BCR-ABL Negative Myeloproliferative Neoplasms Research Articles

The CHIP-clinic as the catalyst of preventive medicine

Clonal Hematopoiesis of Indeterminate Potential (CHIP) is associated with an increased risk of cardiovascular diseases (CVD) and is a precursor stage to the BCR-ABL negative chronic myeloproliferative neoplasms (MPNs). These diseases are acquired stem cell neoplasms, arising due to mutations in the hematopoietic stem cell. The most prevalent is the JAK2V617F (JAK2) mutation, which potently generates reactive oxygen species (ROS), and accordingly contributes greatly to the chronic inflammatory state and the increased risk of thrombosis in MPNs. The MPNs are largely underdiagnosed blood cancers with a long pre-diagnostic phase of several years, when the elevated blood cell counts are considered reactive to smoking, blood clots, infections or chronic inflammatory diseases. Since the JAK2 mutation as CHIP-JAK2 associates with an increased risk of CVD and an increased risk of hematological and non-hematological cancers there is an urgent need to explore and validate the JAK2 mutation as a novel risk factor for CVD and to establish CHIP-clinics, which in an interdisciplinary collaboration between experts from several disciplines, and ensure timely diagnosis of the undiagnosed MPN patient and associated comorbidities. We envisage studies of the JAK2 mutation in large CVD cohorts to deliver the “Proof of Concept” for the JAK2 mutation to be implemented as a novel, highly important risk factor for CVD. These novel preventive strategies are considered to have the potential of reducing morbidity and mortality in a large population of citizens and patients, carrying the thrombosis- and CVD-promoting JAK2 mutation.

Neutrophil-to-lymphocyte and platelet-to-lymphocyte ratio as novel prognostic biomarkers in BCR-ABL negative myeloproliferative neoplasms.

Higher neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) have been associated with increased risk of thrombosis, cardiovascular mortality, but their role in myeloproliferative neoplasms (MPN) remains unclear. We analyzed NLR and PLR as prognostic markers for thrombosis and overall survival (OS) in the study that included 461 consecutive MPN patients who were diagnosed from 2018 to 2022 at University center. Twenty age-matched patients without hematological disorder were used as controls. NLR and PLR were significantly increased in whole MPN group compared to controls. NLR was highest in PV > PMF > ET (p < 0.001) while PLR was highest in ET > PMF > PV (p < 0.001). Thrombosis occurrence during follow-up correlated with NLR, NLR ≥ 4.5, presence of ≥ 2 CV factors and previous thrombosis. Arterial thrombosis was associated with previous thrombosis, NLR and NLR ≥ 4.5. Similarly in venous thrombosis previous thrombosis was risk factor, together with NLR, NLR ≥ 4.5, PLR, but also secondary malignancy and female gender. In multivariate Cox model, most important factors for thrombosis development during follow-up were previous thrombosis, NLR ≥ 4.5 and PLR ≥ 500; for arterial thrombosis, NLR ≥ 4.5 and previous thrombosis; for venous thrombosis PLR ≥ 500 and previous thrombosis. Patients with pre-PMF had significantly higher NLR than ET patients. In multivariate Cox regression model, most important factors associated with survival were NLR ≥ 4.5 and PLR ≥ 500. This study highlights strong prognostic correlation of NLR ≥ 4.5 and PLR ≥ 500 with development of thrombosis and OS in MPN. Besides previous thrombosis, most important factor associated with development of arterial thrombosis is NLR ≥ 4.5 and for venous PLR ≥ 500. Our results revealed that NLR ≥ 4.5 could be used as additional marker to distinguish ET from prePMF.

Momelotinib - a promising advancement in the management of myelofibrosis in adults with anemia.

Myelofibrosis (MF) is a rare BCR-ABL negative myeloproliferative neoplasm characterized by clonal proliferation of stem cells, with mutations in JAK2, CALR, or MPL genes. MF presents in primary and secondary forms, with common symptoms including splenomegaly, anemia, and thrombocytopenia. Diagnostic criteria involve bone marrow examination and mutation studies. Current treatments are limited, with allogeneic stem cell transplant as the only curative option. Recent FDA approval of Momelotinib (MMB) offers new promise for MF patients with anemia. MMB, a JAK1/2 and ACVR1 inhibitor, effectively reduces spleen size, improves hemoglobin levels, and decreases transfusion dependency. The MOMENTUM trial compared MMB to danazol in JAK inhibitor-treated MF patients with anemia, showing MMB's superior symptom relief and transfusion independence rates. Additionally, the SIMPLIFY-1 and SIMPLIFY-2 trials evaluated MMB in JAK inhibitor-naïve and experienced patients, respectively, confirming MMB's non-inferiority to ruxolitinib in spleen volume reduction and highlighting its benefits in transfusion requirements. MMB's unique dual inhibition mechanism addresses anemia by suppressing hepcidin production, thus enhancing erythropoiesis. These trials collectively suggest MMB as an effective treatment for MF, improving quality of life and offering a survival advantage for patients with anemia. Despite challenges, such as trial design limitations and adverse events, MMB represents a significant advancement in MF management, providing a new therapeutic option for a previously underserved patient population.

Myeloproliferative neoplasms in the adolescent and young adult population: A comprehensive review of the literature.

