Apoptosis of haemopoietic cells in the marrow of patients with myelodysplastic syndrome (MDS) has been suggested as a mechanism for peripheral cytopenias. We determined the expression of Fas (CD95), ...Fas‐Ligand (Fas‐L) and TNF‐α factors known to be involved in apoptosis, in the marrow of 44 patients with MDS and characterized their functional relevance in in vitro assays of haemopoiesis. Multidimensional flow cytometry revealed phenotypically aberrant blasts as defined by orthogonal light scatter and CD45 expression in the marrow of 24/44 patients. Among those blasts Fas expression was increased on CD34‐positive cells and on cells co‐expressing HLA‐DR. In addition, Fas‐L was expressed on some CD34+ cells of MDS patients but was never detected on CD34+ cells in normal marrow. Fas and Fas‐L mRNAs as well as mRNA for TNF‐α, known to increase Fas expression in normal marrow, were up‐regulated in patients with MDS. TNF‐α protein and sTNF‐R1 levels in marrow plasma were higher in MDS patients than in controls (P < 0.002 and <0.003, respectively). However, results were dependent upon disease category: TNF‐α levels were significantly higher in patients with refractory anaemia (RA) than in patients with RA with excess blasts (RAEB) or RAEB in transformation (RAEB‐T) (P = 0.043). Conversely, the proportion of Fas‐L‐positive cells was lowest in patients with RA (P = 0.037). In marrow cultures, Fas‐Ig, rhuTNFR:Fc or anti‐TNF‐α antibody, by blocking Fas or TNF mediated signals, respectively, significantly increased the numbers of haemopoietic colonies compared to untreated cells (P < 0.001, P < 0.003, P < 0.001, respectively). These results show significant dysregulation in the expression of TNF‐α, Fas and Fas‐L in the marrow from MDS patients. Altered expression of these molecules appears to be of functional relevance in the dysregulation of haemopoiesis in MDS and may be amenable to therapeutic interventions.
This study was designed to determine the safety of a nonmyeloablative regimen in patients with primary immunodeficiency disorders (PID) who had infections, organ dysfunction or other risk factors ...that precluded conventional hematopoietic cell (HC) transplant. Fourteen patients received HLA-matched related (n=6) or unrelated (n=8) HC grafts from marrow (n=8), peripheral blood mononuclear cells (n=5) or umbilical cord blood (n=1), either without conditioning (n=1), or after 200 cGy total body irradiation alone (n=3) or with 90 mg/m2 fludarabine (n=10). All patients were given postgrafting immunosuppression with mycophenolate mofetil and cyclosporine. Mixed (n=5) or full (n=8) donor chimerism was established in 13 patients, and one patient rejected the graft. Eight patients developed acute grade III (n=1) and/or extensive chronic GVHD (n=8). With a median follow-up of 4.9 (range, 0.7-8.1) years, the 3-year overall survival, event-free survival and transplant-related mortality were 62, 62 and 23%, respectively. Correction of immune dysfunction was documented in 8 of 10 patients with stable donor engraftment. These preliminary results indicated that this approach was associated with stable donor engraftment and a low incidence of early mortality and, thus, can be considered for certain high-risk patients with PID. However, there was a risk of GVHD, which is an undesirable outcome for this group of patients.
Patients with chemotherapy-refractory chronic lymphocytic leukemia (CLL) have a short life expectancy. The aim of this study was to analyze the outcome of patients with advanced CLL when treated with ...nonmyeloablative conditioning and hematopoietic cell transplantation (HCT).
Sixty-four patients diagnosed with advanced CLL were treated with nonmyeloablative conditioning (2 Gy total-body irradiation with n = 53 or without n = 11 fludarabine) and HCT from related (n = 44) or unrelated (n = 20) donors. An adapted form of the Charlson comorbidity index was used to assess pretransplantation comorbidities.
