ALBERT

Description: Introduction The JAK2V617F allele burden at progenitor levels increases over time in polycythemia vera (PV), with the dominance of the JAK2V617F-positive clone at the CD34+ compartment being an important modifier of the disease phenotype. The relationship between clonal dominance and the evolutionary pattern of JAK2V617F granulocytic load is presently unknown. Objective To analyze the relationship between clonal dominance of CD34+ cells with the evolution of the granulocytic JAK2V617F mutant load in PV patients. Methods A total of 37 patients with PV were included in the study. At time of the study, 30 patients corresponded to the chronic phase of the disease, whereas 7 patients had evolved to myelofibrosis (post-PV MF). Seventeen patients were in early chronic phase (less than 5 years from diagnosis) and 13 patients in late chronic phase (more than 5 years from diagnosis). Granulocytes were isolated from peripheral blood by density gradient, whereas CD34+ cells were purified by immunomagnetic positive selection. Stem cells (CD34+, CD38-) and progenitors (CD34+, CD38+) populations were further separated by fluorescence-activated cell sorting. JAK2V617F allele burden was measured by quantitative PCR in all three cell fractions. Clonal dominance was defined as an absolute difference ≤ 10% in JAK2V617F between granulocytes and CD34+ populations. Granulocytic JAK2V617F mutant load was measured every year with the evolutionary pattern of the JAK2V617F allele burden being stratified as follows: stable 〈 50%, stable ≥ 50%, progressive increase and unexplained decrease. The study was approved by the local Ethics Committee and informed consent was obtained according to the Declaration of Helsinki. Results JAK2V617F allele burden in CD34+/CD38- cells was 8%, 24.9% and 71.4% in early chronic phase, late chronic phase and post-PV MF, respectively (p 〈 0.001). Similar results were observed in CD34+/CD38+ cells (JAK2V617F allele burden: 19.1%, 36.1% and 71.8% in early chronic phase, late chronic phase and post-PV MF, respectively, p 〈 0.001). No significant differences were observed among the three groups in granulocyte JAK2V617F allele burden. Six patients (16.2%) presented clonal dominance. According to disease status, clonal dominance was observed in 5 out of 7 (71.4%) patients with post-PV MF and in 1 out of 13 (7.7%) patients in late chronic phase, whereas no patient in early chronic phase showed clonal dominance. JAK2V617F monitoring was performed in 29 patients. According to their granulocytic JAK2V617F evolutionary patterns, 9 patients remained with a stable JAK2V617F below 50%, 13 patients showed a stable JAK2V617F ≥ 50%, 6 patients experienced a progressive increase of the JAK2V617F mutant load and 1 case presented an unexplained decrease of JAK2V617F. Clonal dominance was observed in 5 out of 20 (25%) patients with a stable JAK2V617F 〉50% or a changing profile in comparison to 0 out of 9 patients with stable JAK2V617F 〈 50% (p=0.05). Conclusion Clonal dominance correlates with JAK2V617F allele burden evolutionary pattern.This may be a factor to be taken into account in the therapeutic strategy of PV patients and supports the need to monitor JAK2V617F burden during clinical follow-up. Funding this study was supported by grants from the Ministry of Education and Science of Spain and Instituto de Salud Carlos III FEDER (EC10-136, FIS PI10/018087, RD09/0076/00036 and RD12/0036/0010), 2009SGR929 and by grant from Asociación Española Contra el Cáncer Catalunya. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1738 Introduction: Hydroxycarbamide (HC) is an antimetabolite used as first-line therapy in high risk essential thrombocythemia (ET) and polycythemia vera (PV). In treatment-naive patients, HC achieves complete hematological and partial molecular response in 80% and 50% of patients, respectively. Genetic factors involved in the pharmacokinetics of HC, as well as in the acquisition of the JAK2V617F mutation, could play a role in the variability among these patients in achieving a molecular response. Objective: To assess the influence of 46/1 JAK2 haplotype and urea transporters polymorphisms on the molecular response in JAK2V617F-positive PV and ET patients treated with HC. Methods: JAK2V617F allele burden was measured by quantitative PCR at diagnosis and every 6 months during follow-up in 53 PV patients and in 41 ET patients receiving HC therapy. Molecular response to HC treatment was defined according to European LeukemiaNet (ELN) criteria. SNPs