Abstract: Introduction . Ruxolitinib (RUX) is the only targeted therapy available for the treatment of myelofibrosis (MF)-related splenomegaly and symptoms. Significant clinical responses may be achieved in around 50% of patients (pts). However, half of responding pts lose the response over time. Aims . To report the outcome of a large cohort of MF pts after RUX failure, in terms of disease status, treatment strategies and survival. Methods . A clinical database was created in 23 European Hematology Centers including retrospective data of 537 MF pts treated with RUX from Jan 2011 to July 2018. Updated information at the date of July 15th 2018 was available in 442 pts who were included in the present analysis. Spleen and symptoms response (SR & SyR) to RUX were evaluated according to the 2013 IWG-MRT criteria. RUX-related toxicity and infections were graded according to the WHO scale. Overall (OS) was estimated from the date of RUX discontinuation to the date of death or last contact, using the Kaplan-Meyer method (log-rank test). Results . After a median follow-up of 30.5 months (1.7-84.3), 214 out of 442 evaluable (48.4%) pts had discontinued RUX. 43 (20.1%) died while on therapy because of: MF progression (34.9%), infections (25.6%), heart disease (16.3%), second neoplasia (7%), hemorrhages (7%), other (9.2%). The median follow-up after RUX discontinuation for the remaining 171 pts was 11.3 months (0.5-66.7). Causes of RUX discontinuation were: drug-related toxicity (28.6%), loss/lack of response (23.4%), MF progression (12.3%), acute leukemia (AL) (13.4%), allogeneic stem cell transplantation (ASCT) (11.1%), second solid neoplasia (4.1%), other unrelated causes (i.e. pts decision; 7.1%). After stopping RUX, 68 pts received 1 line of therapy, 21 received 2 lines and 9 received 〉 2 treatments; 73 pts did not receive any therapy. Treatments received after RUX discontinuation, alone or in combination, included hydroxyurea (HU) (n. 61, 62.2%), ASCT (n. 20, 20.4%), second-generation JAK2 inhibitors (momelotinib/fedratinib/pacritinib) (n. 11, 11.2%), splenectomy (n. 7, 7.1%), azacytidine/decitabine (n. 5, 5.1%), chemotherapy (n. 4, 4.1%), investigational agents (imetelstat/PRM151: n. 4), danazole (n. 4), erythropoietin-stimulating agents (ESA) (n. 4). A total of 95 pts (55.6%) died after RUX discontinuation, because of: MF progression (30.5%), AL (25.4%), infections (14.7%), second neoplasia (9.5%), hemorrhages (4.2%), heart disease (4.2%), ASCT (4.2%), thrombosis (2.1%), other (5.2). Median survival time from RUX stop of the 171 evaluable pts was 22.6 mos (95% CI, 13.2-30.7). Among baseline features, survival after discontinuation was significantly influenced by the dynamic international prognostic score (DIPSS) category (p 〈 0.001), transfusion dependency (p 〈 0.001) and driver mutation status (with triple-negative pts having the worst survival compared to JAK2V617F and CALR-mutated pts, p=0.01). During therapy, 45 out of 153 (29.4%) and 123 out of 161 (76.4%) evaluable pts achieved a SR and a SyR at any time. Survival was not affected by the previous response to RUX at any time-point. Conversely, survival significantly differed according to the reason for stopping RUX, with pts discontinuing because of AL evolution/second solid neoplasia having the worst outcome (Figure 1a, p 〈 0.001). In pts who discontinued RUX in chronic phase, the use of second generation TKIs and other investigational agents tended to prolong survival compared to the administration of conventional medical treatments (i.e. HU, danazole, ESA) (Figure 1b, p=0.07) Discussion . After RUX failure, very limited therapeutic options are available and the prognosis of MF pts is dismal, particularly for those pts starting RUX with advanced stage disease (i.e. high DIPSS category and transfusion dependency). Also, disease evolution