Clonogenic assays and engraftment in allogeneic bone marrow transplantation

The significance of clonogenic assays for determining the hematopoietic potential of bone marrow grafts is still a matter of controversy. We determined the number of myeloid (GM-CFU), early erythroid (BFUe) and mixed (CFU-GEM) clones in 23 consecutive allogeneic bone marrow grafts. The growth of GM-CFU was stimulated by placental-conditioned medium, whereas both phytohemagglutinin-stimulated leucocyte-conditioned medium (PHA-LCM) and 'pluripoietin' from the 5637 cell line served as equally efficient stimulators of BFUe and CFU-GEM growth. Plating efficiency (e.o.p.) of GM-CFU and numbers of myeloid and mixed progenitors transplanted per kg body weight were significantly lower in those patients who died in the aplastic phase 2-6 weeks postgrafting (n = 4). These data show that low numbers of clonogenic cells, in particular GM-CFU, indicate a higher risk of death from infection following bone marrow transplantation (BMT) and argue for a contribution of GM-CFU in the seeding of an aplastic bone marrow.     

Genotypic analysis of engraftment in thalassemia following bone marrow transplantation using synthetic oligonucleotides

DNA hybridization with synthetic oligonucleotide probes was used to assess engraftment in 19 thalassemic patients who received bone marrow grafts from their respective healthy HLA-identical siblings. Three oligomers complementary to the tandem repetitive sequences of different hypervariable regions of human DNA were designed so as to produce simple RFLP (restriction fragment length polymorphism) patterns. Each probe hybridizes to one or two bands in HinfI-digested genomic DNA. The combined use of these three probes allowed a discrimination between all the HLA-identical siblings tested. Both donor-specific and recipient-specific DNA fragments existed in 18 out of the 19 sibling pairs studied. One pair possessed only a donor-specific fragment. DNA analysis at an early stage after the graft detected donor-specific fragments in 15 out of 19 patients, recipient-specific fragments in three patients and a mix of recipient and donor fragments in one patient. At a later stage this patient possessed donor-specific fragments only. Follow-up DNA analysis confirmed these findings. Thus 16 patients continued to display donor-specific fragments over 60 days post-transplant. These DNA data showed strong correlation with the clinical status of the patients as well as with other markers of engraftment including cytogenetics and hemoglobin synthesis. The patients who showed donor-specific fragments over 60 days have been free of thalassemic symptoms for over 300 days. Moreover, in 11 cases it was possible to predict the fate of the graft within 15 days after transplantation. In conclusion, the use of the three synthetic oligonucleotide probes provides a powerful tool in documenting engraftment in bone marrow transplantation.     

Characteristics of engraftment after repeated autologous bone marrow transplantation

The rate of engraftment after autologous bone marrow transplantation (ABMT) is extremely variable and largely unpredictable. To identify factors influencing engraftment, we studied 35 patients with refractory germ cell tumors undergoing high-dose chemotherapy with carboplatin (900-2000 mg/m2) and etoposide (1200 mg/m2) with bone marrow rescue. Prior to the initiation of chemotherapy, bone marrow sufficient for two marrow infusions was harvested (range 0.86-4.82 x 10(8) nucleated cells per kg). All 35 patients received half of the collected bone marrow 3 days after the last dose of chemotherapy; 23 responders received a second round of the same chemotherapy followed by infusion of the second half of the bone marrow. Eighteen patients could be compared for the two transplant episodes. The "rate of engraftment" was defined as the unweighted mean of four parameters: 1) the number of days until the absolute granulocyte count surpassed 0.2 x 10(9)/liter, 2) the number of days until the absolute granulocyte count surpassed 0.5 x 10(9)/liter, 3) the number of days until the last platelet transfusion, and 4) the number of days until the reticulocyte count surpassed 25 x 10(9)/liter. No significant correlation was found between rate of engraftment and such factors as the number of nucleated cells per kg infused, the dose of chemotherapy, extent of prior chemotherapy, tumor response to the high-dose chemotherapy, age of the patient, or the days of granulocytopenic fever (all p greater than 0.20). In contrast, a close correlation was found for the number of units of platelets (p = 0.005) and red blood cells (p = 0.006) transfused following each of the two transplants. There was no significant difference between rate of engraftment after first and second transplantation. Comparison of these data with the results obtained in reported ABMT with separate harvests suggests that the characteristics of the infused marrow determine the rate of engraftment after ABMT. This model of repeated transplantation could provide an important tool for assessing the therapeutic efficacy of hematopoietic growth factors.     

