Littérature scientifique sur le sujet « Ph-negative chronic myeloproliferative neoplasms, JAK2-V617F mutation, LAMP »

Abstract Abstract 3892 Poster Board III-828 Background The point mutation G1849T (V617F) of the JAK2 gene occurs at high frequency in several Ph-negative chronic myeloproliferative neoplasms (Ph-neg-CMNs), such as Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Myelofibrosis (MF). The molecular analysis of this mutation is mandatory in the diagnostic work up of these diseases. Several molecular diagnostic techniques are currently used but each of them present some important limitations such as a low sensitivity and the requirement of labor intensive procedures performed with expensive specialized equipment that may not always be readily available in clinical laboratories. Method We have developed a non-PCR method for the identification of the JAK2V617F mutation called Allele Specific (AS)-LAMP, based on the Loop mediated isothermal AMPlification (LAMP) principle (Notomi et al. NAR 2000). LAMP reaction efficiently produces, within one hour, a large amount of amplified DNA and does not require gel separation of the amplified product which is indirectly detected by measuring the loss of intensity of a light beam through the reaction solution in which suspended particles of magnesium pyrophosphate are generated as a result of the DNA amplification process. Pyrophosphate salts produce turbidity which is both visible to the naked-eye and monitorable in Real-Time turbidimetry. AS-LAMP consists of 4 primers suitable for LAMP and a self-annealed primer highly specific for the mutated target sequence. To ensure efficiency and sensitivity a Peptide Nucleic Acid (PNA) probe specific for the wild-type allele was added thus resulting in absence of normal allele amplification within the reaction time. The AS-LAMP assay was optimized on plasmid controls and on human genomic DNA extracted from the HEL and K562 cell lines, respectively carrying or not the JAK2V617F mutation. The level of sensitivity was determined by testing serial dilutions of mutant HEL DNA in K562 DNA at concentrations of 100, 10, 1, 0.5, 0.1, 0.05, 0.01 and 0%. Results This simple, easy to perform and rapid AS-LAMP assay selectively detects the JAK2V617F mutated DNA down to 0.05%. Moreover, when mutant DNA is present in the range of 1%-100% in wild type DNA, we observed a linear relationship between the mutant allele burden and the amplification time. We have validated this AS-LAMP assay on DNA obtained from 87 patient samples previously analyzed by conventional Allele Specific PCR (ASO-PCR): 19 PV, 58 TE, 3 IMF, 1 post ET Acute Myeloid Leukemia (AML), 1 post PV and 1 post ET Myelofibrosis, 2 Idiopathic Erythrocytosis (IE) and 2 unclassified CMNs. All samples which proved positive by ASO-PCR resulted positive with our AS-LAMP assay (100% concordance). In addition, 6 ET and 1 IE previously found negative by ASO-PCR were found to be low-positive (<1%) with AS-LAMP. Interestingly, the molecular monitoring in one patient with post-PV MF achieving complete remission after allogenic transplantation, proved repeatedly negative by ASO-PCR but positive by AS-LAMP. Sequencing analysis after PCR amplification with PNA confirmed the presence of the JAK2V617F mutation in all these LAMP-low-positive samples. None of the negative controls, (1 AML, 2 Acute Lymphoblastic Leukemia, 2 Follicular Non Hodgkin's Lymphoma, 2 Chronic Lymphocytic Leukemia, and 1 healthy donor) gave false positive results. Conclusions This novel, non-PCR based allele-specific LAMP assay is rapid and reduces the risk of contamination related to post amplification manipulations. Most importantly, it is highly specific and sensitive and significantly increases our ability to detect a low JAK2V617F tumor allele burden. For all these reasons, the AS-LAMP assay can be a valid and powerful tool in the routine diagnostic work up and the molecular monitoring of these diseases. Disclosures: Minnucci: Diasorin S.p.A.: Employment. Amicarelli:Diasorin S.p.A: Employment. Salmoiraghi:Diasorin S.p.A.: Consultancy, Honoraria. Spinelli:Diasorin S.p.A: Consultancy, Honoraria. Adlerstein:Diasorin S.p.A.: Employment. Rambaldi:Diasorin S.p.A.: Consultancy, Honoraria.