rs12340895 and rs12343867 were used to determine 46/1 haplotype status and SNPs rs2298720 (SLC14A1), rs9960464 (SLC14A2), were used to genotype urea transporters. The rate of molecular response was compared according to 46/1 JAK2 haplotype status (negative, heterozygous, homozygous), and genotype of 2 urea transporters: SLC14A1 (GG, GA, AA) and SLC14A2 (GG, GA, AA). The study was approved by the local Ethics Committee and informed consent was obtained according to the Declaration of Helsinki. Results: Overall, molecular response (CR or PR) was achieved in 48 patients (51%), corresponding to 46% and 57% in PV and ET patients, respectively (p=0.3). In ET patients, there were no significant differences in the proportion of patients achieving a molecular response according to 46/1 JAK2 haplotype or to the different transporter genotypes. PV patients with the GG genotype in transporter SLC14A2 obtained more frequently a molecular response than those with AA or GA genotype (71% versus 36%, p=0.03). No significant differences in molecular response were observed in PV patients according to transporter SLC14A1 and 46/1 JAK2 haplotype. Conclusion: 46/1 JAK2 haplotype status did not influence molecular response in PV or ET patients treated with HC. Polymorphisms in urea transporters showed a minimal effect on the rate of molecular response in these patients. Disclosures: No relevant conflicts of interest to declare.

Description: Patients with Philadelphia-negative myeloproliferative neoplasm (MPN) are prone to the development of second cancers, but the factors associated with these events have been poorly explored. In an international nested case-control study, we recruited 647 patients with carcinoma, nonmelanoma skin cancer, hematological second cancer, and melanoma diagnosed concurrently or after MPN diagnosis. Up to 3 control patients without a history of cancer and matched with each case for center, sex, age at MPN diagnosis, date of diagnosis, and MPN disease duration were included (n = 1234). Cases were comparable to controls for MPN type, driver mutations and cardiovascular risk factors. The frequency of thrombosis preceding MPN was similar for cases and controls (P = .462). Thrombotic events after MPN and before second cancer were higher in cases than in controls (11.6% vs 8.1%; P = .013), because of a higher proportion of arterial thromboses (6.2% vs 3.7%; P = .015). After adjustment for confounders, the occurrence of arterial thrombosis remained independently associated with the risk of carcinoma (odds ratio, 1.97; 95% confidence interval, 1.14-3.41), suggesting that MPN patients experiencing arterial events after MPN diagnosis deserve careful clinical surveillance for early detection of carcinoma. This study was registered at www.clinicaltrials.gov as NCT03745378.

Description: Introduction: Over half of patients with chronic myeloid leukemia (CML) in sustained deep molecular remission do not lose the major molecular response (MMR) after stopping treatment with tyrosine kinase inhibitors (TKI). This strategy is safe in controlled clinical trials, but there is scarce information on its applicability in the real-life setting. We aimed to assess if treatment cessation was feasible in clinical practice in a large nationwide series of CML patients from Spain. Methods: This retrospective study comprised a series of 236 patients in chronic-phase CML who discontinued TKI treatment outside of clinical trials between April 2009 and February 2018 in 33 Spanish institutions. Inclusion criteria were: a) TKI treatment duration 〉3 years; b) sustained MR4.5 in 〉4 consecutive determinations (one single point in MR4 was acceptable) during 〉2 years; c) molecular monitoring in a reference laboratory expressing the results on the International Scale (IS). Patients who had undergone allogeneic hematopoietic stem-cell transplantation were excluded. Molecular relapse was defined as consecutively detectable BCR-ABL1 transcripts showing a ≥1 log increase or loss of MMR in any single sample. Treatment-free remission (TFR) was estimated by the method of Kaplan-Meier and defined as the time from TKI discontinuation to the date of restarting therapy for any reason or, if treatment was not restarted, the date of last contact. Incidence of molecular relapse was calculated using the cumulative incidence function with resumption of TKI treatment in the absence of molecular relapse and death in MMR as competing events. Analysis of factors predicting molecular relapse was done by the method of Fine and