Detection of genome-wide copy number variants in myeloid malignancies using next-generation sequencing.

AIMS: Genetic abnormalities, including copy number variants (CNV), copy number neutral loss of heterozygosity (CN-LOH) and gene mutations, underlie the pathogenesis of myeloid malignancies and serve as important diagnostic, prognostic and/or therapeutic markers. Currently, multiple testing strategies are required for comprehensive genetic testing in myeloid malignancies. The aim of this proof-of-principle study was to investigate the feasibility of combining detection of genome-wide large CNVs, CN-LOH and targeted gene mutations into a single assay using next-generation sequencing (NGS). METHODS: For genome-wide CNV detection, we designed a single nucleotide polymorphism (SNP) sequencing backbone with 22 762 SNP regions evenly distributed across the entire genome. For targeted mutation detection, 62 frequently mutated genes in myeloid malignancies were targeted. We combined this SNP sequencing backbone with a targeted mutation panel, and sequenced 9 healthy individuals and 16 patients with myeloid malignancies using NGS. RESULTS: We detected 52 somatic CNVs, 11 instances of CN-LOH and 39 oncogenic mutations in the 16 patients with myeloid malignancies, and none in the 9 healthy individuals. All CNVs and CN-LOH were confirmed by SNP microarray analysis. CONCLUSIONS: We describe a genome-wide SNP sequencing backbone which allows for sensitive detection of genome-wide CNVs and CN-LOH using NGS. This proof-of-principle study has demonstrated that this strategy can provide more comprehensive genetic profiling for patients with myeloid malignancies using a single assay.

Concurrent detection of targeted copy number variants and mutations using a myeloid malignancy next generation sequencing panel allows comprehensive genetic analysis using a single testing strategy.

Currently, comprehensive genetic testing of myeloid malignancies requires multiple testing strategies with high costs. Somatic mutations can be detected by next generation sequencing (NGS) but copy number variants (CNVs) require cytogenetic methods including karyotyping, fluorescence in situ hybidization and microarray. Here, we evaluated a new method for CNV detection using read depth data derived from a targeted NGS mutation panel. In a cohort of 270 samples, we detected pathogenic mutations in 208 samples and targeted CNVs in 68 cases. The most frequent CNVs were 7q deletion including LUC7L2 and EZH2, TP53 deletion, ETV6 deletion, gain of RAD21 on 8q, and 5q deletion, including NSD1 and NPM1. We were also able to detect exon-level duplications, including so-called KMT2A (MLL) partial tandem duplication, in 9 cases. In the 63 cases that were negative for mutations, targeted CNVs were observed in 4 cases. Targeted CNV detection by NGS had very high concordance with single nucleotide polymorphism microarray, the current gold standard. We found that ETV6 deletion was strongly associated with TP53 alterations and 7q deletion was associated with mutations in TP53, KRAS and IDH1. This proof-of-concept study demonstrates the feasibility of using the same NGS data to simultaneously detect both somatic mutations and targeted CNVs.