Marrow aplasia developing 13 years after HLA-identical sibling allogeneic transplantation for chronic myeloid leukaemia:successful treatment with antithymocyte globulin and peripheral blood stem cell infusion from the original donor

Secondary or late graft failure has been defined as the development of inadequate marrow function after initial engraftment has been achieved. We describe a case of profound marrow aplasia occurring 13 years after sibling allogeneic bone marrow transplantation for chronic myeloid leukaemia (CML) in first chronic phase. Although the patient remained a complete donor chimera, thereby suggesting that an unselected infusion of donor peripheral blood stem cells (PBSC) or bone marrow might be indicated, the newly acquired aplasia was thought to be immune in aetiology and some immunosuppression was therefore considered appropriate. Rapid haematological recovery was achieved after the infusion of unselected PBSC from the original donor following conditioning with anti-thymocyte globulin (ATG).

Leukaemic transformation of donor cells in a patient receiving a second allogeneic bone marrow transplant for severe aplastic anaemia

Allogeneic blood or bone marrow transplantation is a successful treatment for leukaemia and severe aplastic anaemia (SAA). Graft rejection following transplantation for leukaemia is a rare event but leukaemic relapse may occur at varying rates, depending upon the stage of leukaemia at which the transplant was undertaken and the type of leukaemia. Relapse is generally assumed to occur in residual host cells, which are refractory to, or escape from the myeloablative conditioning therapy. Rare cases have been described, however, in which the leukaemia recurs in cells of donor origin. Lack of a successful outcome of blood or bone marrow transplantation for severe aplastic anaemia (SAA), however, is due to late graft rejection or graft-versus-host disease. Leukaemia in cells of donor origin has rarely been reported in patients following allogeneic bone marrow transplantation for SAA. This report describes leukaemic transformation in donor cells following a second allogeneic BMT for severe aplastic anaemia. PCR of short tandem repeats in bone marrow aspirates and in colonies derived from BFUE and CFU-GM indicated the donor origin of leukaemia. Donor leukaemia is a rare event following transplantation for severe aplastic anaemia but may represent the persistence or perturbation of a stromal defect in these patients inducing leukaemic change in donor haemopoietic stem cells.

Treatment of relapse after allogeneic bone marrow transplantation with reduced intensity conditioning (FLAG +/- Ida) and second allogeneic stem cell transplant

Acute leukaemias in relapse after allogeneic stem cell transplantation (SCT) respond poorly to donor leucocyte infusions (DLI) compared with chronic myeloid leukaemia (CML), at least in part because of faster disease kinetics. Fludarabine-containing 'non-myeloablative' chemotherapy followed by further allo SCT may offer more rapid and effective disease control. We report 14 patients with relapse after allo SCT for acute leukaemia [seven acute myeloid leukaemia (AML), five acute lymphoblastic leukaemia (ALL)] or refractory anaemia with excess blasts in transformation (RAEB-t, n = 2) treated with fludarabine, high-dose cytosine arabinoside (ara-C) and granulocyte colony-simulating factor (G-CSF) with (n = 10) or without (n = 2) idarubicin (FLAG +/- Ida) or DaunoXome (FLAG-X) (n = 2) and second allo SCT from the original donor. Donors were fully human leucocyte antigen (HLA) -matched in 13 cases with a single class A mismatch in one. Actuarial overall survival was 60% and disease-free survival was 26% at 58 months. Remissions after the second SCT were longer than those after the first bone marrow transplantation (BMT) in eight of the 13 assessable patients to date. Haematopoietic recovery was rapid. Transplants were well tolerated with no treatment-related deaths. The major complication was graft-versus-host disease (GvHD, acute >/= grade II-2 cases, chronic - eight cases, two limited, six extensive) although there have been no deaths attributable to this. FLAG +/- Ida and second allo SCT is a safe and useful approach and may be more effective than DLI in the treatment of acute leukaemias relapsing after conventional allo SCT.

