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: Isochromosome 17q [i(17q)], a poor prognostic cytogenetic abnormality is a product of the breakage or inappropriate division of the pericentromere leading to the duplication of the long and loss of the short arm of chromosome 17. The region of the breakpoints maps at 17p11, a region encompassing a key tumor suppressor gene: TP53. I(17q) are detected in myelodysplastic/ myeloproliferative neoplasms (MDS/ MPN), chronic myeloid leukemia (CML), and acute myeloid leukemia (AML). This abnormality can occur as a sole structural abnormality or in combination with other chromosomal defects. The presence of i(17q) is associated with poor therapeutic response, disease progression, and an unfavorable clinical outcome. Elucidation of the molecular architecture of patients (pts) carrying i(17q) may lead to better understanding of disease biology and development of novel compounds that can target this disease. We selected 11 pts with i(17q) to characterize their genomic differences. We applied whole exome sequencing (WES) in order to define latent molecular defects explaining the clinical phenotype of this disease. The index case was a male MDS/MPN pt with isolated i(17q), 27% RS, hypercellular bone marrow (BM), mild splenomegaly, and atypical megakaryocytes. The pt developed 7% BM blasts without clinical response to growth factors. Molecularly this pt was a wild type SF3B1, a gene frequently mutated in RARS-T and associated with lower transformation rate to leukemia, better survival, and good/intermediate risk cytogenetic abnormalities. WES was performed on 2 ug of total DNA extracted from BM cells. Non-clonal CD3+ cells were used as source of germ-line control. Twenty-millions reads were run on an Illumina HiSeq2000 sequencer. Using a stringent bio-informatic algorithm developed in house, all variants were filtered based on a variation score (〉=30) and a coverage (30X) and the tumor nucleotide variation analysis was performed for each pair (tumor vs. germ-line), where only the variants unique to the tumor were retained. Variants were ultimately filtered in order to exclude SNPs by an in-house annotation and importing the hg19 SNP135. We detected 65 unique candidate genes. Four genes were confirmed to be somatic: 3 were novel: ZFP42 (4q35.2), P4HTM (3p21.31), and VPRBP (3p21.2) and 1 includes the newly discovered SETBP1 (18q12.3) gene. Three variants detected on the chromosome 17 had a wild type configuration. The subsequently genotyped all the pts (MDS/MPN/-U 3; AML 4; RCMD 1; CML 1; RAEB-1 2; mean age: 68 years; male/female: 8/3; i(17q)/other abnormalities:3/8) for the above genes and for a panel of genes known to be mutated in MDS/MPN and other diseases in order to find any genetic association explaining the disase phenotype. We applied Sanger sequencing to DNA derived from BM/peripheral blood cells (BM/PB:7/4) for the following genes and respective exons: TP53 (all exons), SF3B1 (13-16), SRSF2 (1-2), U2AF1 (2 and 6), TET2 (all exons), DNMT3A (18-23), IDH1/2 (4), CBL (8-9), N/KRAS (1-2), ASXL1 (12), JAK2 (12 and 14), EZH2 (16, 18 and 19), MPL (exon 10), BCAS3 (12, 15 and 16), FLT3 (11 and 17), and CSF3R (13,14, and 17). In total, we found 16 heterozygous missense mutations and 1 tandem duplication. We found somatic mutations in ZFP42, P4HTM, and VPRBP in 1 pt. The index case reported a mutation in SETBP1 and SRSF2. SF3B1 was detected as a sole abnormality in 1 patient. Of note, the patient with SF3B1 mutation (K700E) had 50% RS and achieved a complete hematologic remission after decitabine therapy. The most frequent mutations were found in SETBP1 and SRSF2. SETBP1 was found to be mutated in 4/11 (36.3%) pts (D868N, I871T, and G870S was common in 2 pts) while SRSF2 mutations (P95H/R) were found in 3/11 (27.2%) pts. Three pts showed concomitant SRSF2 and SETBP1 mutations. NRAS (G12D) was mutated in 1 pt and associated with SRSF2 and SETBP1 mutations. One pt showed mutations in TET2, JAK2, and TP53. Of note, this pt did not respond to treatment. One pt with MDS/MPN showed a mutation in CSF3R (Q741X), a novel gene discovered in chronic neutrophilic leukemia and atypical CML. The pt also has monosomy 7 and i(17q) abnormality. FLT3-ITD was found in 1 pt. As of last follow-up, only 2 pts remain alive. In sum, we found that poor risk molecular mutations in SRSF2 and SETBP1 are frequently found in i(17q) myeloid malignancies and may be the drivers of poor outcomes in this disease. Disclosures: No relevant conflicts of interest to declare.

