Monoclonal gammopathy-associated pure red cell aplasia.

Pure red cell aplasia (PRCA) is a rare disorder characterized by inhibition of erythroid precursors in the bone marrow and normochromic, normocytic anaemia with reticulocytopenia. Among 51 PRCA patients, we identified 12 (24%) patients having monoclonal gammopathy, monoclonal gammopathy of undetermined significance or smouldering multiple myeloma, with presence of monoclonal protein or abnormal serum free light chains and atypical bone marrow features of clonal plasmacytosis, hypercellularity and fibrosis. Thus far, three patients treated with anti-myeloma based therapeutics have responded with reticulocyte recovery and clinical transfusion independence, suggesting plasma cells play a key role in the pathogenesis of this specific monoclonal gammopathy-associated PRCA.

T-cell immune responses to Wilms tumor 1 protein in myelodysplasia responsive to immunosuppressive therapy.

Clinical observations and laboratory evidence link bone marrow failure in myelodysplastic syndrome (MDS) to a T cell-mediated immune process that is responsive to immunosuppressive treatment (IST) in some patients. Previously, we showed that trisomy 8 MDS patients had clonally expanded CD8(+) T-cell populations that recognized aneuploid hematopoietic progenitor cells (HPC). Furthermore, microarray analyses showed that Wilms tumor 1 (WT1) gene was overexpressed by trisomy 8 hematopoietic progenitor (CD34(+)) cells compared with CD34(+) cells from healthy donors. Here, we show that WT1 mRNA expression is up-regulated in the bone marrow mononuclear cells of MDS patients with trisomy 8 relative to healthy controls and non-trisomy 8 MDS; WT1 protein levels were also significantly elevated. In addition, using a combination of physical and functional assays to detect the presence and reactivity of specific T cells, respectively, we demonstrate that IST-responsive MDS patients exhibit significant CD4(+) and CD8(+) T-cell responses directed against WT1. Finally, WT1-specific CD8(+) T cells were present within expanded T-cell receptor Vß subfamilies and inhibited hematopoiesis when added to autologous patient bone marrow cells in culture. Thus, our results suggest that WT1 is one of the antigens that triggers T cell-mediated myelosuppression in MDS.

Effects of granulocyte-colony-stimulating factor on Monosomy 7 aneuploidy in healthy hematopoietic stem cell and granulocyte donors.

BACKGROUND: Reports of Monosomy 7 in patients receiving granulocyte-colony-stimulating factor (G-CSF) have raised concerns that this cytokine may promote genomic instability. However, there are no studies addressing whether repeated administration of G-CSF produces Monosomy 7 aneuploidy in healthy donors. STUDY DESIGN AND METHODS: We examined Chromosomes 7 and 8 by fluorescent in situ hybridization (FISH) in CD34+ cells from 35 healthy hematopoietic stem cell transplant (HSCT) donors after G-CSF administration for 5 days and by spectral karyotyping analysis (SKY) in four individuals to assess chromosomal integrity. We also studied 38 granulocyte donors who received up to 42 doses of G-CSF and dexamethasone (Dex) using FISH for Chromosomes 7 and 8. RESULTS: We found no abnormalities in Chromosomes 7 and 8 in G-CSF-mobilized CD34+ cells when assessed by FISH or SKY, nor did we detect aneuploidy in G-CSF- and Dex-treated donors. CONCLUSION: G-CSF does not promote clinically detectable Monosomy 7 or Trisomy 8 aneuploidy in HSCT or granulocyte donors. These findings should be reassuring to healthy HSCT and granulocyte donors.

Primitive quiescent CD34+ cells in chronic myeloid leukemia are targeted by in vitro expanded natural killer cells, which are functionally enhanced by bortezomib.

