Compartment-specific mutational landscape of clonal hematopoiesis

Abstract

Clonal hematopoiesis (CH) is characterized by somatic mutations in blood cells of individuals without hematologic disease. While the mutational landscape of CH in peripheral blood (PB) has been well characterized, detailed analyses addressing its spatial and cellular distribution in the bone marrow (BM) compartment are sparse. We studied CH driver mutations in healthy individuals (n = 261) across different anatomical and cellular compartments. Variant allele frequencies were higher in BM than PB and positively correlated with the number of driver variants, yet remained stable during a median of 12 months of follow-up. In CH carriers undergoing simultaneous bilateral hip replacement, we detected ASXL1-mutant clones in one anatomical location but not the contralateral side, indicating intra-patient spatial heterogeneity. Analyses of lineage involvement in ASXL1-mutated CH showed enriched clonality in BM stem and myeloid progenitor cells, while lymphocytes were particularly involved in individuals carrying the c.1934dupG variant, indicating different ASXL1 mutations may have distinct lineage distribution patterns. Patients with overt myeloid malignancies showed higher mutation numbers and allele frequencies and a shifting mutation landscape, notably characterized by increasing prevalence of DNMT3A codon R882 variants. Collectively, our data provide novel insights into the genetics, evolution, and spatial and lineage-specific BM involvement of CH.

Fig. 1. Comparison of variant allele frequencies (VAF) in CH among BM and PB samples.

A VAF distribution in BM and PB samples. B Pairwise comparison of VAFs of 43 variants in paired BM and PB samples. The blue line represents equal VAFs in both compartments. C Pairwise comparison of VAFs in paired BM and PB samples, according to mutated gene. Left panel: DNMT3A variants (n = 17); middle panel: TET2 variants (n = 12; solid lines) and ASXL1 variants (n = 2; dashed lines); right panel, non-DTA variants (n = 12).

Fig. 2. Variant allele frequencies (VAF) in CH.

A VAF distributions for the five most frequently mutated genes. B Association between number of mutations and highest VAF per individual.

Fig. 3. Spatial heterogeneity of clonal hematopoiesis in the bone marrow compartment as detected by NGS.

Spatial heterogeneity was observed in two individuals (UPN_2 and UPN_248); mutations with significantly different VAFs (p < 0.01) are shown in red.

Fig. 4. Lineage involvement of ASXL1-mutated CH.

A Overall analysis of allelic burden according to the cell fraction of six ASXL1 mutations in five CH individuals. Symbols: *p < 0.05, **p < 0.01, ***p < 0.001 using Friedman test followed by Nemenyi’s multiple comparison test. B Analysis of individual-specific allelic burden within each sorted cell fraction of the five ASXL1-mutated CH individuals.

Fig. 5. Longitudinal analyses of CH clonal evolution.

A total of 31 variants in 21 individuals were analyzed in sequential PB samples for up to 18 months. Solid lines indicate DTA variants, dashed lines indicate non-DTA variants.

Fig. 6. Driver mutation landscape in individuals with CH compared to patients with MDS or sAML.

A Number of mutations per individual in CH, MDS and sAML. B Variant allele frequency distribution in CH, MDS and sAML.

Fig. 7. Intra-genic localization of DNMT3A variants in CH, MDS and sAML.

Green dots indicate missense mutations, black dots indicate InDels, brown dots indicate nonsense mutations. Plots were generated using the MutationMapper tool by Gao et al. [24] and Cerami et al. [25].