Marrow transplants from matched unrelated donors for aplastic anaemia using alemtuzumab, fludarabine and cyclophosphamide based conditioning.

Graft failure, regimen-related toxicity and graft-versus-host disease (GVHD) are the critical barriers to unrelated donor transplants for aplastic anaemia (AA). We investigated the use of a novel conditioning regimen consisting of alemtuzumab (humanized CD52 antibody), fludarabine and cyclophosphamide in seven patients with AA, who underwent bone marrow transplant procedure using matched unrelated donors. The aetiology of AA was acquired (n=3), Fanconi's (n=3) and congenital (n=1). Median age was 13 years (range 8-35). All the donors were fully matched for HLA class I and II antigens using high-resolution typing. All the patients engrafted at a median of 18 days (range 13-35). Two patients died of transplant-related complications: one of adenovirus disease and the other developed extensive chronic GVHD of skin followed by cytomegalovirus (CMV) disease. Three patients developed Grade II acute GVHD disease (GVHD); none had Grade III-IV acute GVHD. Of the six evaluable patients, only one developed chronic GVHD. We conclude that this conditioning regimen for unrelated donor transplants for AA is sufficiently immunosuppressive to allow stable engraftment and appears to have a favourable impact on the incidence and severity of GVHD, warranting further investigation.

Autologous recovery following non-myeloablative unrelated donor bone marrow transplantation for severe aplastic anaemia.

We report the outcome of nine unrelated bone marrow transplants performed for acquired severe aplastic anaemia at a single centre. Six patients received transplants from fully matched donors. Three donor/recipient pairs were mismatched, two at a single allele on high resolution typing. Pre-transplant conditioning consisted of cyclophosphamide and in vivo Campath-1 monoclonal antibody. One patient also received total body irradiation (TBI), and another patient with a coexisting paroxysmal nocturnal haemoglobinuria (PNH) clone received additional busulphan. Cyclosporin A was given for 12 months as prophylaxis against graft-versus-host disease (GVHD). Six of nine patients are alive and transfusion independent with a mean follow-up of 24 months (range: 1.5-94). All six patients who received fully matched transplants are alive; the three who received mismatched grafts died. Four long-term survivors developed autologous haematological recovery following rejection of their grafts. Acute GVHD grade II+ occurred in two patients. We highlight the importance of high-resolution HLA typing, including Cw matching in reducing the incidence of graft rejection and GVHD, resulting in improved survival in our patient group. This study also shows that autologous recovery with long-term survival can occur following non-irradiation conditioning regimens.

Outcome of 69 allogeneic stem cell transplantations for Fanconi anemia using HLA-matched unrelated donors: a study on behalf of the European Group for Blood and Marrow Transplantation.

Allogeneic stem cell transplantation is the only treatment that can restore a normal hematopoiesis in Fanconi anemia (FA). In this retrospective multicenter study, we analyzed the results of this approach using HLA-matched unrelated bone marrow donors, and tried to identify covariates predicting the outcome of the transplant. From January 1985 to June 1998, 69 FA patients were transplanted with unrelated HLA-matched donors. Patients' characteristics before and after transplant were provided by the European group blood and marrow transplant registry and were analyzed in collaboration with the European Fanconi Anemia Registry. The 3-year probability of survival was 33%. Extensive malformations, a positive recipient cytomegalovirus serology, the use of androgens before transplant, and female donors were associated with a worse outcome. Primary graft failures were observed more frequently when female donors were used, mainly because the grafts contained lower nucleated cell doses per kilogram of recipient body weight compared with grafts coming from male donors. The probability of grade III-IV acute graft-versus-host disease (GVHD) was 34%. Elevated serum alanine/aspartate transaminases before transplantation; limb, urogenital tract, or nephrologic malformations; and non-T-cell-depleted grafts were predictors of severe acute GVHD. This study shows the dramatic impact of preexisting congenital malformations on the outcome of FA patients transplanted with HLA-matched unrelated donors. If the use of T-cell depletion has led to a dramatic reduction of acute GVHD incidence, no significant outcome improvement was observed with this approach, mainly because of an increased risk of graft failure. (Blood. 2000;95:422-429)

Progressive telomere shortening in aplastic anemia.