Sixty-one of 64 patients had sustained engraftment, whereas three patients rejected their grafts. The incidences of grades 2, 3, and 4 acute and chronic graft-versus-host disease were 39%, 14%, 2%, and 50%, respectively. Three patients who underwent transplantation in complete remission (CR) remained in CR. The overall response rate among 61 patients with measurable disease was 67% (50% CR), whereas 5% had stable disease. All patients with morphologic CR who were tested by polymerase chain reaction (n = 11) achieved negative molecular results, and one of these patients subsequently experienced disease relapse. The 2-year incidence of relapse/progression was 26%, whereas the 2-year relapse and nonrelapse mortalities were 18% and 22%, respectively. Two-year rates of overall and disease-free survivals were 60% and 52%, respectively. Unrelated HCT resulted in higher CR and lower relapse rates than related HCT, suggesting more effective graft-versus-leukemia activity.
CLL is susceptible to graft-versus-leukemia effects, and allogeneic HCT after nonmyeloablative conditioning might prolong median survival for patients with advanced CLL.
Approximately 40% of children with acute myeloid leukemia (AML) who respond to initial therapy subsequently relapse. Multidimensional flow cytometry employing a standardized panel of monoclonal ...antibodies enables the detection of small numbers of occult leukemic cells that persist during therapy using technology adaptable by most clinical laboratories. We performed a prospective, blinded evaluation of bone marrow specimens obtained from 252 pediatric patients with de novo AML to determine whether detection of occult leukemia defined as more than or equal to 0.5% blasts with aberrant surface antigen expression as determined by flow cytometry was predictive of subsequent relapse. Occult leukemia was detected in 41 (16%) of the 252 patients who responded to initial induction therapy. In time-dependent multivariate analyses that controlled for allogeneic marrow transplantation, variable intervals between sample submission, age, sex, white blood cell count at diagnosis, presence of splenomegaly or hepatomegaly, and presence of more than 15% blasts in the marrow after the first course of induction, patients harboring occult leukemia were 4.8 times more likely to relapse (95% confidence interval CI = 2.8 to 8.4,P < .0001) and 3.1 times more likely to die (95% CI; 1.9 to 5.1, P < .0001) than those lacking leukemia detectable by flow cytometry. In this analysis, flow cytometric evidence of leukemia after the initiation of therapy emerged as the most powerful independent prognostic factor associated with poor outcome. Among patients in whom a marrow sample was available for analysis at the end of consolidation therapy, overall survival at 3 years was 41% versus 69% for patients with and without occult leukemia, respectively (P = .0058).
Children with neurofibromatosis type 1 (NF1) carry germline mutations in one allele of the NF1 gene and are predisposed to myeloid malignancies, particularly juvenile myelomonocytic leukemia (JMML). ...Disruption of the remaining NF1 allele can be found in malignant cells. Flow cytometric cell sorting techniques to isolate the malignant cell populations and molecular genetic methods to assay for somatic loss of the normal NF1 allele were used to study an unusual child with NF1 and JMML who subsequently had T-cell lymphoma. The data show that malignant JMML and lymphoma cells share a common loss of genetic material involving the normal NF1gene and approximately 50 Mb of flanking sequence, suggesting that the abnormal T-lymphoid and myeloid populations were derived from a common precursor cell. These data support the hypothesis that JMML can arise in a pluripotent hematopoietic cell.
The low frequency of plasma cells and the lack of specific cell surface markers has been a major obstacle for a detailed characterization of plasma cells in normal human bone marrow. Multiparameter ...flow cytometry enabled the identification of plasma cells in normal bone marrow aspirates. The plasma cells were located in a unique position in the correlation of forward light scattering, orthogonal light scattering, and immunofluorescent-labeled CD38. The identity of the sorted cell populations was confirmed by microscopic examination of Wright's stained slides and slides stained for cytoplasmic immunoglobulin using polyclonal antibodies reactive with light chains; ie, anti-κ fluorescein isothiocyanate and antiλ phycoerythrin (PE). The purity of the sorted plasma cells was greater than 97% (n = 4). The average frequency of plasma cells in normal bone marrow aspirates was low—0.25% of the nucleated cells (n = 7)—but surprisingly consistent between individuals (SD = .05; range 0.14% to 0.30%). A detailed analysis showed two distinct populations of plasma cells: (1) A population relatively smaller by forward light scattering expressed CD22, CD35, and slgE and was identified as early plasma cells (ie, lymphoplasmacytoid), and (2) a population larger by forward light scattering lacked these markers and was identified as mature plasma cells. The antigenic profile of the normal plasma cells was determined in two-color immunofluorescence studies. The expression of cell surface immunoglobulin G (IgG), IgA, IgE, IgD, IgM, and the cell surface antigens CD10, CD11 b, CD13, CD11c, CD14, CD15, CD16, CD19, CD22, CD20, CD33, CD35, CD45, and HLA-DR was determined on the plasma cells. A significant heterogeneity in cell surface antigen expression was observed within the plasma cell population. Unexpectedly, myeloid-specific cell surface antigens such as CD33 and CD13 and the early B-cell antigen identified by CD10 were expressed on a proportion of plasma cells. These observations imply that the association of myeloid and early B-cell markers described in multiple myeloma may not be associated with the neoplasia but is a normal phenomenon.