JAK2 (Janus kinase 2) V617F, CALR (Calreticulin) exon 9, and MPL (receptor for thrombopoietin) exon 10 mutations are associated with the vast majority of Ph-negative chronic myeloproliferative neoplasms (MPNs). These mutations affect sequential stages of proliferative signal transduction and therefore, after the emergence of one type of mutation, other types should not have any selective advantages for clonal expansion. However, simultaneous findings of these mutations have been reported by different investigators in up to 10% of MPN cases. Our study includes DNA samples from 1958 patients with clinical evidence of MPN, admitted to the National Research Center for Hematology for genetic analysis between 2016 and 2019. In 315 of 1402 cases (22.6%), CALR mutations were detected. In 23 of these 315 cases (7.3%), the JAK2 V617F mutation was found in addition to the CALR mutation. In 16 from 24 (69.6%) cases, with combined CALR and JAK2 mutations, V617F allele burden was lower than 1%. A combination of JAK2 V617F with MPL W515L/K was also observed in 1 out of 1348 cases, only. JAK2 allele burden in this case was also lower than 1%. Additional mutations may coexist over the low background of JAK2 V617F allele. Therefore, in cases of detecting MPNs with a low allelic load JAK2 V617F, it may be advisable to search for other molecular markers, primarily mutations in exon 9 of CALR. The load of the combined mutations measured at different time points may indicate that, at least in some cases, these mutations could be represented by different clones of malignant cells.

Introduction. Haplotype JAK2 46/1 is associated with more frequent development of Ph-negative myeloproliferative neoplasms (MPN) and with an increased detection rate of the JAK2 V617F mutation. At the same time, the molecular mechanisms of such associations remain unclear. Previously, there were no studies of regional, age and gender aspects of the predictive value of carriage of the 46/1 JAK2 haplotype, which could assess this relationship in some observations.Aim — to analyze the degree of association between 46/1 haplotype and the V617F mutation of the JAK2 gene depending on the sex, age, and place of residence of patients examined for suspected MPN.Methods. The study included 949 DNA samples from patients with suspected MPN. Samples of 150 volunteers and blood donors were included in the control group. Haplotype 46/1 (rs10974944), V617F mutation in the JAK2 gene, mutations in the CALR gene (type 1: c.1092_1143del; L367fs*46, COSV57116546; type 2: c.1154_1155insTTGTC; K385fs*47, COSV57116551) and in the MPL gene (W515K, W515L) were determined for all samples using real-time polymerase chain reaction (PCR-RT).Results. The 46/1 JAK2 haplotype were shown to be associated with a clinically significant level (> 2 %) of the allelic burden of the JAK2 V617F mutation. The odds ratio of the risk of developing a V617F positive MPN when carrying this haplotype variant did not depend on the main place of residence of the patients and was found to be most pronounced in men under 50 years of age. The odds ratio of the risk did not depend on the age of the examined women.Conclusion. The association of 46/1 haplotype with the presence of other drivers of MPN mutations in the CALR or MPL genes was also statistically significant, which confirms the hypothesis of “favorable soil” rather than “hypermutability” of the JAK2 gene.

