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

Abstract

Essential Thrombocythemia (ET) patients at high-risk of thrombosis require cytoreductive treatment, typically with hydroxycarbamide. Many patients are resistant or intolerant to hydroxycarbamide (HC-RES/INT) and are at increased risk of disease progression. MAJIC-ET is a randomized phase 2 study comparing ruxolitinib (RUX) to best available therapy (BAT) in HC-RES/INT ET, which showed no difference between the two arms in rates of hematological response or disease progression. The impact of additional non-MPN driver mutations (NDM) on the risk of disease complications in HC-RES/INT ET patients is unknown. Since the presence of NDM may influence trial outcomes, we expand the primary MAJIC-ET analysis to serially evaluate NDM in MAJIC-ET patients using a targeted myeloid 32-gene panel. NDM at baseline were detected in 30% of patients, most frequently affecting TET2 (11%) followed by TP53 (6.4%) and SF3B1 (6.4%). The presence of a NDM was associated with inferior 4-year transformation-free survival (TFS; 65.4% [95% CI 53.3 – 75%] vs. 82.8% [95% CI 73.2 – 89.1%], p=0.017). Specifically, TP53 (p=0.01) and splicing factor (SF, SF3B1, ZRSR2, SRSF2; p<0.001), but not TET2 mutations were associated with reduced TFS which was not mitigated by RUX treatment. Longitudinal analysis identified new mutations in 19.3% of patients; primarily affecting TET2, TP53 and SF3B1. We report the first comprehensive mutational analysis of HC-RES/INT ET patients and highlight the clinical/prognostic utility of serial mutation analysis for NDM in HC-RES/INT ET, including the importance of SF and TP53 mutations which identify HC-RES/INT ET patients at increased risk of disease transformation.

Figure 1. Baseline mutational analysis and correlation with clinical characteristics and treatment response.

(A) Pie chart showing number of NDM per patient. (B) Balloon plot showing association of driver mutations with NDM with size and colour of bubble corresponding to frequency of association; NDM were more often associated with JAK2V617F mutations. (C) Column and dot plot showing variant allele frequencies (VAF) of each NDM (column) with corresponding driver mutation (blue dot). Red star indicating TN patient; driver mutation VAF was higher in 66.67%, 87.5% and 20% of JAK2, CALR and MPL-mutated patients suggesting driver mutation acquisition first in these, although with the caveat that order of mutation acquisition can only be definitively assigned using single-cell methodologies. (D) Dot and box plots of median age at trial entry in patients with NDM compared to patients without NDM; 71 versus 64 years, p=0.0001 (upper plot) and hemoglobin (Hb) level (mean Hb 115g/l) lower in patients with NDM compared to patients without NDM (mean Hb 125g/l), p=0.01 (lower plot). Dots represent each individual patient and each horizontal line and box represent the median for age/mean for Hb and interquartile ranges respectively using Mann-Whitney U test to compare median ages (non-normal distribution) and Student’s t-test to compare Hb means (normal distribution). (E) Post hoc analysis of 1-year platelet count responses; significantly more patients on RUX who were JAK2-mutated achieved plt <400 than non JAK2-mutated patients (upper bar chart). This difference was not seen within the BAT arm (lower bar chart). BAT=best available therapy; JAK2=JAK2V617F; NDM=non-MPN driver mutation; Plt <400=platelet count of, 400 x 10⁹/l; Plt ≥400=platelet count of ≥400 x 10⁹/l; RUX=ruxolitinib; TN=Triple negative.

Figure 2. Kaplan-Meier curves of transformation-free survival (TFS) stratified by mutational statuses with survival estimates, reported at 4-years.

(A) TP53 mutations were associated with inferior 4-year TFS; TP53-mutated (42.9% [95% CI 9.8 – 73.4%]) versus TP53-wild type (WT) patients (79.8% [95% CI 69.7 – 86.8%]), p=0.011. (B) SF mutations conferred a poorer 4-year TFS; SF-mutated (40% [95% CI 12.3 – 67%]) versus SF-WT (81.5% [95% CI 71.4 – 88.3%]), p=0.00039. (C) Comparing patients with HMR with LMR at 4-years; HMR 41.2% (95% CI 23.3-72.7%) versus LMR 84.6% (95% CI 76.9 – 93.1%), p<0.0001. (D) Stratifying patients with high risk molecular (HMR) mutations in this study by treatment arm demonstrates no amelioration of negative impact of HMR mutation with RUX treatment; patients with HMR on RUX had TFS at 4-years of 36.4% (95% CI 26.2 – 46.6%) and on BAT 50% (29.1 – 67.7%) (p=0.505 between these arms) as compared to those without these mutations (i.e. low molecular risk, LMR) with TFS at 4-years of 84.7% (95% CI 71.6 – 92%) on RUX and of 90.6% (95% CI 78.5 – 96%) on BAT (p=0.101 between these arms). The log-rank test was used to compare survival estimates between groups. (E) Forest plot showing multivariable cox model of TFS. Covariates significant on univariate analysis were included; TP53 mutations, SF mutations, treatment arm, JAK2V617F mutation status, disease duration at trial entry (TE), age and gender. HMR mutations independently retained negative impact on TFS with a hazard ratio (HR) of 4.21, p=0.006. Treatment arm, JAK2V617F status, disease duration at TE and age were not significant but notably male gender was associated with a poorer TFS, HR 4.5, p=0.006. Driver mutation allele ≥50% was independently associated with a poorer TFS, HR 4.11, p=0.016. Age and disease duration at TE were categorized as continuous variables. CI=confidence interval; HR=hazard ratio; HMR=high molecular risk risk (SF and TP53 mutations); LMR=low molecular risk (without SF or TP53 mutations); JAK2=JAK2V617F; NDM=non-MPN (myeloproliferative neoplasm) driver mutation; SF=splicing factor mutation (SF3B1, ZRSR2, SRSF2); WT=wild type.