The poor outcome in high molecular risk, hydroxycarbamide-resistant/intolerant ET is not ameliorated by ruxolitinib

TO THE EDITOR:

Essential thrombocythemia (ET) is a myeloproliferative neoplasm (MPN) defined by thrombocytosis, increased risk of vascular thrombosis,^1,2  hemorrhage,³  and progression to myelofibrosis^4,5  and acute myeloid leukemia.^4,5  Patients are risk-stratified to identify those who might benefit from cytoreduction to reduce the risk of vascular complications.⁶  Resistance/intolerance to hydroxycarbamide (HC-RES/INT), a first-line cytoreductive treatment, develops in 20% of high-risk patients⁷  with increased risk of disease progression and reduced survival.⁸  New approaches are needed to predict disease-transformation risk in these patients, together with development of therapies that reduce this risk.

Following the discovery of the Janus kinase 2 (JAK2) mutation (JAK2V617F), present in ∼50% of ET,⁹  the first approved JAK1/JAK2 inhibitor, ruxolitinib (RUX), is now widely used for treatment of myelofibrosis¹⁰  and polycythemia vera.¹¹  The MAJIC-ET trial explored the role of RUX in HC-RES/INT ET, randomizing patients 1:1 to RUX or best available therapy (BAT), demonstrating similar rates of 1-year complete hematological response.¹²  Mutational status was not comprehensively reported in this paper. This is important as ET patients (29% to 52.5%)^13,14  carry mutations in non-MPN driver genes (NDMs). Inferior prognosis is associated with specific mutations at diagnosis.¹⁴  The impact of NDMs in HC-RES/INT ET is unknown, as is the effect of RUX on disease course in molecularly defined subgroups. We therefore evaluated the mutational status of MAJIC-ET patients and correlated this with clinical outcomes.

Next-generation sequencing was performed at baseline (n = 110) and serially if a later sample was available (see supplemental Methods [available on the Blood Web site] for next-generation sequencing and statistical analysis methodology). Median follow-up was 55 months (95% confidence interval [CI], 49.9-60.4). JAK2, CALR, and MPL mutations were present in 49.1%, 30%, and 4.5% of patients, respectively, and 16.4% of patients were “triple negative” (TN). Baseline NDMs were present in 30% (n = 33) of patients with >1 present in 10% (Figure 1A), most frequently TET2 (n = 12), TP53 (n = 7), and SF3B1 (n = 7) genes (Figure 1B; supplemental Table 1). Driver mutation variant allele frequency (VAF) was higher than NDM VAF in 66.67%, 87.5%, and 20% of JAK2, CALR, and MPL-mutated patients, respectively (Figure 1C). Patients with NDMs tended to be older with lower hemoglobin levels (Figure 1D; supplemental Table 2). TP53 mutations trended toward a higher frequency in TN (17.6%) than in JAK2/CALR/MPL-mutated patients (4.3%; P = .073). In the primary analysis, driver mutation status did not correlate with CHR.¹²  Since platelet count reduction is a key therapeutic goal, we performed a post hoc analysis defining platelet response as <400 × 10⁹/L at 1 year. RUX-treated JAK2V617F-mutated patients had significantly more platelet responses than JAK2V617F wild-type (WT) patients, a difference not seen for BAT-treated patients (Figure 1E). RUX discontinuation more often occurred in non-JAK2V617F–mutated patients (odds ratio, 3.9; 95% CI, 1.2% to 13.1%; P = .027) in whom treatment failure was the most frequent cause (41.7%, n = 10 of 24) followed by treatment toxicity (33.3%; n = 8 of 24). In contrast, in JAK2V617F-mutated patients, the commonest cause for RUX discontinuation was a transformation event (43.8%; n = 7 of 16) followed by treatment failure (31.3%; n = 5 of 16). NDMs did not influence hematological/symptom responses (supplemental Table 3).

Transformation events occurred in 12.7% (supplemental Table 3). TP53-mutated patients had inferior 4-year transformation-free survival (TFS) of 42.9% (95% CI, 9.8% to 73.4%) vs 79.8% (95% CI, 69.7% to 86.8%) for WT patients (P = .011) (Figure 2A). Splicing factor (SF) mutations conferred a poorer 4-year TFS of 40% (95% CI, 12.3% to 67%) vs 81.5% for WT patients (95% CI, 71.4% to 88.3%; P = .00039) (Figure 2B), predominantly attributable to mutated SF3B1 (P = .004). High molecular risk (HMR) mutations in this cohort (defined by SF and TP53 mutations) conferred poorer TFS (P < .0001) (Figure 2C), which was not ameliorated by RUX (Figure 2D). HMR mutations retained their negative impact on multivariable analysis (Figure 2E). Driver mutation VAF ≥50% and male sex independently conferred poorer TFS, findings reported by other groups.^15,16  Mutated TET2 did not correlate with clinical outcomes, comparable to previous findings.¹⁴ 