Abstract Objectives and background: Genetic mutations result in abnormalities of myelopoietic proteins and lie in the basis of Ph-negative myeloproliferative neoplasms (MPNs) development and its subsequent progression. Several somatic mutations in JAK2, MPL, TET2, EZH2, ASXL1, CBL, IDH1, IDH2, IKZF1 genes were detected in chronic and blastic phase MPNs. Recent studies have revealed a number of epigenetic alterations that contribute to Ph-negative MPNs pathogenesis and determine the clinical outcome. Mutations involving the EZH2 gene are thought to result in loss of methyltransferase activity suggesting a potential role of tumor suppressor gene silencing as a mechanism in the disease progression. Decrease in ubiquitin ligase activity caused by mutations CBL gene leads to myeloid proliferation. EZH2, CBL mutations are thought to be of prognostic value in MPN’s at the time of transformation to the blastic phase but data are inconsistent and require the further verification.The goal of our research was to determine the significance of mutations genes EZH2, CBL in the diagnosis and prognosis of Ph-negative MPNs. Methods. We have examined 102 patients with Ph-negative MPNs (45 pts with PV, 30 pts with ET and 27 pts with PMF). For all patients the detection of V617F mutation of JAK2 gene was done. V617F-negative pts with PV and pts with ET or PMF underwent the analysis of mutations in 12-th exon of JAK2 and 515 codone of MPL gene respectively. For 80 pts (30 with PV, 28 with ET and 22 with PMF) cytogenetic analysis and EZH2 mutation status were performed. Identification of CBL mutations was performed in 24 patients with available RNA samples. Mutations in 8, 10, 17, 18, 19 exons of EZH2 and RING-domen of CBL were defined by sequence analysis. V617FJAK2 mutation was detected in 44/45 (97,8%) pts with PV, 16/30 (53,3%) pts with ET and 13/27 (48,1%) pts with PMF. 538-539del-insL in 12-th exon of JAK2 was found in 1/45 (2,22%) patient with PV. W515KMPL mutation was identified in 1/30 (3,33%) pt with ET and 1/27 (3,7%) pt with PMF. 2 mutations of EZH2 gene have been found in 2 individuals with PMF (2/22). Both mutations are located in the 19 exon. The Ile713Thr mutation was detected in the patient with a del(6)(q15) karyotype which is associated with an intermediate cytogenetics risk. This patient subsequently underwent transformation from PMF to myelodysplastic syndrome in 9 months after the disease onset. Another case of mutation harboring (Thr731Asp) was detected in a patient with PMF and poor prognosis karyotype (chromosome 7 monosomy). This patient had transformation PMF to acute myeloid leukemia and died after 20 months. Homozygous mutation Q420R in CBL gene was detected in 1/24 patient with complex karyotype. Disease progression was observed after 16 months from the diagnosis. Conclusion. Mutations in EZH2 and CBL genes could be assessed as additional prognostic markers of unfavourable prognosis in patients with BCR-ABL-negative MPNs with different chromosomal aberrations. The integration of cytogenetic and molecular analyses could be a valuable option for stratification of patients and optimising the treatment strategy. References: Tefferi A. Novel mutations and their functional and clinical relevance in myeloproliferative neoplasms: JAK2, MPL, TET2, ASXL1, CBL, IDH and IKZF1. Leukemia 2010; 24:1128–1138. Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 315 Introduction: Dysregulated JAK-STAT signaling in chronic myeloproliferative neoplasms (MPNs) has primarily been attributed to activating mutations in tyrosine kinases. However, JAK-STAT activation can be demonstrated in some patients lacking JAK2 or MPL mutations, suggesting alteration of other regulatory elements in this pathway. One regulator of JAK-STAT signaling is LNK (SH2B3), an adapter protein that contains a proline-rich N-terminal dimerization domain (Pro/DD), a pleckstrin homology (PH) domain (plasma membrane localization), and an SH2 domain. LNK binds to cytokine receptors (e.g. MPL, EPOR) and JAK2 via its SH2 domain, inhibiting downstream STAT activation and providing critical negative feedback regulation. LNK-/- mice exhibit features consistent with an MPN phenotype. We recently reported the first human disease-related LNK mutations in two JAK2 V617F-negative MPN patients (Oh et al, Blood, Aug 12, 2010). One patient with primary myelofibrosis (PMF) exhibited a 5 base-pair (bp) deletion and missense mutation (DEL) leading to a premature stop codon and loss of the PH and SH2 domains. A second patient with essential thrombocythemia (ET) was found to have a missense mutation (E208Q) in the PH domain. Both mutations conferred aberrant JAK-STAT signaling in cell lines and primary patient samples, indicating that loss of LNK negative feedback regulation contributes to MPN pathogenesis. We now report the results of a comprehensive screen