Edy Hasrouni

Notch-mediated patterning and cell fate allocation of pancreatic progenitor cells

Early pancreatic morphogenesis is characterized by the transformation of an uncommitted pool of pancreatic progenitor cells into a branched pancreatic epithelium that consists of ‘tip’ and ‘trunk’ domains. These domains have distinct molecular signatures and differentiate into distinct pancreatic cell lineages. Cells at the branched tips of the epithelium develop into acinar cells, whereas cells in the trunk subcompartment differentiate into endocrine and duct cells. Recent genetic analyses have highlighted the role of key transcriptional regulators in the specification of these subcompartments. Here, we analyzed in mice the role of Notch signaling in the patterning of multipotent pancreatic progenitor cells through mosaic overexpression of a Notch signaling antagonist, dominant-negative mastermind-like 1, resulting in a mixture of wild-type and Notch-suppressed pancreatic progenitor cells. We find that attenuation of Notch signaling has pronounced patterning effects on multipotent pancreatic progenitor cells prior to terminal differentiation. Relative to the wild-type cells, the Notch-suppressed cells lose trunk marker genes and gain expression of tip marker genes. The Notch-suppressed cells subsequently differentiate into acinar cells, whereas duct and endocrine populations are formed predominantly from the wild-type cells. Mechanistically, these observations could be explained by a requirement of Notch for the expression of the trunk determination gene Nkx6.1. This was supported by the finding of direct binding of RBP-jκ to the Nkx6.1 proximal promoter.

Distinct iron architecture in SF3B1 -mutant myelodysplastic syndrome patients is linked to an SLC25A37 splice variant with a retained intron

Perturbation in iron homeostasis is a hallmark of some hematologic diseases. Abnormal sideroblasts with accumulation of iron in the mitochondria are named ring sideroblasts (RS). RS is a cardinal feature of refractory anemia with RS (RARS) and RARS with marked thrombocytosis (RARS/-T). Mutations in SF3B1, a member of the RNA splicing machinery are frequent in RARS/-T and defects of this gene were linked to RS formation. Here we showcase the differences in iron architecture of SF3B1-mutant and wild-type (WT) RARS/-T and provide new mechanistic insights by which SF3B1 mutations lead to differences in iron. We found higher iron levels in SF3B1 mutant vs WT RARS/-T by transmission electron microscopy/spectroscopy/flow cytometry. SF3B1 mutations led to increased iron without changing the valence as shown by the presence of Fe2+ in mutant and WT. Reactive oxygen species and DNA damage were not increased in SF3B1-mutant patients. RNA-sequencing and Reverse transcriptase PCR showed higher expression of a specific isoform of SLC25A37 in SF3B1-mutant patients, a crucial importer of Fe2+ into the mitochondria. Our studies suggest that SF3B1 mutations contribute to cellular iron overload in RARS/-T by deregulating SLC25A37.

Successful use of very low dose subcutaneous decitabine to treat high-risk myelofibrosis with Sweet syndrome that was refractory to 5-azacitidine.

1 Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, USA, 2 Department of Anatomic Pathology, Cleveland Clinic, Cleveland, OH USA, 3 Department of Clinical Pathology, Cleveland Clinic, Cleveland, OH USA and 4 Leukemia Program, Department of Hematologic Oncology and Blood Disorders, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, USA

SF3B1 mutations are infrequently found in non-myelodysplastic bone marrow failure syndromes and mast cell diseases but, if present, are associated with the ring sideroblast phenotype

The bone marrow failure syndromes (BMFS) are hematologic disorders characterized by impaired production and sometimes function of peripheral blood (PB) cells. While they can be either acquired or inherited, the majority are acquired and include myelodysplastic syndromes (MDS), aplastic anemia (AA),

Impact of allogeneic hematopoietic cell transplant in patients with myeloid neoplasms carrying spliceosomal mutations.

