JAK2 V617 Research Articles

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.

Assessment of relation between JAK2 gene and thrombosis in myeloproliferative neoplasms

Background Thrombotic complications are the most considerable etiology causing morbidity and mortality in patients with philadelphia (Ph) negative myeloproliferative neoplasms (MPN). There are many studies evaluating the association of JAK2 mutation and risk of thrombosis in MPN with inconclusive results. We also investigated the relation between JAK2 mutation in all Ph negative MPN and thrombosis.
 Material and Methods Thrombotic events and demographic features of 177 patients with Ph negative MPN were evaluated retrospectively.
 Results JAK2 V617 F mutation was detected in 57% of patients with essential thrombocythemia (ET), %90.3 of pateints with polycythemia vera (PV), 100% of pateints with primary myelofibrosis (PMF). Thrombotic complications occured more frequently with JAK2 mutation in all MPN patients than without (p=0.014). In JAK 2 mutation positive groups, the median age, thrombosis risk scores and leucocyte values are higher, splenomegaly and arterial and/or venous thrombosis are detected more frequently (p

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.

Clinical, Pathologic Characteristics and Survival Of Adult Patients With Essential Thrombocythemia: Experience In a Reference Center In Mexico

IntroductionEssential thrombocythemia (ET) is a myeloproliferative neoplasm, characterized by sustained thrombocytosis (≥450 x 109/L), vasomotor symptoms and thrombotic / hemorrhagic complications. Treatment and prognosis relies on risk stratification, based on age and thrombosis history. Survival is similar to that of general population, however there is a low risk of transformation to myelofibrosis (MF) and acute myeloid leukemia (AML). ObjectiveTo describe demographic, clinical and hystopathologic characteristics, as well as general outcome of patients with ET treated at INCMNSZ. MethodsDescriptive and retrospective study of patients with ET, diagnosed from January 2000 to December 2012 and reclassified according to WHO 2008 criteria. General data and overall survival were calculated using SPSS v17.0. ResultsForty-nine patients with ET were included, with a median age of 56 years, 65.3% were female. At diagnosis, 98% had an ECOG of 0 to 1, and 42.9% had three or more comorbidities. Table 1 shows laboratory parameters at diagnosis. Forty-nine percent and 16.3% were classified as having high risk according to Tefferi and IPSET classifications respectively. In 60.4% of biopsies, reticulin was not reported; in positive cases, grade 1 fibrosis was the most frequent in 20.8%. Karyotype was normal in 53.1%, and it was possible to determine JAK2 V617F mutation only in 16.4%, reported as positive in 8.2%. Thrombotic events were present in 16.3%, 50% of which were venous. Secondary Von Willebrand syndrome was documented in 10.2%. Hydroxyurea and acetylsalicylic acid were the most used drugs, 91.8% and 89.8% respectively, among others. Of patients who received treatment, 42.9% and 38.8% had partial and complete responses, respectively. Histological transformation was documented in 14.3%, 8.17% to MF and 6.12% to AML, at a median of 7.8 years from diagnosis. At last follow-up, 79.6% were alive, with median overall survival not reached, and 8.2% had died, mainly because of progression of MF/AML.Table 1Laboratory parameters at diagnosis (median)Hemoglobin14.1 g/dLWBC8,600 /uLPlatelets1,043,000 (535,000 – 2,580,000) / uLFerritin57.35 ng/mLVitaminB12713 pg/mLErythrocytesedimentationrate5 mm/hC-reactiveprotein0.15 mg/LProthrombintime / INR11.2” / 1.1Partialthromboplastintime33.4” Discussion and conclusionsIn this cohort thrombosis incidence is similar to international reports (11-25%), and the high rate of transformation to MF and AML perhaps is influenced by the diagnostic criteria used in the early 2000 (PVSG criteria vs WHO criteria). Concerning mutational status of JAK2 V617, the incidence is low and strong conclusions can not be made in this matter because of the retrospective sense of the study and the availability of the test in our center. In conclusion, these results emphasize the importance of adhering to strict clinical and histopatological criteria to diagnose patients with ET, establish the risk and identify those who do not require cytoreductive treatment, in order to reduce the risk of long term complications. Disclosures:No relevant conflicts of interest to declare.