into AL and occurrence of a second solid neoplasia significantly reduced life expectancy. In chronic phase pts, survival probability may be improved by the use of medical therapies that are still in the experimental phase. Novel investigational agents are needed. Disclosures Palandri: Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Abruzzese:BMS: Consultancy; Ariad: Consultancy; Novartis: Consultancy; Pfizer: Consultancy. Vitolo:Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Gilead: Speakers Bureau; Takeda: Speakers Bureau; Sandoz: Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Aversa:Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Basilea: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria; Astellas: Honoraria; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees. Cuneo:Gilead: Other: advisory board, Speakers Bureau; Roche: Other: advisory board, Speakers Bureau; Abbvie: Other: advisory board, Speakers Bureau; janssen: Other: advisory board, Speakers Bureau. Foà:ROCHE: Other: ADVISORY BOARD, Speakers Bureau; AMGEN: Other: ADVISORY BOARD; JANSSEN: Other: ADVISORY BOARD, Speakers Bureau; GILEAD: Speakers Bureau; NOVARTIS: Speakers Bureau; CELTRION: Other: ADVISORY BOARD; ABBVIE: Other: ADVISORY BOARD, Speakers Bureau; INCYTE: Other: ADVISORY BOARD; CELGENE: Other: ADVISORY BOARD, Speakers Bureau. Di Raimondo:Celgene: Honoraria; Takeda: Honoraria, Research Funding. Cavo:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Breccia:Pfizer: Honoraria; Incyte: Honoraria; BMS: Honoraria; Novartis: Honoraria. Palumbo:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Abstract: Introduction. Blast phase (BP) is the terminal and most incurable phase of myelofibrosis (MF) and occurs in a not negligible fraction of patients (pts). In the pre-ruxolitinib (RUX) era, peripheral blasts, thrombocytopenia, unfavorable cytogenetics, and high risk category were identified as predictors of BP. RUX is the standard of care for symptomatic MF; however, information on clinical/laboratory correlates of BP in RUX-treated pts is not available. Aims. The primary objective of the study is to assess real-world data on incidence, risk factors and outcome of BP in RUX-treated MF pts. Methods. A multicentre observational retrospective study on RUX-treated MF pts was conducted in 20 European Hematology Centers. Data were extracted from an electronic database that included consecutive pts treated with RUX from June 2011. Data cut-off was June 2019. Risk category was assessed at RUX start according to the Dynamic International Prognostic Score System (DIPSS) or the Myelofibrosis Secondary to PV and ET Collaboration Prognostic Model (MYSEC-PM) in pts with post-Polycythemia Vera (PV)/post-Essential Thrombocythemia (ET) MF (secondary MF, SMF). A time-to-event (BP) analysis was conducted with Fine & Gray model with death/time of stem cell transplant as competing risks. Variables tested for association with BP were: age≥65yr, sex, transfusion-dependency, PLT 〈 150x109/l, peripheral blasts ≥3%, marrow fibrosis grade, CALR-unmutated genotype, unfavorable karyotype, spleen length (≥10 cm), total symptoms score (≥20), previous hydroxyurea (HU), alkylating agents, and interferon (IFN) use, time from MF diagnosis to RUX start, and PV/ET duration. Cumulative Incidence Function among risk categories for DIPSS and MYSEC-PM was calculated applying the Gray's model. Results . Overall, 589 MF pts were included and observed for 1833 pt-yrs from RUX start (median, 35.4 mos). Diagnosis was PMF in 304 pts (51.6%), PPV-MF in 164 pts (27.8%) or PET-MF in 121 (20.6%); 58.4% males. Molecular status was: JAK2V617F (82.5%), CALR (11.3%) and MPLW515K/L (1.1%); 5.1% were triple negatives. Overall, 368 (62.5%) pts received ≥1 cytoreductive therapy before RUX, specifically: HU, n. 357; alkylating agents, n. 47; anagrelide, n. 