ALBERT

Description: Abstract 1898 Poster Board I-921 Introduction: JAK2V617F mutation is detected in more than 90% of cases of polycythemia vera (PV) and in about 50% of cases of essential thrombocythemia (ET). Recently, JAK2-exon 12 and MPL mutations have been reported in myeloproliferative neoplasms (MPN). All these three mutations have a disease causing potential. There are still 50% of ET and PMF patients negative for JAK2V617F mutation. Thus, further genetic analysis to identify novel disease-related aberrations in MPN is required. Methods: We performed whole genome analysis on granulocytic DNA of 45 MPN patients (19 PV and 26 ET) using the single nucleotide polymorphism (SNP) Array 6.0 platform [Affymetrix 6.0]. Genotypes were analyzed using Genotyping Console 3.0.2. Data were normalized against a commercial and an own set of reference samples. All patients had JAK2V617F screening performed. Clinical and analytical data and results of cytogenetics study performed at diagnosis of MPN were colected from clinical reports. Results: From 45 MPN patients, 41 had normal cytogenetics at diagnosis; there were no data concerning cytogenetics study from the rest 4 patients. Using SNP-A 6.0 platform, we detected aberrations (gain or loss of the molecular material) in the following regions: 1q12, 9p1, 17q21, 4q, 3q26 and 8p12. Aberrations in a region 1q12 (n=14) were presented in 8 of PV (gain in 3 and loss in 5) and in 6 of ET (gain in 2 and loss in 4) patients. Aberrations in a region 9p1 (n=27) were observed in 11 of PV (gain in 6 and loss in 5) and in 16 of ET (gain in 10 and loss in 6) patients. Aberrations in a region 17q21 (n=26) were presented in 11 of PV (gain in 3 and loss in 8) and in 15 of ET (gain in 9 and loss in 6) patients. Aberrations in the region 4q (n=20) were detected in 8 of PV (gain in 5 and loss in 3) and in 12 of ET (gain in 9 and loss in 3) patients. Interestingly, only the gain of the molecular material was detected in a region 3q26 (n=12; 5 PV and 7 ET patients). In case of aberration in a region 8p12 (n=20), all 12 ET patients presented gain of the molecular material, whereas PV patients had gain (n=3) or loss (n=3) of the molecular material. There were no relation between the presence of these aberrations and the status of the JAK2V617F mutation, analytical data or clinical outcome of the patients. Conclusions: 1. As we know, patients with ET have low frequency of cytogenetics aberrations. Nevertheless, using SNP-A 6.0 platform [Affymetrix 6.0], it is possible to detect new genomic aberrations in these group of patients. 2. According to our results, gain in 8p12 region is especially related to ET patients. Recently has been reported that, in a 8p12 region there is localized gen INDOL1 that, may be involved in the inhibition of immune response to tumours. 3. Gain in a 3q26 region can be related with both PV and ET. In this region, there are localized two microRNA: hsa-mir-1263 and has-mir-720. 4. SNP-A 6.0 technology should not replace conventional cytogenetics in study of MPN patients. However, SNP-A 6.0 platform, as a high resolution assay, can be useful in identification of new genomic abnormalities that may be relevant for pathogenesis of MPN. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: The JAK2V617F mutation is frequent in MPN, however its clinical implication is still in debate. There are few publications that analyze changes in JAK2V617F allele burden. Methods: We performed a single centre study on 65 patients (19 polycythemia vera (PV), 42 essential thrombocythemia (ET) and 4 primary myelofibrosis (PMF)) all of them with at least two JAK2V617F determination separated by minimum 12 months. The JAK2 mutation was determinated in DNA from peripheral blood granulocytes (in 3 cases from bone marrow smear) by MutaScreenTMKit (IPSOGEN). Results: The mean follow-up period was 26 months (range 12–176). In 42/65 (64.6%) patients JAK2 mutation was positive and in 24/42 (57%) changes in JAK2V617F allele burden (〉10%) were observed. Interestingly, in 7/18 (39%) patients who showed an increase of JAK2V617F allele burden progression was observed. In this group, one case of PMF with initial increase of JAK2V617F allele burden during transformation to acute myeloid leukemia (AML) and posterior decrease after transformation* and one case of ET with an increase during transformation to myelofibrosis (MF) and posterior conversion to JAK2V617F negativity after allo-HSCT** were observed (table1). 