Primitive quiescent CD34(+) chronic myeloid leukemia (CML) cells are more biologically resistant to tyrosine kinase inhibitors than their cycling counterparts; however, graft-versus-leukemia (GVL) effects after allogeneic stem cell transplantation (SCT) probably eliminate even these quiescent cells in long-term surviving CML transplant recipients. We studied the progeny of CD34(+) cells from CML patients before SCT, which were cultured 4 days in serum-free media with hematopoietic growth factors. BCR-ABL expression was similar in both cycling and quiescent noncycling CD34(+) populations. Quiescent CD34(+) cells from CML patients were less susceptible than their cycling CD34(+) and CD34(-) counterparts to lysis by natural killer (NK) cells from their HLA-identical sibling donors. Compared with cycling populations, quiescent CD34(+) CML cells had higher surface expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors DR4 and DR5. Bortezomib up-regulated TRAIL receptor expression on quiescent CD34(+) CML cells, and further enhanced their susceptibility to cytotoxicity by in vitro expanded donor NK cells. These results suggest that donor-derived NK cell-mediated GVL effects may be improved by sensitizing residual quiescent CML cells to NK-cell cytotoxicity after SCT. Such treatment, as an adjunct to donor lymphocyte infusions and pharmacologic therapy, may reduce the risk of relapse in CML patients who require treatment by SCT.

Targeting immune dysregulation in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are a heterogeneous group of bone marrow disorders characterized by ineffective hematopoiesis and a tendency to develop leukemia. In some patients, laboratory and clinical evidence supports a role for the immune system in the pathogenesis of early MDS. Many younger patients who respond to immunosuppressive therapy with drugs such as antithymocyte globulin and cyclosporine have clonal expansions of cytotoxic CD8(+) T cells that suppress normal hematopoiesis, as well as expansion of CD4(+) helper T-cell subsets that promote and sustain autoimmunity. Immunosuppressive therapy can produce hematologic responses in some patients and may improve survival and halt leukemic progression. In this report, we describe a 56-year-old woman who presented with fatigue and easy bruising, eventually became pancytopenic, and was diagnosed with MDS. After treatment with a clinical protocol using alemtuzumab, an anti-CD52 antibody, her blood cell counts returned to normal and she has remained in complete remission for more than 2 years of follow-up. In this article, we review the pathobiology of immune dysregulation in MDS and summarize the role of immunosuppressive therapy in MDS.

Graft-versus-host disease: role of inflammation in the development of chromosomal abnormalities of keratinocytes.

Graft-versus-host disease (GVHD) is a major risk factor for secondary malignancy after hematopoietic stem cell transplantation. Squamous cell carcinoma (SCC) of the skin and mucous membranes are especially frequent in this setting where aneuploidy and tetraploidy are associated with aggressive disease. The current study is directed at the mechanism of neoplasia in this setting. Unmanipulated keratinocytes from areas of oral GVHD in 9 patients showed tetraploidy in 10% to 46% of cells when examined by florescein in situ hybridization (FISH). Keratinocytes isolated from biopsy sites of GVHD but not from normal tissue showed even greater numbers of tetraploid cells (mean = 78%, range: 15%-85%; N = 9) after culture. To mimic the inflammatory process in GVHD, allogeneic HLA-mismatched lymphocytes were mixed with normal keratinocytes. After 2 weeks, substantial numbers of aneuploid and tetraploid cells were evident in cultures with lymphocytes and with purified CD8 but not CD4 cells. Telomere length was substantially decreased in the lymphocyte-treated sample. No mutations were present in the p53 gene, although haploinsufficiency for p53 due to the loss of chromosome 17 was common in cells exposed to lymphocytes. These findings suggest that in GVHD, inflammation and repeated cell division correlate with the development of karyotypic abnormalities.

Long-term follow-up of patients with moderate aplastic anemia and pure red cell aplasia treated with daclizumab.

BACKGROUND: Pure red cell aplasia and moderate aplastic anemia are marrow failure states with an immune pathogenesis. Previously, we described short-term improvements in blood counts in two pilot studies treating moderate aplastic anemia (mAA) and pure red cell aplasia (PRCA) patients with daclizumab, a humanized monoclonal antibody to the interleukin-2 receptor; we now report our long-term experience with a larger cohort of patients. DESIGN AND METHODS: After a median follow-up period of 4.8 years, 19 of 45 (42%) evaluable mAA patients and 10 of 26 (38%) patients with PRCA responded by three months and 2 additional mAA patients responded by six months following administration of the drug. RESULTS: Seven of 28 (25%) mAA patients achieved long-term packed red blood cell PRBC transfusion independence, and all PRCA responders achieved long-term transfusion PRBC transfusion independence. CONCLUSIONS: Red cell transfusion-independence prior to treatment in mAA patients predicted response. The only significant adverse treatment-related events were transient rashes and arthralgias. Daclizumab is safe and effective, and produces lengthy remissions in patients with PRCA and mAA.

