Acquired aplastic anemia in adults. IV. Histological and CFU studies in transplanted and non-transplanted patients.

A follow-up study of 10 patients suffering from acquired aplastic anaemia, comprising methocrylate-embedded bone marrow biopsies and CFU cultures, is presented. The haematopoietic recovery patterns and changes in the inflammatory infiltration after permanent engraftment could be distinguished from those in non-transplanted patients. After anti-thymocyte globulin treatment followed by allogeneic bone marrow infusion, the recovery pattern resembled that in non-transplanted patients. The persistently low colony-forming capacity in some patients could be explained by the existence of lymphoid inhibitory cells, which suggests an immunologic auto-destructive mechanism.     

The future for treatment by bone marrow transplantation for adrenoleukodystrophy,metachromatic leukodystrophy,globoid cell leukodystrophy and Hurler syndrome

Summary Within the past decade, bone marrow transplantation has been applied to over 200 patients worldwide with the intention of treating storage diseases. Bone marrow transplantation has provided a method for treatment of adrenoleukodystrophy, metachromatic leukodystrophy, globoid cell leukodystrophy and Hurler syndrome. After engraftment, significant improvement in the clinical course of each of these diseases occurs. Survival data of engrafted patients are superior to those of non-transplanted. Engraftment and the resulting enzymatic reconstitution are concordant. Outcomes based on neuropsychological tests indicate continued maintenance and in some cases increase in cognitive function. Magnetic resonance imaging as well as spectroscopic examinations of the brain provide further evidence that positive changes occur in the central nervous system following long-term engraftment. A better quality of life follows engraftment. Greater gains from use of bone marrow transplantation for these particular storage diseases will occur in the future. Earlier diagnosis will allow bone marrow transplantation in the presymptomatic stage at a younger age, providing an enhancement of positive effects noted from such treatment. At the same time, advances in bone marrow technology will serve to reduce the risk factors involved with the bone marrow transplantation process itself. These two factors taken together will be more than additive in providing benefits from use of bone marrow transplantation.     

Morphology and quantitative composition of hematopoietic cells in murine bone marrow and spleen of healthy subjects

Laboratory mice play an outstanding role in modeling human development and disease. In contrast to human leukemia, the spleen is involved in almost all cases, and the bone marrow is only variably involved in murine models. Although mice have been used for medical research for over 100 years, there are only few reports with a small number of cases looking at morphology and quantitative composition of murine hematopoietic cells in the bone marrow of non-transplanted animals of most strains. To our knowledge, there is not even a single report describing the splenogram in C57BL/6J mice, one of the most commonly used strains for medical research. The present study illustrates the morphology of the hematopoietic cells in the bone marrow and spleen of non-treated C57BL/6J mice and establishes the murine myelogram from the largest healthy C57BL/6J cohort reported to date. Furthermore, we present the first murine splenogram described for C57BL/6J mice. Our study supports the acceptance of the presence of >5 % blast cells as providing clear evidence of abnormality in bone marrow like in humans. In addition, we are the first to show <1 % blast cells in the normal spleen. Interestingly, classical dysplastic changes were rare in normal healthy mice. Our study of the bone marrow and spleen of healthy non-transplanted animals provides reference ranges of each cell type and for the myeloid/erythroid ratio, which can be used to interpret preclinical gene therapy data, leukemogenesis, and hematopoiesis studies, and may improve the quality of such analyses.     

Intensive remission induction therapy for chronic myeloid leukemia in blast phase with a goal of post-remission bone marrow transplant →— a pilot study

Abstract: An intensive protocol utilizing mitoxantrone, high-dose cytarabine, vincristine, etoposide and methylprednisolone as induction therapy for chronic myeloid leukemia in blast transformation is described. Fourteen patients were treated, with a remission/second chronic phase achieved in 64%. None of the 3 patients older than 50 yr responded. Complete hematological responses were seen in 9 of the 11 younger patients, 4 of whom also became BCR-ABL negative by Southern Blot analysis. Four patients went on to allogeneic bone marrow transplant. Median remission durations were 4.5 (1–5) and 8.5 (5–16) months in the non-transplanted and transplanted cohorts, respectively. Median survival is 1.5 (0.5–3), 9.5 (7–14) and 17 (14–61 +) months in the non-responding, responding non-transplanted and transplanted cohorts, respectively. Toxicity, particularly gastrointestinal, was significant. This represents an aggressive protocol that should be reserved for patients who are potential transplant candidates.     