Myeloproliferative neoplasms (MPN) are characterized by a clonal proliferation of myeloid lineage cells within the bone marrow. The classical BCR-ABL negative MPNs are comprised of polycythaemia vera, essential thrombocythaemia and primary myelofibrosis. Historically, the majority of MPNs are diagnosed in adults older than 60 years of age; however, in recent years, there has been recognition of MPNs in the adolescent and young adult (AYA) population. AYAs with MPN, typically defined as between the ages of 15 and 39 years old, may comprise up to 20% of patients diagnosed with MPN. They demonstrate unique patterns of driver mutations and thrombotic events and remain at risk for progression to more aggressive disease states. Given the likely long length of time they will live with their disease, there is a significant unmet need in identifying well-tolerated and effective treatment options for these patients, particularly with the advent of disease modification. In this review, we provide a comprehensive overview of the clinical features, disease course and management of AYA patients with MPN and, in doing so, highlight key characteristics that distinguish them from their older counterparts.

JAK2, CALR, and MPL Mutation Profiles in Colombian patients with BCR-ABL Negative Myeloproliferative Neoplasms.

Among the chronic myeloproliferative neoplasms (MPNs) not associated with BCR-ABL mutations are polycythemia vera, primary myelofibrosis, and essential thrombocythemia. These diseases are caused by mutations in genes, such as the JAK2, MPL, and CALR genes, which participate in regulating the JAK-STAT signaling pathway. This study aimed to establish the frequencies of mutations in the JAK2, MPL, and CALR genes in a group of Colombian patients with a negative clinical diagnosis of BCR-ABL chronic myeloproliferative neoplasms. The JAK2 V617F and MPL W515K mutations and deletions or insertions in exon 9 of the CALR gene were analyzed in 52 Colombian patients with polycythemia vera, primary myelofibrosis, and essential thrombocythemia. The JAK2V617F mutation was carried by 51.9% of the patients, the CALR mutation by 23%, and the MPL mutation by 3.8%; 23% were triple-negative for the mutations analyzed. In these neoplasms, 6 mutation types in CALR were identified, one of which has not been previously reported. Additionally, one patient presented a double mutation in both the CALR and JAK2 genes. Regarding the hematological results for the mutations, significant differences were found in the hemoglobin level, hematocrit level, and platelet count among the three neoplasms. Thus, this study demonstrates the importance of the molecular characterization of the JAK2, CALR and MPL mutations in Colombian patients (the genetic context of which remains unclear in the abovementioned neoplasms) to achieve an accurate diagnosis, a good prognosis, adequate management, and patient survival.

Interferon-Alpha Impairs Neoplastic Vasculogenesis in a 3D iPSC-Based Model of JAK2V617F+ Myeloproliferative Neoplasms

Overt Myelofibrosis (MF) is an end-stage subtype of BCR-ABL1 negative myeloproliferative neoplasms (MPN), which is characterized by progressive bone marrow (BM) fibrosis resulting in loss of normal hematopoietic support as well as inflammatory and vascular complications. While aberrant neoangiogenesis is known to participate in the pathogenesis of MF, Erba et al. (Am J Pathol, 2017) suggested the occurrence of endothelial-to-mesenchymal transition (EndMT) in BM and spleen of MF patients (pts). Given the relevance of endothelial cells (EC) for MF development and the occurrence of JAK2V617F-mutated EC in MPN pts, targeting of the vascular niche has emerged as a promising therapeutic strategy. To untangle the impact of the JAK2V617F mutation on human vasculogenesis, we leveraged high-purity and clonal patient-specific induced pluripotent stem cell (iPSC)-derived EC (iEC) harboring either the JAK2V617F mutation in hetero- (JAK2V617F HET) or homozygosis (JAK2V617F HOM). JAK2 WT and CRISPR-repaired isogenic JAK2 WT (both referred to as JAK2 WT) were used as controls. In a first set of in vitro experiments, JAK2V617F mutated iEC showed a significantly increased proliferation rate in comparison to JAK2 WT iEC. We next evaluated the effect of selected drugs on iEC viability to assess their vascular toxicity. While treatment with 1 µM each of fedratinib, anagrelide or venetoclax led to a decrease in iEC viability (all **p ≤ .01), no significant alterations were observed with 1 µM ruxolitinib (RUX) or 1000 U/ml interferon-alpha (IFNa). Overall, no differences between the 3 genotypes were observed, pointing towards a genotype-independent mode of action. Given the favorable vascular toxicity profile of IFNa, as shown above, its anti-angiogenic potential was then assessed via endothelial tube formation assays. Pre-incubation with 1000 U/ml IFNa prior to seeding of the cells onto a reconstituted basement membrane matrix led to a significant inhibition of capillary-like structures formation in JAK2 WT and JAK2V617F HET or HOM iEC. One of the limitations of 2D culture assays is the lack of 3D homotypic cellular organization, compromising its translational value. Therefore, we established an adaptive 3D fibrin-based model to recapitulate, in vitro, the critical EC-pericyte crosstalk required for formation and maturation of blood vessels. Recent studies highlighted the capacity of BM mesenchymal stem cells (MSC) to differentiate into pericytes. Therefore, we seeded JAK2 WT and JAK2V617F HETor HOM iEC with or without MSC in fibrin hydrogels. MSC and iEC were visualized using different PKH dyes as fluorescent cell linkers to enable live tracking of vascular tree formation. Although self-assembly of JAK2 WT and JAK2V617F HET or HOM iEC into vessel-like structures was observed in the generated monocultures, the presence of MSC in the cocultures led to a significant increase of the formed network area (Fig. 1), hence recapitulating the critical support of pericytes. While acute IFNa exposure did not alter vascular tree formation (Fig. 1), a separate pre-treatment of iEC and MSC prior to cocultivation, significantly impaired neoplastic vasculogenesis (Fig. 2). Interestingly, interactive microscopy image analysis revealed an increased MSC/iEC interaction in JAK2V617F HOM pre-treated cocultures (*p ≤ .05). Bulk RNA-sequencing of JAK2 WT and JAK2V617F HET or HOM iEC treated for 24h with 1000 U/ml showed a strong response to IFNa. Future analysis may unravel a JAK2V617F specific transcription signature. Moreover, we induced EndMT in our 3D iEC-MSC coculture model by treatment with pro-inflammatory cytokines, reflected by the loss of EC markers (e.g. Claudin 5) and upregulation of mesenchymal markers (e.g. aSMA) by immunofluorescence. We are currently evaluating the capacity of RUX vs IFNa to inhibit EndMT. In summary, we show that IFNa impairs neoplastic vasculogenesis in a robust in vitro 3D coculture system that enables precise control of cellularity and genotype. Our model is a valuable tool to decipher the poorly investigated impact of IFNa on the BM microenvironment. In addition, its translational and scalable properties (e.g. by integrating primary or iPSC-derived hematopoietic cells) will allow us to study the impact of additional drugs on MPN-associated fibrotic transformation of the BM.

