Classical Philadelphia Chromosome-negative Myeloproliferative Neoplasms Research Articles

A comparative analysis of the clinical and genetic profiles of blast phaseBCR::ABL1-negative myeloproliferative neoplasm and acute myeloid leukemia, myelodysplasia-related.

The classic Philadelphia chromosome-negative myeloproliferative neoplasms (Ph (-) MPNs), have variable potential for progression to the blast phase (MPN-BP) of the disease. Except initiated by distinct driver mutations, MPN-BP frequently carry similar genetic abnormalities defining acute myeloid leukemia myelodysplasia-related (AML-MR). Because of dissimilar initial pathogenesis, MPN-BP and AML-MR are retained under different disease categories. To determine if separately classifying these entities is justified, we compare MPN-BP with AML-MR patients based on mutational landscape and clinical parameters. 104 MPN-BP patients and 145 AML-MR patients were identified with available clinical, cytogenetic, and genetic data. AML-MR patients presented with a higher blast count (median, 51% vs. 30%) while MPN-BP patients had higher WBC counts, platelet counts and bone marrow cellularity (all p<0.0001). Patients with MPN-BP showed similar genetic mutations with similar mutation pattern (functional domain, hotspot and locus involved by the mutations) but a different mutation rate from AML-MR, with more frequent JAK2, CALR, MPL, ASXL1, IDH2, SETBP1 and SRSF2 mutations and less frequent TP53 and DNMT3A mutations. The overall survival (OS) of MPN-BP (OS post-BP-progression) is comparable to that of AML-MR (median OS, 9.5 months vs. 13.1 months, p=0.20). In addition, the subgroups of MPN-BP show similar OS as AML-MR. When harboring certain mutation such as TP53, ASXL1, DNMT3A, TET2, RUNX1, IDH1, IDH2, EZH2, U2AF1, BCOR and SRSF2, MPN-BP and AML-MR patients carrying the same somatic mutation show no difference in OS. MPN-BP and AML-MR harbor similar somatic mutations and clinical outcomes, suggesting a unified clinical disease entity.

Management of classical Philadelphia chromosome-negative myeloproliferative neoplasms in Asia: consensus of the Asian Myeloid Working Group.

Myeloproliferative neoplasms (MPN) are a heterogeneous group of clonal hematopoietic stem cell disorders characterized clinically by the proliferation of one or more hematopoietic lineage(s). The classical Philadelphia-chromosome (Ph)-negative MPNs include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). The Asian Myeloid Working Group (AMWG) comprises representatives from fifteen Asian centers experienced in the management of MPN. This consensus from the AMWG aims to review the current evidence in the risk stratification and treatment of Ph-negative MPN, to identify management gaps for future improvement, and to offer pragmatic approaches for treatment commensurate with different levels of resources, drug availabilities and reimbursement policies in its constituent regions. The management of MPN should be patient-specific and based on accurate diagnostic and prognostic tools. In patients with PV, ET and early/prefibrotic PMF, symptoms and risk stratification will guide the need for early cytoreduction. In younger patients requiring cytoreduction and in those experiencing resistance or intolerance to hydroxyurea, recombinant interferon-α preparations (pegylated interferon-α 2A or ropeginterferon-α 2b) should be considered. In myelofibrosis, continuous risk assessment and symptom burden assessment are essential in guiding treatment selection. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) in MF should always be based on accurate risk stratification for disease-risk and post-HSCT outcome. Management of classical Ph-negative MPN entails accurate diagnosis, cytogenetic and molecular evaluation, risk stratification, and treatment strategies that are outcome-oriented (curative, disease modification, improvement of quality-of-life).

Second malignancies among older patients with classical myeloproliferative neoplasms treated with hydroxyurea.

Patients with classical Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocythemia (ET), and primary and secondary myelofibrosis (MF), are known to have an increased risk of second malignancies (SMs). Hydroxyurea (HU) is a guideline-recommended cytoreductive therapy for patients at high risk for MPNs. Controversy exists as to whether HU use is associated with a higher risk of SMs, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). We conducted a retrospective cohort study of older patients diagnosed with MPN (age ≥66 years) between 2010 and 2017 and included the data in the Surveillance, Epidemiology, and End Results Medicare-linked database. Multivariable competing risk analyses adjusting for patient characteristics were used to assess the impact of HU on the development of SM. We identified 4023 patients (1688 with PV, 1976 with ET, and 359 with MF) with a median age of 77 (interquartile range [IQR], 71-83) years at the time of MPN diagnosis. After a median follow-up of 3.25 (IQR, 2.10-5.00) years, 489 patients developed an SM (346 solid, 73 lymphoid, and 70 myeloid malignancies). The cumulative incidence probability of SM was 19.88% (95% confidence interval [CI], 17.16%-22.75%) among 2683 HU users and 22.31% (95% CI, 17.51%-27.47%) among 1340 nonusers, respectively (Gray's test, P< .01). We did not identify significant differences in the incidence of solid or hematologic SMs, including AML/MDS (hazard ratio, 1.33; 95% CI, 0.77-2.29; P= .30), between HU users and nonusers. Our results suggest that the use of HU does not increase the risk of SM in older patients with MPN.

