53 results on '"Heesun J. Rogers"'
Search Results
2. Metabolic Dysfunctions Driven By PRPF8 Explain Cases with Ring Sideroblasts without SF3B1 Mutations: Similarity/ Differences of Genotype/ Phenotype Association
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Valeria Visconte, Arda Durmaz, Hussein Awada, Ishani Nautiyal, Tariq Kewan, Waled Bahaj, Anjali Advani, Caroline Astbury, Heesun J. Rogers, Jaroslaw P. Maciejewski, and Carmelo Gurnari
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Published
- 2022
3. Vacuolization of hematopoietic precursors: an enigma with multiple etiologies
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Hetty E. Carraway, Valeria Visconte, Laila Terkawi, Eric D. Hsi, Lisa Durkin, Sunisa Kongkiatkamon, Heesun J. Rogers, Hassan Awada, Simona Pagliuca, Carmelo Gurnari, Misam Zawit, and Jaroslaw P. Maciejewski
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0301 basic medicine ,Adult ,Male ,Pathology ,medicine.medical_specialty ,Zinc poisoning ,Immunology ,Bone Marrow Cells ,Ubiquitin-Activating Enzymes ,Biochemistry ,03 medical and health sciences ,0302 clinical medicine ,Erythroid Cells ,Genes, X-Linked ,Medicine ,Humans ,Myeloid Cells ,Child ,Myeloid Progenitor Cells ,Aged ,Aged, 80 and over ,Myeloproliferative Disorders ,business.industry ,Cell Biology ,Hematology ,Syndrome ,Middle Aged ,Settore MED/15 ,Haematopoiesis ,030104 developmental biology ,Vacuolization ,030220 oncology & carcinogenesis ,Mutation ,Vacuoles ,Etiology ,Female ,business - Published
- 2021
4. Molecular Derivation of Extramedullary Myeloid Sarcomas Based on Machine Learning Analysis of Genomic Clusters in AML
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Hassan Awada, Carmelo Gurnari, Hetty E. Carraway, Simona Pagliuca, Tariq Kewan, Jaroslaw P. Maciejewski, Heesun J. Rogers, Anjali S. Advani, Sudipto Mukherjee, Waled Bahaj, Bhumika J. Patel, Valeria Visconte, Suresh Kumar Balasubramanian, and James R. Cook
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Myeloid ,medicine.anatomical_structure ,Computer science ,Immunology ,medicine ,Cell Biology ,Hematology ,Computational biology ,Biochemistry - Abstract
Extramedullary deposits of leukemic blasts, also often called myeloid sarcoma (MS), constitute an unusual presentation of AML, which can occur in isolation (IMS, often in the de novo setting) or in the context of a fully-blown leukemic process (LMS) either at diagnosis or relapse. This peculiar variant of an acute leukemia may be due to distinct phenotypic features promoting homing of blasts to non-hematopoietic locations or/and be a result of specific molecular lesions (perhaps related to a specific genotype). So far, systematic studies of these phenomenon have been scarce with the largest cohorts reporting on 70 pts and less over the last decade. By applying machine learning (ML) to perform latent class analysis of molecular features in AML patients (n=6,788), we have identified and validated 4 molecular clusters characterized by distinct clinical phenotypes (Awada et al Blood 2021). Genomic cluster-1 (GC-1) was characterized by predominantly normal karyotype and NPM1 MT that co-occurred with DNMT3A MT and FLT3 ITD, and IDH2 R140. In GC-2 mostly abnormal karyotype was present with CEBPA Bi, and IDH2 R172K, FLT3 ITD/TKD and lack of NPM1 MT. GC-3 had highest frequency of ASXL1 MT, BCORL1 MT, and DNMT3A MTalong with splicing factor mutations. Finally, GC-4 had the highest percentage of complex karyotype and TP53 MT. We observed a significant difference in survival [medians, months] across the molecularly-defined subgroups: GC-1 [34.1]; GC-2 [26.5]; GC-3 [15.8]; and GC-4 [9.2]. However, the genomic clustering (GCs 1-4) did not favor pAML over sAML (progressed from previous myeloid disorders). These unbiased molecular features were then compared to those found in 91 MS pts (median age 64 years) to discern their possible origins from specific genomic AML clusters vs. an as yet described unique MS genomic signature. Among the 91 pts, 19% of them were IMS while 81% were LMS (of which 43% were de novo vs. 57% refractory/relapsed). Soft tissue (32%), skin (22%), lymph nodes (13%), and gastrointestinal tract (13%) were the most common extramedullary sites involved. An involvement of more than two sites was observed in 15 (16.5%) cases. MS-specific phenotypic features were explored based on immunohistochemical (IHC) stains and flow cytometry studies. MS neoplastic cells showed reactivity with CD43 (54% of cases), muramidase/lysozyme (53%), CD117 (39%), myeloperoxidase (35%), CD33 (32%), CD45 (30%), and CD34 (30%). Flow cytometry of the LMS cases demonstrated that 90% of patients expressed surface CD13, CD33, CD45, and HLA-DR while positivity for CD38 was present in 83% of cases, and CD117 and CD34 in 60%. By comparison to our AML cohort, MS patients had a significant higher median age at the time of diagnosis (64 vs. 62 years p=0.041) and leukocyte counts (WBC 7k vs. 4k/uL, p=0.002), higher percentage of sAML (67% vs. 40% p Disclosures Balasubramanian: Servier Pharmaceuticals: Research Funding. Patel: Apellis: Consultancy, Other: educational talks, Speakers Bureau; Alexion: Consultancy, Other: educational talks, Speakers Bureau. Mukherjee: McGraw Hill: Honoraria, Other: Editor of Hematology Oncology Board Review (ongoing); Acceleron: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Membership on an entity's Board of Directors or advisory committees; AAMDS in Joint Partnership with Cleveland Clinic Taussig Cancer Institute: Honoraria; Genentech: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb Co.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Research/Independent Contractor, Research Funding; Eusa Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Teaching and Speaking; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Research/Independent Contractor, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees; Partnership for Health Analytic Research: Honoraria; BioPharm: Consultancy; Jazz Pharmaceuticals: Research Funding. Advani: Macrogenics: Research Funding; Glycomimetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Research Funding; Kite Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Immunogen: Research Funding; OBI: Research Funding; Abbvie: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Carraway: Celgene, a Bristol Myers Squibb company: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Other: Independent review committee; Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Stemline: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Other: Independent review committee; Astex: Other: Independent review committee; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Maciejewski: Bristol Myers Squibb/Celgene: Consultancy; Novartis: Consultancy; Regeneron: Consultancy; Alexion: Consultancy.
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- 2021
5. Lymphocyte Cytosolic Protein 1 I232F Mutation Impairs Granulocytic Proliferation with a G2/M Block in Severe Neutropenia
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Seth J. Corey, Aron Flagg, Heesun J. Rogers, Upendra Mahat, Andrei I. Ivanov, Hrishikesh M Mehta, Bhavuk Garg, and Chao Yie Yang
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Lymphocyte cytosolic protein 1 ,Block (telecommunications) ,Immunology ,Mutation (genetic algorithm) ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Molecular biology ,Severe neutropenia - Abstract
Introduction: As critical effectors of innate immune response, neutrophils migrate from the vasculature into inflamed tissue, where they engage in phagocytosis and clearance of pathogens and apoptotic cells. Migration, phagocytosis, and granule release require multiple well-coordinated events involving remodeling of the actin cytoskeleton. While mutations of some regulators of actin polymerization, such as Wiskott-Aldrich Syndrome gene (WAS) result in Severe Congenital Neutropenia (SCN), the role of actin cytoskeleton in regulating neutrophil ontogeny and functions remain poorly understood. Lymphocyte Cytosolic Protein 1 (LCP1) is an actin bundling protein encoded by LCP1 gene. A 7-year-old girl presented at age of 2 years with chronic severe neutropenia and frequent infections. Next generation sequencing did not reveal a variant in genes known to cause neutropenia. Whole exome sequencing identified a novel LCP1 p.I232F variant of unknown significance, which was not present in either parent. Bone marrow examination revealed a granulocytic hypoplasia with significant dysmorphic giant granulocytes with complex hypersegmentation and increased apoptosis. (Figure 1A) Based on these observations, we hypothesized that missense mutation in LCP1 (I232F) impairs granulocytic proliferation, and causes neutropenia. Aims: To investigate the role of LCP1 I232F, discovered in a child with severe symptomatic neutropenia, in granulopoiesis. Methods: in vitro experiments involving the expression of wild type and mutant LCP1 I232F in murine myeloblast cell (32D cells) and human cervical cancer HeLa cell. We deployed various biologic and functional studies to investigate the role of LCP1 I232F in granulocyte proliferation, differentiation, survival, and function. Results: To determine the pathophysiologic significance of LCP1 I232F, we stably transduced interleukin 3-dependent murine myeloblast 32D and human cervical cancer HeLa cell lines with a doxycycline-inducible lentiviral constructs containing the cDNA for either wild type LCP1 or LCP1 I232F. The mutant LCP1 I232F expressing 32D cells showed impaired proliferation with the appearance of dysplastic granulocytic cells. (Figure 1A, B) However, we did not observe block in differentiation. A cell cycle arrest at G2/M phase occurred only in the LCP1 I232F expressing cells (Figure 1B) and was associated with an upregulation of markers of cell cycle arrest (Cdkn1a and Tp53). LCP1 I232F overexpression did not lead to increased expression of genes involved in apoptosis or the unfolded protein response. The LCP1 I232F cells with dysplastic features were further analyzed by confocal microscopy, which demonstrated increased level of F-actin and enrichment of F-actin and LCP1 at the cell cortex. Flow cytometric evaluation of these cells further confirmed the presence of increased level of F actin. (Figure 1C) Expression of LCP1 I232F impaired cell motility and invasiveness in both 32D and HeLa cells. However, mutant LCP1 expressing 32D cells demonstrated normal oxidative burst upon treatment with N-formyl-Met-Leu-Phe (fMLP) and phorbol myristate acetate (PMA). Confocal imaging and subcellular fractionation revealed diffuse localization of LCP1, but only the mutant form was found in the nucleus. (Figure 1C) In LCP1, two tandem CH domains (CH1-CH2 and CH3-CH4) formed two actin-binding domains (ABD1 and ABD2) that bind to actin to participate in actin bundling. I232F is located at LCP1-CH1 that interacts with actin. To study how the mutation impacts LCP1-actin binding, we constructed a model of LCP1-ABD1 based on the cryo-EM structure of F-actin/LCP1-ABD2 and performed 40 ns of molecular dynamics (MD) simulations of LCP1-ABD1 and LCP1-ABD1 I232F alone to study their dynamical motions. We found that LCP1-ABD1 I232F adopted a more stable open conformation than LCP1-ABD1 to bind effectively with F-actin. The model implies a prolonged association of LCP1 I232F with actin than wild-type LCP1, and the consequence is the increased stabilization of actin bundles. Conclusions: Like the WAS gain-of-function mutations in SCN, LCP1 I232F resulted in severe neutropenia with dysplastic granulocytic precursors, cell cycle arrest, impaired motility, and increased F actin. We also found decreased proliferation of granulocytic progenitor and precursor cells. Our findings suggest that LCP1 is an important gene involved in granulopoiesis. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
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- 2021
6. Monoclonal IgM gammopathy in adult acquired pure red cell aplasia: culprit or innocent bystander?
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Valeria Visconte, Simona Pagliuca, Carmelo Gurnari, Bhumika J. Patel, Hassan Awada, Heesun J. Rogers, Misam Zawit, Jason Valent, and Jaroslaw P. Maciejewski
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Male ,Pathology ,medicine.medical_specialty ,IgM ,Acquired Pure Red Cell Aplasia ,Monoclonal igm ,Pure red cell aplasia ,Red-Cell Aplasia, Pure ,Monoclonal Gammopathy of Undetermined Significance ,Culprit ,Bone Marrow ,Gammopathy ,medicine ,Bystander effect ,Humans ,Waldenström's macroglobulinemia ,Molecular Biology ,Aged ,Bone marrow failure syndromes ,business.industry ,Cell Biology ,Hematology ,Middle Aged ,Settore MED/15 ,medicine.disease ,Immunoglobulin M ,Bone Marrow failure syndromes ,MGUS ,Molecular Medicine ,Waldenstrom Macroglobulinemia ,business - Published
- 2021
7. Molecular and Clinical Aspects of Acute Myeloid Leukemia with Inv(3)(q21q26)/t(3;3)(q21;q26) Carrying Spliceosomal Mutations
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Heesun J. Rogers, Valeria Visconte, Hassan Awada, Cassandra M Kerr, and Jaroslaw P. Maciejewski
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business.industry ,Immunology ,Cancer research ,Myeloid leukemia ,Medicine ,Cell Biology ,Hematology ,business ,Biochemistry - Abstract
Inversion or translocation of the chromosome 3, specifically inv(3)(q21q26.2/ t(3;3)(q21;q26.2) are present in 1-2% of acute myeloid leukemia (AML) cases and are classified as a distinct entity in the 2016 WHO classification. Hallmark genetic alterations in this entity include mutations in GATA2 and MECOM. In fact, these rearrangements result in over activation of MECOM due to juxtaposition with a distal GATA2 enhancer. Cytomorphologic phenotypes include anemia, normal to elevated platelets and multilineage dysplasia in a hyperplastic bone marrow (BM). Studies have shown the frequent occurrence of NF1, NRAS and RUNX1 mutations. While proceeding towards our molecularly informed AML subtyping (Awada, Blood 2019;1406), we observed a high occurrence of somatic mutations in the splicing factor SF3B1 in inv(3)/t(3;3) AML. We were particularly intrigued by this observation considering several key aspects of SF3B1 mutations in the context of MDS. For instance, SF3B1 mutations are highly associated with clear phenotypic and morphologic features and carry favorable prognosis in MDS. These mutations are often found in patients carrying less deleterious abnormalities [e.g., del(5q)] and their founder clonal nature has been uncovered through experimental studies. Recent studies unveiled the occurrence of SF3B1 mutations in de novo AML and low complete remission rate when combined with other mutations (e.g., DNMT3A). To investigate whether SF3B1 mutations were unequivocally frequent in inv(3)/t(3;3) AML compared to other splicing factor mutations, we moved forward in dissecting the clinical, morphologic and molecular profiles of these cases. We analyzed results from whole exome sequencing and targeted deep sequencing from the Cleveland Clinic and publicly available data of AML with inv(3)/t(3;3) (de novo AML, n=32; secondary AML from antecedent myeloid neoplasms, n=11; t-AML, n=1). In our cohort, mutations in the most common components of the RNA splicing machinery (SF3B1, SRSF2, U2AF1, ZRSR2) were observed in 27% (n=12) of the patients. Among splicing factor mutations, SF3B1 was the most mutated gene (77%; 10/13 total mutations); 7 cases had inv(3) and 3 had t(3;3). Mutations were observed at canonical sites: K700E (70%) and K666N (30%) with no difference compared to the hotspots observed in MDS. Sixty% of patients were female. Median age was 61 years (range, 36-73). Anemia was present in 50%, leukopenia in 10% and thrombocytopenia in 50% of the patients. For 40% of the cases, BM smears for iron staining was available and showed absence of ringed sideroblasts. Complex karyotype (CK) was present in 20% of the patients; -7/del(7q) was present as the only cytogenetic abnormality in 30% or with CK in 10% pf the cases. Variant allele frequency (VAF) of SF3B1mutations in inv(3)/t(3;3) was not different than the those without inv(3)/t(3;3) (42% vs 40%). Survival analysis was performed in 3 subgroups: SF3B1MT AML (n=70), SF3B1MT AML + inv(3)/t(3;3) (n=10), AML + inv(3)/t(3;3) (n=34). SF3B1MT AML + inv(3)/t(3;3) and AML + inv(3)/t(3;3) had similar OS (11.7 vs 9.7 months) which was shorter than the that of SF3B1MT AML without any inv(3)/t(3;3) (19.4 months, P=0.002) suggesting that SF3B1MT in the context of inv(3)/t(3;3) might hold a different prognostic significance strongly due to the presence of inv(3)/t(3;3). Given this observation, we delved into the clonal diversity of SF3B1 mutations and its co-occurrence with other molecular mutations. Comparison of VAFs showed that SF3B1 mutations in relation to other mutations were dominant/founder in 30%, secondary/subclonal in 20% while co-dominant to another gene (VAF differences In sum, we describe that SF3B1 mutations occur in combination with inv(3)/t(3;3) in AML and might represent a subclass of this entity in which lesions in SF3B1 gene could potentially hide a cryptic association between splicing abnormalities and disease phenotypes. Figure 1 Disclosures Maciejewski: Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria.
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- 2020
8. The Clonal Trajectories of SF3B1 Mutations in Myeloid Neoplasia
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Hetty E. Carraway, Heesun J. Rogers, Hassan Awada, Carmelo Gurnari, Mikkael A. Sekeres, Cassandra M Kerr, Vera Adema, Valeria Visconte, Simona Pagliuca, and Jaroslaw P. Maciejewski
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Oncology ,medicine.medical_specialty ,Univariate analysis ,Immunology ,Significant difference ,Context (language use) ,Cell Biology ,Hematology ,Variant allele ,Biology ,Biochemistry ,Somatic evolution in cancer ,Zygosity ,Myeloid neoplasia ,Internal medicine ,medicine ,Overall survival - Abstract
Molecular lesions have diagnostic and prognostic impacts in myeloid neoplasia (MN). The beau idéal is the presence of SF3B1MT in MDS with ringed sideroblasts. Although most of these patients have a classic phenotype, little is known about the factors diverting it during the clonal evolution. Clinical trajectories of patients with SF3B1MT might depend on clonal hierarchy, dynamics and subclonal diversity in relation to other lesions. Herein, we studied the molecular architecture of patients with SF3B1MT to determine whether clonality and rank might infer distinct MDS features. We collected molecular and clinical information of 3561 patients with MN from Cleveland Clinic and publicly shared data. Results of targeted deep sequencing of 176 MDS/ AML genes were included. We applied an in-house variant allele frequency (VAF) based bioanalytic method to resolve clonal hierarchy. VAF (adjusted for copy number and zygosity) was used to classify the mutations into dominant (if a cutoff of at least 5% difference between VAFs existed), secondary (any subsequent subclonal hit) and codominant hits ( SF3B1 MT were detected in 9% of patients (n=335): 140 were SF3B1DOM (42%), 121 were SF3B1SEC (36%) and 74 were codominant (SF3B1COD, 22%). Comparison of SF3B1MT VAFs among the 3 groups showed no significant difference (40 vs 39 vs 44%, P= .6). Due to lack of distinct partition, we delved into the implications of SF3B1COD on mutational stability, disease phenotypes, molecular associations and overall survival (OS) as compared to the other 2 configurations. Serially-collected samples (n=21) showed clear scenarios of dominant clones remaining stable or switching to secondary due to impending acquisition of other hits or secondary clones remaining as such but denoted an ambivalent framework of codominant clones with "sweeping" features. Cases with SF3B1COD had a trend of OS similar to that of SF3B1SEC (median OS: 18 vs 15.9 mo.) and as such a poorer outcome compared to that of SF3B1DOM (39.8 mo.; P= .02). Moreover, the mutational profile of SF3B1CODvsSF3B1DOM was partially similar to that of SF3B1SECvs.SF3B1DOM with significantly higher odd ratios for DNMT3A (P< .0001), ASXL1, FLT3, RUNX1, TET2 (P≤ .05 for each). No variant morphologic features were observed when comparing SF3B1COD to the other 2 groups. By virtue, we strictly compared only cases with SF3B1DOM and SF3B1SEC, further supported by our single cell analysis(n=5), to showan unambiguous distinction between these two statii. Mutational burden analysis revealed that SF3B1DOM patients had a fewer number of mutations than those with SF3B1SEC (median 1.0 vs 2.6). SF3B1DOM was mostly detected in patients with a normocellular bone marrow as compared to SF3B1SEC (46 vs 29%, P= .02) and had a lower proportion of multidysplastic myeloid cells (29 vs 53%, P= .01). On the other side, SF3B1SEC were often present in the context of bilineage dysplasia (47vs. 26%, P= .02). Focused morphologic analysis revealed that SF3B1DOM cases were significantly associated with a higher RS% (median 37 vs 3%, P= .002; frequency of RS ≥15%: 77 vs 44%, P= .003) with higher VAF% significantly correlating with higher RS%. Patients with SF3B1SEC had shorter OS than those with SF3B1DOM (15.9 vs 39.7 mo., P< .0001). Even by dichotomizing according to VAF, the median OS of cases with VAF>40% was significantly shorter than those with VAF40%, the OS was shortened compared to SF3B1DOM (31 vs 11.6 mo., P= .001) and similarly when it laid between 20% and 40% (49.7 vs 25.6 mo., P= .01) suggesting a strong impact of associated hits. In SF3B1SEC, univariate analyses showed significantly higher odds of hits in RUNX1 (43 vs 19%, P< .0001), TET2 (29 vs 11%, P= .0005), FLT3 (22 vs 11%, P= .02), DNMT3A (20 vs 7%, P= .004), ASXL1 (16 vs 5%, P= .06), BCOR/L1 (17 vs 5%, P= .005), IDH1/2 (11 vs 2%, P= .008), CBL (8 vs 1%, P= .009) and CEBPA (7 vs 2%, P= .04) compared to SF3B1DOM. Interestingly, cases with co-existing TET2 mutations had a marked decrease in OS in SF3B1SECvsSF3B1DOM(10.1 vs 96.1 mo., P= .02) suggesting that the mutational ranking in a disease triggered by SF3B1MT can be skewed by stronger hits. In sum, our study suggests that molecular ranking in the context of SF3B1 clonal configuration is a key factor diverting the clinical and phenotypic trajectories of patients with MN and SF3B1MT. Disclosures Carraway: BMS: Consultancy, Other: Research support, Speakers Bureau; Jazz: Consultancy, Speakers Bureau; Stemline: Consultancy, Speakers Bureau; Takeda: Other: Independent Advisory Committe (IRC); ASTEX: Other: Independent Advisory Committe (IRC); Abbvie: Other: Independent Advisory Committe (IRC); Novartis: Consultancy, Speakers Bureau. Sekeres:Pfizer: Consultancy; BMS: Consultancy; Takeda/Millenium: Consultancy. Maciejewski:Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria.