Description: Pharmacologic therapies that target the JAK-STAT pathway are clinically used to alleviate splenomegaly and disease-related constitutional symptoms in MF. However, it is clear that some patients develop intolerance or resistant to this therapy. Furthermore, there are MF related complications especially cytopenias that are not alleviated by these therapies. Therefore, alternative and complementarytherapies are warranted in the management of MF. We hypothesized that other pathways downstream of the JAK-STAT signaling pathway can play a role in the pathophysiology of MF. We used whole exome (WES) and RNA sequencing technologies to interrogate new molecular markers and pathways which can serve as novel targets for this disease. In 4 MF patients [JAK2 mutant (MUT) =2, and wild type (WT) =2], WES was performed using the Illumina platform. All of the variants were filtered based on PHRED score (〉=30) with coverage was set at 30X. Analysis of data in JAK2/MPL WT patients demonstrated the presence of 263 candidate genes. After clarifying the status of tumor nucleotide variants in each gene compared to germline (CD3+) fraction, 7 genes (RBL1, ADSS, ZNF717,MUC4, TUBB4Q and CDC25A) were selected for further somatic confirmation by direct sequencing. Among these genes, only alteration in CDC25A, a regulator of cyclinE/cdk2 (cyclin-dependent kinase-2) and cyclinA/cdk2 kinase, was confirmed to be somatic. This genetic change was previously reported as somatic by WES in lung cancer although not confirmed by direct sequencing (Bartkova et al, Nature, 2005, Apr 14; 434 (7035):864-70). Based on these observations and since CDC25A acts as a downstream effector of JAK-STAT signaling, we hypothesized that, CDC25A phosphatase, may be a driver in MF pathogenesis. The transcriptome of two patients, one MUT and one WT for JAK2 was then analyzed. RNA was isolated from bone marrow (BM) cells of healthy individuals (HI) (N=3). cDNA was made from 1.5-3 ug of RNA and fragmented for library preparation. RNA-sequencing was performed on 20 million sequence reads. Paired-end 90 base pair reads were generated on an Illumina HiSeq2000 sequencer and aligned to the human genome 19. RNA-splicing patterns were analyzed by a bioinformatics algorithm and gene expression analysis was carried out using GSEA (Visconte V; Blood. 2012). By using FDR80%) while JAK2 MUT samples had only a few positive megakaryocytes (

Description: Abstract 312 Prognosis in myelodysplastic syndromes (MDS) is heavily influenced by cytogenetics. Trisomy 8 (+8) is reported in 15–20% of MDS patients (pts) and is one of the most commonly identified karyotypes in this disease. Sole +8 is an intermediate-risk karyotype in MDS by the International Prognostic Scoring System (IPSS). However, pts with +8 myeloid malignancies exhibit wide clinical heterogeneity. In chronic myelomonocytic leukemia (CMML), +8 is considered high-risk, while in MDS functional data suggests an “immunopathologic” cause that is responsive to immunosuppressive agents and confers a good prognosis. Single nucleotide polymorphism array (SNP-A) and molecular technologies (next generation sequencing) have led to further refinement of prognosis in MDS, and subsequent discovery of molecular mutations with distinct effects on outcomes. We hypothesized that the differential outcomes noted in +8 MDS are primarily influenced by both clinicomorphologic features and the acquisition of new cytogenetic (isolated, +1, ≥2) and molecular defects. To further characterize the clinical and molecular behavior of +8 myeloid neoplasms, we analyzed 68 pts seen at the Cleveland Clinic with a +8 clone. Hematologic, bone marrow (BM), cytogenetic (metaphase cytogenetic [MC]/SNP-A) and survival data were collected. Survival comparisons were made by Kaplan-Meier analyses. Cox-proportional hazard ratio was used to determine factors predictive of outcomes. Response was evaluated per International Working Group 2006 criteria. Most (69%) pts were male; median age 69 years (38–89), and median follow-up 15 months. 