JAK2 V617 Mutation Research Articles

A Real World, Single-Centre Study of Young Adult Patients with Philadelphia Negative Myeloproliferative Neoplasms

Classical ‘Philadelphia negative’ myeloproliferative neoplasms (MPNs) are clonal haematopoietic disorders and include essential thrombocythaemia (ET), polycythaemia vera (PV) and myelofibrosis (MF). MPNs have a heterogenous phenotype with an inherent risk of thromboembolic and haemorrhagic complications, and risk of blastic transformation. MPNs are commonly diagnosed in the 6 th decade or later, although up to 20% of patients are diagnosed < 40 years. Current risk stratification and treatment guidance, however, is frequently extrapolated from older cohorts. Here, we present a comprehensive retrospective analysis of 107 MPN adolescent and young adult (AYA) patients diagnosed <40 years, representing 19.6% of the total outpatient MPN cohort at University College London Hospital. Median follow up was 78 months (range (r), 1 - 507). Table 1 displays clinicopathological details of the entire cohort. Median age at diagnosis was 32 years (r,13-40). There was a female preponderance (66%); 57.9% of patients were Caucasian. Most patients had a diagnosis of ET (57.9%), with 24.3% PV and 9.3% MF, respectively. The most common driver mutation was JAK2 V617F, observed in 60.7%, followed by CALR (15.9%) and MPL (1.9%); 17% of patients were classified as triple negative (TN). Where data was available (n=27), median variant allelic frequency (VAF) for JAK2 V617F was 19.7% (r, 4-75%) and for CALR 43% (r, 5.7 - 53). 9/107 (8.4%) of patients had additional pathogenic gene abnormalities, most commonly variants of NRAS (22.2%), TET2 (22.2%) and ASXL1 (22.2%). We observed a high thrombosis rate of 19.6% (21/107); the majority had a diagnosis of ET (61.9%).. In 80% of such cases, this was the heralding event leading to the MPN diagnosis. Venous thrombosis was more frequent than arterial thrombosis (68% vs 33%). In particular, there was a disproportionate number of venous thromboses at unusual sites including cerebral venous sinus and splanchnic venous thromboses, representing 40% and 33% of total events, respectively. Only 3 (2.8%) patients were found to have a co-existent thrombophilia. Younger age at diagnosis appeared to associate with a higher rate of thrombosis (60% in patients aged <20, 18.2% in patients aged 20-30, and 17.2% in patients aged 31-40). Thrombotic events were most common in those with a JAK2V617 mutation (23.4%), followed by those who were TN (20.0%). Only 5.9% of CALR mutated cases had a thrombotic event. In terms of cardiovascular risk factors, 13.0% (14/107) and 6.5% (7/107) of patients had diagnoses of hypertension and type 2 diabetes, respectively. Haemorrhagic events were reported in 8.4% (9/107) of patients; 3.7% (4/107) had grade 3 or 4 bleeding complications, requiring transfusion and/or surgical intervention. All who experienced haemorrhagic events were on treatment with an anti-platelet or anti-coagulant agent. 4.7% (5/107) were diagnosed with acquired von Willebrand's disease, of whom one experienced a grade 3 haemorrhagic event. No patients transformed during follow up. 33 patients reported pregnancies, with 7/33 (21%) reporting antenatal or perinatal medical complications. Over 50% (55/107) of patients were commenced on cytoreduction. Most common agents employed were pegylated interferon (45.5%; 25/55) and hydroxycarbamide (43.6%%; 24/55). No patients required combination therapy. 92/107 (86%) patients were treated with an antiplatelet agent and 9/107 (8.4%) were on anti-coagulant treatment with the majority (88.8%) receiving warfarin rather than a DOAC. AYA MPN patients have unique characteristics and heterogeneous clinico-biological features differentiating them from older MPN cohorts. Our data highlights a female preponderance, higher incidence of venous thrombosis (particularly atypical sites), and lower incidence of arterial thrombosis. Whilst traditionally younger patients are considered to have ‘low risk’ disease, this real-world study highlights a significant thrombosis rate of ~ 20% which may have associated morbidity considering the relatively longer duration of the disease course and need for cytoreductive therapy. Specific AYA MPN directed risk stratification and therapeutic algorithms are required to inform holistic and age-appropriate patient management.

The Combination of Navitoclax and Ruxolitinib in JAK Inhibitor-Naïve Patients with Myelofibrosis Mediates Responses Suggestive of Disease Modification

Background: Myelofibrosis (MF) is a myeloproliferative neoplasm associated with splenomegaly, high symptom burden, bone marrow fibrosis (BMF), and poor prognosis. The Janus kinase inhibitor (JAKi) ruxolitinib is the current standard of care for patients with MF. It improves splenomegaly and disease symptoms with limited impact on disease biology, highlighting an unmet medical need in MF. Driver mutations of JAK2, CALR, and MPL are key to the pathogenesis of MF. Reduction in variant allele frequency (VAF) of driver mutations and reversal of BMF are suggestive of disease modification (Pemmaraju, Cancer, 2022). Additionally, mutations in ASXL1, EZH2, SRSF2, IDH1/2, and U2AF1 p.Q157 are classified as high molecular risk (HMR) mutations associated with a worse prognosis (Arber et al., Blood, 2016). We previously demonstrated evidence of disease modification independent of HMR mutations by adding navitoclax to ruxolitinib in patients with suboptimal responses to ruxolitinib monotherapy (Pemmaraju, Lancet Oncology, 2022). Herein, we investigated whether the type of MF and risk (e.g., age, Dynamic International Prognostic Scoring System [DIPSS], and HMR) correlated with clinical outcomes and responses suggestive of disease modification (e.g., VAF reduction and improvement in BMF) among JAKi treatment-naïve patients with MF treated with the combination of navitoclax and ruxolitinib. Methods: Cohort-3 of the phase-2 multicenter REFINE study (NCT03222609) enrolled JAKi treatment-naïve patients with MF. Enrolled patients had primary or secondary MF with splenomegaly (DIPSS ≥ Intermediate-1) and did not receive prior JAK-2 therapy or bromodomain and extra terminal motif (BET) inhibitors. Patients initiated navitoclax at 100 mg QD or 200 mg QD if baseline platelet count was ≤ 150 × 109/L or > 150 × 109/L, respectively. Ruxolitinib was given BID with starting dose based on baseline platelet count per local label. The primary endpoint was spleen volume reduction of ≥ 35% (SVR35) from baseline at week 24, assessed by MRI conducted by central review. Key secondary and exploratory endpoints evaluated in this analysis were a reduction in BMF obtained from BM biopsies by local evaluation and a reduction in VAF for the driver gene mutations (JAK2V617, CALR, or MPL), respectively. Driver gene VAF and HMR mutations were determined in whole blood with the 50-gene Focus Myeloid next-generation sequencing panel (Interspace Pharma Solutions, Morrisville, NC, USA). Results: As of Feb 07, 2022, all enrolled patients (N = 32) treated with navitoclax and ruxolitinib were evaluable for biomarker analysis. The median duration of follow-up was 6.1 months (range, 1.9 – 18.6); median age was 69 years (range, 44 – 83), and median spleen volume was 1889.08 cm3 (range, 645.6 – 7339.6). At baseline, 22 (69%) patients had JAK2, 6 (19%) had CALR, and 3 (9%) had MPL mutations. One (3%) patient was triple negative. Nineteen (59%) patients had HMR mutations. SVR35 at week 24 was observed in all subgroups known to confer poor prognosis, including age (≥75 years, 50% [n = 4/8]), high DIPSS score (Intermediate-2, 63% [n = 12/19]; high, 33% [n = 1/3]), and HMR mutations (47% [n = 9/19]; Figure). BMF grade improvement was evaluable in 26/32 (81%) patients, of which 9/26 (35%) achieved ≥ 1-grade improvement at any time during treatment with a median time-to-improvement of 12.3 weeks (range, 12.1 – 24.1). Complete resolution of BMF was observed in 2/9 (22%) patients. The baseline BMF grades of these patients were 2 and 3. BMF ≥ 1-grade improvement was achieved by 7/13 (54%) patients with HMR mutations and 2/13 (15%) without HMR mutations. Reduction in driver gene JAK2V617 mutation VAF > 20% from baseline at week 12 or 24 was observed in 14/28 (50%) patients, and 5/14 (36%) patients achieved > 50% VAF reduction from baseline. There were no differences in > 20% VAF reductions from baseline to week 12 or 24 between those with or without HMR mutations (7/15 [47%] versus 7/13 [54%], respectively). Conclusions: Among JAKi treatment-naïve patients with MF, the combination of navitoclax and ruxolitinib reduced splenomegaly in several high-risk groups known to confer poor prognosis. Reductions in BMF and VAF were independent of HMR mutations. The reduction in BMF and VAF for the driver mutation JAK2V617 is encouraging and suggestive of evidence of disease modification with the combination of navitoclax and ruxolitinib. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

