JAK2V617F Allele Burden Research Articles

A higher JAK2V617F allele burden may be a risk factor for hemorrhagic events in younger patients with polycythemia vera

ABSTRACT Objectives: Hemorrhagic events are a rare but potentially fatal complication in patients with polycythemia vera (PV). Methods: We analyzed the characteristics of hemorrhagic events in 267 patients with PV. Results: A median follow-up of 4.8 years revealed that 23 (8.6%) hemorrhagic events occurred. Significantly more hemorrhagic events occurred in younger patients aged below 60 years (n = 72) than in older patients aged 60 years or above (n = 191) (n = 12 [16.7%] vs. n = 11 [5.8%], respectively, P = 0.012). In univariate analysis among the younger patients, white blood cell (WBC) count ≥ 15 × 109/L (hazard ratio [HR] = 7.746, 95% confidence interval [CI] 2.082-28.830, P = 0.002), palpable splenomegaly (HR = 5.629, 95% CI 1.193–26.550, P = 0.029), and JAK2V617F allele burden ≥ 80% (HR = 22.850, 95% CI 2.885–181.00, P = 0.003) were associated with an increased risk of hemorrhagic events. In multivariate analysis, JAK2V617F allele burden ≥ 80% (HR = 9.394, 95% CI 1.046–84.380, P = 0.046) was a significant risk factor. Conclusions: There is an increased risk of hemorrhagic events after diagnosis in younger PV patients with a high JAK2V617F allele burden, high WBC count or palpable splenomegaly. It is important to consider treatment options that aim to avoid hemorrhagic events by reducing the JAK2V617F allele burden in younger PV patients.

JAK2 mutational status and the contribution of TERT and JAK2 polymorphisms to the occurrence of myeloproliferative neoplasms in Eastern Morocco

Background: The JAK2 V617F somatic mutation is a hallmark of myeloproliferative neoplasms (MPN) and is present in some patients with splanchnic venous thrombosis (SVT). Objectives: We investigated for the first time in Eastern Morocco the JAK2 mutational status and germline risk factors, such as the TERT and JAK2 polymorphisms, in MPN and SVT patients. Methods: This study included 38 patients with MPN, 24 patients presenting with SVT and 60 healthy donors from the BRO Biobank. JAK2 mutations were analyzed using qPCR and Sanger sequencing. Predisposing polymorphisms to MPN were evaluated using Sanger sequencing. Results: JAK2 V617F mutation was positive in 64.5% of patients with MPN and 20.8% of patients with SVT. The JAK2 V617F allelic burden ranged from 2% to 97.53%. We found a strong association between the JAK2 rs56241661 polymorphism of the JAK2 46/1 haplotype and the development of MPN. However, no association was detected between the TERT rs2736100 polymorpism and MPN. Conclusion: The JAK2 mutational status and its allelic burden in Eastern Morocco are consistent with previous studies. The JAK2 46/1 haplotype was strongly associated with MPN. However, unlike other previously studied populations, the TERT polymorphism rs2736100 has no effect on the occurrence of MPN in our population. Keywords: Myeloproliferative neoplasms; JAK2 V617F; TERT and JAK2 polymorphisms; genetic predisposition; Morocco.

Relation of JAK2 V617F allele burden and coronary calcium score in patients with essential thrombocythemia.

JAK2 V617F (JAK2) mutation is associated with clonal hemopoiesis in myeloproliferative neoplasms as well as with faster progression of cardiovascular diseases. Little is known about the relationship between allele burden and the degree of atherosclerotic alteration of coronary vasculature. We previously reported that carotid artery stiffness progressed faster in patients with JAK2 positive essential thromocythemia (ET) patients. After a four-year follow-up we investigated whether mutation burden of a JAK2 allele correlates with a higher coronary calcium score. Thirty-six patients with JAK2 positive ET and 38 healthy matched control subjects were examined twice within four years. At each visit clinical baseline characteristics and laboratory testing were performed, JAK2 mutation burden was determined, and coronary calcium was measured. JAK2 allele burden decreased in 19 patients, did not change in 5 patients, and increased in 4 patients. The coronary calcium Agatston score increased slightly in both groups. Overall, there was no correlation between JAK2 allele burden and calcium burden of coronary arteries. However, in patients with the JAK2 mutation burden increase, the coronary calcium score increased as well. The average JAK2 allele burden decreased in our patients with high-risk ET during the four-year period. However, in the small subgroup whose JAK2 mutation burden increased the Agatston coronary calcium score increased as well. This finding, which should be interpreted with caution and validated in a larger group, is in line with emerging evidence that JAK2 mutation accelerates atherosclerosis and can be regarded as a non-classical risk factor for cardiovascular disease.

Long-term safety and efficacy of ropeginterferon alfa-2b in Japanese patients with polycythemia vera.

Ropeginterferon alfa-2b (ropegIFN), a new-generation interferon-based agent, has been approved in Japan for patients with polycythemia vera (PV) who are ineligible for or respond inadequately to conventional treatment. However, long-term outcomes with ropegIFN in Japanese patients have not been reported. This extension of a phase2 study of ropegIFN in Japanese patients with PV aimed to determine its long-term safety/efficacy, and changes over time in JAK2 V617F allele burden. Here, we report data from the phase2 study and subsequent extension over a period of 36months. The primary endpoint was the complete hematologic response (CHR) maintenance rate without phlebotomy (hematocrit value < 45% without phlebotomy during the previous 12weeks, platelet count ≤ 400 × 109/L, and white blood cell count ≤ 10 × 109/L). The CHR maintenance rates were 8/27 (29.6%), 18/27 (66.7%), and 22/27 (81.5%) at 12, 24, and 36months, respectively. No thrombotic or hemorrhagic events occurred. The median allele burden change from baseline was - 74.8% at 36months. All patients experienced adverse events; 25/27 (92.6%) experienced adverse drug reactions (ADRs), but no serious ADRs or deaths occurred. This interim analysis demonstrated the safety and efficacy of ropegIFN over 36months in Japanese patients with PV.

Impact of JAK2 allele burden on MF outcome in the era of ruxolitinib.