69% (47/68) had MDS, 10% (7/68) MDS/MPN, 3% (2/68) MPN and 18% (12/68) AML. 41% of pts had an isolated +8 abnormality (+8 (i)), 16% had one additional abnormality (+8plus1), and 43% had ≥2 additional abnormalities defined as +8complex. By IPSS, Int-1=34%, Int-2=25%, high=42%. SNP-A data were available in 51% of the cases and detected new lesions in 60% of them (gains[46%], losses[43%], UPD[23%]). Clinically, pts with +8complex had higher median peripheral blood blasts (PB) compared to +8plus1 and +8(i) (3.5 vs 0 vs 0%,p=.04). By treatment, pts received high intensity (BMT±high dose chemo=35%), low intensity (hypomethylating agents [HMA]± lenalidomide=37%) or supportive therapies=28%). Overall response rate was 47%, with 14% CR. By cytogenetic grouping, +8complex had lower response rates compared to +8 with additional karyotype (30 vs 60 vs 52%; p=.06). Median overall survival (OS) was 15 months, and median event free survival (EFS) was 8 months, with worse OS noted in +8 complex compared to +8 plus1 and +8(i) (OS=11 vs 22 vs 29 months, p=.02; EFS=5 vs 10 vs 12 months; p=.04). To investigate the biologic rationale of these observations, we performing direct sequencing for poor prognostic genes in myeloid malignancies, such as ASXL1, IDH1/2, EZH2, K/NRAS, CBL and TP53 and for predictors of good outcomes/response like SF3B1/TET2. Molecular mutations were identified in 9/24 (38%) pts, with mutational frequencies within +8(i) (12 pts) of TET2 (42%), ASXL1 (42%), K-/NRAS (17%), SF3B1 (17%), and TP53 (0%). None of these mutations were detected in the +8plus1 or +8complex cohorts except for TP53 in one +8complex pt. No IDH1/2/EZH2/CBL mutations were found in the entire +8 cohort. Interestingly, mutations in TET2 conferred a better OS (88 vs 12 mos; p=.01). In +8 myeloid malignancies, ASXL1 mutations did not impact OS. We are currently performing whole exome sequencing and other immunogenetic studies in +8 pts to help identify factors that contribute to these group outcomes. To further define factors predictive of worse outcomes in +8 pts, univariate and multivariate analyses were performed and a predictive model of OS was designed. Using a simple scoring system, one point each was assigned to LDH ≥240, platelets 5%, and 2 points to an ANC 2], median OS=4.6 months; p

Description: Background: The presence of the lupus anticoagulant (LA) is an established risk factor for thrombosis especially in the post operative setting. The risk of thrombosis among patients testing indeterminate for lupus anticoagulant is unknown. In our study we aim to estimate the incidence of postoperative thrombosis in this population and determine risk factors. Methods: We studied adult patients undergoing LA testing within 3 months of major surgery at the Cleveland Clinic between 2008 and 2013. The International Society for Thrombosis and Hemostasis (ISTH) defines the following criteria for a positive lupus anticoagulant: 1) a prolonged phospholipid dependent screening test, 2) an inhibitor demonstrated on a mixing study with normal plasma, 3) evidence that the inhibitor is phospholipid dependent, and 4) absence of a coexisting factor inhibitor, direct thrombin inhibitor or heparin. Patients fulfilling some but not all of the ISTH criteria were considered to be LA indeterminate. Patients with previous venous thromboembolism (VTE) and hypercoagulable states were excluded. We collected data on patient demographics, surgery and VTE in the first 30 postoperative days. Patients were divided into different groups based upon the presence of concomitant malignant or rheumatological disease, types of surgery and the utilization of thromboprophylaxis. Associations between categorical variables were determined using Fisher’s exact test. Multivariate logistic regression was performed; variables included age, type of surgery and utilization of pharmacologic thromboprophylaxis. Results: Of 791patients undergoing perioperative lupus anticoagulant testing, 176 were diagnosed LA indeterminate. The median age was 55 years with males comprising 52.3% of the population. Twenty six (14.7%) patients had a concomitant malignancy. Eighteen patients (10.2%) were diagnosed with a rheumatological illness. Seventy four (42.1%) patients underwent cardiac and vascular surgical procedures. General surgery including gastrointestinal surgery accounted for 53 (30.1%) patients. Neurosurgical patients (20) comprised 11.4% of the population studied. Fifteen (8.5%) patients underwent orthopedic surgery and fourteen (8%) patients