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

Abstract

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.

Figure 1

Outline of the Karyogene workflow. Genomic DNA was processed to capture target loci using RNA baits and sequenced on a HiSeq 2000 sequencer as described in “Methods.” Sequencing data were mapped to the genome and analyzed through the indicated software to detect the corresponding types of mutations. The bait design underpinning these is described in supplemental Figure 1. HD, high definition; PE, paired-end.

Figure 2

Genomic characterization of myeloid malignancies using Karyogene. Individual AML (n = 62) and MDS (n = 50) samples are represented in columns and genetic mutations in rows. AML samples were unselected whereas MDS samples were pre-selected to harbor chromosomal copy number changes. Mutations are grouped into chromosomal translocations (top), substitutions and indels (middle), CNAs (bottom), and CN-LOH events (bottom row). Clinically relevant CNAs are depicted in separate rows and “other large CNAs” refers to changes affecting regions larger than 3 mbp (described in detail in supplemental Figure 3). The presence of mutations in different contexts is indicated according to the key (bottom left). TF, transcription factor.

Figure 3

Example of cloneHD output for an MDS sample. (A) Read depth of genome-wide SNP loci (top) and the posterior probability of copy number state of the inferred clone (bottom) in sample MDS108; with karyotype 47, XY, +8, add (13q)[12]. Chromosomes 1 to 22 and chromosome X (23) are depicted. For chromosome 8 and for 13q, copy number gains reflect the karyotype as does the reduced coverage for X. (B) Genome-wide BAF for MDS108 (top) and posterior probability of the B-allele state of the inferred clone (bottom). The B-allele states of 0/2 at 2p and 11q indicate a loss of heterozygosity in these regions, thus in keeping with CN-LOH in these regions. This region includes the DNMT3A gene and CN-LOH explains the high VAF (0.97) for the R882C mutation that was also detected in MDS108. BAF, B-allele fraction.

Figure 4

Identification of the novel fusion gene MLL-DIAPH2 in an AML sample with t(X;11)(q13;q23). (A) Structure of the MLL (KMT2A) and DIAPH2 genes indicating the DNA breakpoint regions in MLL intron 10 and DIAPH2 intron 4 in this patient with a t(X;11)(q13;q23). (B) Structure of the MLL-DIAPH2 fusion gene verified using PCR amplification and Sanger sequencing of leukemic DNA using primers 1 and 2 (p1 and p2) and cDNA using primers 3 and 4 (p3 and p4). Gel electrophoresis and Sanger sequencing of the PCR product from each experiment are shown delineating translocation breakpoint in DNA sequence (intron 10 of MLL and intron 4 of DIAPH2), and in cDNA (exon 10 of MLL and exon 5 of DIAPH2). (C) Protein structure of MLL, DIAPH2, and (predicted) MLL-DIAPH2 fusion. AT, adenine-thymine hook DNA-binding; BCR, breakpoint cluster region; bkpt, breakpoint; BRD, bromodomain; CXXC, cysteine-X-X-cysteine; DAD, diaphanous autoregulatory domain; FH1-3, formin homology 1-3; FYRC, phenylalanine (F)/tyrosine (Y)-rich C-terminal; GBD, rho GTPase-binding; PHD, plant homeodomain; SET, Su(var)3-9, Enhancer-of-zeste and Trithorax.