Gray. Results: Table 1 shows the main characteristics of the series. Median follow-up from treatment discontinuation was 21.5 months, and 5 patients died in MMR due to CML unrelated causes. TKI therapy was reinitiated due to molecular relapse (MMR loss: n=52, increase 〉1 log in BCR-ABL transcript level at two consecutive assessments without losing MMR: n=12), patient preference (n=2), and severe withdrawal syndrome (n=1). One additional patient lost MMR after 20 months from treatment cessation but decided not to be retreated, with spontaneous recovery of MMR. The probability of TFR at 4 years was 64% (95% Confidence Interval [CI]: 55%-72%)(Figure 1). The cumulative incidence of molecular recurrence was 33% (95% CI: 26%-38%) at 3 years (Figure 2). Forty-nine relapses (75% of total) occurred in the first 6 months. The latest MMR loss was detected 30 months after treatment stop. One patient restarted treatment 44 months after TKI discontinuation due to ≥1 log increase in BCR-ABL1 transcripts in two consecutive samples without losing MMR. In univariate analysis, duration of TKI treatment of less than 5 years (P=0.005) and time in RM4.5 shorter than 4 years before TKI discontinuation (P=0.003) were both significantly associated with a higher incidence of molecular recurrence. No patient progressed to the advanced phases of CML. At the time of restarting treatment, the median BCR-ABL1 IS was 0.3%, with this value being 〉5% in only 7 instances. Most patients (81%) received the same TKI that they were taking before the trial of treatment cessation. Median follow-up after treatment resumption was 20 months. Among the 64 patients who restarted treatment due to molecular relapse, 46 of 52 cases regained MMR after a median time of 3 months, and 47 of 64 regained MR4.5 after a median time of 5 months. Response status at last control was: MR4.5 (n=196), MR4 (n=15), MMR (n=14), complete cytogenetic response (n=10), and other (n=1). Fifty-one patients (22%) developed musculoskeletal or joint pain after treatment cessation. In patients stopping imatinib, a significant increase in Hb levels, leukocyte counts, total lymphocyte counts, platelet counts, and cholesterol levels was observed. At 6 months, an increase in Hb level 〉2 g/dL was observed in 47% of patients with anemia. By contrast, nilotinib discontinuation was not followed by any relevant change in laboratory values. Conclusions: Our results confirm that treatment discontinuation is feasible and safe in clinical practice in Spain. Duration of TKI treatment of less than 5 years and a time in RM4.5 shorter than 4 years before TKI discontinuation were significantly associated with a higher incidence of molecular recurrence. Disclosures Hernandez Boluda: Incyte: Consultancy; Novartis: Consultancy. García Gutiérrez:Incyte: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Ferrer Marin:Incyte: Consultancy; Novartis: Consultancy, Research Funding. Cervantes:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Hospital Clinic Barcelona: Employment.

Description: Introduction: The dominance of the JAK2V617F-positive clone at the CD34+ compartment is an important modifier of the disease phenotype in myeloproliferative neoplasms (MPN). Recently, mutations in the calreticulin gene (CALR) have been described in around 40-70% of JAK2V617F and MPL wild-type essential thrombocythemia (ET) and myelofibrosis (MF) patients. However, there is limited information regarding the role of CALR mutant clone in hematopoietic progenitor cells. Objective: To study the mutant allele burden at progenitor level in JAK2V617F-positive and CALR-mutated MPN. Methods: Sixty-five patients with MPN including 36 with polycythemia vera (PV) all JAK2V617F-positive, 13 with ET (7 JAK2V617F-positive and 6 CALR-mutated) and 16 with MF (9 JAK2V617F-positive post-PV MF, 4 CALR-mutated primary MF and 3 CALR-mutated post-ET MF) were included in the study. Granulocytes were isolated from peripheral blood by density gradient, whereas CD34+ cells were purified by immunomagnetic positive selection. Stem cells (CD34+CD38-) and progenitors (CD34+CD38+) populations were further separated by fluorescence-activated cell sorting. JAK2V617F and CALR allele burden was measured by quantitative PCR and PCR followed by fragment analysis, respectively, in stem cells, progenitor cells and granulocytes. The study was approved by the local Ethics Committee and informed consent was obtained according to the Declaration of Helsinki. Results: CALR-mutated ET patients harbored a higher mutant load in CD34+CD38- than JAK2V617F-positive ET patients (30.6 vs 6.3%, p=0.01), whereas no significant differences were observed in CD34+CD38+ and in granulocytes allele burdens. Moreover, CALR-mutated ET patients showed a higher mutational load in CD34+CD38- than JAK2V617F-positive PV (30.6% vs 15.7%, p=0.04) but the mutant load in granulocytes was lower (29.6% vs 63.3%, p

