Unlike TET2 and ASXL1, Co-Occurring DNMT3A Mutations Are Not Associated with Increased Age in JAK2-Mutant Myeloproliferative Neoplasms (MPNs)

MPNs including essential thrombocythemia (ET), polycythemia vera (PV), and myelofibrosis (MF) are characterized by JAK2, CALR, and MPL mutations. Additional mutations outside these driver genes also co-occur. To better understand interactions between phenotypic driver mutations and concomitant mutations, we evaluated patterns of somatic mutation acquisition in 1301 MPN (535 ET, 392 PV, 331 MF, and 43 MPN NOS) patients who underwent clinical next generation sequencing (NGS) at our institution. For this analysis, we focused on a single NGS panel obtained closest to MPN diagnosis.

Median age at MPN diagnosis was 59 years, with MF patients older than ET/PV patients (p<4e-11). 67%, 17%, and 4.5% of patients had mutations in JAK2, CALR, or MPL. The average variant allele fraction (VAF) for JAK2, CALR, and MPL was 48%, 34%, and 40%. 52% of patients had pathogenic mutations in an additional gene (range 0-7), most frequently TET2, DNMT3A, and ASXL1. PV patients were more likely to have a TET2 mutation compared with JAK2-mutated ET patients (42% vs 25%). Mutation combinations that occurred together more frequently than expected (p<1e-3) included DNMTA-TET2, ASXL1-TET2, ASXL1-DNMT3A, and ASXL1-SRSF2. We then investigated how these concomitant mutations interacted with the MPN driver mutation and each other.

JAK2-mutated MPN patients with no concomitant mutations were younger than JAK2-mutated patients with TET2 or ASXL1 mutations. In contrast, age distributions were similar between JAK2-only patients and those with JAK2 and DNMT3A mutations (Fig 1A). This observation held when looking within PV and ET, and whether patients with single or multiple concomitant mutations were considered. Since DNMT3A, TET2 and ASXL1 clonal hematopoiesis is associated with increasing age, the similar age distribution of JAK2-only and JAK2-DNMT3A co-mutant patients was an unexpected finding, and worthy of further study.

We evaluated the fraction of JAK2-mutated patients with a given concomitant mutation as a function of age. The fraction of patients with TET2 and ASXL1 mutations increased with age, but not the fraction of patients with DNMT3A mutations (95% CI of slope: 4e-3 to 7e-3 for TET2, 0 to 3e-3 for DNMT3A). Similarly, when we evaluated the fraction of JAK2-mutated patients with a given concomitant mutation as a function of JAK2 VAF, the fraction of patients with TET2 and ASXL1 mutations increased as JAK2 VAF increased, but the fraction of patients with DNMT3A mutations decreased (Fig 1B; 95% CI of slope: 1e-3 to 3e-3 for TET2, -2e-3 to 0 for DNMT3A). One potential explanation for this is that JAK2 and DNMT3A mutations occur as independent clonal acquisitions and that the JAK2-mutant clone “out-competes” the DNMT3A-mutant clone over time. At JAK2 VAFs of 50% and 100%, the ratio of TET2:JAK2 VAFs clustered at 1.0 and 0.5, suggesting frequent presence of a dominant JAK2-TET2 clone in all cells with heterozygous or homozygous JAK2 mutations. ASXL1 mutations occurred mostly in patients with JAK2 VAF > 50%, consistent with it being a subclonal acquisition. In contrast, DNMT3A concomitant mutations were more likely to occur in patients with JAK2 VAF<50% vs >50%, again suggesting that DNMT3A clones diminish as JAK2 clones expand. 6/45 DNMT3A mutations that occurred as the sole concomitant mutation were R882 hotspot mutations. However, DNMT3A was more likely to co-occur at JAK2 VAF>50% in patients with >1 concomitant mutation (most commonly TET2). This suggests that in the context of JAK2-mutant driven MPNs, DNMT3A subclones may require the presence of an additional mutation to expand.

In summary, we found DNMT3A mutations behave differently from TET2 and ASXL1 mutations in JAK2-mutant MPNs. Patients with JAK2-mutant MPN are less likely to have DNMT3A mutations as JAK2 VAF increases. Compared with TET2 or ASXL1, DNMT3A appears more likely to exist as an independent clone in JAK2-mutant MPN, with our data suggesting the JAK2-mutant clone exhibits increased fitness compared to the DNMT3A-mutant clone over time. However, DNMT3A mutations persist when accompanied by other concomitant mutations (particularly TET2).

These results raise interesting questions regarding clonal competition in MPN. Since all our analyses were performed from single clinical NGS panels, our findings require additional validation, including with multi-gene single-cell genotyping, which we are currently pursuing.