JAK2 mutations in polycythemia vera: from molecular origins to inflammatory pathways and clinical implications

Polycythemia vera (PV) belongs to the group of classical myeloproliferative neoplasms (MPNs), also known as BCR::ABL1-negative MPNs. This group also comprises essential thrombocythemia (ET) and primary myelofibrosis (PMF). Together, these clonal hematologic disorders are characterized by increased proliferation of one or more myeloid cell lineages in the absence of dysplasia [1, 2]. PV is characterized by an acquired driver mutation of the Janus kinase 2 (JAK2) gene, which was first discovered in exon 14 (JAK2^V617F) in 2005, with other mutations being described in exons 12 to 15 later on [3‐8]. Clinically, PV is associated with an excessive production of red blood cells, platelets, and neutrophils, coupled with a significant load of disease-specific symptoms including elevated rates of vascular events and a 20-year risk for progression towards myelofibrosis or acute leukemia of approximately 16% and 4%, respectively [1, 9]. In this regard, the variant allele frequency (VAF) of the JAK2^V617F mutation emerges as a critical factor influencing the outcomes of PV patients [10].

The genetic underpinnings of PV have garnered significant attention, as they not only elucidate the pathophysiology of the disease but also guide therapeutic strategies and prognostic assessments. Central to the genetic landscape of PV is the JAK2^V617F mutation in exon 14 that is identified in approximately 97% of patients [1]. This gain-of-function mutation leads to an overactivation of the JAK2 gene resulting in the constitutive activation of the JAK-STAT pathway, a critical driver of erythroid progenitor proliferation [11]. Around 30% of PV patients bear JAK2^V617F mutations in homozygous state, most likely due to loss of heterozygosity [12‐14]. Coexistence of unmutated, heterozygous, and homozygous mutant progenitor cells result in a wide spectrum of quantitative levels of JAK2^V617F that extend from minimal fractions of a percent to complete dominance at 100%, clinically quantified as allele burden or variant allele frequency (VAF) [10, 15]. Of note, the significance of JAK2 mutations extends beyond the V617F variant (Fig. 1), with mutations in exon 12 of the JAK2 gene accounting for most of the JAK2^V617F-negative cases [6, 11, 16]. The outcomes of these patients resemble those of JAK2^V617F-positive PV patients, with similar incidences of thrombotic events, transformation to myelofibrosis or acute myeloid leukemia and death while being associated with markedly elevated hemoglobin levels, diminished serum erythropoietin concentrations, and reduced platelet and leukocyte counts at the time of diagnosis [17, 18].

Besides the canonical and noncanonical JAK2 mutations, myeloid neoplasm-associated mutations/additional (nondriver) mutations were reported in PV patients, which highlights the complexity of the genomic landscape of PV. For completeness, alterations in the following genes have been found: SRSF2, SF3B1, U2AF1, ZRSR2 (mRNA splicing genes); TET2, DNMT3A, IDH1/2, ASXL1, EZH2 (epigenetic regulation genes); SH2B3, NF1, NRAS, KRAS, CBL, FLT3, PPM1D, ERBB (miRNA deregulation, intracellular signaling genes); NF-E2, TP53, RUNX1, CUX1, ETV6 (transcription factors) and in rare cases other MPN-driver genes (CALR, MPL) [11].

The precise detection of driver mutations is crucial for the diagnosis of PV versus secondary erythrocytosis, since the few triple-negative PV cases may harbor other mutations. In general, this can be achieved through highly sensitive single-target techniques such as quantitative reverse transcriptase–polymerase chain reaction and digital droplet PCR, or through multitarget panel assays using next-generation sequencing. These methods should have a minimal sensitivity threshold for VAF of 1% to adequately distinguish between wild-type, triple-negative cases, and those harboring other mutations ([11, 19]; Table 1) lists diagnostic methods for the detection of JAK2^V617F mutations.

Of note, JAK2^V617F mutations are not the rate-limiting step for the development of PV, which was shown by the fairly long latency between the acquisition of the mutation and manifestation of PV and the detection of the clonal hematopoiesis of indeterminate potential (CHIP) [25‐27] Research is ongoing to investigate factors that suppress or promote the expansion of the JAK2-mutated clone, e.g., inflammation mediated by IL-1β [25].

The genetic profile, epigenetic dysregulation, signaling cascades, DNA damage response and inflammatory pathways impact on the effectiveness of JAK inhibitors, which are a cornerstone of the management of PV. This highlights the importance of genetic testing in guiding treatment decisions and risk assessment [1, 20, 28].

