Search

BACKGROUND Cytopenias have been reported in dogs treated with phenobarbital, but detailed descriptions of bone marrow findings and response to treatment are lacking. OBJECTIVES We aimed to characterize the hematologic findings and clinical outcomes of dogs that had been receiving phenobarbital at the time of marrow evaluation. METHODS Archived bone marrow slides and clinicopathologic data were reviewed in dogs undergoing marrow evaluation for any hematologic problems that developed while receiving phenobarbital (2008-2020). Dogs were excluded if marrow samples lacked diagnostic value, phenobarbital was withdrawn >1 day before marrow collection, a same-day complete blood count (CBC) was lacking, or dogs had concurrent illness or therapy known to cause cytopenias. RESULTS Thirteen dogs met inclusion criteria: eight pancytopenic, three anemic/thrombocytopenic, one neutropenic/thrombocytopenic, and one nearly neutropenic. Neutropenia was marked (

Germline mutations in RUNX1 result in autosomal dominant familial platelet disorder with associated myeloid malignancy (FPDMM). To characterize the hematopathologic features associated with a germline RUNX1 mutation, we reviewed a total of 42 bone marrow aspirates from 14 FPDMM patients, including 24 cases with no cytogenetic clonal abnormalities, and 18 with clonal karyotypes or leukemia. We found that all aspirate smears had ≥10% atypical megakaryocytes, predominantly characterized by small forms with hypolobated and eccentric nuclei, and forms with high nuclear-to-cytoplasmic ratios. Core biopsies showed variable cellularity and variable numbers of megakaryocytes with similar features to those in the aspirates. Granulocytic and/or erythroid dysplasia (≥10% cells per lineage) were present infrequently. Megakaryocytes with separate nuclear lobes were increased in patients with myelodysplastic syndrome (MDS) and acute leukemia. Comparison to an immune thrombocytopenic purpura cohort confirms increased megakaryocytes with hypolobated eccentric nuclei in FPDMM patients. As such, patients with FPDMM often have atypical megakaryocytes with small hypolobated and eccentric nuclei even in the absence of clonal cytogenetic abnormalities; these findings are related to the underlying RUNX1 germline mutation and not diagnostic of MDS. Isolated megakaryocytic dysplasia in patients with unexplained thrombocytopenia should raise the possibility of an underlying germline RUNX1 mutation.

Context: Bone marrow abnormalities reported in various stages of HIV infection have not attracted much attention; this is partly because the clinical picture of HIV infection is still dominated by opportunistic infections and malignancies. Aims: 1. To study the bone marrow abnormalities in patients with HIV / AIDS and to find their association with peripheral hematological abnormalities. 2. To draw conclusions and compare with previous studies.Settings and Design: The present Prospective study was conducted at the Department of pathology, Kamineni Institute of Medical Sciences, Narketpalli over a period of 2 years starting from January 2017 to Dec 2018. Methods and Material: The study population included 54 newly detected HIV positive symptomatic or asymptomatic patients. Patients were classified into two clinical groups according to NACO criteria 1) AIDS and 2) Non-AIDS. Bone marrow analysis was done for HIV patients who presented with anemia, leucopenia, Thrombocytopenia and Bi-cytopenia. Results: Patients were classified into two clinical groups: AIDS (55.5%) and NON-AIDS (44.5%). Most common age group involved was 21-40 years and Male to Female ratio is 4:1 in AIDS Group and 1.6:1 in Non-AIDS group. Most common haematological abnormality was Anaemia present in 73.3% of AIDS group and 79.1% of Non-AIDS group. Bone marrow was predominantly normocellular (AIDS: 50%, Non-AIDS: 62.5%) with normoblastic maturation. Myelodysplasia was predominantly seen in Granulocytic series (AIDS: 26.6%, Non-AIDS: 12.5%). Erythroid dysplasia was reported in 23.3% of cases of AIDS group and 8.3% in Non-AIDS group. Megakaryocytic dysplasia was seen in 2 cases of AIDS group. Myelodysplasia was common in patients with CD4 counts < 200/µL. Patients with myelodysplasia presented with anaemia (52.9%), Leukopenia (41.1%) and Thrombocytopenia (11.7%).Conclusions: Bone marrow examination is usually performed in HIV patients to evaluate peripheral cytopenia. Myelodysplasia commonly observed in HIV patients predominantly involves Granulocytic series. Erythroid dysplasia is prominent in AIDS Patients. Myelodysplasia associated with anemia carries poor prognosis and marks progression to advanced disease. Bone marrow abnormalities evolve and increase in frequency during progression of HIV disease and thorough schematic evaluation carries prognostic significance.

A healthy 14-year-old, male neutered, Miniature Poodle was found to have a persistent erythrocyte macrocytosis and reticulocytosis with a normal and stable HCT. The hematologic features of macrocytosis, increased Howell-Jolly bodies, and metarubricytosis, in the absence of anemia or other cytopenias, combined with the cytologic evidence of bone marrow erythroid dysplasia, including megaloblastosis, binuclearity, increased mitotic activity, and nuclear fragmentation, are consistent with previous reports of congenital dyserythropoiesis termed poodle macrocytosis. We speculate that the additional presence of persistent reticulocytosis in the absence of an identifiable stimulus for accelerated erythropoiesis may represent a phenotypic variation of this inherited condition, and the morphologic abnormalities of the dyserythropoiesis are described.

Glucose-6-phosphate isomerase (GPI) deficiency, a genetic disorder responsible for chronic nonspherocytic hemolytic anemia, is the second most common red blood cell glycolytic enzymopathy. We report three patients from two unrelated families of Czech and Slovak origin with macrocytic hemolytic anemia due to GPI deficiency. The first patient had 15% of residual GPI activity resulting from two new heterozygous missense mutations c.478T > C and c.1414C > T leading to substitutions p.(Ser160Pro) and p.(Arg472Cys). Two other patients (siblings) inherited the same c.1414C > T p.(Arg472Cys) mutation in a homozygous constitution and lost approximately 89% of their GPI activity. Erythroid hyperplasia with dysplastic features was observed in the bone marrow of all three patients. Low hepcidin/ferritin ratio and elevated soluble transferrin receptor detected in our GPI-deficient patients suggest disturbed balance between erythropoiesis and iron metabolism contributing to iron overload.

Background: Cytopenias are common in systemic lupus erythematosus (SLE), and it is fundamental to determine their etiology in order to establish an adequate therapeutic strategy. Objectives: To describe the findings in the bone marrow aspirations (BMA) and biopsies of patients with SLE and cytopenias, as well as clinical and laboratory features associated with the etiology of the hematological abnormalities. Methods: We performed a retrospective study in a third-level hospital in Mexico City. We included patients who fulfilled ACR criteria for SLE, presented with cytopenias and had a BMA and biopsy performed between 2000 and 2016. We described the main aspirate and biopsy findings, and also analyzed the final diagnosis and its association with clinical, laboratory and serological features. Results: We included 101 patients; median age was 32 years and 81.2% were women. Leukopenia ( Regarding bone marrow findings, there was erythroid dysplasia in 50.5% of patients, granulocytic dysplasia in 28.7% and megakaryocytic dysplasia in 16%. Myelofibrosis was found in 2.6%. An increase in plasma cells (≥5%) was found in 21.8%. In 72.3% of patients, bone marrow interpretation was conclusive. The most common diagnoses were disease activity (24.8%) and drug-associated myelotoxicity (28.7%). When compared to other etiologies, in patients with cytopenias secondary to disease activity, it was more frequent for the bone marrow be hypercellular (56 vs 23%, p=0.006) and to have increased megakaryocytes (40 vs 17.4%, p=0.048). Conversely, granulocytic dysplasia was less common in this group of patients (17.4% vs 54.3%, p=0.036) We analyzed factors associated with both activity and toxicity as final diagnoses (Table 1). After multivariate analysis, a neutrophil count Conclusion: The main causes of cytopenias in our SLE patients were disease activity and bone marrow toxicity. Our findings suggest that when patients present with less than 1000 neutrophils/µl, it is unlikely for the cytopenias to be secondary to disease activity. Our study reinforces the diagnostic utility of BMA and biopsy, and the associations we described may assist clinicians to determine the etiology of cytopenias in SLE patients, in order to make appropriate therapeutic decisions. Disclosure of Interests: Ana Barrera-Vargas: None declared, Jonathan Campos-Guzman: None declared, Samuel Govea-Pelaez: None declared, Aldo Garcia-Ramos: None declared, Roberta Demichelis-Gomez: None declared, Christianne Bourlon: None declared, Javier Merayo-Chalico Speakers bureau: Pfizer, Jorge Alcocer-Varela: None declared

BACKGROUND Despite bone marrow (BM) immunophenotyping by flow cytometry has progressively been recognized as an important tool for the diagnosis of myelodysplastic syndromes (MDS), the sparse knowledge about normal erythroid maturation and the lack of markers for erythroid characterization is a major shortcoming. METHODS Here, we analyzed the expression of CD43 and CD49d, two markers included in the diagnostic panel for B-cell chronic lymphoproliferative disorders (B-CLPD), in the CD34+ compartment of normal BM and along the normal and dysplastic erythroid maturation. For this, 13 normal BM aspirates and 18 BM aspirates from MDS patients were studied by flow cytometry. RESULTS Normal BM presented a higher expression of CD43 and CD49d among CD34+ erythroid precursors, compared to CD34+ cells committed to the remaining hematopoietic cell lineages. CD43 expression progressively decreased along the normal erythroid maturation, whereas CD49d levels increased from Stage I to Stage II, were maintained in Stages II and III, and then decreased until the last stage of maturation. In MDS, the expression of CD43 and CD49d followed a similar pattern, but with decreased expression levels for both markers, observed in all erythroid maturation stages (P < 0.05). CONCLUSIONS Our results point to the usefulness of CD43 and CD49d, two markers commonly present in B-CLPD diagnosis panels, in the identification of dysplastic phenotypic features in the erythroid lineage. This allows a feasible and inexpensive way to identify patients who would benefit from a more extensive study to evaluate the presence of MDS, during the processing of suspected B-CLPD samples. © 2019 International Clinical Cytometry Society.

International audience; An evaluation of the significance of specified dyserythropoietic features in suspected myelodysplastic syndrome (MDS) and acute myeloid leukaemia with erythroid dysplasia was made by means of evaluation of 100 electronic images of bone marrow erythroblasts from each of 20 subjects 11 with a myeloid neoplasm, six with another condition that could cause erythroid dysplasia and three healthy controls. The evaluation was carried out independently by seven experienced haematologists/haematopathologists who were blinded to the diagnosis. The majority of the dyserythropoietic features listed in the World Health Organization Classification of Tumours of Haematopoietic and Lymphoid Tissues were validated, although karyorrhexis was found to be infrequent and lacking in specificity; multinuclearity and megaloblastosis were more often observed but also lacked specificity. Good majority agreement on the identification of dysplastic features was obtained. Despite this, it was demonstrated that a reliable diagnosis of MDS can often not be made on the basis of erythroid morphology alone. Interpretation of dyserythropoiesis must be carried out with full knowledge of other clinicopathological features and with a constant awareness of the other conditions that can be confused with MDS. An iron stain is essential, as cases with ring sideroblasts may otherwise not be recognised as having MDS. © 2018 John Wiley and Sons Ltd

Refractory anemia with ring sideroblasts (RARS) is a myelodysplastic syndrome (MDS) characterized by isolated erythroid dysplasia and 15% or more bone marrow ring sideroblasts. Ring sideroblasts are found also in other MDS subtypes, such as refractory cytopenia with multilineage dysplasia and ring sideroblasts (RCMD-RS). A high prevalence of somatic mutations of SF3B1 was reported in these conditions. To identify mutation patterns that affect disease phenotype and clinical outcome, we performed a comprehensive mutation analysis in 293 patients with myeloid neoplasm and 1% or more ring sideroblasts. SF3B1 mutations were detected in 129 of 159 cases (81%) of RARS or RCMD-RS. Among other patients with ring sideroblasts, lower prevalence of SF3B1 mutations and higher prevalence of mutations in other splicing factor genes were observed (P < .001). In multivariable analyses, patients with SF3B1 mutations showed significantly better overall survival (hazard ratio [HR], .37; P = .003) and lower cumulative incidence of disease progression (HR = 0.31; P = .018) compared with SF3B1-unmutated cases. The independent prognostic value of SF3B1 mutation was retained in MDS without excess blasts, as well as in sideroblastic categories (RARS and RCMD-RS). Among SF3B1-mutated patients, coexisting mutations in DNA methylation genes were associated with multilineage dysplasia (P = .015) but had no effect on clinical outcome. TP53 mutations were frequently detected in patients without SF3B1 mutation, and were associated with poor outcome. Thus, SF3B1 mutation identifies a distinct MDS subtype that is unlikely to develop detrimental subclonal mutations and is characterized by indolent clinical course and favorable outcome.

