Introduction: Ph-negative myeloproliferative neoplasms (MPNs) are a group of clonal stem-cell disorders with an elevated risk for thrombosis. Leukocytosis is a well-known risk factor for thrombosis. Neutrophils from MPN patients display features of activation. On stimulation, neutrophils produce neutrophil extracellular traps (NETs), which have been implicated in the pathogenesis of thrombosis. Myeloperoxidase-conjugated DNA levels (a specific NET marker) have been found increased in plasma of thrombotic MPN patients (Guy, A, ISTH 2019 OC 77.3). Ex vivo, activated neutrophils from JAK2V617F patients are primed to form NETs, diminishing under ruxolitinib treatment. Mice with conditional knock-in of JAK2V617F have increased NETs formation and thrombotic events. Inhibition of JAK/STAT signaling by ruxolitinib abrogated NETs formation and reduced thrombosis in this murine model (Wolach, O,Sci. Transl. Med. 2018). Thus, a link between JAK2V617F expression, NETs formation and thrombosis has been suggested (Wolach, O, 2018). It remains unclear whether, in MPN patients, JAK/STAT independent pathways are implicated in the NETs formation. With this aim, we explored the effects of different cytoreductors in NETs formation. Patients and Methods: In a multicentric study conducted at Hospital General Universitario Morales Meseguer (Murcia, Spain) and Hospital del Mar (Barcelona, Spain), EDTA plasma samples were collected from MPN patients (n=104). Patients include polycythemia vera (PV, n=35, all of them JAK2V617F), essential thrombocythemia [ET, n=47; 38 JAK2V617F, 4 CALRmut, 5 triple negative (TN)], myelofibrosis (MF, n=9; 4 JAK2V617F, 2 CALRmut, 3 TN), and unclassifiable MPN by WHO-2016 (MPN-u, n=13; 12 JAK2V617F, 1 CALRmut). Samples were collected at different time points following up the disease: at diagnosis or before any cytoreductive treatment, time 0, (n=100), less than 6 months of treatment (time 6; n=60), from 6 to 12 months of treatment (time 12; n=60), and from 12 to 24 months of treatment (time 24; n=49). Among the 104 patients, we had clinical information about treatment on 99 cases, included hydroxyurea (HU, n=69), ruxolitinib (n=15), IFN-α (n=2), and non-treated (n=13, all basal samples). We measured citH3-DNA complexes, as specific marker of NETs, by ELISA using rabbit anti-citH3 (Abcam) and peroxidase-conjugated anti-DNA antibody (Cell Death Detection ELISA). The absorbance at 405 nm (A405) was measured in a plate reader (Biotek®). Thus, we followed up the NETosis marker evolution with treatment over time. One-tailed and paired Wilcoxon test was applied and p<0.05 was taken as statistical significance. Results: Overall, regardless of the treatment, we observed a reduction of citH3-DNA levels over time (Fig. 1A), being these significant between time 0 and times 6 and 12 (p=0.02 for both time-points), Importantly, patients treated with HU showed a substantial reduction of NETs in all time-points (Fig. 1B, left), which was statistically significant between times 0 and 6 (p=0.03) and between times 0 and 24 (p=0.02). Although in patients treated with ruxolitinib, the citH3-DNA levels seem to be reduced at time 12, the difference was not significant (Fig. 1B, right), probably because of the small sample size. Regarding the MPN phenotype, decrease of citH3-DNA levels over time was only observed in PV patients (Fig. 1C, left), specially at time 12 (p=0.03). In contrast, under cytoreductive treatment, ET patients did not show significant differences in NETs levels over time (Fig. 1C, middle). In MF, there were not enough samples to obtain a clear conclusion (Fig. 1C, right). Regarding the driver gene mutations, we detected a significant decrease in the citH3-DNA levels in JAK2V617F patients (Fig. 1D, left), specifically between basal time and times 6 and 12 (p<0.02 for both time-points). Unfortunately, there are not enough samples to obtain a clear conclusion in CALRmut (Fig. 1D, right) and TN patients. Conclusions: Our results showed that hydroxyurea reduces NETosis levels in plasma from MPN patients, suggesting that JAK-STAT independent pathways could be implicated in the NET formation. Hydroxyurea could decrease thrombosis in MPN patients, not just reducing the number of functionally abnormal cells in peripheral blood, but also by abrogating NET formation. Acknowledgements: This study was supported in part by ISCIII (PI18/00316 & PI16/0153) & Fundación Séneca (20644/JLI/18). Disclosures Bellosillo: Qiagen: Consultancy, Speakers Bureau; Roche: Consultancy, Research Funding, Speakers Bureau.