Hemopoietic reconstitution after bone marrow transplantation

Forty-one patients underwent bone marrow transplantation (BMT) for treatment of severe aplastic anemia or hematologic malignancies. Hemopoietic reconstitution after BMT was monitored by peripheral blood counts, counts of bone marrow cellularity, and clonal assays for hemopoietic progenitors (CFUc, CFUe, and BFUe), along with bone marrow morphology. The number of transplanted nucleated cells and the number of transplanted progenitors (CFUc, CFUe, and BFUe) correlated significantly with the time of reticulocyte recovery. The number of transplanted CFUc correlated significantly with the time of granulocyte recovery. Platelet recovery occurred late and showed large variations. No correlation between the transplanted cells and the recovery of nucleated cells or hemopoietic progenitors (CFUc, CFUe, and BFUe) in the bone marrow was found. Bone marrow cellularity and hemopoietic progenitors showed a rapid, but incomplete, recovery during the first 56 days after BMT. Hematologic studies on seven long-term survivors with an uncomplicated posttransplantation course revealed subnormal bone marrow cellularity and hemopoietic progenitor incidence up to three years after BMT, despite normal peripheral blood counts. The low progenitor incidence could be explained by a proliferative defect of the stem cells, compensated for by an amplification in the more differentiated compartment of hemopoiesis.     

Lymphokine-activated killer (LAK) cell purging of leukemic bone marrow: range of activity against different hematopoietic neoplasms

Natural killer cells activated in vitro by incubation with IL-2 display a broad range of cytolytic activity against neoplastic cells. These lymphokine-activated killer (LAK) cells can discriminate between neoplastic and normal bone marrow cells and may represent a useful means of purging bone marrow prior to autologous transplantation. We demonstrate that LAK cells can successfully remove four distinctly different malignant hematopoietic cell types from normal bone marrow grafts. The LAK purging technique is capable of a 2-3 log10 reduction in tumor cells in the bone marrow graft without compromising hematological recovery or survival. Our results also suggest, however, that an inhibitory effect on stem cell function by allogeneic LAK cells exists, and this form of purging may be used only if greater levels of bone marrow are transferred in an allogeneic setting. The ability to detect and eliminate malignant cells in bone marrow prior to use for autologous transplantation suggests that LAK cells, alone or in conjunction with current methods of bone marrow purging, could be useful for the in vitro treatment of bone marrow in patients who require high-dose chemotherapy and autologous bone marrow transplantation.     

Immunomagnetic purging of breast cancer from bone marrow for autologous transplantation

Intensive chemotherapy with autologous bone marrow transplantation is a promising approach for the treatment of breast cancer, provided that clonogenic tumor cells do not contaminate the patient's bone marrow. We have previously demonstrated that a combination of 4-hydroperoxycyclophosphamide (4-HC) and immunomagnetic purging (IMP) with monoclonal antibodies and microspheres could remove 4-5 logs of clonogenic breast cancer cells from a 10-fold excess of human bone marrow cells. In the present report we have evaluated an apparatus for separating tumor cells from a large volume of human marrow. This apparatus will permit preparation of large volumes of purged marrow for use in studies of intensive therapy with autologous marrow support. Bone marrow progenitor cell (CFU-GM) recovery following this IMP technique was 85% of the unpurged control, and suggests that marrow recovery following high dose systemic chemotherapy will not be adversely affected. A phase I study to evaluate marrow reconstitution following IMP is underway. Preliminary data suggest that this IMP method will not delay engraftment in breast cancer patients receiving high-dose chemotherapy and autologous bone marrow support, but further study is required.     