Association of telomere length of peripheral blood leukocytes with hematopoietic relapse, malignant transformation, and survival in severe aplastic anemia.

CONTEXT: Critically short telomeres produce apoptosis, cell senescence, and chromosomal instability in tissue culture and animal models. Variations in telomere length have been reported in severe aplastic anemia but their clinical significance is unknown. OBJECTIVE: To investigate the relationship between telomere length and clinical outcomes in severe aplastic anemia. DESIGN, SETTING, AND PATIENTS: Single institution analysis of 183 patients with severe aplastic anemia who were treated in sequential prospective protocols at the National Institutes of Health from 2000 to 2008. The pretreatment leukocyte age-adjusted telomere length of patients with severe aplastic anemia consecutively enrolled in immunosuppression protocols with antithymocyte globulin plus cyclosporine for correlation with clinical outcomes were analyzed. MAIN OUTCOME MEASURES: Hematologic response, relapse, clonal evolution, and survival. RESULTS: There was no relationship between hematologic response and telomere length with response rates of 56.5% of 46 patients in the first, 54.3% of 46 in the second, 60% of 45 in the third, and 56.5% of 46 in the fourth quartiles. Multivariate analysis demonstrated that telomere length was associated with relapse, clonal evolution, and mortality. Evaluated as a continuous variable, telomere length inversely correlated with the probability of hematologic relapse (hazard ratio [HR], 0.16; 95% confidence interval [CI], 0.03-0.69; P = .01). The probability of clonal evolution was higher in patients in the first quartile (24.5%; 95% CI, 8.7%-37.5%) than in quartiles 2 through 4 (8.4%; 95% CI, 3.2%-13.3%; P = .009), and evolution to monosomy 7 or complex cytogenetics was more common in the first quartile (18.8%; 95% CI, 3.5%-31.6%) [corrected] than in quartiles 2 through 4 (4.5%; 95% CI, 0.5%-8.2%; P = .002) [corrected]. Survival between these 2 groups differed, with 66% (95% CI, 52.9%-82.5%) surviving 6 years in the first quartile compared with 83.8% (95% CI, 77.3%-90.9%) in quartiles 2 through 4 (P = .008). CONCLUSION: In a cohort of patients with severe aplastic anemia receiving immunosuppressive therapy, telomere length was unrelated to response but was associated with risk of relapse, clonal evolution, and overall survival.

Treatment of severe aplastic anemia with a combination of horse antithymocyte globulin and cyclosporine, with or without sirolimus: a prospective randomized study.

BACKGROUND: We hypothesized that the addition of sirolimus to standard horse antithymocyte globulin (h-ATG) and cyclosporine (CsA) would improve response rates in severe aplastic anemia, due to its complementary and synergistic properties to cyclosporine A. DESIGN AND METHODS: To test this hypothesis, we conducted a prospective randomized study comparing hATG/CsA/sirolimus to standard h-ATG/CsA. A total of 77 patients were treated from June 2003 to November 2005; 35 received h-ATG/CsA/sirolimus and 42 h-ATG/CsA. The two groups were well matched demographically and in blood counts prior to therapy. The primary end-point was hematologic response rate at 3 months, defined as no longer meeting the criteria for severe aplastic anemia. The study was powered to show a superior hematologic response rate of h-ATG/CsA/sirolimus compared to standard h-ATG/CsA. RESULTS: The overall response rate at 3 months was 37% for h-ATG/CsA/sirolimus and 50% for h-ATG/CsA and at 6 months 51% for h-ATG/CsA/sirolimus and 62% for h-ATG/CsA. After a planned interim analysis of 30 evaluable patients in each arm, accrual to the h-ATG/CsA/sirolimus arm was closed, as the conditional power for rejecting the null hypothesis was less than 1%. The rate of relapse, clonal evolution, and survival (secondary outcomes) did not differ significantly between patients treated with the two different regimens. CONCLUSIONS: Despite a theoretical rationale for its use, sirolimus did not improve the response rate in patients with severe aplastic anemia when compared to standard h-ATG/CsA.