6514 Background: Myelodepleted myelofibrosis (MF), characterized bycytopenias, lower JAK2V617F allele burden (JAK2%) and shorter benefit from JAKi ruxolitinib (RUX), has worse survival (OS) compared to myeloproliferative MF. Lower JAK2% and inferior OS is more typical for primary MF (PMF) vs MF from essential thrombocythemia or polycythemia vera (PPV/PET-MF). We sought to investigate the impact of JAK2% (&lt;/≥ 50%), cytopenias and the use of RUX in outcome of PMF/PPV-PET-MF patients from our center. Methods: 601 medical charts of JAK2 mutated patients with MF (known JAK2%) were retrospectively reviewed. We divided patients based on the absence (-) or presence (+) of cytopenias (hemoglobin &lt; 10 g/dL or platelets &lt; 100 x10^9/L) and leukocytosis (WBC ≥ 25 x10^9/L) into: Gr1 = (-)/(-) [absence of both]; Gr2 = (-)/(+) [proliferative]; Gr3 = (+)/(-) [cytopenic]; Gr4 = (+)/(+) [cytopenic and proliferative]) and evaluated OS per JAK2 &lt;/≥ 50% and PMF vs PPV/PET-MF. We assessed the tolerance of RUX ≥3 years. We used descriptive statistics, Kaplan-Meier curve with log-rank test and regression analysis for demographics, estimation of OS and its comparison. OS was censored at the time of stem cell transplantation. Results: Median age of the entire cohort was 75 years (64% males). Patients with JAK2 &lt;50%, more likely PMF, had more anemia and thrombocytopenia, higher blasts, less leukocytosis, and smaller splenomegaly. JAK2 &lt;/≥ 50% did not discriminate OS of the entire group, PMF or PPV/PET-MF (median OS of ~ 45 months for each; only PPV/PET-MF with 64 months). OS of Gr1-4 according to JAK2 &lt;/≥ 50% and PMF/ PPV/PET-MF and combined OS of patients with comparable outcome (A-D) are shown in the table. The final groups with distinct outcomes included: A+B) non-cytopenic PET/PPV-MF and PMF irrespectively of proliferation and JAK2% - with one exception of non-cytopenic, proliferative PMF with JAK2 ≥50% - had median OS of 70 months (range, 54-86); D) cytopenic and proliferative PMF with any JAK2% and PPV/PET-MF with JAK2 &lt; 50% of median OS 19 months (range, 13-25), and C) the rest of the cytopenic patients and non-cytopenic, proliferative PMF with JAK2 ≥50% with a median OS of 36 months (range, 32-42). Among the 3 final groups, similar proportion of patients were exposed to RUX during their follow-up: 36% (A+B), 31% (B) and 28% (C), respectively (p =0.13). The most patients in (A+B) group were able to tolerate RUX for ≥3 years (p =0.036). All patients had improved OS with RUX (data to be presented). Conclusions: Cytopenias and/or proliferation, rather than JAK2 &lt;/≥50%, define the outcome of PMF and PPV/PET-MF patients. [Table: see text]

Effective Management of Polycythemia Vera With Ropeginterferon Alfa-2b Treatment.

Polycythemia vera (PV) is a myeloproliferative neoplasm. Ropeginterferon alfa-2b is a new-generation polyethylene glycol-conjugated proline-interferon. It is approved for the treatment of PV at a starting dose of 100 µg (50 µg for patients receiving hydroxyurea (HU)) and dose titrations up to 500 µg by 50 µg increments. The study was aimed at assessing its efficacy and safety at a higher starting dose and simpler intra-patient dose escalation. Forty-nine patients with PV having HU intolerance from major hospitals in China were treated biweekly with an initial dose of 250 µg, followed by 350 µg and 500 µg thereafter if tolerated. Complete hematological response (CHR) was assessed every 12 weeks based on the European LeukemiaNet criteria. The primary endpoint was the CHR rate at week 24. The secondary endpoints included CHR rates at weeks 12, 36 and 52, changes of JAK2V617F allelic burden, time to first CHR, and safety assessments. The CHR rates were 61.2%, 69.4% and 71.4% at weeks 24, 36, and 52, respectively. Mean allele burden of the driver mutation JAK2V617F declined from 58.5% at baseline to 30.1% at 52 weeks. Both CHR and JAK2V617F allele burden reduction showed consistent increases over the 52 weeks of the treatment. Twenty-nine patients (63.0%) achieved partial molecular response (PMR) and two achieved complete molecular response (CMR). The time to CHR was rapid and median time was 5.6 months according to central lab results. The CHRs were durable and median CHR duration time was not reached at week 52. Mean spleen index reduced from 55.6 cm2 at baseline to 50.2 cm2 at week 52. Adverse events (AEs) were mostly mild or moderate. Most common AEs were reversible alanine aminotransferase and aspartate aminotransferase increases, which were not associated with significant elevations in bilirubin levels or jaundice. There were no grade 4 or 5 AEs. Grade 3 AEs were reversible and manageable. Only one AE led to discontinuation. No incidence of thromboembolic events was observed. The 250-350-500 µg dosing regimen was well tolerated and effectively induced CHR and MR and managed spleen size increase. Our findings demonstrate that ropeginterferon alfa-2b at this dosing regimen can provide an effective management of PV and support using this dosing regimen as a treatment option.

Treatment outcome and germline predictive factors of ropeginterferon alpha-2b in myeloproliferative neoplasm patients.

Studies have shown that some single nucleotide polymorphisms (SNPs) could serve as excellent markers in foretelling the treatment outcome of interferon (IFN) in myeloproliferative neoplasms (MPN). However, most work originated from western countries, and data from different ethnic populations have been lacking. To gain insights, targeted sequencing was performed to detect myeloid-associated mutations and SNPs in eight loci across three genes (IFNL4, IFN-γ, and inosine triphosphate pyrophosphatase [ITPA]) to explore their predictive roles in our cohort of 21 ropeginterferon alpha-2b (ROPEG)-treated MPN patients, among whom real-time quantitative PCR was also performed periodically to monitor the JAK2V617F allele burden in 19 JAK2V617F-mutated cases. ELN response criteria were adopted to designate patients as good responders if they achieved complete hematological responses (CHR) within 1 year (CHR1) or attained major molecular responses (MMR), which occurred in 70% and 45% of the patients, respectively. IFNL4 and IFN-γ gene SNPs were infrequent in our population and were thus excluded from further analysis. Two ITPA SNPs rs6051702 A>C and rs1127354 C>A were associated with an inferior CHR1 rate and MMR rate, respectively. The former seemed to be linked to grade 2 or worse hepatotoxicity as well, although the comparison was of borderline significance only (50%, vs. 6.7% in those with common haplotype, p = 0.053). Twelve patients harbored 19 additional somatic mutations in 12 genes, but the trajectory of these mutations varied considerably and was not predictive of any response. Overall, this study provided valuable information on the ethnics- and genetics-based algorithm in the treatment of MPN.

Exposure-efficacy and exposure-safety analyses of ropeginterferon alfa-2b treatment in patients with polycythaemia vera.

To investigate the exposure-response (E-R) relationship, including exposure-efficacy and exposure-safety, of ropeginterferon alfa-2b treatment in patients with polycythaemia vera (PV). Based on the results of the phase II trial A20-202 regarding ropeginterferon alfa-2b in patients with PV, E-R analyses were performed to evaluate the efficacy and safety of the given dosing regimen. The E-R analyses were based on logistic and linear regression and the relationship between exposure to ropeginterferon alfa-2b and key efficacy and safety variables. The key efficacy variables included complete haematologic response (CHR) and reduction of the driver mutation JAK2V617F. The safety variable was treatment-related adverse events (TRAEs). A clear relationship between the exposure to ropeginterferon alfa-2b and CHR was observed, with an increase in drug exposure resulting in an increased probability of achieving CHR. Similar CHR probabilities were observed in the third and fourth quantiles of the average concentration at Week 24. The results from the exposure-JAK2V617F model indicated that the JAK2V617F allele burden decreased with increasing exposure to ropeginterferon alfa-2b and baseline body surface area. Exposure-safety analysis revealed a risk of AEs associated with transaminase abnormalities, which were not associated with clinical significance. Our analyses have shown that patients with PV treated with ropeginterferon alfa-2b had an increased probability of achieving CHR and a molecular response with acceptable safety risks at the 250-350-500 μg titration dosing regimen. This study has provided the relevant data for the application of a biologics licence of ropeginterferon alfa-2b for PV treatment in China.

Recurrent Stroke with Rapid Development of Intracranial Artery Stenosis and Subsequent Successful Mechanical Thrombectomy in Essential Thrombocythemia.