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.

Detection of Three Different MPLW515 Mutations in 10.1% of All JAK2 V617 Unmutated ET and 9.3% of All JAK2 V617F Unmutated OMF: A Study of 387 Patients.

Recently MPLW515 mutations have been reported in osteomyelofibrosis (OMF) and essential thrombocythemia (ET). To further evaluate this marker for routine diagnostics of CMPD we have analyzed 387 patients (pts) by a melting curve assay and subsequent sequencing of pts with aberrant curves. 97 pts had a diagnosis of ET (79 with unmutated JAK2V617 (V617wt) and 18 with JAK2V617F), 164 had V617wt undefined CMPD with thrombocytosis, 101 had OMF (54 with V617wt and 47 with V617F) and 25 had V617wt/exon12wt polycythemia vera (PV). Overall we detected 3 different mutations in 16 pts: W515L (n=10), W515K (n=5) and a so far undescribed W515S mutation in 1 pt. In 8/97 ET MPLW515 mutations were detected. All 8 pts were V617wt. Thus, the frequency of W515 mutations in V617wt ET was 8/79 (10.1%). In addition, 3/164 (1.8%) CMPD with thrombocytosis were mutated (subsequently classified ET). In OMF MPLW515 mutations were detected in 5 of 54 V617wt pts (9.3%). No MPLW515mut was found in 18 ET and 47 OMF with JAK2V617F and in the 25 V617wt/exon12wt polycythemia vera (PV). In 4 pts the mutation was homozygous (3 W515K, 1 W515L) and all 4 pts were at advanced stage of their disease: 3 OMF 2-6 years (y) after initial diagnosis and 1 ET in transformation 9 y after diagnosis. Mutation/wt ratio in the remaining pts were 10–100% (median: 40%) in ET and 40% in OMF. The female/male ratio in the mutated cases was 9/7. The age was between 30 and 80 y (median 64 y) and thus was in the same range as in V617F mutated ET/OMF (median 65.3 y; n=489) but significantly higher than in V617wt ET/OMF (median 55.6 y; n=410; p<0.01). Median peripheral blood counts of the W515 mutated cases were: WBC 6,700/μl, Hb 120 g/L, platelets 755,000/μl in ET and WBC 5,200/μl, Hb 80 g/L, platelets 175,000/μl in OMF. These values are similar to those in the JAK2V617F control group (WBC 9,150/μl, Hb 142 g/L, platelets 719,000/μl in ET and and WBC 11,400/μl, Hb 105 g/L, platelets 195,000/μl in OMF. All pts with available data (not all parameters available for all cases) showed dysplastic and enhanced megakaryopoiesis and elevated erythrocyte count. 50% had spenomegaly and mild hepatomegaly. Cardiac problems were described in 4 pts. Karyotype was available in 8 pts and all had normal karyotypes. In particular, it has to be mentioned that even in 1p34, where the MPL gene resides, no aberration was observed. This has not been anticipated as in JAK2 mutated cases there is a high correlation to trisomy 9 or aberration of 9p (where the JAK2 gene resides). In conclusion, MPLW515 mutated ET and OMF can not be discriminated from JAK2V617F mutated ET or OMF based on clinical parameters. However, MPLW515 may help to support the diagnosis of pts being suspicious for ET or OMF and showing no JAK2V617F mutation. In addition, during follow up homozygousity can indicate progression of disease.

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.

The Thrombopoietin Receptor, MPL, Is a Therapeutic Target of Opportunity in the MPN.