Thrombotic events (19.1%, n = 21 of 110) were not influenced by mutational status overall. This is in contrast to previous studies reporting a greater thrombotic risk in JAK2V617F-mutated patients.⁴  A possible explanation is that this association is not seen in HC-RES/INT patients who have a longer disease course and have undergone treatment, often with multiple lines of therapy. Furthermore, the number of events here is small and should therefore be interpreted with caution. Hemorrhagic events (9.1%; n = 10 of 110) were specifically associated with SF mutations (P = .007) (supplemental Table 3). Grade 3/4 hematological toxicities were not associated with mutational status. Overall survival at 4 years was 91.5% (95% CI, 80.2% to 96.4%) in BAT and 83% (95% CI, 70.4% to 90.5%) in RUX arms (P = .22) and was not influenced by mutational status.

One-year driver mutation molecular responses (MRs) were rare (n = 3), occurring exclusively in the RUX arm: a complete MR in 2 patients (JAK2V617F-mutated and CALR-mutated) and 1 CALR-mutated partial MR. Longitudinal driver mutation analysis was performed in 54% (n = 50 of 93); median analysis time was 48 months (24-60 months) with no significant change in VAF at any time point (supplemental Figure 1A-B). One-year MR was subsequently lost in 2 patients (supplemental Figure 1C-D) in association with clonal evolution of NDMs in both cases. Longitudinal NDM analysis was possible in 52% (n = 57 of 110); median analysis time was 40 months (6-60 months). New NDMs, defined by identification at VAF ≥5%, were detected in 19.3% (n = 11 of 57) at a similar frequency across treatment arms (supplemental Table 4) and no significant correlations were detected with baseline NDMs or clinical/survival outcomes. However, a median follow-up time of 10.7 months (95% CI, 9.05-12.4) after later NDM analysis is not sufficient time for survival analysis. These data highlight the clinical utility of serial molecular analysis in HC-RES/INT ET.

In this analysis, we identify NDMs at baseline in 30% of patients with ET, a higher frequency than most previous analyses, which may relate to the high-risk nature of this cohort.^13-15,17 TP53 and SF3B1 mutations were observed, each at 6.4%, higher than previously reported in ET (∼2% and 2% to 5%, respectively).^13-15,18  This may relate to the fact that this study analyzes a particular high-risk cohort for which there are limited data published on mutation profiles for comparison. The frequent detection of TP53 mutations in TN patients was unexpected but the numbers are too few (n = 3) to draw firm conclusions. Disease transformation was specifically associated with SF (most commonly SF3B1) and TP53 mutations, determining HMR for this cohort. Although prevalence of non-SF3B1 SF mutations in this cohort was low, we included these as HMR as they are established adverse-risk mutations in MPNs.¹⁵  However, this definition of HMR requires independent validation in larger cohorts before being applied in clinical practice. TP53 mutations in MPNs have been associated with acute myeloid leukemia transformation^14,15  but have not been reported to increase myelofibrotic transformation in ET.^14,15  Myelofibrotic transformation has been reported in association with SF mutations in ET, most often mutated SF3B1,^14,19  but a recent large MPN study identified SRSF2, ZRSR2, and U2AF1 but not SF3B1¹⁵  as myelofibrotic transformation predictors in ET. This contrasts with myelodysplastic syndromes in which SF3B1 mutations confer better survival^20-22  with lower risk of disease progression,²⁰  suggesting that disease context and comutations (primarily JAK2V617F here) are relevant.

Importantly, disease transformation in HMR patients was not mitigated by RUX, which is noteworthy as there has been interest in the possibility that early intervention with JAK2 inhibition might attenuate disease progression. We observed a novel association between SF mutations and hemorrhagic events; this finding needs independent corroboration due to low event rate. We also found that JAK2V617F-mutated status correlated with improved platelet responses to RUX, and, notably, more non-JAK2V617F–mutated patients stopped RUX, raising the possibility that JAK2V617F-mutated ET patients might selectively benefit from RUX.

In summary, we report for the first time, comprehensive mutational analysis of HC-RES/INT ET within the context of a prospective randomized clinical trial. We found a particularly high prevalence of TP53 and splicing factor mutations, which were strongly predictive of subsequent disease transformation, and not mitigated by RUX. This highlights the clinical/prognostic utility of serial mutation screening in HC RES/INT ET to allow identification of patients at risk of disease transformation.