ALBERT

Description: Bone marrow (BM) fibrosis is a key pathomorphologic feature of patients (pts) with primary myelofibrosis (PMF) and the fibrotic phases of essential thrombocythemia (post-ET MF) and polycythemia vera (post-PV MF). The degree of BM fibrosis appears to correlate with survival. Indeed worse survival has been associated with increased BM fibrosis. The BM stromal microenvironment is important in the pathogenesis of BM fibrosis. Cellular components (fibroblasts, macrophages, endothelial cells, adipocytes), structural fibrils (collagen, reticulin) and extracellular matrix components are all forming elements of the BM stroma. Increased stromal fibrosis has been linked to abnormalities in the number/ function of megakaryocytes and platelets in hematologic diseases. Several cytokines like Platelet Derived Growth Factor (PDGF) and Transforming Growth Factor-Beta (TGF-b) have been also linked to the pathophysiology of BM fibrosis. PDGF has been shown to increase fibroblast growth in megakaryocytes and platelets although increased PDGF did not correlate with increased production of either reticulin or collagenous fibrosis. Moreover, PMF pts have increased TGF-b levels in platelets, megakaryocytes, and monocytes. Nitric Oxide (NO) is a ubiquitous gas important in physiologic processes particularly vasodilatation. Dysregulation of NO levels has been implicated in pulmonary hypertension (PH), hemoglobinopathies, and cardiovascular diseases. In Peyronie’s disease, a localized fibrosis of the penile tunica albuginea, increased NO production by expression of iNOS decreases collagen deposition by neutralization of profibrotic reactive oxygen species and decreased myofibroblast formation. Aside from its role in maintaining normal vascular tone, NO also plays a role in fibroblast formation and collagen biosynthesis. We previously reported that ruxolitinib, a JAK1/2 inhibitor restores NO levels leading to improvement of PH in MF pts (Tabarroki et al., Leukemia 2014). We now hypothesize that plasma/serum NO level is a key regulator of BM fibrosis in MF and that ruxolitinib treatment (Tx) leads to improvement of BM fibrosis by NO modulation. Using a Sievers 280i NO analyzer we measured the plasma/serum NO level of a large cohort (n=75) of pts with myeloid and myeloproliferative neoplasms (MPN) [MDS, RARS/RCMD=8; MPN, ET=8, PV=8, MF=24, Mastocytosis=7; MDS/MPN, CMML=11, MDS/MPN-U, RARS-T=9]. Healthy subjects (n=10) were used as a control. MPN pts had low NO (nM) levels among the pts studied with the lowest level found in MF pts: MF=30.31±11.8, PV=39.0±16.1, ET=36±20.3, RARS=74.6±41.7 (P=.01), CMML=84.4±89.2 (P=.04), RCMD=163.4±103.8 (P

Description: Background Smoking is a risk factor for development of MDS and for overall survival. The pathogenesis of MDS is a multi-step process with both environmental and genetic influences. The link between smoking and MDS is thought to be mediated by organic solvents in tobacco. We identified specific molecular abnormalities associated with smoking exposure in MDS patients (pts). Methods 151 MDS pts seen from 2000-2012 with complete smoking and molecular data were included. Correlations and univariable comparisons were performed using Fisher’s Exact, Chi-square, Kruskal-Wallis, or Wilcoxon rank sum tests. Poisson regression models were used to assess associations between the number of mutations present and demographic and clinical factors. Analysis was performed using next-generation targeted deep gene sequencing with 22 common gene mutations, selected based on the frequency observed in a cohort of MDS patients analyzed by whole exome sequencing. Mutations were considered individually and in functional groups: methylation (TET2, IDH1, IDH2), histone modification (ASXL1, EZH2), and gene splicing (SRSF2, U2AF1, SF3B1). Paired DNA (tumor vs. CD3+ lymphocytes) produced raw sequence reads aligned using Burrows-Wheeler Aligner (BWA). Variants were detected using the Broad Institute's Best Practice Variant Detection GATK toolkit. Results Median age at MDS diagnosis was 68 years (range 20-85); 42% were female; and 77% had de novo MDS. IPSS (low, Int-1, Int-2, high) and IPSS-R (very low, low, int, high, very high) categories were: 26%, 44%, 22%, 7% and 10%, 40%, 19%, 18%, 13%, respectively; IPSS-R cytogenetic categories were: very good (20 pack years) were also associated with worse survival (p=.03 and .01, respectively), though current or ex-smokers with

Description: Pulmonary hypertension (PH) is an under-recognized complication of myelofibrosis (MF) occurring in 30% of MF patients and associated with poor survival. Echocardiographic diagnostic findings include; elevated right ventricular systolic pressure (RVSP)〉35 mmHg, right atrial (RA) enlargement and increased tricuspid regurgitation velocity (TRV) ≥2.5 m/sec. The pathophysiology of PH in MF has not been elucidated, although in idiopathic PH, the proliferation of pulmonary artery endothelial cells has been linked to activation of STAT3 pathway. Dysregulation of JAK-STAT pathway has been implicated in the pathogenesis of MF. Ruxolitinib, a JAK1/2 inhibitor, was approved for management of splenomegaly and cytokine-mediated symptoms in MF. Furthermore, no specific therapy in the management of MF-associated PH has been established. Given the association between MF and PH and the possible pathophysiologic link mediated by JAK signaling, we prospectively followed 19 patients with MF-associated PH and compared their echocardiographic findings and PH relevant serum biomarker levels (nitric oxide [NO], NT-pro brain natriuretic peptide [NT-proBNP], von Willebrand antigen (vWB), ristocetin co-factor (RCA), and uric acid (UA) pre- and post-ruxolitinib therapy. All categorical data were summarized for frequency, counts and percentages, and the comparison between two groups was performed by two-sample Wilcoxon signed rank test. Among 19 patients (pts), 9 had PMF, 5 post-ET MF, 4 post-PV MF and one CMML-1. In this cohort, 11 were females and 8 were males. The median age of the cohort was 68 years (range, 50-81 years). Fifteen pts were JAK2 V617F positive and 4 were wild-type, 8 were intermediate-1, 4 intermediate-2 and 6 high risk per Dynamic International Prognostic Scoring System-Plus risk grouping. The mean ruxolitinib dose was 10 mg BID (range: 5 mg QOD-20 mg BID]. Median duration of disease was 32 mos (6-164 mos), ruxolitinib duration of treatment was 10 mos (4 -17 mos) and follow-up was 11 mos (6-22 mos). Prior to the initiation of ruxolitinib treatment, NT-pro BNP levels, were measured and found to be elevated in 90% (17/19) of pts. In addition, UA, vWB, and RCA levels were all elevated in 47% (9/19), 24% (4/17), and 12% (2/17) of pts respectively. The strongest correlation among serum biomarkers was between plasma vWB and RCA levels (r2=-0.89, P=