Description: INTRODUCTION The diagnosis of chronic myelomonocytic leukemia (CMML) according to WHO 2017 requires the presence of ≥1x109/L and ≥10% of monocytes in peripheral blood (PB). Presence of dysplasia is frequent but not always present. Recently, Geyer et al. described olygomonocytic CMML (O-CMML) as those MDS cases with relative monocytosis (≥10% monocytes) and monocyte count 0.5-94% of total monocytes is a highly sensitive and specific diagnostic marker for CMML. We assessed peripheral monocyte subsets by FC in 11 O-CMML cases and compared those with 20 CMML cases, 14 D-CMML and 6 proliferative CMML (P-CMML). In addition, we studied the aberrant expression of CD56, CD2 and CD7 in monocytes. As mentioned, O-CMML may be considered an early-phase of D-CMML and some D-CMML may progress to P-CMML, an entity with an ominous prognosis. We compared the percentage of Mo1 and non-classical monocytes (Mo3) among O-CMML, D-CMML and P-CMML in order to evaluate if a progressive accumulation of Mo1 and a progressive decrease in Mo3 could be appreciated among these entities. In our view, Mo1 could be considered as a marker of tumor burden, while Mo3, formerly considered as a specific type of dendritic cell, could be related with immunosurveillance. In order to reinforce this hypothesis we evaluated if the reduction in Mo3 would be also accompanied by a decrease in plasmocytoid dendritic cells (pDC). METHODS Twenty CMML and 11 O-CMML were prospectively studied from 02/2016 to 04/2018. Patients' characteristics are summarized in Table 1. We performed FC study of monocyte subsets in PB describing Mo1 (CD14bright/CD16-), intermediate monocytes (Mo2) (CD14bright/CD16+) and Mo3 (CD14dim or -/CD16bright). In addition, we assessed the expression of CD56, CD2 and CD7 in monocyte population and quantified pDC (CD123bright/HLA-DR+). Comparisons were evaluated by Chi-Square test, Man-Whitney U-test or by Kruskall-Wallis test as appropriate. RESULTS & DISCUSSION 1) 6/11 (55%) O-CMML showed an increase in the fraction of Mo1〉94% of total monocytes. In contrast, 12/14 (86%) D-CMML and 6/6 (100%) P-CMML showed a percentage of Mo1〉94% of total monocytes. Considering together all overt CMML, 18/20 (90%) presented Mo1〉94% of total monocytes. This result was almost identical to that reported in the original study by Selimoglu-Buet. 2) The median percentage of Mo1 and Mo3 monocytes was statistically different among these three entities (Mo1, p=0,005; Mo3, p=0,002). Table 2. Interestingly, the median percentage of Mo1 (% Mo1) was significantly lower in O-CMML when compared to P-CMML (p=0,004) and a clear trend was observed when compared to D-CMML. In the same way, % Mo1 was significantly lower in D-CMML when compared to P-CMML (p=0,017). Moreover, the median percentage of Mo3 (% Mo3) was significantly higher in O-CMML when compared to P-CMML (p=0,002) and a clear trend was observed when compared to D-CMML. In the same line, % Mo3 was significantly higher in D-CMML when compared to P-CMML (p=0,002). Likewise, the median percentage of pDC (% pDC) was significantly higher in O-CMML when compared to P-CMML (p=0,004). A clear trend was observed when O-CMML was compared with D-CMML, and D-CMML with P-CMML. These data reinforce the hypothesis that progression from O-CMML to D-CMML and P-CMML could be guided by a progressive accumulation of Mo1 ("tumor burden increase") and by a progressive reduction of Mo3 and pDC ("immunosurveillance decrease"). 3) Expression of CD56, CD2 and CD7 in monocytes is depicted in Table 3. No aberrant expression of CD7 was observed in any case. In contrast, CD56 expression was observed in 9/11 O-CMML, 7/14 D-CMML and 5/6 P-CMML. Considering together all overt CMML, 12/20 expressed CD56. CD56 expression in monocytes is a common finding in CMML and has been rarely described in other myeloid disorders. This may be interpreted as another indicator that O-CMML is in the continuum of CMML. CONCLUSIONS O-CMML presents flow cytometric immunophenotypic characteristics in line with overt CMML. These data support that O-CMML is in the biological continuum of overt CMML. Disclosures No relevant conflicts of interest to declare.