Traditional hematologic measures accompanied by the detection of genetic markers, particularly the JAK2 mutation which can be characterized by a complex coexistence of multiple clones that evolve over time, are in the center of a definitive PV diagnose and essential for informing personalized treatment strategies [1, 10, 20]. Given that virtually all patients with PV harbor JAK2 mutations and that peripheral blood and bone marrow biopsies are equally informative, the first step in approaching a PV diagnosis should include screening of the JAK2 mutation in exon 14 and exon 12 [1, 29].

Although the two major diagnostic classification systems, the international consensus classification (ICC) and the WHO fifth edition for MPN, are similar, the latter no longer requires red cell mass assessment. In more detail, the ICC diagnosis requires three major criteria to be met (1. hemoglobin/hematocrit, males above 16.5 g/dl/49%, females above 16 g/dl/48%; or red blood cell mass more than 25% above mean normal predicted values; 2. JAK2^V617F or JAK2 exon 12 mutation; 3. age-adjusted hypercellularity with panmyelosis in the bone-marrow biopsy, including prominent erythroid granulocytic, and increase in pleomorphic, mature megakaryocytes without atypia) or the first two major criteria and the minor criterion (i.e., subnormal erythropoietin levels). Of note, bone marrow biopsies may not be required in males and females with hemoglobin/hematocrit levels above 18.5 g/dl/55.5% and 16.5 g/dl/49%, respectively (Table 2). Confirmation by repeat testing is required if the VAF is below 1% [19, 30]. If repeat testing confirms low VAF, careful interpretation in the context of the patient’s full clinical presentation is required, as low levels can be found in otherwise healthy individuals, represent an early stage of the disease, or indicate clonal hematopoiesis of indeterminate potential (CHIP) [31]. In case of a negative result, the PV diagnosis becomes less likely and testing for secondary erythrocytosis should be considered ([19, 30]).

In terms of survival, age remains the most important predictor in PV [32]. However, there are also age-independent factors which should not be overlooked, including abnormal karyotype, leukocytosis, and non-JAK2 mutations (e.g., TET2, SRSF2, IDH2, RUNX1, ASXL1, U2AF1, EZH2, TP53) that are included in the mutation-enhanced international prognostic systems (MIPSS) PV risk stratification tool to estimate survival outcomes [33, 34]. Furthermore, the history of any thrombosis at or prior to diagnosis also impacts the overall survival of PV patients, thus, contributing to the categorization of PV patients as either high-risk (thrombosis history or patient age above 60 years) or low-risk (no history of thrombosis, age ≤ 60 years) [1]. This nuanced approach to risk stratification that takes factors such as patient age, thrombosis history, and JAK2^V617F allele burden into consideration, helps to tailor treatment strategies more effectively. Additionally, acquired mutations not only play an important role in the diagnostic algorithm but could also serve as a marker of minimal residual disease, if assessed [35‐37].

Regarding thrombotic risk stratification, new independent venous (VETS) and arterial (ARTS) predictive risk scores have been proposed based on differences between the molecular profile and the clinical presentation. Whereas the ARTS was shown to improve the currently recommended scores discrimination power by including four independent risk factors (i.e., age above 60 years at diagnosis, prior arterial thrombotic events, cardiovascular risk factors and mutations of TET2 or DNMT3A), the VETS that includes only two risk factors (i.e., JAK2^V617F VAF ≥ 50% and prior venous thrombotic events) did not. This underscores the need for an improved pathophysiological understanding to more accurately identify PV patients at risk for venous thrombosis [38].