Histological alterations in bone marrow in patients with late engraftment after autologous bone marrow transplantation

Bone marrow histology after bone marrow transplantation has rarely been studied. Here, we reviewed the pre- and post-transplant bone marrow biopsies (BMB) of 40 acute myelogenous leukemia (AML) patients autografted in our center, 28 with normal and 12 with delayed peripheral recovery. The two groups were comparable in terms of previous therapy, disease phase and the number of infused cells, and received the same conditioning regimen. In the former group, reduced bone marrow cellularity and mild reticulin abnormalities were usual histological findings; in the latter, five patients had the same pattern, but the other seven had an almost undetectable hematopoietic parenchyma and severe reticulin derangement. One of these seven patients died of reactivated hepatitis B virus infection; the others eventually achieved peripheral recovery, with none of them experiencing a relapse. Autografted AML patients are excellent subjects for histological investigations. They account for the majority of delayed engraftments, the contribution of extramedullary components to the timing of engraftment is minimal, and leukemia relapse cannot be ruled out. These results suggest that BMB is a useful investigation in the work-up of late engraftment. A high degree of reticulin derangement with an almost undetectable hematopoietic parenchyma appear to be the morphological hallmarks of late engraftment.     

Poor engraftment after allogeneic bone marrow transplantation: role of chimerism analysis in treatment and outcome

We analyzed the clinical course and risk factors of 18 patients with poor engraftment after allogeneic bone marrow transplantation (BMT), defined as absolute neutrophil count below 0.1 x 10(9)/l 28 days post-BMT. Significant risks associated with non-engraftment included HLA one antigen mismatch, BMT from matched unrelated donor, and a low dose of colony-forming units-granulocyte-macrophage (<10(4)/kg). Examined by a semiquantitative analysis of polymorphic microsatellite markers, donor DNA chimerism on day 28 was found to be predictive of treatment outcome. Seven patients had detectable donor DNA, varying from 43 to 100%. Five of them responded to granulocyte colony-stimulating factor (G-CSF) and achieved engraftment. Two were given further infusions of peripheral blood hematopoietic stem cells (PBSC) from the same donors, resulting in engraftment in one of them. Eleven patients had no detectable donor DNA, and none responded to G-CSF. Autologous regeneration occurred in six of these patients, four after infusion of backup marrow and two spontaneously. The remaining five patients died despite the administration of PBSC from the same or different donors. Regular monitoring of donor DNA chimerism is useful in the management of patients at high risk of poor engraftment.     

GM-CSF therapy for delayed engraftment after autologous bone marrow transplantation

Based on previous observations that granulocyte-macrophage colony-stimulating factor (GM-CSF) promotes granulocyte recovery following chemotherapy, we evaluated the effect of recombinant human GM-CSF on hematopoietic progenitors and clinical outcome in six patients with delayed engraftment (greater than 55 days) after high-dose therapy and autologous bone marrow transplantation (ABMT). Three patients responded to a 14-day course of GM-CSF (10 micrograms/kg body weight/day) with at least a sevenfold rise in circulating granulocytes and a corresponding increase in granulopoietic activity in the bone marrow. A fourth patient died of infection on the 8th day of GM-CSF therapy with no evidence of response, and the remaining two, one of whom received a lower dose of GM-CSF (5 micrograms/kg/day), did not respond. There was no change in platelet or red cell transfusion requirements in any patient during the treatment. In two of the three responders, the granulocyte counts returned to pretreatment levels by 4 and 7 weeks after stopping the drug, respectively. We observed a marked increase in marrow-derived as well as in circulating granulocyte-macrophage progenitors (granulocyte-macrophage colony-forming units, CFU-GM) by the end of the 14-day course of GM-CSF in the three responders. There was no change in the frequency of circulating or marrow-derived erythroid (erythroid burst-forming units, BFU-E) or multilineage (multilineage colony-forming units, CFU-GEMM) progenitors. The results indicate that GM-CSF therapy in patients with markedly delayed engraftment after ABMT may stimulate granulopoiesis, but the effect is transient in some patients.     