Myeloproliferative Neoplasms, Version 3.2022, NCCN Clinical Practice Guidelines in Oncology.

The classic Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) consist of myelofibrosis, polycythemia vera, and essential thrombocythemia and are a heterogeneous group of clonal blood disorders characterized by an overproduction of blood cells. The NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for MPN were developed as a result of meetings convened by a multidisciplinary panel with expertise in MPN, with the goal of providing recommendations for the management of MPN in adults. The Guidelines include recommendations for the diagnostic workup, risk stratification, treatment, and supportive care strategies for the management of myelofibrosis, polycythemia vera, and essential thrombocythemia. Assessment of symptoms at baseline and monitoring of symptom status during the course of treatment is recommended for all patients. This article focuses on the recommendations as outlined in the NCCN Guidelines for the diagnosis of MPN and the risk stratification, management, and supportive care relevant to MF.

The effects of plasma viscosity in thromboembolic events among patients with essential thrombocytosis: A case-control study.

Essential thrombocythemia (ET) is an entity of classic Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), characterized by thrombocytosis with megakaryocytic hyperplasia where in the thrombocytes are increased with abnormal function.Thrombotic events are seen frequently and represent the main cause of morbidity and mortality in patients with MPNs, mainly polycythemia vera and ET. This study has aimed to research the effects of clonally increased thrombocytes on plasma viscosity (PV) levels among patients with ET and the relationship between PV and thromboembolism history, according to the hypotheses about the effects of PV in thromboembolic events among patients with ET. A total of 55 patients were enrolled in the study group, 18 of who had been newly diagnosed with ET according to 2016 World Health Organization criteria and had not previously been treated. 37 of them had already been diagnosed with ET and had been treated. There were 47 healthy volunteers in the control group. 5 cc blood samples were taken from the patients into tubes including an anticoagulant to measure their PV levels. PV of the control group was found to be lower than in the study group and both each patient groups (p < 0.05). No relationship was found between the patient groups in terms of PV (p = 0.404). The mean PV levels of the 16 patients with a history of thromboembolism and the 39 patients with no such history were 2.42±0.17 cP and 2.33±0.20 cP, respectively. The mean PV levels were found to be similar according to their history of thromboembolism in all patient groups and in treated patients (p = 0.572 vs p = 0.991). We have found that PV levels were increased in clonally increased thrombocytes in patients with ET when compared with the control group. This is the first study in this field according to our knowledge.

Novel therapies vs hematopoietic cell transplantation in myelofibrosis: who, when, how?

Abstract Myelofibrosis is one of the classical Philadelphia chromosome–negative myeloproliferative neoplasms characterized by progressive marrow failure and chronic inflammation. Discovery of the JAK2 mutation paved the way for development of small molecular inhibitors and further facilitated the research in understanding of molecular biology of the disease. Development of novel medications and synergistic combinations with standard JAK inhibitor (JAKi) therapy may have the potential to improve depth and duration of disease control and symptomatic benefit, whereas advancements in allogeneic hematopoietic stem cell transplantation (HCT) have improved tolerability and donor availability, allowing for more patients to pursue this potentially curative therapy. The increase in options for medical therapy and changing risk profile of HCT is leading to increased complexity in counseling patients on choice of management strategy. In this case-based review, we summarize our approach to symptom-directed medical therapy, including the use of novel drugs and combination therapies currently under study in advanced clinical trials. We outline our recommendations for optimal timing of HCT, including risk-adapted selection for early HCT as opposed to delayed HCT after upfront JAKi therapy, as well as the use of pretransplant JAKi and alternative donor sources.