Improved survival in aplastic anemia (AA) has shown a high incidence of late clonal marrow disorders. To investigate whether accelerated senescence of hematopoietic stem cells might underlie the pathophysiology of myelodysplasia (MDS) or paroxysmal nocturnal hemoglobinuria (PNH) occurring as a late complication of AA, we studied mean telomere length (TRF) in peripheral blood leukocytes from 79 patients with AA, Fanconi anemia, or PNH in comparison with normal controls. TRF lengths in the patient group were significantly shorter for age than normals (P < .0001). Telomere shortening was apparent in both granulocyte and mononuclear cell fractions, suggesting loss at the level of the hematopoietic stem cell. In patients with acquired AA with persistent cytopenias (n = 40), there was significant correlation between telomere loss and disease duration (r = -.685; P < .0001), equivalent to progressive telomere erosion at 216 bp/yr, in addition to the normal age-related loss. In patients who had achieved normal full blood counts (n = 20), the rate of telomere loss had apparently stabilised. There was no apparent association between telomere loss and secondary PNH (n = 13). However, of the 5 patients in the study with TRF less than 5.0 kb, 3 had acquired cytogenetic abnormalities, suggesting that telomere erosion may be relevant to the pathogenesis of MDS in aplastic anemia.

Thy-1 expression on blast cells from adult patients with acute myeloid leukemia.

Bone marrow and peripheral blood from 28 adult patients with acute myeloid leukemia (AML) were analyzed for the surface expression of the Thy-1 antigen by dual-colour flow cytometry. The Thy-1 antigen was expressed on greater than 5% of cells from seven patients with the proportions of Thy-1 positive cells ranging from 8.1% to 85.0%. The CD34+ Thy-1+ phenotype was present in all seven cases. The expression of Thy-1 on leukemia cells from patients with AML may need to be considered in the development of methods of normal stem cell isolation from these patients.

Allogeneic murine melanoma cell vaccine: a model for the development of human allogeneic cancer vaccine.

In an attempt to induce an immune response against tumour antigens, several groups are transfecting cytokine and other genes into autologous tumour cells which are given to the patient as a vaccine. This process is labour-intensive, time-consuming and expensive. Allogeneic cells would offer a more convenient vehicle for the delivery of cytokines and other molecules. However, current dogma suggests that MHC-matched cells are a prerequisite for an effective immune response. Using murine melanoma models we compared allogeneic and autologous vaccination and showed that the survival of C56BL/6 mice (H-2b) was prolonged with some degree of protection achieved against an autologous B15-F10 (H-2b) cell challenge when the mice were vaccinated with allogeneic K1735-M2 (H-2k) cells but not when immunized with autologous B16-F10 cells. Both vaccination with live and irradiated allogeneic cells induced an anti-tumour effect using only one immunization and no boost or adjuvant. Protection was not observed after vaccination with another melanoma (S91; H-2d) or with a carcinoma (A9HT; H-2k). Allogeneic vaccination promoted a cytotoxic cellular response against both the allogeneic and the syngeneic melanomas. This allogeneic vaccination model will be useful for studying the underlying mechanisms of protection, in both pre- and post-challenge settings, as well as for developing whole cell vaccination systems using genetically modified allogeneic tumour cells.

In vitro progenitor analysis in a Diamond Blackfan anaemia patient who responded once but not twice to interleukin-3 therapy, using short-term and long-term cultures and c-kit analysis.