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- 2020
9. The Biological Inferences from the Ranking of SF3B1 Mutations in the Clonal Hierarchy of Myeloid Neoplasia
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Jaroslaw P. Maciejewski, Hetty E. Carraway, Vera Adema, Jack Khouri, Bartlomiej P Przychodzen, Manja Meggendorfer, Hassan Awada, Ashwin Kishtagari, Cassandra M Kerr, Sunisa Kongkiatkamon, Torsten Haferlach, Aziz Nazha, Jibran Durrani, Mikkael A. Sekeres, Heesun J. Rogers, Anjali S. Advani, and Valeria Visconte
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Genetics ,Mutation ,Hierarchy (mathematics) ,Immunology ,Disease progression ,Cell Biology ,Hematology ,Biology ,medicine.disease ,medicine.disease_cause ,Biochemistry ,Ranking (information retrieval) ,Myeloid neoplasia ,Leukemia ,medicine ,Platelet Count measurement ,Protein p53 - Abstract
Chromosomal abnormalities can be founder lesions (e.g., t (8; 21), inv (16), inv (3)), initiate or advance disease progression (both founder and secondary hits e.g., ASXL1, TP53, RUNX1) or can be obligatory secondary hits (FLT3, NPM1). Hence, the rank of these mutations may determine the biological properties and clinical outcomes. However, while many mechanistic studies have been undertaken without identifying the key pathogenetic factors resulting from SF3B1 mutations, important biological clues can be derived from the consequences of SF3B1 alterations in the context of the clonal architecture of myeloid neoplasia (MN). SF3B1 mutant patients often have a homogeneous phenotype with isolated erythroid dysplasia, ring sideroblasts (RS) and favorable prognoses. Studies in primary MDS cells have suggested that SF3B1 mutations are initiating lesions and provide a marked clonal advantage to MDS-RS cells by propagating from rare lympho-myeloid hematopoietic stem cells. However, there is significant diversity of clinical phenotypes and outcomes including the observation that the disappearance of RS can be observed during the disease course of clonal MN and might suggest cellular shifts due to acquisition of additional hits. In such scenarios, the cell's fate in the context of SF3B1 mutations is pre-defined by the predominance of expanded hits. We took advantage of our detailed database of molecularly and clinical annotated cases with MN to study the SF3B1 mutatome and describe whether the clonal nature (ancestral vs. secondary) might change the clinical and phenotypic trajectories of MDS cells and whether the concatenation of mutations decreases the competitiveness of SF3B1 clones, leading to the dominance of other driver genes and subsequently to clonal evolution. The clonal hierarchy was resolved using our in-house designed VAF-based bioanalytic method and confirmed by the PyClone pipeline, which showed a high level of concordance. We first assigned clonal hierarchy to SF3B1 mutations by using VAFs (adjusted for copy number and zygosity) and classifying the mutations into dominant (if a cutoff of at least 5% difference between VAFs existed), secondary (any subsequent sub-clonal hit) and co-dominant hits (if the difference of VAFs between two mutations was In conclusion, our study of the clonal architecture of SF3B1 mutations highlights that clonal progression of cases with MN harboring SF3B1 mutations might be inferred by the rank of additional genetic lesions cooperating with SF3B1. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Advani:Abbvie: Research Funding; Macrogenics: Research Funding; Pfizer: Honoraria, Research Funding; Amgen: Research Funding; Glycomimetics: Consultancy, Research Funding; Kite Pharmaceuticals: Consultancy. Nazha:Tolero, Karyopharma: Honoraria; Novartis: Speakers Bureau; MEI: Other: Data monitoring Committee; Daiichi Sankyo: Consultancy; Jazz Pharmacutical: Research Funding; Incyte: Speakers Bureau; Abbvie: Consultancy. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Sekeres:Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion: Consultancy; Novartis: Consultancy.
- Published
- 2019
10. Pharmacologic Normalization of Altered Transcriptome of SF3B1 Mutant Myeloid Neoplasia
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Hetty E. Carraway, Cassandra M Kerr, Hassan Awada, Allison Noe, Yuriy Fedorov, Steffan T. Nawrocki, Daniel J. Lindner, Heesun J. Rogers, Bartlomiej P Przychodzen, Anjali S. Advani, Yvonne Parker, Kevin R. Kelly, James G. Phillips, Vera Adema, Rachel Toth, Valeria Visconte, Drew J. Adams, Anand D. Tiwari, Jennifer S. Carew, Mikkael A. Sekeres, Christina Snider, and Jaroslaw P. Maciejewski
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Immunology ,Cell ,CD34 ,Cell Biology ,Hematology ,Biology ,Pharmacology ,Biochemistry ,Transplantation ,Transcriptome ,chemistry.chemical_compound ,medicine.anatomical_structure ,chemistry ,In vivo ,medicine ,Erythropoiesis ,Bone marrow ,Growth inhibition - Abstract
SF3B1 mutations disrupt normal pre-mRNA splicing to cause disease. Drugs inhibiting the interaction between the SF3b complex and RNA or agents degrading auxiliary splicing factors are being tested as new avenues for targeted therapy in myeloid neoplasia (MN) with SF3B1 mutations. Here we describe the ability of small molecules to restore altered RNA processes in SF3B1MT MN. We previously reported (Visconte, ASH 2018) the identification of the small molecule 4-pyridyl-2-anilinothiazole (PAT) which showed growth inhibition of CRISPR/Cas9 SF3B1+/K700E cells and primary SF3B1MT cells. PAT did not influence the growth of other cell models without (THP1, MOLM13FLT3, OCIAML3DNMT3A, SIGM5TET2/DNMT3A, K562PHF6) and with other splicing factor mutations (K562U2AF1, K562LUC7L2). We now describe data from medicinal chemistry, transcriptome, and in vivo studies to advance drug development for SF3B1MT MN. SAR studies focused on logical and systematic modifications of PAT, e.g., i) replacement of the 2,4-disubstituted thiazole spacer ring with other heteroatom containing rings (5,6,7 membered aromatic or aliphatic ring structures); ii) alternative linking groups for the NH linker of the aniline of the tail region (sulfonamide, amide, substituted amine linkers); iii) alternative substituted aromatic and aliphatic ring structures for the phenyl head region substituent, led us to identify permissive sites for further chemical optimization. For example, a 4-chlorophenyl analog demonstrated activity [IC50, 3μM] similar to PAT. Competitive repopulation assays of bone marrow (BM) cells from dual reporters (ACTBtdTomato; EGFP) B6.GtROSA26 mixed with BM cells from conditional knock-in Sf3b1+/K700E mice injected in pre-lethally irradiated B6.SJL-PtprcaPepcb/BoyJ (CD45.1) recipients (n=18) were used as a preclinical murine model. This model then allowed i) demonstration of drug efficacy in reducing the competitiveness of SF3B1MT cells and ii) evaluation of therapeutic index in normal hematopoiesis. Post-transplant recovery, recipients of B6.GtROSA26 cells underwent PAT treatment (10 mg/Kg/IP/5 days weekly) for a period of 6 weeks without showing any signs of distress or drug intolerance (drop in blood count, weight loss, abdominal swelling, liver or kidney toxicity). Two weeks after transplantation, donor Sf3b1+/K700E cells had an engraftment capability similar to that of donor B6.GtROSA26 cells (83.6 ± 4 vs. 86.4 ± 2.4) when transplanted as a sole graft in CD45.1 recipients. PAT reduced almost half the percentage of Sf3b1+/K700E donor cells at 6 weeks of treatment (47.4%) vs. pre-treatment (83.6%). In mixed (1:1) BM transplants, Sf3b1+/K700E cellshad a repopulative disadvantage against competitors B6.GtROSA26 contributing for 16% of the marrow reconstitution. Similar to single graft transplants, PAT decreased the percentage of Sf3b1+/K700E cells at 6 weeks vs. pre-treatment (average, 6% vs. 16%) in chimeras. Consistent with the lack of toxicity of PAT treatment B6.GtROSA26 cells in chimeras were not affected by PAT and gradually repopulated the host (post-treatment, 80% vs. pre-treatment, 64%). Subsequently, we focused our efforts identifying important genes known to be dysregulated in MDS that were mostly influenced by drug treatment and minimally affected in normal cells. Our approach was based on the analyses of genes linked to erythropoiesis (a key hallmark of low-risk MDS). In normal hematopoiesis TGF-β signaling inhibits terminal erythroid maturation. Out of 13,775 genes, 5% (664/13,775) were found differentially expressed between CRISPR/Cas9 SF3B1+/K700E and parental cells of which 60% of these genes were significantly up-regulated and 40% down-regulated. Pathway analysis showed that the expression levels of SMAD family of genes and GDF factors changed significantly upon drug treatment. SMAD7 mRNA levels are 3-fold lower in MDS CD34+ cells (n=159) compared to the ones of healthy subjects (n=17) (GEO accession GSE58831) leading to TGF-β over activation. PAT treatment normalized SMAD7 expression levels in CRISPR/Cas9 SF3B1+/K700E cells by 3-fold while reducing the levels of GDF11. In summary, we have identified new drug entities that are modulators of transcriptomic changes which decrease the competitiveness of SF3B1MT cells. These results suggest combination therapies with current TGF-β pathway inhibitors. Disclosures Advani: Glycomimetics: Consultancy, Research Funding; Kite Pharmaceuticals: Consultancy; Amgen: Research Funding; Pfizer: Honoraria, Research Funding; Macrogenics: Research Funding; Abbvie: Research Funding. Kelly:Novartis, Bayer, Janssen, Pharmacyclics, Celgene, Astrazeneca, Seattle Genetics: Honoraria, Speakers Bureau; Takeda: Research Funding; Genentech, Verastem: Consultancy. Sekeres:Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Novartis: Consultancy; Alexion: Consultancy.
- Published
- 2019
11. Atypical chronic myeloid leukemia is clinically distinct from unclassifiable myelodysplastic/myeloproliferative neoplasms
- Author
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Sa A. Wang, Rashmi Kanagal-Shamanna, Adam Bagg, Eric D. Hsi, Courtney D. DiNardo, Kathryn Foucar, Heesun J. Rogers, Joseph Hatem, Julia T. Geyer, Ramon V. Tiu, Keyur P. Patel, Jesse Jaso, Daniel A. Arber, Patricia S. Fox, Ken H. Young, Srdan Verstovsek, Francesco C. Stingo, Carlos E. Bueso-Ramos, Devon Chabot-Richards, Meenakshi Mehrotra, Raja Luthra, Attilio Orazi, Robert P. Hasserjian, and Elizabeth Weinzierl
- Subjects
Adult ,Blood Platelets ,Male ,Pathology ,medicine.medical_specialty ,Myeloid ,Clinical Trials and Observations ,Leukocytosis ,DNA Mutational Analysis ,Immunology ,Biology ,Biochemistry ,Gastroenterology ,Leukemia, Myeloid, Chronic, Atypical, BCR-ABL Negative ,Myelodysplastic–myeloproliferative diseases ,hemic and lymphatic diseases ,Internal medicine ,medicine ,Humans ,Granulocyte Precursor Cells ,Myeloproliferative neoplasm ,Aged ,Proportional Hazards Models ,Aged, 80 and over ,Hematology ,L-Lactate Dehydrogenase ,Myelodysplastic syndromes ,food and beverages ,Cell Biology ,Middle Aged ,Prognosis ,medicine.disease ,Myelodysplastic-Myeloproliferative Diseases ,Leukemia ,Treatment Outcome ,medicine.anatomical_structure ,Hematologic Neoplasms ,Karyotyping ,Myelodysplastic Syndromes ,Mutation ,Atypical chronic myeloid leukemia ,Female ,medicine.symptom ,Follow-Up Studies - Abstract
Atypical chronic myeloid leukemia (aCML) is a rare subtype of myelodysplastic/myeloproliferative neoplasm (MDS/MPN) largely defined morphologically. It is, unclear, however, whether aCML-associated features are distinctive enough to allow its separation from unclassifiable MDS/MPN (MDS/MPN-U). To study these 2 rare entities, 134 patient archives were collected from 7 large medical centers, of which 65 (49%) cases were further classified as aCML and the remaining 69 (51%) as MDS/MPN-U. Distinctively, aCML was associated with many adverse features and an inferior overall survival (12.4 vs 21.8 months, P = .004) and AML-free survival (11.2 vs 18.9 months, P = .003). The aCML defining features of leukocytosis and circulating myeloid precursors, but not dysgranulopoiesis, were independent negative predictors. Other factors, such as lactate dehydrogenase, circulating myeloblasts, platelets, and cytogenetics could further stratify MDS/MPN-U but not aCML patient risks. aCML appeared to have more mutated RAS (7/20 [35%] vs 4/29 [14%]) and less JAK2p.V617F (3/42 [7%] vs 10/52 [19%]), but was not statistically significant. Somatic CSF3R T618I (0/54) and CALR (0/30) mutations were not detected either in aCML or MDS/MPN-U. In conclusion, within MDS/MPN, the World Health Organization 2008 criteria for aCML identify a subgroup of patients with features clearly distinct from MDS/MPN-U. The MDS/MPN-U category is heterogeneous, and patient risk can be further stratified by a number of clinicopathological parameters.
- Published
- 2014
12. Clinical, Immunophenotypic and Genomic Findings of Acute Undifferentiated Leukemia and Comparison to AML with Minimal Differentiation: A Study from the Bone Marrow Pathology Group
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Olga K. Weinberg, Ezra Baraban, Julia T. Geyer, Robert P. Hasserjian, Chi Young Ok, Sa A. Wang, Eric D. Hsi, Attilio Orazi, John K Sydney Philip, Valentina Nardi, Jacqueline S. Garcia, Heesun J. Rogers, Adam Bagg, Richard Stone, Daniel A. Arber, and Jason H. Kurzer
- Subjects
Acute leukemia ,Pathology ,medicine.medical_specialty ,Myeloid ,biology ,business.industry ,CD117 ,Immunology ,CD33 ,Myeloid leukemia ,Induction chemotherapy ,Cell Biology ,Hematology ,Biochemistry ,Immunophenotyping ,medicine.anatomical_structure ,hemic and lymphatic diseases ,biology.protein ,Medicine ,Acute Undifferentiated Leukemia ,business - Abstract
Introduction: Acute undifferentiated leukemia (AUL) is a rare type of acute leukemia that shows no evidence of differentiation along any lineage. Clinical, immunophenotypic and genetic data is limited: the largest study to date reported 16 AUL cases but did not use the current WHO classification and included limited genetic data on 5 cases (Ann Hematol 2013 92:747-758). Moreover, it is uncertain if AUL is biologically distinct from acute myeloid leukemia with minimal differentiation (AML MD), which also shows limited myeloid marker expression and has been reported to have a poor prognosis. Methods: A total of 95 cases (36 AUL cases 59 AML MD) were identified from pathology databases of eight academic institutions with available diagnostic flow cytometric data, cytogenetic findings, and clinical data by searching for diagnoses of "AUL" or "AML MD". Diagnosis of AUL required absence of any lineage-defining markers including MPO, CD19 and CD3. Using the WHO classification, diagnosis of AML with MD required expression of at least 2 myeloid markers (CD117, CD13 or CD33), absence of myeloid maturation (CD15) or monocytic markers (CD64, CD11b, lysozyme or non-specific esterase). Next generation sequencing with extensive mutational panel data was available in 78 cases. Outcome analysis for overall survival (OS) and relapse-free survival (RFS) and were performed using Kaplan Meier and log rank test for patients who received induction chemotherapy. Results: Based on cytogenetic abnormalities (N=27) or history of MDS (N=2), according to the 2016 WHO Classification, 28 cases (6 AUL and 22 AML MD) were re-classified as AML with myelodysplasia related changes (AML MRC). The remaining 30 AUL patients presented with similar age, blood counts, bone marrow cellularity, and blast percentage as the 37 AML MD patients (all p > 0.05). Comparison of immunophenotype in the two groups showed that AUL blasts had more frequent expression of TdT (p=0.0003) and lacked myeloid markers (CD117, CD13 or CD33 p0.05) were seen between these two groups. The frequency of abnormal karyotype was similar between AUL and AML MD (16/30 [53%] vs 15/37 [41%], respectively). The most common mutations identified in AUL were PHF6 (7/18), SRSF2 (7/18), RUNX1 (7/23), ASXL1 (6/23) and BCOR (5/16). Compared to AML MD, AUL cases were characterized by frequent mutations in PHF6 (7/18 vs 1/23, p=0.013) and SRSF2 (7/18 vs 2/22 p=0.028). Limiting AUL cases to only those with 1 myeloid marker or less also showed similar findings with more frequent mutations in PHF6 (7/16 vs 1/25, p=0.0031), SRSF2 (6/15 vs 3/26 p= 0.018) and trend towards higher BCOR frequency (5/15 vs 2/26, p=0.078) in AUL patients as compared to AML MD. RUNX1 mutation was seen in 7/23 AUL and 8/29 AML MD (p>0.05). 19/30 AUL patients received induction chemotherapy (AML-type regimen in 18 cases and an ALL-type regimen in 1 case) and 15/30 achieved complete remission. In 10 AUL patients who relapsed, 9 showed identical immunophenotype and one case showed expression of CD13 and CD33. Outcome data in the subset of patients who received induction showed no difference in OS, RFS, or rates of complete remission between AUL and AML MD groups (p>0.05). The 28 AML MRC cases that were originally classified as AUL or AML MD presented with lower WBC (p=0.026), more frequent abnormal karyotype (27/28) specifically complex karyotype (20/28 p=0.002), frequent TP53 mutations (p=0.0002) when compared to the AUL group. AML MRC patients showed worse overall OS (p=0.029) as compared with AUL patients and a trend toward worse outcome as compared with AML MD (p=0.068). Conclusions: In this largest series to date, AUL group shows distinct characteristics from AML MD, including more frequent PHF6 and SRSF2 mutations and expression of TdT. However, clinical outcome is similar between the two groups in patients treated with induction chemotherapy. Cases reclassified as AML-MRC had shorter survival compared to de novo AUL and trend towards worse outcome when compared to AML MD patients. These results suggest a genetic rationale for the separation of AUL as a distinct entity from AML MD and also support the WHO classification of cases with history of prior MDS and/or MDS-type karyotype findings as AML-MRC. Disclosures Garcia: Celgene: Consultancy.