of a large cohort of MPN, overlap myelodysplastic syndrome (MDS)/MPN, and post-MDS/MPN acute myeloid leukemia (AML) patients for LNK mutations. Methods: A total of 341 samples were sequenced (Table 1; polycythemia vera (PV)=34, erythrocytosis=7, ET=61, PMF=75, post-PV/ET MF=25, MPN-U=7, chronic myelomonocytic leukemia (CMML)=71, juvenile myelomonocytic leukemia=20, MDS/MPN=8, MDS with fibrosis=2, refractory anemia with ring sideroblasts and thrombocytosis=4, idiopathic hypereosinophilic syndrome/chronic eosinophilic leukemia=4, systemic mastocytosis=4, and post MDS/MPN AML=19). A deep sequencing approach (Illumina multiplexing system) was used to evaluate 84 samples, in which all exons of LNK were sequenced. For the remainder of the samples, direct sequencing was performed on exon 2, the region containing the previously reported DEL and E208Q mutations. Results: After excluding variants previously reported in SNP databases, a total of 11/341 (3.2%) patients were found to have non-synonymous mutations, including 3/61 (4.9%) ET, 3/75 (4.0%) PMF, and 5/71 (7.0%) CMML patients (Table 1). Each of the mutations localized to exon 2 of LNK, implicating this region as a possible mutational hotspot. This included the aforementioned patients with the DEL and E208Q mutations, which were confirmed by deep sequencing. In two other patients, sequencing of DNA from cultured skin fibroblasts DNA indicated that the mutations were germline. For the remaining seven patients, germline analysis is currently ongoing. In one patient with CMML, a 1 bp deletion leading to a frameshift and premature stop codon was identified (Q72fs). This mutation localized to the Pro/DD, likely resulting in a complete loss of LNK function. Interestingly, this patient who is wild type for the JAK2 and RAS genes, also carries a heterozygous CBL mutation (C396Y), suggesting that LNK and CBL mutations may have cooperative effects. Four patients (one with PMF, three with CMML) were found to have a missense mutation (S186I) at a highly conserved residue in the Pro/DD. The previously reported E208Q mutation was also found in one patient with ET and one patient with CMML. None of the 81 patients known to be JAK2 V617F-positive exhibited somatic LNK mutations, suggesting that LNK mutations may provide an alternative basis for JAK-STAT activation in the absence of JAK2 V617F. Conclusion: Missense and deletion mutations of the LNK gene occur at a low frequency in MPNs and MDS/MPNs and segregate predominantly in exon 2. Further analysis of post-MPN AML samples (represented at a low frequency in the current cohort) and other subtypes of acute and chronic myeloid malignancies is warranted to better characterize the disease spectrum of LNK mutations and whether they are mutually exclusive of JAK2 V617F. We are currently investigating whether loss of negative feedback regulation of JAK-STAT signaling is related to haploinsufficiency of LNK or dominant negative effects of the mutant protein. Disclosures: No relevant conflicts of interest to declare.

Background. Ph-negative chronic myeloproliferative neoplasms (MPNs) are characterized by proliferation of one or more myeloid cell lineages and include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Somatic Jak2, MPL and CALR gene mutations are responsible for more than 90% of NPM cases. These mutations affect sequential stages of prolipherative signal transduction and therefore after the emergence of one type of mutation another types basically should not have any selective advantages for clonal expansion. However, simultaneous findings of these mutations have been reported by different investigators in up to 10% of MPN cases. Aim. To evaluate frequencies of MPL and CALR mutations in Jak2 positive MPN cases for Russian cohort of patients. Methods. Archival DNA samples from MPN patients followed up at the National Research Center for Hematology between 2014 and 2019 included into retrospective study. DNAs and RNAs were extracted from blood using reagent kit from Interlabservice (Russia). Jak2 V617F mutation was quantified by real-time PCR kit from Syntol (Russia) according to manufacturers instructions. CALR exon 9 deletions/insertions were analyzed by fragment analysis (sensitivity >= 3%). MPL W515L/K mutations were assessed by in-house allele specific PCR. All cases were tested for phi-negativity using BCR-ABl p210 PCR kit from Interlabservice (Russia). Results. At least one of the mutations was found in 3863 cases. Jak2 V617F mutation - 3385 cases (87.6%); CALR insertion or deletion - 471 case (12.2%); MPLW515L/K mutation - 31 case (0.8%). We have found 28 cases (0.7%) with Jak2 and CALR mutations combined and 3 cases (0.1%) with Jak2 and MPL mutations in the cohort studied. Matched measures were obtained at least twice at different time points during the course of disease for these cases. No cases