High JAK2V617F allele burden is associated with an increased burden of coronary artery calcification in MPNs

Abstract Background Patients with the haematological cancers Philadelphia-negative Myeloproliferative Neoplasms (MPNs), have an increased risk of coronary artery and aortic valve calcification, and an increased risk of myocardial infarctions. The most common acquired mutation in MPNs, the JAK2V617F mutation, has been linked to increased risk of thrombosis. Aims To exam the association between the JAK2V617F mutation, its allele burden, and coronary artery and aortic valve calcification. Methods One hundred and sixty one patients with MPN disease from one specialized haematological outpatient clinic where included. Information on demographics, smoking, alcohol habits and co-morbidities were registered. Blood samples were drawn for determination of the JAK2V617 mutation and allele burden. Patients were examined by cardiac computer tomography in order to determine their coronary artery calcium score (CACS) and aortic valve calcification (AVC) score. The association between the JAK2V617F mutation, its allele burden and coronary artery and aortic calcification was investigated with univariate and multivariate logistic regression analysis, adjusting for age, sex, ischemic heart disease (IHD), stroke, smoking, obesity, hypertension, hypercholesterolemia, diabetes mellitus, and family history of IHD or stroke in the multivariate analysis. Results Of the 161 patients (52% male, mean age 65.5±10.5 years), 137 (85%) were JAK2V617F positives, and the JAK2V617F allele burden was quantified in 120. The median JAK2V617F allele burden was 12% (IQR range 6–33%). There were 42 (26%) patients with a CACS >400, and 93 (58%) patients AVC. Among the JAK2V617F positive patients, 38 (24%) had a CACS >400 and 81 (59%) had AVC. In the 32 patients with a JAK2V617F allele burden>33%, 19 (59%) had a CACS >400 and 26 (81%) had AVC. In the univariate logistic regression analysis the presence of JAK2V617F mutation was not associated with a CACS >400 (Odds Ratio (OR) 1.92, 95% confidence interval (CI) 0.62–5.98, p=0.26). Similar result was found for the analysis on AVC (OR 1.45, 95% CI 0.61–3.45, p=0.41). In contrast, a JAK2V617F allele burden >33% was significantly associated with a CACS >400 (OR 5.31, 95% CI 2.23–12.66, p=0.0002), and similarly with AVC (OR 4.14, 95% CI 1.55–11.05, p=0.0045). In the multivariate adjusted analysis, a JAK2V617F allele burden >33% was significantly associated with a CACS >400 (OR 2.08, 95% CI 0.43–10.10, p=0.36), but not with AVC (OR 0.90, 95% CI 0.27–3.03, p=0.86). In the adjusted analysis the association between a JAK2V617F allele burden >33% and CACS >400 was significant (OR3.86, 95% CI 1.14–13.09, p=0.031), but the analysis on AVC was not (OR 1.59, 95% CI 0.42–6.04, p=0.50). Conclusion There is a significant association between a JAK2V617F allele burden >33% and the burden of coronary calcification in MPNs, measured as a CACS >400. The association remains significant after adjustment for cardiovascular risk factors. Funding Acknowledgement Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): Region Sjællands Sundhedsvidenskabelige ForskningsfondTømrermester Jørgen Holm og hustru Elisa F. Hansens Mindelegat

MPN-196 Unusual Association Case of Janus Kinase 2 Mutation and BCR-ABL Transcripts: Successful Treatment With Hydroxyurea

It is well known that mutations (like JAK2) are mutually exclusive events in myeloproliferative neoplasms (MPN). The presence of mutations (like JAK2) is seen in BCR-ABL1-negative MPN, but its presence with chronic myeloid leukemia (CML) is a rare finding; only a few cases have been reported (1). In this case, we describe patients who had both mutations (BCR-ABL and JAK2 V617F). We present the case of a 37-year-old man referred to our clinical department in 2017 for thrombocytosis. He had a history of arterial hypertension, was a chronic smoker and occasional alcoholic, and had a right fifth toe amputation in 2013. We discovered an asthenic patient with no splenomegaly and amputation of the third toe on the right foot on clinical examination. A complete blood count revealed that the patient was thrombocythemic (platelet [PL] counts of 1,783,000/mm3), had neutrophil leukocytosis (21,300/L), and had a hemoglobin level of 12 g/L. JAK2 V617 mutation was positive, and transcript BCR-ABL (M-bcr) was found by RT-PCR quantification to be 0.082%. However, karyotype analysis revealed that he was 46 years old, XY. His bone marrow was hypercellular with an increased number of megakaryocytes. The patient was followed in the cardiovascular department for his microangiopathy, which resulted in a second amputation of the right third toe in 2017. When he did not achieve complete hematological remission after one month on imatinib mesylate 400 mg daily, the dose was increased to 600 mg/J. Due to treatment failure with PL counts around 100,000 after 6 months, the treatment was changed to hydroxyurea (1 g/J) and imatinib was discontinued. The patient had been in complete hematologic remission for the previous 3 years. Several cases of concomitant JAK2 myeloproliferative disorders and BCR-ABL transcript have been reported. This hybrid disease is more likely to be misdiagnosed because it is underdiagnosed. Patients with association had a poor response to imatinib (3), which was also the case in our patient, who had a good hematological response to hydroxyurea but a poor response to imatinib.