Description: INTRODUCTION The diagnosis of chronic myelomonocytic leukemia (CMML) according to WHO 2017 requires the presence of ≥1x109/L and ≥10% of monocytes in peripheral blood (PB). Establish an accurate diagnostic is difficult since many clinical situations present persistent monocytosis. The presence of dysplasia, mainly dysgranulopoiesis, is frequent but not always present in CMML. Cytogenetic aberrations are infrequent in this disease (20-25% of cases). Although 85-90% of CMML patients present at least one mutation in TET2, SRSF2 or ASXL1 genes, the use of NGS panels is not widespread. Furthermore, mutations in these genes are among the most frequently observed in age-related clonal hematopoiesis. Therefore, complementary techniques are required to support the diagnosis of this entity. The study of the peripheral monocyte subsets by flow cytometry (FC) has gained special interest due to a high sensitivity and specificity for the diagnosis of CMML (S = 90.6%, E = 95.1%, Selimoglu-Buet et al., Blood, 2015). An increase in the fraction of classical monocytes (Mo1) to 〉94% of total monocytes is an event frequently observed in CMML. There are no specific bone marrow (BM) FC panels for the diagnosis of CMML and very few have been validated for the diagnosis of MDS. "Ogata score", the only multicenter validated score in MDS, has not been applied in CMML. The aim of our study was to evaluate the usefulness of FC in PB and BM for the diagnosis of CMML. METHODS Twenty-two CMML were prospectively studied from 02/2016 to 04/2018. Patients' characteristics are summarized in Table 1. Diagnostic procedure consisted of morphological, cytochemical (Perls, myeloperoxidase, nonspecific esterase), cytogenetic and FC studies in BM, and morphological and FC studies in PB. "Ogata Score" was applied in BM samples (Table 2). Aberrant coexpression of CD2, CD7 and CD56 in BM monocytes was assessed. Immunophenotypic maturation profile of the monocytic elements in BM distinguishes: promonocytes (CD34-/CD117-/CD64++/CD14- or dim/CD45+/HLA-DR+++), mature monocytes (CD34-/CD117-/CD64++/CD14++/CD45++/HLA-DR++) and mature monocytes in terminal stage (CD300e+). In PB, the monocytes FC subsets (Mo1, Mo2 and Mo3) were studied, as well as the aberrant coexpression of CD2, CD7 and CD56 (Table 3). RESULTSThe presence of ≥2 aberrations in Ogata Score predicted properly the diagnosis of CMML in all patients analyzed (100% sensitivity). Due to the study design, we could not obtain results about specificity.An increase in Mo1 (classical monocytes) 〉 94% was detected in 18/20 patients (Table 3). This method predicted the diagnosis of CMML with a sensitivity of 91%, a result almost identical to the original study (Selimoglu-Buet et al., Blood, 2015).A good positive correlation was established between the percentage of BM promonocytes detected by morphology and by FC (Rho Spearman 0.61, P = 0.003).A negative correlation was found between the percentage of promonocytes by FC in MO and the expression of CD56 (Rho Spearman -0.612, P = 0.002). Similarly, CD56+ CMML presented a percentage of promonocytes by FC significantly lower than the CD56- CMML group (median: 24.5% (14-40) vs. 41% (23-71), P = 0.005). The expression of CD56 seems to be related to a more mature immunophenotypic profile of the monocytic population. On the other hand, the correlation between the percentage of CD56+ monocytes in BM and PB was almost perfect (Rho Spearman 0.928, P

Description: Introduction Malignancy can be heralded by unprovoked venous thromboembolism (VTE) but also by arterial thrombosis. To date it is unknown whether this association is present also in myeloproliferative neoplasms (MPN), in which arterial thrombosis is more frequent that venous thrombosis and solid tumors are reported with an increased frequency. The MPN-K nested case-control study addressed the impact of cytoreductive drugs on the risk of developing second cancer in MPN patients (Barbui T et al, Leukemia 2019); here we re-examined the study database to evaluate the frequency and type of vascular complications in MPN patients with second cancer excluding leukemia and to establish whether arterial and venous thrombosis during follow-up after diagnosis of MPN could predict the occurrence of a second cancer. Patients and methods Cases were patients with second cancer diagnosed concurrently or subsequent to the diagnosis of MPN. Controls were