The diagnostic utility of targeted gene panel sequencing in discriminating etiologies of cytopenia

Abstract

The diagnostic utility of somatic mutations in the context of cytopenias is unclear: clonal hematopoiesis can be found in healthy individuals, patients with aplastic anemia (AA), clonal cytopenia of undetermined significance (CCUS) and myelodysplastic syndrome (MDS). We examined a cohort of 207 well-characterized cytopenic patients with a 640-gene next generation sequencing (NGS) panel and compared its diagnostic utility with a "virtual" 41 gene panel. The TET2, SF3B1, ASXL1, and TP53 were the most commonly mutated genes (frequency > 10%). Mutations in the 640-gene panel show high sensitivity (98.3%) but low specificity (47.6%) for diagnosis of MDS. Notably, mutations of splicing factors and genes in the RAS pathway are relatively specific to MDS. Furthermore, high variant allele frequency (VAF) predicts MDS: when the VAF is set at 20%, the positive predictive value (PPV) for MDS is 95.9%, with a specificity of 95.3%. The presence of two or more somatic mutations with ≥10% VAF showed a PPV of 95.2%. While the "virtual" 41-gene panel showed a mild decrease in sensitivity (95.7% vs 98.3%), 100% specificity was observed when either VAF was set at ≥20% (100% vs 95.3%), or two or more somatic mutations had VAFs ≥ 10%. Our study shows targeted gene panel sequencing improves the diagnostic approach and accuracy for unexplained cytopenia, with its high sensitivity and high PPV for MDS when applying VAF cutoffs. Furthermore, a 41-gene panel was shown to have at least comparable performance characteristics to the large 640-gene panel.

The prognostic impact of variant allele frequency (VAF) in TP53 mutant patients with MDS: A systematic review and meta‐analysis

“…The clinical relevance of VAF for the majority of mutations is unknown. Studies have found that VAF aids in the diagnosis of cytopenia, high VAF predicts MDS, with the VAF of gene mutation set at 20%, and the positive predictive value for MDS is 95.9%, with a specificity of 95.3% 27 . More recent studies on the impact of the clonal burden impact of TP53, TET2, DNMT3A, and NPM1 revealed that a high VAF indicates a shorter survival time and is transferred to AML 28 .…”

Cancer spectrum and outcomes in the Mendelian short telomere syndromes

“…As a sensitivity analysis, because the follow-up time for MDS is limited to 2001 and later, the yearly incidence rates for MDS in 2001 were Targeted sequencing for MDS/AML somatic mutations Library preparation We performed somatic mutation analysis on peripheral blood using a custom-designed targeted panel as described. 34 Briefly, DNA hybrid capture libraries were prepared using an Agilent SureSelect-XT target enrichment kit with 200 ng of peripheral blood-derived genomic DNA template as input. The somatic MDS/AML genes queried are listed in supplemental Table 2.…”

CREBBP and STAT6 co-mutation and 16p13 and 1p36 loss define the t(14;18)-negative diffuse variant of follicular lymphoma

“…Using 200 ng of extracted DNA from the above analysis, DNA hybrid capture libraries were prepared using an Agilent SureSelect-XT (Agilent Technologies, Santa Clara, CA) custom-designed target enrichment kit evaluating fullgene sequences of 641 cancer-related genes (see Supplementary Information), as previously described 22 . Following DNA quality control, shearing and library preparation, next generation sequencing was performed on an Illumina HiSeq 2500 (Illumina Biotechnology, San Diego, CA) using 2 × 100 bp Rapid Run v2 paired end chemistry.…”

“…Piccard Tools v1.119 was used for SAM to BAM conversion. The final BAM file was used for variant calling using a custom pipeline MDLVC v.6 22 and HaplotypeCaller v3.3. Variants with low variant allele frequency (VAF) (<5%) and variants found in a reference pool of normal samples were excluded.…”