Clinical Characteristics and Prognostic Risks of Philadelphia-Negative Myeloproliferative Neoplasms at Cipto Mangunkusumo General Hospital.

BackgroundPhiladelphia-negative myeloproliferative neoplasms (Ph-MPNs) are clonal hematopoietic cell malignancy that comprises polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). They prone to develop thrombosis, bleeding, fibrotic progression, and leukemic transformation. We aimed to study clinical characteristics, thrombosis complications, and prognostic risk in Indonesians with Ph-MPNs.MethodsThis was a single-center retrospective cross-sectional study, including patients with Ph-MPNs who attended Hematology and Medical Oncology outpatient clinic at Cipto Mangunkusumo General Hospital, Jakarta, between 2016 and 2021. Demographic data, clinical characteristics, previous antiplatelet treatment, baseline laboratory data, JAK2V617F mutation, and treatment were reviewed for analysis. The prognostic risk model was assessed for PV (IPSS), ET (IPSET-thrombosis), and PMF (IPSS and DIPPS) patients.ResultsOne hundred forty-six patients were classified as PV (31.5%), ET (38.4%), and PMF (30.1%) with median ages of 54, 53.5, and 55 years, respectively. PMF presented with the most diverse clinical presentations. JAK2V617 mutations were detected in 82%, 42.5%, and 76.5% of PV, ET, and PMF patients. PV had the highest thrombosis event pre/at diagnosis (26.1%), with predominantly arterial thrombosis. Bleeding occurred more in PMF (20.5%). Phlebotomy and hydroxyurea are the only treatment available for Ph-MPNs in the government hospital in Indonesia. IPSS intermediate risk in PV (47.8%), IPSET-thrombosis low risk in ET (46.4%), and IPSS and DIPSS intermediate-2 in PMF (38.6% and 50% respectively) were the most common risk groups.ConclusionEssential thrombocythemia was the most prevalent Ph-MPNs in Indonesia. Arterial thrombosis was the highest thrombosis event pre/at diagnosis in PV despite IPSET-thrombosis low risk being the most common risk group. In JAK2V617 mutation-positive MPN population, thrombosis event was also the highest in PV.

Janus kinase 2 (JAK2V617F) mutations in patients with splanchnic vein thrombosis, a single centre experience

JAK2V617F mutation is recurrent in polycythaemia vera and essential thrombocythaemia, which are myeloproliferative neoplasms (MPNs) frequently associated with arterial and venous thromboembolism. It also reported as a marker for occult MPN in patients with splanchnic venous thrombosis. We evaluate frequency of JAK2V617F and CALR mutations in patients’ samples presenting with splanchnic vein thrombosis that are sent to Clinical Referral Haematology Laboratory, Hospital Ampang, the tertiary referral laboratory offering both tests within Ministry of Health Malaysia. We retrospectively investigated JAK2V617F mutations samples from years 2016–2020 with presenting history of splanchnic vein thrombosis confirmed by ultrasound or computed tomography (CT) imaging. JAK2V617F mutation analysis was done by amplification refractory mutation system (ARMS) PCR and CALR mutation analysis by high resolution melting (HRM). There was a total of 93 patients (47 men and 46 women) during 2016 and 2020. 16 patients were found to have the JAK2V617 mutation, and the remaining were negative for JAK2V617F and CALR mutation. Of note, we found that patients with JAK2 V617F mutation presented with higher total white blood cell count and platelet count than those without mutation (p<0.05). Despite thrombosis being a common manifestation of MPN, the role of screening JAK2V617F mutations in all patients presenting with splanchnic vein thrombosis without other features of MPN is unclear. However, the significance of this mutation screening provides an accurate diagnosis and further understanding of the role of JAK2V617F mutation in thrombosis has a great potential for future development of therapeutics intervention.

The Gaisbock syndrome after COVID-19 pneumonia

In 1905, Felix Gaisbock, MD, described a syndrome in patients with hypertension, elevated hematocrit levels, plethoric appearances, and no splenomegaly. He postulated this relative erythrocytosis was due to stress. In this case report, a 40-year-old Caucasian man with obesity was admitted with recurrent deep vein thrombosis and increasing oxygen requirements 2 weeks after hospitalization with COVID-19 pneumonia. This patient had a 10-year history of untreated hypertension and erythrocytosis. He had a ruddy appearance, a normal erythropoietin level, and a negative JAK2 V617 mutation. In this case, Gaisbock syndrome was suspected.

Acute Portal Vein, Superior Mesenteric Vein and Splenic Vein Thrombosis Secondary to JAK2 V617 Mutation- A Case Report

Portal vein thrombosis (PVT) is a rare finding which usually occurs in association with local factors such as cirrhosis, malignancy, pancreatitis, intraabdominal infections or systemic hypercoagulable states. It may present acutely as abdominal pain, ascites, fever or exist in a chronic state which is generally asymptomatic and an incidental finding. With advancement in Imaging and laboratory studies, PVT cases are diagnosed more frequently along with its predisposing factors. The invention of JAK2 mutation and it’s addition to the WHO criteria for Myeloproliferative neoplasm (MPN) diagnosis, has increased the number of MPN cases which were previously labelled idiopathic. We present a case of 54 year old female diagnosed with unprovoked PVT with bowel ischemia and JAK 2 mutation positive, managed surgically and with long term anticoagulation.