Short Report:Engraftment of T-cell-depleted allogeneic haematopoietic stem cells using a reduced intensity conditioning regimen

Graft-versus-host disease (GVHD) remains a significant complication in patients undergoing allogeneic stem cell transplantation (SCT) using a reduced intensity conditioning regimen. Although T-cell depletion (TCD) reduces the risk of GVHD after a myeloablative conditioning regimen, it is associated with an increased risk of graft failure. We have therefore examined whether TCD compromises engraftment using a fludarabine-based conditioning regimen. Fifteen patients have been transplanted using such a regimen of whom 13 underwent ex vivo TCD. All but one patient demonstrated durable engraftment and no patient receiving a TCD product developed severe GVHD. Thus, TCD may play a role in GvHD prophylaxis using such regimens.

Acute skin GVHD following syngeneic BMT for CLL

A 41-year-old woman received a syngeneic BMT for CLL and subsequently developed acute skin GVHD. Transfusion-related allogeneic GVHD was excluded on the basis of an unchanged HLA type in circulating lymphocytes. Short tandem repeat PCR was used to confirm syngeneicity between donor and recipient. The patient had a personal and family history of autoimmune disease which may have made her particularly susceptible to development of syngeneic GVHD. The distinction between allogeneic and syngeneic or autologous GVHD is important because of therapeutic implications.

High frequency of donor chimerism after allogeneic transplantation of CD34+-selected peripheral blood cells

Ex vivo T cell depletion of allogeneic grafts is associated with a high (up to 80%) rate of mixed chimerism (MC) posttransplantation. The number of transplanted progenitor cells is an important factor in achieving complete donor chimerism in the T cell depletion setting. Use of granulocyte colony-stimulating factor (G-CSF) peripheral blood allografts allows the administration of large numbers of CD34+ cells. We studied the chimeric status of 13 patients who received allogeneic CD34+-selected peripheral blood progenitor cell transplants (allo-PBPCTs/CD34+) from HLA-identical sibling donors. Patients were conditioned with cyclophosphamide (120 mg/kg) and total-body irradiation (13 Gy in four fractions). Apheresis products were T cell-depleted by the immunoadsorption avidin-biotin method. The median number of CD34+ and CD3+ cells infused was 2.8x10(6)/kg (range 1.9-8.6x10(6)/kg) and 0.4x10(6)/kg (range 0.3-1x10(6)/kg), respectively. Molecular analysis of the engraftment was performed using polymerase chain reaction (PCR) amplification of highly polymorphic short tandem repeat (PCR-STR) sequences in peripheral blood samples. MC was detected in two (15%) of 13 patients. These two patients relapsed at 8 and 10 months after transplant, respectively. The remaining 11 patients showed complete donor chimerism and were in clinical remission after a maximum follow-up period of 24 months (range 6-24 months). These results were compared with those obtained in 10 patients who were treated with T cell-depleted bone marrow transplantation by means of elutriation and who received the same conditioning treatment and similar amounts of CD3+ cells (median 0.45x10(6)/kg; not significant) but a lower number of CD34+ cells (median 0.8x10(6)/kg; p = 0.001). MC was documented in six of 10 patients (60%), which was significantly higher than in the allo-PBPCT/CD34+ group (p = 0.04). We conclude that a high frequency of complete donor chimerism is achieved in patients receiving allo-PBPCT/CD34+ and that this is most likely due to the high number of progenitor cells administered.

Monitoring of lineage-specific chimaerism allows early prediction of response following donor lymphocyte infusions for relapsed chronic myeloid leukaemia

Donor lymphocyte infusions (DLI) have been shown to enhance the graft-versus-leukaemia (GVL) effect and induce haematological and molecular remission in patients with relapsed CML following allogeneic bone marrow transplantation (BMT). The potent donor cell-mediated cytolysis following DLI may lead to a short period of aplasia before the re-establishment of donor haematopoiesis. The absence of detectable donor cells in patients prior to DLI infusion may result in permanent aplasia in certain patients. We report on four patients who relapsed 1, 3, 6.5 and 7 years post-BMT for chronic phase CML and were treated with DLI from their original BMT donor. Polymorphic short tandem repeats (STRs) were used to assess haematological chimaerism both prior to and following DLI. At the time of relapse, STR-PCR indicated the presence of donor cells in all four patients, at levels ranging from 1-40%. A clinical and molecular response was seen in 4/4 patients following a short period of cytopenia and all patients remain in clinical remission with a follow-up of 2 months-3 years post-DLI. STR-PCR indicated that a response was occurring during the period of pancytopenia when metaphase analysis was unsuccessful. Lineage-specific analysis of the cellular response to DLI was monitored using STR-PCR of peripheral blood (PB) and bone marrow (BM) lymphocyte-enriched fractions and CD2-positive and -negative T cell fractions. In one patient BM and PB CD34-positive and -negative fractions were also assessed. A change in the ratio of donor:recipient cells in the PB lymphocyte fraction was the earliest molecular indication of an anti-leukaemic response. Subsequent conversion to donor chimaerism occurred in the other lineages and the granulocyte fraction was the last lineage to convert. In conclusion, lineage-specific STR-PCR permits detailed monitoring of subtle changes in donor/recipient cell dynamics in specific lineages following DLI during the crucial pancytopenic phase and may be a useful predictor of haematological response to DLI therapy.

