Janis L. Abkowitz

Persistent B19 Parvovirus Infection in Patients Infected with Human Immunodeficiency Virus Type 1 (HIV-1): A Treatable Cause of Anemia in AIDS

OBJECTIVE To determine the role of B19 parvovirus in red cell aplasia of patients infected with human immunodeficiency virus type 1 (HIV-1). DESIGN Uncontrolled clinical trial, with assay of serum, peripheral blood cells, and bone marrow for virus using DNA hybridization and immunocytochemistry techniques; these assays were then correlated with clinical findings, results of immunoassays for antivirus antibodies, and with immunoglobulin (Ig) therapy. SETTING Government medical referral center, and university and private hospitals. PATIENTS Seven patients with pure red cell aplasia and serologic evidence of infection with HIV-1. MEASUREMENTS AND MAIN RESULTS All patients had giant pronormoblasts in the bone marrow (present in transient aplastic crisis caused by acute B19 parvovirus infection). High concentrations of B19 parvovirus were demonstrated in sera, in several cases in samples separated by weeks or months. Viral DNA and capsid protein were present in the bone marrow of three patients studied, and active viral replication was detected by southern analysis. There was no antivirus IgG in capture immunoassay and no or very low levels of antivirus IgM. The patients did not have symptoms of fifth disease, the illness caused by this virus in immunologically normal persons. Six patients were treated with a regimen of intravenous commercial immunoglobulin. In all cases, this therapy resulted in rapid reduction in serum virus concentrations and full recovery of erythropoiesis. Relapses in two cases were predicted by DNA hybridization studies, and these cases were successfully retreated. CONCLUSIONS The B19 parvovirus is a remediable cause of severe chronic anemia in HIV-infected patients. Recognition of and therapy for parvovirus in this population will avoid erythrocyte transfusion and should prevent transmission of the virus to other persons, including immunosuppressed persons and women of child-bearing age.

Identification of a human heme exporter that is essential for erythropoiesis

FLVCR, a member of the major facilitator superfamily of transporter proteins, is the cell surface receptor for feline leukemia virus, subgroup C. Retroviral interference with FLVCR display results in a loss of erythroid progenitors (colony-forming units-erythroid, CFU-E) and severe anemia in cats. In this report, we demonstrate that human FLVCR exports cytoplasmic heme and hypothesize that human FLVCR is required on developing erythroid cells to protect them from heme toxicity. Inhibition of FLVCR in K562 cells decreases heme export, impairs their erythroid maturation and leads to apoptosis. FLVCR is upregulated on CFU-E, indicating that heme export is important in primary cells at this stage. Studies of FLVCR expression in cell lines suggest this exporter also impacts heme trafficking in intestine and liver. To our knowledge, this is the first description of a mammalian heme transporter.

Leukemia of large granular lymphocytes: association with clonal chromosomal abnormalities and autoimmune neutropenia, thrombocytopenia, and hemolytic anemia

Three patients had leukocytosis of large granular lymphocytes and chronic neutropenia. Clonal chromosomal abnormalities (trisomy 8 and trisomy 14) and lymphocytic infiltration of splenic red pulp, hepatic sinusoids, and bone marrow indicated the neoplastic nature of the large granular lymphocytes. Demonstration of a T3+, T8+, HNK-1 + phenotype and low natural killer cell activity that was augmented by interferon treatment showed the leukemic cells to be immature natural killer cells. Multiple autoantibodies were present and included rheumatoid factor and antinuclear, antineutrophil, antiplatelet, and antierythrocyte antibodies, suggesting a defect of B-cell immunoregulation. In addition, in-vitro studies showed impaired suppression of immunoglobulin biosynthesis by abnormal cells from one patient. Antineutrophil antibodies and absence of direct cell-mediated inhibition of granulocyte-macrophage colony formation supported a humoral immune mechanism for the neutropenia. In these patients the syndrome of splenomegaly, multiple autoantibodies with neutropenia, and lymphocytosis of large granular lymphocytes is due to a neoplastic proliferation of immature natural killer cells.

