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.

Immune haemolytic anaemia following T cell-depleted allogeneic bone marrow transplantation for chronic myeloid leukaemia:association with leukaemic relapse and treatment with donor lymphocyte infusions

Immune haemolytic anaemia (IHA) is a recognised complication after allogeneic stem cell transplantation (SCT) and occurs more frequently if marrow cells have been subjected to T cell depletion (TCD). Among 58 consecutive patients who underwent TCD-allogeneic SCT from volunteer unrelated donors for the treatment of CML at the Hammersmith Hospital during a 3-year period (1 March 1996 to 28 February 1999) we identified nine cases of IHA. All patients had a strongly positive direct and indirect antiglobulin test and in eight patients the serological findings were typical of warm-type haemolysis often with antibody specificities within the Rh system. All nine cases had clinically significant haemolysis and were treated initially with prednisolone and immunoglobulin. The onset of IHA coincided with the occurrence of leukaemic relapse in six cases, and the presence of host haemopoiesis confirmed by lineage-specific chimerism in all four cases studied. Five patients received donor lymphocyte infusions (DLI); in three molecular remission and the restoration of full donor chimerism coincided with resolution of haemolysis. We conclude that in the context of leukaemic relapse, DLI is an effective therapy for IHA following allografts involving TCD.

Cyclosporin A and short-term methotrexate versus cyclosporin A as graft versus host disease prophylaxis in patients with severe aplastic anemia given allogeneic bone marrow transplantation from an HLA-identical sibling:results of a GITMO/EBMT randomized trial

A randomized trial was carried out comparing cyclosporin A (CsA) and short-term methotrexate (MTX) versus CsA alone for graft versus host disease (GVHD) prophylaxis in patients with severe aplastic anemia (SAA) undergoing allogeneic bone marrow transplantation (BMT) from a compatible sibling. Seventy-one patients (median age, 19 years; range, 4-46 years) were randomized to receive either CsA and MTX or CsA alone for the first 3 weeks after BMT. Subsequently, both groups received CsA orally, with gradual drug reduction until discontinuation 8 to 12 months after BMT. Patients randomized in both arms had comparable characteristics and received the same preparative regimen (ie, cyclophosphamide 200 mg/kg over 4 days). The median time for neutrophil engraftment was 17 days (range, 11-31 days) and 12 days (range, 4-45 days) for patients in the CsA/MTX group and the CsA alone group, respectively (P =.01). No significant difference was observed in the probability of either grade 2, grade 3, or grade 4 acute GVHD or chronic GVHD developing in the 2 groups. The Kaplan-Meier estimates of 1-year transplantation-related mortality rates for patients given either CsA/MTX or CsA alone were 3% and 15%, respectively (P =.07). With a median follow-up of 48 months from BMT, the 5-year survival probability is 94% for patients in the CsA/MTX group and 78% for those in the CsA alone group (P =. 05). These data indicate that the use of CsA with MTX is associated with improved survival in patients with SAA who receive transplants from compatible siblings. (Blood. 2000;96:1690-1697)

Influence of ultraviolet-B irradiation on engraftment, graft-versus-host disease and graft-versus-leukemia effect in a rat model for allogeneic bone marrow transplantation

Ultraviolet-B (UVB) irradiation is known to inhibit lymphocyte activity and consequently to reduce the incidence of graft-versus-host disease (GVHD) in experimental models for allogeneic bone marrow transplantation (BMT). GVHD is frequently associated with morbidity and mortality, but also with the beneficial graft-versus-leukemia (GVL) effect, demonstrated by a reduction in the incidence of leukemia relapse. In this study, we investigated whether UVB treatment of allogeneic T cells could prevent GVHD while sparing the beneficial GVL effect following allogeneic BMT in the Brown Norway myelocytic leukemia (BNML) rat model analogous to human acute myelocytic leukemia (AML). The dose of UVB required to abolish lethal GVHD in the rat allogeneic BMT model (WAG/Rij donors into BN recipients) was 4000 J/m2. However, this UVB dose simultaneously abrogated all GVL activity mediated by the T cells in the graft, while the radio-protective capacity of rat BM cells was strongly reduced. The number of allogeneic BM cells required to protect lethally irradiated BN rats was increased 50 to 100-fold. It is concluded that UVB acts as a non-selective form of T cell inactivation, and that UVB pretreatment of an allogeneic marrow graft is unlikely to be useful clinically as a preventive measure for GVHD, since other means of reduction of the number of functional T cells are less damaging to bone marrow stem cells.

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.

Multifocal remitting-relapsing cerebral demyelination twenty years following allogeneic bone marrow transplantation

We report a case study of a female who received an allogeneic bone marrow transplantation (BMT) from a sex-mismatched related donor and who, after a twenty-year interval, developed an acute fulminant biopsy-proven demyelinating disorder of cerebral white matter which followed a remitting-relapsing chronic course. In situ hybridization studies using Y-chromosome-specific markers revealed Y-chromosome-positive mononuclear cells in biopsy samples of white matter. Magnetic resonance imaging (MRI) studies of the asymptomatic healthy male donor showed multiple white matter lesions. These observations suggest that donor lymphocytes were sensitized to central nervous system (CNS) antigens prior to or at the time of transplantation but remained dormant for 20 years before becoming activated to cause widespread demyelination.