Targeting Insulin-like Growth Factor/Insulin Receptor Signaling to Overcome JAK2 Inhibitor Persistence in Myeloproliferative Neoplasms

Classic Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) are driven by aberrant activity of the JAK2 tyrosine kinase. While JAK2 inhibitors rarely induce molecular remission, they provide quality of life improvements for patients and thus will be a mainstay for MPN treatment for the foreseeable future. This near ubiquitous upfront ineffectiveness of JAK2 inhibitors must be overcome if they are going to contribute to a significant disease-altering therapy for MPNs. The insulin-like growth factor-1 receptor (IGF1R) can contribute to resistance to kinase inhibitor cancer therapeutics and a recent study demonstrated a requirement of IGF1R in JAK2-V617F-induced MPN in mice. Furthermore, IGF is known to play a role in neoplastic erythroid colony formation of hematopoietic progenitors from MPN patients. Thus, there is evidence for a potential role for IGF1R signaling in the context of JAK2 inhibition, particularly in the setting of JAK2 inhibitor resistance/persistence, a concept that remains underexplored.We observed an increase in activated IGF1R and insulin receptor (IR), which is structurally similar to IGF1R, in JAK2-V617F-driven MPN model cells (UKE1, SET2, and HEL) that are persistently growing in high concentration of the JAK inhibitor ruxolitinib. This suggests cell signaling is re-wired to alter the activity of IGF1R/IR in this state. Interestingly, in ruxolitinib sensitive/drug naïve cells, we observed a rapid increase in IGF1R-beta and IR-beta protein levels in response to ruxolitinib treatment. These observations further suggest IGF1R/IR may contribute to the response of cells to ruxolitinib.To assess the role of IGF1R/IR activation in both ruxolitinib-persistent cells and drug naïve cells, we utilized the IGF1R/IR selective kinase inhibitor OSI-906. OSI-906 weakly inhibited growth of JAK2-V617F-driven cells (GI50s between 1 and 8 uM), and cells continued to proliferate at a reduced rate in the presence of a high concentration of drug. The IGF1R/IR inhibitor had little effect on cell viability, but in combination with ruxolitinib, it synergistically suppressed cell growth in part due to augmented induction of apoptosis. This combination also synergistically suppressed the neoplastic (erythropoietin-independent) growth of erythroid colonies of hematopoietic progenitors from MPN patients, which supports previous work indicating a role for serum IGF1 in this erythroid colony formation.Low concentration of IGF1R/IR inhibitor (0.1 uM) suppressed cell growth and viability of cells persistently growing in ruxolitinib yet had no effect on drug naïve cells. This along with our observation of enhanced activation of IGF1R/IR in these cells suggests that in the presence of ruxolitinib, cells may become more dependent on IGF1R/IR activity. We have always observed that high concentration of ruxolitinib is unable to reduce cell viability to zero in certain cell lines (UKE1 & HEL). Ruxolitinib (10 uM) induces a G1 cell cycle arrest in these cells and a sub-population of persister cells remains viable for several weeks. These persister cells remain competent for cell growth once ruxolitinib is removed. However, OSI-906 completely prevented the development of ruxolitinib persister cells. Culturing UKE1 cells in low serum sensitized cells to ruxolitinib and prevented the survival of persister cells. Supplementation of exogenous IGF-1 partially rescued this sensitivity and allowed for persister cells to survive, suggesting IGF-1 may contribute to the development of ruxolitinib persistence.Finally, in addition to an increase in IGF1R/IR protein, ruxolitinib (10 uM) treatment also led to long-term depletion of IRS1/2 levels, the main signaling docking protein that binds to IGF1R/IR. This correlated with re-activation of the ERK pathway, which has been reported to be due to SHC binding to IGF1R in the absence of IRS1/2. Because these persister cells remain arrested in G1, ERK activation is not sufficient to support cell growth, but perhaps supports cell survival. This may highlight potential therapeutic targets to prevent ruxolitinib persistence. Identifying mechanistic aspects of cell survival in the presence of ruxolitinib could play a significant role in the development of combination therapies for MPNs. Our data support the notion that ruxolitinib treatment may alter the dynamics of IGF1R/IR signaling leading to tolerance of cells to ruxolitinib. DisclosuresReuther:Incyte Corporation: Research Funding.