A heme export protein is required for red blood cell differentiation and iron homeostasis

Hemoproteins are critical for the function and integrity of aerobic cells. However, free heme is toxic. Therefore, cells must balance heme synthesis with its use. We previously demonstrated that the feline leukemia virus, subgroup C, receptor (FLVCR) exports cytoplasmic heme. Here, we show that FLVCR-null mice lack definitive erythropoiesis, have craniofacial and limb deformities resembling those of patients with Diamond-Blackfan anemia, and die in midgestation. Mice with FLVCR that is deleted neonatally develop a severe macrocytic anemia with proerythroblast maturation arrest, which suggests that erythroid precursors export excess heme to ensure survival. We further demonstrate that FLVCR mediates heme export from macrophages that ingest senescent red cells and regulates hepatic iron. Thus, the trafficking of heme, and not just elemental iron, facilitates erythropoiesis and systemic iron balance.

Evidence that hematopoiesis may be a stochastic process in vivo.

To study the behavior of hematopoietic stem cells in vivo, hematopoiesis was simulated by assuming that all stem cell decisions (that is, replication, apoptosis, initiation of a differentiation/maturation program) were determined by chance. Predicted outcomes from simulated experiments were compared with data obtained in autologous marrow transplantation studies of glucose 6–phosphate dehydrogenase (G6PD) heterozygous female Safari cats. With this approach, we prove that stochastic differentiation can result in the wide spectrum of discrete outcomes observed in vivo, and that clonal dominance can occur by chance. As the analyses also suggest that the frequency of feline hematopoietic stem cells is only 6 per 107 nucleated marrow cells, and that stem cells do not replicate on average more frequently than once every three weeks, these large–animal data challenge clinical strategies for marrow transplantation and gene therapy.

Germline ETV6 mutations in familial thrombocytopenia and hematologic malignancy

We report germline missense mutations in ETV6 segregating with the dominant transmission of thrombocytopenia and hematologic malignancy in three unrelated kindreds, defining a new hereditary syndrome featuring thrombocytopenia with susceptibility to diverse hematologic neoplasms. Two variants, p.Arg369Gln and p.Arg399Cys, reside in the highly conserved ETS DNA-binding domain. The third variant, p.Pro214Leu, lies within the internal linker domain, which regulates DNA binding. These three amino acid sites correspond to hotspots for recurrent somatic mutation in malignancies. Functional studies show that the mutations abrogate DNA binding, alter subcellular localization, decrease transcriptional repression in a dominant-negative fashion and impair hematopoiesis. These familial genetic studies identify a central role for ETV6 in hematopoiesis and malignant transformation. The identification of germline predisposition to cytopenias and cancer informs the diagnosis and medical management of at-risk individuals.

The replication rate of human hematopoietic stem cells in vivo

Hematopoietic stem cells (HSCs) replicate (self-renew) to create 2 daughter cells with capabilities equivalent to their parent, as well as differentiate, and thus can both maintain and restore blood cell production. Cell labeling with division-sensitive markers and competitive transplantation studies have been used to estimate the replication rate of murine HSCs in vivo. However, these methods are not feasible in humans and surrogate assays are required. In this report, we analyze the changing ratio with age of maternal/paternal X-chromosome phenotypes in blood cells from females and infer that human HSCs replicate on average once every 40 weeks (range, 25-50 weeks). We then confirm this estimate with 2 independent approaches, use the estimate to simulate human hematopoiesis, and show that the simulations accurately reproduce marrow transplantation data. Our simulations also provide evidence that the number of human HSCs increases from birth until adolescence and then plateaus, and that the ratio of contributing to quiescent HSCs in humans significantly differs from mouse. In addition, they suggest that human marrow failure, such as the marrow failure that occurs after umbilical cord blood transplantation and with aplastic anemia, results from insufficient numbers of early progenitor cells, and not the absence of HSCs.

Relation of Clinical Response and Minimal Residual Disease and Their Prognostic Impact on Outcome in Acute Myeloid Leukemia

PURPOSE Both presence of minimal residual disease (MRD) and achievement of complete remission (CR) with incomplete platelet recovery (CRp) rather than CR after induction therapy predict relapse in acute myeloid leukemia (AML). These results suggest a correlation between response (peripheral count recovery) and MRD at the time of morphologic remission. Here we examine this hypothesis and whether MRD and response provide independent prognostic information after accounting for other relevant covariates. PATIENTS AND METHODS We retrospectively analyzed data from 245 adults with AML who achieved CR, CRp, or CR with incomplete blood count recovery (CRi) after induction therapy. Bone marrow samples were collected on or closest to the first date of blood count recovery, and MRD was determined by 10-color multiparameter flow cytometry. RESULTS The 71.0% of patients who achieved CR had MRD less frequently and had lower levels of MRD than the 19.6% of patients achieving CRp and 9.4% achieving CRi. Although pretreatment covariates such as cytogenetics, monosomal karyotype, relapsed or refractory rather than newly diagnosed AML, and FLT3 internal tandem duplication were associated with relapse, their prognostic effect was much lower once MRD and response were taken into account, the univariable statistical effect of which was not materially affected by inclusion of pretreatment covariates. CONCLUSION Our data indicate that post-therapy parameters including MRD status and response are important independent prognostic factors for outcome in patients with AML achieving remission. MRD status and type of response (CR v CRp or CRi) should play important, and perhaps dominant, roles in planning postinduction therapy.