36/42 (85.7%) of JAK2V617F positive patients were under cytoreductive treatment: 30 with hydroxyurea (HDU), 1 anagrelide (ANA) and 5 mixed. There was no decrease of JAK2-V617F allele burden in patients without cytoreductive treatment. Data of patients with HDU treatment are presented in table 2. 3/5 of patients with mixed cytoreduction showed an increase of JAK2V617F allele burden: all of them presented bad clinical control but no data of progression were observed. None of JAK2V617F negative patients (n=23: 1 PV, 2 PMF, 20 ET) became positive for the mutation with the mean follow-up period of 19.37 months. There were no cases of transformation in this group of patients. Conclusions: According to our study, JAK2V617F allele burden can change during the follow-up. Increase (or stability) of JAK2V617F allele burden are more frequent in transformation from ET to PV or MF; in cases of progression to AML we can observe decrease, stability or initial increase with posterior decrease of JAK2V617F allele burden. Decrease of JAK2 allele burden can be related to cytoreductive treatment. Increase of JAK2V61F in patients with cytoreductive treatment can be related to transformation or bad clinical control. More prospective studies are needed to conclude whether changes in JAK2-V617F allele burden could be useful to predict transformation of MPN patients. Table1. Changes of JAK2V617F allele burden and clinical evolution in JAK2V617F positive patients. JAK2 Clinical evolution (N=42) STABILITY PROGRESSION DECREASE 6/42 (14%) 5 1 [1 PV-AML] STABILITY 18/42 (33%) 15 3 [1 PV-MF; 1 ET-MF; 1 ET-AML] INCREASE 18/42 (43%) 11 7 [3 ET-PV; 3 ET-MF (one case with posterior allo-HSCT**); 1 PMF-AML*] Table2. Changes of JAK2V617F allele burden in JAK2V617F positive patients with HDU and no cytoreductive treatment. Treatment JAK2V617F: changes of allele burden DECREASE STABILITY INCREASE Untreated - 4 2 [ET-PV] HDU newly started 2 5 [PV-MF and ET-AML after 14 and 7 years of treatment respectively] 7 [2 ET-PV; PMF-AML*] HDU already treated 3 [PV-AML] 7 6 [3 ET-MF (one case with posterior allo-HSCT**)]

Description: Background Hepcidin acts as the main regulator of iron homeostasis through regulation of intestinal absorption and macrophage release. Hepcidin deficiency causes iron overload whereas its overproduction is associated with anaemia of chronic diseases. The aims of the study were: to identify genetic variants in the hepcidin gene (HAMP) promoter, to asses the associations between the variants found and iron status parameters, and to functionally study the role on HAMP expression of the most frequent variant. Results The sequencing of HAMP promoter from 103 healthy individuals revealed two genetic variants: The c.-153C 〉 T with a frequency of 0.014 for allele T, which is known to reduce hepcidin expression and the c.-582A 〉 G with a 0.218 frequency for allele G. In an additional group of 224 individuals, the c.-582A 〉 G variant genotype showed no association with serum iron, transferrin or ferritin levels. The c.-582G HAMP promoter variant decreased the transcriptional activity by 20% compared to c.-582A variant in cells from the human hepatoma cell line HepG2 when cotransfected with luciferase reporter constructs and plasmid expressing upstream stimulatory factor 1 (USF1) and by 12-14% when cotransfected with plasmid expressing upstream stimulatory factor 2 (USF2). Conclusions The c.-582A 〉 G HAMP promoter variant is not associated with serum iron, transferrin or ferritin levels in the healthy population. The in vitro effect of the c.-582A 〉 G variant resulted in a small reduction of the gene transactivation by allele G compared to allele A. Therefore the effect of the variant on the hepcidin levels in vivo would be likely negligible. Finally, the c.-153C 〉 T variant showed a frequency high enough to be considered when a genetic analysis is done in iron overload patients.