Bone marrow transplantation from identical twins in the treatment of aplastic anaemia: implication for the pathogenesis of the disease

S ummary . Treatment of aplastic anaemia by bone marrow transplantation from a syngeneic (identical twin) donor has provided insights into the pathophysiology of the disease.
We report from patients with severe anaemia who were treated by syngeneic bone marrow transplantation. None of the patients had sustained recovery of peripheral blood counts. All four received second transplants from the same twin donor after immunosuppressive conditioning treatment. Each had prompt recovery of haematopoiesis. A review of the literature indicates that failure of syngeneic bone marrow transplantation in patients with aplastic anaemia is not uncommon. These data indicate that aplastic anaemia may be caused by a mechanism other than an absence or intrinsic abnormality of haematopoietic stem cells in many patients.     

Autologous bone marrow transplantation in acute leukemia with marrow purged with alkyl-lysophospholipid.

Alkyl-lysophospholipids are anticancer agents that are selectively toxic to leukemic cells and relatively sparing of normal bone marrow cells. Thus, they would be likely candidates for purging remission marrows before autologous bone marrow transplant. One of the more promising agents is edelfosine, which could be safely used for purging without prolonging marrow recovery. Assays for marrow progenitor cells were performed before and after purging and cryopreservation in 64 patients. There was no significant reduction in colony formation after purging when compared with unpurged cryopreserved marrow, but there was a significant reduction after cryopreservation. Twenty-four patients with acute leukemia in second (16 patients) or third remission (3 patients), early relapse (3 patients), or in first remission with successfully treated extramedullary relapse (2 patients) received marrow-ablative chemotherapy and total body irradiation followed by infusion of marrow purged for 4 hours with 50 to 100 micrograms/mL of edelfosine. There were 9 lymphoblastic and 15 myelogenous leukemia patients. The median time to granulocyte recovery to 500/microL was 26 and 33 days for the 50 and 75 microgram/mL doses, respectively. The patient whose marrow was purged at the dose of 100 micrograms/mL failed to engraft. The median time to platelet recovery to 25,000/microL was 45 and 37 days for the 50 and 75 micrograms/mL doses, respectively. Twenty-nine percent of the patients remain disease free from 131 to 1,291 days, with a median of 356 days. These results have established that purging with 75 micrograms/mL of edelfosine is a safe dose and is recommended for a phase II trial.     

Autologous nonfrozen bone marrow transplantation after intensive chemotherapy: a pilot study

15 patients with metastatic, nonhematopoietic neoplasms refractory to conventional means of treatment were given intensive chemotherapy followed by infusion of autologous noncryopreserved bone marrow which had been stored at 10 degrees C. This study has shown that the procurement of bone marrow from patients with advanced disease and reinfusion 12 h after high dose chemotherapy is tolerated without significant patient morbidity. The use of marrow stored at 10 degrees C leads to adequate recovery of granulocyte stem cells. The present data also suggest that autologous bone marrow transplantation is beneficial in shortening hematopoietic recovery time in patients receiving high dose chemotherapy and may improve response rates in patients with refractory neoplasms.     

Impaired cellular and humoral immunity is a feature of Diamond-Blackfan anaemia; experience of 107 unselected cases in the United Kingdom

Diamond-Blackfan anaemia (DBA) is a rare bone marrow failure syndrome characterised by anaemia, congenital anomalies and cancer predisposition. Although infections are the second leading cause of mortality in non-transplanted patients, immune function is largely unexplored. We identified quantitative deficits in serum immunoglobulins and/or circulating T, natural killer and B lymphocytes in 59 of 107 unselected patients (55·1%) attending our centre over a 7-year period. Immune abnormalities were independent of ribosomal protein genotype and arose in both steroid-treated and steroid-untreated patients. In summary, these data highlight the high prevalence and spectrum of infections and immune defects in DBA.     

Autologous bone marrow transplantation in acute leukemia: a phase I study of in vitro treatment of marrow with 4- hydroperoxycyclophosphamide to purge tumor cells