had urologic procedures. Thirty eight (21.6%,CI 16.1-28.3%) patients developed VTE in the first 30 postoperative days. Of the patients with VTE, 16 (42.1%) had isolated deep vein thrombosis (DVT). Six patients had DVT associated with internal jugular vein (IJV) thrombosis (15.8%). Five patients (13.2%) patients had DVT associated with PE. Two patients (5.2%)had IJV DVT and PE in combination. Thrombosis was also reported at the sites of arteriovenous grafts, portal vein and in the central retinal vein. Twelve (31.5%) clots were related to the presence of indwelling central venous catheters. No significant association between presence of cancer or rheumatological disease and incident thrombosis was identified. Of those tested for beta 2 glycoprotein antibodies and anticardiolipin antibodies, no significant association was observed between presence of post-operative thrombosis and the presence of antibodies. While only 70 patients (39.8%) received any form of pharmacological prophylaxis against VTE, there was significant reduction in the incidence of VTE in patients that received prophylaxis as compared to those that did not. A statistically significant increased odds of thrombosis was observed in the neurosurgical population as compared to the patients undergoing other surgical procedures. On multivariate logistic regression analysis, neurosurgical patients had 3.4 fold increased risk of post operative thrombosis (CI 1.3-9.3) when compared to the rest of the surgical population studied, irrespective of the utilization of thromboprophylaxis. Conclusion: The incidence of thrombosis for patients with an incidental finding of LA indeterminate is 21.6%, comparable to that seen in the general population of patients undergoing similar procedures. This implies that standard guidelines should be used in choosing appropriate post-operative thromboprophylaxis in patients with this laboratory finding. More aggressive anticoagulant regimens do not appear to be necessary, although this remains to be confirmed in a controlled randomized study. Disclosures No relevant conflicts of interest to declare.

Description: Background: Sideroblastic anemia (SA) can present as congenital SA (CSA), acquired clonal SA, and acquired reversible SA. Patients (pts) with SA have anemia and ring sideroblasts (RS). Acquired clonal SA is often linked to myelodysplastic syndromes (MDS) or myelodysplastic/ myeloproliferative neoplasms (MDS/MPN). Clinico-pathologic overlap features, unmet morphologic and/or cytogenetic criteria complicate the diagnosis of SA leading to delayed therapies. Currently the diagnosis of SA is based on bone marrow (BM) examination and routine blood tests. There is a need to find easily testable biomarkers that can lead to faster diagnosis of clonal and non-clonal SA. Somatic mutation in splicing factor 3b, subunit 1 (SF3B1) are frequent in MDS-RS and MDS/MPN-RS and have been closely associated with RS. Objective: SF3B1 mutations can be a useful diagnostic biomarker for pts with acquired clonal SA who present with cytopenias and/or minimal morphologic changes suspicious of MDS and MDS/MPN but not sufficient to make a definitive diagnosis. Patients and Methods: Six pts with SA at presentation and seen at Cleveland Clinic were included in this study. The median age was 38 years (range, 6-75). Blood tests and BM biopsy showed persistent anemia [Hgb, 10.5 g/dL (range, 8.8-13)], RS [numerous (3 pts), 15% (1 pt), rare (1 pt) and 〉15% (2 pts)], 3/6 pts had minimal erythrodysplasia with 1 pt having a mild megakaryocytic dysplasia, 3 pts had hypercellular (60-90%), 2 pts had normocellular (50%, 80%) and 1 pt had hypocellular BM (30%) for age, and 〈 5% BM (2%=2 pts; 1%=1 pt; 3%=1 pt). Two pts had PLT count 〉 400 k/uL, 3 pts had 〉 100 k/uL, and 1pt

Description: Abstract 457 Ring sideroblasts (RS) are abnormal nucleated erythroblasts characterized by iron granules in mitochondrial cristae. RS are seen in acquired and congenital sideroblastic anemia. Dysfunction in mitochondrial metabolism has been implicated in the pathogenesis of RS. However, the true mechanism leading to RS formation remains elusive. Clonal sideroblastic anemias are usually acquired in the context of MDS: 15% or more RS in the bone marrow (BM) with appropriate morphologic and cytogenetic criteria in MDS is best classified as RARS, but varying quantities of RS (