Two phases of engraftment established by serial bone marrow transplantation in mice

Serially transplanted bone marrow eventually fails to reconstitute lethally irradiated mice. The reasons for this loss of repopulating ability are unknown. We showed that serial bone marrow transplantation changed the ratio of hematopoietic progenitors in bone marrow. The numbers of granulocyte-macrophage colony-forming units (CFU-GM) in the bone marrow did not change with serial transplantation. Spleen CFU (CFU-S) numbers decreased with serial transfer but remained at levels which should be associated with engraftment, even on the transfers which were unsuccessful. The CFU-S, therefore, did not appear to be the cells responsible for long-term hematopoietic repopulation. The number of successful serial transfers was dependent on the size of the grafts, and prolonging the time interval between transfers reestablished the ability of the serially transplanted marrow to reconstitute lethally irradiated recipients. Serial bone marrow transplantation dissociated two phases of engraftment. The first unsustained phase was maintained with repeated serial transfer and appeared to be produced by committed progenitors. The second sustained phase was eventually lost with repeated serial transfer and was apparently due to the pluripotent stem cell.     

Donor-origin cell engraftment after intraosseous or intravenous bone marrow transplantation in a rat model

We compared the effects of intraosseous BMT with those of standard i.v. BMT on the efficacy on donor-cell engraftment into the BM and lymphoid organs across an MHC barrier in rats. Twenty-four intraosseous and 24 i.v. BMTs were performed from 48 ACI (RT1(a)) donors to 48 Lewis (RT1(l)) recipients. Each transplant group received either intraosseous or i.v. BMT. Groups I and II served as controls without immunosuppression (n=16); groups III and IV received cyclosporine monotherapy (n=16); and V and VI received alphabeta-TCR monoclonal antibody and cyclosporine A (alphabeta-TCR/CsA) for 7 days (n=16). In each group, four rats received 35 x 10(6) transplanted bone marrow cells (BMCs) and four received 70 x 10(6) cells. All animals survived without GVHD. Mean (+/-s.d.) donor-cell engraftment into BM of recipients after intraosseous BMT was 7.9% (+/-1.3%) in recipients receiving alphabeta-TCR-CsA and 70 x 10(6) BMCs, and 4.2% (+/-1.4%) in recipients after i.v. transplantation. The seeding efficacy of donor cells into lymphoid tissue was greater after intraosseous BMT and alphabeta-TCR-CsA than after standard i.v. transplantation. In our model, intraosseous BMT facilitated donor-cell engraftment under short-term immunodepletive alphabeta-TCR/CsA protocol, which resulted in a temporary state of immune unresponsiveness.     

Chemotherapy prior to autologous bone marrow transplantation impairs long-term engraftment in mice

OBJECTIVE: Autologous bone marrow transplantation in cancer patients is often preceded by multiple cycles of chemotherapy. In this study, we assessed in a mouse model whether stem cells were affected by prior chemotherapy. METHODS: Donor mice were treated with three consecutive injections of 150 mg/kg 5-fluorouracil (5-FU). Peripheral blood counts were allowed to recover before the subsequent dose of 5-FU was given. Mice recovered from three doses of 5-FU and showed normal steady-state hematopoiesis. Bone marrow cells from these mice were mixed with congenic competitor cells and transplanted into lethally irradiated recipients. RESULTS: Although in vivo homing of cells from these mice was not impaired, donor leukocyte contribution steadily decreased posttransplantation. In contrast to in vivo homing, both in vitro migration toward stromal-derived factor (SDF)-1 and the average CXC chemokine receptor-4 (CXCR4) expression were lower in 5-FU-treated cells. Moderate reductions in L-selectin and CD11a expression were observed on stem cells of 5-FU-treated mice. CD43, CD44, CD49d, and CD49e were normally expressed and could thus not explain the reduced engraftment of these cells. CONCLUSION: We therefore conclude that 5-FU either directly damages stem cells or that the replicative stress induced by 5-FU causes a decline in stem cell reconstitution potential resulting in lower chimerism levels posttransplantation, that declines in time.     

High-dose cytostatic agents in allogeneic bone marrow transplantation: comparison of the engraftment promoting potential

We investigated the potential of various cytostatic agents for preventing graft rejection following allogeneic bone marrow transplantation. LEW rats received a lethal dose (35 mg/kg) of busulfan followed by injection of 1 x 10(8) F1(CAP x LEW) marrow cells, which are unable to induce a graft-versus-host reaction in LEW recipients. Rejection of the marrow graft was assessed by monitoring haematocrit and granulocyte counts. Due to its weak immunosuppressive activity, busulfan by itself is unable to allow engraftment of allogeneic marrow. Therefore, agents administered in addition to busulfan can be tested for their capacity to prevent marrow graft rejection. 120 mg/kg of cyclophosphamide, 20 mg/kg of ACNU and 240 mg/kg of ifosfamide completely prevented rejection of the allogeneic marrow. Maximum doses of BCNU applicable in conjunction with busulfan reduced the rejection rate to 12% (30 mg/kg) and 17% (40 mg/kg), whereas the antitumour agents thiotepa, melphalan, and carboplatin exhibited a very limited engraftment-promoting potential in this experimental setting. Thus, BCNU (carmustine), ACNU (nimustine), and ifosfamide might be suitable candidates for conditioning of allogeneic bone marrow graft recipients.     