Background: Polycythemia vera (PV), Essential Thrombocytosis (ET) and Primary Myelofibrosis (PMF) are Myeloproliferative Neoplasms (MPNs) with median age at diagnosis of ~56-70 years old. However, around 10%-15% of cases are diagnosed during young adulthood and there are scanty data about their molecular profile and its implications in clinical outcomes. Objective: To analyze the clinical and molecular characteristics of young adult patients (≤45y.o.) with MPNs (Y-MPN) and to correlate them with clinical features and outcomes. Material and Methods: This is a retrospective single-center study including MPN patients diagnosed below the age of 45 years. Molecular characterization was performed using DNA from granulocytes at diagnosis or before the start of cytoreductive therapy. JAK2V617F was assessed by quantitative allele-specific PCR and CALR mutations by fragment analysis of exon 9. Further molecular profiling was performed by next generation sequencing (NGS) with a custom panel of 25 myeloid-associated genes (ASXL1, CALR, CBL, CSF3R, DNMT3A, ETV6, EZH2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NRAS, PRPF8, RUNX1, SETPB1, SF3B1, S2HB3, SRSF2, STAG2, TET2, TP53, U2AF1 and ZRSR2) using Illumina technology. Pathogenic mutations in genes previously related with poor outcomes (ASXL1, EZH2, IDH1, IDH2, SRSF2 and U2AF1) were named as Mutations of Adverse Significance (MAS). Molecular alterations were correlated with diagnosis, progression to PV post ET (PVpET), progression to MF post PV/ET (MFpPV/ET), start of cytoreduction and major thrombotic events (MTE). Time to progression (TTP) and overall survival (OS) were calculated from diagnosis to progression (ELN criteria) and to last visit. Results: From 646 MPN followed in our clinic, 109 (17%) cases were Y-MPN; females 72 (66.1%). At diagnosis the median age was 35 y.o. (9-45). 23 patients (21%) were PV, 91% carried JAK2V617F, 4% (1) carried an exon-12 JAK2 mutation and 1 was JAK2V617F and exon-12 negative. 84 cases (77.1%) were ET, 53.5% (45) JAK2V617F, 25% (21) CALR, (52% type-1 mutation, see table) and 21.4% (18) triple negative (TN). There was 1 PMF with CALR type-1 mutation. No MPL canonical mutations were found. ET was predominantly diagnosed in females (M/F: 26/58). Regarding clinical variables, we found a high proportion of ET-JAK2V617F with high LDH values, higher platelet count for CALR-ET and ET-TN patients (967 and 978 vs 728 for ET-JAK2V617F, p=0.03) and higher frequency of MTE at or before diagnosis for JAKV617F cases (p=0.001). The mean follow-up was 152 months (SD +/-10.4); 16 progressions were registered (PFS 305 months); 8 patients to MFpPV/ET and 8 ET-JAK2V617F to PVpET. An increase in the VAF of JAK2V617F was observed at the time of progression either to PVpET or to MFpET/PV (see table). Seven MTE were registered during this time, 3 in JAK2V617F, 2 in CALR type-1, 1 in exon-12 and 1 in a TN case. 38 (34.8%) cases started cytoreduction, with median time to cytoreductive therapy of 251 (172-330) months; JAK2V617F cases started cytoreduction more often (p=0.04) than patients with other genotypes. No progression to AML nor deaths were recorded. The NGS panel was performed in 102 (93.5%) cases. Pathogenic mutations in non-driver genes were found in 41.2% (42) of cases, being TET2 (7%), ASXL1 (6%) and DNMT3A (5%) the most frequently mutated genes. Also, in 28.4% (29) variants of unknown significance (VUS) were found, involving TET2 (6%), SETBP1 (4%), SH2B3 (5%), and JAK2 (4%) among others. The mutations in SH2B3 (1 pathogenic, 5 VUS) were more frequent in JAK2V617F patients and those in DNMT3A were more common in PV patients. The presence of mutations in non-driver genes (pathogenic or VUS) did not correlate with MTE before or after diagnosis, the start of cytoreduction nor clonal progression. Regarding the 19 TN cases, in 7 (36.8%) one or more non-canonical pathogenic variants implicating MPL, JAK2 and TET2 genes were found. Finally, 8 patients (7.8%) harbored