Myeloproliferative neoplasms including PV are marked by a continuous inflammatory state that is considered a major driving force for clonal evolution and disease progression to the advanced myelofibrotic stage with bone marrow failure, massive splenomegaly, and ultimately leukemic transformation [39, 40]. The JAK2^V617F gain-of-function mutation leads to a constitutive activation of the JAK-STAT pathway, which promotes myeloid and megakaryocyte proliferation as well as differentiation and the production of inflammatory cytokines and chemokines. This aberrant cytokine production that includes interleukins (IL‑6, IL-1β) and tumor necrosis factor-alpha (TNFα) fosters an inflammatory microenvironment that favors disease progression and associated symptoms. These cytokines further amplify JAK-STAT signaling, creating a feedback loop that exacerbates both neoplastic growth and inflammation [41‐43]. Of note, certain inflammatory cytokines that are increased in patients with MPN, including TNFα and interferon α (IFNα), have demonstrated the ability to provide a selective growth benefit to cells harboring the JAK2^V617F mutation compared to wild-type cells in vitro [44, 45]. Multiple studies that investigated cytokine levels in MPNs have shown correlations between reduced survival and elevated levels of GM-CSF, IFNα, IL-1β, IL‑4, IL‑5, IL-10, MCP‑1, MIP-1α, MIP-1β, and TNFα [46]. The cytokine excess may further explain symptoms commonly associated with PV that impact patient quality of life, including fatigue, night sweats, fever, bone pain, and pruritus [47, 48]. Moreover, JAK2^V617F has been demonstrated to promote p53 degradation through the accumulation of HDM2 that in turn increases NFκB activity, and to induce the production of reactive oxygen species (ROS) [41, 49]. The presence of JAK2^V617F, along with elevated levels of ROS, is correlated with a heightened risk of thrombosis [50‐54], premature atherosclerosis [46], the formation of extracellular neutrophil traps [51], ischemic heart disease [55‐57], and cancer [58, 59]. Studies suggested that the inflammatory marker NLR (neutrophil-to-lymphocyte ratio) serves as an independent risk factor and a novel prognostic indicator for thrombosis in patients with PV [60, 61]. The current evidence with respect to the presence of increased levels of cytokines, chemokines, and ROS in MPNs underscores the importance of targeting both the JAK-STAT pathway and inflammation, suggesting that early intervention to control inflammation could potentially delay or prevent transformation to MF.

The biological behaviors of cells harboring the JAK2^V617F mutation exhibit notable differences based on whether they are heterozygous (i.e., presence in a single cell as a single copy) or homozygous (i.e., presence in a single cell as a double copy) for this mutation, since the intensity of JAK2^V617F signal transduction enhancement is directly correlated with the gene dosage of the mutation [15]. Mouse models engineered to express JAK2^V617F have shown that progression and severity of the PV phenotype are influenced by two key factors: the level of JAK2^V617F expression and the cellular context of this expression, specifically whether it occurs in hematopoietic stem cells (HSCs) or in progenitor cells [62‐64]. In this context, an increased JAK2^V617F mutant allele burden has been correlated with pruritus, fibrotic transformation, and venous thrombosis [65, 66]. This dual dependency highlights the complex role of the JAK2^V617F allele burden in the modulation of disease characteristics and underscores the importance of the cellular origin regarding the expression and impact of the mutation on disease progression.

Moreover, the JAK2^V617F allele burden was shown to vary significantly between PV, masked PV (i.e., a JAK2^V617F-positive entity with a phenotype mimicking ET due to isolated thrombocytosis but endogenous erythroid colony formation and histological features of PV) and ET [67]. JAK2^V617F allele burden of ≥ 50% has not only been linked to more prevalent splenomegaly and circulating CD34⁺ cells [10] but also to a higher probability of progression to MF [10, 12, 66, 68, 69], acute leukemia, and death [70]. Interestingly, MPN patients with an elevated JAK2^V617F allele burden (stratified at 26.3%, the median JAK2^V617F allele burden at baseline of the investigated cohort), were shown to have a higher prevalence of concomitant chronic kidney disease and displayed unfavorable dynamics of kidney function over time. This may be attributed to chronic inflammation, which not only drives the expansion of the malignant clone in MPN but could also potentially contribute to kidney function decline through mechanisms such as fibrosis and microvascular damage [71].