Essential thrombocythemia is a myeloproliferative neoplasm. Ischemic stroke is frequently the first manifestation of essential thrombocythemia. We herein report a patient with JAK2V617 mutation-positive essential thrombocythemia who developed recurrent ischemic stroke with rapid development of intracranial artery stenosis and subsequently underwent successful mechanical thrombectomy. The high JAK2V617F allele burden in our patient (58.4%) may have affected the patient's condition. We discuss similar reports in the literature and the possible pathophysiologic mechanism of large artery involvement in these patients.

Exploratory Study on Impact of Comorbid Sleep Apnea Treatment in Patients with Polycythemia Vera and Essential Thrombocythemia

Background: Systemic inflammation has been implicated in the progression of myeloproliferative neoplasms (MPNs), polycythemia vera (PV) and essential thrombocythemia (ET). Obstructive sleep apnea (OSA) is a highly prevalent disorder that can lead to TNF-driven systemic inflammation. OSA leads to oxidative stress and NF-kB driven inflammatory gene expression through the mechanistic pathways of intermittent hypoxia and autonomic activation. We therefore sought to understand the prevalence of OSA in patients with MPNs and the feasibility of understanding the impact of treating comorbid sleep apnea in patients with PV and ET. Methods: Patients with PV and ET diagnosed using the 2008 or 2016 WHO criteria underwent STOP-BANG questionnaires to evaluate for the likelihood of OSA. Based on the finding of 3 or more positive responses on STOP-BANG questionnaire (PLoS One 2015;10:e0143697), patients underwent further evaluation with polysomnography or home sleep apnea test to diagnose OSA at an American Academy of Sleep Medicine-certified sleep laboratory (Figure 1). Patients with apnea-hypopnea index (AHI) of 5 or more events per hour and Eastern Cooperative Oncology Group scale score of 0-2 underwent continuous positive airway pressure therapy (CPAP). Effects of CPAP therapy on MPN-symptom assessment form total symptom scores (MPN-SAF TSS), JAK2V617F, MPL and CALR allelic burden, and exploratory endpoints of changes in inflammatory and thrombotic markers were ascertained (Figure 1). Comparisons with observational cohorts were planned to ascertain changes in the above endpoints at 0-, 3- and 6-months intervals (Figure 1). Based on a 40% reduction in allele burden (SD of 25%) and correlation of 0.5 between paired measurements, it was estimated that using a paired t-test with 27 evaluable patients will provide a 95% power to detect a 20% change in JAK2V617F burden from 40% to 20%. Similarly, assuming a 50% correlation (SD of 16) between pre- and post-therapy MPN-SAF TSS scores, 27 evaluable patients will provide a 80% power to detect a change of 10 at the two-sided 0.025 significance level. Results: Screening for OSA in patients with ET and PV revealed a high prevalence of OSA risk based on STOP-BANG scores. 26/29 males (mean age 63.6±11.8 years; BMI 29±3.3) and 16/26 females (mean age 61.5±12.8 years; BMI 28.2±6.5) had STOP-BANG scores of 3 or more. 8 patients with PV and ET have so far been enrolled into this study. 6/8 patients (age ranges from 41-74 years; 4 females and 2 males) with OSA (AHI 5-56 events/hr; 4% oxygen desaturation index 4.7-30/hr) underwent CPAP therapy. 1/6 patients in the CPAP arm did not tolerate CPAP and was excluded. 5/6 patients completed study procedures as did 2 additional patients without OSA in the observational study arm. Of 5/6 patients undertaking CPAP, 3/5 had the JAK2V617F mutations whereas 2/5 had the CALR mutation. JAK2V617F allele burdens at baseline in 2/5 patients were 80.7% and 4% at baseline and reduced to 73% and 1% at 6 months respectively whereas the third patient had undetectable JAK2V617F burdens throughout the study. All except one patient experienced improvement in MPN-SAF TSS scores after 6 months of CPAP therapy. While all 5 patients treated with CPAP showed improvements in C-reactive protein levels, analysis of NFkB1, TNF and IL-6 transcript levels showed mixed results. Similar mixed results were found with hypoxia-inducible factor (HIF) target gene expression ( SLC2A1, VEGF-A, EDN1) and markers of iron turnover (% transferrin saturation, transferrin, iron and ferritin levels) despite reduction in erythropoietin values in 4/5 patients with CPAP therapy at 6 months. Conclusions: Given the significant likelihood of OSA in patients with PV and ET (as based on STOP-BANG scores of 3 or more), there is need to evaluate and treat for OSA. Preliminary results show abnormalities in inflammatory markers with reductions in C-reactive protein and erythropoietin levels following CPAP therapy even though mixed results were noted in inflammatory and HIF-target gene transcripts, and markers of iron turnover. Larger studies and longer follow-up are required to understand the impact of OSA on not only the thrombotic and inflammatory milieu in patients with MPNs, but also the benefit of treating on prevention of disease progression and thrombotic manifestations common in MPNs. Funding: Huntsman Interventional Trial Grant CTO #HCI-17-HEM 28.

Ruxolitinib Treatment in Polycythemia Vera Results in Reduction in JAK2 Allele Burden in Addition to Improvement in Hematocrit Control and Symptom Burden

Introduction:In the pivotal RESPONSE and RESPONSE 2 trials, the Janus kinase (JAK)1/JAK2 inhibitor ruxolitinib was superior to best available therapy (BAT) at providing hematocrit control and complete hematologic response, as well as in improving disease-related symptoms, in patients with polycythemia vera (PV). Here we report the results of a post hoc pooled efficacy analysis using data from the RESPONSE studies. Methods:RESPONSE and RESPONSE 2 were randomized, open-label, multicenter phase 3 trials that assessed efficacy and safety of ruxolitinib in adults with PV who had resistance or intolerance to hydroxyurea. Splenomegaly was required for enrollment into RESPONSE but not for RESPONSE 2; patients in both studies were randomized 1:1 to ruxolitinib (starting dose, 10 mg twice daily) or BAT (most commonly hydroxyurea [59%, 49%] or interferon formulations [12%, 13%] in RESPONSE and RESPONSE 2, respectively). Patients in either trial who were randomized to BAT could cross over to ruxolitinib at the time of primary analysis (Week 32 in RESPONSE; Week 28 in RESPONSE 2) or later for efficacy or safety reasons. In this pooled analysis, hematocrit control was assessed in the pooled population at Weeks 28 and 80 and was defined as hematocrit &amp;lt;45% that was maintained since Week 16 with ≤1 phlebotomy occurring postrandomization and before Week 4. Symptom control (using the Myeloproliferative Neoplasm Symptom Assessment Form Total Symptom Score [MPN-SAF TSS]) and changes over time in JAK2V617F allele burden were also assessed. Response rate 95% CIs were calculated using the Clopper-Pearson exact method, and the odds ratio (OR) and 95% CI for comparisons of the proportion of patients achieving ≥50% reduction from baseline in MPN-SAF TSS were calculated by the Mantel-Haenszel method. Results:Overall, 371 patients were randomized in RESPONSE and RESPONSE 2 (ruxolitinib, n=184; BAT, n=187). Patient baseline characteristics were similar across trials and treatment groups and have been previously published. Mean (SD) age in the overall pooled analysis population was 61.8 (11.0) years; most patients were male (62.5%) and White (88.1%). At Week 28, 62.0% (95% CI, 54.5%-69.0%) of ruxolitinib patients achieved hematocrit control compared with 18.2% (12.9%-24.5%) of BAT patients (Figure 1). Durable hematocrit control was maintained to Week 80 by 47.3% (95% CI, 39.9%-54.8%) of patients randomized to ruxolitinib (nearly all patients in the BAT arm crossed over to ruxolitinib). The percentage of patients achieving ≥50% reduction from baseline in MPN-SAF TSS at Week 16 (the only common timepoint in both studies) was higher for ruxolitinib vs BAT (48.7% [95% CI, 40.7%-56.8%] vs 18.0% [95% CI, 12.5%-24.6%]; OR, 4.3 [95% CI, 2.6-7.2]). Mean (SD) change from baseline to Week 16 in MPN-SAF TSS was −4.4 (10.0) for ruxolitinib patients and 0.6 (6.9) for BAT patients. JAK2V617F allele burden decreased consistently from baseline to Week 208 in patients randomized to ruxolitinib. Mean JAK2V617F allele burden decreased from 66.1% to 41.4% at 4 years. Best change from baseline in JAK2V617F allele burden is shown in Figure 2, with a decrease of ≥25% in 57.1% of patients randomized to ruxolitinib and ≥50% decrease in 38.9%. Similar reductions in JAK2V617F allele burden were observed following crossover from BAT to ruxolitinib (Figure 2), with a best decrease of ≥25% and ≥50% in 48.7% and 35.3% of patients, respectively. Analysis of the BAT group is limited by small patient numbers after Week 32, as most patients crossed over to ruxolitinib, with a best decrease of ≥25% and ≥50% in 7.2% and 2.6% of patients respectively. Conclusions:In this pooled analysis of RESPONSE and RESPONSE 2, patients with PV treated with ruxolitinib achieved durable hematocrit control through Week 80 and had better symptom control at Week 16 than those who received BAT. Reductions in JAK2V617F allele burden were consistently observed through Week 208 in patients treated with ruxolitinib, including those who crossed over from BAT. Taken together, these results provide further evidence of the patient benefit of ruxolitinib in patients with PV with or without splenomegaly.