The Pan-PIM Inhibitor INCB053914 Displays Potent Synergy at Low Doses in Combination with Ruxolitinib in Pre-Clinical Models of MPNs

Classic Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) are hematopoietic stem cell disorders that affect about 300,000 people in the U.S. and can progress to bone marrow failure and acute myeloid leukemia. MPNs are driven by aberrant activation of the JAK2 tyrosine kinase, which leads to activation of downstream signaling molecules including STAT5, ERK, and AKT, among others. While the JAK inhibitor ruxolitinib is approved for use with patients with intermediate and high risk myelofibrosis and patients with polycythemia vera who are resistant or intolerant to hydroxyurea based on compelling clinical and/or hematologic efficacy, molecular responses to ruxolitinib therapy occur only rarely. In order to identify new MPN regimens that may enhance patient benefits, we investigated the effect of combining the JAK1/JAK2 inhibitor ruxolitinib with a new inhibitor (INCB053914) of the PIM family of Ser/Thr kinases, which are downstream targets of JAK2/STAT signaling. Members of the PIM family of kinases are proto-oncogenes that can cooperate with cMyc to induce lymphomagenesis in mice. PIM kinase activity regulates a variety of cell processes, including cell proliferation and apoptosis. For example, PIM kinases phosphorylate and inactivate the pro-apoptotic protein BAD. In addition, PIM activity can regulate translation initiation via activation of the mTORC1 complex, leading to inactivation of the translational initiation repressor 4EBP1. Triple PIM1/2/3 knockout mice demonstrate no major deleterious abnormalities suggesting specific targeting of PIM kinase activity may have limited adverse effects. However, the elimination of PIM1 in mice has suggested a role of PIM1 in the function of hematopoietic stem cells. Thus, deregulated PIM activity could affect hematopoietic stem cell disorders such as MPNs. We have recently demonstrated that PIM family members are elevated in MPN cells that persistently grow in the presence of ruxolitinib and that exogenous expression of PIM1 can induce ruxolitinib resistance. Taken together, PIMs appear to be strong candidates as therapeutic targets in MPNs.INCB053914 (PIMi) exhibits biochemical IC50s against Pim1/2/3 of 0.24 nM/30 nM/0.12 nM (at 1 mM ATP). In this study, we demonstrate that PIMi exhibits potent combination effects with ruxolitinib in pre-clinical models of MPNs. PIMi displayed synergistic effects in combination with ruxolitinib in MPN model cell lines, including BaF3/EpoR-JAK2-V617F, UKE1, and SET2. These effects include growth inhibition and, importantly, synergistic induction of apoptosis. The phosphorylation of biomarkers for Pim activity, including BAD, p70S6K, S6, and 4EBP1 was significantly diminished with combination of low dose ruxolitinib (0.1 uM) and PIMi (0.1 uM). Cells that were generated to persistently grow in ruxolitinib remained sensitive to PIMi. Importantly, neoplastic growth of erthyroid colonies from JAK2-V617F-positive MPN patients was sensitive to low dose PIMi (5 nM), with colony formation from some patient samples displaying sensitivity to as low as 1 nM of PIMi. In combination with ruxolitinib (25-50 nM), low dose PIMi displayed impressive synergy in inhibiting neoplastic colony formation of primary patient cells. Finally, in an in vivo model of JAK2-V617F-driven MPN cell growth, the combined treatment of ruxolitinib and PIMi suppressed tumor formation in CB17-SCID mice at doses that had little anti-tumor effect as single agents. The combination therapy was well tolerated in mice.Together, these data suggest that the pan-PIM inhibitor INCB053914 may provide a highly effective novel anti-MPN therapeutic, particularly in combination with ruxolitinib. INCB053914 is currently under investigation in a Phase 1/2 study in patients with advanced hematological malignancies (ClinicalTrials.gov: NCT02587598). Our findings support a clinical study investigating the combination of INCB053914 and ruxolitinib in MPN patients. DisclosuresCollins:Incyte Corporation: Employment, Equity Ownership. Koblish:Incyte Corporation: Employment, Equity Ownership. Ruggeri:Incyte Corporation: Employment, Equity Ownership. Stubbs:Incyte Corporation: Employment, Equity Ownership. Reuther:Incyte Corporation: Research Funding.

The Epidemiology of Myeloproliferative Neoplasms in New Zealand between 2010 and 2017: Insights from the New Zealand Cancer Registry.