In PV and ET patients who received long-term treatment with ruxolitinib and who were shown to achieve sustained JAK2^V617F molecular response, this response was linked to a decreased likelihood of progression to MF. Moreover, patients who reached partial molecular response (above 50% VAF reduction) exhibited a decreased likelihood of developing secondary myelofibrosis compared to those whose JAK2^V617F VAF was unaffected [72]. In several studies, peripheral blood JAK2^V617F VAF above 50% was shown to correlate with higher white blood cell (WBC) count, absolute neutrophil count, hematocrit (HCT), and lower platelet count than VAF below 50% [10, 65, 66, 73]. Furthermore, patients with JAK2^V617F VAF above 50% have a greater risk of developing venous thrombosis [10] that persisted even after adjustment for prior events, WBC count, and age [65, 74, 75]. Evidence from randomized perspective trials correlating response and outcomes with JAK2^V617F VAF is piling up, suggesting that a VAF reduction is associated with better blood count control and lower risk of thrombosis as well as disease progression [10]. In contrast, in real-world analyses, primary therapies with phlebotomy and hydroxyurea (HU) failed to demonstrate normalization of blood cell counts in the context of reducing the risk of thrombosis, when the European LeukemiaNet (ELN) response criteria were applied [76, 77]. Taken together, achieving, and maintaining a low JAK2^V617F allele burden serves as a marker for therapeutic efficacy and more favorable patient outcomes [78, 79], which was best shown in the PROUD-PV and CONTINUATION-PV studies [80, 81], PVN1 [82], COMBI [83], COMBI-II [84], and MAJIC [85] trials. This is further supported by data from the PROUD-PV/CONTINUATION-PV trial that have been presented by Kiladjian et al. at EHA 2024 in Madrid. The data indicate that molecular response is associated with prolonged event-free survival in patients with early stage PV and can effectively be achieved with ropeginterferon alfa-2b. Additionally, ropeginterferon alfa-2b treated patients spent more time in molecular response that HU/BAT-treated patients and higher JAK2^V617F allele burden was associated with an increased risk of events, suggesting that depletion of the mutant clone below the current threshold for molecular response may confer further benefit. According to the extended COX proportional hazard model used by the study group, a 10% lower allele burden corresponds with an approximately 34% lower risk of events [86]. Thus, PV treatments that target the clonal expansion of JAK2^V617F are essential in terms of optimal management of the risk of thrombosis and disease progression.

Despite current treatments, the overactivation of the JAK2 signaling pathway accompanied by a thromboinflammatory state and a disordered HSC niche leads to more severe forms of the diseases in a subset of PV patients. The 20-year risk of PV patients transitioning to post-PV MF or AML (acute myeloid leukemia) is approximately 16% and 4%, respectively. This underlines the need for a deeper understanding of the molecular drivers of disease progression and for the concomitant detection of passenger mutations besides JAK2^V617F [10, 87, 88]. Disease progression has been associated with the occurrence or intensification of thromboembolic events, major bleedings, and constitutional symptoms such as pruritus, night sweats, fever, weight loss, and fatigue. At the same time, leukocytosis, advanced age, and history of thrombosis have been found to be independent risk factors for overall survival [89‐92]. Regarding cytogenetics, PV patients with an abnormal karyotype at the time of diagnosis demonstrated a higher risk of disease progression and a shorter period of transformation-free survival compared with those with a normal karyotype [93]. Germline predisposition in MPN (e.g., CHST15 mutation) might also affect the disease evolution to the fibrotic or blast phases [94]. Moreover, progression may be detectable at the cellular or molecular levels as well [89‐92, 95].

According to the IWG-MRT consensus criteria, post-PV MF is defined by bone marrow fibrosis grade ≥ 2 (3-point scale) or ≥ 3 (4-point scale). Additionally, two minor criteria must be met, which are anemia or sustained loss of need for phlebotomy and/or cytoreductive therapy, splenomegaly, leukoerythroblastosis, and development of constitutional symptoms [96]. It has been hypothesized that the development of bone marrow fibrosis results from the progressive replacement of hematopoietic cells by reticulin fibers, which has been attributed not solely to the chronic inflammatory state induced by aberrant JAK-STAT signaling but also to the acquisition of additional somatic mutations within HSC [97, 98]. Of note, the presence of JAK2^V617F mutation alone is not sufficient in a low allele burden to drive progression to MF, while a high allele burden has been linked to an increased risk of transformation [1, 10, 12, 91, 99]. In this context, additional ASXL1 mutations, for instance, which have been reported to be quite rare in PV (less than 7%) but more frequent in post-PV MF (19–40%), are suggested to play a role in disease progression and inferior survival outcomes [14, 100].

Regarding leukemic progression, the WHO defines progression to the accelerated phase in MPNs as 10–19% blasts in peripheral blood or bone marrow and the blast phase as ≥ 20% blasts [30]. In this context, TP53 mutations have been reported to occur in total in 6–8% of PV patients but in approximately 66% of MPN cases with accelerated or blast phases, and they are associated with disease progression and poor overall prognosis [101, 102]. Moreover, mutations in the RUNX1/AML1 gene were linked to leukemic transformation in MPNs [91]. Further genetic factors associated with leukemic transformation in PV include SRSF2 and IDH2 mutations [33, 91, 92, 103].

