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

Abstract

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)

Molecular synergy underlies the co-occurrence patterns and phenotype of NPM1-mutant acute myeloid leukemia

“…In human AML, copy-neutral LOH is common for FLT3-ITD, but less so for mutant NRAS; for example, in a recent study we identified only one such LOH event among 13 RAS-mutant human AMLs. 13 Nevertheless, in keeping with our findings, studies using the Nras G12D/1 model, in combination with retroviral insertional mutagenesis, resulted in high-penetrance AML with frequent LOH for Nras-G12D when combined with overexpression of oncogenes such as Evi1. 6,39 The different incidence of LOH for mutant RAS between murine and human AML may operate through the fact that, in comparison with the acquisition of other oncogenic mutations (eg, Idh1-R132Q in our study), LOH for Nras-G12D may be more expedient in mice given the large numbers of Npm1 cA/1 /Nras G12D/1 preleukemic HSCs.…”

“…Validation of mutations was performed using MiSeq sequencing (Illumina) of amplicon libraries, as was previously described (see supplemental Methods, supplemental Figure 1, and supplemental Tables 6 and 7 for primer sequences). 12,13 Full details of analysis are provided in the supplemental Methods.…”

Integration of Technical, Bioinformatic, and Variant Assessment Approaches in the Validation of a Targeted Next-Generation Sequencing Panel for Myeloid Malignancies

“…20, [35][36][37] These studies have shown the range of application of NGS in the context of molecular diagnostics for hematologic malignancies; here, we add to this body of work by describing, in detail, key considerations around the integration of wet-bench, data analysis, and variant assessment approaches, which are all required for successful test implementation. A comprehensive validation approach needs to establish both test performance characteristics and test limitations.…”

Prognostic tumor sequencing panels frequently identify germ line variants associated with hereditary hematopoietic malignancies

“…4,5 These panels may identify variants in genes associated with HHMs, 4,6 but tumor-only sequencing cannot differentiate between acquired and germ line variants. We hypothesized that we could identify some patients with HHMs using tumor-only NGS panels, and we determined the frequency at which this occurs.…”