Campath-1G in vivo confers a low incidence of graft-versus-host disease associated with a high incidence of mixed chimaerism after bone marrow transplantation for severe aplastic anaemia using HLA-identical sibling donors

We have evaluated the effect of in vivo Campath-1G on engraftment and GVHD in 23 patients with severe aplastic anaemia transplanted from HLA-identical sibling donors. In 14 patients Campath 1g was given pre-transplant for up to 9 days in an attempt to overcome graft rejection (group 1). In nine patients Campath-1G was given pre-transplant, but also continued post-transplant until day +5 to reduce GVHD (group 2). There were three patients with late graft failure in group I following initial neutrophil engraftment, and four cases of grade II+ GVHD. In group II, two patients had early graft failure (no take), and there were no cases of acute GVHD out of seven evaluable patients. One patient in group I developed chronic GVHD of the liver, and two patients (one in each group) had transient localised chronic GVHD. PCR of short tandem repeats was used to evaluate chimaeric status in 13 patients. Of 11 patients with initial neutrophil engraftment, only one had 100% donor haemopoiesis at all times. The remaining patients had either transient mixed chimaerism or persistence of recipient (< 20%) cells. We conclude that in vivo Campath-1G is associated with a high incidence of mixed chimaerism which tips the balance away from GVHD but towards graft rejection.

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.

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.

Ultraviolet irradiation for the prevention of graft-versus-host disease and graft rejection in bone marrow transplantation

This review describes an approach to the prevention of graft-versus-host disease (GVHD) and graft rejection following allogeneic BMT that differs from conventional methods. Ultraviolet (UV) irradiation inhibits the proliferative responses of lymphoid cells to mitogens and alloantigens by inactivation of T lymphocytes and dendritic cells, and in animal models this can prevent both GVHD and graft rejection. It is important that the marrow repopulating capacity of haemopoietic stem cells is not damaged by the irradiation process. We have found that polymorphic microsatellite markers are a sensitive way of assessing the impact of UV irradiation on chimerism after BMT in rodents.

Infection of donor lymphocytes with human T lymphotrophic virus type 1 (HTLV-I) following allogeneic bone marrow transplantation for HTLV-I positive adult T-cell leukaemia

Human T lymphotrophic virus type 1 (HTLV-I) associated leukaemia has a poor prognosis even with chemotherapy. We describe a patient with adult T-cell leukaemia treated with allogeneic bone marrow transplantation from an HTLV-I negative identical sibling donor. During follow-up after bone marrow transplantation, HTLV-I could be repeatedly isolated inspite of anti-viral prophylaxis. The patient died of an acute encephalitis and HTLV-I could be detected in autopsy material from the brain. By a PCR-based technique using short tandem repeats (STRs) it was shown that the patient's haemopoiesis was of donor origin. This shows the infection of donor cells in vivo by an aetiological agent which has been implicated in the leukaemogenic process for adult T-cell leukaemia.

Successful bone marrow transplant for Fanconi anaemia in transformation

We present a patient who was diagnosed as suffering from Fanconi anaemia at the age of 36 years. At the time of diagnosis his bone marrow showed features of pre-leukaemic transformation. He received an allogeneic bone marrow transplant (BMT) from his HLA-identical sibling. The post-transplant course was unremarkable with evidence of trilineage engraftment at day +32 and no acute or chronic GVHD. He is well with sustained engraftment and no haematological evidence of Fanconi anaemia 18 months post-transplant.

Donor leukemia following allogeneic bone marrow transplantation

Although allogeneic bone marrow transplantation has been shown to be a highly effective treatment for acute and chronic leukemia, leukemic relapse remains a significant problem. Leukemic relapse occurs in recipient cells in the majority of cases, but the paucity of donor cell leukemias may reflect the sensitivity of the investigative technique. We have developed a highly sensitive technique to identify the origin of all hematopoietic cells in the post transplant state which is based on PCR amplification of microsatellites, polymorphic tandem repetitive elements. We have identified donor leukemia (AML M5) following a sex matched BMT for severe aplastic anemia, verified a previously reported case of donor leukemia following BMT for chronic granulocytic leukemia and recently identified an acquired cytogenetic abnormality(del 11q23) in donor cells four years following an apparently successful BMT for AML. In all cases the donors have remained healthy. Postulated mechanisms include transfer to the transplanted marrow of a dormant oncogene residing in the DNA of either a virus, the chromosomes of degenerating irradiation damaged host leukemic cells or in the marrow stroma which is radioresistant and host in origin following BMT. Using sensitive techniques donor leukemia has been shown to be a more common event than was previously thought and an understanding of its pathogenesis may allow us to elucidate leukemogenic mechanisms in man.