post-ET MF Research Articles

Development and validation of a model for the early prediction of progression from essential thrombocythemia to post-essential thrombocythemia myelofibrosis: a multicentre retrospective study

Essential thrombocythemia (ET), a myeloproliferative neoplasm (MPN), has a substantial risk of evolving into post-essential thrombocythemia myelofibrosis (post-ET MF). This study aims to establish a prediction nomogram for early prediction of post-ET MF in ET patients. The training cohort comprised 558 patients from 8 haematology centres between January 1, 2010, and May 1, 2023, while the external validation cohort consisted of 165 patients from 6 additional haematology centres between January 1, 2010, and May 1, 2023. Univariable and multivariable Cox regression analysis was performed to identified independent risk factors and establish a nomogram to predict the post-ET MF free survival. Both bias-corrected area under the curve (AUC), calibration curves and concordance index (C-index) were employed to assess the predictive accuracy of the nomogram. Multivariate Cox regression demonstrated that elevated red blood cell distribution width (RDW), elevated levels of lactate dehydrogenase (LDH) and the level of haemoglobin (Hb), a history of smoking and the presence of splenomegaly were independent risk factors for post-ET MF. The C-index displayed of the training and validation cohorts were 0.877 and 0.853. The 5 years, 10 years AUC values in training and external validation cohorts were 0.948, 0.769 and 0.978, 0.804 respectively. Bias-corrected curve is close to the ideal curve and revealed a strong consistency between actual observation and prediction. We developed a nomogram capable of predicting the post-ET MF free survival probability at 5 years and 10 years in ET patients. This tool helps doctors identify patients who need close monitoring and appropriate counselling. This research was funded by the Key R&D Program of Zhejiang (No. 2022C03137); the Public Technology Application Research Program of Zhejiang, China (No. LGF21H080003); and the Zhejiang Medical Association Clinical Medical Research special fund project (No. 2022ZYC-D09).

Do Hematologic Parameters Predict Thrombosis or Disease Transformation in Essential Thrombocythemia?

Background: Patients with essential thrombocythemia (ET) are burdened with an increased risk of thrombosis and disease transformation, both significant contributors to morbidity and mortality. However, based on the available literature, there are limited tools available to predict the development of these complications. Particularly, the association between hematologic parameters and thrombosis is unclear, making risk stratification and disease management challenging. Utilizing a large retrospective database, we assessed the impact of hematocrit (Hct), red blood cell count (RBC), white blood cell count (WBC), and platelet count (Plt) on the subsequent development of thrombosis and disease transformation to myelofibrosis (MF), polycythemia vera (PV), and acute leukemia. Methods: We included ET patients treated at our institution between 2007 and 2021. The index time was date of diagnosis or date of first contact at Mount Sinai Hospital for patients with a prior history of ET. Hematologic parameters (Hct, RBC, WBC, Plt) were documented at three-month intervals after the index time. Patients were included in the study if they had at least three sets of laboratory values prior to the outcome of interest. For each hematologic parameter, we performed univariate cox analysis of time to thrombosis or progression to either MF, PV, or acute leukemia. Each lab parameter was analyzed as a time dependent covariate, taking into account the length of time the patient was above or below the threshold of interest. Hct and plt were specifically evaluated as dichotomous variables, with a cut off of less than or greater than 45% for Hct based on CYTO-PV results. For plt, two cut-off values were used: less than or greater than 400 (based on ELN response criteria) and less than or greater than 600 (based on Cortelazzo et al HU treatment goal) in plt/microliter. Results: We identified 430 patients who met criteria and were included in the analysis. Our cohort of patients was mostly female (61%), non-Hispanic (66%), white (64%), JAK2 mutated (60%), with an average age at diagnosis of 56 years and a median follow-up time of 33 months. 41 (7%) patients had a history of prior thrombosis. There was a total of 23 major thromboses that occurred after the index time with 12 venous and 11 arterial thrombotic events. 38 patients progressed to post ET-MF, 20 patients developed post ET-PV, and 10 patients transformed to acute leukemia. For the assessment of thrombotic risk, there were no significant associations between any of the hematologic parameters and this outcome. We also evaluated the risk of thrombosis in the JAK2 positive subset of patients (n of 15), and did not find any significant association with any of the lab parameters (in particular, for hct < 45%, p > 0.99). For transformation to MF, plt < 400 x10 9/L was associated with an increased risk of the development of post-ET MF (HR 2.2, p 0.016), and the same was seen with plt < 600 x10 9/L (HR 2.2, p 0.044). The development of PV was less likely with a hct lower than 45% (HR 0.03, p < 0.001), with similar results seen for plt < 400 x10 9/L (HR 0.2, p 0.03) and plt < 600 x10 9/L (HR 0.2, p 0.002). For transformation to acute leukemia, plt < 400 x10 9/L (HR 8.3, p 0.008) was associated with an increased risk of this complication. Conclusions: In this retrospective cohort of 430 patients with ET, we found no associations between four different hematologic parameters over time with thromboses. In particular, this data once again argues against aiming to achieve a particular platelet goal to minimize thrombosis in ET patients. The lack of association between hematologic parameters and thrombotic risk was similarly exemplified in our subset of JAK2 mutated ET patients. Pertaining to disease transformation, plt count appeared to be the most predictive out of all of the parameters, with lower plt values corresponding to increased risk of both post-ET MF and acute leukemia, which both warrant further exploration. A hct level of less than 45% appeared to be protective against the development of post-ET PV. Significant limitations of our study include the retrospective nature as well as the small number of events of interest that our patients experienced. Future studies must continue to focus on surrogate endpoint validation in order to more reliably predict thrombotic events as well as disease progression in patients with ET given the significant morbidity and mortality that such events incur.

Type 1/like Calr Mutation in Momelotinib-Treated Patients with Myelofibrosis Is the Most Prominent Predictor of Drug Survival and Longevity without Transplant

Background: Non-transplant treatment options in myelofibrosis MF are palliative in scope and include Janus kinase 2 (JAK2) inhibitors (JAKi). FDA-approved JAKi for the treatment of MF include ruxolitinib, fedratinib, and pacritinib. A fourth JAKi, momelotinib, is currently under review. We have previously reported long-term survival data on momelotinib and drug survival that was significantly longer, retrospectively compared to ruxolitinib ( AJH. 2022;97:E433). In the current study, we focused on molecular predictors of drug survival (i.e., drug retention) and longevity without allogeneic hematopoietic stem cell transplant AHSCT (transplant-censored survival). Methods : The current study included data from Mayo Clinic patients who participated in a phase-1/2 core study of momelotinib (NCT00935987). Study design, drug doses and schedule, and additional details on treatment effect and side effects have previously been published. Drug survival was calculated from time of initiation of momelotinib to time of treatment discontinuation. Survival analysis required censoring of patients at time of AHSCT. Conventional statistical methods were applied using JMP Pro 16.0.0 software (SAS Institute, Cary, NC, USA). Results : 79 JAKi-naïve patients with MF were considered: median age 67 years; males 54%; PMF 65%, post-PV MF 20%, post-ET MF 15%; JAK2 71%, type 1/like CALR ( CALR-1) 14%, CALR-2 3%, MPL 8%, triple-negative 5%. Risk category according to DIPSS was high/intermediate in 80% of the patients and unfavorable karyotype in 29%. Non-driver mutation information was available in a subset of patients and included ASXL1 22% and SRSF2 18%. Median time from diagnosis to study entry was 25.2 months; patients were enrolled between November 2009 and March 2011 with follow-up information updated through July 2023; during this period, 68 (86%) deaths, 13 (16%) leukemic transformations, and 7 (9%) AHSCTs were recorded. Median follow-up for alive patients was 12.5 years (range 11.3-13.1). Anemia response rate was 48% and spleen 47%. Median treatment duration was 25 months (range 0.1-157); the proportion of patients remaining on active therapy was 68% at 1 year, 30% at 3 years, 16% at 5 years, and 7% at 10 years (Figure 1a). At the time of this writing, 5 (6.3%) patients were still receiving momelotinib therapy with treatment durations of 152 to 157 months. In univariate analysis, molecular predictors of drug survival included CALR-1 (p=0.006) but not ASXL1 (p=0.6) or SRSF2 (p=0.3) mutation, or unfavorable karyotype (p=0.08). The 3-, 5-, and 10-year drug survival rates for CALR-1-mutated patients were 64%, 40%, and 18%, respectively, and for CALR-1 wild-type 25%, 11%, and 4% (p=0.01, Figure 1b). CALR-1 independent association with longer drug survival was also appreciated for younger age (p<0.01) and absence of baseline transfusion dependency at time of study entry (p=0.02); all three risk factors remained significant during multivariable analysis with HR (95% CI) rates of 0.4 (0.2-0.87) for CALR-1, 0.5 (0.3-0.89) for age <63 years, and 0.6 (0.3-0.9) for absence of transfusion-dependency. Spleen response (p<0.01) but not anemia response (p=0.23) was also associated with longer drug survival; in multivariable analysis all 4 risk factors retained significance. Multivariable analysis of genetic variables for transplant-censored survival identified absence of CALR-1 (HR 3.5, 95% CI 1.2-10.5; p=0.03) and presence of high molecular risk (HMR; ASXL1/ SRSF2) mutations (HR 2.3, 95% CI 1.1-4.6; p=02) to independently predict inferior survival. Among 11 patients with CALR-1 mutation, median transplant-censored survival was 125 months with 10-year survival rate of 60% (Figure 1c); by contrast, the corresponding figures were 37 months and 12%, in the 68 patients not harboring CALR-1 mutation (Figure 1c). In multivariable analysis, absence of CALR-1 (HR 2.9, 95% CI 1.02-8.1), age ≥63 years (HR 3.0, 95% CI 1.5-6.0), and absence of spleen response (HR 2.6, 95% CI 1.5-4.5) were identified as being most significant in predicting inferior transplant-censored survival. Conclusions : CALR-1 mutation is the most important risk factor for both drug survival and longevity without AHSCT, in momelotinib-treated patients with MF. Such information might help identify MF patients who might benefit from treatment with momelotinib and in whom AHSCT might be deferred or not.