Psychological aspects of bone marrow transplantation: a retrospective study of 17 long-term survivors

In a retrospective study 17 patients were interviewed 1-5 years after bone marrow transplantation (BMT) about their emotional experiences and about the information and support given to them. In all patients the illness, the BMT and its consequences created severe emotional strain; their psychological state was closely linked to their physical condition. Yet, most patients showed a surprising emotional elasticity and affective adjustment. Although the patients were overwhelmingly positive about the information and support they had received, an important number of them felt inadequately prepared for the emotional and sexual problems they had to face in the first period after discharge. The results of this study have led to structural changes in the psychiatric and psychosocial approach to the BMT patients.     

Utility of flow cytometric reticulocyte quantification as a predictor of engraftment in autologous bone marrow transplantation

Flow cytometric reticulocyte quantification with thiazole orange (TO) was used to study erythropoiesis in 20 patients following autologous bone marrow transplantation for the treatment of acute myelogenous leukemia. Flow cytometric reticulocyte analysis provided not only the reticulocyte percentage and absolute reticulocyte count but a quantitative reticulocyte maturity index (RMI) proportional to the amount of RNA in the reticulocytes. The RMI values, but not the reticulocyte percentage or absolute counts, correlated temporally with the rise in the absolute neutrophil counts in the posttransplantation period. In the majority of patients (12/20), the RMI value was the earliest indicator of bone marrow engraftment. The findings of this study demonstrate an important clinical utility of TO reticulocyte analysis by flow cytometry and indicate the diagnostic importance of the RMI measurement in the evaluation of erythropoietic activity in bone marrow transplant patients.     

Low-intensity conditioning is sufficient to ensure engraftment in matched unrelated bone marrow transplantation

OBJECTIVE: Matched unrelated bone marrow transplantation (BMT) for patients with hematological malignancies is associated with a high incidence of transplant-related complications due to high doses of chemoradiotherapy administered pre-BMT to ensure engraftment. The aim of this study was to investigate the feasibility of low-intensity conditioning for BMT from matched unrelated donors. MATERIALS AND METHODS: Sixteen patients with hematologic malignancies underwent non-T-cell-depleted BMT following a low-intensity conditioning regimen consisting of fludarabine monophosphate 30 mg/m(2)/day for 6 days, busulfan 4 mg/kg/day for 2 days, anti-T lymphocyte globulin 10 mg/kg/day for 4 days. Seven of the patients suffered from chronic myelogenous leukemia, four from acute lymphoblastic leukemia, four from acute myelogenous leukemia, and one from Ki-1 non-Hodgkin's lymphoma. Three of the patients had secondary leukemia and two were post-autologous BMT (ABMT). All patients were transplanted from fully matched unrelated donors. RESULTS: Fifteen of the 16 patients had 100% donor chimerism; no graft rejection was observed. None of the patients developed >Grade II veno-occlusive disease, sepsis, multiorgan failure, or renal or pulmonary toxicity. Four patients died posttransplant; one of thrombocytopenia and severe hemorrhagic cystitis, one of central nervous system toxicity, one of Grade IV graft-vs-host disease, and one following relapse (9 months post-BMT). Survival and disease-free survival at 36 months are 75% (95% confidence interval 46-90%) and 60% (95% confidence interval 30-80%), respectively. CONCLUSION: These results indicate that low-intensity conditioning is sufficient to ensure stable engraftment of bone marrow grafts in a matched unrelated setting.     

Allogeneic bone marrow vs. peripheral blood stem cell transplantation: a long-term retrospective single-center analysis in 329 patients

