21 research outputs found
Development and validation of a comprehensive genomic diagnostic tool for myeloid malignancies
No full textet al.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 mainstreamdiagnosis; 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.Wedemonstrate 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.This study was supported by a Wellcome Trust Clinician Scientist Fellowship (100678/Z/12/Z) (T. McKerrell), the Wellcome Trust Sanger Institute (WT098051), and an educational grant from Celgene (ref: 51261). G.S.V. is funded by a Wellcome Trust Senior Fellowship in Clinical Science (WT095663MA), and work in his laboratory is also funded by Bloodwise and the Kay Kendall Leukaemia Fund. A.J.W. is supported by a Specialist Programme from Bloodwise (12048) and by the Medical Research Council (MC_U105161083). I.V. is funded by the Spanish Ministerio de Economía y Competitividad subprograma Ramón y Cajal.Peer Reviewe
JAK2 V617F hematopoietic clones are present several years prior to MPN diagnosis and follow different expansion kinetics
Get PDFMcKerrell, Thomas et al.The JAK2 V617F mutation is the most common somatic mutation in the classical myeloproliferative neoplasms (MPNs), present in >95% of cases of polycythemia vera (PV) and ~50% of essential thrombocythemia (ET) and myelofibrosis (MF).1-4 It is usually the sole identifiable driver mutation in MPNs5 and was recently also identified as a driver of age-related clonal hemopoiesis in healthy individuals.6-9 In order to investigate the preclinical clonal evolution of MPNs, we identified 12 individuals with a JAK2 V617F mutant MPN, who 4.6 to 15.2 years previously (median 10.2 years) had also donated blood to register with the Cyprus Bone Marrow Donor Registry at the Karaiskakio FoundationThis work was supported by the Wellcome Trust Sanger Institute (WT098051). T. McKerrell is funded by a Wellcome Trust Clinician Scientist Fellowship (100678/Z/12/Z). G.S.V. is funded by a Wellcome Trust Senior Fellowship in Clinical Science (WT095663MA), and work in his laboratory is also funded by Cancer Research UK,
Bloodwise, the Kay Kendall Leukaemia Fund, and Celgene. I.V. is supported by the Spanish Ministerio de Economía y Competitividad, Programa Ramón y Cajal.Peer Reviewe
The Neutron Activation Analysis of Halaf and 'Ubaid Pottery from Tell Arpachiyah and Tepe Gawra
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R.I.S.C.L: A Holistic Molecular Diagnostic Tool for Myeloid Malignancies
No full textThe genomic landscapes of acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), myeloproliferative disorders (MPD) and other related myeloid malignancies are now amongst the best characterized cancer genomes. These malignancies share most of their somatic driver mutations, many of which have therapeutic and prognostic significance (Patel et al, NEJM 2012). Patient prognostication and clinical decision-making can be greatly facilitated by testing for these mutations in parallel with established diagnostic assays. Here, we describe and validate RISCL (Rearrangements, Indels, Substitutions, Copy number and Loss-of-heterozygosity), a novel methodological and bioinformatic tool for the molecular diagnosis of myeloid malignancies. This tool employs targeted DNA capture to simultaneously: 1) identify coding mutations in 49 genes, 2) detect the four most important translocations in AML and 3) derive genome-wide copy number and zygosity data.
Samples & methods
1. Samples
Genomic DNA was extracted from bone marrow samples of 62 patients with AML (n=86 samples, including 24 remission samples) and 68 patients with MDS; and from blood granulocytes and mononuclear cells from 5 cord blood samples and 18 adults with normal hematopoiesis.
2. cRNA baits and sequencing
The bait library (Agilent) contained 53,613 probes to capture: 1) all exons from 49 genes 2) intronic breakpoint sites for PML-RARA, CBFb-MYH11 and RUNX1-RUNX1T1 and MLL breakpoints 3) 9958 SNPs (minor allele frequency 0.40-0.45) for genome wide copy number and zygosity analysis. Barcoded sequencing was performed using Illumina HiSeq 2000 (100bp paired-end).
3. Bioinformatic analysis
We used bespoke bioinformatics for detecting coding substitutions and indels (MIDAS; Conte et al, Leukemia 2013), chromosomal translocations (SMALT-FIT), copy number analysis (Avadis software) and detection of specific mutations such as MLL-PTD and FLT3-ITD (in-house scripts).
4. Verification of results
To validate the sensitivity and specificity of our approach, we compared our findings to conventional diagnostic data and are also validating 30% of randomly selected variants.
