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 The Revised International Prognostic Scoring System (IPSS-R) was developed to risk stratify untreated patients (pts) with MDS. It has since been validated in pts treated with a single line of drug therapy, and has been modified in untreated pts to include mutational data; however, these approaches do not reflect typical MDS pts who receive different types of treatment in different sequences. We propose a prognostic model that incorporates mutational data and predicts outcome in pts with primary and secondary MDS regardless of their initial or subsequent treatments. Methods Clinical and mutational data of 333 pts with newly diagnosed MDS who were treated at out institution between 1/2000-1/2012 were analyzed. The IPSS-R was calculated at diagnosis. Survival was calculated from the date of diagnosis to last follow up or death. A panel of 62 gene mutations obtained by next generation targeted deep sequencing selected based on the frequency observed in a separate cohort of MDS patients analyzed by whole exome sequencing (WES). A Cox proportional multivariate analysis including age, IPSS-R score and mutations that are present in 〉/= 10 pts was used to select independent prognostic factors. The fit of the proposed model to the data was assessed by using the Akaike information criterion (AIC). Results Pt clinical characteristics are summarized in Table 1. Median age was 68 years (range, 20-87); 214 pts (64%) had de novo MDS, 39 (12%) had prior antecedentl hematologic disorders, 37 (11%) secondary MDS, and 43 (13%) had chronic myelomonocytic leukemia (CMML). Pts received between 0-7 lines of therapy: 15% did not receive any treatment, 85% received at least one treatment, 40% received 〉/=2 treatments, 20% received 〉/= 3 treatments and 14% of pts eventually underwent hematopoietic cell transplant (HCT). First line therapies included: growth factors (30%), azacitidine +/- combination (32%), decitabine +/- combination (7%), single agent lenalidomide (5%), investigational agents (5%), induction chemotherapy with cytarabine and an anthracycline (7+3, 2%), and immunosuppressive therapy (4%). With a median follow-up of 38 months (mo) (range, 0.4-128.5), 70 pts (21%) progressed to AML and the median OS was 35.1 mo (range, 0.4-128.5). Per IPSS-R risk groups, median OS for very low was 35 mo, low 35 mo, intermediate 22 mo, high 19 mo, and very high 12 mo, Figure 1. Among the 62 gene mutations, 25 were present in 〉/= 10 pts: TET2 (17%), ASXL1 (15%), SF3B1 (14%), STAG2 (11%), DNMT3A (11%), RUNX1 (10%), U2AF1 (9%), GPR98 (8%), ZRSR2 (7%), BCOR (6%), TP53 (5%), NF1 (5%), EZH2 (5%), APC (5%), SUZ12 (5%), BCORL1 (4%), CBL (4%), PRPF8 (4%), NRAS (3%), CUX1 (3%), DDX54 (3%), IDH2 (3%), KDM6A (3%), PHF6 (3%), and SETBP1 (3%). A Cox proportional hazard analysis including age, IPSS-R score, and the 25 genes mutations listed above identified the following as independent prognostic factors: age, IPSS-R, ASXL1, BCOR, BCORL1, EZH2, IDH2, SF3B1,TP53. The linear predictive Cox model score obtained using the fitted coefficients of each prognostic factor was: ASXL1 X 0.65+BCOR X 0.92+BCORL1 X (-1.65)+EZH2 X 0.71+IDH2 X (-1.0)+SF3B1 X (-0.59)+TP53 X 1.24+Age X 0.04+IPSS-R score X 0.43. Four prognostic groups were proposed: low (score 0-3.4, 80 pts, median OS 47.3 mo), intermediate-1 (score 3.5-4, 69 pts, median OS 30.2 mo), intermediate-2 (score 4.1-5.4, 131 pts, median OS 19.9 mo), and high (score 〉/= 5.5, 53 pts, median OS 12.2 mo), p 〈 0.001, Figure 2. The new model demonstrated a markedly better fit, reflected in an AIC of 2026, compared to 2058 for the IPSS-R. Conclusion We propose a new mathematical model that incorporates age, IPSS-R score and several gene mutations that can accurately predict OS in pts with primary and secondary MDS as well as CMML regardless of initial or subsequent treatments, including HCT. This model also highlights the importance of mutational data along with clinical data for risk stratification in MDS. Figure 1 Overall survival by IPSS-R Figure 1. Overall survival by IPSS-R Figure 2 Overall survival by the new prognostic model Figure 2. Overall survival by the new prognostic model Table 1 Patient characteristics No. (%) / [Range] Total 333 Median age, years 68 [20-87] Male 205 (62) Female 128 (38) Median white blood cell (WBC) X 109/L 3.8 [0.69-125.9] Median absolute neutrophil count (ANC) X 109/L 1.62 [0.02-170] Median hemoglobin,g/dl 9.6 [3.9-14.6] Median platelets X 109/L 93 [4-776] Median bone marrow blasts % 2 [0-19] IPSS-R Very low 50 (15) Low 128 (38) Intermediate 59 (18) High 60 (18) Very high 36 (11) Disclosures No relevant conflicts of interest to declare.