with simultaneous CALR and MPL mutations were detected. In 23 from 31 (74%) cases with combined mutations Jak2 V617F allele burden was lower than 3%. Among cases with combined mutations 5 were diagnosed with PV, 8 - with ET, 8 - with PMF and 10 with unclassified MPN. No correlations between diagnosis, mutation combination or allele burden were found. Conclusions. Based on the data, obtained on retrospective DNA samples we cannot state whether combined mutations are present in different clones of myeloid cells or in one. Indirectly, the fact that more often mutations in CALR and MPL genes were found in the cases with a low Jak2 V617F allele burden may indicate that additional mutations occur in the "competing" cell clone. Further prospective studies with mutation monitoring over the therapy are required to assess the value of combined mutations for MPN pathogenesis. Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 1975 Introduction: Telomeres are reliable indicators of previous cell proliferation and cell ageing. Moreover, a marked though variable loss of telomere length (TL) has been observed in several hematological malignancies. In particular, some recent studies have reported a marked TL reduction in patients with Ph-negative Chronic Myeloproliferative Neoplasms (Ph-neg-CMNs) (Ferraris AM et al, Br J Haematol 2005; Bernard L et al, Leukemia 2009). This supports the possible influence of TL in the development of CMNs. Moreover, TL might be of prognostic relevance in these disorders. The present study reports the analysis of TL in a large series of patients with Ph-neg-CMNs, including Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Myelofibrosis (MF), and in a few cases of Secondary Erythrocytosis (SE), as well. Aims of the study were: i. to verify the rate of TL reduction in Ph-neg-CMNs; ii. to compare TL in ET, PV and MF; iii. to investigate the role of TL as a marker of proliferation, evaluating differences in TL compared to healthy subjects and subjects with Secondary Erythrocytosis (SE); iv. to verify telomere dynamics according to the treatment received. Methods: Peripheral blood (PB) samples were obtained from 239 Ph-neg-CMNs patients (median age 68 yrs, range 10–91): 78 had PV, 100 had ET and 61 MF. Most patients were analyzed for JAK2 mutations: among 72 evaluated PV patients, a JAK2V617F mutation was found in 66 (92%) and an exon 12 mutation was identified in 2 patients; a JAK2 V617F mutation was detected in 50 (50%) ET patients and in 44 (73%) MF patients. Samples were obtained either at diagnosis or during follow-up. More than a half of CMNs patients received before TL analysis at least 1 year of cytoreduction (129 patients); 90% received Hydroxyurea (HU) and 26% other cytoreductive drugs with or without HU. As control, PB samples from 202 healthy age-matched subjects and from 14 SE subjects were analyzed. TL was assessed by Southern blot analysis, according to standard procedures (TeloTAGGG Telomere Length Assay Kit, Roche Diagnostic, Mannheim, Germany). JAK2V617F mutation analysis was performed by ASO-PCR and digestion with BSAXI (Guerini et al, Leukemia 2008). Results: PV, ET and MF patients showed individual progressive TL shortening correlated with age as observed in the healthy population. However, CMNs patients had TL significantly shortened (5,890 bp, ± 1,305) compared to healthy age-matched individuals (median: 7,330 bp, ± 1574) (p<.0001). PV and MF showed the most pronounced TL loss among CMNs, significantly different from healthy controls, with TL values respectively of 5,621 bp (± 1,446) (p<.0001) and 5,361 bp (± 1,335 bp) (p<.0001). ET patients had a median TL of 6,422 bp, (± 1131bp). SE patients had a median TL of 6,972 bp (± 1355 bp) with no difference from healthy subjects (p= 1.00). At ANCOVA multivariate analysis of disease characteristics, short TL correlated significantly with JAK2V617F mutation allele burden >50% (p=.0025), age (p=.0132) and diagnosis of PV (p=.0122). No significant correlation was found with disease duration, history of thrombosis and treatment with cytoreductive or anti-aggregants agents. Conclusions: i. Ph-negative CMNs are confirmed to present with TL reduction compared to the age-matched healthy population; ii. PV and MF show higher degree of TL loss than ET; ii. high JAK2V617F mutation burden is strongly correlated to TL loss; iii. SE patients and healthy subjects do not differ in TL, suggesting the possible use of TL as a parameter to distinguish SE from PV, particularly in the few cases lacking JAK2 mutations. iv. no evident influence on accelerated cell ageing is observed in patients receiving cytoreductive treatment with Hydroxyurea. The absence of a marked telomere loss in patients receiving Hydroxyurea, reduces the harm of using this drug when clinically indicated. Disclosures: No relevant conflicts of interest to declare.