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%

The t(8;21)(q22;q22) translocation is specifically observed in acute myeloid leukemia (AML) M2 subtype, whereas del(5q) is one of the most common cytogenetic aberrations in myelodysplastic syndromes (MDS). Thus, t(8;21)(q22;q22) and del(5q) appear to be mutually exclusive, and the association between them has not been characterized yet. Here, we report an 81-year-old woman with coexistent t(8;21)(q22;q22) and del(5q) at initial diagnosis. The bone marrow was infiltrated with 18.4% myeloblasts, and showed marked myeloid and erythroid dysplasia. Myeloblasts were positive for CD19 and CD56 as well as CD13, CD33, CD34 and HLA-DR. G-banding and spectral karyotyping showed 46,XX,del(5)(q?),t(8;21)(q22;q22)[18]/46,XX[2]. Both del(5)(q?) and t(8;21)(q22;q22) were present in a single clone. Fluorescence in situ hybridization (FISH) on metaphase spreads detected a RUNX1/RUNX1T1 fusion signal on the der(8)t(8;21)(q22;q22), and confirmed deletion of CSF1R signaling at 5q33-q34 on the del(5)(q?). Furthermore, FISH on interphase nuclei revealed that the RUNX1/RUNX1T1 fusion signal and deletion of CSF1R signaling were found in 66.0% and 58.0% of interphase cells, respectively, suggesting that del(5)(q?) occurred in cells with RUNX1/RUNX1T1. These results indicated a diagnosis of AML with t(8;21)(q22;q22)/RUNX1/RUNX1T1 rather than MDS, even though the percentage of bone marrow myeloblasts was less than 20%. Based on these findings, together with those of other reported cases, del(5q) seems to be an extremely rare but recurrent secondary aberration in AML with t(8;21)(q22;q22).

Background The only potentially curative therapy for myelodysplastic syndrome is allogeneic hematopoietic cell transplant; unfortunately, there is a high relapse rate. The objective of this study was to perform a detailed clinicopathologic study of patients with relapsed myeloid neoplasm following allogeneic hematopoietic cell transplant for myelodysplastic syndrome. Methods Pre-transplant, post-transplant, and relapse bone marrow and peripheral blood morphologic features (including dysplasia) were retrospectively evaluated by study authors. Clinical features and results of cytogenetic analysis and engraftment/chimerism studies were obtained from the medical record. Results Our study describes 21 patients with a median time to relapse of 6 months (range 2–82). Ten of the patients relapsed with higher grade disease, including six with overt acute myeloid leukemia. Pre-transplant megakaryocyte dysplasia was associated with dysplastic megakaryocytes in the relapse specimen; however, neither erythroid dysplasia nor granulocytic dysplasia were associated with their counterpart in the relapse specimen. Relapse specimens had a lower marrow cellularity and higher blast percentage than pre-transplant disease. Cytogenetic comparisons before and after transplant showed variety, including clonal evolution (22%), the same abnormal clone (33%), or a different abnormal clone (22%). Conclusions Our detailed review of post-transplant marrow biopsies prior to relapse highlights the difficulty in diagnosing relapse and particularly impending relapse.

Mutations (MT) in the 5' untranslated region (UTR) of ANKRD26 (A26) are implicated in ANKRD26- related thrombocytopenia (A26-RT), an autosomal dominant disorder of mild to moderate thrombocytopenia (TP) often presenting in adulthood, although severe and pediatric cases are reported. Erythrocyte and leukocyte counts are normal to increased, with unremarkable morphology. Platelet (plt) size is usually normal, as with ETV6- and RUNX1-mutated TP. Together, A26, ETV6, and RUNX1 germline (GL) MT comprise a separate 2016 WHO category of myeloid neoplasms (MN) with GL predisposition and preexisting platelet disorders. Normal plt size separates A26-RT from other familial TPs with giant plts. No consistent morphological plt aberrations have been reported. Bleeding history is absent or mild, and while TP is not life threating, 8-10% of patients (pts) develop a MN, including a 30-fold increased risk for AML relative to the general population. A26 is an inner membrane adaptor protein with 2 major domains: ankyrin repeats (ANKR) and coil-coil (C-C), both of which interact with signaling and cytoskeletal proteins. A26-RT MT are almost exclusively in the 5'UTR, however, rare A26 coding variants (A26-CV) are reported to segregate with familial TP. Still, some studies on A26-RT limit sequencing to the 5'UTR. As such, A26-CV are not well represented or described. We performed whole-exome sequencing (WES) on 195 pts with MN seen at Cleveland Clinic between 2004 and 2012, and downloaded WES .bam files from online sources, totaling 653 MN cases. Using a standard pipeline for discovery of rare GL variants ( Complete clinical description was available for 8 A26-CV pts, all diagnosed with MDS or MDS/MPN at med age of 64. Two pts had antecedent bruising 1 year prior to diagnosis (dx). No prior bleeding was noted. Three pts had prior TP, two of whom had bicytopenia and pancytopenia. First degree family history (FH) was positive for cancer in 6 pts (75%), including 2 pts with FH of hematologic neoplasms. One pt had a 2nd-degree relative with a non-malignant hematologic condition requiring transfusions. At dx, cytogenetics were normal and complex in 2 pts each (25%). Deletions in chromosomes 5, 17, 20, and Y were observed in 1 pt each, and monosomy 7 in 2 pts. On bone marrow aspirate, 6 pts (75%) had dysmegakaryopoiesis, found in interstitial patterns and clusters. Mild to moderate dyserythropoiesis was observed in 5 pts (63%), and 5 pts had dysgranulopoiesis. The med blast percentage was 1.5% (range 0-8%), with 5 pts having hypercellular marrow, med 65% (range 40-95%). On corresponding CBC, pts were anemic (med hemoglobin of 9.5 g/dL), with the majority (n=5) showing signs of erythroid dysplasia, and also thrombocytopenic (med plt of 99 k/μL), with giant plts observed in 3 pts. Two pts had both giant plts and abnormal erythroid morphology. Hypo and monolobate, hyposegmented, and hypogranular forms were observed in the 3 lineages, as well as detached nuclear lobes, budding, segmentation, and nuclear-cytoplasmic dyssynchrony. On next generation sequencing, co-occurring SRSF2 and ASXL1 MT were observed in 3 and 2 pts, respectively. In sum, we have identified 22 A26-CV in MN, suggesting a role in predisposition as with A26-RT. We have seen in our cohort that A26-CV pts present differently from those with A26-RT. They have a variable past medical history and limited FH of TP, are anemic, with multilineage dysplasia observed not just in bone marrow, but also on peripheral blood smear, especially in megakaryocytes and erythrocytes. This is not surprising, as A26 is expressed in both lineages. The presence of giant plts is noteworthy. The mechanism for hypomorphic A26-CV may differ from that of the A26 5' UTR, which increase A26 levels in late-stage megakaryopoiesis by abrogating RUNX1/FLI1 binding, leading to aberrant proplatelet formation. Given the plt size and presence of nuclear phenotypes, altered interactions with signaling and cytoskeletal proteins could be involved, and may represent a novel A26 phenotype. Further investigation and association of A26-CV with MN ontogeny is under way. Disclosures Mukherjee: Takeda: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Projects in Knowledge: Honoraria; Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Partnership for Health Analytic Research, LLC (PHAR, LLC): Consultancy; McGraw Hill Hematology Oncology Board Review: Other: Editor; Bristol-Myers Squibb: Speakers Bureau. Advani:Kite Pharmaceuticals: Consultancy; Amgen: Research Funding; Macrogenics: Research Funding; Glycomimetics: Consultancy, Research Funding; Pfizer: Honoraria, Research Funding; Abbvie: Research Funding. Nazha:Tolero, Karyopharma: Honoraria; MEI: Other: Data monitoring Committee; Novartis: Speakers Bureau; Jazz Pharmacutical: Research Funding; Incyte: Speakers Bureau; Daiichi Sankyo: Consultancy; Abbvie: Consultancy. Gerds:Imago Biosciences: Research Funding; Sierra Oncology: Research Funding; Roche: Research Funding; Incyte: Consultancy, Research Funding; Celgene Corporation: Consultancy, Research Funding; Pfizer: Consultancy; CTI Biopharma: Consultancy, Research Funding. 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.

Del (9q) is a recurrent cytogenetic abnormality in acute myeloid leukaemia (AML). We report an analysis of 81 patients with del(9q) as a diagnostic karyotypic abnormality entered into the Medical Research Council AML trials 10, 11 and 12. Patients were divided into three groups: (i) Sole del (9q), 21 patients ; (ii) Del(9q) in association with t(8 ; 21), 29 patients ; (iii) Del(9q) in association with other cytogenetic abnormalities, 31 patients. Sole del(9q) was associated with a characteristic bone marrow phenotype at diagnosis: a single Auer rod was found in all cases examined. There was also an association with erythroid dysplasia (74%) and granylocytic lineage vacuolation (90%). The incidence of all three of these features was significantly higher (P < 0.05) in the sole del(9q) group compared with control cases lacking del(9q). The overall survival (OS) of all 81 patients was compared with a control group of 1738 patients with normal cytogenetics entered in the same trials over the period of investigation. The 5-year OS for patients with del(9q) was 45%, compared with 35% for the control group (P = 0.09). Patients with del(9q) in association with t(8 ; 21) had a 5-year OS of 75%, which was significantly better than the groups with either sole del(9q) (40%) and del(9q) with other abnormalities (26% ; P = 0.008). Karyotyping indicated a common area of deletion in the region 9q21-22, which was present in 94% of cases. It is likely that the deletion of single or multiple tumour suppressor genes located in this region may underlie the pathogenesis of del (9q) AML.

