24. Methylation sequencing enhances interpretation of clonal hematopoiesis dynamics

Somatic mutations in hematopoietic stem cells give rise to clonal hematopoiesis (CH), a pre-malignant state that precedes hematologic malignancy. In current clinical practice, CH clone size as quantified by the variant allele fraction (VAF) is serially monitored with DNA sequencing. Increases in VAF are often interpreted as portending progression to malignancy. CH leads to a myeloid bias and VAF measurements can be confounded by cell-type proportions, which also vary according to immune demands. We developed a targeted enzymatic DNA methylation sequencing assay that costs ∼$80/sample (including reagents, library preparation and sequencing) and captures ∼4 million CpGs and applied it to 91 samples from patients with CH. We used the resulting methylation data to infer cell-type proportions. We found that predicted cell-type proportions for lymphocytes and granulocytes correlated highly with complete blood cell counts (R^2 = 0.84 and p-value = 2.65 × 10^-14; R2 = 0.88 and p-value = 4.31 × 10^-16), but predictions for monocytes were much less correlated (R^2 = 0.26, p-value = 2.04 × 10^-3). Furthermore, we observed that as monocyte proportion increased, so did reported percent change in VAF. Correlation was highest for clones driven by mutations in TET2, which have been shown to have more extreme degrees of myeloid bias. This work raises concerns about current methods of monitoring CH based solely on VAF. Given the low cost, cell-type proportion prediction from DNA methylation is a feasible addition to CH assays. Our work suggests that cell-type proportions would provide vital context for accurate interpretation of VAF throughout hematologic malignancy progression and treatment.