Description: Background For decades, cytogenetic analysis has played an essential role in AML risk stratification. Among the 50% of AML patients (pts) with normal karyotype (NK), outcome can vary widely. More recently, whole genome sequencing (WGS) and whole exome sequencing (WES) have identified several recurrent mutations that play an important role in AML pathogenesis and impact outcome. Pts with secondary AML (sAML) have a particularly poor prognosis, are not as responsive to standard induction chemotherapy, and often are referred in first complete remission to hematopoietic stem cell transplantation. We hypothesized that different genomic patterns exist between primary AML (pAML) and sAML that can distinguish the two, and can alter treatment recommendations. To negate the impact of chromosomal abnormalities, we focused our analyses on pts with NK. Methods We performed WES and multi-amplicon targeted deep sequencing on samples from bone marrow and peripheral blood of pts diagnosed with sAML at our institution between 1/2003- 1/2013 and who had NK cytogenetics. We compared them to pts with NK primary AML (pAML) whose data were extracted from The Cancer Genome Atlas (TCGA). A panel of 62 gene mutations that has been described as recurrent mutations in myeloid malignancies was included. Mutations were considered individually and grouped based on their functional pathways: RNA splicing (SF3B1, U2AF1/2, SRSF2, ZRSR2), DNA methylation (TET2, DNMT3A, IDH1/2), chromatin modification (ASXL1, EZH2, MLL, SUZ12, KDM6A), transcription (RUNX1, CEBPA, NPM1, BCOR/BCORL1, SETBP1, ETV6), activating signaling (FLT3, JAK2), cohesion (STAG2, SMC3, RAD21), RAS superfamily (K/NRAS, NF1, PTPN11, CBL) and tumor suppressor genes (TP53, APC, WT1, PHF6). Using deep sequencing methodology for resequencing or targeted sequencing, variant allelic frequency (VAF) was measured for each mutation detected. VAF was adjusted by zygosity evaluated by SNP-array karyotyping. For confirmation of clonal architecture, serial sample sequencing and single colony PCR were applied. Differences were compared using Fisher-exact test and Mann-Whitney U test for categorical and continues variables respectively. Results: Of 143 pts included, 101 (71%) had pAML and 42 (29%) had sAML. Compared to pAML, sAML pts were older (59 vs 69 years, p

Description: Background: African-American (AA) patients (pts) have a younger age at diagnosis and worse outcomes compared to whites (WTs) across many cancers, including acute myeloid and lymphoblastic leukemias. This difference may be related to disease biology rather than access to medical care or socioeconomic status. The incidence of MDS and age at diagnosis in national cancer registries in AAs is lower than in WTs. Detailed biological and clinical characteristics and outcome of AA pts with MDS compared to WTs have not been defined. Methods: We collected mutational and clinical data on MDS pts diagnosed from 1/2000-1/2012. Next-generation gene-targeted deep sequencing of 62 common gene mutations (selected based on frequencies established in a separate cohort of MDS pts studied by whole exome sequencing) were analyzed as individual mutations and then grouped into several functional pathways which were hypothesized to characterize MDS pathogenesis. International Prognostic Scoring System-Revised (IPSS-R) score was calculated as described previously. Overall survival (OS) was measured from the time of diagnosis to time of death or last follow up. Time-to-event analyses were performed by the Kaplan-Meier method, with curves compared by log rank test. Differences among variables were evaluated by the Fisher’s exact test and Mann-Whitney U test for categorical and continuous variables, respectively. Results: Of 341 pts, 44 (13%) were AA. Comparing WTs to AAs, pts had a similar median age (68 for both), absolute neutrophil count (1.6 vs 2.23) X 109/L, hemoglobin (9.7 vs 9.4) g/dL, platelets (93 vs 91) X 109/L, and bone marrow blasts (2% vs 3%), respectively. IPSS-R risk category distribution for WTs and AA was: very low 15% vs 9%, low 35% vs 30%, intermediate 18% vs 18%, high 16% vs 23%, very high 10% vs 18%, and not applicable 6% vs 2%, respectively. Among AA pts, 25% had very poor risk cytogenetics per IPSS-R criteria (complex 〉3) compared to 10% of WTs (p=.008) which led to 41% of AA pts having high and very high risk IPSS-R scores compared to 26% of WTs (p=.035). Further, WTs were more likely to receive a treatment (86% vs 66%, p

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=