IL13 Contributes to Fibrotic Progression of the Myeloproliferative Neoplasms

Pre-fibrotic-primary myelofibrosis PMF (Pre-PMF) is an indolent form of PMF that frequently progresses to overt-PMF. While both stages of the disease are characterized by the presence of dysplastic megakaryocytes, progression to fulminant disease is associated with significantly increased fibrosis in the bone marrow. We have previously shown that megakaryocyte maturation in overt-PMF is impaired due to a GATA1 deficiency. However, in Pre-PMF patients most megakaryocytes express GATA1. This raises the possibility that alterations in megakaryocyte development occur during the progression of the disease and may contribute to fibrosis. This progression in megakaryocyte defects is likely driven not only by the aberrant JAK/STAT signaling but also microenvironmental factors. To identify such factors, we performed studies in two mouse models of PMF (driven by JAK2V617 and MPLW515L mutations) before and after development of fibrosis. Using an unbiased approach, we measured the levels of different cytokines in the bone marrow, plasma, and spleen. In addition, we performed single cell RNAseq in bone marrow populations. We observed extensive changes in the level of cytokines in the bone marrow of the MPLW515L mouse model compared to the JAK2V617F model. We initially focused on those cytokines that are elevated in the bone marrow of both murine models, including IL13 (Figure 1A), because previous studies have shown that IL13 is elevated in PMF patients and that JAK2 inhibitors do not decrease IL13 (1-3). Moreover, elevated IL13 has been identified in patients who progress to secondary AML (2). How IL13 may contribute to the progression of the disease has not been investigated.We assayed the effect of IL13 on megakaryocytes in vitro and discovered that it promoted megakaryocyte differentiation in the absence of thrombopoietin (TPO) and potentiated the effect of TPO. This effect was observed in cultures of both wild-type and MPLW515L megakaryocytes. Next, we assayed for expression of the IL13 receptor (Il13ra1) in the bone marrow of JAK2V617F and MPLW515L mutant mice and found that it was highly upregulated compared to wild-type animals. IL13ra1 expression was particularly intense in the megakaryocyte lineage, and its expression increased with disease progression (Figure 1B). Next, we asked whether IL13 is essential for myeloproliferative neoplasm (MPN) development in vivo. To study this, we transplanted bone marrow cells from Il4/13 f/f Mx1-Cre mice expressing MPLW515L to irradiated recipients, waited until MPN developed, and then excised by pIpC injection. This experiment revealed that loss of IL13 and IL4 led to a profound reduction in disease burden (Figure 1C), decreased splenomegaly, and diminished degree of bone marrow fibrosis. Moreover, loss of IL13 and IL4 decreased the levels of pro-inflammatory cytokines in the bone marrow and spleen (Figure 1D). We attribute this effect to deletion of IL13 because IL4 was only moderately increased in the bone marrow of the MPLW515L mouse model, and because IL4 has been reported to not be altered in the MPNs.Finally, we performed single cell RNA-seq on bone marrow cells from mice transplanted with JAK2V617F or control progenitor cells early and late in the disease process (Figure 1E). Our results revealed that there was decreased myeloid progenitors but an enhancement in the mast cell lineage that tracked with the degree of fibrosis. We confirmed the presence of elevated numbers of mast cells in the bone marrow by immunohistochemistry (Figure 1F). Mast cells produce IL13, and therefore they are the likely source for the increased IL13. Finally, consistent with the observation that IL13 signaling is primarily mediated through STAT6, we found enrichment of STAT6 target genes in megakaryocyte progenitors from the late timepoint in our scRNAseq data (Figure 1G).In summary, our data demonstrate that IL13 is involved in the progression of PMF and that inhibition of the IL13 signaling pathway should be investigated as a therapeutic option in PMF.1. Tefferi A, et al. J Clin Oncol (2011)2. Fisher DAC, et al. Leukemia (2019)3. Chen P, et al. Front Med (2021) [Display omitted] DisclosuresCrispino: Forma Therapeutics: Research Funding; Scholar Rock: Research Funding; MPN Research Foundation: Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Consultancy.

Characteristics and Outcome of Philadelphia(Ph) Negative Myeloproliferative Neoplasms(MPN) in Patients Younger Than 45 Years - a Multicenter Retrospective Study

Introduction: Age is an adverse prognostic factor in Ph-negative MPNs. Most MPN patients are diagnosed in their sixth decade, which may affect their ability to cope with disease symptoms and treatment and thus worsen their outcome. In contrast, in younger patients other challenges such as long term treatment side effects, prevention of disease progression and fertility issues are more prominent. The current study investigates the characteristics and outcome of Ph-negative MPN patients younger than 45 years.Methods: A retrospective study,evaluating patients 18-45 years of age at the time of diagnosis of Ph-negative MPN, between 1985-2017. Primary outcomes were mortality, transformation to secondary myelofibrosis (MF), transformation to acute myeloid leukemia (AML) and undergoing an allogeneic stem cell transplantation (alloSCT). Secondary outcomes were thrombotic events and pregnancy complications. Risk factors for the composite outcome of venous and arterial thrombosis and for the composite outcome of transformation to MF,AML or undergoing AlloSCT were determined by multivariate analysis.Results: 106 patients were included. The median follow-up time was 8 years (range 5 months to 32 years). Diagnosis at presentation was polycythemia vera (PV) (34,32%), essential thrombocytosis (ET) (50,47%), primary MF (15,14%), early MF(4,4%)and MPN not otherwise specified (NOS) (3,3%). Patient baseline characteristics are presented in table 1.At diagnosis, 37 patients (35%) were treated with hydroxyurea,10(10%) with another cyto-reductivetherapy (Interferon,anagrelide,ruxolitinib) and 54(55%) did not receive any cyto-reductive therapy.Within a median follow up of 8 years, 10 patients developed secondary MF (9.4%) ,2 patients developed AML (2%) and 7 patients underwent alloSCT (6.6%).7patients have died (6.6%); 1 due to transformation to AML, 3 due to MF -related complications ,2 due to alloSCT complications and 1 due to sudden death. The only significant risk factor for the composite outcome of transformation to MF, AML or alloSCT was diagnosis of MF at presentation (P=0.05).There were 37(35%) thrombotic events, mostly venous (29 events).Thrombotic events were most frequent in ET patients (16,43%), followed by PV (12,32%), MF (7,19%),early MF(1,3%) and MPD NOS(1,3%). Risk factors for composite thrombosis were JAK2 V617 mutation (P=0.052) and splenomegaly (P=0.003).25 women became pregnant after diagnosis of MPN (46 pregnancies). Spontaneous abortions were documented in 4 pregnancies, fetal malformation in 2(7%) and premature delivery in one(3%).Maternal outcome was uneventful in all pregnant women.Conclusions: Patients diagnosed with Ph-negative MPN at a young age are at comparable risk as the general population of MPN patients of developing secondary MF but at lower risk of developing AML at a median follow up of 8 years. A diagnosis of MF at presentation predicts a worse prognosis. JAK2 mutation and splenomegaly at presentation are associated with an increased risk for thrombotic events. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

JAK2, CALR, and MPL Mutation Profiles in BCR-ABL Negative Myeloproliferative Neoplasms, a Referral Center Experience in the Middle East.

Background & Objective:JAK2, CALR, and MPL genes play pivotal roles in the pathogenesis of BCR-ABL negative myeloproliferative neoplasms. This study was conducted to evaluate the frequency of JAK2, CALR, and MPL mutations in BCR-ABL negative myeloproliferative neoplasms and their association with demographic data and hematologic parameters in a referral center, in the Middle East. Methods:Seventy-one patients with BCR-ABL negative myeloproliferative neoplasms were evaluated for JAK2 V617F, CALR type 1, CALR type 2, and MPL by allele-specific PCR and conventional PCR from 2018 to 2019.Results:Twenty-three patients were categorized as polycythemia vera, JAK2 V617F was observed in 91.3% of these cases. Thirty-eight patients were classified as essential thrombocythemia of which 52.6% showed JAK2 V617F, 18.4% demonstrated CALR type 1, 7.9% denoted CALR type 2 and there was no mutation reported in 21.1%. Seven patients were recognized as primary myelofibrosis and exhibited JAK2 V617F mutation in 57.1%, CALR type 1 in 14.3 %, CALR type 2 in 14.3% and no mutation in 14.3%. Three patients were diagnosed as MPN, unclassifiable and 33.3% revealed JAK2 V617F mutation, and no mutation was found in 66.6%. The age (59.15±13.10) and neutrophil percent (70.78±10.14) were higher in patients with JAK2 V617 mutation compared to other mutations (P=0.000, and P=0.03). Platelet count was significantly higher in patients with CALR type 1 mutation (1240400± 402053) (P=0.000).Conclusion:JAK2 V617F was associated with patients’ higher age and higher neutrophil count in CBC. CALR mutation had an association with higher platelet count. No MPL mutation was found in this study and it seems that its frequency is lower than what is expected in this region.