Interleukin-3 (IL-3) therapy as a treatment for Diamond Blackfan anaemia (DBA) patients has been largely disappointing despite early hope it would be suitable for stimulating arrested erythropoiesis. Initial hope came from in vitro discoveries that IL-3 (+EPO) generated well-haemoglobinized BFU-E colonies in some patients, but was soon tempered by the realization that in vitro and in vivo IL-3 response did not, in the majority of cases, correlate. Nevertheless in vitro testing has been the main focus in analysing the abnormality in the stem and progenitor cell compartment in DBA. Here we report in vitro analysis of a DBA patient who responded once to IL-3 therapy, but not a second time following relapse, using short-term culture, long-term culture and c-kit analysis. Progenitor numbers before and after the first therapy were in the high normal range, but after relapse were much reduced below normal levels. Long-term cultures suggested some arrested progenitors had been reactivated into normal cycle by the first therapy, but may not have been replaced by more immature progenitors. c-kit analysis revealed increased expression in all tested cell populations. These results imply that the first IL-3 therapy reactivated some erythroid progenitors but left the progenitor pool depleted when more immature cells remained arrested.

Aplastic anemia: evidence for dysfunctional bone marrow progenitor cells and the corrective effect of granulocyte colony-stimulating factor in vitro.

We investigated the effects of granulocyte-macrophage colony-stimulating factor, interleukin-3, stem cell factor, interleukin-6, and granulocyte colony-stimulating factor (G-CSF) alone, and in combination, on the clonogenic potential of normal and aplastic anemia (AA) bone marrow mononuclear cells (BMMC and CD34+ cells. AA BMMC consistently produced a significantly lower absolute number of colonies than normal, but, when account was taken of the reduced proportion of CD34+ cells in AA BM, there was no significant difference in terms of cloning efficiency (CE). However, when removed from the influence of accessory cells, the CE of AA CD34+ cells decreased significantly more than normal, indicating a defect in their function, either in terms of dependence on accessory cell-derived factors or susceptibility to cell damage when sorted. Of the factors studied, G-CSF had the most significant effect on the response of CD34+ cells from both groups when removed from their accessory cells. This was particularly true for AA CD34+ cells, whose response to cytokine stimuli containing G-CSF enabled them to match the response of normal CD34+ cells.

Diamond Blackfan anaemia: differential pattern of in vitro progenitor response to macrophage inflammatory protein 1-alpha.

The congenital disorder of erythropoiesis Diamond Blackfan anaemia (DBA) exhibits a defect in the stem/progenitor cell compartment, located at the erythroid progenitor level (CFU-GEMM, BFU-E, CFU-E). Treatment of DBA with interleukin-3 (IL-3) has had limited effect, despite in vitro studies suggesting that progenitor cells were capable of responding to IL-3. Whether IL-3 is not reaching the appropriate defective target cell, the cells cannot respond, or the marrow humoral inhibitory system is overriding it, is not clear. To investigate humoral inhibitory activities we examined the response of 15 DBA bone marrows in vitro to the inhibitory chemokine macrophage inflammatory protein 1-alpha (MIP1-alpha) in the presence of the stimulatory cytokines erythropoietin, granulocyte-macrophage colony-stimulating factor, IL-3, and stem cell factor. In vitro data agreed with our previous work showing that our patients formed three statistically different groups in response to stimulatory cytokines (type I DBA erythroid colony numbers approximately normal > type II DBA > type III DBA). Addition of MIP1-alpha to cultures caused average erythroid and myeloid suppression, which sequentially increased with DBA type (type I inhibition < type II < type III). The differential level of inhibition shown by MIP1-alpha in these DBA patients lends further evidence for the presence of distinct subgroups in this disorder.

The effect of human flt-3 ligand on committed progenitor cell production from normal, aplastic anaemia and Diamond-Blackfan anaemia bone marrow.

We investigated the effect of the human ligand for flt-3 (FL) on the committed progenitor colony formation of normal bone marrow (BM) (n = 9) and BM from four aplastic anaemia (AA) and three Diamond-Blackfan anaemia (DBA) patients. Methylcellulose committed progenitor cell assays were carried out using FL alone and in combinations with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3) and c-kit ligand (KL). FL alone had a limited, though significant, effect on the production of granulocyte-macrophage colony-forming unit (CFU-GM) colonies from normal BM and showed an additive effect with IL-3 and GM-CSF separately, but not in combination. FL did not increase the stimulation of KL and did not have an effect on the production of erythroid progenitor colonies. FL had no effect on the AA and DBA BMs studied.