The Upregulation of the FBLN1 and HS3ST4 Genes May Represent a Potential Mechanism Driving the Fibrotic Progression in Primary Myelofibrosis and Leading to Adverse Prognosis

Myelofibrosis is a group of diseases characterized by increased proliferation of bone marrow fibrous connective tissue and a decrease in hematopoietic cells, leading to impaired bone marrow hematopoietic function. Myelofibrosis includes primary myelofibrosis（PMF）, myelofibrosis secondary to polycythemia vera（post-PV MF）, and myelofibrosis secondary to essential thrombocythemia（post-ET MF）. Studies suggest that certain genes may be responsible for driving the development of myelofibrosis and causing adverse prognosis. By conducting RNA-Seq analysis, we investigated the differentially expressed genes and their impact on prognosis in primary myelofibrosis and secondary myelofibrosis. Furthermore, we analyzed the potential mechanisms driving the progression of fibrosis. Additionally, we performed a comparative analysis of the clinical features among various types of myelofibrosis. In this study, we performed transcriptome sequencing to analyze gene expression differences in 14 overt-PMF samples, 5 post-ET MF samples, and 3 post-PV MF samples.Functional and pathway analyses were conducted on these differentially expressed genes, and cluster heatmaps were used to visualize their expression patterns.Simultaneously, we performed a comparative analysis of clinical characteristics among 370 overt-PMF patients, 96 post-ET MF patients, and 52 post-PV MF patients.The study revealed that compared to post-PV MF, overt-PMF showed 81 upregulated and 2 downregulated differentially expressed genes, while compared to post-ET MF, overt-PMF displayed 19 upregulated and 3 downregulated differentially expressed genes (Figure a) (Figure b). Based on the similarity of gene expression profiles among the samples, a clustering analysis was performed (Figure c).The results demonstrated that in overt-PMF, the FBLN1 gene was significantly upregulated compared to post-PV MF and was enriched in pathways related to extracellular matrix formation and cell adhesion among the differentially expressed genes (Figure d). Additionally, the HS3ST4 gene was significantly upregulated in overt-PMF compared to post-ET MF and was enriched in pathways related to interleukin-8 generation and regulation of cytokine production involved in inflammatory responses.In the analysis of clinical characteristics among different types of myelofibrosis patients, it was observed that overt-PMF was more prone to transform into acute leukemia compared to post-PV MF, had a higher tendency to develop transfusion dependency (Figure e), and exhibited lower hemoglobin levels and hematocrit at the time of diagnosis (Figure f). Moreover, compared to post-ET MF, overt-PMF had a higher likelihood of transforming into acute leukemia and lower platelet counts at the time of diagnosis. The research findings indicate that the expressions of FBLN1 and HS3ST4 genes are significantly upregulated in overt-PMF, and patients with overt-PMF have a worse prognosis compared to those with post-PV MF and post-ET MF. Previous studies have shown that FBLN1 is involved in extracellular matrix assembly, promoting the formation and stabilization of collagen fibers. It can also interact with integrin receptors and other cell adhesion molecules, participating in cell-extracellular matrix signaling processes. On the other hand, HS3ST4 is involved in processes such as cell adhesion, cell migration, and cell signaling. Based on these findings, it is speculated that the upregulation of FBLN1 and HS3ST4 gene expressions may potentially drive the progression of fibrosis and lead to an adverse prognosis in patients with myelofibrosis. Acknowledgement: This research was funded by the Key R&D Program of Zhejiang, No. 2022C03137; Public Technology Application Research Program of Zhejiang, China, No. LGF21H080003; Zhejiang Medical Association Clinical Medical Research special fund project, No. 2022ZYC-D09. Correspondence to: Dr Jian Huang, Department of Hematology, The First Affiliated Hospital of Zhejiang University School of Medicine. No. 79 Qingchun Road. Hangzhou, Zhejiang, PR China. househuang@zju.edu.cn

Internal and External Validation of the Predictive Scoring Systems from Essential Thrombocythemia to Post-ET Myelofibrosis

Essential thrombocythemia (ET) is one of the myeloproliferative neoplasms with a high risk of progression to post-ET myelofibrosis (post-ET MF). However, there still lacked the prediction model of post-ET MF. Here, we conducted a study of 708 ET patients identified by 2016 WHO diagnostic criteria in 13 hematology centers between 01 January 2010 to 01 July 2023, aimed to identify risk factors progression to MF and develop a prediction nomogram for post-ET MF(ChiCTR2200061208). The training cohort included 543 ET patients with ET from 8 hematology centers. The external validation cohort included 165 subjects from another 5 hematology centers. Univariable and multivariable analysis identified the red blood cell distribution width (RDW) level (P=0.002), lactic dehydrogenase (LDH) level (P=0.003), smoking(P=0.003) and splenomegaly (P=0.000) were independent risk factors of post-ET MF(Table A). These independent risk factors were included to develop a prediction model of nomogram (Figure B). The bias-corrected AUC were both high in the training cohort (AUC=0.899) and external validation cohort (AUC=0.892). Analysis of calibration curves showed that the apparent and bias-corrected lines were overlapped with the ideal line in both training and external validation cohorts, and the Hosmer−Leeshawn tests showed that the model were a good fit (P = 0.73 in training cohort, P=0.43 in external validation cohort) (Figure C, D). Decision curve analysis(DCA) was conducted to assess the clinical usefulness of nomogram by quantifying the net benefits at defferent threshold probability of post-ET MF. Threshold probabilities for the standardized net benefit nomogram in predicting the probability of the occurrence of post-ET MF ranged from 0.02 to 0.87 in the training cohort, 0.01 to 1 in the external validation cohort. In conclusion, we developed a nomogram that can predict the risk of progression to MF in ET patients, which can help clinicians adjust therapy timely to prevent disease progression to a great degree. Acknowledgement: This research was funded by the Key R&D Program of Zhejiang, No. 2022C03137; Public Technology Application Research Program of Zhejiang, China, No. LGF21H080003; Zhejiang Medical Association Clinical Medical Research special fund project, No. 2022ZYC-D09. Correspondence to: Dr Jian Huang, Department of Hematology, The First Affiliated Hospital of Zhejiang University School of Medicine. No. 79 Qingchun Road. Hangzhou, Zhejiang, PR China.