Increased soluble urokinase plasminogen activator receptor (suPAR) is associated with thrombosis and inhibition of plasmin generation in paroxysmal nocturnal hemoglobinuria (PNH) patients.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired genetic disorder of the bone marrow that produces intravascular hemolysis, proclivity to venous thrombosis, and hematopoietic failure. Mutation in the PIG-A gene of a hematopoietic stem cell abrogates synthesis of glycosylphosphoinositol (GPI) anchors and expression of all GPI-anchored proteins on the surface of progeny erythrocytes, leukocytes, and platelets. Urokinase plasminogen activator receptor (uPAR), a GPI-linked protein expressed on neutrophils, mediates endogenous thrombolysis through a urokinase-dependent mechanism. Here we show that membrane GPI-anchored uPAR is decreased or absent on granulocytes and platelets of patients with PNH, while soluble uPAR (suPAR) levels are increased in patients' plasma. Serum suPAR concentrations correlated with the number of GPI-negative neutrophils and were highest in patients who later develop thrombosis. In vitro, suPAR is released from PNH hematopoietic cells and from platelets upon activation, suggesting that these cells are the probable source of plasma suPAR in the absence of GPI anchor synthesis and trafficking of uPAR to the cell membrane. In vitro, the addition of recombinant suPAR results in a dose-dependent decrease in the activity of single-chain urokinase. We hypothesized that suPAR, prevents the interaction of urokinase with membrane-anchored uPAR on residual normal cells.

The role of the immune system in myelodysplasia: implications for therapy.

Patients with myelodysplastic syndrome (MDS) have intrinsic, usually acquired genetic defects in their hematopoietic stem cells, but some others exhibit T-cell-mediated inhibition of hematopoiesis and good responses to immunosuppression. In these cases, MDS shares a similar pathophysiology with aplastic anemia (AA). Here, we review the evidence supporting a role of the immune system in the pathophysiology of MDS and the results of clinical trials of immunosuppressive agents.

Successful use of balloon ablation to treat menorrhagia complicating aplastic anemia.

BACKGROUND: Aplastic anemia (AA) complicated by menorrhagia is treated with transfusion and hormonal therapy. When bleeding is life-threatening, balloon endometrial ablation can safely be used to treat menorrhagia in selected patients. CASE: A 56-year-old postmenopausal woman was diagnosed with AA after several weeks of menorrhagia and pancytopenia. She became heavily alloimmunized after extensive platelet transfusion. During treatment with antithymocyte globulin, vaginal bleeding increased and the platelet count fell to 1,000/microl on supportive measures. After bleeding stopped with use of intravenous Premarin, she was examined in the operating room. There, a clot was removed and appeared to be a uterine caste; hemostasis continued. Transvaginal ultrasound revealed a normal endometrial contour and thin endometrium; endometrial histology was benign. After she completed antithymocyte globulin and her platelet count could be maintained over 30,000/microl with matched platelets, endometrial ablation was performed without any complications. CONCLUSION: Thermal balloon endometrial ablation is an effective alternative to hysterectomy for women with persistent menorrhagia and AA when supportive measures fail. Prior to endometrial ablation, evaluation should ensure normal endometrial contour and histology, and that sufficient blood products are available to maintain platelet counts above 30,000/microl during the healing process.

Telomere length in paroxysmal nocturnal hemoglobinuria correlates with clone size.