a MAS, of which 3 progressed to MF (2 CALR to MF and 1 ET-JAK2V617F to PVpET); TTP was similar to the rest of the cohort. Conclusions: Our data show that 41% of Y-MPN patients harbor pathogenic mutations in non-driver genes. There was no correlation between their presence and clonal progression, major thrombotic events or overall survival. Mutations of adverse significance did not predict major clinical outcomes. Monitoring of JAK2V617F allele-burden can help to predict progression to MFpPV/ET or PVpET. Disclosures Andrade-Campos: Sanofi-Genzyme: Consultancy, Speakers Bureau; Takeda-Shire: Speakers Bureau; Celgene-BMS: Consultancy. Fernández:Roche: Consultancy, Speakers Bureau. Salar:Janssen: Speakers Bureau; Roche: Speakers Bureau; Celgene: Speakers Bureau. Bellosillo:Qiagen: Consultancy, Speakers Bureau; Roche: Consultancy, Research Funding, Speakers Bureau.
Background:The pathogenesis of PV‐associated thrombosis is multifactorial. Prior thrombosis, advanced age, leukocytosis, JAK2V617F mutation, and cardiovascular risk factors are the main factors influencing thrombotic risk. However, there is scarce information regarding the role of mutations in non‐driver genes in the development of thrombotic events during clinical follow‐up.Aims:To investigate the influence of the molecular profile characterized at PV diagnosis in the probability of developing thrombosis during clinical evolution.Methods:A total of 127 JAK2V617F PV patients were included in the study. JAK2V617F allele burden was assessed by quantitative allele specific PCR in the peripheral blood granulocytes at the moment of diagnosis. Targeted NGS was performed on DNA extracted from granulocytes also at diagnosis. Amplicon libraries were constructed using QIAseq Targeted DNA custom panel (Qiagen, Hilden, Germany), covering the full exonic regions of 25 commonly mutated genes in myeloid malignancies and were sequenced using either MiSeq or NextSeq (Illumina, San Diego, CA, USA). Time to event curves were drawn by the method of Kaplan and Meier with the log rank test for comparisons.Results:With a median follow‐up of 50 months (range 1–200), fourteen patients developed 20 thrombotic events after diagnosis (10.9%), 17 corresponding to arterial thrombosis (85% of the total thrombotic episodes) and 3 to venous thrombosis. Five patients presented more than one thrombotic event. Cerebrovascular disease was the most frequent condition in the group of arterial thrombosis (41%). Median time between diagnosis and the appearance of the first thrombosis was 28 months (range 5.8–80). Median JAK2V617F allele burden was 54% (range 5–99). The distribution by quartiles of the JAK2V617F mutational load was as follows: 0–24% n = 15 (11.8%), 25–49% n = 43 (33.9%), 50–74% n = 33 (26%), 75–100% n = 36 (28.3%). There was no statistically significant difference in thrombosis free survival according to the baseline mutational load of JAK2V617F. A total of 104 somatic mutations other than JAK2V617F were detected at diagnosis in 61 patients (48%). The mean number of pathogenic additional mutations was 0.8 mutation/patient. The most frequently mutated genes were TET2 (36%), ASXL1 (13.2%) and DNMT3A (12.5%). Overall, no difference was observed in the probability of developing thrombosis during the follow‐up according to the presence or absence of pathogenic non‐driver mutations at diagnosis (p = 0.89)(Figure 1a). Nevertheless, we showed a significant correlation between the presence of ASXL1 mutations and the probability of thrombosis (35% thrombotic events in ASXL1 mutated patients vs 8% in wild‐type ASXL1 patients, p = 0.002) (Figure 1b). No significant difference was observed in thrombosis‐free survival according to mutations in TET2 or DNTM3A genes.Summary/Conclusion:Overall, no significant association was observed between the genetic profile (including JAK2V617F allele burden and pathogenic non‐driver mutations) of PV patients at diagnosis and the probability of thrombosis during clinical evolution. However, the subset of ASXL1 mutated patients showed a higher risk of thrombosis. Additional studies are needed to confirm this finding.imageThis study was supported in part by grants from ISCIII and Spanish Ministry of Health, PI16/0153, 2017SGR205, PT17/0015/0011. Beca Gilead 2016 and Xarxa de Banc de Tumors de Catalunya