Molecular predictors of outcome are increasingly important in determining optimal therapy for myeloid neoplasms. Mutations in the spliceosomal genes (U2AF1 and SRSF2) predict for poor outcomes in myelodysplastic syndromes (MDS) and related diseases. We investigated the effect of hematopoietic cell transplant (HCT) on the negative prognostic impact of U2AF1 and SRSF2 mutations. In total, 122 patients with MDS (30%), acute myeloid leukemia (51%), myeloproliferative neoplasms (MPN) (11%), and MDS/MPN (8%) receiving a HCT from 2003 to 2012 were evaluated for mutations in U2AF1 and SRSF2 by direct sequencing. Median time of follow up was 24 months (range 0.46–110). SRSF2 mutations were detected in 11 (10%) patients and U2AF1 in 3 (3%) patients. There were no significant differences in baseline characteristics between mutated and wild‐type (WT) patients. Patients carrying SRSF2 and U2AF1 mutations had similar overall survival (P = 0.84), relapse mortality (P = 0.50), and non‐relapse mortality (P = 0.72) compared to WT patients. However, taking into account disease status and cytogenetics in a subset of AML patients, SRSF2 and U2AF1 mutations were associated with worse survival (HR 3.71, P = 0.035). Am. J. Hematol. 91:406–409, 2016.

Clinicopathologic and molecular characterization of myeloid neoplasms harboring isochromosome 17(q10).

To the Editor: This is the third in a series of short articles concerning iron deficiency and the role of intravenous (IV) iron. Iron deficiency anemia is nearly ubiquitous in this population [1] and it is the opinion of many key opinion leaders in the field that failure to routinely replace iron intravenously represents and unmet clinical need. In those with uncomplicated iron deficiency without underlying inflammatory disorders which lead to iron restricted erythropoiesis due to hepcidin upregulation, oral iron is effective and inexpensive. Unfortunately, estimates that 70% of those to whom it is prescribed report significant gastrointestinal perturbation, results in significant nonadherence to therapy. Evidence suggests oral iron causes severe adverse invents when inflammatory bowel disease is present and worsens the underlying pathology [2–4]. While some gastroenterologists continue to extol the virtues of low doses of oral iron, even if well tolerated in subjects with quiescent disease, a year of therapy is required to replace stores. European and American guidelines differ. European guidelines state that the preferred route of iron supplementation in inflammatory bowel disease (IBD) is IV, even though some patients will respond to oral iron. IV iron is more effective and better tolerated and improves the quality of life to a greater extent than oral iron supplementation (Grade A) [2]. Absolute indications for IV iron include severe anemia (Hgb <10 g/dl), intolerance of or inappropriate response to oral iron, severe intestinal disease acuity, and concomitant therapy with an erythropoiesis agent or patient preference. Oral iron supplements can be used if absolute indications for IV iron therapy are not met. If oral iron is used, the response and tolerance should be monitored and treatment changed to IV as necessary (Grade C). Because side effects of oral iron are dose related and because its absorption and efficacy are no greater when high doses are used, no more than 100 mg of elemental iron daily should be prescribed (Grade C). American guidelines state, “oral formulations are more convenient and less expensive and may be used as a first-line option for patients whose IBD activity and anemia are mild” [5]. The medical literature is rife with prospective comparative evidence demonstrating superiority of IV iron over oral iron in both efficacy and toxicity. Recent publications support this view [6–8]. With the advent of four new formulations which allow safe and rapid complete replacement dosing in 15–60 min, the failure to routinely administer IV iron to anemic patients with inflammatory bowel disease may represent another unmet clinical need. A recent publication demonstrated that not only does a 15 min infusion of ferric carboxymaltose correct anemia but prevents recurrence of anemia in patients with inflammatory bowel disease [9]. Similar evidence of efficacy has been published with low molecular weight iron dextran [10,11] and ferumoxytol [12]. Although it remains reasonable to try oral iron in those without active disease, considering the litany of gastrointestinal perturbations present in inflammatory bowel disease, even in remission, perhaps it is time for American gastroenterologists to become more congruent with their European colleagues.

A case of mistaken identity: When lupus masquerades as primary myelofibrosis

Introduction: Autoimmune myelofibrosis is an uncommon hematologic disease characterized by anemia, bone marrow myelofibrosis, and an autoimmune feature. Myelofibrosis is often associated with other conditions, including infections, nutritional/endocrine dysfunction, toxin/drug exposure, and connective tissue diseases, including scleroderma and systemic lupus erythematosus. Absence of clonal markers (JAK2) and heterogeneity of the symptoms often complicate the diagnosis. Case presentation: Here, we present two cases of systemic lupus erythematosus–induced autoimmune myelofibrosis. The first case is of a 36-year-old African American female with diagnosis of systemic lupus erythematosus at the age of 12 years. The second patient is a 44-year-old African American male with family history of systemic lupus erythematosus who developed anemia and constitutional symptoms later on. Both patients showed hypercellularity and fibrotic changes of the bone marrow. Moreover, mutational analysis showed that both patients were wild type for JAK2 (V617F and exon 12) and MPL (exon 10). Conclusions: These two cases illustrate that anemic patients with fibrotic changes in the bone marrow without other clinicopathologic features associated with primary myelofibrosis in the presence of clinical manifestations and history of an autoimmune disease should suggest an autoimmune myelofibrosis. These cases demonstrate that a good clinical history combined with molecular technologies and pathomorphologic criteria are helpful in distinguishing between primary myelofibrosis and a nonclonal myelofibrosis from an associated condition.