Introduction Thromboses are a common complication in patients with myeloproliferative neoplasms (MPN) and are reported to occur at a rate of 15- 20% at sea level. In the MPN-Thromboses spectrum which includes both arterial and venous thrombosis, cardiovascular disease (CVD) is noted to be the most common thrombotic event. JAK2 V617F is reported to be the most common driver mutation in MPNs and is associated with increased risk of thromboses. CALR and MPL are other mutations whose contribution to the thrombotic phenotype is not known. Chronic hypoxia from living in moderate or high altitude is reported to be an independent prothrombotic risk factor. The average elevation of New Mexico is 5,700 feet (1,740 meters) above sea level. The goal of this study is to evaluate the frequency of thromboses and prothrombotic risk factors in patients with MPN in patients in this distinct population. Methods We reviewed 134 patients, who were diagnosed with MPN in University of New Mexico Comprehensive Cancer Center between 2001 to 2019. A retrospective chart review was conducted to identify demographics, clinical and molecular risk factors for both arterial and venous thromboses. The mutation analyses for Janus Kinase 2 (JAK2), myeloproliferative leukemia (MPL) gene and calreticulin (CALR) gene were performed by polymerase chain reaction (PCR). Contingency table and logistic regression methods were applied to analyze and compare the distribution of the prothrombotic risk factors between the patients with and without thrombosis. Results In this study, 62 patients (47%) were diagnosed with ET, 47 patients (35%) with PV, and 22 patients (17%) with primary myelofibrosis (PMF). Seventy-five patients (56%) were females. Mean age at diagnosis was 62 years. 102 patients (77%) were living in the Albuquerque metropolitan area with an average elevation of 5312 feet above sea level and others were in areas with an elevation of 6000 feet or higher in New Mexico. Forty-four patients (33%) experienced either arterial (29) or venous thromboses (11) or both (4). A significant percentage (70.4%) of thrombotic events were either ischemic stroke or myocardial infarction. The patients with thromboses were predominantly males (21/36, 57%) while most patients without thromboses were females (56/90, 62%) with p=0.003. Twenty-one (53%) patients with thromboses had ET; however, a higher proportion of patients with PV (20/47, 42.5%) developed thromboses compared to ET or PMF (32.2% and 9% respectively). Also, a significant number of patients (32/44, 76%) with thromboses have JAK2 mutations while only 4 patients (9%) have CALR gene mutation. Although not statistically significant, CALR mutation was associated with lesser thrombotic events than other MPN patients. In univariate logistic regression analysis, PV and ET were significantly associated with increased thrombotic events. Patients with PV showed a 7.7-fold increase and patients with ET have a 5.1-fold increase in odds for thrombosis compared to patients with PMF (p=0.0365). Female gender was associated with decreased thrombotic risk with an odds ratio of 0.46 (p=0.0387). There was no significant difference between patients with and without thromboses, regarding other clinical characteristics such as age, previous aspirin use, leukocytosis, diabetes, hypertension, hyperlipidemia, obesity, and smoking. Conclusion An increased frequency of thromboses was observed among patients with Ph negative MPN in New Mexico which is significantly higher than previously reported studies. This strongly suggests the role of mild to moderate hypoxia as a contributing prothrombotic risk factor for MPN. The role of chronic hypoxia and its influence in thrombotic events in MPN need to be further evaluated in prospective studies. The decreased risk of thrombosis in females and patients harboring CALR mutations compared to other common mutations was consistent with other published studies. JAK2 mutation was not associated with an increased risk of thrombosis. Other genetic factors in this population were not evaluated in this study. Disclosures Arana Yi: Jazz Pharmaceuticals: Other: Advisory Board.