Background/Aim: Abnormal immunophenotypes of haematopoietic cells in myelodysplastic syndromes (MDS) have been identified by flow cytometry (FCM) as a typical characteristic of myeloid dysplasia. Considering that most MDS patients show varying degrees of erythroid dysplasia, we analysed the immunophenotypic feature of erythroblasts to evaluate its diagnostic application in MDS. Methods: Erythroid antigens CD71 and CD105 expression were analysed using FCM. The development index (DI) acquired by log transformation of the CD71/CD105 expression ratio was used to denote the erythroid maturation and distinguish patients with non-clonal cytopenias from low-risk MDS. The diagnostic quality of DI in distinguishing MDS from non-clonal cytopenia patients was evaluated using the receiver–operator characteristic (ROC) curve. Results: Under-expression of CD71 and over-expression of CD105 were detected in erythroblasts of MDS patients compared with non-clonal cytopenias. The diagnostic test showed good diagnostic power that the area under the ROC curve was greater than 0.9. The diagnostic sensitivity and specificity were 75.6% and 92.3%, respectively, according to the DI threshold defined by the ROC curve in low-risk MDS patients with normal karyotypes. Moreover, the DI showed a positive correlation with the haemoglobin level, and the MDS patients with lower DI usually showed frequent red cell transfusion. The patients with a lower DI generally had the HLA-DR15 allele or marrow hypocellularity. Conclusion: Developmental defects and immune-associated factors may contribute to the erythroid dysplasia. The DI derived from ratios of CD71 and CD105 expression is a useful marker to characterise dyserythropioiesis associated with MDS and can be helpful in distinguishing it from dyerythropoiesis associated with non-clonal disorders.

Morphology has dominated the diagnosis and classification of MDS for decades. With the advent of NGS, morphology is used as a gold standard to assess associations of specific disease sub-entities with individual mutations or their combinations. An example is the association between SF3B1 mutations and ringed sideroblasts, or with disease-defining lesions, such as t(8:21), 11q23 translocations, and EVI1 mutations. Beyond these discrete examples, assignments of diverse morphologic features to combinations of specific genotypes has been more challenging. To identify potentially novel paradigms of associations between molecular hits and morphologies we examined 835 patients with MDS. Common somatic mutations, chromosomal abnormalities and morphologic reviews were subjected to blinded, objective standard diagnostic criteria. Abnormalities included individual lineage dysplasia and cytopenias, monocytosis, fibrosis, and increased megakaryocytes, all classified using standard criteria. To correlate the presence of mutations to morphologic criteria, only mutations with a clonal burden >10% were used. In total, 794 out of 835 patients (95%) harbored at least one lineage dysplastic feature. Myeloid, erythroid and megakaryocytic dysplasia occurred in 56% (470/835), 71% (590/835) and, 70% (587/835) of patients, respectively. Bi- and tri-lineage dysplasia were also identified in 36% (297/835) and 33% (278/835). In 90% (753/835) of patients there was cytopenia: 48% (402/835) had neutropenia, 61% (510/835) anemia, and 61% (510/835) thrombocytopenia. More than 50% of patients had multiple-cytopenias; 23% (194/835) had pancytopenia. Monocytosis was present in 20% (168/835) of patients and 31% (256/835) had elevated numbers of megakaryocytes. For analysis all possible combinations (25 pairs in total) were queried and those present in >80 patients were evaluated. Based on NGS among 36 genes, TET2 (27%), SF3B1 (23%), ASXL1 (19%), del(5q) (16%), SRSF2 (14%), DNMT3A (11%) and del(7q) (10%),, were frequently mutated genes/CNAs, each present in >10% of patients. A number of relationships between phenotypic features and molecular context were uncovered. For example, patients with thrombocytopenia also commonly had tri-lineage dysplasia [38% (198/510) vs. 25%, (80/325), p When we performed statistical analyses that sought correlations between clinical features and genetic alterations, 78 significant associations were identified. For instance, patients with erythroid dysplasia more frequently had SF3B1 mutations [in 15% of erythroid dysplasia patients, 90/590] than patients without it [6%, 15/245] (p=.0002, q=.001). In contrast, these patients were less likely to have EZH2 mutations [4.2% (25/590) vs. 11.4% (28/245), p Using various conventional and more advanced strategies, we present the first comprehensive genotype-phenotype association study that linked somatic molecular events to morphologic and clinical features. We demonstrate that the complexities of how molecular context governs morphologic changes can be deciphered. Certain rigorously defined morphologic configurations show specific associations with individual mutations or their combinations. Disclosures Nazha: MEI: Consultancy. 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; Opsona: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy.

Myelodysplastic syndromes (MDS) constitute a form of blood cancer that primarily affects the elderly and is characterized by anemia (or other cytopenias) and a high risk of leukemic transformation.1 The World Health Organization (WHO) system has formally classified MDS as 1 of 5 myeloid malignancies that also include acute myeloid leukemia (AML) and myeloproliferative neoplasms.2 All myeloid malignancies, including MDS, are clonal stem cell diseases. Unlike the case with BCR-ABL1-positive chronic myelogenous leukemia,3 the disease-causing mutation in MDS is currently unknown. It is generally believed that the MDS phenotype is additionally affected by secondary mutations, genetic haploinsufficiency, epigenetic changes, and altered host response that result in cytokine, immune, and bone marrow stromal changes.1 Unfortunately, none of this information has successfully been translated into a robust pathogenetic framework or effective targeted therapy. In routine clinical practice, MDS are suspected when an otherwise unexplained anemia is associated with other cytopenias, increased mean corpuscular volume, or increased red cell distribution width. The peripheral blood smear in MDS shows oval macrocytes, a dimorphic red blood cell population, or hyposegmented/hypogranulated neutrophils. Diagnosis requires bone marrow examination and cytogenetic studies. The consensual minimum criterion for diagnosis is the presence of erythroid, granulocyte, or megakaryocyte dysplasia in 10% or more of informative cells.2 In this regard, one must exclude the possibility of erythroid dysplasia associated with vitamin B12/folate/copper deficiency, viral infections, chemotherapy, or lead/arsenic poisoning. The detection of an abnormal karyotype confirms the morphologic diagnosis of MDS.4 The WHO system further subclassifies MDS into 6 subcategories depending on the percentage of blasts in the bone marrow or peripheral blood, presence or absence of bone marrow multilineage dysplasia, or excess ring sideroblasts: refractory cytopenia with unilineage dysplasia, refractory anemia with ring sideroblasts, refractory cytopenia with multilineage dysplasia, refractory anemia with excess of blasts, MDS associated with isolated 5q–, and MDS unclassifiable.2 Myelodysplastic syndromes are rare in young people, with an estimated incidence rate of less than 1 per 100,000 before age 50 years and approximately 2 per 100,000 between ages 50 and 60 years; however, the incidence rises significantly after age 60 years: approximately 8 per 100,000 between ages 60 and 70 years and greater than 20 per 100,000 after age 70 years.5 In 2004, close to 10,000 new US cases of MDS were registered, and approximately 7000 cases occurred in patients older than age 70 years.5 Prognosis is poor for patients with MDS, with 3-year survival rates estimated at less than 50%.5 The standard prognostic tool in MDS is the International Prognostic Scoring System (IPSS), which classifies patients into low-, intermediate-1, intermediate-2, and high-risk categories on the basis of the percentage of bone marrow blasts, the karyotype, and the number of cytopenias; the respective median survival rates are estimated at 8, 5.3, 2.2, and 0.9 years.6,7 Although additional IPSS-independent prognostic variables have since been described, their added practical value has been modest.8 From the standpoint of both disease biology and prognosis, it is important to distinguish primary MDS from therapy-related MDS; the latter is closely related to therapy-related AML and develops in the setting of prior exposure to chemotherapy (eg, alkylating agents, topoisomerase II inhibitors), radiotherapy, radiation accidents, benzene, or other toxins and is prognostically worse than primary MDS.9 Current management in MDS includes supportive care (eg, red blood cell transfusions), drug therapy, and allogeneic hematopoietic cell transplant (allo-SCT). Currently, allo-SCT is the only treatment modality for MDS with curative potential. However, because most patients with MDS are older than age 70 years,5 they are not good candidates for allo-SCT, and the value of allo-SCT in younger patients is undermined by the substantial risk of treatment-related death and morbidity.10-12 Furthermore, cure is not necessarily essential in the presence of drug therapy that can effectively control disease symptoms and prevent disease-related mortality.13,14 Unfortunately, drug therapy for MDS has had limited success, which reflects our inadequate insight into disease pathogenesis. Regardless, 4 new drugs (azacitidine, decitabine, lenalidomide, and deferasirox) were recently approved by the Food and Drug Administration for use in certain types of MDS. In the following sections, I comment on their true value in current practice.

An 11-year-old female Dachshund was presented with depression, diarrhea, weight loss, and radiographic evidence of masses involving the liver, spleen, and cranial lobe of the right lung. Results of a CBC included severe nonregenerative anemia (HCT 14.2%, hemoglobin, 4.3 g/dL, reticulocytes 66,000/microL) with marked metarubricytosis (nucleated RBCs 6.39 x 10(3)/microL). Examination of the peripheral blood smear revealed marked erythroid dysplasia, including marked anisocytosis with a prevalence of macrocytes, Howell-Jolly bodies, diffuse basophilic stippling, and multinucleated and atypical nucleated RBCs. Neutrophil hypersegmentation and giant forms were also noted. Numerous erythrocytes, particularly polychromatophilic cells, contained inclusions consistent with Cabot rings, which appeared as delicate red-purple ellipsoid or figure 8 structures. Rarely, Cabot rings were observed extracellularly. The dog was treated symptomatically with blood transfusions, prednisone, erythropoietin, and vitamin supplementation, but the anemia progressively worsened. The dog was euthanized 2 months after presentation. Bone marrow aspirate and core biopsy specimens obtained at the time of euthanasia revealed marked dysplastic changes in all cell lines, especially dyserythropoiesis, along with infiltrating carcinoma cells. A necropsy was performed, and histologic examination revealed poorly differentiated adenocarcinoma of the lung with multiple metastases to the marrow, spleen, and liver. The final diagnosis was marked myelodysplasia secondary to metastatic adenocarcinoma. Cabot rings are found rarely in humans with myelodysplasia, but have not been described previously in dogs. Based on the findings in this case, Cabot rings may occur rarely in dogs with severe dyserythropoiesis.

Erythroid dysplasia is a common feature of myelodysplastic syndromes (MDS). Currently available information about the immunophenotypic features of normal and dysplastic erythropoiesis is scarce and restricted to relatively few markers. Here we studied the expression of CD117, CD35 and CD44 throughout the normal (n=16) and dysplastic (n=48) bone marrow erythroid maturation. CD35 emerged as an early marker of CD34+ erythroid-committed precursors, which is expressed before CD105 and remains positive thereafter. MDS patients (with and without morphologic dyserythropoiesis) displayed overall increased expression of CD44, associated with slight alterations on CD35 expression, suggesting that phenotypic alterations in MDS may precede morphologic dysplasia. In turn, MDS patients with anemia showed increased expression of CD117., P.L. is supported by a grant from the Fundação para a Ciência e Tecnologia (FCT) of Portugal (SFRH/BD/32097/2006).

IMDSFlow19: et al., Definite progress has been made in the exploration of myelodysplastic syndromes (MDS) by flow cytometry (FCM) since the publication of the World Health Organization 2008 classification of myeloid neoplasms. An international working party initiated within the European LeukemiaNet and extended to include members from Australia, Canada, Japan, Taiwan and the United States has, through several workshops, developed and subsequently published consensus recommendations. The latter deal with preanalytical precautions, and propose small and large panels, which allow evaluating immunophenotypic anomalies and calculating myelodysplasia scores. The current paper provides guidelines that strongly recommend the integration of FCM data with other diagnostic tools in the diagnostic work-up of MDS.

