between IL7R-JAK mutations and epigenetic modulators in T-cell acute lymphoblastic leukemia

Development and validation of a comprehensive genomic diagnostic tool for myeloid malignancies

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