First-in-Class JAK/Rock Inhibitor Rovadicitinib in Myeloproliferative Neoplasms: A Single Arm, Multicenter, Open-Label, Phase I/Ib Study

Introduction: Rovadicitinib (TQ05105) is a first-in-class, oral, small molecule JAK/ROCK inhibitor which inhibits cell proliferation, induces cell apoptosis and decreases inflammatory cytokines by affecting the JAK-STAT signaling pathway in preclinical studies. Here we report the primary results of the phase I/Ib study of rovadicitinib in Myeloproliferative Neoplasms (MPN) patients. Methods: We conducted a single arm, multicenter, open-label, phase I/Ib trial (NCT04339400) at 9 sites in China. The study included dose-escalation cohort (phase I) and dose-expansion cohort (phase Ib). Aged ≥18 with MPN, dynamic international prognostic scoring system (DIPSS) Int-1 or higher risk of MF, naïve or resistant/intolerant to hydroxyurea and/or interferon therapy of PV or ET, were enrolled. Patients with MF must have palpable splenomegaly (≥5 cm below the left costal margin). Eligible patients received escalating doses of oral rovadicitinib 5 mg QD, 5 mg BID,10 mg BID, 15 mg BID, 20 mg BID of each 28-day cycle, and a modified “3 + 3” design was used in phase I. Treatment continued until progressive disease or unacceptable toxicity. The primary outcomes were the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) in phase I, and the proportion of patients whose reduction of at least 35% in spleen volume (SVR35) at week 24 compared with baseline in phase Ib. The main secondary end point was proportion of patients whose Total Symptom Score decreased ≥ 50% (TSS50) at week 24 in phase Ib. Results: Between November 20, 2018, and September 22, 2022, 102 patients were assessed for eligibility across two cohorts, 79 were enrolled and treated with rovadicitinib including, 15 patients in t phase I and 64 in phase Ib. 50 (63%) patients were male. The median age was 59 years (IQR 50-65), and 16 (20%) patients were aged 65 years or older. All patients had a definite diagnosis during the screening period, 64 (81%) patients were MF (55 were PMF, five were post-PV MF, four were post-ET MF), 10 (13%) patients were ET, five (6%) patients were PV. Mutations of JAK2, CALR, and MPL were analyzed, 60 (76%) of 79 patients with JAK2 V617F mutations, 10 (13%) patients with CALR mutations, four (5%) patients with MPL mutations. The median spleen volume was 1907 cm 3 (IQR 964-2994). In phase I, the number of patients enrolled per dose level was one at 5 mg QD, three at 5 mg BID, four at 10 mg BID, three at 15 mg BID, and four at 20 mg BID. The dose-limiting toxicities (DLTs) were experienced by two patients at 20 mg BID (one patient experienced grade 3 platelet count decrease with bleeding, one patient experienced grade 4 platelet count decrease). Given the safety profile and shrinking spleen, 15 mg BID was identified as MTD and RP2D. Rovadicitinib plasma peak concentrations and areas under the concentration versus time curve increased proportionally with dose. Terminal half-life was 1.3 h. No accumulation trend was noted. In phase Ib, 58 patients were included spleen volume set, 56 patients were included TSS set. 63.79% (37/58) patients achieved SVR35 during the study period (Figure 1A). The proportion of patients who had SVR35 was 37.93% (22/58) at week 24 and 39.66% (23/58) at week 48, respectively. The mean duration of splenic response was approximately 16 months. 87.5% (49/56) patients achieved TSS50 during the study period. The proportion of patients who had TSS50 was 71.43% (40/56) at week 24 and 48.21% (27/56) at week 48, respectively. Patients had durable splenic responses and significant symptom responses. The most common treatment-emergent adverse events (TEAEs) were 1~2 grade (Table 1). Grade ≥3 TEAEs were reported in 39 (49%) patients, the most common grade ≥3 hematological TEAE (≥10%) were platelet count decrease (17.72%) and anemia (16.46%). Occurrence of other grade ≥3 non-hematologic TEAEs was very low. There was only one death, due to disease progression and not related to rovadicitinib. TEAEs were generally manageable. Conclusions: Rovadicitinib was generally safe, well-tolerated and showed meaningful clinical activity in patients with MF, especially with palpable splenomegaly. Rovadicitinib may be a new treatment option for myelofibrosis patients. Furthermore, a randomized double-blind phase 2 study is ongoing, aiming to assess the efficacy and safety of rovadicitinib compared to hydroxyurea in patients with intermediate-2 or high-risk myelofibrosis in China (NCT05020652).

The Efficacy and Safety of Selinexor in Combination with Ruxolitinib in Ruxolitinib-Treated Myelofibrosis Patients： the Interim Analysis of a Prospective, Open-Label, Multicenter, Parallel-Cohort, Phase 2 Study

Background: Myelofibrosis is a chronic hematologic malignancy characterized by constitutional symptoms, bone marrow fibrosis, extramedullary hematopoiesis resulting in splenomegaly and a propensity toward leukemic progression. Although JAK inhibitors have improved patients' splenomegaly and symptom burden, the clinical management of MF is still challenging for its limited treatment options, especially when patients become less responsive or intolerable to Ruxolitinib (RUX) treatment. Selinexor (SEL) is a novel, first-in-class, oral selective inhibitor of nuclear export agent that binds the karyopherin protein exportin 1 (XPO1). Preclinical studies showed SEL decreased viable cells and colony formation both in newly diagnosed and RUX-exposed MF cells. ESSENTIAL trial showed SEL's sustained spleen responses as a single agent in JAKi refractory MF. Besides, a phase I study reported, with SEL plus RUX in treatment-Naïve MF, 92% patients(pts) achieved SVR35, 69% had symptom response and 65% had stable or improved Hgb levels. We have presented promising efficacy and safety data in real-world setting of SEL plus RUX regimen in MF patients in 2023 EHA. Here we are to report the efficacy and safety of the regimen in RUX-treated patients in a prospective clinical trial. Methods: This is a prospective, open-label, multi-center, parallel-cohort phase 2 study, examining SEL plus RUX regimen in RUX-treated MF pts. The study includes MF pts with suboptimal RUX responses or intolerable to RUX treatment. Pts are given SEL 40mg or 60mg QW plus RUX (dosage per investigator judgement, RUX intolerable pts will receive reduced dose of RUX). A total of 40 pts will be included into the study. The primary endpoint is spleen response assessed by palpation or CT/MRI scan per IWG-MRT and ELN response criteria. Key secondary endpoints include anemia response, symptom response defined as symptom resolution or alleviation according to TSS or physician's judgement, and safety. Results: Characteristics As of July 2023, 28 pts had received at least one dose of oral weekly SEL, including 12 suboptimal RUX responses pts and 16 RUX intolerable pts. The median age was 65 years old. Mean duration of prior RUX treatment was 24.5 months (range: 4-77). 18 pts had primary MF, 4 had post-PV MF, and 6 had post-ET MF. Median time from MF diagnosis to study enrollment was 5 years (0.25-29). JAK2, CALR and MPL mutations were present in 18 (64.28%), 5 (17.85%) and 1 (3.57%) pts respectively. All pts presented constitutional symptoms, 25 (89.29%) had splenomegaly, and 7 (25.00%) had packed red blood cell (PRBC) transfusion-dependent anemia. DIPSS risk category was intermediate 23 (82.14%), high risk 3 (10.71%) and unknown 2 (7.14%). Median treatment duration was 134 days (14-525) (Table 1). 15 pts (53.57%) discontinued SEL. Reasons for treatment discontinuation were death in 4 pts (3 from covid-19, 1 sepsis), participation in clinical trial in 2 pts, unsatisfactory response in 2, transplantation in 1, toxicity in 2, economic burden in 1 patient and lost to follow-up in 3. Efficacy Analysis The analysis of spleen response included 21 pts with spleen length assessment data by either palpation or CT/MRI. Spleen response is defined as length reduction more than 50% per IWG-MRT and ELN response criteria. 15 (71.42%) pts had a reduction in spleen volume and 8 (38.09%) achieved spleen response ( Figure 1). For symptom response, 27 pts are with available data. 23 (85.18%) pts had symptom alleviation, 9 (33.33%) achieved ≥50% reduction in TSS or symptom response assessed by physician, of which 1 achieved symptom resolution. There are 12 pts with PRBC transfusion-independent anemia (Hgb≤100g/L) at baseline and had received study treatment for at least 28 days. 10 (83.33%) pts had maintained stable or improved Hgb levels at the last follow up. 5/7 (71.42%) PRBC transfusion-dependent pts became transfusion independent (1 pt) or had reduced transfusion frequency (4 pts), and 2 pts remained stable. Safety The most common TEAEs were nausea 14 (50.00%), vomiting 9 (32.14%), decreased appetite 8 (28.57%), and anemia 7 (25.00%). The most frequent TEAEs ≥ Grade 3 were anemia 3(10.71%) and thrombocytopenia 2(7.14%). Conclusion: In this preliminary analysis, the combination of SEL and RUX showed encouraging efficacy and manageable safety in RUX-treated pts with myelofibrosis.