33; and IFN, n. 29. Median time from MF diagnosis to RUX start was 1.3 yrs. DIPSS for the whole cohort was: INT-1 (52.9%), INT-2 (40.1%), and HIGH (7%). DIPSS distribution in PMF pts was: INT-1 (47.8%), INT-2 (45.7%), and HIGH (6.5%), while SMF pts were categorized at LOW (11.1%), INT-1 (43.1%), INT-2 (31.2%) and HIGH (14.6%) risk according to the MYSEC-PM. Overall, 65 (11%) developed BP. In 61 pts, BP caused RUX withdrawal after a median time of 1.2 yrs (0.7-6.2); in 4 pts BP occurred after RUX stop (median time: 2.4 yrs). BP incidence rate was 3.6 x100 pt-yrs and was comparable in PMF and SMF (p=0.1). In univariate analysis, the probability of BP evolution for the PMF cohort was significantly reduced by previous IFN use (p=0.001). In SMF, predictors for BP in univariate analysis were PLT 〈 150 x109/l (p=0.001), blasts ≥3% (p=0.002), grade 3 marrow fibrosis (p=0.03) and PV/ET duration ≥ 10 yrs (p=0.02); previous IFN significantly reduced the risk of BP (p=0.02). In multivariable analysis, PLT 〈 150 x109/l (HR 2.4, 95% CI 1.1-5.4, p=0.03), blasts ≥3% (HR 3.3, 95% CI 1.4-7.5, p=0.004) and previous IFN (HR 0.1, 95% CI 0.02-0.8, p=0.04) remained significant. High DIPSS risk significantly predicted BP in PMF (p=0.04, HR [95% CI]: 2.6 [1.1-6.5] ) but not in SMF (p=0.40). In this latter cohort, only the MYSEC-PM was associated with BP (p=0.02, HR 1.7 [95% CI]: [1.1-2.8] ) (Fig.1). Estimated HRs, in reference to the lower score category, were: 1.10 for INT-1, 1.82 for INT-2, and 4.04 for HIGH risk. HR for HIGH risk, comparing to all lower risk groups, was 3.53 (95% CI: 1.53-8.11). Overall, 54 (81.8%) BP pts died and median survival was 2.8 mos. Survival after BP was not influenced by type of MF, previous response to RUX, and type of salvage treatment. Conclusions. Thrombocytopenia and peripheral blasts at RUX start identified pts at higher risk of BP in SMF, while previous IFN use was associated with reduced BP evolution in both PMF and SMF, suggesting a possible disease-modifying action of this agent. Also, this analysis supports the ability of MYSEC-PM in predicting BP in pts with SMF. Despite RUX use, outcome after BP remained dismal, confirming the need for newer treatment strategies. Disclosures Palandri: Novartis: Consultancy, Honoraria. Breccia:Incyte: Honoraria; Celgene: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; BMS: Honoraria. Tiribelli:Incyte: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. Benevolo:Novartis Pharmaceuticals: Consultancy. Bonifacio:Novartis: Honoraria; Amgen: Honoraria; Pfizer: Honoraria; Incyte: Honoraria; BMS: Honoraria. Iurlo:Pfizer: Honoraria; BMS: Honoraria; Incyte: Honoraria; Novartis: Honoraria. Elli:Novartis: Membership on an entity's Board of Directors or advisory committees. Abruzzese:BMS: Consultancy; Incyte: Consultancy; Novartis: Consultancy; Pfizer: Consultancy. Sgherza:Novartis: Honoraria; BMS: Honoraria; Pfizer: Honoraria; Incyte: Honoraria. Cavazzini:Pfize: Honoraria; Incyte: Honoraria; Novartis: Honoraria. Crugnola:Novartis: Honoraria; Incyte: Honoraria. Isidori:Janssen: Honoraria; Novartis: Honoraria; Gilead: Honoraria. Heidel:Novartis: Consultancy, Research Funding; Celgene: Consultancy; CTI: Consultancy. Latagliata:Janssen: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; Celgene: Honoraria. Trawinska:Novartis: Consultancy, Honoraria. Krampera:Janssen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Cuneo:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Honoraria, Speakers Bureau; Abbvie: Honoraria, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Cavo:takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; bms: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; novartis: Honoraria. Palumbo:Novartis: Honoraria; Janssen: Honoraria; Celgene: Honoraria; Amgen: Honoraria; Hospira: Honoraria; Teva: Honoraria.