New Insights into the Pathophysiology of Acquired Cytopenias

This review addresses three related bone marrow failure diseases, the study of which has generated important insights in hematopoiesis, red cell biology, and immune-mediated blood cell injury. In Section I, Dr. Young summarizes the current knowledge of acquired aplastic anemia. In most patients, an autoimmune mechanism has been inferred from positive responses to nontransplant therapies and laboratory data. Cytotoxic T cell attack, with production of type I cytokines, leads to hematopoietic stem cell destruction and ultimately pancytopenia; this underlying mechanism is similar to other human disorders of lymphocyte-mediated, tissue-specific organ destruction (diabetes, multiple sclerosis, uveitis, colitis, etc.). The antigen that incites disease is unknown in aplastic anemia as in other autoimmune diseases; post-hepatitis aplasia is an obvious target for virus discovery. Aplastic anemia can be effectively treated by either stem cell transplantation or immunosuppression. Results of recent trials with antilymphocyte globulins and high dose cyclophosphamide are reviewed. Dr. Abkowitz discusses the diagnosis and clinical approach to patients with acquired pure red cell aplasia, both secondary and idiopathic, in Section II. The pathophysiology of various PRCA syndromes including immunologic inhibition of red cell differentiation, viral infection (especially human parvovirus B19), and myelodysplasia are discussed. An animal model of PRCA (secondary to infection with feline leukemia virus [FeLV], subgroup C) is presented. Understanding the mechanisms by which erythropoiesis is impaired provides for insights into the process of normal red cell differentiation, as well as a rational strategy for patient management. Among the acquired cytopenias paroxysmal nocturnal hemoglobinuria (PNH) is relatively rare; however, it can pose formidable management problems. Since its first recognition as a disease, PNH has been correctly classified as a hemolytic anemia; however, the frequent co-existence of other cytopenias has hinted strongly at a more complex pathogenesis. In Section III, Dr. Luzzatto examines recent progress in this area, with special emphasis on the somatic mutations in the PIG-A gene and resulting phenotypes. Animal models of PNH and the association of PNH with bone marrow failure are also reviewed. Expansion of PNH clones must reflect somatic cell selection, probably as part of an autoimmune process. Outstanding issues in treatment are illustrated through clinical cases of PNH. Biologic inferences from PNH may be relevant to our understanding of more common marrow failure syndromes like myelodysplasia.

PARVOVIRUS B19 AS A CAUSE OF ACQUIRED CHRONIC PURE RED CELL APLASIA

Parvovirus B19 infection causes chronic anaemia in immunodeficient individuals by selective suppression of erythropoiesis. The bone marrow morphology is character‐istic of pure red cell aplasia (PRCA). To determine the frequency of B19‐induced PRCA we retrospectively analysed a series of 57 PRCA patients. B19 DNA was present in serum of eight patients (14%) and could be extracted from bone marrow aspirate slides from five of these patients. Recent exposure to the virus was confirmed by the presence of anti‐B19 IgM in sera from four and by the finding of giant pronormoblasts in marrow aspirates from five of the B19 DNA‐positive patients. The sensitivities of anti‐B19 IgM and of giant pronormoblasts were only 50% and 63%, respectively; specificites were 90% and 92%. Unexpectedly, PRCA in two B19 DNA‐positive patients remitted after antilymphocyte globulin or cyclosporin A therapy, suggesting that the clinical course of B19‐induced PRCA may be indistinguishable from other forms of PRCA. As therapy with immunoglobulin is uniformly effective for treatment of B19‐associated anaemia, our data suggest that all patients with acquired PRCA should be evaluated for evidence of B19 infection. B19 DNA analysis is the most reliable method to demonstrate infection.