This phase I study was conducted to determine the maximal safe concentration of 4-hydroperoxycyclophosphamide (4HC) that could be used for in vitro treatment of bone marrow from patients with acute leukemia undergoing autologous bone marrow transplantation. Concentrations of 40 to 120 micrograms/mL of 4HC were used in 30 patients with relapsed or high-risk acute leukemia and in six patients with nonleukemic malignancies. All patients received marrow-lethal cytoreductive therapy followed by infusion of the 4HC-treated marrow. Complete inhibition of granulocyte and macrophage colony-forming cells was obtained at 80 micrograms/mL. Nevertheless, only one transplant-related death and otherwise full hematologic recovery was observed at concentrations of 4HC up to 100 micrograms/mL. At 120 micrograms/mL, there were three transplant-related deaths, including two of the three patients who required the infusion of reserve marrow. Among the acute leukemia patients, three remain in complete remission at 1,337, 1,017, and 967 days after transplant. Among the nonleukemic patients, two remain in complete remission at 1,081 and 1,017 days after transplant. At the maximum safe concentration of 4HC (100 micrograms/mL), satisfactory hematologic recovery can be obtained, despite elimination of detectable hematopoietic progenitors.     

Bone marrow-derived CD45+ and CD45- cells reside in skeletal muscle

In this report we examined the kinetics of accumulation in muscle tissue of donor-derived CD45+ and CD45- cells from one to several weeks post-bone marrow transplantation. Only after hemopoiesis is fully reconstituted are CD45+ cells recovered from muscle tissue in proportions similar to ones we reported earlier in non-transplanted mice. Furthermore, using marked donor bone marrow cells, we document that in addition to CD45+ cells, donor-derived CD45- cells with certain phenotypic characteristics progressively accumulate in muscle post-transplantation. The turnover rate and physiologic roles of these populations in muscle are unclear and require further study.     

A study of disturbances in granulocytopoiesis in humans using a diffusion chamber system

Data are presented demonstrating that bone marrow granulocytic cells from normal persons continuously proliferate and mature during 5 d of cultivation in a diffusion chamber system (DC). Bone marrow granulocytopoiesis in 3 patients with different granulocytopoietic disorders was investigated using DC. In patient 1, with drug-induced agranulocytosis, bone marrow aspirated on the "day of recovery" revealed intensive proliferation in DC resulting in a highly significant increase of mature granulocytes. Patient 2 suffered from chronic neutropenia of unknown aetiology. After 5 d cultivation of bone marrow in DC an increase of metamyelocytes was observed, indicating delayed maturation of granulocytic cells. Patient 3 had neutropenia probably due to the splenic sequestration. Data obtained with DC suggest stimulated granulocytopoiesis in this patient. The results obtained using DC reflect the particular phase of the disease in which bone marrow was aspirated. It may be concluded that this method could provide additional data in quantitative and functional disorders of granulocytes.     

Allogeneic bone marrow transplantation for severe aplastic anaemia-the London experience

Using the Seattle protocol with minor modifications, 23 patients with severe aplastic anaemia received allogeneic bone marrow transplants from HLA/ mixed leucocyte culture matched sibs in three London centres between 1973 and 1977. Ten patients (43.5%) are alive 6 months to 5 years after transplantation, and are well with full haemopoietic reconstitution, two with autologous bone marrow recovery following the graft procedure. A failure of the marrow graft to take, or take followed by rejection occurred in 12 patients (52%). Failure of marrow recovery was associated with a high early mortality from bacterial or fungal infection. The only survivors amongst those who rejected the first graft were four patients in whom a subsequent graft from the same donor was successful, and two in whom autologous recovery occurred. Graft versus host disease (GVHD) occurred in seven patients, and was fatal in one case. The most frequent complication after successful engraftment was varicella-zoster infection which occurred in five patients and was fatal in one patient. The overall results compare favourably with those from other transplant centres, but the high rate of graft rejection and low incidence of GVHD differ from other series. The results should encourage further referral of patients with severe AA for bone marrow transplantation.     

Relative sensitivity of blood and bone marrow cultures in typhoid fever.

In a prospective study of typhoid fever in Ahmadu Bello University Hospital, Zaria, the relative diagnostic sensitivities of blood culture and bone marrow culture were studied. The results in 64 patients with proved diagnosis of typhoid fever (either by recovery of S. typhi from stool, blood and/or bone marrow or by a positive Widal agglutination test) are presented. Forty-four per cent and 59% of the patients yielded S. typhi on blood and bone marrow cultures, respectively. In 31 patients who were investigated by both blood and bone marrow cultures, the yields of S. typhi were 35% and 61% respectively. This difference is statistically significant (P less than 0.05). In this study bone marrow culture proved to be the most sensitive diagnostic test for typhoid fever. A simple technique of bone marrow aspiration is described and its use is recommended for large general teaching hospitals.     