- Published
- 2018
13. Development of a Novel Class of Agents Targeting the RNA-Splicing Machinery in Myeloid Malignancies
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Hetty E. Carraway, Allison Noe, Vera Adema, Drew J. Adams, Bartlomiej P Przychodzen, Yvonne Parker, Daniel J. Lindner, Steffan T. Nawrocki, Tomas Radivoyevitch, Kevin R. Kelly, Mikkael A. Sekeres, Yuriy Fedorov, Richard A. Padgett, Hassan Awada, Cassandra M. Hirsch, Rachel Toth, Jaroslaw P. Maciejewski, Valeria Visconte, James G. Phillips, Heesun J. Rogers, Amy C Graham, Anjali S. Advani, Christina Snider, and Jennifer S. Carew
- Subjects
RNA Splicing Factors ,Mutation ,Myeloid ,Immunology ,Cell Biology ,Hematology ,Computational biology ,medicine.disease_cause ,Biochemistry ,Exon ,Splicing factor ,medicine.anatomical_structure ,Drug development ,RNA splicing ,Myeloid cells ,medicine ,Business - Abstract
SF3B1 is a splicing factor gene whose mutations are pathognomonic of MDS with ring sideroblasts. Because of the ubiquitous importance of splicing, a major barrier in targeting cells with spliceosomal mutations is the discovery of agents decreasing the competitiveness of mutant cells while preserving the integrity of wild type cells. To date no specific therapies are FDA approved for SF3B1 mutant (SF3B1MT) MDS and few agents are in early clinical testing. We describe a novel targeted approach to drug development for SF3B1MT malignancies. Our investigative strategy started with a high throughput drug screen. We introduced K700E mutation into myeloid cells using CRISPR/Cas9. We then subjected K562+/K700E and matched-parental K562 cells to high throughput drug screen of a library of 3,000 mechanistically annotated, non-redundant, bioactive compounds. Top hits were validated by dose response testing (8 concentrations in half-log dilutions). Our interest focused on compounds with cytostatic activity towards K562+/K700E cells. Among these, a 4-pyridyl-2-anilinothiazole (PAT) showed preferential inhibition of growth in K562+/K700E cells with an IC50 of 3uM. Dose response showed that K562+/K700E cells were significantly sensitive to PAT with a growth inhibition of 20%, 32%, 51%, 65%, 95% at .3uM (P=.01), 1uM (P=.002), 3uM (P Using PAT as our lead, we employed a fragment based reiterative medicinal chemistry approach to synthesize selective compounds and improve therapeutic index. Libraries were constructed following Lipinski rules with ease of synthetic construction in mind. Pilot libraries were constructed via the classic Hantsch thiazole synthesis which involves condensation of α-halo ketones with substituted thioureas. This enterprise investigated SAR modifications of PAT by considering features such as regiochemistry and basicity of the nitrogen of the pyridine ring in the head region; replacement of the spacer 2,4-disubstituted thiazole ring with heteroatom containing rings (5,6,7 membered aromatic or aliphatic ring structures); alternatives for the aniline of the tail region e.g., sulfonamide, amide, and substituted amine linkers and substituted aromatic and aliphatic ring structures for the phenyl substituent. A major challenge in drug development of agents targeting spliceosomal mutations is the identification of key clusters of aberrantly spliced genes restored by drug treatment, e.g., identification of a pharmacodynamic endpoint that could be used to prove that that the drug reached its target. SF3B1 mutations induce aberrant 3′-ss selection by promoting use of alternative branch points. To identify biomarkers of splicing changes to screen our libraries, K562+/K700E and parental cells were treated with PAT (3uM) and RNA libraries were subjected to RNA Seq. The splicing pattern of parental cells was used as reference. We identified 328 cassette exons (±25% splicing inclusion difference, pFDR In sum, we described novel classes of compounds that inhibit the expansion of SF3B1MT cells by restoring the splicing defects intrinsically associated with SF3B1. Disclosures Carraway: Novartis: Speakers Bureau; Jazz: Speakers Bureau; FibroGen: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Balaxa: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Speakers Bureau. Sekeres:Opsona: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy.
- Published
- 2018
14. Cyclosporine dependent pure red cell aplasia: A case presentation
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Karam Al-Issa, Vikas Dembla, Valeria Visconte, Alan E. Lichtin, Ramon V. Tiu, and Heesun J. Rogers
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Male ,medicine.medical_specialty ,business.industry ,Anemia ,Pure red cell aplasia ,Cell Biology ,Hematology ,Case presentation ,Middle Aged ,Red-Cell Aplasia, Pure ,medicine.disease ,Gastroenterology ,Blood Cell Count ,Discontinuation ,Bone Marrow ,Internal medicine ,Cyclosporine ,Humans ,Molecular Medicine ,Medicine ,Erythrocyte Transfusion ,business ,Molecular Biology ,Immunosuppressive Agents - Published
- 2015
15. Modified Array-based Comparative Genomic Hybridization Detects Cryptic and Variant PML-RARA Rearrangements in Acute Promyelocytic Leukemia Lacking Classic Translocations
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Mark Fesler, Roger A. Schultz, Jacqueline R. Batanian, Raymond R. Tubbs, Blake C. Ballif, Heesun J. Rogers, Aaron M. Gruver, and James R. Cook
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Male ,Acute promyelocytic leukemia ,Receptors, Retinoic Acid ,Translocation Breakpoint ,Chromosomal translocation ,Promyelocytic Leukemia Protein ,Biology ,Translocation, Genetic ,Pathology and Forensic Medicine ,Young Adult ,Promyelocytic leukemia protein ,Leukemia, Promyelocytic, Acute ,immune system diseases ,medicine ,Humans ,Pathology, Molecular ,neoplasms ,Molecular Biology ,Gene Rearrangement ,Genetics ,Comparative Genomic Hybridization ,medicine.diagnostic_test ,Retinoic Acid Receptor alpha ,Tumor Suppressor Proteins ,Nuclear Proteins ,Cell Biology ,Gene rearrangement ,Middle Aged ,medicine.disease ,Retinoic acid receptor alpha ,biology.protein ,Female ,Transcription Factors ,Comparative genomic hybridization ,Fluorescence in situ hybridization - Abstract
Acute promyelocytic leukemia (APL) is typically defined at the molecular level by a reciprocal translocation of the promyelocytic leukemia (PML) and retinoic acid receptor α (RARA) genes. An accurate diagnosis of APL is critical for appropriate choice of therapy and prognostic assessment. Cryptic and variant rearrangements in APL are discoverable by a variety of molecular methods including fluorescence in situ hybridization (FISH), reverse transcriptase polymerase chain reaction, or gene sequencing. Rare reports of FISH-negative APL harboring cryptic rearrangements of PML-RARA detected by reverse transcriptase polymerase chain reaction or sequencing have been described. Here, we describe the detection of cryptic or variant PML-RARA rearrangements by translocation-based comparative genomic hybridization (tCGH), a recently described modification of traditional CGH technology that facilitates the detection of balanced translocations by means of the linear amplification of a potential translocation breakpoint region(s), in 2 unusual cases of APL. One tumor lacked detectable t(15;17) by karyotype and FISH, and the other tumor lacked the typical morphologic and immunophenotypic features of APL and had a variant 3-way translocation involving PML and RARA. PML-RARA translocations were identified by tCGH in both cases providing confirmation of the diagnosis of APL. These data emphasize the benefit of using complementary molecular methods including tCGH for detecting cryptic and variant PML-RARA translocations in unusual cases of APL.
- Published
- 2013
16. SF3B1 haploinsufficiency leads to formation of ring sideroblasts in myelodysplastic syndromes
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John Barnard, Jarnail Singh, Manoj Bupathi, Heesun J. Rogers, Hideki Makishima, Anjali S. Advani, Edward A. Copelan, Hadrian Szpurka, Sami Osman, Anna M. Jankowska, Christine L. O'Keefe, James E. McMahon, Richard A. Padgett, Kazunori Koide, Kyoichi Isono, Mikkael A. Sekeres, Fabiola Traina, Valeria Visconte, Yogen Saunthararajah, Andres Jerez, Jaroslaw P. Maciejewski, Ramon V. Tiu, and Haruhiko Koseki
- Subjects
Adult ,Male ,Spliceosome ,Adolescent ,Immunology ,Haploinsufficiency ,Biology ,Real-Time Polymerase Chain Reaction ,medicine.disease_cause ,Biochemistry ,Mice ,Young Adult ,Biomarkers, Tumor ,medicine ,Animals ,Humans ,RNA, Messenger ,Exome ,Cells, Cultured ,Aged ,Oligonucleotide Array Sequence Analysis ,Mice, Knockout ,Mutation ,Reverse Transcriptase Polymerase Chain Reaction ,Gene Expression Profiling ,Myelodysplastic syndromes ,Cell Biology ,Hematology ,Middle Aged ,Ribonucleoprotein, U2 Small Nuclear ,Phosphoproteins ,medicine.disease ,Molecular biology ,Phenotype ,Anemia, Sideroblastic ,Mice, Inbred C57BL ,Gene expression profiling ,Myelodysplastic Syndromes ,Refractory anemia with ring sideroblasts ,Cancer research ,Female ,RNA Splicing Factors ,K562 Cells - Abstract
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.
- Published
- 2012
17. Acute parvovirus B19 infection detected in bone marrow biopsy
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Heesun J. Rogers and Patrick Feasel
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Pathology ,medicine.medical_specialty ,Anemia ,Biopsy ,Immunology ,Parvoviridae Infections ,Biochemistry ,Bone Marrow ,hemic and lymphatic diseases ,medicine ,Parvovirus B19, Human ,Humans ,biology ,medicine.diagnostic_test ,Parvovirus ,business.industry ,Cell Biology ,Hematology ,Middle Aged ,biology.organism_classification ,Normocellularity ,medicine.disease ,medicine.anatomical_structure ,Erythema Infectiosum ,Acute Disease ,Female ,Bone marrow ,Hemoglobin ,business - Abstract
[Figure][1] A 45-year-old woman with a history of renal transplant and immunosuppressive therapy presented with worsening anemia (hemoglobin, 6.1 g/dL). The bone marrow (BM) demonstrated normocellularity (50%) and erythroid hypoplasia with lack of erythroid maturation. Several giant
- Published
- 2015
18. Genetic and Epigenetic Defects in the Autophagy Machinery in Myelodysplastic Syndromes
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Heesun J. Rogers, Hetty E. Carraway, Valeria Visconte, Swapna Thota, Bhumika J. Patel, Bartlomiej P Przychodzen, Yvonne Parker, Kevin R. Kelly, Mikkael A. Sekeres, Cassandra M. Hirsch, Jennifer S. Carew, Steffan T. Nawrocki, Jaroslaw P. Maciejewski, and Daniel J. Lindner
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0301 basic medicine ,Mutation ,LAMP2 ,Immunology ,Autophagy ,RPTOR ,Cell Biology ,Hematology ,Gene mutation ,Biology ,medicine.disease_cause ,Biochemistry ,Germline ,03 medical and health sciences ,030104 developmental biology ,medicine ,Cancer research ,Haploinsufficiency ,ATG16L1 - Abstract
Autophagy is a degradation process for the turnover of damaged organelles and long-lived proteins that also plays an important role during erythropoiesis. Accordingly, knockout of the essential autophagy gene Atg7 in mice leads to clinico-morphologic features of MDS. To date, no study has determined the prevalence and impact of defects (mutations, aberrant expression) in the autophagy machinery in MDS. We interrogated the occurrence of alterations in 180 autophagy genes by analyzing WES of patients with MDS (N=120). For comparison, we analyzed results from other hematologic neoplasms (N=103) and TCGA (N=202). We detected somatic mutations in autophagy genes in 40/425 patients (9%). Mutations were enriched in MDS (12%;14/120) and prevalent in higher risk MDS patients (30%;12/40). Mutations were found in: ATG2A, ATG4C, ATG14, ATG16L1, BCL2, CDKN2AIPNL, COG8, DNM1L, DNM2, GYS1, HIF1A, KIF1B, LAMP2, MLST8, MTOR, NOD2, PIK3CB, PIK3C2A/B, PIK3C2G, PPP2R2A/B, PPP2R3A, PRKAA1/2, PRKACB, PRKAG1/G2, PTPN2, RICTOR, RPTOR, SEC22B, SMURF1, SQSTM1, STAT3, SUPT20H, TAB2, TNFSF10/13B, ULK4, USP10, VPS11/33B, VTI1A, WDFY3/4, WAC. Twenty-five mutations had a cut-off >20% VAF. Most patients (31/40;78%) had a sole mutation, 4 patients carried 2 mutations each (ULK4, WDFY3), (KIF1B, SEC22B), (VIT1A, TAB2), (DNM2, WAC). PRKACB mutations were found in 3 patients. NOD2, PTPN2, PRKAG1, SEC22B, ULK4, VPS11 and WAC mutations were found in 2 patients. inces autophagy genes has been Loss of function mutations were observed in ULK4, NOD2 and WAC. Four genes mapped to commonly deleted regions e.g., 5q (CDKN2AIPNL, SQSTM1) or 7q (SMURF1, PRKAG2) and coincided with haploinsufficient expression, while 3 genes had hemizygous configuration (SMURF1, PPP2R3A, PIK3C2G). Reactome analysis clustered the mutations in effectors/inhibitors and early stage. The analysis of 263,973 germline variants detected that PIK3C2G, NOD2 and HIF1A were also associated with exonic germline variants predicted to be significantly deleterious. Mutations or aberrant expression of core components of the autophagy network have been associated with poor outcomes in multiple diseases. In our cohort, 21 patients died. Most (57%;21/37) of patients had abnormal karyotype with 4 patients having complex karyotype including -17 and -7. Among the cases with abnormal karyotype, 4 cases had del(5q). Mutant patients had worse survival trending toward significance compared to WT patients (MUTvs. WT=20 vs. 90; median 14 vs. 20 months; LogR=.09). Among disease groups, autophagy mutations were associated with significantly inferior survival in MDS (MUT vs. WT=13 vs. 61; 17 vs. 35 months; LogR=.018) and MDS/MPN (MUT vs. WT=4 vs. 31; 12 vs. 30 months; LogR=.037). Seven mutant patients who received hypomethylating agents had no response. Comparative analyses (Sanger, TruSeq, WES, TCGA) identified that autophagy gene mutations were significantly associated with TET2 (28%;11/40; P=.02) among other mutations [RUNX1, STAG2 (20%;8/40), SRSF2 (18%;7/40), DNMT3A, ASXL1 (15%;6/40)]. Clonal hierarchy showed that autophagy gene mutations were mainly secondary events, were ancestral events in 7 (ATG2A, DNML1, PRKACB, PRKAG1, PTPN2, SEC22B, STAT3) and co-dominant in 2 patients (NOD2, MLST8). When autophagy genes mutations were secondary, the most represented ancestral mutationswere in splicing factors (N=9; SRSF2, PRPF8, U2AF1) and DNA methylation (N=4; TET2, DNMT3A). RNA sequencing determined that changes in autophagy gene expression are overrepresented in specific MDS subtypes with distinct mutational profiles. The expression levels of 2 ULK family members commonly elevated during erythroid maturation were found in SF3B1K700E compared to SF3B1WT MDS patients (N=6; ULK1, FC=2; ULK3, FC=4; P=.05). Erythroid cells of these SF3B1K700E patients showed increased autophagosomes compared to SF3B1WT cells. In vivo administration of the mTOR inhibitor, temsirolimus (10 mg/kg i.p. 5d/week for 2 wk) improved the erythropoiesis of a transgenic Sf3b1 mouse model by increasing CD71+ cells (10-20% vs. 5%) and ameliorating anemia (Hgb: 7.9 vs. 6.6 g/dL; P=.07; MCV: 43.5 vs. 42.4 fL; P=.08). In sum, defects in autophagy genes are present in MDS, co-occur with other mutations and impact survival. Changes in expression levels of autophagy genes may be associated with MDS phenotypes and modulated by autophagy inducing drugs as evidenced in models of SF3B1 mutations. Disclosures Kelly: Novartis: Consultancy, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Consultancy, Speakers Bureau. Maciejewski:Alexion Pharmaceuticals Inc: Consultancy, Honoraria, Speakers Bureau; Apellis Pharmaceuticals Inc: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Speakers Bureau.
- Published
- 2016
19. Recombinant β2-Glycoprotein I (β2GPI) Produced Using a Novel Lentiviral Approach Functions at Least As Well As Plasma-Derived β2GPI in Detection of Anti-β2GPI Antibodies
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Suman Kundu, Mohamed Samour, Sergei Merkulov, Caner Saygin, Heesun J. Rogers, Keith R. McCrae, and Shruti Chaturvedi
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Chromatography ,biology ,Serial dilution ,Chemistry ,Immunology ,Cell Biology ,Hematology ,Biochemistry ,law.invention ,Standard curve ,Sepharose ,Galveston Orientation and Amnesia Test ,Antigen ,IgG binding ,law ,biology.protein ,Recombinant DNA ,Antibody - Abstract
Introduction β2-glycoproteinI (β2GPI) is the primary antigen for antiphospholipid antibodies (Ab), and Ab to β2GPI are associated with thrombosis and recurrent fetal loss.β2GPIis comprised of 5 "sushi" domains. Complex disulfide bonding renders β2GPI a challenging protein to producerecombinantlyin high yield and most studies have utilized domain-deletion mutants produced on a lab scale for structure-function analyses.β2GPIalso has a complex tertiary structure, and is reported to circulate in a "circular" form that may "open" to expose the antigenic site for β2GPIAbunder specific conditions. We developed a novel method to produce recombinant β2GPI in which replacement of the leader peptide allows large scale expression using a lentiviral system with one-step purification on heparin-sepharose. The ability of this protein to bind anti-β2GPI Ab was compared with that of plasma-derived (wild type, WT) β2GPI. Methods β2GPIcDNAwas cloned into pLentiCMV DEST. The β2GPI containing vector was used to transduce HEK293 cells with selection using puromycin. β2GPIwas purified from conditioned medium using HiTrapHeparin HP. Plasma β2GPI was purified using a protocol employing perchloric acid precipitation followed by heparin-sepharose and Mono-S chromatography. To measure anti-β2GPI Ab, we analyzed plasma from 32 patients referred to the Cleveland Clinic Special Coagulation Laboratory for anti-β2GPI testing using the InovaQuanta-Lite ELISA. Normal plasma samples (n=15) were also analyzed at 1:100 dilution to determine cutoffs for anti-β2GPI positivity. Briefly, 96-well plates were coated overnight at 4° C with 2 µg/ml WT or recombinant β2GPI. After blocking β2GPI-coated plates with BSA, 50 µl of patient plasma at 1:10 and 1:100 dilutions were added to individual wells in quadruplicate. A standard curve for IgG binding to each plate was created using affinity-purified rabbit anti-β2GPI IgG at concentrations of 15, 31.25, 62.5, 125, and 250 ng/ml. After incubation for 30 minutes at room temperature, plates were washed three times and 100 µl of a 1:5000 dilution of goat anti-human IgG was added. After 30 minutes, wells were again washed prior to adding 100 µl/well of o-phenylenediamine dihydrochloride. Plates were read at 490 nm after 15 minutes following addition of 25 µl/well H2SO4. Results from different plates were standardized by extrapolating the amount of boundAb from the standard curve prepared on each plate. To compare performance of recombinant β2GPI against WT β2GPI in ELISA we first evaluated correlation using recombinant and WT β2GPI by Spearman's test. The two sets of ELISAs were also compared using the Wilcoxon matched pairs test. ELISA readings were considered positive if they were >90th percentile on a curve established using 15 normal plasmas. Sensitivity and specificity of the assays was determined with respect to the results of the clinical assay. Results Recombinant β2GPI was produced in high yield (10-20 mg/L) and purified to homogeneity with a single heparin chromatography step; the purified protein migrated as a single band of ~50 kDon SDS-PAGE with a characteristic increase in Mr upon reduction. Anti-β2GPI IgG ELISA using WT and recombinant β2GPI demonstrated excellent correlation at both 1:10 (Spearman's rho 0.70, P Conclusion Recombinant β2GPI can be produced in high yield by this novel method and purified with a single heparin chromatography step. It is recognized by anti-β2GPI Abat least as well as WT β2GPI. Further studies focused on site-directed mutagenesis of the intact molecule to optimize assays for detection of pathologic anti-β2GPI antibodies are underway. Disclosures No relevant conflicts of interest to declare.