Non-t(6;9) and Non-Inv(3) Balanced Chromosomal Rearrangements Are Associated With Poor Survival Outcomes in Myelodysplastic Syndromes

BACKGROUND Cytogenetics is an important predictor of survival in patients with myeloid malignancies including myelodysplastic syndromes (MDS). The roles of balanced chromosomal rearrangements (BCR) specifically balanced translocations and inversions in MDS are less established. We hypothesized that BCR are commonly found in MDS and might confer important prognostic and therapeutic effect. PATIENTS AND METHODS Cytogenetic, hematologic, clinical, and survival data from a total of 302 MDS patients seen at the Cleveland Clinic between the years 2002 and 2011 were collected. Direct sequencing for genes relevant in MDS pathophysiology was performed on patients with BCR. Categorical data were analyzed using the χ2 test and survival outcomes were analyzed using the Kaplan-Meier method. RESULTS Twenty-three patients (8%) carried non-t(6;9) and non-inv(3) BCR. These patients had short overall survival (OS) similar to patients with poor risk cytogenetics, defined according to the International Prognostic Scoring System, which was not altered by new single-nucleotide polymorphism arrays or acquired somatic uniparental disomy lesions. Among the detected somatic mutations, only patients harboring mutations in the RNA splicing gene serine/arginine-rich splicing factor 2 (SRSF2) conferred worse outcomes in this group of patients. Therapeutically, patients who received allogeneic hematopoietic cell transplantations had better OS compared with patients treated with pharmacologic therapies or best supportive care only. CONCLUSION BCR are not common in MDS and are associated with poor survival, which might be influenced by the presence of SRSF2 mutation.

Molecular and phenotypic heterogeneity of refractory anemia with ring sideroblasts associated with marked thrombocytosis

Refractory anemia with ring sideroblasts associated with marked thrombocytosis (RARS-T) is a provisional entity within myelodysplastic/myeloproliferative neoplasm (MDS/ MPN), unclassifiable in the 2008 World Health Organiza tion classification. This category includes patients that at the time of initial presentation manifest some diagnostic features (cytopenias and dysplasia) of MDS and others (overproduction of one or more myeloid cell lineages) of MPN. This dual clinical nature limits the distinct clas sification as either MDS or MPN. The diagnostic criteria include peripheral blood (PB; anemia, absence of blasts, platelets  450  10 9 /L) and bone marrow (BM) findings (erythroid dysplasia,  15% of erythroid precursors with RS,  5% blasts, and megakaryocytic hyperplasia with large atypical megakaryocytes) [1,2]. The value of RARS-T as a diagnostic category has remained in question over the years. There is a persistent debate on whether RARS-T is a disease evolving from MDS, is part of the MPN (an essential thrombocythemia (ET) that acquires RS) or is a result of two distinct disorders occurring simultaneously [3]. Data on survival outcomes have also been discordant. RARS-T patients have usually worse outcomes compared to MPN patients and are more likely to develop leukemia although RARS-T patients harboring JAK2 [4,5] or SF3B1 mutations appear to have better survival than wild-type RARS-T patients [5,6]. The presence of DNMT3A and ASXL1 mutations is noted in 17% and 10% of cases and confers an unfavorable prognosis [6,7]. In recent years, molecular analysis has unraveled an overlapping mutational spectrum of RARS-T with MPN or MDS. With the exception of SF3B1 mutations, which have been associated to the RS phenotype in RARS and RARS-T patients [6,8] and linked to the development of erythroid dysplasia, macrocytic anemia, splenomegaly and thrombocytosis in an SF3B1 /2 mouse model [9], the vast majority of mutational events and frequencies have not explained either the MPN or other features of the disease. Indeed mutations in JAK2, MPL, and TET2 are common (60%, 5–20%, and 26%,