Results
A mean of 94% of targeted bases were covered at least by 30x. In AML samples, the four most common coding mutations identified affected NPM1 (n=10), CEBPA (n=8), IDH1 (n=8) and NRAS (n=7). By comparison to conventional diagnostics, we detected 5/5 IDH1R132, 4/4 CEBPA, 1/1 IDH2R172K and 8/9 NPM1 mutations. In MDS samples, the top four mutations affected TP53 (n=15), TET2 (n=13), SRSF2 (n=9), ASXL1 (n=8) and mutations affecting spliceosome genes (n=18) that were mutually exclusive, as previously described (Yoshida et al, Nature 2011).
RISCL detected 100% of known translocations (28/28) in AML patients, namely CBFb-MYH11 (n=8/8), PML-RARA (n=9/9), RUNX1-RUNX1T1 (n=4/4) and rearrangements of MLL (n=7/7). In every case of MLL rearrangement the gene partner was identified and in one case with t(X;11) we identified a novel gene partner to MLL, DIAPH2. Furthermore, we identified one patient with an MLL rearrangement not identified at diagnosis.
Copy number analysis efficiently detected known large chromosomal deletions or monosomies in chromosome 5 (18/18) and 7 (10/12). Overall 47/54 large deletions were detected using Avadis software. Furthermore in one MDS patient we were able to detect a submicroscopic heterozygous deletion in chromosome 4 which included TET2. However this method was much less sensitive for detecting trisomies (13/27 trisomies detected overall). The reasons for this disparity between detection of deletions and amplifications using a standardized depth of coverage algorithm are unclear, but may include subclonal mutations, selection of karyotypically abnormal cells during metaphase preparation or limitations of our bioinformatic analysis, which we are currently investigating.
Figure 1 shows an example of the results of our holistic analysis using RISCL from an informative case of AML.
In summary we describe RISCL, a novel powerful holistic NGS tool for detailed characterization of myeloid malignancies that can be used for patient stratification and a personalized approach to malignancy in the molecular era. The same approach can be extended to other malignancies
Leukemia-associated somatic mutations drive distinct patterns of age-related clonal hemopoiesis
Get PDFOpen Access funded by Wellcome Trust. Under a Creative Commons license.-- Understanding Society Scientific Group: et al.Clonal hemopoiesis driven by leukemia-associated gene mutations can occur without evidence of a blood disorder. To investigate this phenomenon, we interrogated 15 mutation hot spots in blood DNA from 4,219 individuals using ultra-deep sequencing. Using only the hot spots studied, we identified clonal hemopoiesis in 0.8% of individuals under 60, rising to 19.5% of those ≥90 years, thus predicting that clonal hemopoiesis is much more prevalent than previously realized. DNMT3A-R882 mutations were most common and, although their prevalence increased with age, were found in individuals as young as 25 years. By contrast, mutations affecting spliceosome genes SF3B1 and SRSF2, closely associated with the myelodysplastic syndromes, were identified only in those aged 70 years, with several individuals harboring more than one such mutation. This indicates that spliceosome gene mutations drive clonal expansion under selection pressures particular to the aging hemopoietic system and explains the high incidence of clonal disorders associated with these mutations in advanced old age.This project was funded by a Wellcome Trust Clinician Scientist Fellowship (100678/Z/12/Z; to T. McKerrell) and by the Wellcome Trust Sanger Institute (grant number WT098051). G.S.V. is funded by a Wellcome Trust Senior Fellowship in Clinical Science (WT095663MA), and work in his laboratory is also funded by Leukaemia Lymphoma Research and the Kay Kendal Leukaemia Fund. I.V. is funded by Spanish Ministerio de Economía y Competitividad subprograma Ramón y Cajal. C.S.G. is funded by a Leukaemia Lymphoma Research Clinical Research Training Fellowship. We thank Servicio Santander Supercomputación for their support. We acknowledge use of DNA from The UK Blood Services Collection of Common Controls (UKBS collection), funded by the Wellcome Trust grant 076113/C/04/Z, by the Juvenile Diabetes Research Foundation grant WT061858, and by the National Institute of Health Research of England.Peer Reviewe
Characterization of gene mutations and copy number changes in acute myeloid leukemia using a rapid target enrichment protocol
Get PDFPrognostic stratification is critical for making therapeutic decisions and maximizing survival of patients with acute myeloid leukemia. Advances in the genomics of acute myeloid leukemia have identified several recurrent gene mutations whose prognostic impact is being deciphered. We used HaloPlex target enrichment and Illumina-based next generation sequencing to study 24 recurrently mutated genes in 42 samples of acute myeloid leukemia with a normal karyotype. Read depth varied between and within genes for the same sample, but was predictable and highly consistent across samples. Consequently, we were able to detect copy number changes, such as an interstitial deletion of BCOR, three MLL partial tandem duplications, and a novel KRAS amplification. With regards to coding mutations, we identified likely oncogenic variants in 41 of 42 samples. NPM1 mutations were the most frequent, followed by FLT3, DNMT3A and TET2. NPM1 and FLT3 indels were reported with good efficiency. We also showed that DNMT3A mutations can persist post-chemotherapy and in 2 cases studied at diagnosis and relapse, we were able to delineate the dynamics of tumor evolution and give insights into order of acquisition of variants. HaloPlex is a quick and reliable target enrichment method that can aid diagnosis and prognostic stratification of acute myeloid leukemia patients.This project was funded by the Wellcome Trust. NB is a fellow of the European
Hematology Association and was supported by the Academy of Medical Sciences.