Recent advances in therapies for primary myelofibrosis.

Primary myelofibrosis (PMF), polycythemia vera (PV) and essential thrombocythemia (ET) form the classical BCR-ABL1-negative myeloproliferative neoplasms (MPNs) that are driven by a constitutive activation of JAK2 signaling. PMF as well as secondary MF (post-ET and post-PV MF) are the most aggressive MPNs. Presently, there is no curative treatment, except allogenic hematopoietic stem cell transplantation. JAK inhibitors, essentially ruxolitinib, are the therapy of reference for intermediate and high-risk MF. However, presently the current JAK inhibitors behave mainly as anti-inflammatory drugs, improving general symptoms and spleen size without major impact on disease progression. A better understanding of the genetics of MF, the biology of its leukemic stem cells (LSCs), the mechanisms of fibrosis and of cytopenia and the role of inflammatory cytokines has led to new approaches with the development of numerous therapeutic agents that target epigenetic regulation, telomerase, apoptosis, cell cycle, cytokines and signaling. Furthermore, the use of a new less toxic form of interferon-α has been revived, as it is presently one of the only molecules that targets the mutated clone. These new approaches have different aims: (a) to provide alternative therapy to JAK inhibition; (b) to correct cytopenia; and (c) to inhibit fibrosis development. However, the main important goal is to find new disease modifier treatments, which will profoundly modify the progression of the disease without major toxicity. Presently the most promising approaches consist of the inhibition of telomerase and the combination of JAK2 inhibitors (ruxolitinib) with either a BCL2/BCL-xL or BET inhibitor. Yet, the most straightforward future approaches can be considered to be the development of and/or selective inhibition of JAK2V617F and the targeting MPL and calreticulin mutants by immunotherapy. It can be expected that the therapy of MF will be significantly improved in the coming years.

Introduction and Genetic Correction of Calreticulin Mutations in Human Hematopoietic Stem and Progenitor Cells Sheds Light on MPN Pathogenesis

Introduction:Recurrent mutations in calreticulin (CALR) are present in 70% to 80% of essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients without a JAK2 or MPL mutation. Despite recent advances in understanding mutant CALR, the detailed mechanisms are not fully elucidated, and current knowledge is mainly based on transgenic mouse models or human cancer cell lines. Thus, to more faithfully model MPN pathogenesis, we first aimed to introduce heterozygous type-1 and type-2 CALR mutations into healthy human hematopoietic stem and progenitor cells (HSPCs) via targeted CRISPR/Cas9-mediated gene knock-in (KI) and investigate its impact on HSPC function in vitro and in vivo. Second, we aimed to correct CALR mutations in patient-derived HSPCs to study their dependence on the initial driver event to exert an MPN phenotype.Methods:We used CRISPR/Cas9 to introduce heterozygous CALR mutations into the endogenous gene locus of healthy cord blood-derived HSPCs. Our approach is based on homologous recombination using DNA repair templates delivered by adeno-associated virus serotype 6 (AAV6). Briefly, Cas9-sgRNA ribonucleoprotein (RNP) was used to cut the DNA. Simultaneously AAV6, carrying either a mutation-bearing or a wildtype control cDNA, was co-delivered to allow for targeted in-frame integration. This way, mutant CALR remains under the control of the endogenous promoter. Concurrent integration of a fluorescent reporter downstream of the mutated exon, enabled purification and tracking of modified cells via flow cytometry. Purified CRISPR-modified HSPCs were used for in vitro collagen-based colony-forming assays, proliferation and differentiation assays in liquid culture, and intrafemoral transplantation into immunodeficient NSG mice to assess their pathogenic potential.Results:Our CRISPR/Cas9 KI strategy enabled us to efficiently generate and enrich for heterozygous CALR mutant human HSPCs. Modified cells harbor the mutation at the endogenous CALR locus with intact gene regulatory regions. Correct integration and transcript expression were confirmed on DNA and RNA level by sanger sequencing. Additionally, CALR mutant protein expression was confirmed via immunohistochemistry using a diagnostically approved mutant-specific antibody.Type-1 and type-2 CALR mutations led to TPO-independent growth of CD34 + HSPC-derived cells and a two-fold (p<0.01) increase of megakaryocyte colonies in collagen-based media compared to wildtype control KI. These findings were corroborated by significantly enhanced CD41 + CD42b + megakaryocyte formation of CALR mutant HSPCs upon liquid culture differentiation.When transplanted into sublethally irradiated immunodeficient NSG mice, CALR mutant HSPCs showed robust engraftment in the bone marrow with a myeloid lineage skewing, outcompetition of wildtype cells and increased formation of CALR mutant CD41 + megakaryocyte progenitors.To investigate, if removal of type-1 and type-2 CALR mutations can ameliorate MPNs, we utilized our KI strategy to correct both CALR mutations in MPN patient-derived HSPCs by replacing them with wildtype sequences. A successful correction was confirmed on DNA and RNA level and by the absence of mutant CALR protein. Opposite to the results from introducing CALR mutations, correcting the mutations led to a two-fold decrease in megakaryocyte colony formation. Interestingly this was only seen in ET and post-ET MF samples, whereas primary MF samples were unaffected, underscoring the importance of other secondary genetic driver events in the pathogenesis of primary MF.Conclusion:Our system allows us to investigate human MPN pathogenesis prospectively and shed light on the transforming mechanisms of mutant CALR in primary HSPCs. We could show that CALR mutations prime HSPCs toward the formation of platelet-producing megakaryocytes. Genetic correction of CALR mutations in MPN patient-derived HSPCs revealed a dependence on the oncogenic mutant CALR driver event in ET and post-ET MF patients, opening the possibility of an ex vivo gene correction approach to remove mutant CALR in patient-derived HSPCs . Lastly, since MPN patient-derived cells have notoriously low engraftment potential in mice, our CRISPR/Cas9-engineered CALR mutant model also provides a powerful new strategy to generate MPN xenotransplants with defined genotypes for the evaluation of novel therapies. DisclosuresGreinix: Celgene: Consultancy; Therakos: Consultancy; Takeda: Consultancy; Sanofi: Consultancy; Novartis: Consultancy. Sill: Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees. Zebisch: Novartis: Consultancy; AbbVie: Consultancy; Celgene: Consultancy, Honoraria. Reinisch: Celgene: Research Funding; Pfizer: Consultancy.

Leukemic Transformation of Post-Essential Thrombocythemia Myelofibrosis: A Unique Case Presenting with Double MPL and CALR Mutations

Leukemic Transformation of Post-Essential Thrombocythemia Myelofibrosis: A Unique Case Presenting with Double MPL and CALR Mutations

Effects of Tamoxifen on the Mutant Allele Burden and Disease Course in Patients with Myeloproliferative Neoplasms - Results of the Tamarin Study

Effects of Tamoxifen on the Mutant Allele Burden and Disease Course in Patients with Myeloproliferative Neoplasms - Results of the Tamarin Study

Pathological characteristics of megakaryocytes in myeloproliferative neoplasms and their correlation with driver gene mutations