The mechanism that leads to hemopoietic failure in patients with myelodysplastic syndrome (MDS) is not well understood. There is evidence, however, that regulatory molecules such as tumor necrosis ...factor (TNF)-α, Fas (CD95), and Fas-ligand, which negatively affect hemopoiesis by way of apoptosis are upregulated. Here we analyzed marrow samples from 80 patients with MDS in regard to TNF-α and Fas-ligand levels and a possible correlation with various disease parameters and risk factors. TNF-α levels were elevated in comparison to samples from normal marrow donors, however, no significant correlation with FAB subtype, cytogenetic risk group or score by the International Prognostic Scoring System (IPSS) was observed. However, there was an inverse correlation between the cytogenetic risk category (low, intermediate, high) and levels of soluble Fas-ligand. The major source of TNF-α were mononuclear (non-stromal) cells which appeared to produce TNF-α at maximum levels. Limiting dilution analysis of CD34+ precursor cells showed that individually assayed cells, removed from companion cells that presumably provided negative signals such as TNF-α or Fas-ligand, were able to generate progressively increasing numbers of colonies. Stromal layers derived from MDS marrow did not have an inhibitory effect. In fact, higher colony numbers were obtained from both normal and MDS marrow derived hemopoietic precursors propagated on irradiated stromal layers from MDS marrow than on stromal layers from normal marrow. These results show that substantial numbers of normal hemopoietic precursors persist in MDS marrow. However, differentiation into mature cells is inhibited by negative signals originating from accessory or abnormal hemopoietic precursors in the non-adherent marrow fraction.
We present photometric and spectroscopic observations of SN 2007if, an overluminous (M_V = -20.4), red (B-V = 0.16 at B-band maximum), slow-rising (t_rise = 24 days) type Ia supernova in a very faint ...(M_g = -14.10) host galaxy. A spectrum at 5 days past B-band maximum light is a direct match to the super-Chandrasekhar-mass candidate SN Ia 2003fg, showing Si II and C II at ~9000 km/s. A high signal-to-noise co-addition of the SN spectral time series reveals no Na I D absorption, suggesting negligible reddening in the host galaxy, and the late-time color evolution has the same slope as the Lira relation for normal SNe Ia. The ejecta appear to be well mixed, with no strong maximum in I-band and a diversity of iron-peak lines appearing in near-maximum-light spectra. SN2007 if also displays a plateau in the Si II velocity extending as late as +10 days, which we interpret as evidence for an overdense shell in the SN ejecta. We calculate the bolometric light curve of the SN and use it and the \ion{Si}{2} velocity evolution to constrain the mass of the shell and the underlying SN ejecta, and demonstrate that SN2007 if is strongly inconsistent with a Chandrasekhar-mass scenario. Within the context of a "tamped detonation" model appropriate for double-degenerate mergers, and assuming no host extinction, we estimate the total mass of the system to be 2.4 +/- 0.2 solar masses, with 1.6 +/- 0.1 solar masses of nickel-56 and with 0.3-0.5 solar masses in the form of an envelope of unburned carbon/oxygen. Our modeling demonstrates that the kinematics of shell entrainment provide a more efficient mechanism than incomplete nuclear burning for producing the low velocities typical of super-Chandrasekhar-mass SNeIa.
PDF