Mean Corpuscular Hemoglobin Optimizes the Thrombocytosis Risk Stratification in Polycythemia Vera: A Retrospective Multi-Center Study

Thrombosis was a common complication and the main cause of mobility of polycythemia vera (PV) with a lifetime incidence of 22%. Thrombosis risk was correlated with age, prior thrombosis, hypertension, red blood cell distribution width (RDW), leukocytosis, and JAK2V617F allele burden in previous studies, but only age≥65 years and prior thrombosis were included in thrombosis risk stratification. Finding independent thrombosis factors is significant to depict the landscape of PV and decrease the probability of thrombosis. Here, we aimed to find potential predictors to optimize the thrombocytosis risk stratification. Retrospectively reviewed 763 patients diagnosed with PV according to the 2016 WHO criteria from 17 hematology center in China from Jan 1, 2010, to July 1, 2023. Analyzed clinical characteristics and genetic features of thrombosis risk and compared the potential predictor in different risk stratification. The patients' clinical features are in table A.A total of 508(66.8%) subjects were male, the median age was 62 years (IQR, 53-70 years). The media follow-up was 45 months (IQR, 25-77 months). 84 (11.1%) subjects had thrombotic event pre-diagnosis, 25 (3.3%) of which were venous and 59 (7.9%) were arterial. 72 (9.6%) subjects had thrombotic events after diagnosis, 10 (1.3%) of which were venous and 62 (8.3%) were arterial (Figure B). Univariable analysis found thrombosis was associated with the thrombosis risk stratification (P=0.011), hypertension (P=0.017), diabetes (P=0.010), leukocytosis (P=0.026), mean corpuscular hemoglobin (MCH) (P=0.001), triglyceride (P=0.025), cholesterol (P=0.022), and splenomegaly (P=0.000). Thrombosis risk stratification (HR=1.74 [1.00, 3.02], P=0.026) and MCH (HR=0.92, [0.86, 0.98], P=0.010) were independently risk factors in multivariable variable analysis. We divided by MCH with cut-off point of 28.5pg, patients with MCH&amp;lt;28.5pg were associated with worse thrombosis-free survival (TFS) (Figure E). We further analyzed the effect of MCH in different thrombosis risk stratification. Results showed MCH &amp;lt;28.5pg was also meaningful in both low (P=0.0164) and high risk groups (P=0.0014) (Figure F, G). High risk stratification and MCH&amp;lt;28.5pg showed higher probability of thrombosis during the follow-up period (Figure C, D). In our study, potential risk factors from other studies such as JAK2V617F allele burden, hyperlipidemia, hypertension, leukocytosis, and RDW were not significant independent predictors of thrombosis. However, we found MHC&amp;lt;28.5pg was correlated with worse TFS both in low thrombosis risk stratification group and high thrombosis risk stratification group. Our study suggested that MCH&amp;lt; 28.8pg was an independent predictor and can increase the accuracy of predicting thrombosis. Acknowledgement: This research was funded by the Key R&amp;D Program of Zhejiang, No. 2022C03137; Public Technology Application Research Program of Zhejiang, China, No. LGF21H080003; Zhejiang Medical Association Clinical Medical Research special fund project, No. 2022ZYC-D09. Correspondence to: Dr Jian Huang, Department of Hematology, The First Affiliated Hospital of Zhejiang University School of Medicine. househuang@zju.edu.cn

Individualized Dosing of Ropeginterferon Alfa-2b Ensures Optimal Response in Patients with Low-Risk Polycythemia Vera (PV)

Introduction: Ropeginterferon alfa-2b at a fixed dose of 100 µg/2 weeks was efficacious in patients with low-risk polycythemia vera (PV) in the Low-PV trial, but the response rate was lower in a group of patients who switched to ropeginterferon alfa-2b after failing to respond to phlebotomy alone (Barbui et al., 2023). The finding that this group had a higher proportion of males and higher baseline values for body-mass index, phlebotomy demand, blood counts and JAK2V617F allele burden suggests that these characteristics might predict a higher dose requirement of ropeginterferon alfa-2b. We analyzed low-risk PV patients from the PROUD-PV/CONTINUATION-PV (NCT01949805/NCT02218047) cohort to examine the impact of baseline characteristics and individually optimized dose levels of ropeginterferon alfa-2b on response status at 12, 24 and 72 months. Methods: Patients diagnosed with PV (WHO 2008 criteria) were randomized 1:1 and received ropeginterferon alfa-2b or standard care for a total of ≥6 years during the 12-month PROUD-PV study and its extension CONTINUATION-PV. Low-risk patients (aged ≤60 years with no history of thrombosis) allocated to the ropeginterferon alfa-2b arm were analyzed. Ropeginterferon alfa-2b was started at a dose of 100 µg/2 weeks (50 µg if transitioning from hydroxyurea) and escalated by 50 µg/2 weeks until blood counts normalized to a maximum of 500 µg/2 weeks. Grade 2-3 drug-related toxicity mandated dose reduction/interruption. During maintenance treatment the dosing interval could be extended to 3-4 weeks. Complete hematologic response (CHR) defined by modified ELN criteria (without the spleen criterion) and safety data at 12, 24 and 72 months were assessed. Results: In the setting of individualized dosing, 33/56 (58.9%) low-risk PV patients allocated to ropeginterferon alfa-2b in PROUD-PV achieved a CHR at month 12. Response rates increased with longer treatment duration; of the 46 low-risk patients enrolled in CONTINUATION-PV; 80.4% and 73.2% of patients with available data achieved a CHR at months 24 and 72, respectively. Baseline characteristics by response status at month 12 indicated that males were overrepresented among non-responders (15/23 non-responders [65.2%] were male vs 11/33 responders [33.3%] [p=0.0291]), and baseline median JAK2V617F allele burden was non-significantly higher in non-responders at 12 months (45.6% vs 35.5%: p=0.1860). However, no significant differences in these baseline parameters were observed by long-term response status (24 months or 72 months), nor were there consistent differences in baseline body mass index or blood counts according to response status at any time point evaluated. The most frequent reason for non-response was failure to meet the CHR criterion hematocrit &amp;lt;45% without phlebotomy in the past 3 months, accounting for the majority of non-responders at months 12, 24 and 72 (13/23 [56.5%], 6/9 [66.7%], and 6/11 [54.5%], respectively), followed by discontinuation for any reason, which accounted for 30.4% of non-responders at month 12 and 36.4% at month 72. Only one low-risk patient discontinued due to ropeginterferon alfa-2b-related toxicity. Despite response-driven dosing, the median 4-weekly derived dose of ropeginterferon alfa-2b in the first year was only somewhat higher in non-responders compared with responders (809 µg [range: 209-898] vs 717 µg [188-898]; p=0.6954); 10/23 of non-responders at month 12 did not escalate the dose immediately according to protocol on ≥1 occasion during the first year, e.g. due to responses already observed for leukocyte or platelet counts. In the second year, the median 4-weekly doses were 997 µg (range: 211-997) vs 690 µg (88-1036) respectively (p=0.2024), indicating that non-responders received and tolerated the maximum dose (500 µg/2 weeks). Conclusions: High long-term response rates (80.4% at 24 months; 73.2% at 72 months) were achieved with ropeginterferon alfa-2b treatment in the low-risk PV population when the dose was optimized on an individual basis. No specific baseline characteristics among low-risk PV patients appear to be associated with long-term response to ropeginterferon alfa-2b. However, the PROUD-PV/CONTINUATION-PV studies show that - as hypothesized based on the Low-PV trial data - some low-risk patients require and can tolerate high doses, and that the optimal dose of ropeginterferon alfa-2b varies substantially between patients.

