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.

FIGURE 1

Spectrum of the mutated gene identified in 201 patients with cytopenias. A, The frequency and patterns of 79 recurrent mutated gene identified in 201 cases, which are shown in indicated colors (640-gene NGS panel). B, Distribution of mutations of 41 genes ordered by gene function which are commonly mutated in MDS and AA as a “virtual panel” in 201 cases. Aplastic anemia (AA); myelodysplastic syndrome (MDS)

FIGURE 2

Range plot of the variant allele frequencies of 201 cases with different NGS panels Patients are in columns, sorted by “gold standard” diagnosis. VAFs are shown as dots within columns; a dot at VAF = 0 indicates that the patient had no somatic mutations. The min and max VAFs of the cases are the boundaries of each column. A, Range plot of the VAFs by the 640 gene panel. B, Range plot of the VAFs by the 41 gene panel. VAF, variant allele frequency