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- 2016
20. Targeting Autophagy in Myelodysplastic Syndromes
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Hetty E. Carraway, Heesun J. Rogers, Anjali S. Advani, Daniel J. Lindner, Mei Yin, Mikkael A. Sekeres, Bartlomiej P Przychodzen, Yvonne Parker, John Barnard, Valeria Visconte, Kevin R. Kelly, Jaroslaw P. Maciejewski, Steffan T. Nawrocki, Jennifer S. Carew, and Michael J. Clemente
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Atg1 ,Kinase ,Immunology ,Cell ,Autophagy ,Cell Biology ,Hematology ,Biology ,Protein degradation ,Biochemistry ,medicine.anatomical_structure ,Apoptosis ,Cancer research ,medicine ,Erythropoiesis ,ATG4A - Abstract
Autophagy is a conserved mechanism of protein degradation that plays a physiologic role in iron homeostasis. The RARS subtype of MDS is characterized by inefficient erythropoiesis and the presence of erythroblasts with iron-laden mitochondria (ring sideroblasts, RS). Patients with RARS exhibit an accumulation of iron due to the inability of their erythroid precursors to properly process iron intended for hemoglobin synthesis. RARS erythroid cells display increased autophagy compared to cells from healthy subjects and mice lacking the essential autophagy genes Atg1 and Atg7 develop progressive anemia due to the inefficient removal of defective mitochondria. We previously showed that the presence of SF3B1 mutations, which are frequent in RARS patients, are associated with the presence of increased mitochondrial iron content and low levels of apoptosis. This finding suggests that cell death-independent mechanisms may temper excess iron by triggering the clearance of mitochondria during erythroid maturation. RNA sequencing analyses have shown that the autophagy pathway is upregulated in SF3B1MUT cells. We investigated the role of mitochondrial autophagy in the alleviation of iron-related damage in RARS erythroid cells and the potential therapeutic benefit of autophagy-stimulating agents for the selective improvement of erythropoiesis and iron homeostasis in SF3B1MUT RARS cells. Transmission electron microscopy (TEM) demonstrated an accumulation of iron in the mitochondria of SF3B1MUT (K700E, n=4) compared to SF3B1WT RARS erythroblasts. Flow cytometry confirmed increased mitochondrial iron in SF3B1MUT (n=10) compared to SF3B1WT (n=10) RARS cells (82% ± 10 vs. 24% ± 5; P = 0.004). Autophagic vacuolization of the cytoplasm and an increased number of autophagosomes were found in SF3B1MUT (K700E) compared to SF3B1WT erythroblasts (n=2). The mRNA expression levels of numerous autophagy genes were elevated in SF3B1MUT vs. SF3B1WT RARS cells: ATG complexes (ATG2A/B, FC=2; ATG4A, FC=2; ATG9A, FC=5; ATG4C, P=0.05; ATG18 (FC=4.8; P= 0.02), autophagy-initiating kinases (ULK1, FC=2; ULK3, FC=3.9; P=0.05), and cathepsins involved in late stage autophagy (CTSL1, FC=20; CTSD, FC=5.8; P=0.05; CTSB, FC=2.1; CTSE, FC=5.9; CTSD, FC=2; P=0.01). qRT-PCR confirmed that SF3B1MUT cells expressed elevated mRNA levels of selected genes. We next administered 3 FDA-approved drugs with established autophagy-stimulating properties [temsirolimus, metformin, arsenic trioxide (ATO)] to a transgenic SF3B1 mouse model (SF3B1+/-) that exhibits anemia, dysplasia, and RS (Visconte, J Hematol Oncol 2014) to evaluate drug-mediated improvement of erythropoiesis and autophagic clearance of excess iron. Eight-month-old SF3B1+/- mice (n = 10 per group) were treated with vehicle control and the following: temsirolimus (10 mg/kg i.p. 5d/week for 2 wk), metformin (250 mg/kg/d, gavage for 2 wk), and ATO (10 mg/kg i.p. 5 d/wk for 2 wk). All agents were well tolerated and triggered morphologic features of autophagy including increased autophagosome-like structures by TEM. No effects on BM cellularity and/or dysplasia were noted, although changes in the morphology of myeloid cells (numerous swollen nuclei) were detected by Wright stain. Mitochondria that were engulfed in autophagosomes were frequently seen. After 2 weeks, temsirolimus-treated SF3B1+/- mice showed an incremental increase in hemoglobin by 1.2 g/dL (7.9 vs. 6.6 g/dL; P=0.07) and in mean corpuscular volume (43.5 vs. 42.4 fL; P=0.08). Erythropoiesis was improved as shown by increased levels of CD71-positive cells by immunohistochemistry in cells post-temsirolimus treatment compared to pre-treatment (10-20% vs. 5%). Increased dividing erythroid cells with binucleation and budding were also observed in cells following temsirolimus treatment. These hematologic changes were not detected with ATO or metformin. In sum, our data support a role for activated autophagy in the pathogenesis of RARS and indicate that stimulating autophagy with approved existing drugs or novel investigational drugs may yield therapeutic benefit in SF3B1MUT RARS patients. Disclosures Kelly: Pharmacyclics: Consultancy, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Speakers Bureau. Advani:Pfizer: Consultancy, Research Funding. Carraway:Novartis: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Baxalta: Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Research Funding, Speakers Bureau.
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- 2016
21. Multiple 'doughnut' granulomas in Coxiella burnetii infection (Q fever)
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Heesun J. Rogers and Grant W Herndon
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Male ,medicine.medical_specialty ,Immunology ,Q fever ,Biochemistry ,Gastroenterology ,Diagnosis, Differential ,Bone Marrow ,hemic and lymphatic diseases ,Internal medicine ,medicine ,Humans ,Platelet ,Multiple myeloma ,Leukopenia ,Granuloma ,medicine.diagnostic_test ,business.industry ,Complete blood count ,Cell Biology ,Hematology ,Middle Aged ,medicine.disease ,Coxiella burnetii ,Chills ,Hemoglobin ,medicine.symptom ,business ,Q Fever - Abstract
[Figure][1] A 54-year-old man with a history of multiple myeloma presented with intermittent fevers, chills, fatigue, and weight loss for 1 month. A complete blood count showed leukopenia (white blood count: 1.4 × 109/L), mild anemia (hemoglobin: 12.4 g/dL), and thrombocytopenia (platelets
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- 2013
22. Elevated Basal Autophagy in SF3B1 Mutated Myelodysplastic Syndromes: Relationship with Survival Outcomes and Therapeutic Implications
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Hetty E. Carraway, Yingchun Han, Jaroslaw P. Maciejewski, Valeria Visconte, Mikkael A. Sekeres, Steffan T. Nawrocki, Heesun J. Rogers, Anjali S. Advani, Kevin R. Kelly, and Jennifer S. Carew
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Myelodysplastic syndromes ,Immunology ,Autophagy ,Proteolytic enzymes ,Cathepsin D ,Cell Biology ,Hematology ,Biology ,Protein degradation ,medicine.disease ,Biochemistry ,Cathepsin B ,Refractory anemia with ring sideroblasts ,medicine ,Cancer research ,ATG4A - Abstract
About 60-80% of patients with myelodysplastic syndromes (MDS) manifest with anemia. Red blood cell (RBC) transfusions are the most commonly used therapy to alleviate anemia in patients that are ineligible for other curative approaches. Transfusion-dependent patients frequently develop iron overload, which correlates with infections, mortality, leukemia, and organopathy. At the subcellular level, long-term iron exposure produces iron-catalyzed hydroxyl radicals that induce oxidative damage to mitochondria and disrupt bioenergetic homeostasis. The use of iron-chelating drugs to counter transfusion-related iron overload remains controversial due to the significant side effects that these agents cause. New therapies that effectively address iron overload in transfusion-dependent MDS patients are clearly needed. Mitochondrial dysfunction frequently occurs during MDS cell maturation and leads to abnormal iron distribution. However, the mechanistic basis of this biological phenomenon has not been rigorously studied. We previously linked the presence of Splicing factor 3b subunit 1 (SF3B1) mutations, which are frequent in patients with refractory anemia with ring sideroblasts (RARS), with abnormalities in mitochondrial iron. Transmission electron microscopy and flow cytometry showed that mitochondria of SF3B1 mutant RARS patients have higher iron content than those of wild type (WT) RARS patients and expressed an increased mRNA level of the iron transporter, Mitoferrin 1. Despite these prevalent mitochondrial abnormalities and transfusional dependence, SF3B1 mutant lower-risk MDS patients experienced significantly longer median survival compared to SF3B1 WT lower-risk MDS patients (34 mos vs. 13 mos; P = .002; N=16 vs. 101). Autophagy is an evolutionarily conserved lysosomal mechanism of protein degradation that plays a critical role in the elimination of damaged mitochondria and other organelles. We hypothesized that autophagic clearance of defective mitochondria may contribute to the superior survival of SF3B1 mutant patients suffering from transfusion-mediated iron overload. We conducted RNA sequencing analyses on a group of fresh bone marrow (BM) cells of SF3B1 mutant and WT RARS patients and healthy donors (n = 11) to investigate the basal autophagy status in this distinct patient population. In addition to confirming increased levels of mitochondrial transporters such as Mitoferrin 1 and 2 (FC = 2.0), we detected a striking increase in multiple genes involved in the proximal and distal regulation of autophagy in cells of SF3B1 mutant RARS compared to WT RARS patients. Genes controlling the early stages of autophagy including the protein kinases ULK1 (FC = 2.0) and ULK3 (FC = 3.9; P=.05), ATG complexes ATG2A/B (FC = 1.9), ATG9A (FC = 5.5; P=.05), ATG18 (FC = 4.8; P=.02) and the cysteine proteases ATG4A/C (FC = 2.0) were all elevated in SF3B1 mutant RARS vs. SF3B1 WT RARS patients. Key components of the late stages of the autophagic degradation cascade, including multiple members of the cathepsin family of lysosomal proteases [CTSL1: FC = 20.9; CTSD: FC = 5.8 (P=.06); CTSB: FC = 2.1; CTSE: FC = 5.9 (P=.01); CTSD: FC = 1.8], were also significantly increased. qRT-PCR confirmed higher expression levels in the BM cells of RARS patients carrying sole SF3B1 mutations compared to cells of SF3B1 WT RARS patients. The link between SF3B1, mitochondrial iron, and elevated autophagy was specific as evidenced by the unmutated status and lack of significant mRNA changes in any other splicing factor genes including PRPF8. Our data demonstrate that autophagy may play an important, previously unreported role in SF3B1 mutant RARS. Based on our findings, we hypothesize that SF3B1 mutant RARS cells stimulate autophagy to eliminate damaged mitochondria and alleviate iron overload and that further stimulation of autophagy will diminish the pathogenic effects of chronic transfusions. We are currently investigating the therapeutic benefit and pharmacodynamics of autophagy-modulating drugs (temsirolimus, metformin, arsenic trioxide) in in vitro (primary cells) and in vivo (SF3B1 haploinsufficient mice) models of MDS to facilitate the design of an investigator-initiated clinical trial that will test autophagy modulation as a new precision strategy for the treatment of transfusion-dependent patients with low-risk MDS carrying SF3B1 mutations. Disclosures Sekeres: TetraLogic: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Carew:Boehringer Ingelheim: Research Funding.
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- 2015
23. Platelet Function Testing Is Commonly Performed in Patients with Known Confounding Factors
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Paul Elson, Heesun J. Rogers, Dharmesh Gopalakrishnan, and Keith R. McCrae
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medicine.medical_specialty ,Wilcoxon signed-rank test ,business.industry ,Medical record ,Immunology ,Confounding ,Cell Biology ,Hematology ,Biochemistry ,Surgery ,McNemar's test ,Internal medicine ,medicine ,Platelet ,Forty Nine ,Abnormality ,business ,Two Hundred Fifty - Abstract
Introduction: Commonly used platelet function tests include Platelet Function Analyzer -100 Closure Time (PFA-100), used as a screening test, and platelet aggregation testing by Light Transmission Aggregometry (LTA), which can help differentiate between various types of platelet function defects and guide further testing. In this study, we assessed the indications for platelet aggregation testing at our center, and the effect of factors that may interfere with the outcomes of testing. Methods: This was a retrospective electronic medical records based study in which we included all patients in our healthcare network who had platelet aggregation testing done using LTA between August 2008 and August 2013. Routine descriptive measures were used for frequencies and proportions. McNemar test was used for comparison of paired nominal variables and Wilcoxon signed-rank test was used for comparison of paired continuous variables. P value < 0.05 was considered significant. Results: Four hundred ninety seven patients had platelet aggregation testing performed during the 5-yr study period. Among these, 315 (63%) patients had antecedent screening with PFA-100 with 157 (31%) showing at least one abnormality. Two hundred forty nine (50%) patients underwent some form of further testing after LTA, including VWF analysis (42%), platelet flow cytometry (19%) and/or electron microscopy (1%). Two hundred fifty six (51%) patients had at least one factor previously known or suspected to interfere with platelet aggregation testing (platelet count Conclusions: A majority of the patients in our study did not have abnormalities in PFA-100 testing despite abnormal results on subsequent platelet aggregometry. Fifty one percent of patients who underwent aggregometry were on medications that may affect platelet function or had other factors known or suspected to interfere with aggregation testing. Eighty percent of these patients had abnormal results. Repeat testing after correction of the known factor(s) led to a significant improvements in the results of LTA. The most common indication for platelet aggregation testing was a current or past history of abnormal spontaneous bleeding and the most common sites of bleeding in these patients were the skin and mucosal surfaces. Disclosures McCrae: Momenta: Consultancy; Janssen: Membership on an entity's Board of Directors or advisory committees; Syntimmune: Consultancy; Halozyme: Membership on an entity's Board of Directors or advisory committees.
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- 2015
24. Diastolic but Not Systolic Heart Failure Is Associated with Multiple Abnormalities on Platelet Aggregation Testing
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Heesun J. Rogers, Keith R. McCrae, Paul Elson, and Dharmesh Gopalakrishnan
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medicine.medical_specialty ,business.industry ,Immunology ,Diastole ,Diastolic heart failure ,Impaired platelet aggregation ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Surgery ,chemistry.chemical_compound ,Exact test ,chemistry ,Internal medicine ,Aortic valve stenosis ,Heart failure ,medicine ,Cardiology ,Platelet ,business ,Ristocetin - Abstract
Introduction: The effect(s) of co-morbid medical conditions on platelet function is poorly understood. In this retrospective EMR-based study, we analyzed the influence of various diseases on in vitro measures of platelet function - platelet function analyzer-100 (PFA-100) closure times, platelet aggregation (using light transmission aggregometry (LTA)), platelet dense granule release (using lumi-aggregometry), and platelet flow-cytometry for surface glycoproteins. We also examined their influence on VWF testing. Methods: Four hundred ninety seven patients who had platelet aggregation testing performed using LTA between August 2008 and August 2013 were included in our study. Co-morbidities at the time of testing were recorded. Propensity score matching for each individual disease was used to adjust for relevant covariates. We used a 1:1 nearest neighbor match without replacement, with caliper width set to 0.2 times the standard deviation of the logit of the propensity score. Following matching, Fisher's exact test or Chi square test was used as appropriate to assess the association between categorical variables, while the Mann-Whitney test was used to test the association between categorical and continuous measures. Pearson co-efficient was used to assess the correlation between continuous variables. P < 0.05 was considered significant. Results: 1) Congestive heart failure (n = 44) was associated with impaired platelet aggregation in the presence of arachidonic acid (p = 0.001) and collagen (p = 0.009), as well as impaired dense granule release in the presence of collagen (p = 0.002) and epinephrine (0.012). It was also associated with abnormal aggregation (p = 0.024) and release (p = 0.028) in the presence of ≥ 2 agonists in the respective panels. Diastolic heart failure (n = 25) was found to be associated with impaired aggregation in the presence of ADP (p = 0.007), collagen (p = 0.001), or arachidonic acid (p = 0.007), and to ≥ 2 agonists in the aggregation panel (p = 0.008). Systolic heart failure (n = 26) was not associated with abnormalities in aggregation or release. 2) Severe aortic stenosis (n = 17) was associated with prolonged collagen/ADP (p = 0.003) and collagen/epinephrine (p Conclusion: Diastolic heart failure was associated with impaired platelet aggregation in the presence of multiple agonists. Though the mechanism remains unclear, we postulate that this could be related to shear stress to which the platelets are subjected in the non-compliant ventricles. Severe aortic stenosis was associated with prolonged collagen/ADP as well as collagen/epinephrine PFA-100 closure times and with lower ristocetin co-factor/VW antigen ratio suggesting functional impairment of VWF. Though diabetes mellitus was associated with impaired platelet aggregation in the presence of collagen and impaired dense granule release in the presence of epinephrine, no correlation was found between these abnormalities and HbA1C levels, making the significance of the association unclear. Disclosures McCrae: Syntimmune: Consultancy; Momenta: Consultancy; Janssen: Membership on an entity's Board of Directors or advisory committees; Halozyme: Membership on an entity's Board of Directors or advisory committees.
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- 2015
25. Suboptimal Antiplatelet Therapy Suggested By Platelet Aggregation Studies Does Not Correlate with a Change in Clinical Management
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Paul Elson, Heesun J. Rogers, Keith R. McCrae, and Dharmesh Gopalakrishnan
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Aspirin ,education.field_of_study ,Antiplatelet drug ,business.industry ,Cost effectiveness ,medicine.medical_treatment ,Immunology ,Population ,Cell Biology ,Hematology ,Pharmacology ,Clopidogrel ,Biochemistry ,Cilostazol ,Dipyridamole ,P2Y12 ,medicine ,business ,education ,medicine.drug - Abstract
Introduction: With the expanding repertoire of antiplatelet drug targets and therapies, quantifiable parameters to assess their efficacy can prove to be useful in clinical decision-making. In this retrospective analysis we examined patients who had platelet aggregation testing done at our center between August 2008 and August 2013, focusing on those who were on some form of antiplatelet therapy during testing. Our goal was to define the impact of platelet aggregation testing on decision-making regarding continuation or change in antiplatelet therapy. Methods: Light transmission aggregometry (LTA) was used to assess efficacy of treatment with antiplatelet agents. Inhibition of platelet aggregation in response to ADP and arachidonic acid are reflective of the therapeutic effect of aspirin, while inhibition of platelet aggregation in response to ADP reflects the effect of P2Y12 receptor antagonists. As per parameters developed at our center, the combination of arachidonic acid aggregation Results: We studied results of platelet aggregometry in 117 patients who were on some form of antiplatelet therapy - 81 on a single agent (72 on aspirin alone, 9 receiving P2Y12 antagonist alone), 34 on dual therapy (33 on aspirin + P2Y12 antagonist, 1 on aspirin + cilostazol), and 2 patients on triple therapy (1 on aspirin + P2Y12 antagonist + cilostazol, 1 on aspirin + dipyridamole + cilostazol). None of our patients were on Gp IIb/IIIa inhibitors. In total, 108 patients were on aspirin therapy and 43 patients were on P2Y12 inhibitors. In 65 out of these 117 patients, the primary indication for platelet aggregation testing was to monitor the efficacy of antiplatelet therapy, while in the remaining 52, testing was done for other indications. Fifty-nine of these 65 patients were tested in the setting of a recent thrombotic event in the cerebral, coronary, peripheral, or other vascular bed. While 68 (58%) patients had optimal therapeutic response, 49 (42%) patients - 38 of the 108 (35%) patients on aspirin, and 14 of the 43 (32%) patients on a P2Y12 inhibitor - had evidence of suboptimal response to the respective agent. However, antiplatelet therapy was changed or adjusted in only 8 of these 49 patients following these sub-optimal test results, and only 3 had repeat testing following the change (all three of whom were shown to have complete response). Among the 108 patients on aspirin therapy, the total daily dose did not correlate either with the PFA-100 closure times (Collagen/ADP or Collagen/epinephrine) or with the degree of platelet aggregation in response to any of the agonists (ADP, arachidonic acid, collagen, epinephrine or ristocetin). Conclusions: Most of the patients who underwent platelet aggregation testing to monitor the efficacy of antiplatelet therapy had a recent thrombotic event that prompted the test. Though 42% of patients on antiplatelet agent(s) had in vitro evidence of sub-optimal platelet inhibition, antiplatelet therapy was changed or adjusted in only 16% of these individuals, and only 6% had repeat testing following the change. This suggests that, though platelet aggregation testing was potentially useful in monitoring efficacy of platelet inhibition, clinical changes in antiplatelet therapy were guided more by other factors, casting uncertainty upon the cost effectiveness of platelet function testing in this population. No significant increment was found in the in vitro antiplatelet effect of aspirin with increasing daily doses, suggesting lack of a dose-response beyond 81 mg per day. Disclosures McCrae: Syntimmune: Consultancy; Janssen: Membership on an entity's Board of Directors or advisory committees; Halozyme: Membership on an entity's Board of Directors or advisory committees; Momenta: Consultancy.