Assessing Epidemiological Differences in Myelofibrosis: A Comparative Study of a Single-Center Cohort and the SEER National Database

Background Myelofibrosis (MF) is a rare class of BCR-ABL negative myeloproliferative neoplasms characterized by clonal proliferation of hematopoietic stem cells and progressive fibrosis of the bone marrow. This study attempts to elucidate epidemiological discrepancies in survival outcomes between MF patients at a single tertiary care center from an underserved population in the Bronx (which ranks last in health outcomes amongst 62 New York state counties) versus the Survival, Epidemiology, and End Results (SEER) national cancer registry by analyzing different population parameters. Methods Our retrospectively constructed single center database included 96 patients with biopsy confirmed primary or secondary MF who were age ≥20 diagnosed after 2000. 75 remained for analysis after excluding subjects without known race or date of diagnosis. The SEER [SEER research plus data, 17 registries, Nov 2022 Sub (2000-2020)] dataset comprised of 5254 patients with microscopically confirmed primary MF (ICD code: 9661.3) diagnosed between 2000 and 2015, had age of diagnosis ≥20, and whose deaths were attributable to MF diagnosis. Both cohorts were independently analyzed via log-rank testing and/or cox proportional hazards (cox-PH) regression with death as event outcome. Results Mean age of our single-center cohort was 65.87 years. Males [37 (49.3%)] and females [38 (50.7%)] were approximately evenly distributed. Races included non-hispanic white (NHW) [26 (34.7%)], NH black (NHB) [20 (26.7%)], NH asian and pacific islanders (NH-API) [6 (8.0%)], and hispanics of all races [23 (30.7%)]. Primary [40 (53.3%)] and secondary [35 (46.7%)] MF were approximately even. Log rank testing detected significance in race (p=.05) but not sex (p=.2) or type (p=.7). Significant univariate cox-PH findings include increased mortality within the NHB group [hazard ratio (HR) 2.69, p=.04] and increased mortality with age (HR 1.07, p=.005) (Table 1). Low event size precluded multivariate analysis. In the SEER cohort, age was grouped into 4 categories: ≤44 years old [214 (4.1%)], 45-64 [1,590 (30.3%)], 65-84 [3,051 (58.1%)], and ≥85 [399 (7.6%)]. There were more females [3,159 (60.1%)] than males [2,095 (39.9%)]. NHW [3,995 (76.0%)] comprised the overwhelming majority of races reported, followed by NHB [441 (8.4%)], hispanic [420 (8.0%)], and NH-API [398 (7.6%)]. Median household income (MHI) data (inflation adjusted to 2021) was divided into &amp;lt;$70,000 and ≥$70,000 (table 2). Univariate cox-PH noted improved survival in NH-APIs (HR .75, p&amp;lt;.001) and MHI ≥$70K (HR .92, p=.05). However this significance disappeared upon adjustment for age and race. In multivariate cox-PH, age demonstrated significance for each category (45-64 HR 2.20; 65-84 HR 4.45; ≥85 HR 6.59, p &amp;lt; .001). Female patients had better survival compared to males (HR .73, p&amp;lt;.001) (Table 2). Conclusion In this comparison study of MF cohorts from an underserved tertiary center and SEER, we found differences in demographic features and their impact on disease mortality. NHW patients constitute the overwhelming majority of subjects in the SEER dataset while the tertiary cohort has nearly equal distribution of NHW, NHB, and hispanic patients. NHB patients from the single-center cohort tended to do worse while SEER showed no differential survival based on race. Possible explanations for this include differences in racial distribution, healthcare accessibility, and other socioeconomic factors. Median income in the Bronx is 36% lower than the national average and the poverty rate is 2.3x higher. Unexpectedly, no significant association was found between MHI and survival in SEER when adjusted for age and race. Also notable is that improved survival in NH-APIs per SEER data disappeared upon adjustment for age, which may suggest that the NH-API group tends to be younger at diagnosis given that age serves as a negative prognostic marker. Further research is warranted. Regarding sex, SEER data suggests that female patients have a decreased mortality risk compared to males which is consistent with literature. Caution in interpretation is necessary given the low tertiary center sample size and the inherent limitations of the SEER dataset.