Of note, when treatments or environmental factors only partially suppress the JAK2^V617F-mutated hematopoietic stem cells, a selective environment may be created that favors the emergence of cells with additional mutations. These new mutations may confer a competitive advantage to the cells, enabling them to proliferate more aggressively, and potentially lead to disease progression or the development of AML [104]. In the REVEAL study presented at ASH 2023, a multivariate analysis with stepwise model selection showed a significant association of the time from PV diagnosis to enrollment, VAF, history of thromboembolic events and HCT in JAK2^V617F-positive patients with an elevated risk of progression. Of note, the WBC count was excluded as VAF and WBC counts are correlative [105]. This underscores the importance of strategies aimed at effectively controlling or eliminating the mutated clones to prevent disease progression. Considering these factors, phlebotomy, as a solitary intervention, fails to target the fundamental pathophysiological mechanisms underlying the disorder and, consequently, does not mitigate the risk of long-term sequelae, including the progression to myelofibrosis or AML which is predominantly fueled by the evolutionary expansion of the malignant hematopoietic stem cell clone [106]. Thus, comprehensive mutational screening at diagnosis and during follow-up has considerable potential to identify patients at high risk of disease progression and highlights the imperative need for innovative therapeutic strategies to address this disease more effectively.

Traditional cardiovascular (CV) risk factors are prevalent among patients with Philadelphia chromosome-negative MPNs, and JAK2^V617F has been associated with a 12-fold increased risk of developing coronary artery disease [53, 107]. Specifically, the incidence of arterial hypertension ranges from 39% to 70% in patients with PV, while the prevalence of diabetes mellitus has been shown to be between 7% and 16%. Dyslipidemia is observed in 15–38% of this patient cohort, obesity in approximately 7.5%, and smoking is reported in 10–15% of PV cases. Notably, about three-quarters of patients with PV exhibit at least one CV risk factor, and 37.7% are affected by multiple CV risk factors [108‐111]. Thrombosis, predominantly arterial thrombosis, represents the most severe complication associated with cardiovascular disease (CVD) and is a critical complication in the context of PV [112, 113], underscoring the importance of comprehensive risk assessment and management in this patient population. Interestingly, the prevalence of the JAK2^V617F mutation in the general population is higher than initially expected, with an approximate prevalence of 5% in individuals aged 60 years and older [114]. Crucially, the presence of JAK2^V617F-positive clonal hematopoiesis has been associated with heightened rates of thrombotic events among individuals who do not have an established myeloid disorder [114, 115]. This association underscores the importance of recognizing JAK2^V617F-positive clonal hematopoiesis as a significant risk factor for thrombotic events, even in the absence of MPNs, and highlights the need for further research into screening and management strategies for individuals harboring this mutation.

In a Danish case–control study with 538 ischemic stroke patients, 11.3% had a JAK2^V617F mutation. In comparison to age- and sex-matched controls (1613 patients) without ischemic cerebrovascular disease, there was a 2.4-fold increase in the odds of harboring the JAK2^V617F mutation [116]. These observations emphasize the necessity of implementing systematic screening for the JAK2^V617F mutation in patients experiencing ischemic stroke and other thrombotic events [116‐118]. Overall, such an approach has the potential to significantly benefit individuals with undiagnosed or precursor stages of MPNs which CHIP precedes for decades, as these patients may endure recurrent, debilitating, and potentially fatal thrombotic episodes several years prior to receiving an MPN diagnosis. In this context, it must also be mentioned that the appropriate management of conventional cardiovascular risk factors, such as arterial hypertension, dyslipidemia, smoking, obesity, and diabetes, is critical, especially since about three-quarters of PV patients possess at least one CV risk factor and approximately 38% have more than one CV risk factor [110, 111, 119]. As highlighted by Benevolo et al., these modifiable risk factors can significantly influence thrombotic outcomes, and early intervention may therefore help reduce the additional cardiovascular risk posed by the hematological disease [119].