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- 2015
26. A History of Abnormal Bleeding Correlates with Platelet Dysfunction in Aggregation Studies but Not PFA-100 Analyses
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Keith R. McCrae, Paul Elson, Dharmesh Gopalakrishnan, and Heesun J. Rogers
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medicine.medical_specialty ,biology ,business.industry ,Abnormal bleeding ,PFA-100 ,Immunology ,Impaired platelet aggregation ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Gastroenterology ,Bleeding diathesis ,chemistry.chemical_compound ,chemistry ,Von Willebrand factor ,Internal medicine ,biology.protein ,Medicine ,Platelet ,business ,Ristocetin ,Blood Platelet Disorders - Abstract
Introduction:In this retrospective medical records-based study we analyzed the association between abnormal spontaneous or procedure-related bleeding and patterns of abnormality in tests of platelet function. Commonly used platelet function tests include Platelet Function Analyzer -100 Closure Times (PFA-100 CT), platelet aggregation testing using Light Transmission Aggregometry (LTA), and platelet dense granule release assay (by lumi-aggregometry). Other related tests include Von Willebrand factor (VWF) analysis, platelet flow-cytometry for cell surface glycoprotein expression, and electron microscopy. LTA in our center uses five different agonists - ADP, arachidonic acid, collagen, epinephrine and ristocetin, while lumi-aggregometry is performed using four agonists - ADP, arachidonic acid, collagen and epinephrine. Methods:This study included 497 patients who had platelet aggregation testing done using LTA between August 2008 and August 2013. Sixty-nine percent (n = 354) of these patients had a history of abnormal bleeding. Since the data were not normally distributed, Wilcoxon rank-sum test (for continuous variables) and Fisher's exact test (for categorical variables) were used wherever appropriate. P value of < 0.05 was considered as significant. Results:Of the patients with a history of abnormal bleeding, 81% had spontaneous bleeding, 29% had surgery/procedure-related bleeding, and 13% had history of both types of abnormal bleeding. Three hundred nine of these patients had a recent (< 4 weeks) bleeding event. Abnormal bleeding, recent or historical, was found to associate significantly with impaired platelet aggregation, as well as platelet release in response to ADP, arachidonic acid, collagen or epinephrine (P A history of a recent bleeding event ( No significant association was found between a history of abnormal bleeding (either recent or historical) and prolonged PFA-100 closure times (collagen/ADP or collagen/epinephrine), or any abnormality in VWF analyses or platelet flow-cytometry studies. Conclusions:While PFA-100 closure times, VWF analyses and platelet flow-cytometry panel failed to show an association with abnormal bleeding (historical or recent), impaired platelet aggregation or release in response to several agonists was found to correlate with abnormal spontaneous and/or procedure-related bleeding, recent as well as historical, suggesting that platelet aggregation and release assays may be useful in diagnosis of a bleeding diathesis in patients with an appropriate clinical history Disclosures McCrae: Halozyme: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Syntimmune: Consultancy; Momenta: Consultancy.
- Published
- 2015
27. Impact of Non-JAK2 Molecular Mutations and Cryptic SNP Lesions in Myelofibrosis Patients Treated with Ruxolitinib
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Eric D. Hsi, Yasmine Hirbawi, Ramon V. Tiu, Ahmad Zarzour, Karam Al-Issa, Christopher Gerace, Ali Tabarroki, Jing Ai, Evan Hsi, Heesun J. Rogers, Betty K. Hamilton, and Valeria Visconte
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medicine.medical_specialty ,Ruxolitinib ,Pathology ,IDH1 ,business.industry ,Myelodysplastic syndromes ,Immunology ,Cytogenetics ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Gastroenterology ,Lesion ,Internal medicine ,Cohort ,medicine ,Chromosome abnormality ,medicine.symptom ,business ,Myelofibrosis ,medicine.drug - Abstract
The identification of the JAK2V617F mutation in myeloproliferative neoplasms (MPN) paved the way for the pivotal studies that led to the FDA approval of a JAK1/2 inhibitor, ruxolitinib (rux) in patients (pts) with myelofibrosis (MF). Improvement in splenomegaly and debilitating disease-related symptoms were the primary clinical responses observed with rux. Although JAK2 mutational status did not impact response/survival in MF pts, cytogenetics had an impact on prognosis. In a related myeloid neoplasm specifically myelodysplastic syndromes, molecular mutations (TET2/DNMT3A) predict for better therapeutic response to DNA methyltransferase inhibitors. We hypothesized that somatic mutations and single nucleotide polymorphism array (SNP-A) lesions are frequent in MF pts treated with rux and may affect their clinical outcomes. To further investigate the predictive and prognostic impact of SNP-A lesions and somatic mutations in MF pts in the rux era, we studied 54 MF pts who received at least 12 weeks of rux therapy (tx) using a modified dose escalation approach (Tabarroki A et al. 55th ASH; Abstract 1586). Clinical (total symptom score [TSS], spleen size), cytogenetic (metaphase cytogenetics [MC], SNP-A), hematologic and survival data were collected before and 12-weeks post rux tx. Categorical data were analyzed using X2 test. A p-value of Disclosures Tiu: Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees.
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- 2014
28. Somatic Mutations in Splicing Factor 3b, Subunit 1 (SF3B1) Are a Useful Biomarker to Differentiate Between Clonal and Non-Clonal Causes of Sideroblastic Anemia
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Karam Al-Issa, Alan E. Lichtin, Ramon V. Tiu, Mikkael A. Sekeres, Eric D. Hsi, Ali Tabarroki, Valeria Visconte, Bernard J. Silver, Heesun J. Rogers, Sudipto Mukherjee, and Christopher Gerace
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medicine.medical_specialty ,Pathology ,Anemia ,business.industry ,Myelodysplastic syndromes ,Immunology ,Cell Biology ,Hematology ,Erythroid dysplasia ,Neutropenia ,medicine.disease ,Biochemistry ,Gastroenterology ,Sideroblastic anemia ,Dysplasia ,hemic and lymphatic diseases ,Internal medicine ,medicine ,Leukocytosis ,Macrocytic anemia ,medicine.symptom ,business - Abstract
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 Results: All pts presented with sustained anemia, RS w/o or with minimal BM dysplasia, and normal karyotype. SF3B1 mutations (K666N and K700E) were detected in 2 pts (pts#2 and 6) before the clonal disease manifested. The first pt was a 75-year-old man with leukocytosis, transfusion-independent anemia, and fatigue. At the time of SF3B1 molecular testing the BM was hypercellular (60%) with Conclusion: This case series although conducted in few pts provides important insights in the diagnostic use of molecular genetics in clinical practice. Often SA pts come to the clinic with inconclusive morphologic features of MDS or MDS/MPN. The presence of SF3B1 mutations may serve as an additional tool that can help differentiating between clonal and non-clonal cases of SA. In both pts, SF3B1 mutations anticipated the subsequent overt manifestation of clinical MDS or MDS/MPN phenotype. The collection of a larger cohort of pts is ongoing in order to further confirm this finding. Disclosures No relevant conflicts of interest to declare.
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- 2014
29. Risk of Arterial and Venous Thrombosis in Patients with 'Indeterminate' Lupus Anticoagulant Testing
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Suchitra Sundaram, Heesun J. Rogers, Natasha Catherine Edwin, Keith R. McCrae, and Bernard J. Silver
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Lupus anticoagulant ,medicine.medical_specialty ,business.industry ,Immunology ,Warfarin ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Thrombosis ,Surgery ,Venous thrombosis ,Antiphospholipid syndrome ,Internal medicine ,medicine ,Factor V Leiden ,Prothrombin G20210A ,Protein S deficiency ,business ,medicine.drug - Abstract
Background: Laboratory criteria for antiphospholipid antibody syndrome (APS) include lupus anticoagulant (LAC), anti cardiolipin antibodies, and anti-β2 glycoprotein antibodies. International Society of Thrombosis and Hemostasis (ISTH) criteria for the diagnosis of LAC include a positive screening test, positive mixing study, phospholipid dependence and absence of a co-existing factor inhibitor or anti-coagulant drug effect. Patients with test results that fulfill some but not all of the above criteria are considered to have indeterminate LAC. While a positive LAC is an established risk factor for thrombosis, there are limited data about the significance of indeterminate LAC test results. Objective: Our study aimed at determining the prevalence of venous and arterial thrombotic events in patients with indeterminate LAC. Results: We reviewed the clinical course of 500 patients that underwent LAC testing at Cleveland Clinic from 1/11/2008 to 12/28/2010; 346 had indeterminate results. Patients with indeterminate results while on warfarin (n=52) and enoxaparin (n=4) were excluded. Patients treated with heparin at the time of testing (n=50) were included, as heparin in the plasma was neutralized prior to testing. Multivariate logistic regression modeling was performed using XLSTAT software to study the value of various thrombotic risk factors as predictive factors for venous and arterial thrombosis. Associations between categorical variables were tested using the χ2 test or Fisher's exact test. Differences were considered statistically significant at an alpha level of 0.05. In our final cohort of 281 patients with indeterminate results, there were 110 males (39.1%) and 171 females (60.8%), with a median age of 52.5 yrs. Thrombotic manifestations of patients with indeterminate LAC are summarized in Table 1. There were a total of 170 venous thrombotic events in 119 patients (42.5%). Seventy-nine patients had one or more (≥1) instances of deep vein thrombosis (DVT); 45 patients had pulmonary embolism. There were 17 patients with porto-mesenteric thrombosis and 6 patients with other venous thrombotic events. Forty-five patients had concurrent malignancy (16 %) and 22 (8%) had other prothrombotic risk factors including factor V Leiden (n=14), prothrombin G20210A mutation (n=6), elevated homocysteine (n=1), and protein S deficiency (n=1). On multivariate analysis, a relationship was confirmed between the presence of associated prothrombotic disorders and venous thrombosis (OR = 3.36, 95% CI: 1.31- 8.61, P=0.012) (Table 2). Arterial thrombosis was noted in 63 patients, with stroke, myocardial infarction and acute limb ischemia being most common (Table 1). On multivariate analysis, taking into account atherosclerotic risk factors, co-existing hypertension, hyperlipidemia and smoking history were associated with arterial thrombosis (Table 2). Conclusion: Indeterminate results are common among patients referred for LAC testing. In this retrospective cohort, patients with indeterminate LAC had significant rates of venous and arterial thrombosis. Close observation, analysis of other prothrombotic risk factors, and repeat testing of these individuals at a later date should be considered. Disclosures No relevant conflicts of interest to declare.
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- 2014
30. Nitric Oxide As a Mediator of Bone Marrow Fibrosis in Patients with Myelofibrosis
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Kristin Colaluca, Nikolaos Papandantonakis, Heesun J. Rogers, Matt Kalaycio, Anjali S. Advani, Gina Rupp, Tracy Cinalli, Jing Ai, Daniel J. Lindner, Valeria Visconte, Mikkael A. Sekeres, Ali Tabarroki, Ramon V. Tiu, and John Desamito
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Pathology ,medicine.medical_specialty ,Ruxolitinib ,Myeloid ,business.industry ,Essential thrombocythemia ,Immunology ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Leukemia ,Polycythemia vera ,medicine.anatomical_structure ,Fibrosis ,medicine ,Platelet ,business ,Myelofibrosis ,medicine.drug - Abstract
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 Disclosures Tiu: Gilead: Membership on an entity's Board of Directors or advisory committees; Novartis: Speakers Bureau; Incyte : Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.
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- 2014
31. Post Operative Thrombosis Among Patients Testing Indeterminate for Lupus Anticoagulant
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Bernard J. Silver, Keith R. McCrae, Heesun J. Rogers, Suchitra Sundaram, and Natasha Catherine Edwin
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medicine.medical_specialty ,Lupus anticoagulant ,education.field_of_study ,business.industry ,medicine.drug_class ,Deep vein ,Immunology ,Population ,Anticoagulant ,Cell Biology ,Hematology ,Perioperative ,medicine.disease ,Biochemistry ,Thrombosis ,Surgery ,medicine.anatomical_structure ,Antiphospholipid syndrome ,Medicine ,Risk factor ,business ,education - Abstract
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.
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- 2014
32. Burden of Disease and Clinical Responses in Low and Intermediate-1 Risk Myelofibrosis Patients Treated with Ruxolitinib
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Brady L. Stein, Jing Ai, Rokana Taftaf, Alan E. Lichtin, Yogen Saunthararajah, Valeria Visconte, Ahmad Zarzour, Ali Tabarroki, Heesun J. Rogers, Ramon V. Tiu, and Nikolaos Papadantonakis
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medicine.medical_specialty ,Ruxolitinib ,Constitutional symptoms ,business.industry ,Immunology ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Extramedullary hematopoiesis ,Surgery ,medicine.anatomical_structure ,International Prognostic Scoring System ,Internal medicine ,Cohort ,medicine ,Bone marrow ,Myelofibrosis ,business ,Myeloproliferative neoplasm ,medicine.drug - Abstract
Myelofibrosis (MF) is a hematologic neoplasm characterized by variable degrees of cytopenias/ myeloproliferation, extramedullary hematopoiesis, disease-related constitutional symptoms and an increased risk for acute myeloid leukemia (AML) transformation. Ruxolitinib, a JAK1/2 inhibitor is FDA approved for the management of intermediate to high-risk MF. However, intermediate-2 and high-risk MF patients accounted for 99% of patients (pts) in the 2 pivotal trials that led to the approval of ruxolitinib in North America, Australia and Europe (COMFORT 1 and COMFORT 2 studies). However, even low and intermediate-1 (int-1) risk MF pts could have a high burden of disease. Here we report our experience of using ruxolitinib in MF pts stratified as low and int-1 risk by the Dynamic International Prognostic Scoring System (DIPSS). A total of 25 pts with low and Int-1 risk disease were treated with ruxolitinib at the Cleveland Clinic and Northwestern University. The median age of the cohort was 61 yrs (range=33-87; males=9, females=16). The median total symptom score (TSS) by Myeloproliferative Neoplasm Symptom Assessment Form (MPN SAF) was 20. The median pre-treatment spleen size by palpation was 13 cm below the left costal margin. Baseline median bone marrow (BM) fibrosis grading was 2 (on a scale 0-3; grade 1 N=3, grade 2 N=11, and grade 3 N=11). The indications for starting ruxolitinib were constitutional symptoms (fatigue N=20, night sweats N=4, pruritus N=1) and splenomegaly (N=13). The median starting dose was 5 mg twice daily, 10mg twice daily at 3 months and 6 months, and 5mg twice daily at 12 months. The median dose at last follow up was 10 mg twice daily. The median duration of treatment was 12 months. There was a median 73% reduction in the MPN SAF TSS compared to baseline (P< 0.001). There was improvement in each of the parameters of the TSS. The percentage of reduction in spleen size by palpation at 3, 6, 12 months was 49, 57, and 64%, respectively. In 5 patients who had a repeat BM biopsy, there was an improvement in their BM fibrosis by at least 1 grade (N=3), stable fibrosis (N=1) or increased fibrosis (N=1). Hematologic adverse events were reported at 3, 6, 12 months of treatment. Using Common Terminology Criteria for Adverse Events (CTCAEv4.0), there were seven with grade 3/4 anemia and six with grade 3/4 thrombocytopenia. Non-hematologic adverse events reported at 3, 6, 12 months of treatment included dizziness (N=1), headaches (N=1) and infections (pneumonia N=2, cellulitis N=2, perineal abscess N=1, Herpes Zoster N=2, and gluteal abscess after BM biopsy N=1; only one required hospitalization). Ruxolitinib was discontinued in 5 patients due to lack of clinical response (N=3) and grade 3/4 anemia/thrombocytopenia (N=2). The remaining 20 patients are still on treatment. None of the pts progressed to AML or died. The median follow up of our cohort was 27 months. Further, using a multi-Analyte ELISArray Kit, plasma concentrations of interleukins (IL):IL1A, IL1B, IL2, IL4, IL6, IL8, IL10, IL12, IL17A, INFg, TNFa and GM-CSF were measured in low/int-1 risk pts (N=3) and intermediate-2/high risk patients (N=4) and no statistically significant difference was found between the two risk groups (P>0.05) further supporting the high burden of disease observed even in low and int-1 risk MF patients. In conclusion, pts with low/int-1 risk MF have a high burden of disease and can achieve meaningful clinical responses to ruxolitinib similar to int-2/ high risk MF pts without severe toxicity. Larger studies are needed to further evaluate the safety and efficacy of ruxoltinib in this patient population. Disclosures No relevant conflicts of interest to declare.