Description: Ruxolitinib, is a JAK1/2 inhibitor approved for the treatment of intermediate and high risk myelofibrosis (MF). The recommended starting doses for ruxolitinib, are 20 mg PO BID or 15 mg PO BID if platelet (PLT) counts are ≥200 x109/L or 100 to 200 x109/L, respectively. However, drug related side effects which includes grade 3/4 anemia (45.2%), thrombocytopenia (12.9%), fatigue (25.2%) and diarrhea (23.2%) are common using standard dosing. Moreover 70% of patients (pts) started on the standard regimen require dose reduction because of drug related toxicities. Although hematologic side effects are generally expected during early phases of ruxolitinib therapy and reversible in most cases, a therapeutic approach that can avoid myelosuppression while still providing adequate clinical responses is warranted. Therefore, to obviate toxicities and achieve symptom improvement in pts with MF, we employed a dose escalation (DE, starting at 5 mg QOD) approach for ruxolitinib and compared outcomes with another group that previously received standard regimens (SR, 15 or 20 mg BID). In addition to baseline characteristics, clinical data including hematologic parameters, bone marrow (BM) results, Dynamic International Prognostic Scoring System-Plus (DIPSS-plus) risk, and transfusion (TF) requirement were collected. Hematologic and non-hematologic side effects based on CTCAEv.4 and evidence of clinical response (reduction of splenomegaly by palpation) were assessed at 8, 12 and 16 weeks after starting treatment (post-Tx). Descriptive data were summarized as frequency of counts and percentages and continuous data were presented as mean and ranges and the comparison between two groups was performed. Survival outcomes were analyzed using the Kaplan-Meier method. Plasma concentrations for interleukins (IL) including IL1A, IL1B, IL2, IL4, IL6, IL8, IL10, IL12, IL17A, INFg, TNFa and GM-CSF were also measured pre and post- ruxolitinib treatment using a Multi-Analyte ELISArray Kits (SABiosciences) A total of 42 pts seen in our MPN clinic were studied, including 23 females (54%) and 19 males (46%). The median age of the cohort was 69 (43-83 years). Disease subtypes include 23 PMF, 10 post-PV MF, 6 post-ET MF and, 1 CMML-1. Based on the DIPSS-plus risk score, there were 17 high, 16 intermediate-2, and 8 intermediate-1 risk pts. Thirty-two pts (76%) were JAK2V617F mutant and 10 were wild type. The mean duration of treatment was 8 mos (range, 3-17) and follow up was 11 mos (4-36). Twenty-six pts started on DE and 16 received SR. The mean ruxolitinib dose in the whole cohort was 10 mg BID (range; 5mg QOD-25mg BID). The mean maintenance dose in the DE cohort was 5mg BID while in the SR cohort was 15mg BID. In terms of clinical response, the mean pre and post-Tx spleen size was 13.69 cm and 6.8 cm in the DE cohort compared to 17.68 cm and 13.07 cm in SR cohort (p = .03). In terms of hematological side effects, the DE approach resulted in less frequent grade 3-4 or any grade anemia compared to SR (G3-4: 3% vs. 18%, p =.02; overall anemia: 14% vs. 50%, p =.04). In pts treated with DE and SR, 57% and 75% were TF dependent pre-Tx. Post-Tx, 35% of pts treated with DE became transfusion independent vs 0% in the SR group. The DE approach also resulted in less frequent grade 3-4 or any grade thrombocytopenia compared to SR (G3-4: 11% vs. 31%, p= .04; overall thrombocytopenia: 23% vs. 43%, p=.06). There was no difference in the frequency or degree of neutropenia between the DE and SR treated pts. Non-hematologic side effects were also less frequent in the DE group compared to the SR group (bruising: 36 vs 75%, diarrhea: 28 vs 56%, lower extremity edema: 21% vs 50%, and dizziness: 15 vs 31%). In 3 pts treated with DE, the degree of reticulin fibrosis reduced from MF3 to MF2 confirmed with bone marrow biopsy performed 6-12 months post-Tx. We also compared the differences in cytokine profile between the patients treated with DE (n=2) and SR (N=2). No difference in the degree of reduction of MF relevant cytokines including IL-1A, IL-1B, IL-2, IL-4, IL-6, IL-12, TNF-α and GM-CSF were observed between both groups (p=0.2). At the time of analysis, 38 pts are still alive and no difference in survival were observed between DE and SR treated patients (log rank p=0.69). In conclusion, a dose escalation approach for ruxolitinib therapy is better tolerated and with preserved clinical responses compared to the standard dosing regimen in MF patients. Disclosures: No relevant conflicts of interest to declare.