IL-3 is produced by normal stroma in long-term bone marrow cultures.

Interleukin-3 (IL-3) has been shown to have significant effects on haemopoiesis in vitro, but early investigations of normal human long-term bone marrow cultures (LTBMC) have failed to demonstrate IL-3 production by stromal cells, either by Northern blotting for mRNA, or assaying for bioactivity in culture supernatants. One recent report, using reverse transcription-polymerase chain reaction (RT-PCR), demonstrated IL-3 expression in only one of eight cultures. We have developed a sensitive bioassay for the detection of IL-3 production from normal stroma in LTBMC. LTBMC were grown until confluent, irradiated, and stroma harvested by trypsinization to yield single-cell suspensions. These cells were then cocultured with target bone marrow mononuclear cells (BMMC), or CD34+ cells in clonogenic assays, either in the presence or absence of anti-IL-3 neutralizing antibodies. We have demonstrated IL-3 production in 32/34 cases. In addition, by separating stroma from target cells using cell culture inserts, we have shown that direct stroma:stem cell contact is not necessary for colony growth, suggesting that IL-3 diffuses into the supernatant. However, when supernatants from LTBMC were assayed by enzyme-linked immunoassay (ELISA), no IL-3 was detected. This suggests that IL-3 is probably produced at low levels and has a short-range interaction. Stroma production of IL-3 was confirmed by the detection by RT-PCR of IL-3 mRNA in 3/3 cases. The simultaneous detection of CD2 mRNA demonstrated that T cells are part of the bone marrow stroma. It is therefore possible and probably likely that these cells are the source of IL-3.

Diamond-Blackfan anaemia: three patterns of in vitro response to haemopoietic growth factors.

Culture of bone marrow from patients with Diamond-Blackfan anaemia (DBA) has previously shown a variable progenitor response to growth factor stimulation. An extensive standardized study has now been undertaken to investigate the presence of distinct sub-groups in this disorder. In vitro response of bone marrow progenitors to recombinant human growth factors, including stem cell factor, was examined in 18 DBA patients and five normal donors, assessing BFU-E, CFU-GM and CFU-GEMM development. In 16 of the DBA patients a synergistic response to combinations of growth factors was observed with optimal growth in cultures containing erythropoietin, interleukin-3 and stem cell factor. Growth factor induced erythroid response formed three distinct groups, based on BFU-E numbers: type I (mean age 4.87 years) showed > 70% normal erythroid response; type II (mean age 13.87 years) showed < 70% normal; and type III (mean age 15.29 years) < 5% normal. CFU-GM response also followed the trigrouping. The results suggest more than one pathogenic mechanism for the erythroid failure in DBA, indicating DBA may be composed of more than one distinct disorder, and further suggest the defect in DBA may not be confined to the erythroid series.

Genetic mapping of the FACC gene and linkage analysis in Fanconi anaemia families.

Fanconi anaemia is an autosomal recessive disorder associated with increased chromosome breakage and progressive bone marrow failure. The gene for complementation group C (FACC) has been cloned and mapped to chromosome 9q22.3, but neither its genetic location nor the proportion of patients belonging to group C is known. We have used a polymorphism within the FACC gene to localise it within a 5 cM interval on chromosome 9q, bounded by D9S196/D9S197 and D9S176. The genes for Gorlin's syndrome and multiple self-healing squamous epitheliomata have also been mapped to this interval. Linkage analysis with the three highly informative microsatellite polymorphisms flanking FACC in 36 Fanconi anaemia families showed that only 8% of families were linked to this locus. This indicates that the genes for the other Fanconi anaemia complementation groups must map to one or more different chromosomal locations. The markers also allowed rapid exclusion of 56% of the families in our panel from complementation group C, thus substantially reducing the number of patients who need to be screened for FACC mutations.

SLe(x) expression of normal CD34 positive bone marrow haemopoietic progenitor cells.

We investigated sialylated Lewis x (sLe(x)) antigen expression on CD34 positive (CD34+) haemopoietic progenitors in the bone marrow of eight healthy volunteers using monoclonal antibodies. We found that in all the samples examined, CD34+ bone marrow progenitors strongly expressed the sLe(x) antigen. This contradicts previous publications which reported sLe(x) expression on malignant blast cells but not on normal CD34+ progenitor cells.