Background: There is a paucity of data on ethnic disparities in patients with the classical Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs): polycythaemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF). Methods: This study analysed the demographic data for PV, ET and PMF collected by the New Zealand Cancer Registry (NZCR) between 2010 and 2017. Results: We found that the NZCR capture rates were lower than average international incidence rates for PV and ET, but higher for PMF (0.76, 0.99 and 0.82 per 100,000, respectively). PV patients were older and had worse outcomes than expected, which suggests these patients were reported to the registry at an advanced stage of their disease. Polynesian patients with all MPN subtypes, PV, ET and PMF, were younger than their European counterparts both at the time of diagnosis and death (p < 0.001). Male gender was an independent risk factor for mortality from PV and PMF (hazard ratios (HR) of 1.43 and 1.81, respectively; p < 0.05), and Māori ethnicity was an independent risk factor for mortality from PMF (HR: 2.94; p = 0.006). Conclusions: New Zealand Polynesian patients may have increased genetic predisposition to MPN, thus we advocate for modern genetic testing in this ethnic group to identify the cause. Further work is also required to identify modifiable risk factors for mortality in MPN, in particular those associated with male gender and Māori ethnicity; the results may benefit all patients with MPN.

A Young Woman with Thrombocytosis

The diagnostic approach to thrombocytosis involves consideration of reactive, hereditary, and neoplastic causes. Once reactive causes of thrombocytosis, such as iron deficiency, infections, solid tumors, and other obvious causes such as post-splenectomy thrombocytosis, have been ruled out, the focus shifts to myeloid malignancies, such as chronic myeloid leukemia (CML), the classic Philadelphia chromosome-negative (Ph−) myeloproliferative neoplasms (MPNs), essential thrombocythemia (ET), primary myelofibrosis (PMF), polycythemia vera (PV), myelodysplastic syndrome (MDS) with isolated deletion 5q and the rare MDS/MPN “overlap” syndrome, MDS/MPN with ring sideroblasts, and thrombocytosis (MDS/MPN-RS-T).

Hypoxia-inducible factor 1 (HIF-1) is a new therapeutic target in JAK2V617F-positive myeloproliferative neoplasms.

Classical Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) are a heterogeneous group of hematopoietic malignancies including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The JAK2V617F mutation plays a central role in these disorders and can be found in 90% of PV and ~50-60% of ET and PMF. Hypoxia-inducible factor 1 (HIF-1) is a master transcriptional regulator of the response to decreased oxygen levels. We demonstrate the impact of pharmacological inhibition and shRNA-mediated knockdown (KD) of HIF-1α in JAK2V617F-positive cells. Inhibition of HIF-1 binding to hypoxia response elements (HREs) with echinomycin, verified by ChIP, impaired growth and survival by inducing apoptosis and cell cycle arrest in Jak2V617F-positive 32D cells, but not Jak2WT controls. Echinomycin selectively abrogated clonogenic growth of JAK2V617F cells and decreased growth, survival, and colony formation of bone marrow and peripheral blood mononuclear cells and iPS cell-derived progenitor cells from JAK2V617F-positive patients, while cells from healthy donors were unaffected. We identified HIF-1 target genes involved in the Warburg effect as a possible underlying mechanism, with increased expression of Pdk1, Glut1, and others. That was underlined by transcriptome analysis of primary patient samples. Collectively, our data show that HIF-1 is a new potential therapeutic target in JAK2V617F-positive MPN.

Epidemiology of Patients with Classical Philadelphia-Chromosome Negative Myeloproliferative Neoplasms at a Single Academic Medical Center in Singapore