The myeloproliferative neoplasms, polycythemia vera, essential thrombocytosis and primary myelofibrosis share driver mutations that either activate the thrombopoietin receptor, MPL, or indirectly activate it through mutations in the gene for JAK2, its cognate tyrosine kinase. Paradoxically, although the myeloproliferative neoplasms are classified as neoplasms because they are clonal hematopoietic stem cell disorders, the mutations affecting MPL or JAK2 are gain-of-function, resulting in increased production of normal erythrocytes, myeloid cells and platelets. Constitutive JAK2 activation provides the basis for the shared clinical features of the myeloproliferative neoplasms. A second molecular abnormality, impaired posttranslational processing of MPL is also shared by these disorders but has not received the recognition it deserves. This abnormality is important because MPL is the only hematopoietic growth factor receptor expressed in hematopoietic stem cells; because MPL is a proto-oncogene; because impaired MPL processing results in chronic elevation of plasma thrombopoietin, and since these diseases involve normal hematopoietic stem cells, they have proven resistant to therapies used in other myeloid neoplasms. We hypothesize that MPL offers a selective therapeutic target in the myeloproliferative neoplasms since impaired MPL processing is unique to the involved stem cells, while MPL is required for hematopoietic stem cell survival and quiescent in their bone marrow niches. In this review, we will discuss myeloproliferative neoplasm hematopoietic stem cell pathophysiology in the context of the behavior of MPL and its ligand thrombopoietin and the ability of thrombopoietin gene deletion to abrogate the disease phenotype in vivo in a JAK2 V617 transgenic mouse model of PV.

JAK2 Negative Erythrocytosis Associated with JAK2 GGCC_46/1 Haplotype, Calr rs1049481_G, and Normal Erythropoietin Level: Is This a New Entity?

Introduction. The JAK2 haplotype known as "GGCC or 46/1 haplotype" consists of a germline combination of single nucleotide polymorphisms (SNPs) that are inherited together and are frequently associated with the onset of myeloproliferative neoplasms (MPNs) positive for both JAK2 V617 and exon 12 mutations. It has been reported a significant association between JAK2 negative erythrocytosis and the simultaneous occurrence of JAK2 haplotype GGCC_46/1 and CALR rs1049481_G allele, mapping in the 3' UTR of the gene. In the present study, we investigated the presence of JAK2 haplotype GGCC_46/1 and CALR rs1049481_G allele in a more extensive series of erythrocytosis patients and evaluated a possible correlation with serum erythropoietin (EPO) level. Methods. Fifty-nine erythrocytosis patients, diagnosed at our Center from 2009 to 2020, negative for canonical JAK2 mutations, and secondary causes, were included in this study. Forty-nine (83%) out of 59 cases met only two major WHO 2016 diagnostic criteria for polycythemia vera (PV); the remaining 10 cases were "JAK2 negative PV", as they also showed the minor criterion with subnormal serum EPO. Genomic DNA from all patients was extracted from peripheral blood granulocytes and analyzed for the presence of both SNPs. The occurrence of the JAK2 haplotype was investigated by analyzing rs10974944 tagging SNP, using distal JAK2 intron 12 primers (forward: 5'-CTGTGCAGTCCAAACCCATG-3' and reverse: 5'-TTCTCTGCTTGCTAGTGGGT-3'). This amplification generated a PCR product of 272 bp, revealing a C/G substitution by Sanger sequencing. PCR investigated the occurrence of the G/T allele in the SNP rs1049481 in the CALR gene with primers CALR-UTR-F 5'-GACAAGGAGGATGATGAGGACAAA-3' and CALR-UTR-R 5'- AAAAATGAAAGTTCTCGAGTCTCACAGA-3' generating a 205 bp product. In silico data from 2504 healthy individuals of the 1000G Project (1000G) were used as a control group. Fisher's exact test was used to compare the differences of both SNPs and the frequency of their alleles between cases and controls, respectively; the c-square test was employed for evaluating the distribution of JAK2 GGCC haplotype in erythrocytosis patients and controls. Results. Thirty-eight (64.4%) and 21 (35.6%) cases resulted in being positive and negative for the JAK2 GGCC haplotype, respectively. The JAK2 GGCC haplotype occurred to be associated with erythrocytosis as a statistically significant difference in frequency was detected as respect to 2504 healthy individuals from the 1000G Project (p=0.0013). This finding was also confirmed considering the Genome Aggregation Database (gnomAD v2.1.1, https://gnomad.broadinstitute.org/)(p=0.0024). The association was also demonstrated in terms of allelic frequency (p=0.0131) and genotype distribution (p=0.0030). Regarding CALR rs1049481 SNP, a significant difference in the CALR rs1049481_G allelic rate was confirmed in our cohort compared to 1000G controls (p=0.0360). Overall, the association between JAK2 GGCC haplotype and CALR rs1049481_G was observed in 28/59 (47.5%) erythrocytosis cases compared to healthy individuals (628/2504, 25%), and this difference resulted in being statistically significant (p=0.0002). Based on the EPO level, erythrocytosis patients were divided into two groups: normal (49 cases) or subnormal (10 cases). Interestingly, the simultaneous presence of JAK2 GGCC haplotype and CALR rs1049481_G was statistically significantly associated with the erythrocytosis group showing normal EPO (p=0.0133). Conclusions. This study suggests that the JAK2 GGCC haplotype and the presence of the CALR rs1049481_G allele were significantly associated with erythrocytosis cases, negative for canonical JAK2 mutations. Our findings are in line with recent literature evidence showing that germline predisposition factors such as SNPs and haplotypes may play a crucial role in MPN pathogenesis. Moreover, this study shows that patients showing two major WHO 2016 diagnostic criteria (erythrocytosis and panmyelosis) without JAK2 mutations and with normal EPO levels can benefit from the search for germline polymorphisms combination in JAK2 and CALR driver genes for a better diagnostic classification. Therefore, the presence of these polymorphisms could represent a novel minor criterion for the diagnosis of "JAK2 negative PV". Disclosures No relevant conflicts of interest to declare.