In this regard, an interesting patient case of a 50-year-old male who suffered from daily angina pectoris attacks despite optimal medication for CVD (i.e., aspirin and atorvastatin) after the implantation of three stents in 2019 should be mentioned. In 2021, he received another stent and balloon angioplasty but angina pectoris persisted. Thus, the JAK2^V617F allele burden was determined at 0.018%, whereupon an off-label ropeginterferon alfa-2b treatment was initiated at 125 µg every other week. After 2 weeks, the cardiac symptoms decreased, and after 7 weeks, the JAK2^V617F mutation was no longer detectable [117]. However, it is a matter of debate whether such a small change in VAF could explain this outcome. Nevertheless, another 5 patients with MPNs including 3 cases of PV, 1 case of masked PV, and 1 case of ET presented with severe ischemic heart disease and treatment-refractory angina pectoris [120]. Here, too, ropeginterferon alfa-2b treatment resulted in rapid improvement of angina pectoris.

Together these studies/patient cases highlight the potential impact of ropeginterferon alfa-2b on cardiovascular disease, especially angina pectoris, both in individuals with CHIP and those diagnosed with MPNs.

The genetic complexity of PV underscores the need for personalized medicine approaches, with the aim of tailoring treatment based on individual genetic profiles to optimize patient outcomes and minimize complications. Regarding the thromboembolic complications which represent a major clinical challenge in the treatment of PV patients, cytoreductive treatment in addition to phlebotomy and aspirin is justified even in low-risk patients, as there is an elevated risk of thrombosis compared to the general population with or without cardiovascular risk factors (Fig. 2; [92, 121‐125]).

During the last 40 years, IFN-alfa has been shown to be extremely effective in lowering cell counts as well as to achieve disease modification in MPNs [126‐129]. This is further supported by the European LeukemiaNet 2021 recommendations that suggest that the initiation of cytoreductive therapy is warranted under certain conditions in individuals under 60 years of age who have no history of thrombotic events. These conditions include a clearly defined intolerance to phlebotomy, the presence of symptomatic and progressive splenomegaly, persistent leukocytosis (exceeding WBC of 15 × 10⁹/L), an observed progressive increase in leukocyte count (i.e., a 100% increase from baseline if initial counts are below 10 × 10⁹/L or a 50% increase of initial counts above 10 × 10⁹/L), marked thrombocytosis (exceeding platelets of 1500 × 10⁹/L), failure to maintain HCT levels necessitating frequent phlebotomies, ongoing high-risk of cardiovascular events, and a sustained high burden of symptoms. For those patients, recombinant IFN-alfa, specifically ropeginterferon alfa-2b and pegylated IFN alfa-2a, is endorsed as the preferred cytoreductive agent. Furthermore, in patients currently managed with HU who require an alternative therapeutic approach, the panel recommends considering treatment with either IFN-alfa or ruxolitinib [130, 131].

In a subgroup analysis of high- and low-risk PV patients enrolled in the REVEAL study, HCT above 45%, WBC count (above 11 × 10⁹/L) and platelet count (above 400 × 10⁹/L) were significantly associated with increased thromboembolic risk in high-risk patients, whereas only WBC count (above 11 × 10⁹/L) remained associated with thromboembolic risk in low-risk patients. Thus, it is important to lower the HCT and WBC counts to optimize PV management and to reduce thrombotic complications [132]. Within this framework, the European LeukemiaNET (ELN) response criteria for cytoreductive treatment have demonstrated their effectiveness in assessing disease progression, yet raised concerns about their applicability to agents aimed at reducing thrombotic events [76]. Here, a retrospective analysis revealed that the ELN criteria for cytoreduction therapy start can identify an increased risk phenotype in both low-risk and high-risk patients treated with HU. This finding was further confirmed in HU-naïve patients [133]. Since the thrombotic burden is the major source of morbidity and mortality in PV patients, there is an unmet need for new surrogate endpoints that accurately predict the thrombotic risk. Still, the pivotal therapeutic objective in PV patients involves the regulation of HCT. Here, the CYTO-PV study underscored the significance of HCT management, demonstrating a 4-fold reduction in cardiovascular incidents and mortality when the HCT target was set below 45% compared to a 50% threshold, primarily employing phlebotomy and HU for this purpose [134]. However, the impact of controlling WBC or platelet counts on PV outcomes have been difficult to define, which is in part due to the predominant effect of HCT levels on the thrombotic risk. In real-world scenarios, where phlebotomy and HU constitute the primary therapies, the normalization of blood cell counts has not shown a clear benefit with regard to thrombosis risk reduction, even when the ELN response criteria were applied [76, 77].