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- 2014
33. Somatic Mutations in the Wiskott-Aldrich Syndrome Protein Family Member 3 (WASF3) Are Associated with Poor Prognosis in Myeloid Malignancies
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Heesun J. Rogers, Aaron T. Gerds, John Barnard, Ramon V. Tiu, Valeria Visconte, Li Zhang, Ali Tabarroki, and Velizar Shivarov
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Mutation ,Myeloid ,Wiskott–Aldrich syndrome ,Myelodysplastic syndromes ,Immunology ,Myeloid leukemia ,Single-nucleotide polymorphism ,Cell Biology ,Hematology ,Biology ,medicine.disease_cause ,medicine.disease ,Biochemistry ,medicine.anatomical_structure ,Germline mutation ,hemic and lymphatic diseases ,medicine ,Cancer research ,Exome sequencing - Abstract
Myelodysplastic syndromes (MDS) encompass a clinically and pathologically heterogeneous group of blood cancers characterized by dysplasia, cytopenias, and a significant risk for transformation to acute myeloid leukemia (AML). The understanding of MDS biology is the key to deciphering disease pathogenesis and offers the ability to identify specific pathways that may be important for diagnosis, prognosis, and most importantly for designing new targets amenable to therapies. Genetic factors specifically molecular mutations in genes involved in many important cellular processes such as epigenetics, signaling, and RNA splicing have been identified in MDS. These mutations affect disease pathogenesis and are prognostic. Whole exome sequencing (WES) technologies have advanced genomic discoveries in MDS. We applied WES on 2 ug of DNA of 14 patients (pts) (MDS=11 and MF=3). Non clonal CD3+ cells served as germline control. 20 million reads were run on an Illumina HiSeq2000 sequencer. A stringent bio-informatic algorithm filtered all variants based on a variation score (>=30) and a coverage (30X). Tumor nucleotide variation analysis was performed for each pair (tumor vs. germline), where only variants unique to the tumor were retained after the exclusion of SNPs. Using this tool, we discovered the first recurrent somatic mutation (R178H) in a Wiskott-Aldrich syndrome protein (WASP) family of genes specifically Wiskott-Aldrich syndrome protein family member 3 (WASF3) also known as WAVE-3 in a pt with MDS. Further analysis by Sanger sequencing on a larger cohort of pts with myeloid neoplasms [n=241; (MDS= RARS/RCMD: n=30; RAEB1/2: n=49); primary AML (n=42), CMML1/2 (n=30), MPN (MF, ET, PV, CML: n=90)] showed WASF3 mutations in 5/49 (10%) higher risk MDS and 2/25 (8%) JAK2WT PV pts. MDS pts with WASF3 mutations (WASF3MUT) had higher peripheral (3.7% vs. 0.5%, P = .002) and bone marrow blasts % (4.7% vs. 1.4%, P = .005), and lower PLT count (71 vs. 191 k/uL, P = .005) compared to WASF3WT pts. Molecular analysis for a panel of genes known to be mutated in MDS and MPN like ASXL1, U2AF1, SRSF2, SF3B1 and IDH1/2 showed that 2/7 WASF3MUT carried also SRSF2 mutations (P95R/L). WASF3 mutations had a negative impact on the survival outcomes. Indeed WASF3MUT pts had a shorter overall survival (OSMut 10.5 mos vs. OSWT 29.1 mos, P=.01) and progression free survival (PFSMUT 5.1 mos vs. 15.9 PFSWT mos, P = .03) compared to WASF3WT pts. WASF3MUT MDS pts also carried worse OS compared to pts with MDS and normal karyotype (14 mos vs. 41.8 mos, P =.019) and SF3B1MUT which are known to carry a good prognostic outcome in MDS (14 mos vs. 54.1 mos, P =.003). Western blotting showed increased WASF3 protein expression in the index case compared to WASF3WT pts (n=2). We performed microarray analysis on 6 higher risk MDS and 6 MPN pts, with one pt harboring the WASF3 mutation. Illumina Human HT-12 v4 expression beadchips were used to estimate mRNA expression levels. The WASF3R178H mutant pt had a higher WASF3 mRNA expression compared to 2 matched WT pts (Fold Change [FC] =1.44, P=.16). Further, protein complex pathways, specifically, Arp2/3, which has been shown to interact with WASF3 and other WASF family members also showed similar patterns of expression. ARP2 (ACTR2) and ARP3 (ACTR3) were both elevated in WASF3MUT vs. WT (ACTR2: FC=1.79, P=.029; ACTR3: FC=1.60, P=.144). We also found that WAS, which encodes the WASP protein, was expressed in our MDS samples and shows elevated expression in WASF3MUT vs. WT (FC=1.58, P = .05). This suggests that other members of the WASP family of proteins may be involved in MDS pathogenesis. WASP was originally found to be mutated in Wiskott Aldrich syndrome (WAS), an X-linked recessive immunodeficiency disorder. Mutations in this gene are important in actin polymerization, a process essential for cytokinesis, chromosome segregation, endocytosis, migration, and formation of filopodia and lamellipodia. In prostate and breast cancers, overexpression of WASF3 has been associated with increased disease metastasis and tumor invasion and treatment resistance. In conclusion, WASF3 somatic mutations are found in MDS and MPN pts and associated with increased mRNA/ protein expression. The presence of WASF3 mutations is associated with poor survival in myeloid malignancies. Understanding the mechanism by which WASF3 gene act may provide new targeted therapeutic options for myeloid cancers which harbor this mutation. Disclosures No relevant conflicts of interest to declare.
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- 2014
34. Somatic Mutations of the Breast Cancer Amplified Sequence-1 (BCAS1), a Novel Leukemogenic Driver in Myelodysplastic Syndromes with Del(20q)
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Reda Z. Mahfouz, Ali Tabarroki, Heesun J. Rogers, Anjali S. Advani, John Barnard, Matt Kalaycio, Juraj Bodo, Mikkael A. Sekeres, Shashirekha Shetty, Velizar Shivarov, Yogen Saunthararajah, Li Zhang, Ramon V. Tiu, Betty K. Hamilton, and Valeria Visconte
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Candidate gene ,Mutation ,Myeloid ,Myelodysplastic syndromes ,Immunology ,Single-nucleotide polymorphism ,Cell Biology ,Hematology ,Biology ,medicine.disease ,medicine.disease_cause ,Biochemistry ,Somatic evolution in cancer ,medicine.anatomical_structure ,medicine ,Cancer research ,Chromosome 20 ,Exome sequencing - Abstract
Deletion of the long arm of chromosome 20 [del(20q)] is a recurrent clonal abnormality seen in myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), and acute myeloid leukemia (AML). Del(20q) is believed to originate in a pluripotent stem cell of both myeloid and lymphoid cells and contribute to clonal evolution by the loss/inactivation of ≥1 tumor suppressor genes. Commonly deleted regions (CDRs) of del(20q) contain genes that are overexpressed in hematopoietic progenitor cells of del(20q) patients (pts). Although associated with good prognosis when present as a sole abnormality in MDS, the natural history may vary within similar disease subtypes. Next-generation sequencing technologies have accelerated the discovery of genetic mutations implicated in MDS pathogenesis. To date, the vast majority of these mutations are neither specific to a disease subtype nor to a cytogenetic abnormality. We hypothesized that molecular mutations are the main drivers of leukemic progression in MDS with del(20q). Whole exome sequencing (WES) was performed on 6 pts with del(20q): MDS=3, MPN=3. DNA (3ug) was used for WES. 20 million reads were run on an Illumina HiSeq2000 sequencer. A bioinformatic algorithm filtered all tumor variants based on variation score (≥30) and coverage (30X). Tumor nucleotide variants were counted if unique to the tumor excluding SNPs and germline variants. We detected 42 candidate genes. Only 1 nucleotide variation was detected on the chromosome (chr) 20 of 1 high risk MDS with del(20q) pt. The alteration resulted in a proline to a glutamine substitution at position 488 of exon 11 (P488Q) of a gene called Breast Cancer Amplified Sequence-1 (BCAS1). BCAS1 gene is located in chr 20q13.2 region. BCAS1 overexpression has been implicated in aggressive breast and colon cancer subtypes but the mechanisms for this overexpression have not been clarified. Of note, BCAS1 mutations have not been previously identified in any hematologic malignancy. A larger cohort of pts: N=395; AML=70, MPN=205, MDS=90, other bone marrow failure diseases=30, and hematologic cell lines were tested for BCAS1 mutations. We found recurrent BCAS1P488Q mutation in 10/56 (18%) of MDS with del(20q) pts who evolved to high risk disease and AML (sAML). All BCAS1 mutant (BCAS1MUT) pts have an associated del(20q) abnormality. BCAS1MUT pts have a median age of 67 yrs (range, 45-87); male/female=9:1, had more leukopenia (3 vs. 11 x109/L, P=.04), and neutropenia (1.3 vs. 8.3 x109/L, P=.04) compared to other del(20q) pts without mutation. Metaphase cytogenetics, FISH and SNP-A showed that the CDRs of the 20q region of all BCAS1MUT pts did not contain the 20q13.2 region. Screening for 12 genes relevant to MDS pathophysiology showed that BCAS1MUT pts harbored unfavorable genetic mutations: SRSF2 (1/10), TP53 (2/10) and U2AF1 (3/10) with 1 pt having both TP53 and U2AF1. BCAS1P488Q was mutually exclusive in 4 pts. BCAS1MUT had a dismal prognosis compared to BCAS1WT (PFS: 5.1 vs. 15.9 mos, P=.03). Bioinformatic analysis of publicly available gene expression data showed increased BCAS1 mRNA level in AML, CML and in the K562 cells. RT-PCR found a higher (5-fold) BCAS1 mRNA level in the index case compared to one WT pt and no expression in healthy subjects. K562 and CML primary cells (N=6) were used as positive controls. Presence of BCAS1 protein was confirmed by immunohistochemistry and western blotting. CGH revealed that 10% of breast tumors have copy-number gains of a 1-Mb region of 20q13 where BCAS1 maps. Amplification of this region was seen in aggressive subtypes of squamous cell carcinoma. We applied FISH analysis for BCAS1 finding no amplification of the 20q13 region in MUT vs WT (2 vs 2) pts suggesting that other mechanisms possibly other genetic mutations lead to BCAS1 overexpression in sAML. In the K562 cells, 3 copies of BCAS1 were identified which may explain increased BCAS1 expression in these cells. In sum, recurrent BCAS1 mutations are found specifically in MDS with del(20q) pts who transform to AML. They are exclusively found at the time of AML transformation. mRNA and protein overexpression are observed in BCAS1MUT pts and lead to inferior survival outcomes and poor response to chemotherapy. This is the first genetic mutation solely found in a specific MDS cytogenetic subset and may represent a novel mechanism for leukemogenesis. Disclosures No relevant conflicts of interest to declare.
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- 2014
35. Molecular Characterization Of Myeloid Neoplasms Harboring Isochromosome 17q Abnormality
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Shashirekha Shetty, Ali Tabarroki, Edy Hasrouni, Ramon V. Tiu, Matt Kalaycio, Heesun J. Rogers, Eric D. Hsi, Robyn Frum, Anjali S. Advani, Chris Gerace, Mikkael A. Sekeres, Yogen Saunthararajah, Valeria Visconte, Li Zhang, and Hien K. Duong
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Genetics ,Chromosome 7 (human) ,Candidate gene ,Myeloid ,Immunology ,Isochromosome ,Chronic neutrophilic leukemia ,Myeloid leukemia ,Cell Biology ,Hematology ,Biology ,medicine.disease ,Biochemistry ,Chromosome 17 (human) ,Leukemia ,medicine.anatomical_structure ,medicine ,Cancer research - Abstract
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.
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- 2013
36. BCL-2 Family Of Genes Is a Key Regulator In The Pathogenesis Of SF3B1 Mutant and Wild Type MDS With Ring Sideroblasts and Represents a Novel Drug Target In This Disease
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Reda Z. Mahfouz, Valeria Visconte, Matt Kalaycio, Brian T. Hill, Ali Tabarroki, Ramon V. Tiu, John Barnard, Edy Hasrouni, Yogen Saunthararajah, Heesun J. Rogers, and Mikkael A. Sekeres
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Myeloid ,Immunology ,Wild type ,Cell Biology ,Hematology ,Biology ,medicine.disease ,Biochemistry ,Molecular biology ,ETV6 ,Exon ,Leukemia ,medicine.anatomical_structure ,Refractory anemia with ring sideroblasts ,Gene expression ,medicine ,Gene - Abstract
Myelodysplastic syndromes (MDS) are a group of neoplastic diseases characterized by impairment in hematopoietic cell differentiation which leads to disease related consequences like cytopenias and cellular dysplasia. Mutations in splicing factor 3b subunit 1 (SF3B1) are the most frequently identified genetic abnormalities in refractory anemia with ring sideroblasts (RARS) and RARS with thrombocytosis (RARS-T). However, the mechanisms whereby cells acquire clonal dominance and evolution, cytopenias and impairment in differentiation remain unknown. To understand the role of SF3B1 mutations in the pathogenesis of RARS/-T, we analyzed the transcriptome of SF3B1 mutant (MUT) and wild type (WT) RARS/-T patients (pts). RNA was isolated from bone marrow of healthy subjects (n=3), SF3B1 MUT (n=3) and WT (n=3) RARS/-T, and other MDS (n=5). cDNA was made from RNA (1.5-3ug) and fragmented for library preparation. RNA-sequencing was performed on 20 million sequence reads on an Illumina HiSeq2000 and aligned to the human genome 19. RNA-splicing patterns were analyzed by a published bioinformatics algorithm at three levels (exon usage, gene expression, and pathway analysis) (Visconte V; Blood. 2012). Differential exon usage identified 271 and 71 genes with at least 1 exon alternatively spliced in SF3B1 MUT, WT, and healthy subjects (P Disclosures: No relevant conflicts of interest to declare.
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- 2013
37. Splicing Factor 3b Subunit 1 (SF3B1) mediates Mitochondrial Iron Overload In Myelodysplastic Syndromes With Ring Sideroblasts By Alternative Splicing Of Mitoferrin-1 (SLC25A37)
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John Barnard, Matt Kalaycio, Valeria Visconte, Nanthawan Avishai, Li Zhang, Alan E. Lichtin, Heesun J. Rogers, Anjali S. Advani, Edy Hasrouni, Ramon V. Tiu, Yogen Saunthararajah, Reza Sharghi-Moshtaghin, Eitan Fibach, Reda Z. Mahfouz, Midori Hitomi, Arthur H. Heuer, Mikkael A. Sekeres, Ali Tabarroki, Jonathan E. Cowen, Daniel J. Lindner, and Eliezer A. Rachmilewitz
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biology ,medicine.diagnostic_test ,Immunology ,Alternative splicing ,Wild type ,Cell Biology ,Hematology ,Biochemistry ,Molecular biology ,ABCB7 ,Ferritin ,Exon ,GLRX5 ,Total iron-binding capacity ,Gene expression ,biology.protein ,medicine - Abstract
Splicing factor 3b subunit 1, SF3B1 contributes to the formation of ring sideroblasts (RS) in Myelodysplastic syndromes (MDS). Abnormalities in iron trafficking have been implicated in the pathogenesis of refractory anemia with RS (RARS) and RARS with thrombocytosis (RARS-T). In RARS/-T, light microscopy and traditional iron profiles do not accurately reflect intracellular iron status. SF3B1 mutant (MUT, n=25) and wild type (WT, n=8) RARS patients (pts) have no difference in iron profiles (ferritin (ng/mL): 1244 ng/mL ± 926 vs 1215 ± 1065; total iron binding capacity (ug/dL): 252 ± 80 vs 234 ± 50). We previously reported distinct differences in iron distribution between SF3B1 MUT and WT pts based on transmission electron microscopy (EM). Coarse iron deposits are present in the mitochondria of SF3B1 MUT while smaller iron deposits are found in WT. We hypothesized that SF3B1 mutations affect distinct downstream targets leading to the iron phenotype differences in MUT vs WT. To study these differences, we performed a series of EM, flow cytometric (FC) and RNA-sequencing experiments. Quantification of iron by scoring deposits of iron/grid showed higher amount of mitochondrial iron in MUT vs WT by EM. We used a FC based-approach to quantify the cytoplasmic and mitochondrial iron utilizing the properties of two cell-permeant compounds (calcein-AM and rhodamine-B) being retained in the cytoplasm and the mitochondria, respectively. In keeping with our EM studies, we found that SF3B1 MUT have a higher % of rhodamine-B+ cells compared to WT RARS pts and healthy subjects (77 vs 19 vs 0.87%; n=6) indicating that MUT accumulates more mitochondrial iron compared to WT. Cytoplasmic iron was not different between MUT and WT (57 vs 43%) but higher than healthy subjects (1.5%; p=0.02), suggesting that MUT store more mitochondrial iron compared to WT pts. Sf3b1+/- mice also showed higher iron stores in the mitochondria rather than in the cytosplasm (85 vs 16%; n=4). To understand the factors leading to increased mitochondrial iron, we analyzed the transcriptome of BM cells derived from a homogeneous group of SF3B1 MUT (n=3), WT RARS (n=3), and healthy subjects (n=3). Total RNA (1.5-3ug) was subjected to RNA-sequencing using Illumina HiSeq2000. Twenty-million sequencing reads were interrogated. A comprehensive bioinformatic analysis was conducted on three levels (exon usage, gene expression, pathway analysis). Global gene expression analysis detected significant gene expression changes in 59 genes (FDR < 0.2) between MUT and healthy subjects. Mitochondrial genes linked to iron pathophysiology were well represented in the analysis. A total of 354 genes with exon usage/ gene expression difference in at least 1 exon were investigated. One of the top candidate genes showing alternative splicing is SLC25A37 (chr:8p21.2), a mitochondrial iron importer that mediates Fe2+ incorporation into the mitochondria. SLC25A37 was associated with a 2-4 fold higher expression in MUT vs WT and healthy individuals (mean base=4145 vs 2451 vs 1058). We also investigated other genes important in iron metabolism including SLC25A38, PUS1, and GLRX5 but no differences in both exon usage and gene expression were noted supporting the different nature of acquired and congenital sideroblastic anemia (SA). Some mitochondrial genes important in iron trafficking showed no changes in exon usage but exhibited differences in gene expression suggesting that they are pathways independent of SF3B1 mutations but still contributing to iron metabolism. ALAS2 was down-regulated in MUT vs WT RARS (fold change (FC): 0.39) while both MUT and WT pts exhibited an increased level compared to healthy subjects (FC: 5.0 and 2.0). ABCB7 was down-regulated compared to healthy subjects in both groups (FC:0.45 and 0.42). Using bisulfite sequencing we found that MUT have significantly higher hypermethylation of ALAS2 compared to WT RARS pts (FDR Disclosures: No relevant conflicts of interest to declare.
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- 2013
38. Spliceosome Gene Mutations Are Frequently Found In JAK2 Negative Myelofibrosis and Associated With Worse Clinical Outcomes
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Juraj Bodo, John Barnard, Ali Tabarroki, Heesun J. Rogers, Alan E. Lichtin, Mikkael A. Sekeres, Ramon V. Tiu, Edy Hasrouni, Li Zhang, and Valeria Visconte
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Spliceosome ,Myeloid ,Immunology ,Wild type ,Cell Biology ,Hematology ,Biology ,Gene mutation ,medicine.disease ,Biochemistry ,Molecular biology ,Exon ,medicine.anatomical_structure ,hemic and lymphatic diseases ,RNA splicing ,medicine ,Myelofibrosis ,Myeloproliferative neoplasm - Abstract
Myelofibrosis (MF), a Philadelphia chromosome negative myeloproliferative neoplasm (MPN) requires clonal markers for accurate diagnosis according to the 2008 World Health Organization classification scheme. However, molecular mutations in Janus Kinase 2 (JAK2) are found in only 50% of MF patients (pts). Various somatic mutations identified in other myeloid cancers have also been found in MF with pre-existing JAK2 V617F mutations. We previously reported in 57 pts diagnosed with Triple Negative MF (TN-MF: JAK2 exon12/14 and wild type (WT) MPL) the absence of mutations in genes commonly identified in other myeloid malignancies, specifically TET2, DNMT3A, CBL, IDH1/2, SH3B2 (LNK), N/KRAS and EZH2. Only somatic mutations in ASXL1 were identified in a small cohort of TN-MF (16%). Mutations in components of the RNA splicing machinery have been recently identified in varying frequencies in different myeloid malignancies. We performed RNA-sequencing on 2 samples (JAK2 mutant (MUT) and 1 JAK2 WT) and found that 2 spliceosome genes (U2AF1 and SF3B1) had higher mRNA expression levels in the JAK2 WT (MFC=0.76 and 0.82, respectively) compared to JAK2 MUT patient. We hypothesized that somatic mutations in RNA splicing factor genes occur in TN-MF pts and may be helpful in the biological characterization of this group of pts. Therefore, we isolated DNA from bone marrow (BM) or peripheral blood mononuclear cells from a cohort of MF pts (N=132; JAK2 MUT:75 and JAK2 WT:57) and performed Sanger sequencing for SF3B1 (exons 13-16), U2AF1 (exons 2, 6 and 7), and SRSF2 (exons 1 and 2). Baseline characteristics of pts and clinical data including hematologic parameters, BM results, and presence of splenomegaly by palpation were collected. Pts were stratified based on the Dynamic International Prognostic Scoring System-Plus risk as high=40, Int-2=51, Int-1=11 and low-risk=5 pts. The mean duration of follow-up was 16.5 months in JAK2 MUT and 12.8 months in JAK2 WT. Spliceosome mutations were found in 35/132 (27%) (SRSF2=17%, U2AF1=8% and SF3B1=2%) of pts. Interestingly, the frequency of spliceosome mutations in JAK2 WT was higher (31%; SRSF2=26%, U2AF1= 5%, SF3B1=0) compared to JAK2 MUT (18%; SRSF2=10%, U2AF1=6%, SF3B1=2%) pts. Of note, the 2 pts with SF3B1 mutations (K700E) had ring sideroblast in the BM (occasional and 50%). A large number of WT JAK2 patients (50%) had concomitant SRSF2 and ASXL1 mutations. Both SRSF2 and ASXL1 have been associated with poor prognosis in MF. Indeed, in our JAK2 WT cohort, pts who harbored SRSF2 mutations had a higher mean percentage of BM blasts compared to the ones that were SRSF2 WT (5.6% vs 1.8%; P=.006). Moreover, JAK2 WT pts carrying spliceosome mutations had more severe anemia as shown by lower hemoglobin (8.98 g/dL vs 10.5) and higher leukocyte counts (27.5 x109/L vs 20.5) compared to WT cases. Furthermore, we noticed that they also had higher frequency of RBC (Red Blood Cells) transfusions compared to WT (60% vs 40%) cases. Spleen examination by palpation below the left sub-costal margin showed that JAK2 WT cases carrying spliceosome mutations had larger splenomegaly compared to WT cases [10.1 cm (range 4-26), vs 8.7cm (range 2-20); P=0.05]. In conclusion, molecular alterations in the spliceosome machinery are frequently found in MF pts who are WT for JAK2/MPL and are associated with higher BM blast percentage, more severe anemia, higher leukocyte counts, transfusion dependence and more prominent splenomegaly. The observation of appreciable frequency of spliceosome mutations in JAK2 WT MF pts opens the possibility of using spliceosome inhibitors in the management of this disease group. Disclosures: No relevant conflicts of interest to declare.