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

Lenalidomide Is Effective Treatment Option for Patients with Refractory Anemia with Ring Sideroblasts and Thrombocytosis

CONTEXT AND OBJECTIVE:Refractory anemia with ring sideroblasts and thrombocytosis (RARS-T) is a provisional entity included in the MDS/MPN overlap syndromes, with features of refractory anemia with ring sideroblasts (RARS) and thrombocytosis (platelet count ³ 450 x 10/L), recently renamed MDS/MPN with ring sideroblasts and thrombocytosis. Management of RARS-T is extrapolated from MDS and MPN treatment options namely for alleviating symptoms of anemia and, occasionally, for thrombocytosis. Individual case reports have suggested that lenalidomide may be effective in treating RARS-T. In non-del 5q, low-risk MDS, 25% of patients (pts) became red blood cell transfusion independent (RBC-TI). In the phase II study of lenalidomide in non-del 5q lower risk MDS, 35% of pts with RARS treated with lenalidomide achieved RBC-TI. The primary objective of this study is to evaluate a series of pts with RARS-T and compare hematological improvement (HI) responses among different treatment options.METHODS:This is a retrospective, single-center study. Patients with RARS-T were identified from the MDS Database at Moffitt Cancer Center. Samples for somatic mutation testing were obtained at time of referral. Descriptive statistics were used to describe baseline characteristics and response rates to treatment. The primary endpoint was HI using international working group criteria (IWG) 2006. The median overall survival (OS) was calculated from time of diagnosis.RESULTS:We identified 33 pts with RARS-T. The mean age was 67 years (38-80), 15 pts were male (45.5%), and majority were Caucasians (n=27, 82%). According to international prognostic scoring system (IPSS), 24 patients (73%) were low risk and 9 pts (27%) were int-1 risk. By revised IPSS, 11 pts (33%), 18 (55%), 3 (9%), and 1 (3%) were very low, low, intermediate, and high risk, respectively. The mean WBC was 7.7 (2.8-25.7), the mean Hemoglobin was 9.9 g/dl (7.3-12.4), and the mean platelet count was 682 (372-1644). Among 20 pts who had Next generation sequencing for somatic mutations at time of referral, 19 pts (95%) had SF3B1 mutation and 4 pts (20%) had JAK-2 V617 F mutation.The median OS was 90 mo (95% CI 47-134). For pts with JAK-2 V617F mutations, the median OS was 110 mo versus 74 mo for those with wild type (p=0.24). The rate of AML transformation was 3% (one patient only).Among the 33 pts, 8 pts had no treatment (observation only), 22 pts (67%) received erythroid stimulating agent (ESA) prior to any active therapy, 2 patients received lenalidomide with no prior ESA, and 1 pt ruxolitinib with no prior ESA. Twelve pts (36%) received lenalidomide (7 as first line active therapy after ESA and 2 without prior ESA exposure) and 13 pts (33%) received azacitidine (6 as first line active therapy after ESA).The HI rate for ESA was 36% (12/22) with a median duration of ESA treatment of 33mo. The HI rate for lenalidomide was 50% (6/12) with a median duration of lenalidomide treatment of 10.3mo. Among the 9 pts who received lenalidomide as first line active therapy (2 pts without prior ESA exposure) HI rate was 56%. The HI rate for azacitidine was 15% (2/13) with a median duration of azacitidine treatment of 6.3mo.CONCLUSIONS: Pts with RARS-T had favorable outcomes, with long overall survival and a low rate of AML transformation. Rates of hematological improvement with the use of azacitidine were lower than expected. Lenalidomide is an effective treatment option and should be considered when treating patients with RARS-T. [Display omitted] DisclosuresSallman:Celgene: Research Funding, Speakers Bureau. List:Celgene: Research Funding. Komrokji:Novartis: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Celgene: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Speakers Bureau.