Description: Whole exome/genome sequencing has been fundamental in the identification of somatic mutations in the spliceosome machinery in myelodysplastic syndromes (MDSs) and other hematologic disorders. SF3B1, splicing factor 3b subunit 1 is mutated in 60%-80% of refractory anemia with ring sideroblasts (RARS) and RARS associated with thrombocytosis (RARS-T), 2 distinct subtypes of MDS and MDS/myeloproliferative neoplasms (MDSs/MPNs). An idiosyncratic feature of RARS/RARS-T is the presence of abnormal sideroblasts characterized by iron overload in the mitochondria, called RS. Based on the high frequency of mutations of SF3B1 in RARS/RARS-T, we investigated the consequences of SF3B1 alterations. Ultrastructurally, SF3B1 mutants showed altered iron distribution characterized by coarse iron deposits compared with wild-type RARS patients by transmission electron microscopy. SF3B1 knockdown experiments in K562 cells resulted in down-regulation of U2-type intron-splicing by RT-PCR. RNA-sequencing analysis of SF3B1 mutants showed differentially used genes relevant in MDS pathogenesis, such as ASXL1, CBL, EZH, and RUNX families. A SF3B pharmacologic inhibitor, meayamycin, induced the formation of RS in healthy BM cells. Further, BM aspirates of Sf3b1 heterozygous knockout mice showed RS by Prussian blue. In conclusion, we report the first experimental evidence of the association between SF3B1 and RS phenotype. Our data suggest that SF3B1 haploinsufficiency leads to RS formation.

Description: Abstract 1701 Patients with features of MDS and MDS/MPN who do not fulfill diagnostic criteria for a specific subtype of MDS and MDS/MPN are categorized by the WHO 2008 diagnostic criteria as MDS-U and MDS/MPN-U. MDS includes RCUD, RCMD, RARS, RAEB-1, RAEB-2, MDS-U and 5q- syndrome while MDS/MPN includes CMML, JMML, atypical CML and MDS/MPN-U. The natural history of patients who belong to these disease subtypes are hetergeneous. Although included in currently accepted prognostic scoring schemes like the International Prognostic Scoring System (IPSS) in MDS, Revised IPSS, and MD Anderson prognostic scoring schemes, they represent a minority of patients in the cohort. Within MDS/MPN cases, the clinical heterogeneity of diseases that belong to this group has been recognized and has led to the development of the MD Anderson prognostic Scoring System for CMML. Similarly, a prognostic scoring system for JMML has also been devised to help in risk stratification and treatment decisions. However, there are no prognostic scoring systems for unclassified cases of MDS and MDS/MPN. Clinically, we observe stark differences in treatment responses and clinical outcomes between MDS/MPN-U and other MDS/MPN-subtypes, and MDS-U with other subtypes of MDS. In total, we studied 92 patients with unclassifiable cases seen at the Cleveland Clinic, including MDS/MPN-U (n=52 [57%]) and MDS-U (n=40 [43%]). Hematologic, bone marrow (BM), cytogenetic (metaphase cytogenetic [MC]/SNP-A) and survival data were collected. Survival comparisons were made by Kaplan-Meier analyses. Cox-proportional hazard ratio was used to determine factors predictive of outcomes. A p-value of ≤0.05 was considered statistically significant. In this cohort, median age at diagnosis was 69 years (20–88), 65% (60/92) were male, and 35% (32/92) were female. Median follow-up was 21 months. Median absolute neutrophil count (ANC) was 2.69k/uL (0–87), peripheral blood (PB) blasts 0% (0–70%), hemoglobin 9.6g/dL (5–15), and LDH 260 U/L (105–2113). SNP-A karyotyping was completed for 65 patients, and new cytogenetic mutations were detected in 72% (47/65): (gains [64%], losses [57%], UPDs [25%]). In 52% (49/92) of patients, we sequenced molecular mutations that typically confer poor prognosis in myeloid neoplasms, such as ASXL1, IDH1/2, EZH2, K/NRAS, CBL and TP53. This sequencing revealed a mutational frequency of 18% (9/49) in TET2, 14% (7/49) in ASXL1, 6% (3/49) in EZH2 exons 18–19, 2% (1/49) in CBL, 2% (1/49) in NRAS, and 4% (2/49) in TP53. No mutations were found in IDH1/2 and KRAS. In univariate analysis of clinciopathologic factors, the following factors were found to be associated with overall survival: ANC (≥8.5 vs