Diagnostic Performance of Erythropoietin Levels in Polycythemia Vera: Experience at a Comprehensive Cancer Center

IntroductionConsidering the evolving diagnostic criteria of polycythemia vera (PV), we analyzed the utility of serum erythropoietin (EPO) as a predictive marker for differentiating polycythemia vera (PV) from other etiologies of erythrocytosis. Patients and MethodsWe conducted a retrospective study after a review of electronical medical records from January 2005 to December 2016 with diagnosis of erythrocytosis using International Classification of Disease–specific codes. To evaluate the diagnostic performance of EPO levels and JAK2-V617F mutation, we constructed a receiver-operated characteristic curve of sensitivity versus 1-specificity for serum EPO levels and JAK2-V617F mutation as predictive markers for differentiating PV from other causes of erythrocytosis. ResultsWe surveyed 577 patients with erythrocytosis. Median patient age was 59.2 years, 57.72% (n = 329) were male, 86.3% (n = 491) were white, and only 3.3% (n = 19) were African American. A total of 80.88% (n = 351) of those diagnosed with PV had a JAK2-V617F mutation compared to only 1.47% (n = 2) whose primary diagnosis was secondary polycythemia. When comparing JAK2-V617 mutation to the EPO level, the area under the curve of JAK2-V617 (0.8970) was statistically larger than that of EPO test (0.6765). Therefore, the PV diagnostic methodology using JAK2-V617 is better than the EPO test. An EPO level of < 2 mIU/mL was > 99% specific to predict PV but was only 12% sensitive. ConclusionIn the appropriate clinical setting, cytogenetic and molecular studies such as JAK2 mutation status prevail as the most useful tools for PV case identification. The use of isolated EPO to screen patients with erythrocytosis is not a good diagnostic approach.

MPN-376: The Clinical Impact of the Burden of JAK2V617F Mutated Allele in Philadelphia-Negative Chronic Myeloproliferative Neoplasms

Context: Several studies reported the impact of JAK2V617F allele burden on phenotypic properties and clinical course in Philadelphia-negative myeloproliferative neoplasms (Ph-negative MPNs). We aimed to analyze the association of mutated JAK2V617F allele burden with clinical phenotype and laboratory characteristics in essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients. Objective: A total of 202 consecutive Philadelphia-negative MPN patients, 118 with ET and 84 with PMF, were included in this study. Methods: We used a real-time, semi-quantitative polymerase chain reaction (PCR) with JAK2 MutaScreen assay (Ipsogen, Luminy Biotech, Marseille, France) to screen JAK2V617 mutations and mutant allele burden. Results: According to the JAK2V617F status, ET patients were stratified into three groups: JAK2V617F mutation-negative (n=54) and JAK2V617F-positive with mutant allele burden in the lower quartile (n=59) and upper quartile (n=5) ranges. ET patients with upper quartile JAK2V617F allele burden had significantly lower Hgb levels (p=0.001), lower Htc levels (p=0.001), and higher LDH levels (p=0.008) as compared to the other two groups. Also, ET patients with upper quartile JAK2V617F allele burden had significantly increased spleen size and higher bleeding complications (p=0.034; 0.002; respectively). We observed significant association between ET patients with high JAK2V617F allele burden and higher death rates (p=0.007). There was a trend towards higher incidence of venous thrombosis in ET patients with upper quartile JAK2V617F allele burden (p=0.057). According to JAK2V617F status, PMF patients were stratified into three groups: JAK2V617F mutation-negative (n=25) and JAK2V617F-positive with mutant allele burden in the lower quartile (n=41) and upper quartile (n=18) ranges. Comparison across all three groups revealed significant associations between upper quartile allele burden and higher leukocyte counts (p=0.001). Also, PMF patients with upper quartile JAK2V617F allele burden had significantly increased spleen size compared to the other two groups (p=0.018). We observed significant association between PMF patients with low JAK2V617F allele burden and higher incidence of venous thrombosis (p=0.035). Conclusion: Our study reported that presence of higher V617F allele burden in ET is associated with defined haematological and clinical markers indicative of a more aggressive behavior while, in PMF, low allele burden was found to be associated with higher incidence of venous thrombosis.

Efficacy analysis of decitabine monotherapy or combined with arsenic trioxide in treatment of myelodysplastic syndromes

Objective To analyze the therapeutic effect and tolerability of decitabine monotherapy or combined with arsenic trioxide for the treatment of patients with myelodysplastic syndromes (MDS). Methods Clinical characteristics of 32 patients with primary MDS in North Hospital of Ruijin Hospital Affiliated to Medical College of Shanghai Jiaotong University from January 2014 to April 2017 were retrospectively analyzed. The clinical data of these patients were collected, and the patients were followed up. Decitabine combined with arsenic trioxide was used in 23 cases, decitabine (20 mg·m-2·d-1) and arsenic trioxide (0.16 mg/kg) were administrated from day 1 to day 5 and was repeated every 4-6 weeks. For the remaining 9 cases, only decitabine was applied, decitabine(20 mg·m-2·d-1) was administrated from day 1 to day 5 and was repeated every 4-6 weeks. The clinicopathological characteristics and the effect of genetic mutations on the efficacy of treatment were investigated. Results Of the 32 patients with primary MDS, 18 were male and 14 were female. The patients were 17-72 years old with a median age of 56 years old. Genetic analysis revealed 10 cases with TP53 mutations, 8 cases with TET2 mutations, 4 cases with U2AF1 mutations, 3 cases with RUNX1 mutations, 3 cases with ASXL1 mutations, 2 cases with NRAS mutations, 2 cases with DNMT3A mutations and 1 case with JAK2 V617 mutation. The follow-up time was 2-23 months with a median follow-up time of 8 months. A total of 21 cases (65.6%) attained treatment response. Among them, there were 10 cases (31.3%) with complete remission (CR), 5 cases (15.6%) with bone marrow complete remission (MCR), and 6 cases (18.7%) with hematological improvement. There was no significant difference in the efficiency and CR rate between the combination group and the monotherapy group (P = 0.441, P = 0.681). Ten cases were found to have TP53 mutations, of which 7 cases had CR. Multivariate analysis demonstrated that TP53 mutation was an independent risk factor for CR (P = 0.037). All patients developed myelosuppression after treatment, of which 16 cases developed pulmonary infection. Conclusions Decitabine combined with arsenic trioxide in the treatment of MDS is effective and well tolerated. The therapeutic effects of decitabine monotherapy or decitabine combined with arsenic trioxide for treatment of patients with TP53 mutations are better than the average levels. Key words: Myelodysplastic syndromes; Decitabine; Arsenic trioxide; Curative effect