Donor chimaerism is a strong indicator of disease free survival following bone marrow transplantation for chronic myeloid leukaemia

Although Chronic Myeloid Leukaemia (CML) can be treated successfully with allogeneic bone marrow transplantation (BMT), leukaemia relapse remains a significant clinical problem. Molecular monitoring of the post transplant marrow can be useful in predicting relapse particularly in CML patients where the Philadelphia chromosome or its molecular counterpart, the BCR-ABL fusion messenger RNA can be used as a leukaemia specific marker of minimal residual disease (MRD). We have investigated chimaerism (using polymerase chain reaction of short tandem repeat sequences (STR-PCR)) and MRD status (using reverse transcriptase PCR of the BCR-ABL fusion mRNA) in a serial fashion in 18 patients who were in clinical and haematological remission post allogeneic BMT for chronic phase CML. Eleven patients exhibited complete donor chimaerism with no evidence of minimal residual disease. Five patients had transient or low level stable MC. Late MC and MRD was observed in two patients who relapsed > 6 years after T cell depleted BMT for CML. Thus STR-PCR is an appropriate screening test in the post transplant setting for CML patients, but those patients exhibiting mixed haemopoietic chimaerism should also be monitored using a leukaemia specific sensitive molecular assay.

Patterns of hematopoietic chimerism following bone marrow transplantation for childhood acute lymphoblastic leukemia from volunteer unrelated donors

Hematopoietic chimerism was analyzed in serial bone marrow samples taken from 28 children following T-cell depleted unrelated donor bone marrow transplants (UD BMT) for acute lymphoblastic leukemia (ALL). Chimeric status was determined by polymerase chain reaction (PCR) of simple tandem repeat (STR) sequences (maximal sensitivity, 0.1%). At least two serial samples were examined in 23 patients. Of these, two had evidence of complete donor engraftment at all times and eight showed stable low level mixed chimerism (MC) (<1% recipient hematopoiesis). All 10 of these patients remain in remission with a minimum follow-up of 24 months. By contrast, 13 patients demonstrated a progressive return of recipient hematopoiesis. Five of these relapsed (4 to 9 months post BMT), one died of cytomegalovirus pneumonitis and seven remain in remission with a minimum follow-up of 24 months. Five children were excluded from serial analysis as two serial samples were not collected before either relapse (3) or graft rejection (2). We conclude that as with sibling transplants, ex vivo T depleted UD BMT in children with ALL is associated with a high incidence of MC. Stable donor engraftment and low level MC always correlated with continued remission. However, detection of a progressive return of recipient cells did not universally correlate with relapse, but highlighted those patients at greatest risk. Serial chimerism analysis by PCR of STRs provides a rapid and simple screening technique for the detection of relapse and the identification of patients with progressive MC who might benefit from detailed molecular analysis for minimal residual disease following matched volunteer UD BMT for childhood ALL.

