title: Detailed molecular characterisation of acute myeloid leukaemia with a normal karyotype using targeted DNA capture.
abstract: Advances in sequencing technologies are giving unprecedented insights into the spectrum of somatic mutations underlying acute myeloid leukaemia with a normal karyotype (AML-NK). It is clear that the prognosis of individual patients is strongly influenced by the combination of mutations in their leukaemia and that many leukaemias are composed of multiple subclones, with differential susceptibilities to treatment. Here, we describe a method, employing targeted capture coupled with next-generation sequencing and tailored bioinformatic analysis, for the simultaneous study of 24 genes recurrently mutated in AML-NK. Mutational analysis was performed using open source software and an in-house script (Mutation Identification and Analysis Software), which identified dominant clone mutations with 100% specificity. In each of seven cases of AML-NK studied, we identified and verified mutations in 2-4 genes in the main leukaemic clone. Additionally, high sequencing depth enabled us to identify putative subclonal mutations and detect leukaemia-specific mutations in DNA from remission marrow. Finally, we used normalised read depths to detect copy number changes and identified and subsequently verified a tandem duplication of exons 2-9 of MLL and at least one deletion involving PTEN. This methodology reliably detects sequence and copy number mutations, and can thus greatly facilitate the classification, clinical research, diagnosis and management of AML-NK.
authors.affiliation: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK.
authors.forename: N
authors.initials: N
authors.lastname: Conte
authors.forename: I
authors.initials: I
authors.lastname: Varela
authors.forename: C
authors.initials: C
authors.lastname: Grove
authors.forename: N
authors.initials: N
authors.lastname: Manes
authors.forename: K
authors.initials: K
authors.lastname: Yusa
authors.forename: T
authors.initials: T
authors.lastname: Moreno
authors.forename: A
authors.initials: A
authors.lastname: Segonds-Pichon
authors.forename: A
authors.initials: A
authors.lastname: Bench
authors.forename: E
authors.initials: E
authors.lastname: Gudgin
authors.forename: B
authors.initials: B
authors.lastname: Herman
authors.forename: N
authors.initials: N
authors.lastname: Bolli
authors.forename: P
authors.initials: P
authors.lastname: Ellis
authors.forename: D
authors.initials: D
authors.lastname: Haddad
authors.forename: P
authors.initials: P
authors.lastname: Costeas
authors.forename: R
authors.initials: R
authors.lastname: Rad
authors.forename: M
authors.initials: M
authors.lastname: Scott
authors.forename: B
authors.initials: B
authors.lastname: Huntly
authors.forename: A
authors.initials: A
authors.lastname: Bradley
authors.forename: G S
authors.initials: GS
authors.lastname: Vassiliou
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D005091:Exons; D005260:Female; D020440:Gene Duplication; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D059785:Karyotype; D015470:Leukemia, Myeloid, Acute / Q000175:diagnosis* / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D020080:Tandem Repeat Sequences