Drivers and high-molecular-risk mutations in Argentine patients with primary myelofibrosis

Primary myelofibrosis (PMF), a BCR-ABL negative myeloproliferative neoplasm, is a heterogeneous clinical and genetic disorder with a poor prognosis. We aimed to study the mutational profile of driver genes (JAK2, CALR, and MPL) and high-molecular-risk (HMR) genes (ASXL1, EZH2, IDH1/2, and SRSF2) and their prognostic impact in 65 Argentine patients with PMF. Mutually exclusive driver mutations were identified in 88% of the cases and HMR mutations were detected in 37% of patients, including 37.5% of triple negative cases. ASXL1 and SRSF2 were the most frequently mutated HMR genes, and a significant association between these mutations was observed (P = 0.04). Additionally, age &gt;65 years, lower hemoglobin level, constitutional symptoms, male sex, and bone marrow (BM) fibrosis grade ≥ 2 were correlated with ASXL1 mutations, and circulating blasts with SRSF2 mutations. HMR mutations were significantly associated with inferior overall survival (P &lt; 0.001). These mutations were more frequent with increasing risk of DIPSS score and allowed us to identify patients with poor survival in intermediate DIPSS groups. The application of the MIPSS70 score stratified our patients into three risk groups, with significant differences in overall survival (P &lt; 0.001). Interestingly, 85% of the patients who were upgraded to a higher risk category compared to the DIPSS score showed a HMR profile. In conclusion, our study contributes to the understanding of the mutational landscape in MFP, supports the integration of molecular data to improve prognostic models and reinforces the adverse prognostic impact of HMR mutations, particularly in ASXL1 and SRSF2 genes.

Moving toward disease modification in polycythemia vera

AbstractPolycythemia vera (PV) belongs to the BCR-ABL1–negative myeloproliferative neoplasms and is characterized by activating mutations in JAK2 and clinically presents with erythrocytosis, variable degrees of systemic and vasomotor symptoms, and an increased risk of both thromboembolic events and progression to myelofibrosis and acute myeloid leukemia (AML). Treatment selection is based on a patient’s age and a history of thrombosis in patients with low-risk PV treated with therapeutic phlebotomy and aspirin alone, whereas cytoreductive therapy with either hydroxyurea or interferon alfa (IFN-α) is added for high-risk disease. However, other disease features such as significant disease-related symptoms and splenomegaly, concurrent thrombocytosis and leukocytosis, or intolerance of phlebotomy can constitute an indication for cytoreductive therapy in patients with otherwise low-risk disease. Additionally, recent studies demonstrating the safety and efficacy (ie, reduction in phlebotomy requirements and molecular responses) of ropegylated IFN-α2b support its use for patients with low-risk PV. Additionally, emerging data suggest that early treatment is associated with higher rates of molecular responses, which might eventually enable time-limited therapy. Nonetheless, longer follow-up is needed to assess whether molecular responses associate with clinically meaningful outcome measures such as thrombosis and progression to myelofibrosis or AML. In this article, we provide an overview of the current and evolving treatment landscape of PV and outline our vision for a patient-centered, phlebotomy-free, treatment approach using time-limited, disease-modifying treatment modalities early in the disease course, which could ultimately affect the natural history of the disease.

MPN-058 Diagnostic and Prognostic Utility of Flow Cytometric Enumeration of Aberrant hMICL–Expressing Circulating Leukemic Stem Cells in BCR-ABL1–Negative Myeloproliferative Neoplasms

MPN-058 Diagnostic and Prognostic Utility of Flow Cytometric Enumeration of Aberrant hMICL–Expressing Circulating Leukemic Stem Cells in BCR-ABL1–Negative Myeloproliferative Neoplasms

Concomitant BCR-ABL and JAK2 V617F in a Patient with Myeloproliferative Neoplasm: A Case Report

Myeloproliferative neoplasm (MPN) is a clonal proliferation of the haematopoietic stem cells leading to activated tyrosine kinase signaling activity. Myeloproliferative neoplasm is classified into BCR-ABL positive chronic myeloid leukemia (CML) and BCR-ABL negative MPN which harbors JAK2 V617F mutation in most cases. The genetic combination of BCR-ABL and JAK2 V617F mutation is rare with estimated frequency of 0.4% based on recent study. Herein, we reported a case of a man diagnosed with CML detected by fluorescence in-situ hybdridisation (FISH) showing atypical BCR-ABL fusion pattern in 29% nucleated cells (cut-off levels ≥5% for positive signals) in the presence of JAK2 V617F mutation. However, the BCR-ABL transcript was not detected by the conventional reverse transcriptase-polymerase chain reaction (RT-PCR) method which was specific for major fragments. Interestingly, complete hematological remission was not achieved despite initiation of tyrosine kinase inhibitor (Imatinib). In conclusion, it is imperative to scrutinise CML cases for concomitant JAK2 V617F mutation especially patients with atypical clinical or laboratory findings. Therefore, close monitoring with clinical and ancillary technique especially FISH and molecular methods such as DNA sequencing were crucial to help achieve complete hematological, cytogenetic and deep molecular response alongside targeted therapy.

Abstract A38: Pediatric Germline CSF3R T618I mutation gives insight into disease progression and long-term therapy options of Chronic Neutrophilic Leukemia