Risk Factors for Secondary Cancer in a Case-Control Study on 1,259 Patients with Myeloproliferative Neoplasms

INTRODUCTIONThe incidence of secondary cancer (SC) in patients with myeloproliferative neoplasms (MPN) is high and comparable to that of thrombosis. However, the identification of patient subgroups that might be at increased susceptibility of developing SC has not been systematically addressed. We report here the results of an international case-control study (MPN-K) aimed at comparing the frequency of exposure to possible causes of SC in patients with classical MPN, polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF).METHODSThis European Leukaemia Network (ELN) study reports MPN patients from 28 sites of 5 European countries and Israel, diagnosed in the period from 2000 to 2016. Cases were MPN patients with concomitant diagnosis of a non-myeloid SC (n=15) or its presentation during the course of the disease (n=412). Controls were MPN patients cancer-free, matched to the paired case for sex, age (±5 years), date of MPN diagnosis (±5 years), and MPN disease duration (±6 years). A multivariable conditional logistic regression model was used to estimate the effect of selected variables on total SC risk and in different types of SC.RESULTSAmong 1,259 MPN patients, there were 427 cases and 832 matched controls. Cases presented melanoma (n=20; 4.7%), non-melanoma skin cancer (n=69; 16.2% - basal/squamous cell carcinoma), non-skin solid cancer (n=290; 67.9%) including breast, ovary/uterus, colorectal, upper gastrointestinal, liver/pancreas, lung, prostate/urinary, other and lymphoproliferative diseases (n=48; 11.2%) including multiple myeloma, chronic lymphocytic leukemia, low and high grade B- and T-lymphoma.At diagnosis, there were slightly more patients with PV among SC cases (n= 152; 35.6%) than controls (n=256; 30.8%), while conversely there were slightly less ET patients among cases (n=196; 45.9%) than controls (n=426; 51.2%). Cases and controls presented similar proportion of MF diagnosis (n=79 cases, 18.5% and 150 controls, 18.0%). Driver mutations (JAK2 V617, EXON-12, CALR, MPL), non-driver mutations and abnormal karyotype were equally represented in cases and controls. Other variables such as cardiovascular risk factors, exposure to cancerogens, family history of cancer and chronic inflammatory diseases were reported with similar frequency in cases and controls.After MPN diagnosis, exposure to first and other lines of treatments until the index event, with Phlebotomy (n=193; 15.3%), Hydroxyurea (n=814; 64.7%), Anagrelide (n=14; 1.1%), Interferon (n=30; 2.4%), Pipobroman (n=8; 0.6%), Busulphan (n=13; 1.0%), Ruxolitinib (n=11; 0.9%), was similar in the two groups except for aspirin that was used less frequently (p=0.043) in cases (n=320; 74.9%) compared to controls (n=664; 79.9%). In particular, the lower use of aspirin was circumscribed to non-skin solid tumors. A multivariable analysis was carried out in all patients and stratified by different type of tumors (Table). In non-skin solid cancers, the time to exposure of the MPN disease > 5 years (OR=2.95; 95% CI 1.54-5.66, p=0.001) and the PV phenotype (OR=2.40, 95% CI 1.15-5.01, p=0.020) were more burdened by the incidence of events than the reference ET group. No difference in SC risk was found for MF patients compared to patients with ET.Interestingly, the independent protective role of aspirin retained its statistical significance only in non-skin SC. In non-melanoma skin cancer, multivariable analysis revealed that the presence of JAK2 mutation was less associated with SC (OR=0.32, 95% CI 0.13-0.81, p=0.016) and confirmed that exposure to HU and other cytotoxic agents was associated with a significantly higher risk of SC (OR=6.00, 95% CI 1.23-29.28, p=0.027 and OR=9.80, 95% CI 1.24-77.78, p=0.031, respectively). This finding was not seen in non-skin SC and in lymphoma.CONCLUSIONThe considered clinical and biological features, at MPN diagnosis, were not different in cases with SC and controls. During the course of the disease, three factors significantly and independently affected the risk of SC in these MPN patients: 1) patients with PV had a 77% higher risk than those with ET, 2) patients with MPN duration of more than 5 years had a twice higher risk than those with lower duration, 3) for the first time, we documented that in non-skin solid cancers, aspirin treatment reduced SC risk of 38%. Exposure to HU and other cytoreductive drugs was confirmed as a risk factor for non-melanoma skin cancer. [Display omitted] DisclosuresPalandri:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Marchetti:Gilead: Consultancy; takeda: Speakers Bureau; amgen: Speakers Bureau; janssen: Speakers Bureau. Griesshammer:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Symptomatic Hemoglobin New Mexico and a Case of Therapeutic Benefit with Acetazolamide