PB2209 RISK FACTORS OF THROMBOSIS AFTER MPN DIAGNOSIS: A RETROSPECTIVE COHORT OF MPN PATIENTS IN BRAZIL

Background:Myeloproliferative Ph‐negative neoplasms (MPN) are a group of heterogeneous clonal hematopoietic disorders with high risk for thrombotic events. Hemorrhage can also occurs, particularly associated with the treatment.Aims:Describe risk factors for thrombosis and bleeding in patients with Ph‐negative MPN in a single center in Brazil.Methods:We analyzed retrospectively data of 334 patients older than 18 years old, with diagnosis of polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF) and unclassifiable MPN (U‐MPN) defined by the 2008 WHO criteria from 2012 to 2017. Unadjusted and adjusted analyses were conducted by using Logistic Regression models to determine the association of covariates with binary outcome. All statistical analyses were performed using SAS software (SAS Institute Inc, Cary, NC). The primary endpoint was risk of thrombotic event at any time and secondary endpoints were risk of thrombotic event after the diagnosis and the incidence of major bleeding (MB) events defined by the ISTH criteria.Results:Overall, 334 patients with Ph‐negative MPN were recruited. The majority was diagnosed with TE (159/47,6%), followed by PV (92/27,54%), PMF (71/21,26%) and U‐MPN (12/3,6%). The female sex was prevalent (66%) and, at the time of diagnosis, the median age was 56 years. The median follow‐up was 4.9 years (1.3 – 7.7). At least one thrombotic event was found in 42% of the patients during the follow‐up. Among these events, 19% occurred after the MPN diagnosis and arterial thrombosis was more prevalent (61%) being stroke the most frequent event followed by acute coronary syndrome. A subsequent event occurred in less than one third of patients. MB events were seen in just 5%, with 67% related with gastrointestinal bleedings. The incidence of thrombosis and MB was similar among the PV, ET, PMF and U‐MPN as shown in table 1. In a multivariable logistic regression model for thrombosis, age ≥60y (OR 3.01, 95%CI 1.83‐4.95) and JAK2 V617 mutation (OR 3.03 CI 1.81‐5.08) were associated with increased risk of thrombosis at any time. For thrombosis after the diagnosis of MPN, WBC ≥ 7x109/L (OR 2.44, 95%CI 1.03‐5.8), age ≥60y (OR 2.58, 95% CI 1.04‐6.4) and 2 or more CV risk factors (OR 2.4 95% CI 1.02‐5.66) had statistical significance. In our center, all patients with PV have received cytoreductive therapy and none were exclusively treated with phlebotomy, although some patients were defined as low‐risk. Only 4% of the patients with ET received only low‐dose aspirin. Also 43% of the patients harboring a JAK2 V617F mutation already had a thrombotic event before the MPN diagnosis and were treated as high risk.Summary/Conclusion:The incidence of thrombosis and MB were similar among all subtypes of MPN as reported by previous studies. In our population, we confirmed that age ≥ 60 years and JAK2 V617F mutation were associated with increased risk of thrombosis at any time. Seemingly, the presence of JAK2 V617F mutation did not increase the risk of thrombosis after the diagnosis of MPN. Since these patients were mostly treated with cytoreductive therapy, we hypothesize that this strategy may abrogate the negative impact of JAK2 mutation reported in previous cohorts. To the best of our knowledge, this is one of the largest cohorts of patients with MPN ever reported in Latin America. Organizing cooperative study groups in Latin America is an urgent need for improving the knowledge in this field.image

Comparative Analysis of Proinflammatory Cytokine Levels in Peripheral Blood and Bone Marrow of Patients with Myeloproliferative Neoplasms

BackgroundMyeloproliferative neoplasms (MPNs) are characterized by marker somatic mutations involving JAK2, MPL, and CALR, which lead to constitutive activation of tyrosine kinase signaling cascades, subsequently dysregulated proliferative of myeloid linages, and eventually myelofibrosis or leukemic transformation. Recently, it has been argued that proinflammatory processes are crucial to the pathogenesis and progression of MPNs. A number of proinflammatory cytokines including lipocalin, IL-1, IL-2R, IL-6, IL-8, IL-12, IL-15, and IP-10 have been found elevated in the peripheral blood (PB) of MPN patients. However, there has been limited data on the levels of proinflammatory cytokines in the bone marrow (BM) of MPN patients. The present study determined and compared 40 proinflammatory cytokine levels in the PB and BM plasma of MPN patients with unequivocal molecular background, thus intending to illustrate the proinflammatory features of BM microenvironment as well as to evaluate the credibility of PB cytokine profiles.MethodsNewly diagnosed MPN patients (n=12, 8 had JAK2 V617F mutation, 4 had CALR mutations) were included in the present study. PB samples were taken within 48 hours of BM samples. Paired PB and BM plasma cytokine profiles were measured as well as 10 health control PB plasma samples in a single procedure by Quantibody Human Inflammatory Array 3 (RayBiotech, Norcross, GA) which permitted detection of 40 inflammation-associated cytokines.ResultsAmong 12 MPN patients, 8 had JAK2 V617 mutation (6 males, median age 61.5 years), and 4 had CALR mutations (3 males, median age 53.5 years). Positive linear correlations between PB and BM levels were found in 12 proinflammatory cytokines including BLC (r=0.613, p=0.034), I-309 (r=0.872, p<0.001), IL-1α (r=0.666, p=0.018), IL-1β (r=0.929, p<0.001), IL-12p40 (r=0.642, p=0.024), IL-15 (r=0.608, p=0.036), M-CSF (r=0.906, p<0.001), MIG (r=0.596, p=0.041), MIP-1α (r=0.787, p=0.002), MIP-1δ (r=0.648, p=0.023), sTNFRI (r=0.827, p=0.001), and sTNFRII (r=0.644, p=0.024). Compared to health controls, BM levels of G-CSF, IL-2, IL-4, IL-6R, IL-7, IL-8, IL-10, IL-13, MIP-1β, PDGF-BB, RANTES, and TIMP-1 were markedly elevated (all p<0.01), among which IL-2, IL-4, and IL-7 levels were even higher in patients with CALR mutations than those with classic JAK2 V617F mutation (all p<0.05).ConclusionsOptimal linear correlation could only be found in limited species of proinflammatory cytokines, especially I-309, IL-1β, M-CSF, and sTNFRI, between PB and BM plasma of MPN patients. Therefore, caution should be recommended during attempts to illustrate the status of inflammation in MPN patients by circulating cytokine markers. A stronger inflammatory component might exist in MPN patients with CALR mutations than those with classic JAK2 V617F mutation. DisclosuresNo relevant conflicts of interest to declare.