目的探讨骨髓增殖性肿瘤（MPN）巨核细胞病理特征及其与起始基因突变的相关性。方法收集2012年2月至2017年10月于中国医学科学院血液病医院就诊的160例初诊MPN患者，根据世界卫生组织（WHO）2016年MPN诊断标准对患者骨髓活检组织切片进行重新评估。结果160例患者中男72例（45.0％），女88例（55.0％），中位年龄59（13～87）岁。重新评估后真性红细胞增多症（PV）39例，原发性血小板增多症（ET）33例，纤维化前/早期原发性骨髓纤维化（pre-PMF）37例，明显期原发性骨髓纤维化（overt PMF）37例，真性红细胞增多症后骨髓纤维化（post-PV MF）2例，原发性血小板增多症后骨髓纤维化（post-ET MF）1例，MPN-未分类（MPN-U）11例。按PV、ET、pre-PMF及overt PMF疾病亚型顺序，密集成簇分布、少分叶核及裸核巨核细胞逐渐增加，红系增生程度正常及增高的比例逐渐降低，1级及以上网状纤维、胶原及骨硬化的比例逐渐升高。相关性分析示密集成簇分布、少分叶核及裸核巨核细胞占比与红系增生程度呈负相关，与粒系增生程度及纤维化程度呈正相关。对pre-PMF及overt PMF患者病理特征的相关性分析示密集成簇分布及裸核巨核细胞占比与纤维化程度呈正相关。JAK2V617F突变MPN患者红系增生程度明显增高（P＝0.022），CALR突变MPN患者疏松成簇、密集成簇、骨小梁旁分布及裸核巨核细胞明显增多（P＝0.055，P＝0.002，P＝0.018，P＝0.008），1级及以上网状纤维、胶原及骨硬化比例增高（P＝0.003，P<0.001，P＝0.001）。伴JAK2V617F突变pre-PMF及overt PMF患者骨髓增生程度较高（P＝0.037），伴CALR突变患者巨大体积及密集成簇分布巨核细胞明显增多（P＝0.059，P＝0.055），1级及以上胶原及骨硬化的比例明显增高（P＝0.011，P＝0.046）。结论不同亚型MPN患者骨髓巨核细胞病理改变特征各异，其病理异常特征与纤维化水平相关。CALR突变可能与巨核细胞病理异常特征及骨髓纤维化水平相关。

Determinants of Long-Term Outcome in Type 1/like Calreticulin-Mutated Myelofibrosis

Background:Type 1 calreticulin (CALR) variants comprise ~70% of all CALR mutations in primary myelofibrosis (PMF) and form a distinct phenotypic and prognostic disease subset (Leukemia. 2014;28:1568). Determinants of long-term outcome have not, however, been systematically appraised in this population. The current study documents the natural history, molecular correlates, and independent predictors of overall (OS), leukemia-free (LFS), and thrombosis-free (TFS) survival in CALR type 1/like-mutated myelofibrosis.Methods:Patients were recruited from the Mayo Clinic, Rochester, MN, USA. Diagnoses were consistent with World Health Organization (PMF, fibrotic/leukemic transformations) (Blood. 2016;127:2391) and International Working Group for Myeloproliferative Neoplasms Research and Treatment criteria (post-essential thrombocythemia (ET) MF) (Leukemia. 2008;22:437). Laboratory and clinical data were retrospectively abstracted corresponding to time of referral (PMF) or myelofibrotic transformation (post-ET MF). Conventional prognostic scoring was as previously outlined (Blood. 2010;115:1703; J Clin Oncol. 2018;36:1769). Recipients of allogeneic stem cell transplant were censored at the time of transplant. Standard statistical methods were used for all analyses using the JMP® Pro 13.0.0 software package (SAS Institute, Cary, NC, USA).Results:A total of 162 consecutive patients with CALR type 1/like-mutated myelofibrosis were identified: 139 (86%) with PMF and 23 (14%) with post-ET MF with median age 55 years (range 23-85), 62% male. The PMF and post-ET MF cohorts displayed similar phenotypic features with the exception of higher platelet counts (median 443 vs 340 x 109/l; P=0.02) in post-ET MF (Table 1). The most frequent co-existing mutations were ASXL1 (n=47; 37%) and SRSF2 (n=4; 3%), with ASXL1 seen more frequently in PMF (39% vs 11% post-ET MF; P=0.07). Over a median follow-up of 6 years (range 0-25 years), a total of 20 (12%) leukemic transformations and 66 (41%) deaths were recorded, with no significant differences between the PMF and post-ET MF cohorts (P=0.16 and 0.11, respectively). Kaplan-Meier survival estimates revealed comparably favorable median OS in both CALR type 1/like-mutated variants: not yet reached and 13 years in post-ET MF vs PMF, respectively (P=0.7) (Figure 1A).Multivariable analysis disclosed moderate to severe sex-adjusted anemia (P<0.001), ≥2% circulating blasts (P<0.001), very high risk (VHR) karyotype (P<0.001), age >70 years (P=0.006), and constitutional symptoms (P=0.008) to be independent predictors of inferior OS in CALR type 1/like-mutated PMF (Table 2). LFS was significantly shortened in the presence of IDH1 mutations (P=0.01) and platelets <100 x 109/l (P=0.02) while IDH2 mutations (P=0.02), leukocytosis ≥11 x 109/l (P=0.03) and history of arterial thrombosis (P=0.04) were independent predictors of shortened TFS. Importantly, myelofibrosis variant (primary vs post-ET) did not influence survival (P=0.7) or complication rates (P=0.8 for LFS, P=0.09 for TFS) (Table 2).The karyotype- and mutation-enhanced international prognostic scoring system (MIPSS70+ version 2.0), was effective in risk stratifying type 1/like CALR-mutated PMF (P-values 0.01 to <0.0001), with the exception of low vs intermediate (P=0.22) and intermediate vs high risk (P=0.49) (Figure 1C). The detrimental influences of unfavorable/VHR karyotype and ASXL1 mutations were confirmed (Figure 2A-B) while borderline adverse and prognostically neutral effects were seen on OS for U2AF1 (n=116; P=0.05) and SRSF2 (n=120; P=0.98) respectively (Figure 2C-D).Conclusions:The current study documents analogous disease patterns in primary and post-ET CALR type 1/like-mutated MF and provides information on determinants of long-term survival. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Driver Mutation Profile By MLPA in Myelofibrosis Patients

Myelofibrosis is the rarest and most severe Ph- myeloproliferative neoplasm and can present de novo or post Polycythemia Vera or Essential Thrombocythemia. It is characterized by bone marrow fibrosis, extramedullary hematopoiesis and abnormal expression of inflammatory cytokines resulting in several atypical events and may progress to Acute Leukemia. The disease arises from clonal expansion of a single hematopoietic stem cell (HSC), driven by a somatic mutation of JAK2, CALR or MPL genes combined with dysregulation of hematopoietic microenvironment, additional mutations and cytogenetic abnormalities.The aims of this study are to assess driver mutations status in primary or secondary myelofibrosis patients and to correlate their mutational profile with clinical outcomes. The search for JAK2V617F, exon 12 JAK2, calreticulin exon 9 c.1092_1143del52 and c.1154_1155insTTGT, MPLW515K and MPLW515L mutations was performed in 31 subjects using MLPA technique, a method of DNA analysis that allows simultaneous appraisal of different mutations in multiple samples. 48.4% of patients present the JAK2V617F mutation,indel CALR mutations in 38.7% of patients (of these, 66.7% with del52 bp, 33.3% harbored insTTGTC),MPL W515L in 3.2% of patients and 9.7% of patients were triple-negative. From the mutational profile information obtained by MLPA,the clinical-molecular risk score was calculated for each of the individuals in the sample, according Rumi et al.. Being at 12,9% (4) as very low risk, 9.7% (3) as low risk,35.4% (11) as intermediary risk, 29.1% (9) and of high risk, and 12.9%(4) as very high risk. Patients with mutated JAK2 were older, with minor degree of anemia and more leukocytosis, whereas those with CALR mutations had less frequency of leukocytosis and thrombocytopenia. Triple-negative subjects displayed the lowest median age at diagnosis (49.3 years), and bone marrow failure phenotype, similar to Myelodysplastic Syndrome. Risk stratification provided by DIPSS was similar to other centers.Individuals with PMF present constitutional symptoms significantly more often than those with post-ET MF(p = 0.0365).Mortality rate was 29%, and mean survival after diagnosis was 68.3 months. CALR mutated individuals presented higher average survival and median survival according to DIPSS was higher than predicted by the prognostic model, possibly due to the higher frequency of CALR mutations reported.Median follow-up time was 32 months (ranging from 10 months to 13 years).Thromboembolic phenomena were recorded in 19.3% of patients, and evolution to AML in 6.4% of patients and it was verified that 75% of the individuals with Myelofibrosis Post-ET presented thrombotic events at some point in the disease. The association between the DIPSS clinical-laboratory parameters and the demand of transfusion at diagnosis, with the occurrence of Acute Leukemia was assessed using Fisher's exact test but have no significant difference in these parameters between patients who evolved or not for Acute Leukemia. The JAK2 V617F mutation is expected to be present in 60 to 65% of individuals with Myelofibrosis, but this mutation has been identified in only 48% of patients. In contrast, the observed frequency of indel of the CALR, of 38%, was higher than the classically described, from 25 to 30%. The rate of mutation type 2 (ins 5-bp) was also higher than expected, 33.6%.Regarding the mutation subtypes of CALR, mutation type 1 (52-bp deletion) is observed in up to 80% of MFP cases, but has a similar frequency as type 2 (5-bp insertion) in patients with ET. The grouping of patients with primary MF and post-ET may have contributed to the higher incidence of type 2 CALR mutation observed in this sample, although much higher than the 13% frequency described in the literature in a mixed population. The type 1 mutation was observed in 66% of our patients with post-ET and mutated CALR, but the small number of individuals in the study does not allow to estimate the impact of this mutation in the evolution of the disease. Our fraction of CALR-mutated patients is much higher than those described in Asian, European, North American and Argentinean populations. The complex genetic landscape involved in initiation and progression of Myelofibrosis, instigates the adoption of integrative prognostic stratification models. In this scenario, MLPA is a powerful tool for molecular study, and a promising ally for MPN molecular characterization. DisclosuresNo relevant conflicts of interest to declare.

