A total of 136 adult PV patients were recruited with an average age of 61.1 years (52.9% were aged 60 or above) and 51.5% were males (Supplementary Table 1). Of these, 74 patients had a thrombotic event recorded at any time in their medical history (56.1%) and 58 had no thrombotic event (43.9%, information on vascular events was not available from the medical history of 4 patients).
Seventy-seven patients (77/136, 56.1%) presented ≥ 1 additional mutation (i.e. pathogenic non-driver mutation). The most frequently mutated genes were TET2 27.9%, ASXL1 11.8%, and DNMT3A 11% (termed “DTA”; Fig. 1). As expected, patients with DTA mutations were older than those without DTA mutations (65.4 vs. 57.8 years, p = 0.002; Student t-test).
With a median follow-up of 9.4 years, the incidence density of vascular events was 2.83 per 100 person-years. Patients classically stratified as high-risk had shorter thrombosis-free survival as expected, although statistical significance was not reached (Fig. 2A, p = 0.142).
Thirty-seven events (52.9%) were pre-diagnostic (including the cause for diagnosis) and 44 (62.9%) were post-diagnostic; 11 patients had both a pre- and post-diagnostic event. Of the post-diagnostic events with information available (n = 37), 25.7% were venous and 50.0% arterial. The most frequent events were cerebral (30.9%), acute myocardial infarction (25.5%), and portal thrombosis (7.3%).
Comparing the group of PV patients with a thrombotic event at any time in their medical history (n = 74) to those without (n = 58), there was no significant difference in patient age or the VAF of JAK2 mutation, but the group with an event had a higher leukocyte count at diagnosis and more additional mutations (mean 1.1 vs. 0.7, p = 0.004) and DTA mutations (0.53 vs. 0.31, p = 0.001; Supplementary Table 2). Patients with a larger JAK2-mutated clone size (≥ 50% VAF) were not more likely to harbor a DTA mutation (p = 0.858). Patients with a pre-diagnostic event were more likely to have a post-diagnostic event (p = 0.036) and also more likely to have a DTA mutation (p = 0.038).
In univariable analysis, thrombotic event was not associated with age ≥ 60 years or VAF of JAK2 mutation but was associated with the presence of any additional mutation (OR 3.4, p = 0.001) and with any DTA mutation (OR 2.5, p = 0.014). Statistical significance was lost when DTA mutations were not included (non-DTA additional mutation, Supplementary Table 3). The association with any “adverse mutation” (SRSF2, IDH2 or ASXL1, as defined by Tefferi et al. based on impact on overall survival [13]) was marginal (p = 0.064).
DTA mutations were significantly associated with arterial events (OR 4.6, p < 0.001) and marginally with venous events (OR 2.7, p = 0.06). Notably, DTA mutation was predictive of thrombosis-free survival (p = 0.007, Fig. 2B).
Data on cardiovascular risk factors (CVRF) and hypertension was available for 115 patients. Seventy-three of 115 patients (63.5%) were hypertensive. The DTA and CVRF variables are closely related (OR 6.8, p = 0.009), in particular the TET2 mutation with hypertension (p = 0.025). Nevertheless, DTA mutation and additional mutation remained as risk factors for a thrombotic event in hypertensive patients (OR 4.4, p = 0.052; OR 5.8, p = 0.026, respectively; Fisher exact test), with additional mutations losing statistical significance when the DTA genes were removed from the analysis (p = 1.0). The association between thrombotic event and CVRF or hypertension was confirmed in multivariable analysis (OR 3.8, p = 0.030; OR 3.85, p = 0.002, respectively) while the association with DTA mutation was marginal (OR 2.1, p = 0.075; Supplementary Table 4).
To confirm these observations, two homogeneous groups of 47 gender- and age-matched PV patients were formed with (case, n = 47) and without thrombotic events (control, n = 47) in their medical history to exclude selection bias.
In the case-control study, both CVRF and hypertension lost significance in multivariable analysis while DTA mutation was confirmed as a risk factor for thrombotic event (OR 2.9, p = 0.027; Supplementary Table 4), observing a higher number of additional mutations in the group of cases (cases 1.0 vs. controls 0.68, p = 0.026). Importantly, the association between thrombotic event and DTA mutation remained in the group of PV patients aged < 60 years (n = 44; OR 6.67, p = 0.033; Supplementary Table 5) from the case-control study and was predictive for thrombosis-free survival in younger patients (p = 0.024, Supplementary Fig. 1). When all patients in the cohort were dichotimized according to age at diagnosis, the association between thrombosis and DTA mutation was of borderline significance in both age groups (≥ 60 years OR 2.44, p = 0.064; <60 years OR 2.89, p = 0.065). Moreover, when data were dichotimized according to the VAF of mutated JAK2, the association between DTA mutations and thrombosis was significant for the smaller JAK2-mutated clones (VAF < 50%: OR 3.34, CI 1.11–13.67, p = 0.034) and borderline for the larger JAK2-mutated clones (VAF ≥ 50%: OR 3.50, CI 0.82–14.91, p = 0.091).