Abstract: Introduction . The 2016 WHO criteria identified early primary myelofibrosis (early-PMF) as an individual entity with different clinical/laboratory presentations and a significantly better outcome compared to overt PMF. No information is available on the therapeutic effects of ruxolitinib (RUX) in the context of each disease separately. Aims . To report the differences between early and overt PMF patients (pts) treated with RUX in terms of baseline clinical/laboratory characteristics, response to treatment and toxicity. Methods . A clinical database was created in 23 European Hematology Centers including retrospective data of 537 MF pts treated with RUX from Jan 2011 to July 2018. Spleen and symptoms response (SR & SyR) to RUX were evaluated according to the 2013 IWG-MRT criteria. Hematologic toxicity and infections were graded according to the WHO scale. Overall survival (OS) and progression-free survival (PFS) were estimated from diagnosis using the Cox proportional hazards regression model, with adjustment for the dynamic international prognostic score system (DIPSS) and left-truncation. Results . A total of 199 pts had a diagnosis of early (n. 59, 29.7%) or overt (n. 140, 70.3%) PMF confirmed by bone marrow biopsy at RUX start and were included in this analysis. At RUX start, median age was 68.4 yrs (26.5-88.9) and 66.3% of pts had a spleen palpable at ≥10 cm below the left costal margin (LCM) (median spleen length: 12 cm). Median hemoglobin value and total symptoms score (TSS) were 10.5 g/dL and 20 (0-80), respectively. DIPSS distribution was: intermediate-1 (50.5%), intermediate-2 (42.1%), high (7.4%). Molecular status was: JAK2V617F 72.3%, CALR 13.7%, MPLW515K/L 3.1%, triple-negative 5%. Median time from diagnosis to RUX start was 22.4 mos (0.1-394). Compared to overt PMF pts, pts with early PMF started RUX with higher hemoglobin levels (median, 11.6 vs 10.4 g/dl, p=0.01) and lower circulating blast counts (p 〈 0.001), and were more frequently at intermediate-1 DIPSS risk (69.6% vs 42.5%, p 〈 0.001). RUX starting and 12-weeks titrated doses were comparable in the two groups. At 3 and 6 months, 43.1% and 48.9% of early-PMF pts achieved a SR, compared to 27.9% and 31.3% of overt-MF pts (p=0.04 and p=0.04, respectively). The rate of SyR was also higher in early-PMF pts at 3 months (82.5% vs 68.8%, p=0.05) and at 6 months (90.0 vs 73.7, p=0.02). In the first 12 months from RUX start, anemia/thrombocytopenia of all grades occurred in 75.6%/43.1% and 86.3%/60.0% of early and overt PMF pts, respectively (p=0.11 and p=0.03). At 3 months, anemia was more frequent in overt PMF pts (94.7% vs 80.0%, p=0.01), with 32.6% of pts having a grade 3-4 anemia compared to 17.8% in early PMF (p=0.02). The incidence of thrombocytopenia was also higher in overt PMF at 3 (51.5% vs 36.2%, p=0.05) and 6 (52.9% vs 35.8%, p=0.04) months, with only 2.2% and 2.5% of pts having a grade 3-4 thrombocytopenia, respectively. Seventy-five pts had at least one grade ≥2 infectious episode during RUX therapy. Considering death as competing risk, the cumulative risk of infections grade ≥2 was comparable in the two cohorts (p=0.4). Overall, 108 pts discontinued RUX (52.5% and 55.0% of early and overt PMF pts, p=0.7). Evolution into acute leukemia (AL) occurred in 21 pts. After a median follow-up of 23 months, 69 pts died (19 early), specifically because of progression of myelofibrosis (38%), AL (16.9%), infections (11.3%), hemorrhage/thrombosis (12.6%), second neoplasias (8.5%) or transplant-associated toxicity (2.8%), other causes (9.9%). OS (p=0.88) and PFS (p=0.86) were comparable in early and overt PMF pts. Conclusions . This study indicates for the first time that early PMF represents a category of pts that is projected to have better responses and lower toxicities from RUX treatmemt. In the setting of RUX therapy, a WHO-defined diagnosis may contribute to