The Utility of a Coagulation Laboratory Profile in Addition to Clinical Risk Factors for Thrombosis Risk Evaluation in Patients with Myeloproliferative Neoplasms: A Pilot Study

Background: Patients (pts) with myeloproliferative neoplasms (MPN) have a significantly increased risk of arterial and venous thrombotic events (TEs), while some pts may paradoxically suffer from bleeding manifestations. Thrombosis risk evaluation in polycythemia vera (PV) and essential thrombocythemia (ET) has important implications for patient management, however, the currently used risk stratification models are extremely limited. Methods: We designed a pilot study to assess the utility of an expanded coagulation laboratory profile in addition to other clinical variables for thrombosis and bleeding risk evaluation in a heterogenous MPN patient population. Our laboratory profile included PT, aPTT, fibrinogen, D-dimer, von-Willebrand Factor (vWF) antigen and activity testing, platelet (PLT) aggregation studies and thrombin generation assay (TGA, ST-Genesia, Stago, France), along with CBC and comprehensive biochemistry studies. Blood was drawn from pts visiting the hematology clinics or day care at Hadassah Medical Center. Clinical and demographic data were collected retrospectively from electronic medical records. The study was approved by the local IRB committee. Results:74 pts were included in the study; the mean age was 59.7 years and 51% were women. Sixty percent had a diagnosis of PV, 20% had ET, 17.6% had myelofibrosis (MF) and 2.7% had MPN unclassifiable. 78% of the pts harbored a JAK2V617F mutation, 8% and 2.7% harbored CALR and MPL mutations, respectively, and 11% were negative to all three MPN driver mutations. 63% of the pts had at least one cardiovascular risk factor, most commonly hypertension (HTN) and dyslipidemia. Most of the pts were treated with antiplatelet agents (62%), anticoagulants (16%) and/or cytoreductive agents (58%). Of 74 pts, 31% had a history of TEs (20% arterial, 11% venous), 22% reported suffering from microvascular symptoms, and 20% had experienced bleeding. The high rate of bleeding manifestations in our study population suggests an underestimation of the bleeding burden in MPN, as proposed by our group previously [Rottenstreich, Eur J Intern Med 2017]. Other patient characteristics were overall comparable to MPN patient populations in other studies (Table 1). In multivariate analyses, history of TEs was significantly associated with older age, type 2 diabetes mellitus (DM2), HTN and dyslipidemia, but not with gender, MPN subtype, driver mutation status or smoking. Pts with history of TEs had significantly lower hematocrit and prolonged PT/INR, associated with a higher rate of treatment with cytoreduction and anticoagulation, respectively, in these pts. Interestingly, pts with TEs had significantly higher vWF antigen levels (p=0.007), which were independent of age, blood counts and treatments, a finding not previously reported in MPN. No association was observed between TEs and PLT or WBC counts, D-dimer levels and vWF activity (Table 2), however, vWF activity was decreased in pts with higher PLT counts. TGA was of key interest in this study. In 62 pts not receiving anticoagulation, we observed an association between prolonged lag time (suggesting decreased coagulability) and older age, male gender and (near significant trend) JAK2V617 negative status, but did not find a correlation to TEs, blood counts and other variables. PLT aggregation indices were not associated with TEs, bleeding history or blood counts. In multivariate logistic regression analyses, history of DM2 and HTN, and vWF antigen levels, showed the strongest association to TEs, which was independent of age, blood counts and all other variables. A risk-model incorporating these variables showed a sensitivity of 77% and specificity of 92% for history of TEs in our study population. Conclusions: Despite being limited by a small and heterogenous study population and retrospective data collection; these findings support the utility of a coagulation laboratory profile in the evaluation of thrombosis and bleeding risk in MPN. The potential of TGA in risk evaluation in MPN is intriguing but merits further research. The multivariate risk-model incorporating history of DM2 and HTN along with vWF antigen levels merits validation in larger studies. Based on this pilot, a prospective study following over 250 pts with PV and ET (excluding overt MF) was initiated. This study will include NGS mutational profiling and JAK2V617F allele burden testing and omit the use of PLT aggregation studies.

Tamoxifen for the treatment of myeloproliferative neoplasms: A Phase II clinical trial and exploratory analysis

Current therapies for myeloproliferative neoplasms (MPNs) improve symptoms but have limited effect on tumor size. In preclinical studies, tamoxifen restored normal apoptosis in mutated hematopoietic stem/progenitor cells (HSPCs). TAMARIN Phase-II, multicenter, single-arm clinical trial assessed tamoxifen’s safety and activity in patients with stable MPNs, no prior thrombotic events and mutated JAK2V617F, CALRins5 or CALRdel52 peripheral blood allele burden ≥20% (EudraCT 2015-005497-38). 38 patients were recruited over 112w and 32 completed 24w-treatment. The study’s A’herns success criteria were met as the primary outcome ( ≥ 50% reduction in mutant allele burden at 24w) was observed in 3/38 patients. Secondary outcomes included ≥25% reduction at 24w (5/38), ≥50% reduction at 12w (0/38), thrombotic events (2/38), toxicities, hematological response, proportion of patients in each IWG-MRT response category and ELN response criteria. As exploratory outcomes, baseline analysis of HSPC transcriptome segregates responders and non-responders, suggesting a predictive signature. In responder HSPCs, longitudinal analysis shows high baseline expression of JAK-STAT signaling and oxidative phosphorylation genes, which are downregulated by tamoxifen. We further demonstrate in preclinical studies that in JAK2V617F+ cells, 4-hydroxytamoxifen inhibits mitochondrial complex-I, activates integrated stress response and decreases pathogenic JAK2-signaling. These results warrant further investigation of tamoxifen in MPN, with careful consideration of thrombotic risk.