Abstract Chronic Neutrophilic Leukemia (CNL) is a rare BCR-ABL negative myeloproliferative neoplasm that affects approximately 1 in a million individuals. The typical age of onset and disease presentation is in elderly patients with a median age of 63 and a range of 9-84. The oncogenic driver of CSF3R, the G-CSF receptor protein, in CNL has been classified over the last decade and has improved therapies for patients with targeted Tyrosine Kinase Inhibitors, such as Ruxolitinib (a potent JAK1/2 inhibitor). Currently, the only curative option is an allogeneic hemopoietic stem cell transplant with typical median overall survival of 2 years. Because of the low occurrence rate of CNL, there have only been 3 germline cases of CSF3R classified in literature. With these patients, they exhibit a lifelong skewing of the myeloid lineage causing gross neutrophilia. Our patient presented at 13 months with leukocytosis and neutrophilia, resulting in genetic testing revealing the diagnosis of the youngest documented case of CNL. A germline CSF3R T618I constitutive activating mutation was documented in our patient and in her father. Her father had documented lifelong leukocytosis of indeterminate origin and it was not until 33 that genetic testing was done, and he was diagnosed with CNL. He underwent an allogeneic stem cell transplant after being admitted with gross hepatosplenomegaly with a WBC of 322, the highest recorded in a germline CNL patient in literature. Our patient presented to clinic with leukocytosis ranging from 45-98 and hepatosplenomegaly. After a bone marrow aspirate and biopsy was taken to determine disease stage and severity, we immediately began Ruxolitinib therapy. We began 20 mg BID dosing based on platelets and monitored response to therapy while conducting targeted RNA-seq for myelodysplastic and myeloid leukemia genes on the bone marrow, which did not detect any secondary mutations. Ruxolitinib dosing was tapered down in a stepwise fashion, until the patient arrived at the current dose of 7.5 mg BID Ruxolitinib, resulting in stable leukocyte counts around 25. As part of monitoring, we are currently conducting 6-month BM procedures and NGS for AML/MDS genes and Ruxolitinib resistance mutations alongside monthly CBCs. Our patient has been on Ruxolitinib for 9 months and has not developed any secondary contributing mutations and remains sensitive to therapy. We continue to monitor our patient’s response to Ruxolitinib to determine if germline predisposition alters the transformative nature of the disease. Within T618I germline CNL cases, we are investigating the difference in phenotype and disease burden that we observe in our patient’s family compared to literature. Based on current cases, it seems that germline CNL does not transform during childhood and adolescence into AML without the influence of a secondary mutation. Retrospective studies into previous cases of CNL to determine if there is a higher frequency of germline origin could pave the way for potential early-stage therapies and detection of CNL prior to transformation. Citation Format: Kevin Zhang, Matthew Rees, Mahsa Khanlari, Laura Rooms, Raul Ribeiro, Marcin Wlodarski. Pediatric Germline CSF3R T618I mutation gives insight into disease progression and long-term therapy options of Chronic Neutrophilic Leukemia [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A38.

Study of JAK2 V617F allele Burden By Droplet Digital PCR in BCR-ABL1 Negative Myeloproliferative Neoplasms and Its Implications on Disease Phenotype

JAK2V617F mutation is the most common mutation seen among the BCR-ABL1 negative myeloproliferative neoplasms, the incidence of which is 90-95% in PV, 50-70% in ET and 40-50% in PMF. The discovery of the association of JAK2 V617F mutation with all the above mentioned myeloproliferative neoplasms and MPN-U boggled the minds of many scientists on how a single gene mutation can cause different disorders, and it was later reported that the allele burden determines the clinical phenotype of the disease. With the advent of droplet digital polymerase chain reaction(ddPCR), even a small quantity of JAK2 V617F allele burden could be detected and measured, facilitating an earlier definitive diagnosis and hence early treatment. In addition, with the rise in use of JAK2 inhibitors such as Ruxolitinib for treating high risk patients, the quantification of allele burden could probably play a role in defining treatment guidelines for usage of these drugs. Method: After examination of bone marrow aspirate smears and biopsies in clinically suspected cases of MPN, molecular testing and allele burden quantification of JAK2 V617F mutation was done using ddPCR. The objectives of our study was to correlate the allele burden with the clinical phenotype, it's implication on the symptom burden and hematological parameters and it's correlation with the prognosis of patients. The symptom burden was measured using MPNTEN Score, whereas the prognosis for PV and ET was done based on Prognostic risk score for thrombosis. Prognostication of PMF was done using IPSS and DIPSS. Results: In our study, among 50 patients, 50% (n=25) were diagnosed as PV, 30% (n=15) were diagnosed as Primary myelofibrosis, 16 % as ET (n=8) and 4% (n=2) as MPN-U. The mean allele burden of PV was highest (64.5%), closely followed by PMF at 61.65% and lowest in ET (31.17%). The allele burden showed significant (p<0.05) positive correlation with the symptom burden in patients. It was also found that a significant association between presence of para trabecular megakaryocyte localization and allele burden exists (p value of 0.02). It was observed that a significant correlation exists between previous history of thrombosis with increased allele burden at presentation (p<0.05). However, the allele burden quantification at presentation and prognostic scores of PV, ET and PMF did not show significant correlation. Conclusion: To conclude, our study reports that the allele burden varies according to the clinical phenotype, has a bearing on the symptom burden of patients, has morphological implications and has a strong correlation with prior thrombotic episodes. Follow up of these patients based on remission criteria for PV, ET and PMF are being done and will be presented.

