Utility of Detecting Rare Clonal Hematopoiesis for Predicting Leukemic Transformation in Healthy Individuals

Abstract Clonal hematopoiesis is a recognized feature of the aging hematopoietic compartment (Busque L, et al., Blood 2009). Recent next-generation sequencing (NGS) studies have identified the epigenetic regulators DNMT3A and TET2 as genes mutated frequently in clonal hematopoiesis (Busque L, et al., Nat Genet 2012; Xie M, et al., Nat Med 2014; Jaiswal S, et al., NEJM 2014; Genovese G, et al. NEJM 2014). While intrinsically benign, these hematopoietic clones-termed clonal hematopoiesis of indeterminate potential (CHIP)-are detected in approximately 10% of individuals 70 years of age and associated with an increased risk of hematological malignancy and all-cause mortality (Jaiswal S, et al., NEJM 2014; Genovese G, et al., NEJM 2014; Steensma DP, et al., Blood 2015). However, these studies could only detect common clones-greater than 0.02 variant allele fraction (VAF)-due to the intrinsic error rate of NGS. To characterize the prevalence and mutation profile of rare hematopoietic clones below this threshold, we developed methods for error-corrected sequencing (ECS) targeting 54 AML-associated genes, which improved the limit of detection by two orders of magnitude. Using ECS, we identified clonal mutations ranging in frequency from 0.0003-0.1451 VAF in peripheral blood samples banked in 10-year intervals from 20 healthy 50-70 year-old participants in the Nurses' Health Study (NHS), a longitudinal study of >100,000 US registered nurses. Unexpectedly, we identified hematopoietic clones in the peripheral blood of 95% of these healthy individuals (Young AL, et al. Nat Commun 2016), redefining physiologic clonal hematopoiesis of aging. Interestingly, DNMT3A and TET2 mutations comprised 64% of the clonal mutations identified. These clonal mutations were stable longitudinally and present in both myeloid and lymphoid compartments, suggesting that they arose in long-lived hematopoietic stem and progenitor cells. While clonal hematopoiesis harboring AML-associated somatic mutations is ubiquitous in healthy adults, the clonal characteristics that predict leukemic transformation are still unknown. To address this question, we conducted a blinded case-control study with NHS and Health Professionals Follow-up Study (HPFS) participants. We compared rare clonal variants in peripheral blood samples collected pre-diagnosis from 34 individuals who later developed de novo AML, and from 69 healthy controls. Controls were matched to cases for gender, age, ethnicity and time of blood collection (1 case: 2 controls). Using ECS, we identified 598 total clonal variants in 100/103 individuals at 0.0002-0.35 VAF. On average, we found 7.4 clonal variants per case and 5.0 clonal variants per control. The most frequent mutations in both cases and controls occurred in DNMT3A and TET2 . In conditional logistic regression analysis, only DNMT3A R882H/R882C mutations (detected in 8 cases, 3 controls) were significantly associated with future AML risk (OR 6.0, 95% CI 1.3-28.9, p=0.02), with a median of 12.5 years (range 1-22 years) from blood draw to case diagnosis. Recurrent variants not significantly associated with future AML risk were DNMT3A I780T (1 case, 3 controls), DNMT3A R326C (1 case, 3 controls), ASXL1 E1183K (4 cases, 1 control) and JAK2 V617F (4 cases, 1 control). These findings further support that clonal hematopoiesis is a ubiquitous phenomenon in healthy individuals and merely detecting low frequency clonal hematopoiesis may not be a sensitive, specific, or timely predictor for developing AML. Only, DNMT3A R882H and R882C mutations were significantly associated with long-term risk for developing AML in this study. Future studies must identify additional factors that improve AML risk prediction. Disclosures No relevant conflicts of interest to declare.