MPN patients without second cancer. For each case with second cancer, up to 3 cancer-free controls were matched by each center for sex, age at MPN diagnosis, date of MPN diagnosis, and MPN disease duration. Each set consisting of one case and their matched-controls had a similar observational period (from MPN diagnosis until the index date of diagnosis for the second cancer). The study included 647 cases with second cancer (carcinoma, non-melanoma-skin cancers, hematological secondary cancer and melanoma). The most frequent category was carcinoma (n=426, 65.8%). Cases were comparable with the 1,234 matched controls for demographics, type of MPN, and exposure to potential confounders such as mutational profile, abnormal karyotype and cardiovascular risk factors. The thrombotic events of interest were ischemic stroke, transient ischemic attacks, acute coronary syndromes, peripheral arterial thrombosis, deep venous thrombosis (including thrombosis of cerebral and splanchnic veins) and pulmonary embolism. Thrombosis had to be concurrent with or in the 2 years before MPN diagnosis or occurring after MPN diagnosis. The cumulative incidence of either arterial or venous thrombosis from MPN diagnosis was estimated by the Kaplan-Meier method and was compared between cases and controls using the log-rank test. A conditional logistic regression model estimated the Odds Ratio (OR) with 95% Confidence Interval (CI) of second cancer associated with the occurrence of thrombosis before/at diagnosis of MPN and during follow-up. Other covariates were patient age, cardiovascular risk factors, the JAK2V617F mutation, and treatment during follow-up. Results Approximately 20% of either MPN cases or controls had thrombosis before MPN or at diagnosis (19.8% vs. 21.1%, respectively, p=0.462). After a median observation time from diagnosis of MPN to an index date of 4.5 years (interquartile range 1.5-8.2) in cases and 3.7 years (interquartile range 1.5-7.5) in controls, cases showed a percentage of thrombosis higher than in controls (75/647, 11.6% vs. 100/1234, 8.1%, respectively, p=0.013). Approximately one-third of thrombosis preceding cancer occurred in the 12 months before the diagnosis of second cancer (22/75, 29.3%). The excess of thrombosis in cases was due to a higher frequency of arterial thrombosis (6.2% vs. 3.7%, p=0.015), whereas no significant difference was found for venous thrombosis (5.4% vs. 4.3%, p=0.277). While the cumulative incidence of venous thrombosis over time was similar among cases and controls (p=0.864), the cumulative incidence of arterial thrombosis was higher in cases with second cancer (p=0.006) (Figure 1). The excess of arterial thrombosis after MPN diagnosis was limited to cases with carcinoma (6.8% vs 3.9%, p=0.027). In a multivariable model, arterial thrombosis during the follow-up was confirmed to be an independent predictor factor for carcinoma, with an odds ratio of 1.97 (95%CI 1.14-3.41, p=0.015). Conclusions. These findings reveal an association of arterial thrombosis with subsequent second cancer (namely carcinoma) in MPN patients. A possible biological plausibility for this link may be related to an underlying common pathogenic mechanism such as an aberrant inflammatory response consistently found in MPN. This observation may have practical implications and suggests careful clinical surveillance for early diagnosis of second cancer in MPN patients with arterial thrombosis during the follow-up. Disclosures Palandri: Novartis: Consultancy, Honoraria. Iurlo:Novartis: Other: Speaker Honoraria; Incyte: Other: Speaker Honoraria; Pfizer: Other: Speaker Honoraria. Bonifacio:Incyte: Honoraria; Novartis: Honoraria; Amgen: Honoraria; Pfizer: Honoraria; BMS: Honoraria. Rumi:novartis: Honoraria, Research Funding. Elli:Novartis: Membership on an entity's Board of Directors or advisory committees. Lunghi:Pfizer: Honoraria; Novartis: Honoraria; Incyte: Honoraria. Benevolo:Novartis Pharmaceuticals: Consultancy. McMullin:Italopharma: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Speakers Bureau; Daiko Sanyo: Membership on an entity's Board of Directors or advisory committees. Griesshammer:Novartis: Consultancy, Honoraria, Speakers Bureau. Vannucchi:CTI: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees; Italfarmaco: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. Rambaldi:Pfizer: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau.