OBJECTIVE: To study if telomere length can be used as a surrogate marker for the mitotic history in normal and affected hematopoietic cells from patients with paroxysmal nocturnal hemoglobinuria (PNH). METHODS: The telomere length was measured by automated multicolor flow fluorescence in situ hybridization in glycosyl-phosphatidyl-inositol anchored protein (GPI)-negative and GPI-positive peripheral blood leukocytes. Eleven patients were studied, two with predominantly hemolytic PNH and nine with PNH associated with marrow failure. RESULTS: Telomere length in GPI-negative cells was significantly shorter than in GPI-positive cells of the same patient (p < 0.01, n = 11). The difference in telomere length (telomere length in GPI-positive minus telomere length in GPI-negative cells) correlated with the percentage of GPI-negative white blood cells. CONCLUSION: Our results support the hypothesis that telomere length is correlated to the replicative history of GPI-positive and GPI-negative cells and warrant further studies of telomere length in relation to disease progression in PNH.

Brief communication: Successful treatment of pure red-cell aplasia with an anti-interleukin-2 receptor antibody (daclizumab).

BACKGROUND: Pure red-cell aplasia (PRCA) is a rare hematologic disease characterized by anemia, reticulocytopenia, and absence of bone marrow erythroid precursors. Most patients respond to some form of immunosuppressive treatment, but few prospective clinical trials have been performed. OBJECTIVE: To examine the efficacy of a humanized monoclonal antibody to the interleukin-2 receptor (daclizumab) for treating PRCA. DESIGN: Pilot study. SETTING: Federal government research hospital. PATIENTS: 15 patients with idiopathic PRCA. INTERVENTION: Daclizumab, 1 mg/kg of body weight, every 2 weeks for a total of 5 infusions. Pure red-cell aplasia was defined as transfusion-dependent anemia with a reticulocyte count of 60 x 10(9) cells/L or less and bone marrow showing absent or diminished erythroid precursors. MEASUREMENTS: Response to therapy was assessed by transfusion independence, increments in reticulocyte count, and nontransfused hemoglobin. RESULTS: Daclizumab had little toxicity. Of the 15 patients, 6 patients (40%) responded to treatment. All responders became transfusion-independent and achieved normal or near-normal hemoglobin values and normal reticulocyte counts. LIMITATIONS: The study was unblinded, and the number of patients was too small to assess drug safety reliably. CONCLUSIONS: Daclizumab seems safe. Its efficacy in this pilot protocol suggests that expanded study in PRCA and other bone marrow failure syndromes is warranted.

Hematopoietic-specific microRNA expression in human cells.

We examined expression profiles of hematopoietic tissue-specific microRNAs (miRNAs; miR-142, miR-155, miR-181 and miR-223) in 17 commercially available malignant hematopoietic cell lines and compared to those in highly purified normal human B, T, monocytic and granulocytic lineages. Although malignant cell lines examined showed miRNA expression patterns similar to normal human hematopoietic lineages, the levels of miRNA expression among cell lines and normal cell lineages were considerably different, indicating the significance of miRNAs in human hematopoietic diseases. Further our results showed differences in miRNA expression between mouse and human hematopoietic cells, suggesting important regulatory roles of miRNAs in human hematopoiesis and oncogenesis.

Hematologic complications of HIV infection.

Treatment of HIV-infected patients with highly active antiretroviral therapy (HAART) has altered the natural history of human immunodeficiency virus (HIV) infection by decreasing the frequency of opportunistic infections and altering the expected frequency of hematologic complications and AIDS-related malignancies. Thrombotic thrombocytopenic purpura and thrombosis resulting from protein S deficiency are relatively rare complications of HIV in the United States in patients taking HAART, but are frequent in the developing world where these drugs are not available. Cytopenia, particularly anemia, are more common and result both from bone marrow failure and peripheral destruction. Hodgkin's and non-Hodgkin's lymphoma are still problematic in patients with advanced disease with high viral loads. This review will examine and discuss the diagnosis and management of the hematologic complications of HIV.

Late presentation of dyskeratosis congenita as apparently acquired aplastic anaemia due to mutations in telomerase RNA.

Aplastic anaemia in adults is usually acquired, but rarely constitutional types of bone marrow failure can occur late in life. We assessed two families with onset of pancytopenia in adults and detected two novel point mutations in the telomerase RNA gene (TERC) in each family. This gene is abnormal in some kindreds with dyskeratosis congenita. Individuals in our families with mutated TERC did not have physical signs of dyskeratosis congenita, and their blood counts were nearly normal, but all had severely shortened telomeres, reduced haemopoietic function, and raised serum erythropoietin and thrombopoietin. Bone marrow failure of variable severity due to dyskeratosis congenita, historically characterised by associated physical anomalies and early pancytopenia, may be present in otherwise phenotypically normal adults, and can masquerade as acquired aplastic anaemia.