PD-1-Mediated T Cell Exhaustion Is Prevalent Among Patients with MPN-Associated Myelofibrosis Independent of JAK1/2 Inhibition

Background: Primary myelofibrosis (PMF), post-polycythemia vera MF (post-PV MF) and post-essential thrombocythemia MF (post-ET MF) are characterized by expansion of the neoplastic clone and by progressive bone marrow (BM) fibrosis. Like in other hematologic malignancies, in most patients with MF the immune system is significantly deregulated: MF patients' plasma cytokine and chemokine levels are markedly increased and their normal T cell subset distribution is significantly altered. Although treatment with the Janus kinase (JAK)-1/2 inhibitor ruxolitinib significantly decreases cytokine/chemokine levels, reduces spleen size, and improves symptoms and quality of life, it does not reverse BM fibrosis nor does it halt the propagation of the neoplastic clone. The T cell immune checkpoint programmed cell death protein-1 (PD-1) promotes immune tolerance by binding to the tumor's cell surface PD-1 ligand (PD-L1). Whereas the importance of T cell-mediated immune tolerance in MF has been documented and trials evaluating clinical benefits of PD1/PD-L1 checkpoint inhibition are ongoing, little is known about the effect of ruxolitinib on PD-1 expression in T cell subsets. Therefore we systematically analyzed MF patients circulating T cells' surface marker expression prior to and during ruxolitinib treatment.Methods: Peripheral blood cells were obtained from well-characterized PMF, post-PV MF and post-ET MF patients prior to and during the course of treatment with ruxolitinib (n=47) and, as control, from age-matched healthy donors (n=28). The proportion of PD-1-expressing CD4+ and CD8+ cells was assessed using multiparameter flow cytometry. Naïve, central memory, effector memory, and effector T cell subsets were defined based on CD45RO and CD27 cell surface antigen expression.Results: A significantly high number of circulating T cells co-expressing CD4+/PD-1+ and CD8+/PD-1+ was found in MF patients compared to age-matched healthy individuals (5.3±4.1% vs. 3.4±1.7%, P=0.028; 7.1±4.4% vs. 3.8±2.3%, P=0.001). Whereas MF patients' naïve T cells harbored an increased number of cells co-expressing CD8+/PD-1+ (P=0.007), but not CD4+/PD-1+, their T central memory cells had a high proportion of cells co-expressing CD4+/PD-1+ and CD8+/PD-1+ (P<0.001; P<0.001). Similarly, a high proportion of circulating PD-1+ T effector memory cells (P<0.001; P<0.001), and T effector cells (P=0.013; P<0.001) was found in MF patients compared to the same cell subsets in healthy age-matched individuals. The proportions of PD-1+ T cells significantly correlated with LDH level and DIPSS score (CD4+ T cells), monocyte count (CD8+ T cells), and total leukocyte count and spleen size (both subsets). Remarkably, the percentage of PD-1+ cells within naïve and central memory CD8+ T cell populations was significantly higher in MF patients with circulating blasts (P=0.036). To determine the effects of ruxolitinib administration, we performed repeated flow cytometry analyses on MF patients' T cells prior to and during treatment (median duration: 4.3 years). Overall, no significant change in PD-1 expression levels in any of the different T cell subsets was detected over the entire treatment period. However, a significant reduction in percentage of cells co-expressing CD4+/PD-1+ and CD8+/ PD-1+ compared to treatment baseline (4.4±0.4% vs. 7.6±2.0%, P=0.011; 6.3±0.6% vs. 10.4±2.7%, P=0.021) was found in patients whose spleen size was reduced after 6 months of treatment.Conclusions: In patients with MF, circulating T cells express high levels of PD-1. While not restricted to a particular stage of T cell differentiation, the correlation between PD-1-expressing T cells and distinct clinical parameters suggests that increased PD-1 levels might induce immune exhaustion in T cell subsets in different ways. Although ruxolitinib significantly inhibits the JAK1/2 signaling pathway in a variety of hematopoietic cells, thereby lowering cytokine/chemokine levels in almost all MF patients, treatment with ruxolitinib did not affect PD-1 expression nor did it alter its distribution among the T cell subsets. Yet, the proportion of PD-1-expressing CD4+ and CD8+ cells was markedly reduced in patients who experienced a superior response to ruxolitinib as assessed by significant spleen size reduction. How disease burden and MF microenvironment affect PD-1 expression in T cells of patients with MF warrants further investigation. DisclosuresVerstovsek:Incyte: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; Italfarmaco: Membership on an entity's Board of Directors or advisory committees.

Real-World Risk Assessment and Treatment of Patients with Myelofibrosis at Community Oncology Practices in the United States

Real-World Risk Assessment and Treatment of Patients with Myelofibrosis at Community Oncology Practices in the United States

RAS Pathway Mutations Are Associated with Proliferative Features and Frequently Co-Occur with TET2 mutationsin Philadelphia Negative MPN Subtypes

Introduction: The Philadelphia-chromosome negative myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell disorders, which include polycythemia vera, essential thrombocythemia and myelofibrosis. These disorders are characterized by activation of the JAK-STAT pathway via somatic mutations in JAK2, MPL, and CALR. However, a number of recurrent somatic mutations outside the JAK-STAT pathway have been described. Many of these mutations, such as ASXL1 and EZH2, are know to confer risk for disease progression. Other mutations, such as TET2 mutations, appear to alter the biology of disease in preclinical and clinical studies. Thus, further investigation of co-occurring mutation events is important to further the understanding of disease biology. Recurrent activating mutations in the RAS signaling pathway have been described in patients with MPNs. We have sought to determine the clinical impact of RAS mutations in patients with MPN, and to assess the pattern of co-mutational events in patients with RAS mutations.Methods: A targeted 28-gene, amplicon based next-generation sequencing assay was used to sequenced bone marrow aspirate or peripheral blood samples from 211 pts with Philadelphia negative MPN including: ET=61, PV=35, PMF=54, Post ET MF=16, Post PV MF=17, Post MPN AML=20, MPN unclassified=5 and systemic mastocytosis=3. Pts with MDS/MPN overlap syndromes were excluded. NRAS and KRAS mutations were grouped together. FisherÕs exact test and Wilcoxon rank-sum test were used to compare categorical and continuous variables, respectively.Results: N/KRAS mutations occurred at a frequency of 6 % in this cohort (Fig 1a). The median variant allele frequency (VAF) of RAS mutations was 15.6%. This was lower than the VAF of JAK2 V617F (44.6%) (p<0.001), TET2 (43.0%) (p=0.011) and ASXL1 (29.5%) (p=0.029) suggesting that RAS mutations are sub-clonal relative to other MPN associated mutations. By contrast, mutant RAS VAF did not significantly differ from VAFs of genes frequently enriched at the time of leukemic transformation, such as IDH (33.7%) (p=0.079) and TP53 (18.2%) (p=0.582) (Fig 1b). The presence of concurrent JAK-STAT activating mutations with RAS mutations was associated with distinct clinical features. RAS mutations occurred more frequently in patients with fibrotic MPN and post MPN AML and were less frequent in patients with ET and PV (table 1). RAS mutant patients had clinical features of more proliferative disease with higher total WBC, absolute monocytes, percentage circulating blasts, higher frequency of splenomegaly, and were associated with higher DIPSS score in pts with myelofibrosis (Table 1).RAS mutations were associated with mutations in TET2 with 4/32 (13%) with mut-TET2 vs 6/152 (4%) wild type TET2 having RAS mutations, though this result did not reach statistical significance (p=0.074) (Fig 1d). No other gene mutation was positively associated with mutations in RAS. 3/6 pts with TET2/RAS co-mutation had post MPN AML, while the remaining 3 had PMF or post ET MF (Fig 1c). The VAF of mut-RAS was always lower than mut-TET2 in patients where these were co-mutated, a pattern not seen for RAS VAF relative to JAK2 V617F VAF. This suggests that RAS mutations were acquired after TET2 in all cases assessed. Patients with RAS/TET2 co-mutation had the highest incidence of post-MPN AML. In those with wild type TET2 and mutant RAS proliferative disease features were noted including high WBC, monocyte count and splenomegaly. Finally, patients with myelofibrosis and RAS mutations were significantly more likely to have high DIPSS scores.Conclusions:RAS mutations occur in 6% of pts with Philadelphia negative MPN. Patients with co-occurring JAK-STAT pathway activating mutations and RAS mutations had more proliferative disease and a higher incidence of post MPN AML. RAS mutations were more frequent in patients with mutant TET2 and in this context they were associated with post MPN AML. The RAS VAF was always lower relative to TET2 and other MPN driver mutations suggesting they are acquired later in disease evolution. These data suggest the presence of RAS mutations may alter the disease biology of MPNs, and raise the question of the possible clinical efficacy of utilizing therapeutic agents, such as MEK inhibitors, in MPN patients with RAS mutations. Further preclinical and clinical evaluation of the role of this pathway in MPN pathobiology is warranted. [Display omitted] [Display omitted] [Display omitted] DisclosuresLevine:Qiagen: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy.