Abstract Background: Diagnosis of a prefibrotic state (MF0) presents histological diagnostic difficulties. MF0 has a worst prognostic impact than Essential Thrombocythemia (TE) as regard the thrombotic risk and a higher risk towards Idiopathic Myelofibrosis (MI) and acute leukemia evolution. For this reason it is very useful to identify this group of patients. Aims: The aim of this study was to evaluate clinical and hematological impact of the mutational status in patients with an MF0 diagnosis. Methods: A retrospective chart review was performed from January 2010 to July 2015 in a single center in 317 patients affected by Ph negative chronic myeloproliferative neoplasms. Polycythemia vera cases were excluded. Thirty-three patients have been classified as MF0 on the base of the bone marrow histological examination. Onset features include cytogenetics, blood counts, peripheral CD34-positive cells, spleen and liver size, thrombotic events (prior to and post-diagnosis) and thrombotic risk. Fragment analysis was performed to study exon 9 CALR mutation, Real-time PCR was applied for exon 14 JAK2 V617F mutation and Sanger sequencing was used to identify exon 10 MPL mutations for 505 and 515 codons. According to genetic results, patients were identified into four groups according to a positivity for JAK2, MPL, CALR and for triple negativity. Results: The 33 patients with MF0 were: 11 (33%) JAK2 positive, 10 (30%) CALR positive, (10) 30% triple negative and 2 (7%) MPL positive. The latter group was not further considered for analysis due to the low number of cases. Eighty percent of CALR positive patients had a deletion on the exon 9 of the gene (8 del52bp and 2 del46bp), while 20% had the type 2 mutation (ins5bp). The average of JAK2 allelic burden was 20%. The 3 groups of patients were comparable for age, white blood counts and hemoglobin values. There was a female prevalence (23 vs 10 males). Platelet count was higher (median 875.500 103 µl, interquartile range 303.000 103 µl , p = 0.008) in CALR positive patients compared to JAK2 positive (median 569.000 103 µl, interquartile range 433.000 103 µl) and to triple negative patients (median PLT count 629.500 103 µl, interquartile range 378.250 103 µl). Noteworthy, bone marrow cytogenetic exam was normal in all patients. JAK2 positive patients had a larger spleen compared to the other two groups. Pre-diagnosis thrombotic events were exclusive for JAK2 positive patients and absent in the other groups. Triple negative patients do not have a negative prognostic impact for the thrombotic risk. Conclusions: CALR deletion could be considered as an MF0 marker and should be included in diagnostic work-up. JAK2 positivity in MF0 is associated with a high thrombotic risk. Disclosures No relevant conflicts of interest to declare.

Abstract Background Treatment of Philadelphia chromosome negative chronic myeloproliferative neoplasms (MPNs) with recombinant pegylated interferon alpha2a/b (rIFN-alpha) has proven effective. It is well known that prolonged therapy with recombinant type 1 interferons (IFN-alpha and IFN-beta) may induce neutralizing antibodies (nAbs) against the drug leading to treatment failure. Most data on type 1 IFN immunogenicity are available from studies of patients with multiple sclerosis treated with rIFN-beta, and patients with hepatitis C treated with rIFN-alpha. A few reports have demonstrated nAbs in MPN patients not responding adequately to rIFN-alpha treatment, but the phenomenon has not been thoroughly investigated in MPNs. Patients and Methods Newly diagnosed patients with MPNs enrolled in the Danish multicenter trial - DALIAH (Low-dose rIFN-alpha versus Hydroxyurea in The Treatment of Ph-Negative MPNs) were included. Patients were randomized to treatment with either rIFN-alpha 2a or 2b at a starting dose of 45 and 35 mikrograms once weekly, respectively. The occurrence of neutralizing Abs (nAbs) against rIFN-alpha was investigated at baseline, month 12 and month 24 by reporter gene assays (iLiteTM alphabeta and iLiteTM antialpha, Biomonitor A/S, Copenhagen, Denmark). JAK2 V617F quantitative mutation analyses were performed as previously described (Larsen TS, BJH 2007). Statistical analyses were performed using STATA version 9.0. Results Ninety-two patients on sustained treatment with rIFN-alpha2a (n=48) and rIFN-alpha2b (n=44) for 12 months were analyzed for this study. Forty-five patients had ET, 39 patients had PV and 8 patients had proliferative PMF. Thirty-six