INTRODUCTIONInherited disorders due to rare structural Hb variants are not included in national, neonatal screening tests and therefore likely underdiagnosed. Hemoglobin New Mexico is one such rare variant with only one case ever reported in 1985 in conjunction heterozygosity for Hb S.1 This Hb variant involves a missense mutation of the β chain substituting the hydrophobic Proline β100 with the hydrophilic Arginine.1 We present here the second case of Hb New Mexico variant ever reported to our knowledge.CASE PRESENTATIONAn 18-year-old athletic, Latin American female initially presented with episodic chest pain, palpitations, fatigue, headaches, and dyspnea on exertion to the point that she could no longer exercise for one year. Work up revealed erythrocytosis persistent for >1 year with hemoglobin level of 17.3 g/dL, and hematocrit of 47.2 % (Hb went as high as 19g/dL) with normal erythropoietin level, hemoglobin electrophoresis, and JAK2 V617 with reflex to JAK3 exon 12-15 negative. She had extensive evaluation to rule out acquired causes including cardiac, pulmonary, and neurologic evaluation which was all normal. She underwent therapeutic phlebotomies with no clinical improvement over 3 months and symptoms worsened as well as new iron deficiency from phlebotomies so this was discontinued. Evaluation with p50 oxygen dissociation curve revealed high oxygen affinity. Further gene sequencing revealed heterozygous inheritance of Hb New Mexico with HbA: 54.5%, HbA2: 3.6%, and Hb New Mexico: 41.9%. Beta Globin Gene sequencing identified the following mutation: Beta 100, CCT>CGT, Pro>Arg. Her symptoms worsened as well as an episode of syncope but no new neurologic, cardiac, or pulmonary etiologies were identified and at the time Hg 18g/dL. A trial of low dose acetazolamide 125mg daily was given to the patient and her symptoms improved by a 6 week follow up with notable reduction of headache, fatigue, dyspnea, and chest pain.CONCLUSIONSGiven the rarity of this hemoglobin variant in a patient with severe symptoms, data is limited. Acetazolamide, a carbonic anhydrase inhibitor often used for migraines, is known to illicit mild acidosis and therefore potentially reduce hemoglobin-oxygen affinity and perhaps allow for relief of our patient's symptoms. Further therapeutic evaluation is warranted but likely challenging given rarity of the condition. 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.

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.

The JAK2 GGCC (46/1) Haplotype in Myeloproliferative Neoplasms: Causal or Random?