Prevalence of JAK2 V617F in Individuals That Meet World Health Organization Erythrocytosis Criteria for Polycythemia Vera

Introduction:The diagnostic criteria for polycythemia vera (PV) has recently been updated by the World Health Organization (WHO). The criterion for erythrocytosis has been modified downwards: hemoglobin (hb)> 16.5 g/dL or hematocrit (hto)> 49% in men and hb> 16 g/dL or hto> 48% in women. This reduction increases the potential number of patients that would be test for JAK2 V617F mutation if PV is suspected. The V617F mutation in the JAK2 gene is present in 95% of cases of PV. It is estimated that the prevalence of this mutation in the general population is around 0.2%. Our aims are to determine the prevalence of JAK2 V617F in individuals with erythrocytosis according to WHO2016 criteria and to find prognostic factors that could help to identify patients with PV.Methods:We prospectively studied all hemograms performed in our laboratory during 7 nonconsecutive days. Variables studied were hb, hto, leukocytes, neutrophils, platelets, MCV, MCH, MCHC and RDW. JAK2 V617F mutation was studied in all males that had hb> 16.5 g/dl or hto> 49% or females that had hb> 16 g/dl or hto> 48%. JAK2 V617F mutation was studied by PCR assay in which an amplification control fragment and the JAK2 mutant allele were simultaneously amplified. All positive samples were confirmed by quantitative real-time PCR in a reference laboratory. Positive results were considered when the JAK2 V617F allele ratio was ≥ 0.7.The variables collected were correlated with the result of the JAK2 test in a univariate way. The T-Student test was used for the quantitative variables and the Chi-square test for the categorical variables. For the cell count variables, the Mann-Whitney U test was used.Results:A total of 15366 HG were analyzed. 1271 (8.3%) met the inclusion criteria for erythrocytosis. JAK2 V617F was performed on 1001 samples (270 samples were not suitable for the PCR assay due to low quality). Twelve samples (1.2%) were positive for JAK2 V617F mutation. However, 5 samples were excluded due to a known diagnosis of myeloproliferative neoplasm. Therefore, finally prevalence of JAK2 V617 mutation in 996 patients that met WHO erythrocytosis criteria was 0.8% (8/996). Medians for all parameter studied for each group are shown in table 1. In order to find out parameters that could increase the incidence probabilities to identify patients with JAK2 V617F we performed an univariate analysis of the variables included, according to JAK2 mutational status. We found that patients with JAK2 V617F had higher levels of leukocytes, neutrophils, platelets and RDW than patients with negative JAK2 (p <0.001), while the levels of MCV (p = 0.033) and MCH (p = 0.015) were lower in patients with JAK2 V617F (table 2). For the variables that have been statistically significant, the area under the ROC curve (AUC) and the optimal cut-off point that maximizes sensitivity and specificity were calculated (Youden index). Of interest, neutrophils, platelets and RDW show high AUC (≥ 0.79), with high sensitivity and specificity (table 3). Finally, in order to find out the potential interest of these findings, we studied medical records of the 8 patients with JAK2 V617F mutations, finding that 4 patients (50%) had previously suffered a vascular event.Conclusion:The prevalence of JAK2 V617F mutation in subjects with elevated Hb or hto according to WHO2016 criteria is increased with respect to that of the general population.Among this group of subjects, those with JAK2 V617F show significantly different levels of leukocytes, neutrophils, platelets, MCV, CMH and RDW. Interestingly, neutrophils, platelets and RDW show high sensitivity and specificity. Therefore, it is necessary to explore combinations of these parameters and their optimal cut-off points to elaborate efficient strategies for an early diagnostic approach. [Display omitted] DisclosuresMartinez Lopez:Bristol Myers Squibb: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Janssen: Research Funding, Speakers Bureau. García Gutiérrez:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Incyte: Honoraria, Research Funding.

Philadelphia chromosome-negative classical myeloproliferative neoplasms: revised management recommendations from European LeukemiaNet.

This document updates the recommendations on the management of Philadelphia chromosome-negative myeloproliferative neoplasms (Ph-neg MPNs) published in 2011 by the European LeukemiaNet (ELN) consortium. Recommendations were produced by multiple-step formalized procedures of group discussion. A critical appraisal of evidence by using Grades of Recommendation, Assessment, Development and Evaluation (GRADE) methodology was performed in the areas where at least one randomized clinical trial was published. Seven randomized controlled trials provided the evidence base; earlier phase trials also informed recommendation development. Key differences from the 2011 diagnostic recommendations included: lower threshold values for hemoglobin and hematocrit and bone marrow examination for diagnosis of polycythemia vera (PV), according to the revised WHO criteria; the search for complementary clonal markers, such as ASXL1, EZH2, IDH1/IDH2, and SRSF2 for the diagnosis of myelofibrosis (MF) in patients who test negative for JAK2V617, CALR or MPL driver mutations. Regarding key differences of therapy recommendations, both recombinant interferon alpha and the JAK1/JAK2 inhibitor ruxolitinib are recommended as second-line therapies for PV patients who are intolerant or have inadequate response to hydroxyurea. Ruxolitinib is recommended as first-line approach for MF-associated splenomegaly in patients with intermediate-2 or high-risk disease; in case of intermediate-1 disease, ruxolitinib is recommended in highly symptomatic splenomegaly. Allogeneic stem cell transplantation is recommended for transplant-eligible MF patients with high or intermediate-2 risk score. Allogeneic stem cell transplantation is also recommended for transplant-eligible MF patients with intermediate-1 risk score who present with either refractory, transfusion-dependent anemia, blasts in peripheral blood > 2%, adverse cytogenetics, or high-risk mutations. In these situations, the transplant procedure should be performed in a controlled setting.

Ruxolitinib for Myelofibrosis Patients Relapsing after Allogeneic Hematopoietic Transplantation