better identify pts who may deserve a strict monitoring during treatment. Disclosures Palandri: Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Palumbo:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Abruzzese:BMS: Consultancy; Pfizer: Consultancy; Novartis: Consultancy; Ariad: Consultancy. Foà:INCYTE: Other: ADVISORY BOARD; GILEAD: Speakers Bureau; JANSSEN: Other: ADVISORY BOARD, Speakers Bureau; ROCHE: Other: ADVISORY BOARD, Speakers Bureau; CELTRION: Other: ADVISORY BOARD; ABBVIE: Other: ADVISORY BOARD, Speakers Bureau; AMGEN: Other: ADVISORY BOARD; NOVARTIS: Speakers Bureau; CELGENE: Other: ADVISORY BOARD, Speakers Bureau. Vitolo:Takeda: Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Gilead: Speakers Bureau; Sandoz: Speakers Bureau. Aversa:Basilea: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees. Cuneo:Roche: Other: advisory board, Speakers Bureau; Abbvie: Other: advisory board, Speakers Bureau; Gilead: Other: advisory board, Speakers Bureau; janssen: Other: advisory board, Speakers Bureau. Di Raimondo:Celgene: Honoraria; Takeda: Honoraria, Research Funding. Cavo:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Breccia:Incyte: Honoraria; Pfizer: Honoraria; Novartis: Honoraria; BMS: Honoraria.

Abstract: The development of different generations of BCR-ABL1 tyrosine kinase inhibitors (TKIs) has led to the high overall survival of chronic myeloid leukemia (CML) patients. However, there are CML patients who show resistance to TKI therapy and are prone to progress to more advanced phases of the disease. So, implementing an alternative approach for targeting TKIs insensitive cells would be of the essence. Dihydroorotate dehydrogenase (DHODH) is an enzyme in the de novo pyrimidine biosynthesis pathway that is located in the inner membrane of mitochondria. Here, we found that CML cells are vulnerable to DHODH inhibition mediated by Meds433, a new and potent DHODH inhibitor recently developed by our group. Meds433 significantly activates the apoptotic pathway and leads to the reduction of amino acids and induction of huge metabolic stress in CML CD34+ cells. Altogether, our study shows that DHODH inhibition is a promising approach for targeting CML stem/progenitor cells and may help more patients discontinue the therapy.

Abstract: This observational, retrospective study investigates the frequency and reasons for ruxolitinib rechallenge, its therapeutic effects, and its impact on overall survival in a cohort of 219 patients with myelofibrosis discontinuing ruxolitinib for ≥14 days and surviving for ≥30 days. In comparison with 159 patients discontinuing ruxolitinib permanently, discontinuation due to a lack/loss of spleen response is lower ( P = .004) in 60 patients in whom ruxolitinib is rechallenged for ≥14 days (RUX‐again patients): there is a significant increase in the number of patients with large splenomegaly and a high Total Symptom Score ( P 〈 .001) between the first ruxolitinib stop and restart, and there is a significant increase in the number of patients with a Total Symptom Score reduction ( P = .01) during the rechallenge. The use of a ruxolitinib dose 〉 10 mg twice daily is associated with spleen improvements ( P = .05) and symptom improvements ( P = .02), and overall survival is significantly longer in RUX‐again patients ( P = .004).

Abstract: In 794 chronic‐phase myelofibrosis patients treated with ruxolitinib, the impact of the baseline percentage of peripheral blasts (PB) on response and outcome was evaluated. Three subgroups were compared: PB‐0 (no PB, 61.3%), PB‐4 (PB 1%‐4%, 33.5%), and PB‐9 (PB 5%‐9%, 5.2%). At 3 and 6 months, spleen responses were less frequently achieved by PB‐4 ( P = .001) and PB‐9 ( P = .004) compared to PB‐0 patients; ruxolitinib discontinuation‐free, leukemia‐free, and overall survival were also worse for PB‐4 and PB‐9 patients ( P = .001, P = .002, and P 〈 .001, respectively).