The germline JAK2 haplotype known as “GGCC or 46/1 haplotype” (haplotypeGGCC_46/1) consists of a combination of single nucleotide polymorphisms (SNPs) mapping in a region of about 250 kb, extending from the JAK2 intron 10 to the Insulin-like 4 (INLS4) gene. Four main SNPs (rs3780367, rs10974944, rs12343867, and rs1159782) generating a “GGCC” combination are more frequently indicated to represent the JAK2 haplotype. These SNPs are inherited together and are frequently associated with the onset of myeloproliferative neoplasms (MPN) positive for both JAK2 V617 and exon 12 mutations. The association between the JAK2 haplotypeGGCC_46/1 and mutations in other genes, such as thrombopoietin receptor (MPL) and calreticulin (CALR), or the association with triple negative MPN, is still controversial. This review provides an overview of the frequency and the role of the JAK2 haplotypeGGCC_46/1 in the pathogenesis of different myeloid neoplasms and describes the hypothetical mechanisms at the basis of the association with JAK2 gene mutations. Moreover, possible clinical implications are discussed, as different papers reported contrasting data about the correlation between the JAK2 haplotypeGGCC_46/1 and blood cell count, survival, or disease progression.

Morphologic and Molecular Characteristics of De Novo AML With JAK2 V617F Mutation.

Background:JAK2 V617F mutation (mut) in acute myeloid leukemia (AML) is rare. We describe the clinicopathologic findings of a single-institution series of 11 de novo AML cases with JAK2 V617. Methods: We identified cases of de novo AML with JAK2 V617F over a 10-year period. We reviewed diagnostic peripheral blood and bone marrow (BM) morphologic, cytogenetic, and molecular studies, including next-generation sequencing. The control group consisted of 12 patients with JAK2 wild-type (wt) AML matched for age, sex, and diagnosis. Results: We identified 11 patients (0.5%) with JAK2 V617F, with a median age at diagnosis of 72.5 years (range, 36-90 years). Ten neoplasms were classified as AML with myelodysplasia-related changes and 1 as AML with t(8;21)(q22;q22). All JAK2mut AML cases showed at least bilineage dysplasia, 7 of 11 showed fibrosis, 8 of 11 had an abnormal karyotype, and 5 had deletions or monosomy of chromosomes 5 and 7. Using the European LeukemiaNet (ELN) classification, 9 patients (82%) with JAK2mut AML were intermediate-2 and adverse risk. Cases of JAK2mut AML did not have mutations in other activating signaling pathways (P=.013); 7 (64%) showed additional mutations in at least one gene involving DNA methylation and/or epigenetic modification. Patients with JAK2mut AML had a significantly higher median BM granulocyte percentage (12% vs 3.5%; P=.006) and a higher frequency of ELN intermediate-2 and adverse risk cytogenetics (P=.04) compared with those with JAK2wt AML. JAK2mut AML showed higher circulating blasts, but this difference was not significant (17% vs 5.5%; P=not significant). No difference was seen in the median overall survival rate of patients with JAK2mut AML versus those with JAK2wt AML (14 vs 13.5 months, respectively). Conclusions: De novo JAK2mut AML is rare and frequently found in patients with dysplasia, BM fibrosis, and abnormal karyotype with intermediate- or high-risk features; gene mutations in DNA methylation and epigenetic-modifying pathways; and absence of gene mutations in activating signaling pathways.

An Inhibitor of TGF-β Promotes Proliferation of Normal but Not MPN Hematopoietic Cells and Is a Candidate Therapeutic Agent for the Treatment of MPN Patients Carrying JAK2 V617F or Calr pQ365fs Mutations