OBJECTIVE: To evaluate the diagnostic utility of bone marrow (BM) sampling in HIV positive patients. DESIGN: Retrospective cohort analysis. SETTING: Specialist HIV/AIDS service in London. SUBJECTS: 215 consecutive HIV infected patients undergoing 246 BM samplings for investigation of pyrexia without localising signs, haematological abnormalities, or staging/investigation of lymphoma. MAIN OUTCOME MEASURE: Diagnostic yield from (and impact on management of) BM sampling. RESULTS: Of 122 BM samples taken to investigate pyrexia, 33 (27%) revealed the cause on microscopy: unexpected lymphoma in seven (6%), mycobacteriosis in 25 (20%), and toxoplasmosis in one (1%). Marrow infiltration was confirmed in 11 of 38 BM samples taken for staging/investigation of lymphoma/leukaemia. In afebrile patients, of 22 with pancytopenia, BM samples showed HIV associated changes in 17 and specific diagnoses in five (mycobacterial infection in three, haemophagocytic syndrome in one, and megaloblastic change due to vitamin B-12 deficiency in one); of 21 with isolated thrombocytopenia, 20 (95%) BM samples showed immune thrombocytopenic purpura to be the cause and the remaining patient had BM changes of aplasia; of 29 with isolated anaemia, 28 had BM changes of HIV associated dysplasia/erythroid dysplasia and one had unsuspected iron deficiency; all 10 with isolated leucopenia/neutropenia had BM changes ascribed to HIV infection exacerbated by concurrent sepsis or medication; of four BM samples taken for other reasons, one showed mycobacterial infection. CONCLUSIONS: BM sampling has diagnostic utility in HIV infected patients with pyrexia without localising signs, pancytopenia, and staging/investigation of lymphoma; this test has little value in the investigation of afebrile patients with isolated thrombocytopenia, anaemia, or leucopenia as HIV is usually the underlying cause.

Secondary dyserythropoiesis has been associated with vincristine administration in dogs. Evaluation of bone marrow aspirates for the presence of morphologic abnormalities in the erythroid lineage aids in the diagnosis. However, morphologic features of circulating erythroid precursors in these cases have not been described previously. The purpose of this report was to describe the cytologic features of dyserythropoiesis in peripheral blood and also bone marrow smears in a case series of dogs with immune-mediated thrombocytopenia (IMT) treated with vincristine sulfate. Nineteen dogs receiving vincristine for treatment of IMT were identified by retrospectively searching a computerized medical record system. There were 5 dogs that had dysplastic erythroid precursors in peripheral blood smears within 7 days of vincristine treatment. Two of those 5 dogs also had evidence for erythrodysplasia in modified Wright's-stained bone marrow smears obtained postvincristine administration. Morphologic changes included bizarre or inappropriate mitotic figures, abnormal nuclear configurations (fragmentation, elongation, indentation, and binucleation), atypical nuclear remnants (Howell-Jolly bodies), or nuclear and cytoplasmic asynchrony within the erythroid precursors. A brief review of the literature with discussion of the etiologies for dyserythropoiesis is provided. The dyserythropoiesis was clinically insignificant in all 5 cases and resolved. However, pathologists and clinicians should be aware of these potential findings to prevent misdiagnosis of other conditions.

TO THE EDITOR: Transformation from myelodysplastic syndrome (MDS) to chronic myelomonocytic leukemia (CMML) is rarely observed. However, this has been reported in cases of refractory anemia with ring sideroblasts or excess of blasts [1-4]. Moreover, MDS patients may present with monocytosis that does not meet the diagnostic criteria of CMML, which makes diagnosis and classification of these atypical mixed disorders a challenge [5, 6]. These difficulties in diagnostic classification and prognostic stratification may be concerning with regard to decision-making, particularly in this new era of effective disease-modifying therapies, such as hypomethylating agents [6, 7]. Recently, we faced such concerns during the management of a patient who developed CMML 7 years after having been diagnosed with refractory anemia (RA). The full clinical onset of CMML was preceded by progressive loss of response to ongoing treatment with an erythropoiesis-stimulating agent (ESA), worsening anemia, thrombocytopenia, leukocytosis, and increasing monocytosis. A morphological study of the peripheral blood (PB) and bone marrow (BM) revealed the coexistence of myelodysplastic and myeloproliferative syndromes. A cytogenetic alteration (45, X0,-Y), which was not present at diagnosis of RA 7 years earlier, was also detected during the CMML phase. The patient received azacitidine and showed a good response. Here, we describe this rare case and its implications in disease classification and management. On January 2005, a 74-year-old man presented with moderately macrocytic slight thrombocytopenia. Five years earlier (in 2000), he had received postoperative radiotherapy after radical prostatectomy for prostate cancer. Apart from this prostatic neoplasm, for which a regular oncological follow-up had confirmed a persistent complete remission until then, he mentioned one previously cured gastric ulcer and well-controlled hypertension. He complained of fatigue and general unease for the past few weeks. A complete blood count prescribed by his general practitioner had revealed macrocytic anemia with a low reticulocyte count, mild thrombocytopenia, and slight neutropenia. On admission, he appeared pale and fatigued. A comprehensive laboratory evaluation did not reveal any remarkable abnormalities. His coagulative profile and renal and hepatic function were normal. Suspicions of hemolytic disorders, virus infections, and iron and vitamin deficiencies were also dismissed. Morphological examination of PB smears showed isolated erythrocyte macrocytosis but did not provide any other diagnostic findings. BM aspiration and trephine biopsy were performed. BM examination revealed hypercellular BM with evident erythrodysplasia and 6% of blasts without fibrosis. Conventional karyotyping and fluorescence in situ hybridization analyses did not show abnormalities. Therefore, the patient was diagnosed with MDS, RA, according to the French-American-British classification. The patient had an International Prognostic Scoring System score of 1 (intermediate-1 risk). On admission and during the diagnostic phase, he received 4 units of red blood cell (RBC) concentrates. Thus, the patient was scheduled to receive ESA at a weekly dose of 40,000 units by subcutaneous injection (in January 2005). Since then, the patient was regularly followed-up at our clinic and continued to receive erythropoietin treatment. Normal PB values were maintained without the requirement for transfusion, clinical complications, or side effects until June 2012, when his hematologic status slowly began to deteriorate. Worsening anemia (requiring RBC transfusions), thrombocytopenia, and leukocytosis with absolute monocytosis became prominent. A comprehensive medical examination was performed. The examination of PB smears showed absolute monocitosis with 2% of circulating blasts. BM examination showed marked erythroid dysplasia and monocytosis with 15% blasts. A karyotype abnormality, such as 45, X0,-Y, which was not detected at initial diagnosis of RA 7 years earlier, was also found; however, the JAK2 V617F mutation was absent. Therefore, the patient was diagnosed with RA, coexisting with type II CMML. The patient had a MD Anderson Prognostic Score of 3 (intermediate-2 risk) [8]. After a brief course of hydroxycarbamide, administered in order to reduce leukocytosis, the patient received 6 cycles of azacitidine (75 mg/m2, schedule 5+2+2, each cycle every 4 weeks), according to approved indications [9]. After the second course, transfusion independence was achieved and the PB counts significantly improved with disappearance of monocytosis. Complete remission of CMML was achieved after 6 courses of hypomethylating therapy, without any adverse events. In conclusion, this case represents an atypical presentation of CMML secondary to MDS. In addition to its rarity and anecdotal interest, we believe that this report should be discussed with regard to several topics, such as the possible evolution of low-risk MDS in CMML, the existence of secondary CMML as a distinct disease, its absence in the current classification systems, and, finally, the efficacy of hypomethylating therapy [4, 10].

The reproducibility of the World Health Organization 2008 classification for myelodysplastic syndromes is uncertain and its assessment was the major aim of this study. The different peripheral blood and bone marrow variables required for an adequate morphological classification were blindly evaluated by four cytomorphologists in samples from 50 patients with myelodysplastic syndromes. The degree of agreement among observers was calculated using intraclass correlation coefficient and the generalized kappa statistic for multiple raters. The degree of agreement for the percentages of blasts in bone marrow and peripheral blood, ring sideroblasts in bone marrow, and erythroid, granulocytic and megakaryocytic dysplastic cells was strong (P

The initial clinical presentation of T-cell large granular lymphocytic leukemia (T-LGL) and myelodysplastic syndromes (MDS) can be similar, each characterized by unexplained peripheral cytopenias. However, these diseases are pathobiologically distinct and associated with stark differences in prognosis and therapy. T-LGL is a clonal lymphoid disorder defined by phenotypically abnormal cytotoxic T cells and an indolent clinical course, while MDS is a clonal disorder of hematopoietic stem cells defined by ineffective hematopoiesis, morphologic dysplasia, and an elevated risk of acute leukemia. Despite these differences, distinction between T-LGL and MDS can be challenging and misdiagnosis can significantly delay initiation of appropriate therapy. The recent identification of STAT3 mutations in LGL may facilitate this distinction: activating STAT3 mutations occur in 40-70% of T-LGL cases, primarily within the SH2 domain, but have not been reported in patients with MDS without concomitant T-LGL. STAT3 is included within our clinical next generation sequencing (NGS) panel, which is used to evaluate patients with known or suspected hematologic malignancies, primarily acute myeloid leukemia, MDS and myeloproliferative neoplasms, as well as various lymphocytic leukemias. We report the frequency and type of STAT3 mutations within our patient population and assess the impact of this information on diagnosis. Between 1/1/2015 and 6/30/2016, 3414 samples (primarily peripheral blood (PB) or bone marrow (BM)) from 2530 unique patients evaluated at Brigham and Women's Hospital and/or Dana-Farber Cancer Institute underwent clinical NGS with a custom, 95-gene, amplicon-based panel (PMID: 27339098). Exons 2-17 and 21-23 of the STAT3 gene were analyzed in each sample using reference transcript 1 (NM_139276). We identified 40 patients with 40 candidate STAT3 mutations (Figure 1). Based on domain localization, variant allele fraction, and population allele frequency, we classified these sequence variants as somatic SH2 domain mutations (n = 21), somatic non-SH2 domain mutations of unknown significance (n = 5) or germline variants (n = 14). Of the 21 patients with somatic SH2 domain mutations, 9 carried a prior diagnosis of T-LGL and 8 were concurrently diagnosed with T-LGL by conventional diagnostic criteria (clonal aberrant T cells in the setting of neutropenia, anemia, or lymphocytosis). The final 4 patients with STAT3 SH2 domain mutations were unexpected diagnoses of T-LGL. These 4 patients were initially referred from outside institutions for MDS based on the reported presence of unilineage erythroid dysplasia (n=2), unquantified ring sideroblasts (n=1), or pancytopenia with unspecified marrow findings (n=1). In 3 of these cases, the STAT3 mutation discovery prompted T-cell flow cytometric analysis of peripheral blood, which revealed an aberrant immunophenotype, and T-cell receptor gamma gene rearrangement studies, which were clonal; these tests are pending in the 4th case. BM evaluation was performed in 12 of 21 patients, including the 4 with suspected MDS; in all cases, the findings did not meet diagnostic criteria for MDS by expert hematopathology review and all showed a normal karyotype. Five additional cases demonstrated somatic non-SH2 domain STAT3 mutations of unknown pathobiologic significance: 3 myeloid neoplasms, 1 chronic lymphocytic leukemia, and 1 autoimmune hemolytic anemia. Additional non-STAT3 mutations were also frequently identified in tumors other than T-LGL. Our experience demonstrates that STAT3 sequencing is a critical component of the evaluation of unexplained cytopenias, and identification of a mutation can clarify ambiguous phenotypes thus averting the consequences of misdiagnosis or diagnostic delay. Notably, several series have reported that MDS and T-LGL can infrequently occur concurrently and thus identification of a STAT3 mutation and a clonal T-LGL population does not exclude the possibility of concomitant MDS. In our cohort, however, no T-LGL patient with a STAT3 mutation demonstrated pathologic evidence of MDS and the majority (19 of 21) showed no other myeloid-associated somatic mutations. In addition, 5 cases in our cohort had likely somatic, non-SH2 domain STAT3 mutations in the context of disparate clinical scenarios, suggesting that these mutations may have a pathogenic role in other hematologic malignancies, a subject of future study. Disclosures DeAngelo: Ariad: Consultancy; Pfizer: Consultancy; Novartis: Consultancy; Amgen: Consultancy; Baxter: Consultancy; Incyte: Consultancy; Celgene: Consultancy. Stone:Celator: Consultancy; Jansen: Consultancy; ONO: Consultancy; Agios: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics: Consultancy; Amgen: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Consultancy; Merck: Consultancy; Roche: Consultancy; Seattle Genetics: Consultancy; Sunesis Pharmaceuticals: Consultancy; Xenetic Biosciences: Consultancy. Lindsley:MedImmune: Research Funding; Takeda Pharmaceuticals: Consultancy.