Mutation Landscape in Myelofibrosis: Assessment of Frequency and Correlation with Clinical Factors and Outcomes

▪Background: Somatic mutations occur frequently in patients with MF and have been shown to correlate with clinical presentation and outcome. Newer prognostic models integrate molecular data to better identify high-risk patient populations. We attempted to characterize the mutational landscape and correlate mutations with clinical presentation, response to treatment and outcomes.Methods: This was a single institution, retrospective study of patients with a diagnosis of MF seen at our center between 2/2001 and 6/2016. Somatic mutation data was assessed in patients who had undergone next generation sequencing with targeted myeloid panels at the time of referral to our center. Gene classes were defined as previously reported in the literature (Patnaik, Blood Cancer J, 2016).Results: We identified 104 eligible MF patients (median age at diagnosis 72 years; 60% males). The cohort was comprised of PMF (80%), post-PV MF (14%) and post-ET MF (7%) patients. DIPPS-plus risk score was high in 27% of patients, intermediate-2 in 41%, intermediate-1 in 28%, and low in 4%. Frequencies of driver mutations were 65% for JAK2V617F, 16% for CALR (14/17 type 1 or type 1-like)and 8% for MPL with 11% of patients classified as "triple-negative."The frequencies of mutations were 34% for TET2, 42% ASXL1, 12% EZH2, 19% IDH1/IDH2, 11% DNMT3, and 23% SRSF2 (figure 1). All other mutational frequencies occurred in less than 10% of patients. No somatic mutations were seen in 12%, 1 mutation in 22%, 2 mutations in 22%, 3 mutations in 20%, and 4+ mutations in 24%. When grouped in terms of gene function, 45% had a mutation in an epigenetic regulation gene (TET2, DNMT3, IDH1/2), 46% in a gene involved in histone modification/chromatin regulation (ASXL1, EZH2), 41% in a splicing gene (SF3B1, SRSF2, U2AF1, ZRSR2), 30% in a signal transduction gene (KRAS, NRAS, CBL, FLT3, RUNX1, SETBP1), and 7% in a DNA damage response gene (TP53 and PHF6). Mutations in a single gene group were seen in 33% of patients, with 27% having mutations in 2 classes, 16% in 3 classes, and 9% involving 4 separate gene classes.Mutations in epigenetic regulatory genes correlated with increased monocyte count (p = 0.03) and transformation to blast phase (BP) (p = 0.04). Mutations in genes involved in histone modification correlated with increased number of immature myeloid cells (p = 0.001), decreased hemoglobin (p = 0.02), thrombocytopenia (p = 0.009), increased LDH (p = 0.02), increased DIPSS and DIPSS+ (p = 0.002 and 0.001) and were more likely to be transfusion dependent (OR: 3.67 [1.47-9.04]; p = 0.007). Splicing machinery mutations correlated with older age at presentation (p = 0.002). Mutations in signal transduction genes correlated with increased monocyte count (p = 0.006), increased peripheral blasts (p = 0.02) and BP transformation (p = 0.001). ASXL1 was associated with a better response to erythropoiesis-stimulating agents (p = 0.04). The number of individual mutations and affected gene classes correlated with age at diagnosis (p = 0.01 and 0.007).CALRmut MF was associated with improved overall survival (OS) as compared to JAK2V617F or MPLmut MF (HR 0.38 [0.17-0.85], p = 0.02). Triple-negative MF had inferior 2-yr survival of 65% as compared to 83% in JAK2/MPL mutated MF.SRSF2 mutations were associated with inferior OS (HR: 2.99 [1.03-8.69]; p = 0.04), whereas ASXL1 had a trend towards inferior OS (HR 2.09 [0.90-4.84]; p = 0.09. In multivariate regression analysis, controlling for DIPSS, SRSF2 remained significantly associated with OS (p = 0.02). An increased number of somatic mutations was associated with worse OS (HR 1.21 [1.03-1.43], p = 0.02) as was an increased number of affected gene classes.Conclusion: Somatic mutations correlate with clinical features of MF and few carry prognostic significance. SRSF2 mutations have prognostic effects independent of currently utilized risk scores, while the impact of ASXL1 was less clear and potentially captured in standard risk models. Additionally, we showed that mutation burden holds prognostic significance both in terms of quantity of mutations as well as number of affected classes. [Display omitted] [Display omitted] DisclosuresLancet:Amgen: Consultancy; Novartis: Consultancy; Karyopharm: Consultancy; Kalo Bios: Consultancy; Jazz Pharmaceuticals: Consultancy; Biopath Holdings: Consultancy; ERYtech: Consultancy; Pfizer: Research Funding; Quantum First: Consultancy; Boehringer-Ingelheim: Consultancy; Baxalta: Consultancy; Celgene: Consultancy, Research Funding; Seattle Genetics: Consultancy. Sweet:Pfizer: Speakers Bureau; Incyte Corporation: Research Funding; Ariad: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Karyopharm: Honoraria, Research Funding. Komrokji:Novartis: Consultancy, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Validation of Genomic-Based Risk Models in Myelofibrosis and Proposal of Age-Independent Disease-Specific Models