Description: Background: Non-driver mutations and JAK2V617F allele burden have been involved in progression to myelofibrosis (MF) or acute myeloid leukemia (AML) in patients with polycythemia vera (PV) and essential thrombocythemia (ET). It is unknown if both mechanisms play a different role in disease transformation and if they are useful in routine clinical practice. Methods: JAK2V617F allele burden was monitored in 208 patients (PV n=106, ET n=102) for a median of 6.5 years (range: 1-13). Quantification of JAK2V617F allele burden was assessed on the first available sample and every year thereafter. The evolutionary pattern of JAK2V617F allele burden was categorized as persistently low (50%), progressive increase (〉 25% from baseline) or unexplained decrease (not therapy related). Next generation sequencing (NGS) analysis of 51 myeloid-related genes was performed in 100 patients with a median molecular follow-up of 10 years including all cases with transformation to AML or MF. Detected mutations by NGS in the last sample were studied in first paired sample obtained in the chronic phase (median time from diagnosis: 1.6 years). Time to myelofibrosis and time to AML were calculated according to the presence of non-driver mutations or the JAK2V617F evolutionary pattern. Multivariate analysis was performed by Cox regression. Results: With a median follow-up of 13 years (range: 1-30) 32 patients died whereas 24 and 12 patients progressed to MF and AML, respectively. Median age at diagnosis was 63 years (range: 20-94), 115 were women (55%) and 173 (83%) received cytoreduction. A persistently low JAK2V617F allele burden was observed in 62% of patients whereas the remainder presented a persistently high (25%), a progressive increase (11%) or a non-therapy-related decrease of JAK2V617F allele burden (2%). Non-driver mutations were detected in last sample in 48% of patients. Median number of mutations was 1 (range: 1-5). Mutational frequencies were: TET2 12%, DNMT3A 12%, TP53 9%, ASXL1 7%, RUNX1 4%, SF3B1 4%, SRSF2 4%, IDH1/2 4%, SH2B3 3 % and

Description: Abstract 4086 Definition of resistance/intolerance to hydroxyurea (HU) in essential thrombocythemia (ET) has been proposed by the European LeukemiaNet (ELN) however its clinical utilility has not been validated yet. We have retrospectively evaluated such criteria in 166 ET patients treated with HU for a median of 4.5 years. Response to HU treatment was categorized using the ELN criteria. The ELN definitions of resistance/intolerance to HU required the fulfillment of at least one of the following criteria: platelet count greater than 600 × 109/L after 3 months of at least 2 g/day of HU (2.5 g/day in patients with a body weight over 80 kg); platelet count greater than 400 × 109/L and leukocytes less than 2.5 × 109/L or hemoglobin (Hb) less than 100 g/L at any dose of HU; presence of leg ulcers or other unacceptable mucocutaneous manifestations at any dose of HU; HU-related fever. Survival and time-to-event curves were estimated using the Kaplan-Meier method.Variab les attaining a significant level at the univariate analysis were included in a Cox proportional hazards model. Overall, 134 patients achieved a complete clinicohematologic response (CR) and 25 a partial response. Thirty-three patients met at least one of the ELN criteria defining resistance (n=15) or intolerance (n=21) to HU. Fifteen cases developed anemia with thrombocytosis. Other definitions of resistance were less useful. When compared with the others, resistant patients were more likely to display hyperproliferative features at ET diagnosis, such as higher levels of leukocytes (p= 0.05), platelets (p=0.004) and serum LDH (p=0.02). Eleven patients developed leg ulcers leading to a permanent discontinuation of the drug in 8 cases. No distinctive clinical profile could be ascribed to patients exhibiting leg ulcers, with the exception of a high prevalence of cardiovascular risk factors. Other unacceptable mucocutaneous manifestations occurred in 9 patients. Hematologic and mucocutaneous complications were unrelated, with only two patients presenting both types of toxicities. With a median follow-up from ET diagnosis of 7 years (range: 0.5–23), 38 patients (23%) had died, resulting in a survival probability of 65% at 10 years from HU start. The risk of death from any cause was increased by 6.2-fold (95%CI: 2.3–16.7, P0.001). In conclusion, the best discriminating ELN criterion of resistance to HU is based on the detection of anemia. Moreover, such criterion is particularly useful since it also identifies a subset of ET patients with a poor prognosis. Disclosures: No relevant conflicts of interest to declare.