Most patients with myelodysplastic syndrome eventually become dependent on regular red cell transfusions. This dependency has a negative impact on clinical outcome, primarily because it may be associated with more severe marrow failure. In addition, however, transfusion dependency may involve clinical consequences of chronic anemia and iron overload. Although transfusion iron is primarily taken up by the reticuloendothelial cells, the metal is later redistributed to parenchymal cells. This redistribution is modulated by several factors, including the degree of ineffective erythropoiesis through its suppressive effect on hepcidin production. Body iron status is routinely assessed by serum ferritin and transferrin saturation, but there is a need of reliable tools for locating iron accumulation in patients. Magnetic resonance imaging T2* provides a non-invasive method for detecting and quantifying both liver and myocardial iron overload. Clinical consequences of parenchymal iron overload have been reported not only in thalassemia major, but also in patients with myelodysplastic syndrome. Transfusion-dependent patients with isolated erythroid dysplasia and low risk of leukemic evolution are more likely to develop parenchymal iron overload and its toxicity, and therefore may benefit from chelation therapy. There may also be a benefit of chelation therapy in patients with transfusion iron overload undergoing allogeneic stem cell transplantation. Deferoxamine and deferasirox are currently available for treatment of transfusion iron overload in patients with myelodysplastic syndrome.

We present a retrospective analysis of 137 patients with early MDS without excess of blasts that revealed transfusion dependency in 87% of the cases. A significant difference in overall survival was noted between patients receivingor=2 units and those receiving2 units of RBC transfusions/month (65.0 vs. 35.3 months, respectively, P=0.02). Univariate statistical analysis identified the presence of disease progression to advanced MDS (chi(2)=26.4, P=0.001) and the administration of1 or2 units of RBC per month (chi(2)=15.9 and 14.6, respectively, P=0.001) as the most important parameters affecting survival. Nevertheless, even the administration of 1 RBC unit every 4-8 weeks had a significantly adverse impact on survival compared to non-transfused patients. Transfusion dependency itself did not affect disease progression as determined by the presence of multilineage dysplasia and adverse karyotype (expressed by the IM-1 or IM-2 score). Multivariate analysis confirmed disease progression towards leukemia as a highly significant independent variable affecting survival (P=0.0001). None of the other evaluated parameters had a significant impact on survival in patients with progressive disease. In non-transplanted patients without MDS progression, administration of2 units of RBC transfusions/month was the only independent variable with adverse impact on survival in patients with unilineage erythroid dysplasia (P=0.02). In patients with multilineage dysplasia, only heavy transfusion dependency (3TURBC/month) and serum ferritin2000 microg/l adversely affected survival (P=0.03). Modification of the WPSS by replacing transfusion dependency with initial Hb level80 g/l retained its prognostic relevance and allowed the identification of a potential risk subset of early MDS patients with intermediate and high scores and limited survival (40% at 5 years) as early as at the time of diagnosis. Our results confirm a significant negative impact of transfusion dependency on survival in patients with early MDS without excess of blasts. The main risk subgroup is characterized by unilineage dysplasia limited to erythropoiesis in combination with dependency on2TU of RBC per month. These patients usually have prolonged survival that leads to the development of heavy transfusion iron overload and they thus represent the most important target group for intensive chelation therapy.

The diagnostic criteria of acute erythroid leukemias (AEL) has been revised by WHO in 2001. The National Cancer Institute (NCI) published a set of standardized diagnostic and response criteria for acute myeloid leukemia in 1990, which was revised in 2003. The aim of the present study was to establish the best criteria for therapeutic response assessment in the newly classified AEL and evaluate patient outcomes. Fifty-two patients with AEL as defined by the new WHO criteria were evaluated in this study. The following seven indices for therapeutic response assessment were evaluated: (i) NCI criteria (myeloblast percentage among total nucleated cells (TNC) and cellularity); (ii) myeloblast percentage among non-erythroid cells (NEC) and cellularity; (iii) erythroid series percentage among TNC; (iv) pronormoblast percentage among erythroid cells; (v) ratio of pronormoblasts and blasts; (vi) maturation arrest index; and (vii) disappearance of erythroid dysplasia. Complete remission (CR) patients with5% of myeloblast/NEC (NEC-CR) showed significantly longer overall survival periods (mean 55.8 months) compared to CR patients with5% myeloblast/NEC (mean 11.7 months, P = 0.006). NEC-CR patients also had longer event-free survival (median 16.4 months) compared to patients with5% and20% of myeloblast/NEC (median 6.2 months) (P = 0.044). The other indices for therapeutic response assessment are not significant for predictability of relapse and outcomes. Therefore, we recommend that the myeloblast percentage among NEC be used instead of myeloblast percentage among TNC for therapeutic response assessment in AEL.

Bone marrow aspiration smears from 15 dogs naturally infected with leishmania were evaluated. Three of the dogs showed no clinical signs, six had up to three clinical signs and six had more than three. The most common pathological features of the bone marrow were megakaryocytic dysplasia in 10 of the dogs, erythrophagocytosis in eight, erythroid dysplasia in two and emperipolesis in two. The megakaryocytic and erythroid dysplasia were probably related to an increased number of marrow macrophages producing high levels of tumour necrosis factor alpha and interferon gamma. Six of the dogs with clinical signs showed bone marrow dysplastic features and erythrophagocytosis, suggesting that leishmaniosis could be the unique cause of both conditions.

Erythroid dysplasia is the pathologic hallmark of myelodysplastic syndromes (MDS). To develop a quantitative flow-cytometry approach to its evaluation, we analyzed the expression of CD71, CD105, cytosolic H-ferritin (HF), cytosolic L-ferritin (LF) and mitochondrial ferritin (MtF) in erythroblasts from 104 MDS patients, 69 pathologic control patients and 19 healthy subjects. Six-parameter, 4-color flow cytometry was employed, and data were expressed as mean fluorescence intensity. Compared with pathologic and healthy controls, MDS patients had higher expression of HF (P0.001) and CD105 (P0.001), and lower expression of CD71 (P0.001). MtF was specifically detected in MDS with ringed sideroblasts, and there was a close relationship between its expression and Prussian blue staining (r = 0.89, P0.001). In vitro cultures of myelodysplastic hematopoietic progenitors showed that both HF and MtF were expressed at a very early stage of erythroid differentiation, and that MtF expression is specifically related to mitochondrial iron loading. A classification function based on expression levels of HF, CD71 and CD105 allowed us to correctly classify95% of MDS patients. This flow-cytometry approach provides an accurate quantitative evaluation of erythroid dysplasia and allows a reliable diagnosis of sideroblastic anemia, and may therefore be a useful tool in the work-up of patients with MDS.

We retrospectively studied 89 consecutive patients diagnosed with primary myelodysplastic syndrome (MDS) over a period of 10 years to (1) identify prognostic factors for overall survival (OS) and leukemia-free survival (LFS); (2) to assess and compare the Bournemouth-, Spanish-, Düsseldorf-, Lille-, and the International prognostic scoring systems (IPSS); and to (3) compare the French-American-British (FAB) and World Health Organization (WHO) classifications. The median age of patients was 63 years (range, 26-85). Karyotype analyses were done in 85 patients (96%). Median OS was 3 years; 67 patients (75%) have died, and 28 (31%) had progression to acute myeloid leukemia (AML). Major independent prognostic variables for both OS and LFS (multivariate analysis) were percentage of bone marrow (BM) blasts (P0.0001), and in patients with cytogenetic data available, cytogenetic risk groups by Lille-score (OS, P = 0.031/LFS, P = 0.002) and IPSS (OS, P = 0.024). All five prognostic scoring systems successfully discriminated risk groups as regards OS and LFS, but in patients with cytogenetic data available, the major independent prognostic score for OS (P0.0001) and LFS (P = 0.006) was the IPSS. The FAB and WHO classifications also successfully discriminated between risk groups. The new WHO subgroups [refractory cytopenia with multilineage dysplasia (RCMD), with (RCMD-RS) or without ringed sideroblasts] showed a significantly (P = 0.0454) different prognosis for OS, but not for LFS (P = 0.0839), in comparison to the subgroups having erythroid dysplasia only (RA/RARS). Risk stratification into refractory anemia with excess blast-I (RAEB-I) and RAEB-II tended to yield different prognoses for OS and LFS. The 5q-minus syndrome strongly predicted for a good prognosis. In patients treated with the demethylating agent decitabine (n = 24), IPSS "poor risk" cytogenetics were unable to predict for the expected worse prognosis when compared to "intermediate-risk" cytogenetics. In conclusion, we confirm in a single-center patient cohort that the use of the WHO classification improves the predictive value of the FAB classification and that, in patients with cytogenetic data available, the IPSS can be used for clinical decision-making.

We describe a case with a translocation between the two chromosomes 3 with breakpoints q21 and q26, associated with a unique constellation of hematologic abnormalities. Megakaryocytic dysplasia and peripheral thrombocytosis are the most common abnormalities associated with this chromosome abnormality. Our patient had acute myeloid leukemia (AML), thrombocytopenia, dyserythropoiesis, and an increased myeloid/erythroid ration but no mekakaryocytic dysplasia. Although multilineage involvement has been reported, erythrocyte dysplasia associated with thrombocytopenia and without megakaryocyte dysplasia appears to be extremely rare. Our case supports the speculation that #3 abnormalities with breakpoints q21 and q26 affect a pluripotent stem cell and suggests that megakaryocytic involvement may not be pathognomonic in hematologic abnormalities with t(3;3)(q21;26).

A small number of blood cells with the paroxysmal nocturnal hemoglobinuria (PNH) phenotype are occasionally detected in patients with aplastic anemia (AA) and those with myelodysplastic syndrome (MDS). In an attempt to determine the clinical significance of PNH cells in bone marrow failure syndrome, we examined peripheral blood red blood cells (RBCs) and granulocytes of AA patients and MDS/refractory anemia (RA) patients, for the presence of CD55CD59 (PNH) cells using a sensitive flow cytometry. A total of 100 AA patients and 105 RA patients with varying disease duration were studied. A significant increase (≥0.003%) in the percentage of PNH cells compared to normal individuals was detected in 52% of AA patients. The frequency of patients with increased PNH cells was particularly high as 88.6% in newly diagnosed AA patients. On the other hand, the increased PNH cells were detected only in 15 of 105 (14.3%)) RA patients. None of the 15 patients with increased PNH cells showed apparent trilineage dysplasia; in these patients, RA was diagnosed mainly based on the presence of erythroid dysplasia and/or mild trilineage dysplasia as well as hypercellularity in the bone marrow aspirates from the sternum or the iliac bone. None of them exhibited karyotypic abnormalities. Pancytopenia of these patients improved with cyclosporine or anabolic steroids, or remained stable without treatments. Thus, the presence of PNH cells in peripheral blood probably serves as a marker for good prognosis in RA patients.