Background: Prognostication in MF has historically been based on age, symptoms and hematologic features at presentation. Newer models include cytogenetic and genomic data given their independent prognostic value. In 2014, two prognostic models incorporating gene mutation data were proposed: a "mutation enhanced" IPSS (MIPSS) and a genetics-based prognostic scoring system (GPSS) model; the former using mutation data to enrich standard prognostic inputs, while the latter eschewed classic prognostic inputs for a score comprised solely of cytogenetics, mutation data, and age (Vannucchi, Blood, 2014 and Tefferi, Blood, 2014). In this study, we aimed to validate these risk models as well as propose age-independent models to better capture disease-specific prognosis.Methods: This was a single institution, retrospective study of confirmed MF patients seen between 2/2001 and 6/2016. Definitions of primary myelofibrosis (PMF), post-essential thrombocythemia myelofibrosis (post-ET MF) and post-polycythemia vera myelofibrosis (post-PV MF) were according to World Health Organization 2016 criteria and the International Working Group for Myeloproliferative Neoplasms, Research and Treatment, respectively. Mutation and cytogenetic data were assessed from review of electronic medical record. Prognostic scores were calculated on first presentation to our facility. Survival curves were generated using Kaplan-Meier method and compared using the log-rank test.Results: We identified 103 and 97 patients with available data to calculate MIPSS and GPSS scores respectively. The cohorts were 60% male, with median age 72 years at presentation. The majority of patients had a diagnosis of PMF (80%), with a small component of post-ET MF (13%) and post-PV MF (7%). Assigning scores based on MIPSS, 4% of patients were low-risk (LR), 8% intermediate-1 (IR1), 60% intermediate-2 (IR2), and 28% high-risk (HR). The model successfully differentiated between IR2 and HR with median overall survival (OS) 134 and 26 months, respectively (p = 0.0001) (figure 1A). No significant difference in OS was seen between LR/IR1 groups and IR2 (p = 0.58). Using GPSS, 1% of patients were classified as LR, 13% IR1, 25% IR2, and 61% HR. GPSS performed well in delineating between HR and intermediate risk (combined IR1 and IR2) (median OS 44 vs 134 mo, p = 0.02); however it did not distinguish HR from IR2 (p = 0.09) nor between IR1 and IR2 (p = 0.71) (figure 1B).To more specifically assess disease-specific prognosis and better divide risk scores among our patient population, new models were created. Given that age is a negative risk factor for OS independent of disease state, this input was removed. First, an age-independent MIPSS score was created with four risk groups defined: LR (score 0-0.5), IR1 (score 1-1.5), IR2 (2-3), and HR (score 3.5+). Fifteen percent of patients were classified as LR, 32% IR1, 48% IR2, and 6% HR. This system distinguished significantly between IR2 and HR (median OS 98.0 vs 13.5 mo, p = 0.006), while providing improved differentiation between IR1 and IR2 risk groups (median OS not reached vs. 98 months, p = 0.09) (figure 1C).Next, we aimed to create a truly disease-specific model based on the GPSS. Age was omitted and the impact of SRSF2 mutation was enhanced from 1 point to 1.5 points given its greater weighted impact on OS compared to ASXL1 mutation in our database. Values for other inputs remained consistent with GPSS. Using cytogenetic information (CY) and mutation data (M) as the sole inputs a CYM score was created. This yielded four risk groups defined as LR (0 points), IR1 (1-2.5 points), IR2 (3-5 points) and HR (5.5+ points). Seven percent of patients were classified as LR, 28% IR1, 56% IR2, and 9% HR. This model differentiated between IR2 disease and HR disease effectively (median OS 98 vs 15.2 mo., p < 0.0001). No significant difference was observed between IR1 and IR2 disease (figure 1D).Conclusion: Using previously proposed molecular-based models, we were able to validate their utility in the setting of advanced disease; however, insufficient capture of lower risk categories impaired its validity in that setting. We propose age-independent models to better characterize disease-specific risk in an effort to identify patients who many benefit from allogeneic stem cell transplant and/or clinical trial referral. These models merit testing in larger cohorts with longer follow-up to determine their clinical validity. [Display omitted] DisclosuresLancet:Celgene: Consultancy, Research Funding; Amgen: Consultancy; Seattle Genetics: Consultancy; Quantum First: Consultancy; Biopath Holdings: Consultancy; Baxalta: Consultancy; Pfizer: Research Funding; Kalo Bios: Consultancy; Karyopharm: Consultancy; Jazz Pharmaceuticals: Consultancy; ERYtech: Consultancy; Boehringer-Ingelheim: Consultancy; Novartis: Consultancy. Sweet:Pfizer: Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau; Incyte Corporation: Research Funding; Karyopharm: Honoraria, Research Funding. Komrokji:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Speakers Bureau.

Exploring the Potential of JAK1/2 Inhibitor Ruxolitinib with Reduced Intensity Hematopoietic Cell Transplantation (HCT) for Myelofibrosis: Stage I Results of a Prospective Trial Conducted through the Myeloproliferative Disorders - Research Consortium (MPD-RC)

We conducted a two-stage Simon phase II, multi-center study in two groups: related and unrelated donor transplants. Based on the results of a previously conducted prospective study (MPD-RC 101, Rondelli et al, Blood, 2014) demonstrating relatively poor outcome in the unrelated donor arm due to high non-relapse mortality and graft failure (GF), we sought to improve this noted disparate outcome. The hypothesis driving this trial was that pre-transplant JAK1/2 inhibition would result in an increase in performance status, reduction in splenomegaly and inflammatory cytokine expression leading to improved HCT outcome. In the unrelated arm in the first 11 patients, the trial was to be terminated if 6 or more failures (GF or death due to any cause by 100 days post-transplant) were observed. In the related arm in the first 11 patients, the trial was to be terminated if 3 or more failures were observed.Key eligibility criteria included diagnosis of primary MF or post-PV MF or post-ET MF, age 18-70 years, and DIPSS int-2/high risk disease or int-1 risk disease with at least one additional risk factor. Patients could have received up to 6 months of ruxolitinib prior to enrollment provided that there was no evidence of loss of response to ruxolitinib during this period.Ruxolitinib was given for 56 days followed by a 4-day taper ending day -6. The conditioning regimen utilized was IV fludarabine 40 mg/m2 IV and IV busulfan 2.0 mg/KBW IV once daily for 4 days from days -5 to -2. GVHD prophylaxis consisted of cyclosporine or tacrolimus and methotrexate. Patients undergoing unrelated donor HCT also received low dose thymoglobulin (0.5 mg/KBW on day -3/2.0 mg/KBW on day -2 & day -1).The study has enrolled 21 patients (related, 7; unrelated, 14) with a median age of 59 years (range 39-70). 17 (81%) patients had palpable splenomegaly with median spleen size of 11 cm, and 16 patients had int-2/high risk disease. Of the 21 patients, 19 patients received transplantation (related 5/7 [71%], unrelated 14/14 [100%] underwent transplant). Two patients in the related arm did not proceed with transplantation as one progressed to acute myeloid leukemia (baseline blast count 14%) and another experienced sudden death of unknown cause the day after starting ruxolitinib. In addition, there was one GF in the related arm. Due to these 3 failures in the related arm, further enrollment was stopped in this arm per protocol criteria. In the unrelated arm, there were 4 failures (Table 1), and the study met the protocol criteria for further enrollment.Mean percent reduction from baseline to day -9 in palpable spleen length below the left costal margin was 30% (p=0.04) in 12 patients with palpable spleen. 5/12 (42%) had any reduction, and 4/12 (33%) had ≥50% reduction. From baseline to best score on day -9 or day -5, 11/19 (58%) patients with data had any improvement in MPN-SAF Total Symptom Score, and 7/19 (37%) had ≥50% reduction.Of the 19 patients undergoing transplant, ruxolitinib was tapered off successfully in all patients with no significant withdrawal syndrome or delay in transplant noted. In the pre-transplant phase, most common Grade 3 or higher toxicity considered to be related to ruxolitinib was anemia (6/21, 29%), and one patient had legionella pneumonia deemed not related to ruxolitinib. Median time to neutrophil recovery (> 0.5 x 109/L) and platelet recovery (>20 x 109/L) was 19 and 19.5 days. By day 100, 7/19 (37%) experienced Grade 2-4 acute GVHD (G2 in 5, G3 in 2). In the first 30 days post HCT, most common Grade 3 or higher toxicity was febrile neutropenia seen in 5/19 (26%) patients.In all 21 patients from time of registration with median follow-up of 5.8 months, 6-mo overall survival was 75% (95% CI 44-90%). For the 19 transplant recipients, 6-mo OS was 83% (95% CI 55-94%) from date of transplant.Conclusions: Stage 1 trial results highlight important observations. First, ruxolitinib may not be suitable for all myelofibrosis patients pre-transplant, eg, alternative approaches might be more appropriate in the context of accelerating disease. Second, we demonstrated a safe approach for tapering ruxolitinib prior to transplant which allowed patients to commence conditioning with reduced spleen size and symptoms. Third, pre-transplant ruxolitinib does not appear to have any adverse impact on early post-HCT outcomes following unrelated donor transplant, although Stage 2 enrollment will be required to better assess the potential benefits of this approach. [Display omitted] DisclosuresGupta:Novartis: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Research Funding. Yacoub:Incyte: Consultancy, Honoraria, Speakers Bureau; Seattle Genetics: Consultancy, Honoraria, Speakers Bureau; Alexion: Honoraria. Mesa:Promedior: Research Funding; Celgene: Research Funding; CTI: Research Funding; Gilead: Research Funding; Incyte: Research Funding; Galena: Consultancy; Ariad: Consultancy; Novartis: Consultancy. Mead:Novartis: Honoraria, Research Funding, Speakers Bureau. Mascarenhas:Novartis: Other: DSMB , Research Funding; Roche: Research Funding; Promedior: Research Funding; Janssen: Research Funding; CTI Biopharma: Research Funding; Incyte: Other: Clinical Trial Steereing Committee, Research Funding.