Introduction:Myeloproliferative Neoplasms (MPNs) represents a disorder that involves abnormal proliferation of cells originating from the myeloid line. The proliferation of these cells can lead to complications that are at times fatal. Despite its potential to cause life threatening complications, there is little data on this disease in Southeast Asia.As Singapore is a multiracial country in Southeast Asia, there may be some disease characteristics exclusive to patients here due to its unique population composition. The data from this Southeast Asian cohort would be useful to determine disease homogeneity in Asian countries.Methods:A retrospective review of the MPN database from National University Hospital, Singapore (NUHS) revealed 320 patients who were clinically diagnosed with MPN from 2008 to 2017. This data included patients with Essential Thrombocythemia (ET), Polycythemia Rubra Vera (PRV), Primary Myelofibrosis (PMF), Myeloproliferative Neoplasm, unclassifiable (MPN-U) and Chronic Eosinophilic Leukemia (CEL) (Figure 1A) as per the 2017 WHO classification. For this analysis, we included only the classical Philadelphia chromosome negative MPN and focused on the epidemiology, transformation and overall survival rate.Results:There was a slight male predominance with a male to female ratio of 1.3:1. The ethnic groups within this cohort consisted of 65.8% ethnic Chinese, 20.7% Malay, 5.5% Indian and the rest were made up of other ethnic groups within the region such as Eurasians, Thai, Filipinos, Burmese, Indonesians, Bangladeshi, Vietnamese and Arabian patients.The mean age at diagnosis for this group was 60.5. The mean age was 59.2 years for ET, 61.2 for PRV and the mean age of PMF was the oldest at 63.8. The mean age of diagnosis for ET and PMF patients in our cohort was slightly older compared to the Korean cohort (55.4 and 59.5 years) (Byun, et al., 2016).The majority of this cohort was made up of ET patients (53.1%) followed by PRV (35.3%) and PMF (11.6%).77.5% of these patients were JAK2 V617F mutation (JAK2) positive. The percentage of patients who were JAK2 positive for ET, PRV and PMF were 69.2%, 96.9% and 56.3% respectively. The percentage of JAK2 positive patients for the three subtypes were higher in our local population compared to the Chinese and Japanese cohorts.Only 120 patients were tested for Calreticulin Exon 9 (CALR) mutations as this molecular test was only available in our institution from 2015 onwards. ET patients make up 68.4% of CALR positive patients. It was noted that CALR positive patients had comparatively higher mean platelet counts of 925.2 than CALR negative patients with mean platelet counts of 691.7. This phenomenon is seen in both CALR positive ET and CALR positive MF patients.In the 10-year period, 25 patients were lost to follow up and 8 patients transferred their care to another institution. Overall, 27 patients were deceased, with a mean survival of 3.5 years. The death-to-case ratio was 11.5 per 100 cases. The death-to-case ratio for ET, PRV and PMF is 6.1 per 100, 8.2 per 100 and 31.3 per 100 respectively.During this period, only 6 patients had transformation. Three patients progressed to post-ET myelofibrosis and 3 had leukemic transformation. Those who had leukemic transformation were initially diagnosed with PRV (1 patient) and PMF (2 patients). All patients who had leukemic transformation were deceased and had a mean survival of 1.4 years from the transformation event.Conclusion:Whilst there were some observable differences between our data and existing Asian data, there is still insufficient information to determine disease homogeneity. This is partly due to the rapid growth of molecular knowledge in this field and the regular revision of the WHO diagnostic criteria of MPNs over the last decade or so. There needs to be coordinated efforts within the region to ensure that our patients have equal access to these diagnostic platforms and that they receive an accurate diagnosis. DisclosuresChng:Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Aslan: Research Funding; Merck: Research Funding; Takeda: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Celgene: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Amgen: Consultancy, Honoraria, Other: Travel, accommodation, expenses.

Development of a Targeted Next-Generation Sequencing Assay to Detect Diagnostically Relevant Mutations of JAK2, CALR, and MPL in Myeloproliferative Neoplasms.

The classical Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), consisting of polycythemia vera, essential thrombocythemia, and primary myelofibrosis, are a heterogeneous group of neoplasms that harbor driver mutations in the JAK2, CALR, and MPL genes. The detection of mutations in these genes has been incorporated into the recent World Health Organization (WHO) diagnostic criteria for MPN. Given a pressing clinical need to screen for mutations in these genes in a routine diagnostic setting, a targeted next-generation sequencing (NGS) assay for the detection of MPN-associated mutations located in JAK2 exon 14, JAK2 exon 12, CALR exon 9, and MPL exon 10 was developed to provide a single platform alternative to reflexive, stepwise diagnostic algorithms. Polymerase chain reaction (PCR) primers were designed to target mutation hotspots in JAK2 exon 14, JAK2 exon 12, MPL exon 10, and CALR exon 9. Multiplexed PCR conditions were optimized by using qualitative PCR followed by NGS. Diagnostic genomic DNA from 35 MPN patients, known to harbor driver mutations in one of the target genes, was used to validate the assay. One hundred percent concordance was observed between the previously-identified mutations and those detected by NGS, with no false positives, nor any known mutations missed (specificity = 100%, CI = 0.96, sensitivity = 100%, CI = 0.89). Improved resolution of mutation sequences was also revealed by NGS analysis. Detection of diagnostically relevant driver mutations of MPN is enhanced by employing a targeted multiplex NGS approach. This assay presents a robust solution to classical MPN mutation screening, providing an alternative to time-consuming sequential analyses.

Patient characteristics and outcomes in adolescents and young adults with classical Philadelphia chromosome-negative myeloproliferative neoplasms.