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- 2013
39. Triple Negative (JAK2 exon 12 /14 and MPL wild type) Myelofibrosis Have Higher Expression Of CDC25A and Greater Sensitivity To CDC25A Inhibition Compared To JAK2 Mutant Cases
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John Barnard, Alan E. Lichtin, Ali Tabarroki, Ramon V. Tiu, Juraj Bodo, Felicitas Lacbawan, Li Zhang, Daniel J. Lindner, Valeria Visconte, Mikkael A. Sekeres, Heesun J. Rogers, and Edy Hasrouni
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Somatic cell ,Immunology ,Mutant ,Wild type ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Molecular biology ,Transcriptome ,Exon ,Gene expression ,Gene ,Exome sequencing - Abstract
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 ( Disclosures: No relevant conflicts of interest to declare.
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- 2013
40. Modified Dose Escalation Of Ruxolitinib: A Feasible Therapeutic Approach In The Management Of Myelofibrosis
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Ali Tabarroki, Daniel Lindner, Valeria Visconte, Heesun J. Rogers, John Desamito, Hien K. Duong, Alan E. Lichtin, Anjali S. Advani, Ronald M. Sobecks, Tracy Cinalli, Kristin Dodd, Jing Ai, Yogen Saunthararajah, Matt Kalaycio, Brady L. Stein, Mikkael A. Sekeres, and Ramon V. Tiu
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medicine.medical_specialty ,Ruxolitinib ,Anemia ,Maintenance dose ,business.industry ,Immunology ,Cell Biology ,Hematology ,Neutropenia ,medicine.disease ,Biochemistry ,Gastroenterology ,Surgery ,Log-rank test ,Regimen ,Internal medicine ,Cohort ,medicine ,Myelofibrosis ,business ,medicine.drug - Abstract
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.
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- 2013
41. Splicing Factor 3b Subunit 1 (SF3B1) Heterozygous Mice Manifest a Hematologic Phenotype Similar To Low Risk Myelodysplastic Syndromes With Ring Sideroblasts
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Kyoichi Isono, Valeria Visconte, Yvonne Parker, Yogen Saunthararajah, Daniel J. Lindner, John Barnard, Heesun J. Rogers, Ali Tabarroki, Anjali S. Advani, Reda Z. Mahfouz, Edy Hasrouni, Ramon V. Tiu, Haruhiko Koseki, Mikkael A. Sekeres, Quteba Ebrahem, and Li Zhang
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NPM1 ,Mutation ,Myelodysplastic syndromes ,Immunology ,Cell Biology ,Hematology ,Biology ,medicine.disease ,medicine.disease_cause ,Biochemistry ,Molecular biology ,Phenotype ,Transcriptome ,Exon ,Gene expression ,medicine ,Gene - Abstract
The link between SF3B1 mutation and the ring sideroblast (RS) phenotype in myelodysplastic syndromes (MDS) was solidified by the identification of RS in Sf3b1 heterozygous (Sf3b1+/-) mice. The identification of SF3B1 mutations in refractory anemia with RS (RARS) and RARS with thrombocytosis (RARS-T) showed the importance of RNA splicing in MDS biology. Furthermore, it opened the possibility of targeted therapy using spliceosome inhibitors in RARS/-T. However, many questions remain unanswered in linking SF3B1 dysfunction to MDS biology like the downstream targets of this gene. The identification of a robust murine model is essential to study a specific molecularly defined disease-phenotype and develop targeted therapies. We identified occasional RS in the bone marrow (BM) of Sf3b1+/- which are rarely found in current mouse models of MDS (Beachy SH, Hematol Oncol Clin North Am, 2010). However, aside from RS in the BM no other MDS features were found. Sf3b1+/- mice were originally engineered as a means to study the interaction between polycomb (PcG) genes and other protein-complexes (Isono K, Genes Dev, 2005). Homozygous Sf3b1-/- mice died at the stage of pre-implantation of the embryos while Sf3b1+/- appear healthy. Several tools have been tested to model MDS in genetically engineered mice targeting key genes in MDS. However, the creation of an ideal mouse model resembling distinct morphologic MDS subtypes is still lacking. To define a mouse model useful for preclinical therapeutic studies, we evaluated the hematologic features of Sf3b1+/- and Sf3b1+/+ mice during a long term follow-up. Five Sf3b1+/- and 5 Sf3b1+/+ mice were followed over time until 12 months of age. Blood was drawn from the retro-orbital vein every month starting from 6 months of age. Using two-sample Wilcoxon test we compared standard hematologic parameters finding a significant difference over the time between Sf3b1+/- and Sf3b1+/+: hemoglobin (g/dL) 6.9 ±0.73 vs 10.0 ±1.6 (P=0.008), red blood cells (M/uL) 8.3±0.5 vs 5.9±1.0 (P=0.008), platelets (K/uL) 731±105 vs 579±93 (P=0.008), and mean corpuscular volume (fL) 47.8±1.5 vs 45.1±1.1 (P=0.032). We did not detect any significant difference in other parameters although lymphocytes were more represented vs neutrophils, eosinophils and monocytes in Sf3b1+/- vs Sf3b1+/+ (6.3K/uL ±3.1 vs 5.8 ±1.8; P=1). Analysis of the BM, showed no difference in cell number between Sf3b1+/- (n=7) and Sf3b1+/+ (n=7) (44.1±9.1 vs 43.2 ±11; P=0.62). However, distinct dyserythropoiesis such as nuclear budding or irregular nuclei in Wright-Giemsa and occasional RS in Prussian blue stains were noted in Sf3b1+/- which were not present in Sf3b1+/+. In support of the iron overload seen in SF3B1 mutant patients (pts), a similar observation was made in Sf3b1+/- by light microscopy and rhodamine based- flow cytometry to quantify mitochondrial iron (Visconte, Abstract #64897). We also characterized the transcriptome of Sf3b1+/- and Sf3b1+/+. Total RNA was isolated from BM of age/gender matched mice, polyA cDNA was prepared from 3ug of RNA and Mouse RNA-sequencing was run on Illumina HiSeq2000. 200 exons were found differentially used in Sf3b1+/- vs Sf3b1+/+. Chromosome 1 contains the highest number of genes with at least 1 exon alternatively used similar to what we observed in SF3B1 mutant pts. In total 22 genes showed stronger differential expression in Sf3b1+/- vs Sf3b1+/+. Sf3b1 was down-regulated as expected (MFC: 0.74) in Sf3b1+/-. Studies in Sf3b1+/- mice show that Sf3b1 protein physically interacts with Class II PcG proteins (PRC1) which are relevant in MDS. When we interrogated PcG genes and others, we found lower mRNA levels of ezh2 (MFC: 0.06) and npm1 and tpr53 (MFC: 0.01 and 0.28) and no difference in asxl1 and runx1 (MFC:1.22 and 1.1) in Sf3b1+/- vs Sf3b1+/+. Jak2, dock8, and uhrf2 showed significant (P=.0003) higher expression in Sf3b1+/-. MDS is a heterogeneous disease characterized by genetic and non-genetic causes. Introduction of secondary events implicated in MDS pathogenesis can modify the phenotype of Sf3b1+/- mice. In sum, Sf3b1+/- mice after 6 months of follow-up developed macrocytic anemia, thrombocytosis, RS and dyserythropoiesis akin to human RARS/-T. Furthermore, transcriptome analysis shows exon usage/ gene expression changes similar to human SF3B1 mutants lending support that Sf3b1+/- can serve as a mouse model for studying the biology of human low risk MDS specifically that of RARS/-T. Disclosures: No relevant conflicts of interest to declare.
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- 2013
42. Inhibition Of JAK-STAT Pathway As a Therapeutic Option For Myelofibrosis Associated Pulmonary Hypertension
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Daniel J. Lindner, Mikkael A. Sekeres, Ramon V. Tiu, Yogen Saunthararajah, Ali Tabarroki, Gustavo A. Heresi, Kristin Dodd, Sudipto Mukherjee, Tracy Cinalli, Stavros E. Mountantonakis, Betty K. Hamilton, Edy Hasrouni, Alan E. Lichtin, Heesun J. Rogers, Anjali S. Advani, Valeria Visconte, Yvonne Parker, Gina Rupp, Matt Kalaycio, Li Zhang, and Hien K. Duong
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medicine.medical_specialty ,Ruxolitinib ,Endothelium ,business.industry ,Immunology ,Cell Biology ,Hematology ,medicine.disease ,Brain natriuretic peptide ,Biochemistry ,Pulmonary hypertension ,Gastroenterology ,Pathophysiology ,Pathogenesis ,medicine.anatomical_structure ,Internal medicine ,Medicine ,business ,Myelofibrosis ,Lenalidomide ,medicine.drug - Abstract
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= Disclosures: No relevant conflicts of interest to declare.
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- 2013
43. A Prognostic Scoring System for Unclassifiable MDS and MDS/MPN
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Ali Tabarroki, Betty K. Hamilton, Valeria Visconte, Yang Liu, Rami S. Komrokji, Manoj Bupathi, Fabiola Traina, Edy Hasrouni, Ramon V. Tiu, Yogen Saunthararajah, Heesun J. Rogers, Mikkael A. Sekeres, and Hien K. Duong
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Oncology ,Neuroblastoma RAS viral oncogene homolog ,medicine.medical_specialty ,Univariate analysis ,Pathology ,Myeloid ,business.industry ,Immunology ,Hazard ratio ,Cell Biology ,Hematology ,medicine.disease_cause ,Biochemistry ,medicine.anatomical_structure ,International Prognostic Scoring System ,hemic and lymphatic diseases ,Internal medicine ,Cohort ,medicine ,Absolute neutrophil count ,KRAS ,business - Abstract
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 3% v. ≤3%) (p3.6g/dL) (p11.5g/dL) (p85 vs ≤85%) (p Disclosures: No relevant conflicts of interest to declare.
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- 2012
44. Molecular Mutations in U2AF1 Are Most Commonly Found in Del20q Myelodysplastic Syndromes but Do Not Lead to Poor Prognosis in This Karyotypic Subtype
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Heesun J. Rogers, Hideki Makishima, Hien K. Duong, Fabiola Traina, Edward A. Copelan, Anjali S. Advani, Mikkael A. Sekeres, Li Zhang, Michael J. Clemente, Paula de Melo Campos, Betty K. Hamilton, Valeria Visconte, Ramon V. Tiu, Edy Hasrouni, Ali Tabarroki, and Manoj Bupathi
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Oncology ,medicine.medical_specialty ,Pathology ,IDH1 ,Myelodysplastic syndromes ,Immunology ,Hazard ratio ,Cytogenetics ,Cell Biology ,Hematology ,Biology ,medicine.disease ,medicine.disease_cause ,Biochemistry ,Internal medicine ,medicine ,Progression-free survival ,KRAS ,Myelofibrosis ,Exome sequencing - Abstract
Abstract 3804 Del20q is a commonly detected chromosomal abnormality in myelodysplastic syndromes (MDS) and myelofibrosis (MF). In MDS, del20q is a favorable cytogenetic lesion while in MF it is considered an intermediate risk karyotype. We hypothesized that the heterogeneity observed in del20q patients (pts) stems from the acquisition of various mutations during the pts' disease course. These additional chromosomal and/or new molecular defects may help explain these differences. We studied a total of 127 pts with del20q identified either by MC or single polymorphism array (SNP-A). Most pts are male (N=86) and the median age of the cohort is 70 years. The median follow-up time is 24 months. Most pts have MDS (N=76) while the rest are MDS/MPN (N=10), MPN (N=16), sAML (N=18), and other hematological malignancies (N=7). Hematologic parameters, bone marrow (BM) results and clinical data including IPSS, cytogenetics, transfusion requirements, treatment response, overall survival (OS) and progression free survival (PFS) as defined by IWG criteria were collected. We utilized chi-square and Fischer-exact test to compare descriptive variables. Kaplan-Meier statistics were used to compare survival differences between groups and Cox proportional hazard ratio to determine factors that are predictive of outcomes in these pts. A p-value of ≤0.05 is considered statistically significant. Phenotypically, pts with isolated del20q (iso-del20q) have higher ANC, WBC, less splenomegaly and BM blasts. Survival differences were noted between pts with del20q± additional chromosomal defects with better OS noted in pts with iso-del20q compared to del20q with 1 additional cytogenetic abnormality (del20q+1), and del20q with 2 or more cytogenetic abnormalities (del20q+≥2) (OS: 93 vs 52 vs 64 mos, p=.04; PFS: 24 vs 17 vs 6 mos, p=.005; and EFS: 42 vs 38 vs 12 mos, p=.002). Furthermore, PFS is better in iso-del20q compared to del20q+1 and del20q+≥2 pts (24 vs 17 vs 8 mos, p =.005). After defining key differences in survival in pts with del20q, we sought to investigate the biological rationale for these differences. Whole exome sequencing was performed in 4 pts with del20q. Initial candidate genes, SULF2 and PMEPA1 were WT. Analysis showed involvement of U2AF1 which was further confirmed as somatic. Direct sequencing of U2AF1 in this cohort revealed mutations in 25% of pts. Interestingly, among chromosomal subtypes, U2AF1 mutations are most frequently found in pts with del20q (25% (del 20q) vs 3% (−7/-7q) vs 1.6% (normal) vs 0.5% (+8 and −5q). Moreover, in our cohort, U2AF1 is the most frequently mutated gene in the iso-del20q suggesting a strong correlation between this gene and the pathogenesis of del20q. However, U2AF1 has been associated with poor outcomes. In fact, in our MDS cohort (N=340) (Hasrouni E et al. Abstract # 53469), the median OS of U2AF1 mutants (MUT) vs WT are 7 mos vs 20 mos, p =.010 for the whole cohort and remained poor even in normal karyotype MDS (median OS=4 vs 28 mos; p =.0004). Pts with del20q and U2AF1 mutations have excellent survival (median OS=85 mos) suggesting an alternative mechanism whereby U2AF1 modulates this MDS subtype or the presence of another pathway that suppresses the negative effects of U2AF1 mutations in this karyotypic subtype. These findings are reminiscent of the good outcomes correlated with SF3B1 mutations, which we recently found to be associated with lower levels of DNA damage (Visconte V. et al. Abstract # 55089). Interestingly, U2AF1 MUT del20q have lower gamma-H2AX levels compared to U2AF1 MUT in other choromosmal subtypes (5.17±4.68 vs 34.53±30.51) and this may explain decreased propensity for AML transformation in this group. Also, we performed direct sequencing for genes associated with poor (CBL, IDH1/2, DNMT3A, ASXL1, NRAS/KRAS, TP53, EZH2) and good (SF3B1) prognostic outcomes in MDS and found a lower frequency of poor prognostic molecular defects in iso-del20q compared to del20q + ≥2 (22% vs 81%; p=.004). Elucidation of downstream targets by RNA sequencing in del20q cases particularly those with U2AF1 mutations are underway. In addition, screening for mutations involving genes reported to be important in del20q malignancies, like PTPN1 and L3MBTL1 was negative. In summary, pts with del20q have a characteristic clinical profile associated with low risk disease. Iso-del20q have better OS compared to pts with del20q+1 and del20q+≥2 even in the presence of U2AF1 mutations. Disclosures: No relevant conflicts of interest to declare.
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- 2012
45. Biological Rationale for the Favorable Clinical Outcomes of Patients Carrying SF3B1 Mutations in Myelodysplastic Syndromes with Ring Sideroblasts
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Anna M. Jankowska, Yang Liu, John Barnard, Reda Z. Mahfouz, Manoj Bupathi, Yogen Saunthararajah, Jaroslaw P. Maciejewski, Nanthawan Avishai, Arthur H. Heuer, Edward A. Copelan, Ali Tabarroki, Edy Hasrouni, Mikkael A. Sekeres, Fabiola Traina, Ramon V. Tiu, Heesun J. Rogers, Hideki Makishima, Bodo Juraj, Daniel J. Lindner, Betty K. Hamilton, Valeria Visconte, and Reza Sharghi-Moshtaghin
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Myelodysplastic syndromes ,Immunology ,EZH2 ,Cell Biology ,Hematology ,Biology ,Gene mutation ,medicine.disease ,Biochemistry ,Molecular biology ,Chromatin remodeling ,Chromatin ,Refractory anemia with ring sideroblasts ,medicine ,Epigenetics ,Gene - Abstract
Abstract 922 SF3B1, a RNA splicing factor is frequently mutated in refractory anemia with ring sideroblasts (RARS) and RARS with thrombocytosis (RARS-T). Aside from its phenotypic importance, SF3B1 has been associated with good outcomes in MDS. Consistent with others, we found favorable survival outcomes in SF3B1 mutants. To explain the clinical phenotypes (good outcomes, anemia, and less progression to acute myeloid leukemia (AML)), we delved into the consequences of SF3B1 dysfunction in MDS. We hypothesized that SF3B1 mutations result in changes in RNA splicing of key genes in erythropoiesis resulting in anemia. Moreover, we postulated that SF3B1 is a founder mutation in RS formation but insufficient as a sole abnormality to induce a deleterious phenotype. The acquisition of genetic/ epigenetic aberrations may trigger the final phenotype. We reported abundant iron deposits in the mitochondria of SF3B1 mutants. We hypothesized that SF3B1 mutations change the composition/ chemical valence of iron leading to free radicals and DNA damage. We performed Energy-dispersive X-ray/ Electron-Energy Loss Spectroscopy on WT and SF3B1 mutants (n=2) finding that Fe2O3 is the most prevalent iron form. Changes in iron might predispose to DNA damage. We used flow cytometry to measure gamma-H2AX, a marker of DNA damage finding higher gamma-H2AX levels in bone marrow of WT (n=4; 26.35±28.36) vs SF3B1 mutants (n=8; 4.19±4.34), healthy subjects (n=4; 6.15±3.74) and other WT low-risk MDS (n=5; 7.6±1.6). Since increased DNA damage induces chromatin remodeling, we found that WT have more condensed chromatin with granular cytoplasm vs SF3B1 mutants (n=2). Further, RNA-sequencing showed alterations in histone deacetylases (HDAC1/HDAC2) and modification (ASXL1) in SF3B1 mutants. This links splicing mutations to epigenetic events and may represent the 2nd hit to induce the disease phenotype. Other targets were interrogated. Since SF3B1 interacts with members of Class II polycomb group (PcG) in Sf3b1+/− mice and PcG genes, like ASXL1/ EZH2 are mutated in MDS, we performed sequencing on PcG Class II genes (RNF2/ PCGF2). No mutation was found in RARS/RARS-T pts (n=34; SF3B1 mutant=26; WT=8). mRNA levels might be changed in these pts. We also elucidated the role of SF3B1 in the phenotype of anemia. RNA-sequencing has been instructive. Ribosomal proteins (RPS14 and RPS17) are implicated in the pathogenesis of 5q- MDS and Diamond-Blackfan anemia. Both RPS14/ RPS17 expression was different in SF3B1 mutant vs WT (n=2). Further, cytokine analysis showed a trend to higher IL-6 levels in WT vs mutants (n=3). IL-6 is associated with anemia and poor response to therapy in some diseases. We assessed the response to erythropoietin (EPO) and found that none of the WT RARS/-T (n=4) responded to EPO while 15/19 (79%) of SF3B1 mutants responded (p=0.008). It's suggested that SF3B1 mutants have better outcomes due to decreased AML transformation. To dissect this point, we looked at chromosomal defects in SF3B1 mutants (n=27) and WT (n=11), finding that 45% of WT carried complex karyotype (del(5q), del(17p) del(7), and i(17p); p=0.0009) while only 18% of mutants carried one additional abnormality (del(20q), inv3, and 16p). We also checked for concomitant mutations in genes of methylation (TET2, DNMT3A, IDH1/2), histone (ASXL1, UTX, EZH2), transcription (RUNX1, JAK2, TP53), signaling (CBL, NRAS, KRAS), and splicing (U2AF1, SRSF2). We analyzed the global mutational status in RARS/RARS-T (n=38; WT, n=11 and SF3B1 mutants, n=27) finding that 5/11 (45%) WT carried in total 6 other methylation (n=2), transcription (n=1), and splicing (n=3) gene mutations. Further, 15/27 (56%) of SF3B1 mutants carried a total of 16 mutations excluding SF3B1 in: methylation (n=9), transcription (n=4), splicing (n=1), histone (n=2). The most prevalent mutated genes (TET2, DNMT3A) predict for good treatment response. In-vitro treatment with decitabine (0.2 uM) showed changes in chromatin condensation, DNA damage, and response in SF3B1 mutants vs WT (n=2) suggesting that the pathogenetic effects of SF3B1 may be elicited through the methylation pathway. Lastly, SF3B1 predicts for good outcomes. SF3B1 may mediate effects on anemia through alterations in ribosomal function, cytokines, and epigenetic changes. Conversely, SF3B1 mutants have better survival and lower risk to AML evolution because of lower tendency to acquire poor prognostic defects. Disclosures: No relevant conflicts of interest to declare.