In vitro response of normal and aplastic anemia bone marrow to mast cell growth factor and in combination with granulocyte-macrophage colony-stimulating factor and interleukin-3.

We have examined the effect of mast cell growth factor (MGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-3 (IL-3), singly or in combination, on the growth of normal and aplastic anemia (AA) bone marrow in clonogenic assay and long-term bone marrow culture (LTBMC). MGF stimulated colony-forming unit-granulocyte/macrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), and mixed colony-forming unit (consisting of granulocyte-macrophage and erythroid elements) (CFU-GEM) colony formation from both normal and AA marrow. The three-factor combination stimulated the greatest number of colonies. Marrow from less severely affected AA patients was stimulated to produce the highest number of colonies, and a normal response was possible if progenitors were present. When added to LTBMC, MGF alone had little effect. GM-CSF and IL-3 stimulated increased numbers of progenitor cells harvested each week from normal and AA LTBMC. This resulted in normal colony numbers in some patients, the majority of whom were less severely affected than the patients who did not respond in LTBMC. The three-factor combination was additive for normal CFU-GM production. However, no further increases in AA LTBMC resulted from the addition of MGF to GM-CSF and IL-3. The partial correction in clonogenic assay with MGF in some AA patients raises the possibility of therapeutic benefit. We failed to demonstrate increased progenitor cell numbers in AA LTBMC, however. Further studies may overcome possible limitations to progenitor cell proliferation.

Haemopoietic progenitor cells are reduced in aplastic anaemia.

We investigated the frequencies of early populations of progenitors in aplastic anaemia (AA) bone marrow, from patients with a range of disease severity, compared with normal. Double-colour immunofluorescent staining for CD34 and CD33 was carried out on bone marrow mononuclear cells (BMMC) and analysed using fluorescence activated cell sorting (FACS). AA CD34+ cells were reduced by 68% compared to normal. In addition, AA CD33+ cells and the three progenitor subsets (CD34+/CD33-, CD34+/CD33+ and CD34-/CD33+) were reduced by 44-80%. Our data lend further support for an early stem cell deficiency in AA.

Pseudomonal supraglottitis occurring in a patient with profound neutropenia secondary to virus-associated haemophagocytic syndrome.

We present a case of virus-associated haemophagocytic syndrome following Epstein-Barr virus infection in which a fulminant pseudomonal supraglottitis developed. Increasingly, unusual pathogens have been found in immunocompromised patients. This is the first reported case of pseudomonal supraglottitis.

Relative expression of cytochrome P450 isoenzymes in human liver and association with the metabolism of drugs and xenobiotics.

Cytochrome P450s play a central role in the metabolism and disposition of an extremely wide range of drugs and chemical carcinogens. Individual differences in the expression of these enzymes may be an important determinant in susceptibility to adverse drug reactions, chemical toxins and mutagens. In this paper, we have measured the relative levels of expression of cytochrome P450 isoenzymes from eight gene families or subfamilies in a panel of twelve human liver samples in order to determine the individuality in their expression and whether any forms are co-regulated. Isoenzymes were identified in most cases on Western blots based on the mobility of authentic recombinant human cytochrome P450 standards. The levels of the following P450 proteins correlated with each other: CYP2A6, CYP2B6 and a protein from the CYP2C gene subfamily, CYP2E1 and a member of the CYP2A gene subfamily, CYP2C8, CYP3A3/A4 and total cytochrome P450 content. Also, the levels of two proteins in the CYP4A gene subfamily were highly correlated. These correlations are consistent with the relative regulation of members of these gene families in rats or mice. In addition, the level of expression of specific isoenzymes has also been compared with the rate of metabolism of a panel of drugs, carcinogens and model P450 substrates. These latter studies demonstrate and confirm that the correlations obtained in this manner represent a powerful approach towards the assignment of the metabolism of substrates by specific human P450 isoenzymes.