Development and validation of a comprehensive genomic diagnostic tool for myeloid malignancies.
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.
Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom; Department of Haematology, Haemato-oncology Diagnostic Service, Cambridge University Hospitals National Health Service Trust, Cambridge, United Kingdom; Department of Haematology, University of Cambridge, Cambridge, United Kingdom;
Thomas
T
McKerrell
Instituto de Biomedicina y Biotecnología de Cantabria, Cantabria, Spain;
Thaidy
T
Moreno
Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom;
Hannes
H
Ponstingl
Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom; Division of Haematology, Fondazione IRCCS Istituto Nazionale dei Tumori, University of Milan, Milan, Italy;
Niccolo
N
Bolli
Cancer Molecular Diagnosis Laboratory, National Institute for Health Research Cambridge Biomedical Research Centre, Cambridge, United Kingdom;
João M L
JM
Dias
Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom;
German
G
Tischler
Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom; National Research Council, Institute of Genetics and Biophysics, Naples, Italy;
Vincenza
V
Colonna
Department of Haematology, Haemato-oncology Diagnostic Service, Cambridge University Hospitals National Health Service Trust, Cambridge, United Kingdom;
Bridget
B
Manasse
Department of Haematology, Haemato-oncology Diagnostic Service, Cambridge University Hospitals National Health Service Trust, Cambridge, United Kingdom;
Anthony
A
Bench
Department of Haematology, Haemato-oncology Diagnostic Service, Cambridge University Hospitals National Health Service Trust, Cambridge, United Kingdom;
David
D
Bloxham
Agilent Technologies, Cheadle, United Kingdom;
Bram
B
Herman
Agilent Technologies, Cheadle, United Kingdom;
Danielle
D
Fletcher
Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom;
Naomi
N
Park
Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom;
Michael A
MA
Quail
Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom; Department of Haematology, University of Cambridge, Cambridge, United Kingdom;
Nicla
N
Manes
Department of Haematology, University of Cambridge, Cambridge, United Kingdom;
Clare
C
Hodkinson
Department of Haematology, University of Cambridge, Cambridge, United Kingdom;
Joanna
J
Baxter
Department of Haematology, Hospital de la Santa Creu I Sant Pau, Barcelona, Spain;
Jorge
J
Sierra
Department of Haematology, Haemato-oncology Diagnostic Service, Cambridge University Hospitals National Health Service Trust, Cambridge, United Kingdom;
Theodora
T
Foukaneli
Department of Haematology, Haemato-oncology Diagnostic Service, Cambridge University Hospitals National Health Service Trust, Cambridge, United Kingdom; Department of Haematology, University of Cambridge, Cambridge, United Kingdom; Cambridge Institute for Medical Research, Cambridge, United Kingdom; Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge, United Kingdom;
Alan J
AJ
Warren
The Center for the Study of Haematological Malignancies, Nicosia, Cyprus; The Karaiskakio Foundation, Nicosia, Cyprus;
Jianxiang
J
Chi
The Center for the Study of Haematological Malignancies, Nicosia, Cyprus; The Karaiskakio Foundation, Nicosia, Cyprus;
Paul
P
Costeas
Department of Medicine II, Klinikum Rechts der Isar, Technische Universität München, Munich, Germany; German Cancer Consortium, German Cancer Research Center, Heidelberg, Germany;
Roland
R
Rad
Department of Haematology, Haemato-oncology Diagnostic Service, Cambridge University Hospitals National Health Service Trust, Cambridge, United Kingdom; Department of Haematology, University of Cambridge, Cambridge, United Kingdom; Cambridge Institute for Medical Research, Cambridge, United Kingdom; Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge, United Kingdom;
Brian
B
Huntly
Department of Haematology, PathWest/Sir Charles Gairdner Hospital, Perth, Australia; and Pathology and Laboratory Medicine, University of Western Australia, Perth, Australia.
Carolyn
C
Grove
Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom;
Zemin
Z
Ning
Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom;
Chris
C
Tyler-Smith
Instituto de Biomedicina y Biotecnología de Cantabria, Cantabria, Spain;
Ignacio
I
Varela
Department of Haematology, Haemato-oncology Diagnostic Service, Cambridge University Hospitals National Health Service Trust, Cambridge, United Kingdom; Department of Haematology, University of Cambridge, Cambridge, United Kingdom;
Mike
M
Scott
Department of Haematology, Hospital de la Santa Creu I Sant Pau, Barcelona, Spain;
Josep
J
Nomdedeu
Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom;
Ville
V
Mustonen
Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom; Department of Haematology, Haemato-oncology Diagnostic Service, Cambridge University Hospitals National Health Service Trust, Cambridge, United Kingdom; Department of Haematology, University of Cambridge, Cambridge, United Kingdom;
George S
0000-0003-4337-8022
GS
Vassiliou
D002352:Carrier Proteins / Q000235:genetics; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics; D000090205:DNA Methyltransferase 3A; D005260:Female; D000080865:Formins; D023281:Genomics / Q000379:methods*; D019337:Hematologic Neoplasms / Q000175:diagnosis / Q000235:genetics; D011495:Histone-Lysine N-Methyltransferase / Q000235:genetics; D006801:Humans; D007951:Leukemia, Myeloid / Q000175:diagnosis / Q000235:genetics; D008297:Male; D009154:Mutation; D009190:Myelodysplastic Syndromes / Q000175:diagnosis / Q000235:genetics; D051788:Myeloid-Lymphoid Leukemia Protein / Q000235:genetics; D015514:Oncogene Proteins, Fusion / Q000235:genetics; D051941:fms-Like Tyrosine Kinase 3 / Q000235:genetics
D002352:Carrier Proteins; C491462:DIAPH2 protein, human; C000717967:DNMT3A protein, human; D000080865:Formins; C497422:KMT2A protein, human; D015514:Oncogene Proteins, Fusion; D051788:Myeloid-Lymphoid Leukemia Protein; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; D011495:Histone-Lysine N-Methyltransferase; C497970:FLT3 protein, human; D051941:fms-Like Tyrosine Kinase 3