HMR Mutations Drive Poor Prognosis in Myelofibrosis Patients with Lower JAK2V617F Allele Burden but Not in Those with Higher Allele Burden: Results of a Multicenter Study

JAK2V617F is the most common driver mutation in myelofibrosis (MF), followed by CALR and MPL alterations. Clinical variables included in the International Prognostic Scoring System (IPSS), Dynamic IPSS (DIPSS), and DIPSS plus, as well as JAK2V617F allele burden are all well-established prognostic factors influencing the survival of MF patients. In addition, ASXL1, EZH2, SRSF2, IDH and U2AF1 mutations are classified as high-molecular risk (HMR) mutations in this context because of their close association with disease progression and leukemic transformation. However, the relationship between JAK2V617F allele burden and HMR mutations remains unclear. To address this, we recruited 122 MF patients (discovery cohort) diagnosed at the National Taiwan University Hospital (Taipei, Taiwan) who had available mutation profile determined by next-generation sequencing. For external validation, 82 MF patients from the Christie Hospital (Manchester, U.K.) and 51 MF patients from the Cleveland Clinic (Ohio, U.S.A.) and Vilnius University Hospital Santaros Klinikos (Vilnius, Lithuania) were enrolled. The median age of the discovery cohort was 61 years. JAK2V617F was the most common driver mutation (64.8%), followed by CALR (18%) and MPL mutations (9%). ASXL1 mutation was the most common (37%) genetic alteration other than driver mutations, followed by TET2 (16%) and EZH2 mutations (12%). Overall, 55 patients (45%) had at least one HMR mutation ( ASXL1, EZH2, SRSF2, IDH and U2AF1). With a median follow-up of 28.2 months, the median overall survival (OS) of the discovery cohort was not reached (NR) and was well risk-stratified by current prognostication systems, including IPSS, DIPSS plus, MIPSS70, and GIPSS. Notably, patients with at least one HMR mutation had significantly shorter leukemia-free survival (LFS) and OS than those without (both p&amp;lt;0.001). Next, we divided the 79 JAK2-mutated patients into lower- and higher-allele burden groups ( JAK2low and JAK2high) using the median (75%), as the cutoff. Concurring with previous reports ( Tefferi et al, Leukemia 2008; and Guglielmelli et al, Blood 2009), JAK2low patients had significantly inferior OS than JAK2high patients (median, 53 months vs NR, p=0.037). Among all JAK2-mutated patients, those with HMR mutations had a significantly shorter OS than those without (50 months vs NR, p&amp;lt;0.001). When considering HMR mutations and JAK2V617F allele burden together, the presence of HMR alterations conferred worse LFS and OS in JAK2low but not in JAK2high patients (HMR vs without HMR among JAK2low patients, p&amp;lt;0.001 and p&amp;lt;0.001; and JAK2high, p=0.822 and p=0.699, respectively). By and large, patients with concurrent HMR mutations and lower JAK2V617F allele burden had a distinctively inferior OS than those with other constellations of genomic lesions ( p&amp;lt;0.001, Figure 1A). This group of patients also had the shortest survival among the total cohort including both JAK2-mutated and wild patients ( p&amp;lt;0.001). The discriminating power of integrating HMR/ JAK2 allele burden for LFS and OS remained valid in the UK cohort (HMR vs without HMR: JAK2low, p=0.013 and p=0.012; and JAK2high, p=0.337 and p=0.600, respectively) and the US/Lithuania cohort (HMR vs without HMR: JAK2low, p=0.016 and p=0.013; and JAK2high, p=0.643 and p=0.446, respectively). Co-occurrence tests in the discovery and validation cohorts revealed that there was no shared mutational co-occurrence pattern among the 3 cohorts, excluding the potential confounding influence of specific concurrent mutations. More strikingly, time-dependent ROC curve analyses suggested that integrating JAK2 allele burden with HMR mutations could be complementary to current risk stratification systems in predicting LFS and OS of MF patients (Figure 1B) across 3 cohorts. Finally, HMR/ JAK2low was identified as an independent adverse prognostic factor by multivariable analysis, irrespective of age, risk stratification systems, and other mutations. In summary, we observe that HMR mutations accompanied by a lower mutant JAK2V617F allele burden identified a distinct MF population with significantly reduced survival and high risk of leukemic transformation, with independent prognostic relevance extrapolated to and validated in two independent cohorts. Experimental studies are ongoing to ascertain the underlying mechanistic basis and resolve the clonal architecture of this distinct MF genomic grouping.

Final Analysis of the Daliah Trial: A Randomized Phase III Trial of Interferon-α Versus Hydroxyurea in Patients with MPN

Background: Hydroxyurea (HU) is the most commonly used first-line cytoreductive treatment option for patients with myeloproliferative neoplasms (MPN) worldwide. However, increasing evidence on the efficacy and safety of pegylated interferon-alpha2 (IFNα) is emerging, and optimal first-line treatment is to be established. Aims: To compare the efficacy and safety of HU vs. low-dose IFNα in patients with MPN over five years. Methods: DALIAH (NCT01387763) was a randomized phase III trial of HU vs. IFNα in newly diagnosed or untreated patients with MPN (essential thrombocythemia (ET), polycythemia vera (PV), prefibrotic myelofibrosis (PreMF), and primary myelofibrosis (PMF)). Patients &amp;gt; age 60 were randomized (1:1:1) to HU, IFNα-2a, or IFNα-2b whereas patients ≤ age 60 were randomized to IFNα-2a or IFNα-2b. The primary outcome was the JAK2V617F molecular response (MR) rate at 18, 36, and 60 months per 2009 European LeukemiaNetwork (ELN) (ET, PV, PreMF) or 2005 European Myelofibrosis Network (EUMNET) (PMF) criteria. Secondary outcomes included the complete clinicohematologic response (CHR) rate at 12 months. The JAK2V617F allele burden was measured using quantitative polymerase chain reaction (qPCR) on peripheral blood (assay sensitivity: 0.1%). Primary and secondary outcomes were compared between groups (HU vs. IFNα or HU vs. IFNα &amp;gt; 60 years) using Fisher's exact test (categorical variables) or Wilcoxon rank-sum test (continuous variables). Paired comparisons (within groups) were made using Wilcoxon signed-rank test. Serial JAK2V617F measurements were compared by unadjusted mixed-effects linear regression analysis. Results: We included 203 patients (ET: 73 (36%), PV: 89 (44%), PreMF: 16 (8%), and PMF: 25 (12%)) in the modified intention-to-treat (ITT) population. Baseline characteristics were well balanced except for median age (HU: 68 years vs. IFNα: 59 years, p&amp;lt;0.0001) ( Table 1). The MR rate by ITT analysis was similar between HU and IFNα (18 months: 19% vs. 21%, p=1.00; 36 months: 19% vs. 26%, p=0.64; 60 months: 23% vs. 24%, p=1.00) (Figure 1A). However, the JAK2V617F allele burden was significantly lower in the IFNα group at month 36 and beyond (Figure 1B) and the absolute median (IQR) change in JAK2V617F allele burden (baseline to 60 months) was greater with IFNα (-20% (-9;-49) vs. -7% (3;-15), p=0.0053) (Figure 1C). Two patients (IFNα: n=2) were in complete molecular remission (undetectable JAK2V617F) at 60 months. The CHR rate by ITT analysis was higher with HU at 18 months (58% vs. 38%, p=0.03) but similar at all other time points (12 months: 50% vs. 36%, p=0.21; 60 months: 24% vs. 22%, p=0.83) ( Figure 1D). A post hoc subgroup analysis comparing HU with IFNα &amp;gt; age 60 showed comparable efficacy results. Among patients remaining on treatment (per-protocol analysis), the MR and CHR rates were superior in the IFNα group compared to the HU group at 36 months and beyond. The MR and CHR rates (HU vs. IFNα) by per-protocol analysis were: MR at 36 months: 23% vs. 56%, p=0.01; MR at 48 months: 27% vs. 59%, p=0.02; MR at 60 months: 35% vs. 67%, p=0.03; CHR at 36 months: 33% vs. 67%, p=0.002; CHR at 60 months: 38% vs. 62%, p=0.05. Overall treatment discontinuation at 60 months was 60% (HU: 37% vs. IFNα: 65%, p=0.0019). The most common cause of treatment discontinuation was adverse events (HU: 6/38 (16%); IFNα: 71/165 (43%)). Adverse events ≥ grade 3 occurred in 46% (HU: 58% vs. IFNα: 45%, p=0.15). Nineteen major thrombotic events were reported in 16 patients (HU: 4 events in 4 patients; IFNα&amp;gt;60: 12 events in 10 patients; IFNα≤60: 3 events in 2 patients), corresponding to an incidence rate of 2.6 per 100 person-years for HU and 3.4 per 100 person-years for IFNα (IFNα&amp;gt;60: 6.2; IFNα≤60: 1.2). None of the patients transformed into secondary acute myeloid leukemia. Five patients died during follow-up (HU: 2; IFNα: 3). Bone marrow histologic remission rates at 36 and 60 months will be presented at the meeting. Conclusion: ITT analysis detected no significant difference in the MR or CHR rates between HU and IFNα with long-term treatment (5 years) among patients with MPN, reflecting a higher treatment discontinuation rate in the IFNα group (65%). Thus, using the per-protocol principle, the MR and CHR rates were superior in the IFNα group at 36 months and beyond.