The focus on HCT management was a key endpoint in the RESPONSE‑1 and RESPONSE-2 trials that compared ruxolitinib against the best available therapy, which was predominantly HU. These studies included patients requiring phlebotomy, with or without splenomegaly (RESPONSE‑1 and RESPONSE‑2, respectively). In both trials, patients receiving ruxolitinib treatment achieved superior HCT control and experienced a greater likelihood of normalized blood counts [37, 135‐137]. Building on these findings, the final analysis of the PROUD-PV/CONTINUATION-PV trial confirmed higher complete hematologic response rates for ropeginterferon alfa-2b versus control treatment (i.e., HU) at 6 years. Regarding the clinical response according to the ELN criteria, complete hematologic response (CHR) rates were 54.5% vs. 34.9%; a sensitivity analysis utilizing imputation of the last observation carried forward for CHR showed significantly higher response rates in the ropeginterferon alfa-2b treated patients (72.6% vs. 47.3%). This was accompanied by significantly longer treatment time in CHR (60.9% vs. 41.2%) and time with normal WBC counts (93.7% vs. 80.5%). Molecular response was achieved in 66.0% of patients treated with ropeginterferon alfa-2b compared to 19.4% of HU-treated patients. Notably, the JAK2^V617F allele burden in patients treated with ropeginterferon alfa-2b was 8.5% compared to 50.4% in those treated with HU, and homozygous JAK2^V617F mutations were observed in 11.6% versus 50.0%, respectively. In line with the favorable safety profile of ropeginterferon alfa-2b, these study results provide the first evidence of significantly improved probability of event-free survival in ropeginterferon alfa-2b treated patients compared to HU (0.94 vs. 0.82; log-rank test; p = 0.04) given that rates for thrombotic events, progression to myelofibrosis, AML and death were lower (5.4% vs. 16.2%) in addition to durable hematologic and molecular responses [126]. In the DALIAH trial, the 5‑year CHR rate was 22% in the pegylated IFN-treated patients compared to 24% in those receiving HU [138], which might be attributed to the intention-to-treat analysis and the high dropout rate. The American MPN-RC trial that included much more advanced PV patients with a high number of previous thrombotic events had slightly lower CHR rates (1-year CHR, 35% in pegylated IFN-treated patients vs. 37% in HU-treated patients) [139] compared to the ones reported in the PROUD-PV/CONTINUATION-PV trial (1-year CHR, 43% in ropeginterferon alfa-2b-treated patients vs. 46% in those receiving HU) [80, 126]. The higher rates of hematologic remission in PROUD-PV/CONTINUATION-PV are further supported by data of the Low-PV trial that reported a 1-year CHR rate of 84% in ropeginterferon alfa-2b-treated patients compared to 66% in patients receiving phlebotomy [78, 80, 126]. In addition to these findings on CHR, recent findings indicate that early initiation of cytoreduction in low-risk patients does not alter thrombosis-free survival (TFS), and the selection of cytoreductive agents does not affect TFS in high-risk individuals, among contemporary young ET and PV patients diagnosed before the age of 25. Crucially, the data indicate that IFN, in comparison to other cytoreductive treatments (hydroxycarbamide, anagrelide), yields significantly better myelofibrosis-free survival (MFS) [140]. These findings endorse IFN-alfa as an effective disease-modifying therapy for improving long-term MFS. Furthermore, they prompt a re-evaluation of early intervention with IFN-alfa in PV patients with the aim of potentially prioritizing the improvement of MFS [140]. In the meantime, with respect to primary cytoreductive therapy, the Onkopedia Guidelines and the NCCN Guidelines recommend IFN-alfa for all eligible patients and HU only for those who do not meet the criteria for IFN-alfa treatment [141, 142]. Based on the absence of a disease-modifying treatment, the associated burden on patients and caregivers, and the development of novel, alternative approaches, the role of phlebotomy as a primary and exclusive treatment for low-risk PV patients is diminishing. Extended follow-up is required to determine if early intervention and the significant hematologic and molecular responses observed with IFN-alfa–based therapies result in improved long-term clinical outcomes including prevention of thrombotic events and progression to MF and AML. Nevertheless, we feel free to already propose a potential treatment approach that is depicted in Fig. 3.