Little is known about the outcomes of Philadelphia-negative myeloproliferative neoplasms (MPNs) in adolescents and young adults (AYA). We reviewed all patients with essential thrombocythemia (ET), polycythemia vera (PV), and myelofibrosis (MF) treated at our institution from 1988 to 2016 who were aged 16 to 39years (AYA) and described their outcomes in comparison to older MPN population. Of 2206 patients, 185 (8.3%) were identified as AYA: 105 (57%) ET, 43 (23%) PV, and 37 (20%) MF. The median age was 33years [range, 16-39], and median follow-up time 3years [range, 0.04-25]. JAK2 allele burdens were significantly lower among AYA JAK2V617F-mutated patients in both PV (p = 0.001) and MF (p = 0.005). Seven percent of MPN AYA patients were diagnosed with a thrombotic event at, or prior to, diagnosis. Over the short median follow-up, 4 thrombotic (PV = 1, MF = 3) and 3 leukemia (ET = 2, MF = 1) events occurred. In multivariate analysis, AYA did not predict for thrombotic or transformational events across three cohorts. In the MF cohort, there was a reduced frequency of negative prognostic variables of anemia (p = 0.011) and leukocytosis (p = 0.048) in AYA when compared with non-AYA. Overall survival was significantly superior in the AYA cohorts in all three MPN groups, namely MF (p < 0.001), PV (p < 0.001), and ET (p = 0.002). Our findings suggest that MPN AYA patients exhibit an indolent clinical phenotype characterized by favorable survival outcomes.

Showing one’s true CALRs: spleen size and platelet count help refine the molecular diagnostic pathway for patients with splanchnic vein thrombosis

The classical Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) of polycythaemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) are myeloid disorders characterized by clonal expansion of mature hematopoietic cells, extra-medullary hematopoiesis and clinically by increased incidence of thrombosis, hemorrhage and the potential to transform to acute leukemia (1).

3q26.2/EVI1 rearrangement is associated with poor prognosis in classical Philadelphia chromosome-negative myeloproliferative neoplasms.

Classical Philadelphia chromosome-negative myeloproliferative neoplasms are a group of closely related myeloid disorders with different histologic features and clinical presentations at an early stage, but all later develop into a similar fibrotic stage with variable risk of acute transformation. The significance of 3q26.2/EVI1 rearrangement has been well recognized in acute myeloid leukemia, myelodysplastic syndrome, and chronic myeloid leukemia. However, the clinical importance of 3q26.2/EVI1 rearrangement in classical Philadelphia chromosome-negative myeloproliferative neoplasms is unknown. Here we reported 15 patients with classical Philadelphia chromosome-negative myeloproliferative neoplasms showing 3q26.2 rearrangement, including inv(3)(q21q26.2) (n=6), t(3;21)(q26.2;q22)(n=4), t(3;3)(q21;q26.2)(n=3), inv(3)(q13.3q26.2)(n=1), and t(3;12)(q26.2;p13)(n=1). In addition to 3q26.2 rearrangement, 9 of 15 cases had other concurrent karyotypical abnormalities, including -7/7q- and -5/5q-. There were 8 men and 7 women with a median age of 59 years (range, 35-79 years) at initial diagnosis of myeloproliferative neoplasms: 8 patients had primary myelofibrosis, 4 had polycythemia vera, and 3 had essential thrombocythemia. JAK2 V617F mutation was detected in 8/14 patients, including 4/4 with polycythemia vera. The median interval from the initial diagnosis of myeloproliferative neoplasms to the detection of 3q26.2 rearrangement was 44 months (range, 1-219 months). At time of emergence of 3q26.2 rearrangement, 11 patients were in blast phase and 2 patients had increased blasts (6-19%). Dyspoiesis, predominantly in megakaryocytes, were detected in all patients with adequate specimens at time of 3q26.2 rearrangement. Following 3q26.2 rearrangement, 12 patients received chemotherapy, but none of them achieved complete remission. Of 14 patients with follow-up information, all died with a median overall survival time of only 3 months (range 0-14 months) after the emergence of 3q26.2 rearrangement. In summary, 3q26.2 rearrangement in classical Philadelphia chromosome-negative myeloproliferative neoplasms is associated with other concurrent cytogenetic abnormalities, a rapid disease progression and blast transformation, a poor response to chemotherapy and a dismal prognosis.

The evolution and clinical relevance of prognostic classification systems in myelofibrosis.