Real-World Experience of Ropeginterferon-Alfa Treatment of PV and ET - Two Centers Experience

BACKGROUND AND AIM: Interferon alpha is an effective treatment for Polycythemia Vera (PV) and Essential Thrombocythemia (ET) and is the only treatment known to induce sustained molecular responses by decreasing JAK2 mutation allelic burden. Long-acting pegylated interferons are better tolerated. Ropeginterferon-alfa is a monopegylated interferon with a longer half-life and is the first interferon to be FDA-approved for the treatment of PV. It is not yet approved for ET, but clinical trials are ongoing. Given the similar disease biology between PV and ET and the efficacy of pegylated interferon-alfa in both PV and ET, this study aims to look at a real-world experience of treating PV and ET with ropeginterferon-alfa since its approval. METHODS: We conducted a retrospective chart review of all our PV and ET patients treated with ropeginterferon-alfa at Huntsman Cancer Institute, University of Utah (SLC cohort), and at the University of North Carolina at Chapel Hill (UNC cohort), since the drug's approval for PV from November 2021 through June 2023. The complete hematologic response (CHR) was defined as the normalization of blood counts within the reference range of the local lab standards. Prior to ropeginterferon-alfa, the participants were managed with therapeutic phlebotomies, or cytoreduced with either hydroxyurea, pegylated interferon-alpha 2a ( Pegasys), JAK2 inhibitors, or were newly diagnosed and previously untreated. RESULTS: A total of 120 patients (76 in SLC cohort and 44 in UNC cohort) were included in this study. Of those, 75 (62.5%) were diagnosed with PV, 37 (31%) with ET, and 1 with congenital polycythemia due to germline JAK2 mutation. Twenty-three (19%) patients had a prior history of thrombosis. JAK2 mutation was present in 100 (83%) patients, while 9 (7.5%) were CALR mutated, 1 with MPL mutation, and 1 with triple-negative ET. Extended myeloid gene panel sequencing was available in 64 (53.3%) patients and 35 of them (54.6%) patients had additional somatic mutations. Most common somatic mutations include TET2 (11 patients) and DNMT3A (8 patients) and CBL (3 patients), among others. Of the cohort, 15 (12%) patients discontinued ropeginterferon-alfa due to various adverse events, which included: fatigue, joint aches, skin rash, and increased anxiety and depression. Of the 105 patients who are on ropeginterferon-alfa, 9 patients were previously untreated patients while 96 (91.4%) were pretreated with other therapies and switched to ropeginterferon-alfa. In many patients, the switch to ropeginterferon-alfa was done because of the increased frequency and aggressivity of squamous cell skin cancers while on hydroxyurea treatment. All of them started ropeginterferon-alfa at a dose of either 50 mcg or 100 mcg every two weeks and were increased at an increment of 50 mcg to achieve a goal of CHR. As most of the patients were switching from prior therapies, 51 (49%) were already in CHR at the time of initiating ropeginterferon-alfa. The median ropeginterferon-alfa dose to maintain CHR was 50mcg in the SLC cohort and 100mcg in the UNC cohort. (See table for more details). We have 6 patients with DNMT3A mutation, 4 (66.6%) have achieved CHR at a median ropeginterferon-alfa dose of 100 mcg (range 50 mcg - 150 mcg), and 2 patients are undergoing dose titration. Of the 11 patients with TET2 mutation, 7 (63.3%) achieved CHR at a median ropeginterferon-alfa dose of 150 mcg (range 50 mcg - 500 mcg). The analysis of JAK2 V617F and CALR allelic burden, transcripts of inflammatory, prothrombotic, and hypoxia-regulated (HIFs) genes, and evaluation of females for reversal of clonal to polyclonal myelopoiesis is ongoing. CONCLUSION: Ropeginterferon-alfa is effective in PV and ET, however, the dose that achieves CHR is highly individualized, necessitating an incremental titration approach. The patients already in CHR from prior therapy needed relatively lower doses of ropeginterferon-alfa to maintain CHR. Clinical trials are ongoing with an alternate accelerated dosing scheme and results are awaited.

Preclinical Evaluation of INCB160058 - a Novel and Potentially Disease-Modifying Therapy for JAK2V617F Mutant Myeloproliferative Neoplasms