Objectives: Granulocyte colony‐stimulating factor‐mobilized peripheral blood hematopoietic stem cell transplantation (HSCT) provides a valuable and increasingly used alternative to bone marrow transplantation (BMT). This retrospective study aimed at determining whether the stem cell source is predictive for outcome, relapse incidence, non‐relapse mortality, and severity and incidence of both, acute and chronic graft‐versus‐host disease (GVHD) in patients undergoing allogeneic HSCT. Patients and methods: Between 1983 and 2007, 329 adult patients (median age 40, range 18–76) received a first allogeneic HSCT from either sibling (n = 203) or volunteer unrelated donors (n = 126) at our institution. The source of stem cells was bone marrow in 177 (54%) and peripheral blood in the remaining 152 (46%) patients. Results: Overall survival was 37% (31–43%, 95% confidence interval, CI), the relapse incidence was 30% (25–36%, 95% CI), and the non‐relapse mortality was 43% (38–49%, 95% CI) for the entire cohort with no significant differences between peripheral blood stem cell or BMT. In patients receiving myeloablative conditioning, peripheral blood stem cell transplantation (PBSCT) was associated with a significantly lower non‐relapse mortality (32% vs. 46%, P = 0.05), which, however, was restricted to standard‐risk disease (23% vs. 42%, P = 0.02). The overall cumulative incidences of acute GVHD II–IV were 51% and 54% following bone marrow and PBSCT, respectively. Severe acute GVHD III–IV was significantly more frequent after BMT (24% vs. 14%, P = 0.04), whereas chronic GVHD was significantly more frequent following PBSCT (48% vs. 24%, P = 0.0001). By multivariate analysis, PBSCT was only predictive for chronic GVHD (RR 2.29, P = 0.02). Conclusion: Although we failed to demonstrate any advantage of PBSCT over conventional BMT with regard to overall survival, relapse incidence and non‐relapse mortality PBSCT were associated with a significantly higher incidence of chronic graft‐versus‐host disease. Therefore, and by virtue of observations, that some patient groups might benefit from either stem cell source, there is still need for prospective randomized trials with special emphasize on quality of life in long‐term survivors.     

Serum transcobalamin levels as an early indicator of bone marrow engraftment following transplantation

Three B12 binding proteins, the transcobalamins TCI, TCII and TCIII, were determined serially in the serum of five patients who underwent bone marrow transplantation. The increase in TCII, followed by the increase in TCI, proved to be an early indicator of bone marrow regeneration, reaching a peak of up to twice its normal levels at least 5 d prior to the rise of the peripheral white blood cell count.     

Supernatant cell counts from long-term bone marrow culture correlate with the speed of engraftment following autologous bone marrow transplantation.

This study evaluates the relationship between bone marrow growth in a long-term bone marrow culture (LTBMC) system and speed of engraftment of the same marrow following autologous bone marrow transplantation (ABMT). Bone marrow from 21 patients transplanted with unmanipulated, non-cryopreserved autologous marrow was cultured. Samples from 21 normal donors were cultured to establish the normal supernatant cell count range. Supernatant counts from LTBMCs established from marrow taken from patients at the time of bone marrow harvest were compared with the time to neutrophil and platelet engraftment. Supernatant counts, particularly after 1 week in culture, showed close correlation with time to neutrophil and platelet engraftment following ABMT (r = 0.733, p less than 0.01; r = 0.735, p less than 0.01 respectively). Where supernatant cell counts were within the normal range rapid engraftment was predicted (neutrophils greater than 0.5 x 10(9)/l within 21 days, platelets greater than 50 x 10(9)/l within 28 days) and if supernatant counts were below this range, engraftment was predicted to be delayed. After 1 week in culture, the speed of neutrophil and platelet engraftment were correctly predicted in 19 and 18 cases respectively. Preliminary data suggest that LTBMC of marrow obtained 2-6 weeks before harvesting provides similar data, thus allowing the opportunity to intervene, for example with growth factors, in selected patients.     

Cytomegalovirus infection after autologous bone marrow transplantation: occurrence of cytomegalovirus disease and effect on engraftment

Epidemiologic and clinical characteristics of cytomegalovirus (CMV) infection and disease were analyzed retrospectively in 159 autologous marrow transplant recipients. The probability of CMV infection by day 100 after transplant was 22.5% in patients seronegative to CMV before transplant versus 61.1% in seropositive patients (P less than .0001 by logrank test). Multivariate analysis identified positive pretransplant CMV serology as the only definable risk factor for CMV infection (relative risk 1.4, P less than .0001). CMV pneumonia developed in 11 patients at a median time of 100 days after transplant and was fatal in nine cases. CMV pneumonia was associated with significantly decreased probability of survival by day 100 after transplant (relative risk of death of 16.7, P less than .0001). In contrast to earlier reports, CMV infection had no significant effect on the rapidity of platelet or neutrophil recovery after transplant as assessed by time-dependent multivariate analysis. Because the incidence of severe CMV disease is not negligible after autologous marrow transplantation, preventive measures against CMV infection are warranted, as in allogeneic marrow transplantation.