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.

Background and Objectives : Human immunodeficiency virus can involve almost any organ system. Bone marrow changes include varying degree of dysplasia in one or more cell lines, plasmacytosis, granuloma and bone marrow necrosis. Anaemia has been found to be the most common hematological manifestation in HIV/AIDS patients. Hence, present study was aimed to study the bone marrow morphology in HIV infected anaemic patients and to evaluate iron stores. Materials and Methods : Seventy five HIV positive patients haemoglobin ? 10 gm/dl and aged between 18-58 years were taken. Complete hemogram and CD4+ counts were done in all patients and were classified as those having AIDS and Non AIDS, according to NACO criteria. Written informed consent was taken from patients and bone marrow aspiration was done from posterior superior iliac spine. Smears were stained with Leishman stain for morphological study and Perls Prussian blue for iron stores. Results: Total patients included in the study were 75. Majority of patients had normocytic normochromic anaemia. Normocellular bone marrow was most common seen in 63.66% patients. Dysplasia was seen in 76%, being more common in AIDS than Non AIDS. Erythroid dysplasia was the most common dysplasia seen in 68%. Granuloma was seen in one case. Iron stores were found to be adequate in majority of patients. Conclusion: Normocytic normochromic anaemia was the most common peripheral smear finding. Hypocellular bone marrow was more common than hypercellular marrow in advanced stage of disease. Dysplastic changes were more common in AIDS than Non AIDS. Erythroid dysplasia was the most common type of dysplasia. Iron stores were adequate in most of the cases as in anaemia of chronic disease.

HMGA2 is frequently overexpressed in hematopoietic cells of patients with paroxysmal nocturnal hemoglobinuria (PNH) and primary myelofibrosis (PMF), and occasionally in myelodysplastic syndromes/myeloproliferative neoplasms (MDS/MPN). We recently reported that transgenic mice overexpressing truncated Hmga2 cDNA (ΔHmga2 mice) showed increased numbers in all lineages of peripheral blood (PB) cells, hypercellular bone marrow (BM), and splenomegaly in young adults of 3 months old (Ikeda et al, Blood, 2011). ΔHmga2 mice also showed growth advantage of hematopoietic stem cells (HSCs) in serial BM transplants and of committed progenitors in colony-replating assays. Thus, overexpression of HMGA2 may contribute to clonal expansion in PNH or myeloproliferation in PMF and MDS/MPN. Although these disorders show disease progression and development of leukemia in the long term, consequences of long-term overexpression of HMGA2 in hematopoiesis is unknown. Therefore, in this study, we investigated hematopoiesis in old ΔHmga2 mice at 15 months old. We found an increase in PB neutrophils in 16 old ΔHmga2 mice compared with 8 old wild-type (WT) mice (mean ± SD; 4.3 ± 1.7 vs. 2.4 ± 0.6 x109/L, p Disclosures: No relevant conflicts of interest to declare.

Background Bone marrow (BM) fibrosis is commonly recognized in the setting of primary myelofibrosis (PMF) and other myeloproliferative neoplasms. However, a variety of non-malignant conditions are associated with myelofibrosis (MF). While PMF is associated with a limited survival, other causes of MF such as AIMF appear to have a favorable clinical course (Pullarkat et al, Am J Hematol 2001;116:211). We conducted a retrospective chart review of patients diagnosed with non-malignant MF to investigate clinical presentations, therapies, and outcomes of AIMF. Methods Patients diagnosed with MF were identified from pathology records at Los Angeles County-USC Medical Center from 1999-2013. Histories were reviewed for the clinical indication for BM biopsy, medical conditions known to be associated with MF, and clinical course. Patients were excluded if they met diagnostic criteria for PMF or other neoplastic hematologic disorders associated with MF. BM specimens were assessed as described by Pullarkat et al: (1) Presence of diffuse reticulin fibrosis (European Consensus Score; Thiele et al, Haematologica, 2005;90:1128).; (2) Lack of clustered or atypical megakaryocytes; (3) Lack of myeloid or erythroid dysplasia, eosinophilia, or basophilia; (4) Lymphocyte infiltration; (5) Lack of osteosclerosis; (6) Absent or mild splenomegaly; and (7) Presence of autoantibodies. Results Thirteen patients with MF were identified (11 F, 2 M; 23-73 y.). The mean follow up was 3.7 years. 12 presented with both anemia and thrombocytopenia, and represented the most common indication for BM biopsy. 1 of 12 also had severe leukopenia. The remaining patient presented with thrombocytosis. Clinical presentation included symptoms of fatigue, fever, lightheadedness, chest pain, and dyspnea. The spleen was enlarged in 1 patient, borderline enlarged in 4, normal in 6, and unknown in 1. 1 patient had a history of splenectomy. All patients had one or more autoantibodies. These included: ANA (8 patients), dsDNA (1), RF (4), CCP (2), DAT (7), lupus anticoagulant (3), anticardiolipin (2), atypical pANCA (1), anti-MPO (1), anti-SMA (3), anti-SSA (2), and anti-SSB (2). Medical co-morbidities included systemic lupus erythematosus (2 patients), rheumatoid arthritis (2), autoimmune hepatitis and primary sclerosing cholangitis (1), dermatomyositis (1), antiphospholipid antibody syndrome (1), and autoimmune hemolytic anemia (AIHA) (7). 3 AIHA patients had Evan’s syndrome. BM cellularity ranged from normocellular to markedly hypercellular. The reticulin stain showed mild to moderate increase in fibrosis (MF1-2/3). Megakaryocytes were increased in 8 patients, normal in 3 patients, and decreased in 2 patients. Megakaryocytes were normal in appearance and distribution. No myeloid or erythroid dysplasia, eosinophilia, or basophilia were noted. All cases demonstrated increased small lymphocyte infiltrates. IgG4 immunostaining was negative in 4 of 4 specimens tested. In 1 patient with Evan’s syndrome, MF was attributed to chronic romiplostim administration. 1 patient with thrombocytosis was treated as JAK2-negative essential thrombocythemia with normalization of his platelet count after interferon therapy. 4 of 11 remaining patients had a resolution in their cytopenias with steroid treatment with or without additional immunosuppressive agents. Resolution of MF was seen on post-steroid therapy BM biopsy in 2 of these patients. 3 had partial response in their cytopenias when treated with similar immunosuppressive regimens. Thrombocytopenia improved in 1 patient after splenectomy done for an unclear indication. 3 patients died due to unrelated sepsis. Conclusion AIMF is an under recognized cause of MF in patients presenting with cytopenias that responds to steroids and has a good clinical outcome in the majority of patients. While the spectrum of clinical presentations is broad, the BM findings are similar between cases and different from those of PMF. The prognostic and therapeutic differences between AIMF and PMF make distinction between these entities imperative for the treating physician. Disclosures: No relevant conflicts of interest to declare.

Abstract 5051 While clinical studies and guidelines provide important data regarding the safety and efficacy of new drugs, their application to daily practice can be limited by the inclusion of selected patient populations. Population-based studies can provide additional insight into the complex process of implementing treatment guidelines in daily practice. The objective of the MyDyS registry was to collect and analyze data regarding the diagnosis and management of patients with myelodysplastic syndrome (MDS) in Slovakia. Material and methods: five clinical centers collected data regarding demographics, staging, history and cytopathology from newly diagnosed patients (pts) with MDS from 2009 to 2011. Patients were classified according to the French American British (FAB) and World Health Organization (WHO) systems and the International Prognostic Scoring System (IPSS). The database allowed online pts registration. Results: one hundred thirty pts were registered to MyDyS in the yrs 2009–2011 and data from 128 pts were analyzed. Male/ female: 53,1%/ 46.9%; average age 64,8 yrs (range 22–89 yrs). The most frequent diagnosis according to FAB classification was RA (53.9%) and RAEB (21.9%), and according to WHO classification RCMD (27,3%) and RAEB II (22,7%). IPSS Low risk was in 16,4% pts, INT-1 in 43%, INT-2 in 19,5% and High risk in 11,7%. The majority of pts were de novo (89,8%). Patients with secondary MDS (10,2%) had a history of malignancy (breast cancer, 23,1%; hematologic malignancy, 2,6% only) and history of combination chemo and radiation therapy in 61,5%. In these pts, bone marrow (BM) was hypercellular in 49,2%, hypocellular in 24,2% of patients. Number of blasts less than 5% in 60,2% patients. Erythroid dysplasia was observed in 88,3%, myeloid dysplasia in 56,3% and dysplasia of megakaryocytes in 71,9% of patients. BM biopsy was performed at diagnosis in 87,5% and reported as hypercellular in 56,6%, with fibrosis in 62,8% and abnormal localization of precursors (ALIP) in 8%. Peripheral cytopenia in 2–3 lineages was observed in 67,2% pts. Growth factor therapy was given to 63 pts and red blood cell transfusions (average 20,7; range 2–80 RBC units) were given to 35 patients. Deferasirox (mostly 1500mg/ d) was given to 25 patients, and serum ferritin level decreased after 8 months in 27,3% pts, after 12 months in 57,4% pts. Chelation therapy was associated with gastrointestinal intolerance in 1 case, and skin reaction in 2 patients. Conclusion: The presented data document the first steps in the complex process of improvement of management of MDS in daily clinical practice in Slovakia. Expansion of the registry is necessary for determination of best practices for discussion with health care authorities and health insurance institutions. Disclosures: No relevant conflicts of interest to declare.

5q syndrome was first described in 1974 by Van den Berghe who reported the consistent association of the deletion of the long arm of chromosome 5 [del(5q)] with haematological abnormalities: macrocytosis, anaemia, normal or high platelet count and hypolobulated megakaryocytes in the bone marrow. The 5q deletion is typically a large interstitial deletion and contains a number of genes involved in haematopoiesis. The del(5q) is also commonly found in other MDS and AML (10–15% of all patients) and is widely believed to mark the location for a tumour suppressor gene(s), the loss of which may affect important processes such as growth control and normal haematopoiesis. However, studies have failed to identify mutations within the retained alleles, or potential submicroscopic homozygous deletions in the commonly deleted region (CDR) suggesting that either haploinsufficiency promotes the 5q syndrome, or that a retained tumour suppressor allele has undergone epigenetic inactivation. To enable progress towards understanding the 5q− syndrome and to provide a model for testing novel therapeutics we generated a mouse model for the human 5q− syndrome. Using Cre-loxP recombination to delete the syntenic regions in the mouse equivalent to the human 5q CDR we have generated a mouse model (deletion mice), with a deletion on chromosome 18 flanked by the Cd74 gene and Nid67 gene (removing an interval harbouring Nid67, Dctn4, Rbm22,Myoz3, Synpo, Ndst1, Rps14, Cd74). The mice display macrocytic anemia, prominent erythroid dysplasia and monolobulated megakaryocytes in the bone marrow, similar to the human disease. Notably, intercrossing of deletion mice with p53 mice resulted in a reversal of the observed deficit in hematopoietic progenitor cell development, thereby demonstrating for the first time the mechanistic involvement of p53 in 5q syndrome. Recent studies have indicated a role for the ribosomal protein Rps14 in the development of 5q syndrome, and taken in combination with our deletion of Rps14, suggest that 5q may be a new member of the expanding group of ribosomopathies. Our work also suggests a prominent role for p53 in promoting the 5q phenotype in mice and we have recently shown p53 up-regulation in bone marrow trephines from del(5q) patients. This novel mouse model will allow the investigation of new and existing therapeutics, specifically targeted against del(5q) abnormalities, and give further mechanistic insight into 5q− syndrome. 2 Morphological differentiation of hypocellular refractory cytopenia of childhood and severe aplastic anemia and clinical outcome I. Baumann, C. Niemeyer, M. Fuhrer, S. Behrendt, V. Campr, J. Csomor, I. Furlan, V. de Haas, G. Kerndrup, R.J. Leguit, P. De Paepe, P. Noellke, S. Schwarz. Department of Pathology, Hospital Boeblingen, Boeblingen, Department of Pediatrics and Adolescent Medicine, University of Freiburg, Freiburg im Breisgau, Department of Hematology and Oncology, Ludwig-Maximilians-University Munich, Munich, Germany; Department of Pathology, University Hospital Motol, Prague, Czech Republic; Department of Pathology, Semmelweis University, Budapest, Hungary; Dutch Childhood Oncology Group, The Hague, The Netherlands; Department of Pathology, Vejle Hospital, Vejle, Denmark; Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands; Department of Pathology, Ghent University Hospital, Ghent, Belgium; Department of Pathology, University Medical Center Erlangen, Erlangen, Germany