The inhibitory effects exerted by transforming growth factor-β (TGF-β) on adult erythropoiesis has been well established. Canonical SMAD-depedent TGF-β signaling inhibits erythroid differentiation at multiple levels: it induces hematopoietic stem cells into quiescence (Chabanon et at Stem Cells. 2008;26:3150), it elicits a Smad5-dependent inhibition of progenitor cell proliferation (Bruno et al, Blood 1998; 91:1971) by increasing the length of G1 through reduction of G1 cyclin and cyclin-dependent protein kinases (Jacobsen et al, Blood 1995; 86:2957; Geng and Weinberg, PNAS 1993; 90:10315) and triggers Smad4-signaling accelerating terminal erythroid maturation (Zermati et al, Exp hematol 2000; 28:885; Choi et al MCB 2005;271:23). TGF-β is expressed by almost every cell type, but is produced in the greatest amount by megakaryocytes (Assonian et al, JBC 1983;258:7155; Massague, Cell 2008;134, 215).TGF-β has been implicated in the pathogenesis of primary myelofibrosis (PMF) by Schmitt et al (Blood. 2000;96:1342). Plasma, bone marrow and spleen washes of PMF patients, as well as those from murine PMF models, contain 2 fold-greater levels of total and bioactive TGF-β than those from normal controls (Zingariello et al, Blood 2013;121:3345). In addition, megakaryocytes from PMFpatients contain greater amounts of TGF-β than those from other MPNs such as polycythemia vera (PV) and essential thrombocythemia (Ciurea et al, Blood 2007;110:986). Published data, however, also suggest that malignant MPN cells are insensitive to TGF-β. Microarray analyses of PMF bone marrow and spleen cells revealed TGF-β signaling abnormalities which predict activation of non-canonical p38/ERK-dependent rather than canonical SMAD-dependent signaling (Ciaffoni et al, BCMD 2015;54:234). In addition, by phosphoproteomic profiling of PV erythroblasts (Erys) expanded in vitro express lower levels of pSmad2 as compared to Erys from healthy controls (Hricik et al. AJH 2013;88:723).The hypothesis that malignant MPN cells are insensitive to TGF-β was tested here by evaluating the effect of SB431542 [1,3,10 and 26µM], a small molecule inhibitor of TGF-β receptor 1, on ex vivo erythropoiesis in cultures generated by peripheral blood mononuclear cells from patients with JAK2 V617+-PV (n=2) and JAK2 V617F+ or CALR pQ365f+-PMF. Identical experiments were performed with Erys generated from adult peripheral blood (AB, n=3) and cord blood (CB, n=2) of healthy controls. All cultures were stimulated with SCF, IL-3 and EPO with and without dexamethasone (Dex).In cultures of JAK2 V617F+ -PV, SB431542 increased by 2-fold the numbers of progenitor cells observed by day 6, but had no effect on that of Erys observed by day 12-17 [fold increase (FI) ~4 fold in all cases]. Moreover, neither the number of progenitor cells nor that of Erys were affected by SB431542 treatment in cultures generated from JAK2 V617F+ (n=1) and CALR pQ365fs+ -PMF (n=1) patients (Fig. 1). This lack of effects was observed in cultures with and without Dex.By contrast, as expected, in cultures of AB, SB431542 significantly increased by 2.5-fold the number of progenitor cells observed by day 6 and that of Erys observed by day 14-17 (Fig. 1). This increase was associated with greater retention of an immature erythroid phenotype (CD36+ CD235a+ cells 16% vs 3%) and an increased proliferative index (>3 Erys in metaphases per field vs 0). This was observed up to day 17 in cultures both with and without Dex. The effects of SB431542 in cultures of CB were, however, affected by the presence of Dex. In cultures without Dex, SB431542 increased by 2-fold the number of progenitor cells by day 6 but had no effect on that of Erys by day 12-17 (FI=10-15 and CD36+CD235a+ cells >60%). In the presence of Dex, SB431542 did not affect the number of progenitor cells at day 6 but reduced that of Erys by 3-fold on day 12-17. These results suggest that in the case of CB, TGF-β promotes erythroid maturation in synergy with Dex.In conclusion, SB431542 promoted proliferation and maturation of normal adult progenitor cells but had no effect on PMF progenitor cells suggesting that treatments with TGF-β receptor 1 inhibitors may reactivate normal hematopoiesis in PMF patients by providing a proliferative advantage to the resident non-diseased hematopoietic stem cells over the malignant clone. This therapeutic approach will be explored in a MPD-RC, multi-center, phase II trial in patients with PMF. [Display omitted] DisclosuresHoffman:Geron: Consultancy, Membership on an entity's Board of Directors or advisory committees; All Cells, LLC: Consultancy, Membership on an entity's Board of Directors or advisory committees; Promedior: Research Funding. Mascarenhas:Roche: Research Funding; Incyte Corporation: Research Funding; Kalobios: Research Funding; CTI Biopharma: Research Funding; Novartis Pharmaceuticals Corporation: Research Funding; Promedior: Research Funding.