Effect of ultraviolet-B light on lymphocyte activity at doses at which normal bone marrow stem cells are preserved

Ultraviolet B (UVB) light is known to be immunosuppressive, but, probably because of a small UVC component in the emission spectra of some of the UVB lamps used, reports vary on effective dose levels. To prevent potentially lethal graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation, alloreactive donor T-cell activity must be suppressed. In this study, a narrow wavelength UVB lamp (TL01, 312 nm peak emission) was used to determine what doses of UVB were required to abolish rat lymphocyte proliferation while simultaneously preserving rat bone marrow progenitor cell and primitive hematopoietic stem cell viability. Lymphocyte proliferation, as measured by 3H-Thymidine incorporation, in response to lectin stimulation was abolished below detection at doses greater than 3,500 J/m2. When T-cell clonogenicity was measured in a limiting dilution assay, a small fraction (0.6%) was maintained at doses up to 4,000 J/m2. Cytotoxic T-lymphocyte (CTL) activity was reduced after treatment with 4,000 J/m2, but a significant level of cytotoxicity was still maintained. Natural killer cell cytolytic activity was not affected by doses up to 4,000 J/m2. At 4,000 J+m2 there was a 10% survival of colony-forming units-granulocyte-macrophage; a 1% and 4% survival of day-8 and day-12 colony-forming units-spleen, respectively; and 11% survival of marrow repopulating ability cells. Up to 25% of late cobblestone area forming cells (4 to 5 weeks), reflecting the more immature hematopoietic stem cells, were preserved in bone marrow treated with 4,000 J/m2, indicating that early stem cells are less sensitive to UVB damage than are more committed progenitor cells. Thus, a potential therapeutic window was established at approximately 4,000 J/m2 using this light source, whereby the potentially GVHD-inducing T cells were suppressed, but a sufficient proportion of the cells responsible for engraftment was maintained.

PCR amplification of short tandem repeat sequences allows serial studies of chimaerism/engraftment following BMT in rodents

Animal models of bone marrow transplantation (BMT) allow evaluation of new experimental treatment strategies. One potential strategy involves the treatment of donor marrow with ultra-violet B light to allow transplantation across histocompatibility boundaries without an increase in graft rejection or graft-versus-host disease. A major requirement for a new experimental protocol, particularly if it involves manipulation of the donor marrow, is that the manipulated marrow gives rise to long-term multilineage engraftment. DNA based methodologies are now routinely used by many centres to evaluate engraftment and degree of chimaerism post-BMT in humans. We report the adaptation of this methodology to the serial study of engraftment in rodents. Conditions have been defined which allow analysis of serial tail vein samples using PCR of short tandem repeat sequences (STR-PCR). These markers have been used to evaluate the contribution of ultraviolet B treated marrow to engraftment following BMT in rodents without compromising the health of the animals under study. Chimaerism data from sequential tail vein samples and bone marrow from selected sacrificed animals showed excellent correlation, thus confirming the validity of this approach in analysing haemopoietic tissue. Thus the use of this assay may facilitate experimental studies in animal BMT.

Polymerase chain reaction based methods for assessing chimerism following allogeneic bone marrow transplantation

It is important to be able to assess the contribution of donor cells to the graft followmg bone marrow transplantation (BMT), as complete engraftment of marrow progenitors that can give rise to long term donor derived hemopoiesis may be important in long-term disease-free survival. The contribution of the donor marrow, both in terms of filling the marrow "space" created by the intense conditioning regimen and in its ability to mediate a graft versus leukemia effect may be assessed by studying the kinetics of the engraftment process. As BMT involves repopulation of the host hemopoietic system with donor cells, recipients of allogeneic marrow are referred to as hemopoietic chimeras. A donor chimera is an individual who exhibits complete donor hemopoiesis and we would imagine that donor chimertsm carries the best long-term prognosis. A patient who has both donor and recipient cells coexistmg in a stable fashion post-BMT without hematological evidence of relapse or graft rejection is referred to as a mixed chimera. Mixed chimerism may be a prelude to graft rejection or leukemic relapse; therefore, it is important to be able to monitor the presence of these cells in a precise manner.

Studies on hemopoietic chimerism following allogeneic bone marrow transplantation in the molecular biology era

Donor hematopoiesis or donor chimerism in the host following allogeneic bone marrow transplantation (BMT) has appeared crucial to the engraftment process. However, as molecular techniques exploiting neutral variation in human genetic material have been used in the study of chimerism, the detection of residual host cells or mixed hemopoietic chimerism has indicated that donor chimerism is not obligatory following BMT. This review focuses on the detection and significance of mixed chimerism (MC) in patients transplanted for both malignant and non-malignant hemopoietic disease and attempts to tease out the contribution of MC to engraftment, leukemia relapse, graft rejection and long-term disease-free survival.