5q syndrome was first described in 1974 by Van den Berghe who reported the consistent association of the deletion of the long arm of chromosome 5 [del(5q)] with haematological abnormalities: macrocytosis, anaemia, normal or high platelet count and hypolobulated megakaryocytes in the bone marrow. The 5q deletion is typically a large interstitial deletion and contains a number of genes involved in haematopoiesis. The del(5q) is also commonly found in other MDS and AML (10–15% of all patients) and is widely believed to mark the location for a tumour suppressor gene(s), the loss of which may affect important processes such as growth control and normal haematopoiesis. However, studies have failed to identify mutations within the retained alleles, or potential submicroscopic homozygous deletions in the commonly deleted region (CDR) suggesting that either haploinsufficiency promotes the 5q syndrome, or that a retained tumour suppressor allele has undergone epigenetic inactivation. To enable progress towards understanding the 5q− syndrome and to provide a model for testing novel therapeutics we generated a mouse model for the human 5q− syndrome. Using Cre-loxP recombination to delete the syntenic regions in the mouse equivalent to the human 5q CDR we have generated a mouse model (deletion mice), with a deletion on chromosome 18 flanked by the Cd74 gene and Nid67 gene (removing an interval harbouring Nid67, Dctn4, Rbm22,Myoz3, Synpo, Ndst1, Rps14, Cd74). The mice display macrocytic anemia, prominent erythroid dysplasia and monolobulated megakaryocytes in the bone marrow, similar to the human disease. Notably, intercrossing of deletion mice with p53 mice resulted in a reversal of the observed deficit in hematopoietic progenitor cell development, thereby demonstrating for the first time the mechanistic involvement of p53 in 5q syndrome. Recent studies have indicated a role for the ribosomal protein Rps14 in the development of 5q syndrome, and taken in combination with our deletion of Rps14, suggest that 5q may be a new member of the expanding group of ribosomopathies. Our work also suggests a prominent role for p53 in promoting the 5q phenotype in mice and we have recently shown p53 up-regulation in bone marrow trephines from del(5q) patients. This novel mouse model will allow the investigation of new and existing therapeutics, specifically targeted against del(5q) abnormalities, and give further mechanistic insight into 5q− syndrome. 2 Morphological differentiation of hypocellular refractory cytopenia of childhood and severe aplastic anemia and clinical outcome I. Baumann, C. Niemeyer, M. Fuhrer, S. Behrendt, V. Campr, J. Csomor, I. Furlan, V. de Haas, G. Kerndrup, R.J. Leguit, P. De Paepe, P. Noellke, S. Schwarz. Department of Pathology, Hospital Boeblingen, Boeblingen, Department of Pediatrics and Adolescent Medicine, University of Freiburg, Freiburg im Breisgau, Department of Hematology and Oncology, Ludwig-Maximilians-University Munich, Munich, Germany; Department of Pathology, University Hospital Motol, Prague, Czech Republic; Department of Pathology, Semmelweis University, Budapest, Hungary; Dutch Childhood Oncology Group, The Hague, The Netherlands; Department of Pathology, Vejle Hospital, Vejle, Denmark; Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands; Department of Pathology, Ghent University Hospital, Ghent, Belgium; Department of Pathology, University Medical Center Erlangen, Erlangen, Germany

We retrospectively re-classified according to WHO criteria our series of consecutive 650 MDS cases, in order to validate the prognostic role of this classification in a unicentric series of patients. Based on FAB criteria, 196 patients had been diagnosed as RA, 41 as RARS, 242 as RAEB, 74 as RAEB-t and 83 as CMML. The new WHO classification could be applied to 479 of the 650 patients; of 196 FAB-RA only 76 were classified as “pure” RA, with only anemia and erythroid dysplasia, whereas 87 patients were re-evaluated as RCMD, for the presence of peripheral cytopenias and dysplasia in ≥ 10% of 2 cell lines. Twenty-nine patients had cytopenias but with only unilineage dysplasia in granulocytes or megakaryocytes, and were re-interpreted as MDS, unclassified (MDS-U). Of 41 FAB-RARS, 32 maintained their diagnosis, whereas 9 patients were classified as RCMD-RS, based on criteria listed above. One-hundred-one patients were identified as RAEB-1, for the presence of 5–9% blasts in bone marrow, and < 5% peripheral blasts, whereas 141 patients were classified as RAEB-2 for the evidence of 10–19% and of 5–19% blasts in bone marrow and peripheral blood respectively. We tested the WHO classification in univariate analysis with respect to several clinical features at presentation. Significant correlations were found as to hemorrhagic symptoms in RCMD and RAEB-2 patients (p=0.014), acute transformation, with higher frequency in RCMD and RAEB-2 categories (p=0.002), transfusion dependence, with higher requirement in RAEB-2 (107/141 RAEB-2 vs 59/101 RAEB-1) and in RCMD (63/87 patients) categories; also the application of different scoring prognostic systems, such as the Bournemouth and Spanish, appeared to possess stastical significance (p=0.001 and p=0.002, respectively). No differences were found as to sex, age, infection occurrence and cytogenetic abnormalities among various subgroups. Kaplan-Meier survival test showed high significance, with median overall survival ranging from 55.8 months for pure RA to 22.6 months for RAEB-2 category (p=0.00001). In conclusion, the retrospective application of WHO classification to our series of MDS patients clearly identified prognostic correlations in various disease subtypes. Categorization of MDS entities according to WHO criteria may distinguish parameters of prognostic importance in large groups of patients with morphological similar features.

We attempted to overcome a limitation of WPSS at the time of diagnosis by replacement of transfusion dependency with initial Hb level. Hb < 80 g/l was scored 1, Hb ≥ 80 g/l was scored 0. This adjustment was tested in a retrospective analysis of 107 non-transplanted patients with early MDS without of excess of blasts diagnosed between 1980 and 2004. Kaplan Meier estimates for 5 years survival were 58%, 57%, 36% and 12% for patients with very low, low, intermediate and high risk scored at the time of diagnosis according to modified WPSS. A significant difference in overall survival was present between patients with score < 2 and ≥ 2 and in leukemia free survival between patients with score < 1 and ≥1 (P=0.02). An analysis of individual WPSS parameters revealed a significant difference in median survival between patients receiving ≤ 2 units and those receiving > 2 units of RBC transfusions/month (46.1 vs. 26.9 months, respectively, P=0.03), between patients with RA+RARS+5q-syndrome and those with RCMD+RCMD-RS (45.4 vs. 27.8 months, respectively, P=0.02) and between patients with good karyotype and intermediate or poor karyotype according to IPSS (48.0 vs. 22.0 months, respectively, P=0.02). An impact of transfusion dependency on survival was validated by univariate and multivariate regression analysis of different clinical and laboratory variables. Leukemic progression was the most important parameter affecting survival in both univariate (χ2=47.7, P=0,001) and multivariate (P 2 units of RBC transfusions/month was the only independent variable with adverse impact on survival in patients with unilineage erythroid dysplasia (P=0.02). In patients with multilineage dysplasia only heavy transfusion dependence (> 3 TU RBC/month) in combination with serum ferritin > 2000 μg/l adversely affected survival (P=0.03). In conclusion, modification of WPSS by replacement of transfusion dependency with initial Hb level < 80 g/l retained its prognostic relevance and allowed to identify even at the time of diagnosis a potentially risk subset of early MDS patients with intermediate and high score and limited survival (< 40% at 5 years). Our results also confirmed a significant negative impact of transfusion dependency on survival in at least a subset of patients with early MDS. This subgroup is characterized by unilineage dysplasia limited to erythropoiesis in combination with dependency on > 2 TU of RBC per month. These patients have usually a prolonged survival enabling development of heavy transfusion iron overload and thus represent the main target group for intensive chelation therapy. The study was supported by scientific grant NR 9235-3 from the Czech Ministry of Health.

Introduction: Copper deficiency can rarely present as anemia and leucopenia. Copper and iron are essential trace elements through their functions as co-factors in numerous biochemical reactions. Hephaestin is a transmembrane copper-dependent ferroxidase necessary for effective iron transport from intestinal enterocyte into the circulation. Copper is hence required for the efficient utilization of iron in the mammalian system. Copper enzymes such as superoxide dismutase and cytochrome oxidase have an essential role in red cell development and heme maturation. We present 4 patients of copper deficiency. The first patient presented with anemia and leucopenia and the bone marrow showed increased vacuolated erythroblasts with and erythroid dysplasia. This patient had a bone marrow picture resembling erythroleukemia. However, as this patient had undergone a gastric bypass, further studies in the form of vitamin B12, red cell folate and copper were performed. Patient was found to have low copper and low ceruloplasmin levels, with normal B12 and red cell folate levels. Three patients had anemia and neutropenia and showed dysplasia in erythroid lineage, ring sideroblasts, and abnormal accumulation of iron granules in plasma cells. In all four cases, repletion of copper resulted in normalization of peripheral counts and bone marrow showed normal trilineage hematopoiesis. Discussion: Copper deficiency is known to present with microcytic anemia. However we report three cases in which patients presented as normocytic anemia and neutropenia.Bone marrow findings in addition to dysplasia, also showed abnormal iron granules in plasma cells as well as ring sideroblasts. Presence of ringed sideroblasts in myelodysplasia is associated with numerous point mutations in mitochondrial DNA. Mutations identified to date involve among others the cytochrome oxidase subunits I and II. Cytochrome c oxidase is involved in oxidizing FE++ to Fe+++ and incorporation into heme. Copper is a component of cytochrome c oxidase and superoxide dismutase and its deficiency leads to low levels of these enzymes. We propose that copper deficiency plays role in causing dysplastic changes in bone marrow and increase in ringed sideroblasts. Any patient presenting with unexplained anemia or leucopenia, with dysplasia and ringed sideroblasts should be evaluated for copper deficiency. Abnormal iron granules in plasma cells have been reported in other conditions like ethanol toxicity, however we report two patients with copper deficiency who were found to have iron granules in plasma cells. We consider copper an important component of the work-up of patients thought to have myelodysplasia especially if they have undergone a gastric bypass and have normal cytogenetics which is not suggestive of clonal hematopoiesis. Hematological abnormalities in patients with copper deficiency patient 1 anemia neutropenia normocytic patient 2 anemia neutropenia normocytic patient 3 anemia neutropenia microcytic patient 4 anemia neutropenia normocytic bone marrow findings in patients with copper deficiency erythroids myeloid ring sideroblasts iron granules in plama cells patient 1 dysplastic hypoplasia none abnormal iron granules in plama cells patient 2 dysplastic dysplasia 15% abnormal iron granules in plasma cells patient 3 dysplastic hypoplasia none none patient 4 dysplastic dysplasia none none