EP is a European Hematology Association Advanced Research Fellow. GV is a
Wellcome Trust Senior Fellow in Clinical Science. IV is funded by Spanish
Ministerio de Economía y Competitividad subprograma Ramón y Cajal
Dislocation and relocation: clarsach and live electronics
No full textIn writing The Salutation (clarsach and electroacoustic sound) for harpist Catriona McKay in 2002, I wanted to create something which celebrated the history and music of the instrument itself, using studio technology to create a bridge between idiomatic writing for the instrument and environmental/anecdotal sound, including the voice, which referenced place and time.
This paper will chart and reflect on the collaborative work we have done together since then, describing our methodology for interrogating and relocating the clarsach. It will explore how we consciously play with musical, sonic and conceptual distances in our improvised performances, and examine aspects of this shared ‘performance ecosystem’ (Waters, 2007), which enables us to function in multiple contexts.Publisher PD
Clonal haematopoiesis is not prevalent in survivors of childhood cancer
Get PDFClonal haematopoiesis driven by leukaemia-associated somatic mutations is a common feature of ageing (Link & Walter, 2016). This phenomenon, termed clonal haematopoiesis of indeterminate potential (CHIP), is associated with an increased risk of haematological malignancies and all-cause mortality (Link & Walter, 2016). Recent empirical evidence and computational models suggest that mutation acquisition may not be the major rate-limiting factor in the emergence of CHIP (Altrock et al, 2015; McKerrell et al, 2015; Link & Walter, 2016; Young et al, 2016). Instead, clonal expansion of mutant haematopoietic stem cells (HSCs) probably reflects the interaction between the effects of driver mutations and selection pressures prevailing in the bone marrow microenvironment (Link & Walter, 2016). Notably, cytotoxic therapies have been shown to favour expansion of pre-malignant haematopoietic clones (Link & Walter, 2016). Furthermore, both adult and paediatric cancer patients treated with intensive chemoradiotherapy display an earlier onset of ageing-associated morbidities and an elevated risk of therapy-related myeloid neoplasms (t-MN) and other secondary malignancies (Rowland & Bellizzi, 2014). A recent study in adult cancer patients found that CHIP was more prevalent than in the general population and was strongly associated with t-MN and overall mortality (Gibson et al, 2017). Although CHIP is extremely rare in healthy young individuals, its prevalence and prognostic significance in paediatric cancer patients has not been studied. We therefore performed targeted deep sequencing of peripheral blood DNA from 84 childhood cancer survivors to search for subclonal mutations common in t-MN and adult clonal haematopoiesis. No individuals with somatic variants at these loci were identified. Whilst our findings could be explained by a rarity of driver mutations, the fact that human HSCs accrue somatic variants from the first decade of life (Welch et al, 2012) proposes the alternative possibility that such mutations may not confer clonal advantage in the young.This project was funded by the Wellcome Trust Sanger Institute (grant number WT098051). G.S.V. is funded by a Wellcome Trust Senior Fellowship in Clinical Science (WT095663MA). F.F. is funded by Compagnia di San Paolo Grant: “Le cellule staminali del sangue nei guariti di leucemia” Codice SIME 2013-0958 (codice ROL 4201). I.V is funded by the Spanish Ministerio de Economía y Competitividad, Programa Ramón y Cajal
Niche heterogeneity impacts evolution of myeloproliferative neoplasms driven by the same oncogenic pathway
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