Diagnostic and Prognostic Utility of Flowcytometric Enumeration of Circulating Stem Cells with h-Micl Expression in BCR-ABL1-Negative Myeloproliferative Neoplasms

Background: The classical BCR-ABL1-negative Myeloproliferative Neoplasms (MPNs) are clonal haemopoietic stem cell disorders, include Primary Myelofibrosis (PMF), Polycythemia Vera (PV) and Essential thrombocythemia (ET).The lack of specific discriminator makes the subcategorization of BCR-ABL1 negative Myeloproliferative Neoplasms very challenging, especially in distinguishing ET from prefibrotic MF cases. The emphasis on the need for an accurate diagnosis is because of differences in clinical outcome and therapy. Recently, h-MICL (also termed CLEC12A, CLL-1, or CD371) is emerging as a promising marker of leukemic stem cells (LSC) in AML. Its most prominent features is its lack of expression on CD34+CD38-cells in normal bone marrow, regenerating bone marrow and G-CSF stimulated peripheral blood stem cells, making it a highly specific marker for LSCs. Recent studies claim that its specificity for LSCs makes it a promising target for future targeted therapy as well. Thus, h-MICL can turn out to be a crucial marker in the diagnostics of MPN for discriminating MPN phenotypes. Aims & Objectives:To evaluate the utility of flowcytometric enumeration of circulating CD34+CD38- hMICL+ cells in subcategorisation of BCR-ABL1 negative MPNs.To define a discriminatory cut-off of aberrant h-MICL expression at the stem cell level for accurate identification of PMF patients and to especially differentiate early prefibrotic PMF and ET.To correlate aberrant h-MICL+ stem cell subset with Dynamic International Prognostic scoring System (DIPSS score). Materials & Methods: A total of 54 BCR-ABL1 negative MPN patients, including 15 PV, 13 ET ,13 Overt MF, 8 early prefibrotic stages of Primary MF, and 5 Post-PV/ET MF, who presented in the clinics of Hematology department of a tertiary care center in North India were included in the study. Bone marrow examination and mutational analysis were carried out as part of diagnostic workup. Peripheral blood flowcytometric evaluation was done. The samples were processed using stain-lyse-wash method. Flowcytometric analysis was done on BD FACS Canto II (Becton Dickinson, San Jose, CA, USA) using BD FACSDiva™ v8.1.3 software.The subsets analysed were:CD34+ (hematopoietic stem cell enriched population)CD34+CD38+ (committed progenitors)CD34+CD38- (stem cells)The aberrant CD34+CD38-hMICL+ population The aberrant hMICL expression on CD34 + CD38- subsets were correlated with DIPSS score too. Results: Our study included 54 BCR-ABL1 negative MPN patients (42 males, 12 females) with a median age of 49.5 years. A significant difference in distribution of proportion of CD34+CD38-hMICL+ among the MPN sub-groups was found. The median of CD34+CD38-hMICL+cells subset in Overt MF, early stage/ pre PMF and Post-PV/ET MF group was 2.8%, 4.15% and 3% respectively, which was significantly more than PV and ET (p<0.001). Discriminatory cut off of Proportion of CD34+CD38-hMICL+ subset ≥0.9 predicted MF with sensitivity and specificity of 88% and 89%respectively. Cut off to differentiate early/ prefibrotic MF from ET was found to be ≥2 (sensitivity:87.5%;specificity:100%) We found a statistically significant correlation (rho = 0.41, p = 0.036) between proportion of CD34+CD38-hMICL+ cells and DIPSS score. For every 1 unit increase in DIPSS Score, CD34+CD38-hMICL+ cells increased by 1.72%. Discussion & Conclusion: Subcategorization of BCR-ABL1 negative MPNs by morphology alone can be perplexing due to overlapping features and interobserver reproducibility. Driver mutations do not differentiate subtypes. In this study we conclude that CD34+ CD38- hMICL+ subset is reliable for subcategorization of BCR ABL1-negative MPNs. We defined a cut-off to discriminate MF from non-MF MPNs, also prefibrotic MF from ET. To the best of our knowledge, there is only one similar study by Herborg LL, et al (2018). They proposed a cut off of >0 cells as discriminator, with 80% sensitivity and 97% specificity. However, they did not distinguish early stage/prefibrotic PMF and Post-PV/ ET MF. Moreover, study of the correlation of aberrant hMICL expression with any prognostic scoring system is not done. Hence our study is unique and very first of its kind in this regard. We conclude that flowcytometric enumeration of circulating CD34+CD38- h-MICL+ cell subset holds a potential as a robust diagnostic test, tool for prognostication, an indicator of disease progression and a potential target for therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Micheliolide Exerts Therapeutic Effect in Myeloproliferative Neoplasms through Binding to STAT3 and STAT5 Proteins and Inhibiting Their Activities