Background:Diagnosis of CMML according to WHO 2017 requires the presence of ≥1x109/L and ≥10% of monocytes in peripheral blood (PB). Recently, Geyer et al. described oligomonocytic CMML (O‐CMML) as those MDS cases with relative monocytosis (≥10% monocytes) and monocyte count 0.5<1x109/L. The authors showed that molecular signature and outcome of OCMML were similar to overt CMML, suggesting that this represents an earlyphase of CMML. The study of peripheral monocyte subsets by flow cytometry (FC) has gained interest for CMML diagnosis. As showed by SelimogluBuet et al., the increase in the fraction of classical monocytes (Mo1) to >94% of total monocytes is a highly sensitive and specific diagnostic marker for CMMLAims:Since O‐CMML seems to be in the continuum of CMML, it is crucial to determine if these patients show a PB monocyte subset distribution similar to CMML. CD56 expression in monocytes is a typical feature of CMML and is rarely observed in MDS, therefore it seems interesting to assess this feature in O‐CMML. Finally, we evaluate if O‐CMML showed a high incidence of TET2/SRSF2 co‐mutation, the gene signature of CMMLMethods:42 CMML, 20 O‐CMML, 8 MDS without relative monocytosis and 70 reactive monocytosis with ≥1x109/L monocytes (N = 140) were prospectively studied from 02/2016 to 02/2019. Patient characteristics are summarized in Table 1. We performed FC study of monocyte subsets in PB describing Mo1 (CD14bright/CD16‐), Mo2 (CD14bright/CD16+) and Mo3 (CD14dim or ‐/CD16bright). In addition, we assessed the expression of CD56 in monocyte population (cutoff positivity ≥ 20%). Finally, we applied a custom NGS gene panel focusing on TET2/SRSF2 co‐mutation (VAF detection sensitivity: 0.02). Comparisons were evaluated by Chi‐Square test, Fisher exact test or Man‐Whitney U‐test as appropriateimageResults:a)The median percentage of classical monocytes (Mo1) was significantly inferior when comparing O‐CMML with CMML (96% vs 98%; P = 0.009) but O‐CMML showed a significant higher median percentage than MDS (88%; P = 0.001) and reactive monocytosis (89.9%; P < 0.001). No significant differences were observed between MDS and reactive monocytosis group (P = 0.296).b)The percentage of patients with >94% Mo1 was no significantly different when comparing O‐CMML and CMML (74% vs 86%; P = 0.294), nevertheless the percentage of O‐CMML patients showing this feature was significantly higher in comparison with MDS (0%; P = 0.001), the group where these patients are currently included following WHO 2017 criteria, and reactive monocytosis (20%; P < 0.001). Table 2.c) No differences were observed in the percentage of patients showing CD56 expression in monocytes when comparing O‐CMML and CMML (68.4% vs 69%; P = 0.92), nevertheless MDS (12.5%; P = 0.001) and reactive monocytosis (8.6%; P < 0.001) showed this feature in very few cases. Table 2.d)We observed no significant difference in the presence of TET2/SRSF2 co‐mutation between O‐CMML and CMML (42% vs 35%; P = 0.73). This was not detected in MDS.e)The sensitivity (S) and specificity (SP) of the Mo1>94% test was lower in our series than the originally reported by the GFM group (S: 90%; SP: 95%). Our S and SP was 86% and 71% with a PPV of 56% and a NPV of 92%. This could be explained by a high number of false positives since O‐CMML patients are currently considered as MDS. When considering these as CMML the test performed better with a S and SP of 82% and a PPV and NPV of 78% and 85% respectively.Summary/Conclusion:Our data support the diagnosis of O‐CMML as a distinctive entity with biological features similar to CMML
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