The Janus kinase 2 ( JAK2) mutation, JAK2V617F, is the most common oncogenic driver in myeloproliferative neoplasms (MPNs), with nearly all cases of polycythemia vera (PV) and over half of primary myelofibrosis (MF) and essential thrombocythemia (ET) patients positive for the somatic mutation. Approved therapies for MPNs such as ruxolitinib, which act by directly inhibiting activity of the kinase domain (JH1) of JAK2, have demonstrated impressive clinical efficacy and safety in patients with MPNs; however, they do not address JAK2V617F allelic burden or achieve molecular remission of disease. A targeted JAK2V617F selective agent sparing wild-type (WT) JAK2 activity has potential to eliminate mutant cells, induce molecular remission, and theoretically lead to functional cure of MPNs. We report herein the preclinical development of pseudokinase (JH2)-targeting INCB160058, a first-in-class, orally bioavailable small molecule with the ability to selectively target JAK2V617F + cell populations derived from patients with JAK-mutant MPNs. Using structure- and function-guided molecular design, INCB160058 was designed to bind with picomolar affinity to the JH2 domain of JAK2V617F at the canonical ATP-binding site, with high specificity (&amp;gt;2500-fold) relative to binding at the active kinase domain (JH1) targeted by currently approved JAK inhibitors. Live cell single-molecule fluorescence microscopy showed that INCB160058 binding to JAK2V617F blocked ligand-independent thrombopoietin receptor dimerization induced by the mutation, and consequently led to loss of JH1 domain kinase activity. X-ray crystallography analysis indicates that the observed inhibition is likely driven by conformational disruption of the ⍺C helix motif at Phe 594 and Phe 595 in conjunction with a shift of the upstream region from Leu 583 to Asn 589 upon INCB160058 binding to the JH2 domain of JAK2V617F. We utilized both CD34 + human multipotent hematopoietic stem cells derived from patients with JAK2-mutant MF, engineered JAK2-mutant human hematopoietic cancer cell lines (eg, SET2 and UKE-1), and murine BA/F3 cell lines to explore the selective effects of INCB160058 on JAK2V617F compared with WT JAK2. INCB160058 treatment selectively reduced pathogenic phospho-STAT5 levels, decreased abnormal megakaryopoiesis, and suppressed colony formation only in JAK2V617F + CD34 + cells but not in CD34 + cells from healthy volunteers. Importantly, continuous exposure of mutant and WT JAK2 cells to INCB160058 in co-cultures at concentrations below IC 50 resulted in progressive elimination of JAK2V617F + cells without affecting WT cells. At the end of the testing period, the JAK2V617F-harboring population was no longer detectable in the co-culture assay. In NSG mice subcutaneously inoculated with JAK2V617F-expressing SET2 cells, INCB160058 was tolerated and exhibited significant antitumor activity. In addition, following INCB160058 treatment, a significant reduction in the engraftment of total human cells, particularly human erythroid progenitors (hCD45 − mCD45 − Ter119 − hCD71 + hCD235a +), was observed in NSGS mice xenotransplanted with JAK2V617F + CD34 + cells. Moreover, INCB160058 treatment also led to the normalization of various pathogenic cytokines, such as interleukin (IL)-6 and IL-8. Importantly, these observations were absent in NSGS mice engrafted with CD34 + cells from healthy volunteers following INCB160058 treatment, further demonstrating the selectivity of INCB160058 for JAK2V617F. In summary, our results indicate a novel mechanism of action of INCB160058, a high-affinity pseudokinase (JH2) binding inhibitor of JAK2V617F that blocks cytokine-independent activity of JAK2V617F while preserving cytokine-dependent signaling. Extended treatment with INCB160058 at low therapeutic doses results in the specific elimination of mutant JAK2V617F-harboring cells in mouse models and human cancer cells with minimal impact on WT counterparts. Clinical testing of INCB160058 may allow patients with MPNs to achieve molecular remission by eliminating cells with the main genetic aberration and afford an opportunity to overcome the disease.

Red Cells Distribution Width and Prognosis in Myelofibrosis Patients Treated with Ruxolitinib

Introduction Myelofibrosis (MF) is a BCR::ABL1-negative myeloproliferative neoplasm characterized primarily by genetic abnormalities and an aberrant production of pro-inflammatory cytokines (including IL-2, IL-6, TNF-α, and TGF-β among others) released from pathological hematopoietic stem cells leading to the deposition of reactive fibrosis in the bone marrow (BM), at the same time the development of extramedullary hematopoiesis, mainly in the spleen and liver, and finally the production of red blood cells (RBC) of often bizarre shape, i.e. dacryocytes. Consequently, increased variability in RBC volume, so-called anisocytosis, is a hallmark of MF. A small number of studies have already demonstrated that quantitative measurement of anisocytosis in terms of red cells distribution width (RDW) may have a negative prognostic role in newly diagnosed MF patients. Aim In this study we further evaluate the prognostic impact of RDW in a series of patients with primary (PMF) and secondary myelofibrosis (SMF) at diagnosis and during treatment with ruxolitinib (RUX). Methods Between October 2014 and May 2023, the RDW of 49 consecutive patients with PMF or SMF (32 males and 17 females; median age at RUX initiation, 70.5 years) were retrospectively assessed at MF diagnosis, at RUX start and after 3, 6, 12, 24 and 36 months from therapy initiation. All patients were treated with RUX according to current indications, requiring an IPSS risk of at least intermediate-1. Red cells distribution width was routinely reported by automated cell counters as part of RBC count, with an upper limit of normal of 14.5% used as a cut-off in our laboratory. At the time of RDW evaluation, complete blood cells count and spleen size [expressed as the distance from the left costal margin (BCM) in cm] were available for all patients. Results Among 49 patients, 24 had PMF (10 prefibrotic and 14 overt fibrotic) and 25 SMF [15 had post-polycythemia vera (PPV)- and 10 post-essential thrombocythemia (PET)-MF]. The JAK2V617F mutation was detected in 38 (77.6%) cases, CALR mutations in 8 (16.3%), and MPL in 1 (2%), while 2 patients (4.1%) were “triple-negative”. Elevated RDW values were present in all but 3 patients at MF diagnosis with a median RDW of 18.7% (range, 13.3% - 25.7%). RDW was higher in patients with splenomegaly ( p=0.04) and with JAK2V617F mutation ( p=0.09). Furthermore, looking specifically at JAK2V617F-mutated cases, the greater the RDW values, the higher the JAK2V617F allele burden. Interestingly, a higher RDW value at diagnosis also correlated with a shorter time from MF diagnosis to RUX start (-3.5 months per one RDW unit, 95%CI: -7.5 to +0.4; p=0.08). We observed a modest (&amp;lt;1%) increase in RDW during the first 6 months of RUX treatment. In a multivariable random-intercept model that considers all time points and contains the covariates time, RUX dose, and hemoglobin (Hb), we also observed a clear decrease in RDW with increasing Hb (slope: -0.35% per g/dL of Hb, 95%CI: -0.56 to -0.14; p=0.001). Median overall survival (OS) from MF diagnosis was 6.5 years with a median duration of RUX treatment of 33.3 months. The median RDW at diagnosis of 18.7% was used as a cut-off to identify two subgroups of patients [Group 1 (N=22): RDW 18.7% to 25.7%; Group 2 (N=23): RDW 13.3% to 18.6%] showing a difference in OS [Group 1 vs. 2: Hazard Ratio (HR) 2.74, 95%CI: 1.13 to 6.65; p=0.03] ( Figure 1); the median OS was 8.5 (14 deaths) and 11.8 (9 deaths) years for the Group 1 and 2, respectively. Using continuous RDW at diagnosis, the crude HR was 1.21 per one RDW unit (95%CI: 1.06 to 1.37; p=0.004). In a Cox model adjusted for gender, age, and Hb, the HR was 1.13 (95%CI: 0.99 to 1.29; p=0.06). Notably, RDW at diagnosis was associated with a 35% increase in infectious disease mortality in a crude analysis (HR 1.35, 95%CI: 1.07 to 1.71, 8 deaths; p=0.01) and a 27% increase (HR 1.27, 95%CI: 0.99 to 1.63; p=0.07) in the adjusted analysis. Conclusions Red cells distribution width may represent a good integrative measure of several disease processes and also has prognostic significance at the time of MF diagnosis and during RUX treatment. Even though our preliminary observations are limited by the small number of patients and need to be further confirmed by larger studies, we can speculate that RDW may help in the rapid detection of MF patients with a poor prognosis, which can only partially be improved from currently available targeted therapies with JAK inhibitors.