Regression of adjuvant-induced arthritis in rats following bone marrow transplantation.

Total body irradiation followed by bone marrow transplantation was found to be an effective treatment for adjuvant arthritis induced in rats. This treatment is most effective when applied shortly after the clinical manifestation of arthritis--i.e., 4-7 weeks after administration of Mycobacterium tuberculosis. Transplantation of bone marrow at a later stage results in a limited recovery, in that the inflammatory reaction regresses but the newly formed excessive bone is not eliminated. Local irradiation of the affected joints had no effect on the disease. It could also be excluded that the recovery of arthritis following marrow transplantation is due to lack of available antigen. Transplantation of syngeneic bone marrow is as effective as that of allogeneic bone marrow from a rat strain that is not susceptible to induction of adjuvant arthritis. The beneficial effect of this treatment cannot be ascribed to the immunosuppressive effect of total body irradiation, since treatment with the highly immunosuppressive drug Cyclosporin A resulted in a regression of the joint swelling but relapse occurred shortly after discontinuation of the treatment.     

Chemotherapy-induced mesenchymal stem cell damage in patients with hematological malignancy

Hematopoietic recovery after high-dose chemotherapy (HDC) in the treatment of hematological diseases may be slow and/or incomplete. This is generally attributed to progressive hematopoietic stem cell failure, although defective hematopoiesis may be in part due to poor stromal function. Chemotherapy is known to damage mature bone marrow stromal cells in vitro, but the extent to which marrow mesenchymal stem cells (MSCs) are damaged by HDC in vivo is largely unknown. To address this question, the phenotype and functional properties of marrow MSCs derived from untreated and chemotherapeutically treated patients with hematological malignancy were compared. This study demonstrates a significant reduction in MSC expansion and MSC CD44 expression by MSCs derived from patients receiving HDC regimens, thus implicating potential disadvantages in the use of autologous MSCs in chemotherapeutically pretreated patients for future therapeutic strategies. The clinical importance of these HDC-induced defects we have observed could be determined through prospective randomized trials of the effects of MSC cotransplantation on hematopoietic recovery in the setting of HDC with and without hematopoietic stem cell rescue.     

Recovery of Normal Hematopoiesis after Severe Bone Marrow Aplasia Induced by Interferon-α in a Patient with Chronic Myelogenous Leukemia

We describe an interesting case of a patient with chronic myelogenous leukemia (CML) who developed sustained severe bone marrow aplasia after 2 years and 11 months of interferon-alpha (IFN-alpha) therapy but demonstrated recovery of normal hematopoiesis when treated with immunosuppressive therapy with granulocyte-colony stimulating factor (G-CSF). Administration of G-CSF resulted in a partial recovery of hematopoiesis, and after starting immunosuppressive therapy, the patient was no longer dependent on blood transfusions. Moreover, her bone marrow had no Philadelphia chromosome-positive clones. According to the results of the present case, bone marrow recovery can be achieved with immunosuppressive therapy and a fatal outcome avoided, even in CML patients suffering from sustained bone marrow aplasia during IFN-alpha treatment.     

Monoclonal antibodies and magnetic microspheres for the depletion of leukaemic cells from bone marrow harvested for autologous transplantation

The use of a panel of monoclonal antibodies and anti-mouse immunoglobulin-coated microspheres is described for the depletion of leukaemic blasts from bone marrow. Marrow treated in this way rapidly reconstitutes haemopoietic function after high-dose consolidation chemoradiotherapy. The recovery of cells from bone marrow is similar but not identical to results obtained on removal of neuroblasts from marrow to be used for autologous transplant. This is probably a reflection of the cross-reactivity of 'anti-leukaemic' antibodies with a variety of haemopoietic progenitor cells. The study described here demonstrates the feasibility of using this method to purge leukaemic cells from bone marrow. A much larger randomised study between patients receiving either purged or non-purged bone marrow would be necessary to validate the need to remove small numbers of tumour cells from bone marrow.