The JAK1/2 inhibitor ruxolitinib is a cornerstone of management for some subsets of BCR-ABL negative myeloproliferative neoplasms (MPN). While many patients benefit with reduced splenomegaly and symptoms, some patients respond suboptimally to ruxolitinib, and even patients with a good initial response often develop resistance to ruxolitinib after several years of therapy. Here, we evaluated the efficacy of micheliolide (MCL), a natural guaianolide sesquiterpene lactone, in the JAK2V617F positive MPN entities. We firstly evaluated the effect of MCL treatment on cell growth and survival in human MPN cell lines expressing the JAK2V617F mutation. MCL alone reduced cell viability and induced apoptosis of UKE1 and SET2 cells in a dose-dependent manner, and obviously improved the effect of ruxolitinib as monotherapy. Importantly, treatment with MCL also induced significant apoptosis of UKE1 cells which were selected for resistance against ruxolitinib. We further explored the therapeutic potential of MCL in vivo using a Jak2V617F knock-in (Jak2VF) mouse model, and found that DMAMCL, an orally available derivative of MCL, significantly increased ruxolotinib efficacy in Jak2VF mice, manifested as amplified hematological and spleen responses. What's more, DMAMCL, whereas not ruxolitinib, contributed to reduce the in vivo Jak2V617F mutant allele burden reflected by the percentage of mutant hematopoietic cells in peripheral blood and bone marrow. To determine the molecular mechanisms by which MCL exerts efficacy in MPN, we firstly performed western blot (WB) anlyses to evaluate the effect of MCL on hyper-activated JAK-STAT signaling, which is the cornerstone of MPN pathogenesis. We observed a dose-dependent inhibition of STAT3 and STAT5 phosphorylation caused by MCL treatment in UKE1 and SET2 cells (Figure 1A), and as expected, combination MCL with ruxolitinib provided an enhanced inhibitory effect. By next performing the affinity pull-down experiment, we confirmed that MCL suppressed the activation of STAT3 and STAT5 mainly through directly binding to the proteins (Figure 1B). We further conducted RNA-sequencing analysis in UKE1 cells treated with MCL and/or ruxolitinib to gain insights into mechanisms involved in MCL effect. Gene set enrichment analysis (GSEA) showed that genes related to TNF-α-NF-κB signaling and inflammatory response pathway were significantly downregulated upon treatment with MCL, that were verified by WB analyses and real-time quantitative PCR. Additionally, GSEA also revealed significant upregulation of genes related to reactive oxygen species (ROS) and unfolded protein response (UPR) pathway, respectively. Both flow cytometry and fluorescent microscopy imaging confirmed the ROS accumulation by MCL treatment. We also determined the activation of PERK branch of UPR pathway caused by MCL, as assessed by increased phosphorylation of eIF2α, and upregulation of pro-apoptotic genes, including ATF4, CHOP and GADD34. Finally, by addition of antioxidant agent, the activation of UPR pathway and increase of apoptosis in UKE1 cells induced by MCL were significantly rescued, indicating that MCL exerts cytotoxic effects mainly through triggering the oxidative stress in MPN cells. In summary, our results demonstrate that MCL is an effective agent with multiple biological activities in MPN, especially inhibition of STAT3/5 activity via directly binding to the proteins, and may be a promising drug combined with ruxolitinib to benefit patients who have suboptimal response or acquire resistance to ruxolitinib. Figure 1. MCL inhibits STAT3 and STAT5 phosphorylation through binding to the proteins directly. (A) Western blot analyses are shown for phosphorylated-STAT3 (p-STAT3), STAT3, phosphorylated-STAT5 (p-STAT5) and STAT5 levels in total cell extracts from UKE1 and SET2 cells treated with MCL (5, 10, 20μM) for 3 hours. (B) The biotin-conjugated MCL (biotin-MCL) and its inactive analogue S-MCL (biotin-S-MCL) were prepared as the positive- and negative- probe respectively. UKE1 and SET2 cell lysates were incubated with 20μM biotin-MCL or biotin-S-MCL for 1 hour at room temperature, then the mixtures were separately pulled down with streptavidin-coated agarose beads, and STAT3/STAT5 were detected with WB analysis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Polycythemia Vera leading to Jaundice and Marantic Endocarditis, A Case Presentation

Polycythemia vera is an uncommon myeloproliferative neoplasm characterized by the abnormal proliferation and maturation of hematopoietic stem or progenitor cells. It is also known as one of the BCR-ABL1 negative myeloproliferative neoplasms, along with essential thrombocythemia and myelofibrosis. Incomparison to essential thrombocythemia and myelofibrosis, polycythemia is driven by JAK2 mutations, specifically JAK2V617F, an activating mutation encoding a tyrosine kinase [1]. This variant mutation is correlated with increased erythropoiesis and myelopoiesis, lower platelet count, higher incidence of splenomegaly, pruritus and thrombotic events. A 58-year-old male presented to the Emergency Department complaining of pruritus and painless jaundice. Comprehensive blood count revealed an elevated white blood cell count, hemoglobin/ hematocrit, and platelets. Total bilirubin was elevated at 30.3g/dL, with a direct bilirubin of 27g/dL. A Jak- 2 mutation was positive, which suggested a diagnosis of polycythemia vera although atypical given his jaundice. An Erythropoietin level was found to be less than 1, suggesting a primary cause of polycythemia vera. In addition, the patient was found to have marantic nonbacterial thrombotic endocarditis on transesophageal echocardiogram. Patient was treated with aspirin to prevent thrombotic events, hydroxyurea and cholestyramine. The patient’s physical examination of profuse jaundice along with an elevated direct bilirubin count of 27g/dL typically does not correlate to a presentation of polycythemia vera. Through further workup, the patient did not demonstrate hemolytic or intrahepatic causes, both intrinsic and extrinsic to the liver, which would of explained the jaundice presentation. This led to a conclusion that the patient’s polycythemia vera most likely caused the jaundice presentation.