Ribavirin is a synthetic nucleoside analogue given with pegylated interferon alfa-2a for the treatment of hepatitis C. Hemolytic anemia has been reported in 10–15% of patients, usually within 1–2 weeks of the initiation of therapy. The pathogenesis of this phenomenon is unclear. Pegylated interferon alfa-2a has been associated with pancytopenia. We report two cases of ribavirin-induced anemia that are not due solely to hemolysis. Case A is a 56 year old African-American man, status post liver transplant in July 2005 for hepatitis C. He had a recurrence in February 2006, and was placed on ribavirin 1200 mg daily and pegylated interferon 1.8 micrograms weekly. Within 1 month, his hemoglobin dropped to 8.1 g/dL, his MCV was 107mm3 and his reticulocyte count was 57K per mm3, while maintaining a normal white blood cell and platelet count. His Coombs test was negative. There was no response to the administration of erythropoietin. Bone marrow aspirate and biopsy showed marked erythroid dysplasia - including multi-nucleated precursors and megaloblastosis. There was no morphological evidence of myeloid or megakaryocytic dysplasia. Cytogenetic studies revealed a normal male karyotype (46XY). Case B is a 58 year old African-American man with hepatitis C. In January 2006 he was started on ribavirin 1200 mg daily, in combination with pegylated interferon. Eight weeks later, he was found to have a hemoglobin of 8.1 g/dl - decreased from 12.2 g/dL. The MCV was 104.4 mm3, and the reticulocyte count was 56K per mm3. His Coombs test was negative. Bone marrow biopsy performed at that time showed similar findings to Case A. Ribavirin and pegylated interferon were discontinued; a repeat bone marrow after six weeks showed only minimal dyserythropoiesis. He is currently maintaining his hemoglobin between 12 and 13 g/dL, with no transfusion or erythropoietin support. There are no reported associations between ribavirin and erythroid dysplasia in humans. Ribavirin has been shown to have cytotoxic effects on bone marrows in animal models. In one study, ribavirin was given intraperitoneally to rats in doses ranging from 10 to 200 mg/kg 1. Bone marrow samples obtained at 24 and 48 hours demonstrated micronuclei in erythroid precursors, with recovery at 72 hours. Rhesus monkeys given ribavirin at doses of 30 or 100 mg/kg/day intramuscularly for 10 days developed a normocytic, normochromic anemia, which was severe in the high-dose group 2. The bone marrows demonstrated a marked decrease in late erythroid precursors, while the early precursors remained unchanged. There was also vacuolization of the erythroid precursors as well as erythrophagocytosis. No changes were noted in the myeloid precursor cells, and the megakaryocytes appeared increased. After discontinuation of treatment the hemoglobin returned to normal, demonstrating a reversible effect. The bone marrows of the patients demonstrate ineffective erythropoiesis with marked erythroid dysplasia and megaloblastosis. These findings are associated with a decreased proportion of cells in the DNA synthesis of the cell cycle, an increase in the fraction of late precursor cells undergoing apoptosis, and an interruption in mitosis. These findings may be due to ribavirin’s interference in the maturation of erythroid cells. These observations need to be confirmed in a prospective manner in a large cohort of patients; they also suggest the need for a more complete evaluation of anemia in patients treated with ribavirin.

According to evidence- and consensus-based practice guidelines, the majority of patients with myelodysplastic syndrome (MDS) should receive supportive therapy at present. Once anemia is symptomatic, red cell transfusions are the mainstay of therapy. The life expectancy of patients with isolated erythroid dysplasia aged 70 years or older is not significantly shorter than that of the general population; avoiding disease complication is therefore very important in these individuals. Recently we demonstrated that the development of red cell transfusion-dependency significantly affects survival of MDS patients (Malcovati et al. J Clin Oncol 2005; 23: 7594–603). This partly reflects the severity of bone marrow failure; however, development of secondary iron overload significantly worsens survival of transfusion-dependent patients. Considering patients with transfusion needs, iron overload (defined as serum ferritin above 1000 ng mL−1) is developed after receiving a median number of 20–25 RBC units. The effect of iron overload is mainly noticeable among patients with refractory anemia, who have a longer survival and therefore are more prone to develop the adverse effects of iron overload. In contrast, secondary iron overload does not affect the survival of patients with refractory cytopenia, who have a median survival of about 50 months. The negative effect of iron overload is significantly associated with serum ferritin levels, with a 40% increase in hazard for every 500 ng mL-1 of increase in serum ferritin. According to the currently available therapeutic guidelines iron chelation should be considered once a patient has received 20–50 units of red cells, but only in patients for whom long-term transfusion therapy is likely, or when an allogeneic stem cell transplantation is planned. The target serum ferritin concentration should be

Introduction: Bariatric Surgery is promoted as a successful method for weight loss and is performed with increasing frequency. The benefits and the short term risks of the surgical procedure are well understood. However, its long term pathophysiological consequences are still for the most part unknown. They may include metabolic changes that result in malnourishment and malabsorption. Deficiency disorders of iron, B-12 and folate are commonly encountered. However hematological problems from copper deficiency due to malabsorption have not been well studied. Background: Copper and Iron are essential trace elements through their functions as co-factors in numerous biochemical reactions. Hephaestin, a transmembrane copper-dependent ferroxidase, is necessary for effective iron transport from intestinal enterocyte into the circulation. Copper is hence required for iron absorption. Copper enzymes such as superoxide dismutase and cytochrome oxidase have an essential role in red cell development and heme maturation. Patients and Method: We present data on five patients referred for treatment of myelodysplasia whose evaluation revealed copper deficiency and low ceruloplasmin level. All five patients were female and had undergone gastric bypass surgery for weight reduction. The hematological features included pancytopenia, erythroid hyperplasia and dysplasia with increased blasts mimicking erythroleukemia, multilineage dysplasia with ring sideroblasts, bilineage megakaryocytic and erythroid dysplasia. One patient presented with neutropenia and anemia with a normal bone marrow; she was found to have copper deficiency. No cytogenetic abnormalities were observed and the patients had normal B-12 and folate. Copper gluconate was started at 20 mg/day by mouth, with weekly monitoring, till normal levels were reached. Patients were subsequently maintained at 5–10 mg/day with regular monitoring. Upon repletion of copper, all the patients recovered with normalization of the bone marrow and peripheral counts. Discussion: We conclude that Gastric bypass especially Roux-en-Y gives rise to copper deficiency due to malabsorption in the duodenum and can present as pseudomyelodysplasia. We consider measurement of copper and ceruloplasmin levels an important component of the evaluation of patients presenting with myelodysplasia with normal cytogenetics, especially after gastric bypass.

In the last few years, several studies have evaluated the clinical utility of flow cytometry immunophenotyping in myelodysplastic syndromes (MDS). We developed a flow cytometry approach for the evaluation of marrow dysplasia in MDS using discriminant analysis to generate erythroid and myeloid classification functions (Hematol J2004; vol 5 S2: pag S91). However, a major problem in the immunophenotypic analysis of MDS is evaluation of erythroid dysplasia because of the limited availability of specific antibodies (Ab). In order to define new tools, we prospectively analyzed the expression of mitochondrial ferritin (MtF) and H ferritin subunit (HF) in bone marrow cells from 69 consecutive patients with MDS and 40 pathologic controls. Six-parameter, 4-color flow cytometry was performed with an EPIX XL flow cytometer (Beckmann Coulter, Fullerton, CA). Erythroid bone marrow cells were analyzed for the expression of HF using a monoclonal mouse Ab (rH02), expression of MtF using a polyclonal mouse Ab, and expression of Glycophorin A, CD71 and CD105 using monoclonal Abs (Instrumentation Laboratory, Milan, Italy). Data on HF and MtF expression obtained by flow cytometry were compared with those obtained on bone marrow smears by an immunocytochemical method (LSAB kit, Dakopatts, Glostrup, Denmark). Discriminant function analysis was performed to identify erythroid dysplasia, with the aim of distinguishing MDS without ringed sideroblasts (RS), MDS with RS, and controls. A significant correlation was found between immunocytochemically and cytometrically detected HF expression (r=.46, P=.016). Overall, the expression of HF was higher in MDS than in controls (P \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \[Y=\ 12.44573{\ast}(HF_{X-mean})\ {-}\ 2.7698{\ast}(CD71_{X-mean})\ +\ 13.33752{\ast}(CD105_{X-mean})\ {-}\ 4.38426.\] \end{document} A patient will be classified as having erythroid dysplasia if the Y value obtained is greater than zero, or as not having dysplasia if the value is lower than zero. This function was integrated with the immunophenotypic evaluation of MtF (Xmean) in order to differentiate between MDS with and without RS. This function allowed us to classify 96% of MDS patients correctly with no false positive cases. Two MDS patients were incorrectly assigned to the RS subgroup by cytometric evaluation of MtF. Based on these findings, we conclude that flow cytometry analysis of MtF and HF expression is reliable and reproducible. Analysis of the expression of these antigens, together with CD71 and CD105, provide an accurate evaluation of erythroid marrow dysplasia and could be usefully included in the work-up of patients with MDS.

The protein-tyrosine-phosphatase (PTPase) SHP-1 is expressed almost exclusively in hematopoetic cells. Most growth factor receptors rely on the Jak/Stat pathway for intracellular transduction, to drive specific gene expression which are involved in cell proliferation and differentiation. Abnormal signaling in some of these pathways has been linked to hematopoietic malignancies, like the ALL tel-Jak2 gene fusion. In some other cases activation is mediated by kinases not normally associated with Stats, like abl. Recently several authors have linked dysregulation of PTPases, especially SHP-1, to non-Hodgkin lymphoma, familial polycythemia, chronic neutropenia and AML. There are no reports on expression of SHP-1 in myelodysplastic syndromes. Here we hypothesize that SHP-1 downregulation may play a role in the leukemic transformation of myelodysplastic syndromes and may impact survival. Retrospectively, from January 1990 to January 2004, we studied 45 patients (29 men, 16 women; median age 70 yrs) with myelodysplastic syndromes (5 RA, 3 RAS, 3 RCMD, 9 RAEB-I, 11 RAEB-II, 2 5q-, 2 Unclass, 3 AML, 6 SMDS). Bone marrow biopsies were examined from each patient. 5 μm sections were dewaxed, heated for antigen retrieval in sodium citrate buffer, and immunostained by routine methods. Antibodies against SHP-1 and Jak-2p were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and Cell Signaling (Beverly, MA) respectively. SHP-1 and Jak-2p expression were evaluated on bone marrow samples as a percentage of positive cells. A percentage between 33 and 66 was considered normal for SHP-1 expression. Jak-2p positivity was defined as normal, if less or equal to 50 % of cells stained positively. Ranges of normal values were derived from healthy controls. With a median follow-up of 65 months, 13 (28%) showed disease progression, 9 of them towards AML. Median OS and EFS were 15 and 12 months respectively. 25 patients have died, mostly of disease related complications. The U-Mann-Whitney test comparing means between patients that progressed and those that did not was only significant (p