Research of JAK2^V617F pathophysiology has identified numerous new therapeutic targets for inhibiting clonal expansion in PV. Although lower in number, there are ongoing phase 2 and phase 3 trials in low-risk PV patients, e.g., those investigating ruxolitinib (NCT04644211), or ropeginterferon alfa-2b (NCT05481151, ECLIPSE PV trial). A very interesting direction of research in high-risk patients is the assessment of molecules that interfere with iron homeostasis like sapablursen, an antisense oligonucleotide against TMPRSS6 mRNA (NCT05143957), PPMX-T003, a human monoclonal antibody for transferrin receptor 1 (NCT05074550), and rusfertide, a hepcidin mimetic (NCT04767802, PACIFIC trial; NCT05210790, VERIFY trial). In the PACIFIC trial, HCT levels were successfully reduced below 45% without phlebotomy in all 16 phlebotomy-naïve PV patients [143]. Furthermore, in patients whose HCT levels were not adequately controlled by phlebotomy with or without cytoreductive therapy, rusfertide significantly decreased the average number of phlebotomies from 4.63 in the 28 weeks prior to enrollment to 0.43 during treatment [144]. These promising results suggest that consistent HCT control with rusfertide may decrease thrombotic risk compared to intermittent phlebotomy [143, 144]. However, as these agents primarily affect the iron metabolism, more comprehensive studies with extended follow-up are necessary in PV patients. Moreover, studies are testing the effect of an MDM2 inhibitor (idasanutlin, NCT03287245; KRT-232, NCT03669965), an HDAC inhibitor (givinostat, NCT01761968), an LSD1 inhibitor (IMG-7289, NCT04262141) and the combination of a PI3Kδ inhibitor (TGR-1202) with ruxolitinib (NCT02493530). Ruxolitinib is further tested in the RUxO-BEAT trial (NCT02577926), as well as the MITHRIDATE trial (NCT04116502). More information on ropeginterferon alfa-2b is obtained in PV patients with intolerance or resistance to HU (NCT05485948), and the long-term safety and efficacy of this drug is tested in PV patients who have already been treated with ropeginterferon alfa-2b for 52 weeks (NCT04655092). Interestingly, the impact and the risks of using direct oral anticoagulants (rivaroxaban or apixaban) instead of aspirin is tested in the AVAJAK trial (NCT085198960).

Promising findings have also emerged from in vivo studies evaluating an anti-IL-1β antibody alone or in combination with ruxolitinib, which suppresses key inflammatory signaling pathways and could, thus, have beneficial effects on the clinical course [146]. In this context, anti-IL-1R1 antibodies markedly decreased WBC counts, splenomegaly, and bone marrow fibrosis in homozygous JAK2^V617F-positive mice [147]. Direct elimination of JAK2^V617F stem cells is being evaluated in vitro and in vivo in mouse models [148].

The discovery of the JAK2^V617F mutation in 2005 has shed light on the mechanisms behind the overproduction of red blood cells, platelets, and neutrophils, as well as the distinct characteristics of JAK2^V617F-heterozygote and -homozygote cells and their clonal growth characteristics in polycythemia vera (PV). This has finally led to a shift in how medical professionals are approaching the assessment and management of PV. Today, there is an emphasis on risk assessment in PV that goes beyond traditional blood count metrics to also include the specific impact of the JAK2^V617F allele burden, which influences both thrombosis risk and disease progression.

Several clinical trials highlighted in this review have directly linked reduction in variant allele frequency (VAF) to improved patient outcomes in PV. Specifically, lowering VAF has been shown to improve blood count control, reduce the risk of thrombosis, and slow disease progression, thus, underscoring the critical role of VAF reduction in the management of PV. Vice versa, treatments that neither target the clonal expansion of JAK2^V617F nor decrease JAK2^V617F VAF are not fully addressing the risks of thrombosis and disease progression. Here, critical opportunities for the implementation of disease-modifying therapies are missed. However, PV treatment requires prolonged administration to achieve molecular responses, which raises concerns about off-target effects, tolerability, immune suppression, and the potential for the development of cancer. In this context, continued exposure to treatments that place JAK2^V617F hematopoietic stem cells (HSCs) under selection stress without effectively suppressing clonal growth may select for mutations that drive disease progression or even the development of AML.

Moreover, considering the association of JAK2^V617F-mediated clonal hematopoiesis with various types of cardiovascular diseases that carry high incidence risks, the identification of JAK2^V617F-positive individuals could serve as a novel precision medicine approach to stratify and reduce the risk of cardiovascular events.

Ultimately, through the understanding of these molecular origins we will pave the way for more effective therapies giving PV patients and those with clonal hematopoiesis of indeterminate potential (CHIP) the best chance to live a long and normal life without the burden brought about by their medical condition.