out of 39 (92%) PV patients, 22 out of 45 (49%) ET patients and 4 out of 8 (50%) PMF patients were JAK2V617F mutated. Hematological responses at 12 months: ET: 67% CR, 29 % PR; PV: 64% CR, 31% PR (ELN 2009 criteria); PMF: 50% had at least a minor response (EUMNET). The median serum concentration of bioactive IFN-alpha at 12 months was 12,4 (range <2,4-86,4) and 2,6 (range <2,4-12,8) IU/mL serum, for patients treated with rIFN-alpha 2a and -2b respectively. No significant association between hematological or molecular response and serum IFN-alpha activity was found. Serum from 92 patients was analyzed at 12 months and 33 patients were analyzed at both 12 and 24 months and no occurrence of nAbs was seen during treatment. Twenty-four patients had pre-treatment levels of IFN nAbs measured. Notably, one patient was tested positive for the presence of nAbs before rIFN-alpha exposure. This autoAb-positive patient remained positive throughout the study and has shown low IFN serum activity (< 2,4 IU/mL) and only partial hematological and molecular response after 24 months of treatment. Conclusions Development of nAbs in MPN patients completing treatment for 12 months with rIFN-alpha seems exceedingly rare as no patients, neither complete responders nor patients not meeting criteria for complete hematological remission developed nAbs after 12 (24) months of therapy. Its apparent rarity does not justify a routine investigation of nAbs in patients not responding to rIFN-alpha treatment. There was no significant correlation between serum concentration of rIFN-alpha 2a and -2b and clinical or molecular responses. The intriguing finding that one of 24 patients had pre-existing cross reacting nAbs against rIFN-alpha 2a and 2b before commencing rIFN-alpha treatment is interesting and was associated with an insufficient response. Disclosures Off Label Use: Recombinant interferon-alpha 2a and -2b in the treatment of chronic Philadelphia-negative myeloproliferative neoplasms.. El Fassi:Novartis Denmark: Honoraria, Other: Have conducted an educational session for Novartis Denmark, regarding MPNs and ruxolitinib, for this a honorarium was received.. Bjerrum:Bristoll Myers Squibb, Novartis and Pfizer: Other: educational activities. Hasselbalch:Novartis: Research Funding. Bendtzen:Pfizer: Honoraria; Eurodiagnostica AB: Equity Ownership; Novo-Nordisk: Equity Ownership.

Myeloproliferative Neoplasms (MPNs) are haematologic disorders of myeloid progenitor cells characterized by the frequent presence of an acquired activating mutation in exon 14 of the Janus kinase 2, consisting in a Valine to Phenilalanine substitution at codon 617 (JAK2V617F). The kinase activity of mutated JAK2 is constitutively activated, inducing uncontrolled cell proliferation and resistance to apoptosis. JAK2V617F is found in 95% of Polycythemia Vera (PV), 50% of Idiopathic Myelofibrosis (IMF), and 20-40% of Essential Thrombocythemia (ET), the three diseases belonging to MPNs, as described in the World Health Organization (WHO) classification (REF). Thus, the JAK2V617F mutation represents the key clonal marker for diagnosis of MPNs. The identification of the JAK2V617F is mandatory in the diagnostic work up of PV, ET, and IMF. Several molecular techniques are currently available but each of them presents some limits. We developed a novel non-PCR molecular method for the identification of the JAK2V617F mutation based on an Allele Specific-Loop mediated AMPlification (AS-LAMP). This innovative technique amplifies DNA targets under isothermal conditions with high specificity, efficiency, and rapidity. The method neither requires a thermal cycler equipment nor gel separation and the DNA amplification reaction is visible to the naked eye, monitorable by turbidimetry and fluorescence. This method was validated on DNA from both cell lines as well as patients with chronic myeloproliferative neoplasms. Results were compared with those obtained by conventional PCR methods. The AS-LAMP allows a rapid and robust identification of DNA samples harboring the JAK2V617F mutation. No false positive or false negative results were registered on clinical samples previously tested by the reference assay ASO-PCR. This new assay proved also remarkably sensitive since the mutated JAK2V617F DNA could be detected down to 0.05% on clinical samples. AS-LAMP is a simple, robust and easily applicable tool for the molecular diagnosis and monitoring of JAK2V617F mutation in chronic myeloproliferative neoplasms.