P1020: SCREENING FOR PORTAL HYPERTENSION IN PATIENTS WITH PHILADELPHIA NEGATIVE MYELOPROLIFERATIVE NEOPLASMS

Background: Portal hypertension (PH) is a well-recognized complication of BCR-ABL negative myeloproliferative neoplasms (MPNs) and is associated with significant morbidity and mortality. However, the prevalence of PH and its associated risk factors have not been studied systematically in this patient population. Screening by esophagogastroduodenoscopy (EGD) can facilitate early diagnosis of PH and opportunity for intervention to prevent complications. Aims: To investigate the prevalence of PH and its patient and disease-related risk factors. Methods: From May 2013 to March 2019, MPN patients ≥ 18 years old with spleen >5 cm below the costal margin in myelofibrosis (MF) or any size in Polycythemia Vera (PV) or Essential Thrombocythemia (ET) were enrolled prospectively (NCT01816256). Patients with a known history of portal or hepatic vein thrombosis, liver cirrhosis, and esophageal varices were excluded. Screening for PH was done by EGD to evaluate for esophageal varices (EV) or gastric varices (GV), and portal hypertensive gastropathy (PHG). Abdominal Doppler ultrasonography (USG) was performed to evaluate for portal vein thrombosis and spleen size. PH severity was based on the size of varices or severity of PHG. For sample-size calculation, a precision approach was used, considering scenarios where PH prevalence ranged from 5 to 10% and an upper limit for sample size of 100. At a prevalence estimate of 8%, the 90% confidence interval (CI) was 4-14%, with an acceptable lower limit for moving forward with a screening program. Prevalence of PH was calculated as a proportion and CI are exact using Clopper-Pearson method. Results: Among 102 enrolled patients, EGD and USG were completed in 85 and 84, respectively. Primary and Post-PV/ET MF comprised the majority of the study population (93% [79/85]). Evidence of PH was found in 32 patients (38%, [90% CI 29-47]) of whom 12 (14% [90% CI 9-22]) had moderate to severe PH. EV, GV, and PHG were found in 81% (26/32), 13% (4/32), and 31% (10/32) of patients, respectively. The prevalence of PH among MF and PV/ET patients was 35% (28/79) and 67% (4/6), respectively. In MF patients, PH was moderate-to-severe in 13% (90% CI 8-20). No cases of portal vein thrombosis were identified on USG. Apart from a palpable liver observed more frequently in non-PH patients, there were no other significant differences in baseline characteristics among patients with and without PH. The frequency of JAK2, MPL and CALR mutations among PH patients was 78% (25/32),13% (4/32), and 6% (2/32). There was a trend towards more JAK2 mutations in PH patients (78% [25/32] vs 64% [34/53], p=0.23), but no difference in DIPSS risk or frequency of high molecular-risk mutations. Overall survival was similar in both groups. PH-directed interventions were initiated in 59% (19/32); 28% (9/32) were started on beta blocker (BB) prophylaxis, 16% (5/32) were treated with BB and variceal banding, and 16% (5/32) continued on surveillance EGDs alone. Three patients with moderate PH at baseline developed variceal bleeding (2) and ascites (1). All 3 patients were on BB prophylaxis, and 1 had prior prophylactic banding. There were no adverse events related to study procedures EGD or USG. Image:Summary/Conclusion: Given the study population, our results are generalizable to MF patients. Our study shows that clinically significant PH is common in MF patients, and led to additional clinical interventions in significant number of patients. These data support the inclusion of asymptomatic PH screening in the work up of MF patients.

The Use of Allogeneic Hematopoietic Stem Cell Transplantation in Primary Myelofibrosis.

Primary myelofibrosis (PMF) is a BCR-ABL1 negative myeloproliferative neoplasm characterized by clonal proliferation of myeloid cells. This leads to reactive bone marrow fibrosis, ultimately resulting in progressive marrow failure, hepatosplenomegaly, and extramedullary hematopoiesis. PMF is considered the most aggressive of the BCR-ABL1 negative myeloproliferative neoplasms with the least favorable prognosis. Constitutional symptoms are common, which can impact an individual’s quality of life and leukemic transformation remains an important cause of death in PMF patients. The development of the Janus kinase 2 (JAK2) inhibitors have provided a good option for management of PMF-related symptoms. Unfortunately, these agents have not been shown to improve overall survival or significantly alter the course of disease. Allogenic hematopoietic stem cell transplantation (allo-HSCT) remains the only curative treatment option in PMF. However, allo-HSCT is associated with significant treatment-related morbidity and mortality and has historically been reserved for younger, high-risk patients. This review examines patient, disease, and transplant-specific factors which may impact transplant-related outcomes in PMF. Through the vast improvements in donor selection, conditioning regimens, and post-transplant care, allo-HSCT may provide a safe and effective curative option for a broader range of PMF patients in the future.

Renal disease associated with chronic myeloproliferative neoplasms

Patients with BCR-ABL negative chronic myeloproliferative neoplasms (MPNs) present with chronic kidney disease (CKD) in up to one third of cases. These patients are known to have increased cardiovascular risk and usually present with features of stronger myeloproliferation. In recent years CKD has gained attention in MPN patients as a potential renal manifestation of the disease termed MPN-related glomerulopathy. This view has been supported mainly by evidence from kidney biopsies and renal post-mortem autopsy findings of MPN patients, as well as by clinical observations of improvement/slower deterioration of renal function after institution of specific therapies in some MPN cohorts. All current evidence is based only on retrospective datasets which are known to be susceptible to different types of bias. Prospectively designed interventional studies are needed to properly understand true effects of CKD and its control by differently targeted (CKD and MPN directed) therapies.

Transcriptional configurations of myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPNs) is an umbrella term for several heterogenous diseases, which are characterized by their stem cell origin, clonal hematopoiesis and increase of blood cells of the myeloid lineage. The focus will be on BCR-ABL1 negative MPNs, polycythemia vera (PV), primary myelofibrosis (PMF), essential thrombocythemia (ET). Seminal findings in the field of MPN were driven by genomic analysis, focusing on dissecting genomic changes MPN patients. This led to identification of major MPN driver genes, JAK2, MPL and CALR. Transcriptomic analysis promises to bridge the gap between genetic and phenotypic characterization of each patient's tumor and with the advent of single cell sequencing even for each MPN cancer cell. This review will focus on efforts to mine the bulk transcriptome of MPN patients, including analysis of fusion genes and splicing alterations which can be addressed with RNA-seq technologies. Furthermore, this paper aims to review recent endeavors to elucidate tumor heterogeneity in MPN hematopoietic stem and progenitor cells using single cell technologies. Finally, it will highlight current shortcoming and future applications to advance the field in MPN biology and improve patient diagnostics using RNA-based assays.