IntroductionThe Janus-Activated Kinase (JAK) 1/2 -inhibitor ruxolitinib is approved for the treatment of symptomatic myelofibrosis (MF) regardless of the JAK2 mutational status. Ruxolitinib can reduce constitutional symptoms and spleen size resulting in an improvement of the performance status and also in a potential prolonged survival. Here we investigated the efficacy and toxicity of ruxolitinib in MF patients who relapsed after allogeneic stem cell transplantation.Patients and methodsBetween the years 2012 and 2016, 10 myelofibrosis patients of our department who relapsed after allogeneic stem cell transplantation received ruxolitinib. Stem cell donor was matched related (MRD) in two patients, matched unrelated (MUD) in six and mismatch unrelated MMUD in two patients. The median age of the patients was 61 years (r: 41-72). The median time from allogeneic transplantation until the start of ruxolitinib treatment was 23.8 months (r: 2.7-85.7). The median treatment duration was 143.5 days (r: 12-369). The median dose of ruxolitinib was 10 mg bid (r: 5-20mg) according to toxicity. Two patients had already received ruxolitinib prior to allogeneic transplantation. All patients had mixed chimerism at start of ruxolitinib. Two patients had already received donor lymphocyte infusions (DLI) prior to ruxolitinib. Two patients started with DLI beginning at day +30 and at day +147 within ruxolitinib treatment. Seven patients were positive for the JAK2V617 mutation and one patient for CALR mutation. Constitutional symptoms were observed in seven patients and splenomegaly in six patients before treatment start. One patient had aGvHD (skin) and three had cGvHD (oral mucosa, skin, liver) at the time of treatment start. In seven patients bone marrow histology was available before and during ruxolitinib treatment.ResultsWe observed an improvement of constitutional symptoms in six patients (86%). The palpable spleen size was reduced in five patients (83%) with splenomegaly (≥50% n=4, <50% n=1). In five of six patients who were dependent on blood and/or thrombocyte transfusions a reduction of the transfusion frequency was seen. Due to thrombocytopenia (CTC grade 3 and 4) dose reduction was necessary in two patients. One patient newly developed leukocytopenia within ruxolitinib treatment (CTC grade 3); before ruxolitinib one patient had grade 1 leukocytopenia and worsened to grade 3. Prior to ruxolitinib the median chimerism was 57% (r: 0-99.7) whereas during ruxolitinib treatment the median chimerism dropped to a median of 0% (r: 0-93.8). The median JAK2-alllele-level before ruxolitinib was 0.87 (r: 0.03-91.3) versus 8.35 (r: 1.83-29.9) during ruxolitinib treatment. The one patient harboring the CALR mutation had an increase of the allelic burden from 0.56 to 1.68. In five of the patients with an available bone marrow biopsy an aggravation of bone marrow fibrosis could be seen whereas no progression could be seen in two patients. The peripheral blast count increased in three patients, in one patient peripheral blasts newly developed. Progressive disease could be seen in all patients (100%) despite of reduction of spleen size, improvement of constitutional symptoms or a prolonged transfusion interval. Three patients transformed to AML. All patients with GvHD had an improvement and or even complete resolution of their GvHD. One patient developed aGvHD of the skin during ruxolitinib treatment but in this patient immunosuppressive therapy had already been stopped at day +30 due to mixed chimerism. No severe infectious complications were seen. None of the patients had to stop ruxolitinib due to side effects.ConclusionThe Janus-Activated Kinase (JAK) 1/2 -inhibitor ruxolitinib can lead to a reduction of spleen size, an improvement of MF-associated constitutional symptoms and a reduction of the blood transfusion-interval in patients with relapse after allogeneic transplantation. However, ruxolitinib does not lead to an increase of donor chimerism. It seems to have little impact on the JAK2V617F-allele level. A further aggravation of bone marrow fibrosis was seen in most of the patients. Despite of clinical improvement all patients experienced further progression of myelofibrosis and three patients even transformed to AML. Furthermore in those patients with GvHD an improvement or even complete resolution of the GvHD was seen. DisclosuresKroeger:Novartis: Honoraria, Research Funding.

Bone Marrow Reticulin Fibrosis in 579 Patients with Polycythemia Vera and Essential Thrombocythemia: Effect on Clinical Outcome

Background:Prognostic value of bone marrow (BM) fibrosis grading in myeloproliferative neoplasm (MPN) is still debated. Polycythemia Vera (PV) and Essential Thrombocythemia (ET) are long term outcome MPN; however, they could evolve to adverse secondary myelofibrosis-MF or acute leukemia-AL.Aims and Methods:We retrospectively analyzeda cohort of 579 World Health Organization-defined PV (n=180) and ET (n=399) patients, and examined the prevalence and prognostic relevance of BM reticulin fibrosis.All patients were diagnosed between 1990 and 2013 and were recruited in Turin (n= 436) and Bologna (n=143), Italy. BM biopsy sample were reviewed by local pathologist and fiber scoring was performed according to a 3-graded system. Eligibility criteria included the availability of BM samples at diagnosis. Patients with grade 2 or 3-fibrosis were excluded.Overall survival (OS) was evaluated from diagnosis to death using Kaplan Meyer method and Hazard Ratio were estimated with the Cox Model. Cumulative incidence of MF and AL evolution were estimated considering death from any cause as a competing event and compared between groups using the Gray's test.Results:Overall, we observed 115 (63%) grade 0 and 65 (36%) grade 1 fibrosis and 291 (72%) grade 0 and 108 (27%) grade 1 among PV and ET patients, respectively (p= 0.028) We analyzed effect on clinical outcome separately.PVWith a median follow up of 110 months (IQR:70-170), 5 and 10-years OS were 96% and 87%, respectively.Stratifying patients based on fibrosis degree, we observed 15 (13%) and 16 (25%) deaths for grade 0 and 1 fibrosis respectively, with 5 and 10-years OS of 98% vs 90% and 92% vs 82% for grade 0 and grade 1, respectively (p 0.076). Neither clinical findings nor thrombosis were significantly different between fibrosis degree. JAK2 V617F or exon 12 mutation status and allele burden was similar into subgroup. Cumulative incidence of MF evolution at 5 and 10-years was 2,8% and 7,2% vs 3,8% and 18,7% for grade 0 and grade 1 respectively (p 0.123). Cumulative incidence of AL evolution at 10- year was 4,2 % for both grade whereas at 15-years was 4,2% vs 19% for grade 0 and 1, respectively.ETAt a median follow up of 75 months (IQR:39-120), 5 and 10-years OS were 98% and 90%, respectively.We observed 22 (8%) and 16 (55%) deaths for grade 0 and 1 respectively, with 5 and 10-years OS of 98% and 90% for grade 0 vs 97% and 89% for grade 1, respectively (p 0.358).The mutation status was analyzed in 379 patients and showed: 62% JAK2V617F, 19% CALR (type-1/1-like 14% and type2/2-like 5%), 3% MPLW515, 63 patients were triple negative for the above mutations.During follow-up, patients with grade 0 fibrosis showed more thrombotic events, 41 cases (14%; the 71% JAK2V617F-positive) vs 17 (16%). At multivariate analysis MPL mutation showed a higher risk of MF evolution compared to triple negative with an HR of 5,8 (p 0.0014) and to JAK2V617 mutation with an HR of 9,5 (p 0.002).Cumulative incidence of MF evolution was at 5-years 0,5% and 9% and at 10-years 6,2% and 18% for grade 0 and 1, respectively (p 0.0001). Cumulative incidence of AL evolution at 5 and 10-years was 0% for grade 0 and 13% and 7,3% for grade 1, respectively (p 0.096). However grade 0 showed a higher cumulative risk of AL evolution at 15-years (6,9% vs 10% for grade 0 and 1, respectively) (p=0.096).Conclusion:According to data recently published, in ET patients grade 1 BM fibrosis seems to correlate with a higher cumulative risk of MF and AL evolution, whereas in PV patients seems to correlate to a trend of higher mortality, even if not statistically significantly.Data reaffirm the importance of BM examination as part of diagnostic criteria in all MPN. DisclosuresCavo:Janssen-Cilag: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Millennium: Consultancy, Honoraria. Vitolo:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria; Gilead: Honoraria; Celgene: Honoraria; Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.