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- 2012
46. SF3B1, a Splicing Factor Gene, Is Infrequently Mutated in Rare Bone Marrow Failure Diseases but Still Associated with Ring Sideroblast Phenotype
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Heesun J. Rogers, Jaroslaw P. Maciejewski, Mikkael A. Sekeres, Ramon V. Tiu, Edy Hasrouni, Ali Tabarroki, Yang Liu, Fred H. Hsieh, Valeria Visconte, Fabiola Traina, and Christine L. O'Keefe
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Myeloid ,Myelodysplastic syndromes ,Chronic lymphocytic leukemia ,Immunology ,Chronic myelomonocytic leukemia ,Pure red cell aplasia ,Cell Biology ,Hematology ,Biology ,medicine.disease ,Biochemistry ,Leukemia ,Hypocellularity ,medicine.anatomical_structure ,hemic and lymphatic diseases ,medicine ,Myelofibrosis - Abstract
Abstract 3485 Bone marrow failure syndromes (BMFS) are clonal diseases characterized by inefficient hematopoiesis leading to cytopenias. The clinical and biological heterogeneity often complicates therapy. A number of biological/genetic causes determine the pathogenesis of BMFS (immunological factors, cytokine, telomeres length, T-cell repertoire, epigenetic, apoptotic dysregulation, and chromosomal instability). Whole exome/genome sequencing identified novel mutations in myeloid disorders. SF3B1, a splicing factor gene is mutated primarily in myelodysplastic syndromes (MDS) with ring sideroblasts (RS). SF3B1 mutations brought to light the potential role of spliceosomes in MDS. Although, infrequent in other myeloid malignancies, SF3B1 mutations are relatively frequent in Fludarabine-resistant chronic lymphocytic leukemia (CLL) patients (pts). We previously reported 2 cases: myelofibrosis and paroxysmal nocturnal hemoglobinuria (PNH) with SF3B1 mutations and concomitant RS. To investigate the potential role of SF3B1 in the pathogenesis of rare BMFS, we screened a cohort of BMFS and other rare diseases (N=107): PNH, n=25, aplastic anemia (AA, n=17), T-large granular lymphocytic leukemia (T-LGL, n=17), pure red cell aplasia (PRCA, n=16), and mast cell disease (MCD, n=32) for SF3B1 mutations (exons 13–16) by Sanger sequencing. We identified SF3B1 mutations in 4 pts (MCD; n=2, A711D & K666T; PNH; n=1; K666Q; PRCA, n=1; K666N). Clinical history of the mutated cases showed that the 2 MCD pts fulfilled the criteria for cutaneous and indolent MCD. In the cutaneous MCD pt, skin biopsy revealed typical urticaria pigmentosa highlighting a dermal inflammation with increased MC. No infiltration of MC was found in the BM and no dysplasia was noted, except for RS (6%). In the 2nd pt, the BM was hypocellular with clonal infiltration by MC. No other morphologic features were reported. Mutational analysis of genes implicated in diseases related to MCD, (c-KIT, TET2, IDH1/2, DNMT3A, EZH2, ASXL1, and CBL) showed a wild type configuration in both cases. The close association of MCD with chronic myelomonocytic leukemia (CMML) might explain SF3B1 mutations in the MCD pt as mutations in SF3B1 were reported in 6% of CMML. SF3B1 was also mutated in a pt with 10-year history of hemolytic PNH. BM pathology showed erythroid hyperplasia, no dysplasia, and increased RS (17%) in the BM. Perforin staining showed Disclosures: No relevant conflicts of interest to declare.
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- 2012
47. Clinicopathologic Characterization of Acute Myeloid Leukemia and Myelodysplastic Syndrome with Inv(3)(q21q26.2)/t(3;3)(q21;q26.2) Reveals That Complex Karyotype but Not Blast Percentage Is Associated with Poor Survival; A Bone Marrow Pathology Group Study
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Athena M. Cherry, Adam Bagg, Erika Moore, Daniel A. Arber, Pei Lin, Jennifer J.D. Morrissette, Kathryn Foucar, Robert P. Hasserjian, Susan Mathew, Natasha M. Savage, Sa A. Wang, John Anastasi, Attilio Orazi, Eric D. Hsi, James W. Vardiman, Yen-Chun Liu, Carlos E. Bueso-Ramos, Gordana Raca, and Heesun J. Rogers
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Pathology ,medicine.medical_specialty ,Leukopenia ,business.industry ,Immunology ,Myeloid leukemia ,Cell Biology ,Hematology ,Normocytic anemia ,medicine.disease ,Biochemistry ,Lymphoma ,medicine.anatomical_structure ,International Prognostic Scoring System ,Dysplasia ,hemic and lymphatic diseases ,Complex Karyotype ,Medicine ,Bone marrow ,medicine.symptom ,business - Abstract
Abstract 3847 Acute myeloid leukemia (AML) with inv(3)(q21q26.2)/t(3;3)(q21;q26.2); RPN1-EVI1 [inv3/t3] is a distinct type of AML with recurrent genetic abnormalities (RGA) in the 2008 WHO classification, with poor response to therapy and poor prognosis. The resulting dysregulation of EVI1 plays an important role in stem cell self-renewal and leukemogenesis. Although myelodysplastic syndrome (MDS) with inv3/t3 has a high risk of progression to AML, inv3/t3 is not among the genetic abnormalities sufficient for diagnosis of AML, irrespective of blast percentage (%) in the WHO classification. The revised International Prognostic Scoring System (IPSS-R) includes comprehensive cytogenetic subgrouping to better define prognosis in MDS patients. In this system, inv3/t3 is included in a poor risk karyotype group. The objective of this multicenter study was to evaluate a series of patients with MDS/AML and inv3/t3 in order to characterize their clinicopathologic features and outcome, and to apply the IPSS-R to inv3/t3 MDS patients. 111 patients (40 MDS and 71 AML with inv3/t3) were gathered from 8 medical centers. The median age at diagnosis was 56.5 years and was significantly older in MDS than AML with inv3/t3 patients (65 vs 54.5, p=0.03). Patients typically presented with normocytic anemia, thrombocytopenia and mild leukopenia (median Hb 9.1 g/dL, platelet 91 x109/L, WBC 3.6 x109/L). MDS with inv3/t3 patients had lower WBC than AML with inv3/t3 (median 3.1 vs 5.5, p There was no significant difference in overall survival (OS) between MDS and AML with inv3/t3 (12.9 vs 8.0 mo, Cox PH p=0.11, Figure 1). There was no OS difference between MDS and AML after excluding Ph+ cases (Cox PH p=0.17) nor between de novo and therapy related MDS/AML with inv3/t3 (Cox PH p=0.89). Patients with isolated inv3/t3, one additional cytogenetic abnormality, and a complex karyotype showed progressively shorter OS (12.9, 10.0 and 4.3 mo, Cox PH p MDS with inv3/t3 patients were classified into IPSS Intermediate (Int)-1 (21), Int-2 (13), and high (6) risk groups. IPSS-R categorized MDS patients into low (3), Int (6), high (14) and very high (17) risk groups. 57% of IPSS Int-1 risk group patients (expected OS 3.5 year) were reclassified to high or very high risk group in IPSS-R (expected OS The IPSS-R better reflects the OS of inv3/t3 than IPSS but may not fully reflect the generally dismal prognosis. Patients with MDS and AML with inv3/t3 follow a similarly aggressive clinical course, supporting classification of MDS with inv3/t3 as an AML with RGA irrespective of blast%. Additional cytogenetic abnormalities are associated with shorter OS in AML/MDS with inv3/t3 and our data suggest that aggressive therapy with SCT should be considered in these patients. Disclosures: Vardiman: Celgene Corporation: review of slides for clinical trials not relevant to this abstract Other. Foucar:e. Honoraria–Scientific Symposium Pathology Education: ASCP Press; ARP, Amirsys, ASCP Press; ARP, Amirsys Patents & Royalties, Honoraria, Not relevant to this abstract Other.
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- 2012
48. Newly Acquired Molecular Mutations and SNP-A Lesions Alters the Natural History of Trisomy 8 Myeloid Neoplasms
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Ali Tabarroki, Yang Liu, Yogen Saunthararajah, Manoj Bupathi, Rami S. Komrokji, Paul Elson, Ramon V. Tiu, Edy Hasrouni, Mikkael A. Sekeres, Heesun J. Rogers, Betty K. Hamilton, Valeria Visconte, Hien K. Duong, and Fabiola Traina
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Pathology ,medicine.medical_specialty ,Myeloid ,IDH1 ,Myelodysplastic syndromes ,Immunology ,Hazard ratio ,Chronic myelomonocytic leukemia ,Cell Biology ,Hematology ,Biology ,medicine.disease ,Trisomy 8 ,Biochemistry ,Gastroenterology ,medicine.anatomical_structure ,International Prognostic Scoring System ,Internal medicine ,medicine ,Lenalidomide ,medicine.drug - Abstract
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 Disclosures: No relevant conflicts of interest to declare.
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- 2012
49. The Molecular and Cytokine Profile of Triple-Negative (JAK2 V617F, JAK2 exon 12, MPL negative) Myelofibrosis, a Myeloproliferative Neoplasm with Distinct Clinico-Pathologic Characteristics
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Edy Hasrouni, Ramon V. Tiu, Matt Kalaycio, Mikkael A. Sekeres, Hien K. Duong, Ali Tabarroki, Edward A. Copelan, Valeria Visconte, Brady L. Stein, Heesun J. Rogers, and Anjali S. Advani
- Subjects
Mutation ,Myeloid ,Immunology ,Myeloid leukemia ,Cell Biology ,Hematology ,Biology ,medicine.disease_cause ,medicine.disease ,Biochemistry ,Exon ,medicine.anatomical_structure ,Polycythemia vera ,medicine ,Myelofibrosis ,Myeloproliferative neoplasm ,Exome sequencing - Abstract
Abstract 3805 Myelofibrosis (MF) is a clonal Philadelphia chromosome negative myeloid neoplasm characterized by cytopenias with or without proliferative hematologic changes, extramedullary hematopoiesis and an increased propensity for transformation to acute myeloid leukemia. Identification of somatic mutations in Janus kinase 2 (JAK2) has revolutionized our understanding of the pathogenesis of some MF patients. However, there is a wide range of clinical heterogeneity in MF patients and somatic mutations of JAK2 (exon 14 or exon12) and MPL are only found in 50–60%/1–2% and 5–10% of MF patients respectively. Therefore, a large proportion of MF patients remain molecularly unaccounted after sequencing for these commonly analyzed mutations. We hypothesized that MF patients who are lack of JAK2 and MPL mutations, collectively called “triple-negative MF,” have a distinct clinicopathologic profile compared to their mutant counterparts. Our study included 76 MF patients (primary MF=48, post-essential thrombocythemic-MF=21, post-polycythemic-MF=7), with amedian age of 65 years (25–80) and predominantly intermediate 1 or 2 risk by the D-IPSS grouping (low=8, Int-1=20, Int-2=42, high=6). We compared hematologic and clinical variables between JAK2 mutant (N=40) and triple-negative MF (N=36) and found that triple-negative MF patients had a higher WBC count (14.4±23.6. vs 11±14.9) and higher platelet counts (365±434.1 vs 252±220.5) but similar hemoglobin levels, LDH levels and D-IPSS plus risk profiles. No difference in overall survival was noted (25.5 months in triple-negative vs 24 months in JAK2 positive). Newly discovered molecular mutations in myeloid malignancies have provided insight into the biology of MDS and some MPN cases. We performed direct sequencing on 9 genes including TET2 (Exon 3), DNMT3A (Exon 18/19, 20, 21, 22 and 23), IDH1/2 (Exon4), LNK (exon2–8), EZH2 (Exon 18/19, N/KRAS (Exon 1–2), CBL (Exon 8–9) and did not find any mutation in the triple-negative cohort. Recently, L3MBTL1 has been reported to be important in the pathogenesis of polycythemia vera, but sequencing of this gene in MF patients did not yield any mutations. Next generation sequencing technologies have been instrumental in the discovery of new genes important in disease pathogenesis in myeloid neoplasias. We performed whole exome sequencing in the triple-negative cases and found somatic mutations in the following genes, ODZ1 (cellular signal transducer), ZNFE391 (transcriptional regulator), DENND3 (protein-coding), ARHGEF11 (modulator of cellular process through G protein) and PIK3CD (Phosphorylate Inositol Lipids regulator involving in Immune response) in one patient and subsequently confirmed as somatic by direct sequencing. This is the first time, which these genetic mutations were reported in hematologic malignancies specifically in MF. However, these mutations did not recur when screening, a limited cohort of patients (N=20). For better understanding of this disease subtype, we compared the cytokine profile in these two groups based on the fact that, cytokines have been crucial in the pathogenesis of MF, and we found marked elevation in the levels of IL-1B (1.63 vs 0.42), IL-8 (2.89 vs 0.56), IL-17A (2.56 vs 1.12) and IFN-δ (2.07 vs 0.37) in the JAK2 positive patients compared to the triple-negative cases. Comparison of both groups showed similar responses to JAK inhibitors by spleen size shrinkage and constitutional symptoms. In conclusion, despite the discovery of JAK2 and MPL mutations, a subset of MF patients remains molecularly uncharacterized. We found that triple negative MF patients had higher leukocyte and platelet counts, but a similar prognosis. Moreover, we identified novel somatic mutations in 1 triple-negative MF patient, which has not been previously described interestingly, despite lower cytokine levels; responses to JAK-inhibitors were still noted. Whole exome sequencing and other immunogenetic studies may reveal novel genes which may elucidate the pathogenesis of these MF cases. Disclosures: No relevant conflicts of interest to declare.
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- 2012
50. Pathway Analysis of Molecular Mutations Can Modify Morphologic, Cytogenetic and Prognostic Risk Stratification Schemes in Myelodysplastic Syndromes (MDS), Myelodysplastic Syndromes/Myeloproliferative Neoplasms (MDS/MPN), and Secondary Acute Myeloid Leukemia (AML)
- Author
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Yogen Saunthararajah, Matt Kalaycio, Anna M. Jankowska, Edward A. Copelan, Edy Hasrouni, Fabiola Traina, Michael A. McDevitt, Ramon V. Tiu, Mikkael A. Sekeres, Betty K. Hamilton, Valeria Visconte, Ali Tabarroki, Manoj Bupathi, Jaroslaw P. Maciejewski, Heesun J. Rogers, Hideki Makishima, Anjali S. Advani, Andres Jerez, and Yang Liu
- Subjects
Oncology ,Neuroblastoma RAS viral oncogene homolog ,medicine.medical_specialty ,Myelodysplastic syndromes ,Immunology ,EZH2 ,Cytogenetics ,Cell Biology ,Hematology ,Gene mutation ,Biology ,medicine.disease ,Bioinformatics ,medicine.disease_cause ,Biochemistry ,Internal medicine ,medicine ,Progression-free survival ,KRAS ,Exome sequencing - Abstract
Abstract 3791 Molecular mutations of genes important in MDS pathogenesis have been identified using next generation sequencing technologies. These mutations have variable effects on prognosis: results from individual studies are conflicting, and the impact of combining mutations based on pathogenetic pathways has not been explored. Furthermore, most studies do not take into account the types of therapies received by patients (pts). We hypothesized that analyses of mutations incorporating pathways would yield more meaningful results, especially if studied in the context of therapies. We studied 340 pts with MDS (N=176), MDS/MPN (N=88) and sAML (N=76). Median age was 69 years (range, 19–92), 118 (35%) were female. Direct sequencing of 14 genes important in MDS pathogenesis originally identified using whole exome sequencing and involved in methylation (TET2/IDH1/2/DNMT3A), histone modification (ASXL1/EZH2/UTX), signaling (CBL/KRAS, NRAS, JAK2), transcription (RUNX1/TP53) and RNA splicing (SRSF2/U2AF1) was performed. Pts were classified by 2008 WHO criteria. Prognosis was assigned by IPSS (low=67, Int-1=85, Int-2=56, high=64). Karyotypes by metaphase cytogenetics (MC) and SNP-A were stratified by IPSS cytogenetic risk group (Low=82, intermediate=87, poor=106). Treatment received included high intensity (cytarabine+ anthracycline, high dose ARA-C,) (N=75), low intensity (N=124) (hypomethylating therapy, IMiDs, low dose ARA-C), and supportive therapy (N=141) (growth factors, hydroxyurea). Categorical variables were analyzed using X2 or Fischer-exact test. Overall survival (OS) and progression-free survival (PFS) were defined using IWG criteria and analyzed by Kaplan-Meier and log-rank statistics. P-values0.05 were considered statistically significant. Univariate and multivariate analyses were performed by Cox Proportional Hazards, covariates included were age, BM blasts, cytopenias, karyotype, jointly with molecular pathway data. A total of 258 mutations were detected in 158 pts. Mutations involving genes key to methylation (41 vs 37%, p=.01), and histone modification (52 vs 25%, p=.008) were more common in MDS/MPN compared to MDS pts. Mutations in methylation (37 vs 23%, p=.05), transcription (38 vs 14%, p=.01) and splicing (38 vs 17%, p=.007) were more common in MDS compared to sAML pts, and mutations of genes involved in methylation (41 vs 23%, p=.02), signaling (60 vs 7%, p=.005), histone modification (52 vs 23%, p=.004), transcription (49 vs 13%, p=.001) and splicing (45 vs 17%, p=.0005) were more frequent in MDS/MPN compared to sAML pts. Worse outcomes were noted in pts with mutations in histone modification (OS: 17 vs 21 mos, p=.04), transcription (PFS: 7 vs 13 mos, p=.02) and splicing (OS: 11 vs 23 mos, p=.01) genes. This was evident in pts with MDS who had transcription factor-related gene mutations (OS: 9 vs 30 mos, p= Disclosures: No relevant conflicts of interest to declare.
- Published
- 2012
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