Absence of donor-derived keratinocyte stem cells in skin tissues cultured from patients after mobilized peripheral blood hematopoietic stem cell transplantation.

OBJECTIVE: Recent studies suggest that primitive bone marrow-derived cells contribute to regeneration of many tissues, including muscle, endothelium, myocardium, neural tissues, liver, and skin. Conversely, primitive cells resident in muscle and other tissues have been reported to reconstitute hematopoiesis. We investigated the contribution of cells with a primitive hematopoietic phenotype to human epidermal skin formation in recipients of allogeneic mobilized peripheral blood hematopoietic stem cell (HSC) transplantation. PATIENTS AND METHODS: Our study population included female patients who had received granulocyte colony-stimulating factor mobilized peripheral blood HSC transplants from male donors for a variety of benign and malignant hematologic disorders at least 6 months before study entry, with a history of skin graft-vs-host disease. Epidermal skin cells (keratinocytes) obtained from punch biopsies of the skin were cultured under conditions specific for growth and expansion of homogenous populations of keratinocytes from keratinocyte stem cells. After multiple passages, DNA was extracted from cultured cells and evaluated by two different polymerase chain reaction (PCR) method for detection of Y chromosome specific sequences. RESULTS: Neither sensitive PCR-based technique revealed the presence of male donor-derived keratinocyte stem cells in keratinocytes cultured from skin biopsies of female allogeneic transplantation recipients. CONCLUSIONS: We could not confirm the contribution of donor mobilized peripheral blood hematopoietic stem cells to keratinocyte stem cell populations after HSC transplantation. These results cannot explain the presence of donor-derived cells with keratinocyte phenotypic markers in tissue sections of HSC transplant recipients.

The relationship of aplastic anemia and PNH.

Bone marrow failure has been regarded as one of the triad of clinical manifestations of paroxysmal noctumal hemoglobinuria (PNH), and PNH in turn has been described as a late clonal disease evolving in patients recovering from aplastic anemia. Better understanding of the pathophysiology of both diseases and improved tests for cell surface glycosylphosphatidylinositol (GPI)-linked proteins has radically altered this view. Flow cytometry of granulocytes shows evidence of an expanded PNH clone in a large proportion of marrow failure patients at the time of presentation: in our large NIH series, about 1/3 of over 200 aplastic anemia cases and almost 20% of more than 100 myelodysplasia cases. Clonal PNH expansion (rather than bone marrow failure) is strongly linked to the histocompatability antigen HLA.-DR2 in all clinical varieties of the disease, suggesting an immune component to its pathophysiology. An extrinsic mechanism of clonal expansion is also more consistent with knock-out mouse models and culture experiments with primary cells and cell lines, which have failed to demonstrate an intrinsic proliferative advantage for PNH cells. DNA chip analysis of multiple paired normal and PIG-A mutant cell lines and lymphoblastoid cells do not show any consistent differences in levels of gene expression. In aplastic anemia/PNH there is surprisingly limited utilization of the V-beta chain of the T cell receptor, and patients' dominant T cell clones, which are functionally inhibitory of autologous hematopoiesis, use identical CDR3 regions for antigen binding. Phenotypically normal cells from PNH patients proliferate more poorly in culture than do the same patient's PNH cells, and the normal cells are damaged as a result of apoptosis and overexpress Fas. Differences in protein degradation might play a dual role in pathophysiology, as GPI-linked proteins lacking an anchor would be predicted to be processed by the proteasome machinery and displayed in a class I H.A. context, in contrast to the normal pathway of cell surface membrane recycling, lysosomal degradation, and presentation by class II HLA. The strong relationship between a chronic, organ-specific immune destructive process and the expansion of a single mutant stem cell clone remains frustratingly enigmatic but likely to be the result of interesting biologic processes, with mechanisms that potentially can be extended to the role of inflammation in producing premalignant syndromes.