Primary myelofibrosis, the most aggressive of the classic Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), is a clonal disorder characterized by often debilitating constitutional symptoms and splenomegaly, bone marrow fibrosis and resultant cytopenias, extramedullary hematopoiesis, risk of leukemic transformation, and shortened survival. Post-polycythemia vera and post-essential thrombocythemia myelofibrosis represent similar entities, although some differences are being recognized. Attempts to classify patients with myelofibrosis into prognostic categories have been made since the late 1980s, and these scoring systems continue to evolve as new information becomes available. Over the last decade, the molecular pathogenesis of MPNs has been elucidated considerably, and the Janus kinase (JAK) 1/2 inhibitor ruxolitinib is the first drug specifically approved by the US Food and Drug Administration to treat patients with intermediate-risk and high-risk myelofibrosis. This article reviews the evolution of prognostic criteria in myelofibrosis, emphasizing the major systems widely in use today, as well as recently described, novel systems that incorporate emerging data regarding somatic mutations. Risk factors for thrombosis and conversion to MPN blast phase also are discussed. Finally, the practical usefulness of the current prognostic classification systems in terms of clinical decision making is discussed, particularly within the context of some of their inherent weaknesses. Cancer 2016;122:681-692. © 2015 American Cancer Society.

Protein kinase Cɛ inhibition restores megakaryocytic differentiation of hematopoietic progenitors from primary myelofibrosis patients.

Among the three classic Philadelphia chromosome-negative myeloproliferative neoplasms, primary myelofibrosis (PMF) is the most severe in terms of disease biology, survival and quality of life. Abnormalities in the process of differentiation of PMF megakaryocytes (MKs) are a hallmark of the disease. Nevertheless, the molecular events that lead to aberrant megakaryocytopoiesis have yet to be clarified. Protein kinase Cɛ (PKCɛ) is a novel serine/threonine kinase that is overexpressed in a variety of cancers, promoting aggressive phenotype, invasiveness and drug resistance. Our previous findings on the role of PKCɛ in normal (erythroid and megakaryocytic commitment) and malignant (acute myeloid leukemia) hematopoiesis prompted us to investigate whether it could be involved in the pathogenesis of PMF MK-impaired differentiation. We demonstrate that PMF megakaryocytic cultures express higher levels of PKCɛ than healthy donors, which correlate with higher disease burden but not with JAK2V617F mutation. Inhibition of PKCɛ function (by a negative regulator of PKCɛ translocation) or translation (by target small hairpin RNA) leads to reduction in PMF cell growth, restoration of PMF MK differentiation and inhibition of PKCɛ-related anti-apoptotic signaling (Bcl-xL). Our data suggest that targeting PKCɛ directly affects the PMF neoplastic clone and represent a proof-of-concept for PKCɛ inhibition as a novel therapeutic strategy in PMF.

Historical views, conventional approaches, and evolving management strategies for myeloproliferative neoplasms.

The classical Philadelphia chromosome-negative myeloproliferative neoplasms (MPN), which include essential thrombocythemia, polycythemia vera, and myelofibrosis (MF), are in a new era of molecular diagnosis, ushered in by the identification of the JAK2(V617F) and cMPL mutations in 2005 and 2006, respectively, and the CALR mutations in 2013. Coupled with increased knowledge of disease pathogenesis and refined diagnostic criteria and prognostic scoring systems, a more nuanced appreciation has emerged of the burden of MPN in the United States, including the prevalence, symptom burden, and impact on quality of life. Biological advances in MPN have translated into the rapid development of novel therapeutics, culminating in the approval of the first treatment for MF, the JAK1/JAK2 inhibitor ruxolitinib. However, certain practical aspects of care, such as those regarding diagnosis, prevention of vascular events, choice of cytoreductive agent, and planning for therapies, present challenges for hematologists/oncologists, and are discussed in this article.

Considerations and recommendations for a new molecular diagnostic algorithm for the myeloproliferative neoplasms.

The classical Philadelphia chromosome-negative myeloproliferative neoplasms consist of three main pathological and clinical entities with the recurrent JAK2 V617F mutation present in ∼98% of patients with polycythemia vera and ∼50% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). The recent discovery of mutations within the CALR gene in up to 80% of JAK2 V617F-negative ET and PMF patients compels employment of CALR mutational analysis for the molecular diagnosis of these diseases. Evidence derived from a retrospective audit of JAK2 V617F testing provides a framework for the proposal of a diagnostic algorithm that incorporates CALR mutation assessment. It is recommended that relevant clinical details, including a provisional morphological and clinical diagnosis, are provided and that exclusion of alternative causes of erythrocytosis, leucocytosis, or thrombocytosis is required before molecular testing. It should be acknowledged that over-requesting such investigations can impact on the clinical predictive value of these tests when considering the disease specificity of such mutations, with adherence to the diagnostic algorithm necessary to ensure appropriate use of resources.