M. N. Potter

Unrelated donor bone marrow transplantation for children with relapsed acute lymphoblastic leukaemia in second complete remission

Allogeneic sibling bone marrow transplantation (BMT) is the recommended treatment for relapsed childhood acute lymphoblastic leukaemia (ALL), but appropriate donors are only available in 30% of cases. Unfortunately, BMT from unrelated donors (UD) has been associated with high rates of severe graft‐versus‐host disease (GvHD) and transplant‐related mortality (TRM). In an attempt to improve outcome in UD‐BMT we have assessed the impact of T‐cell depletion using CAMPATH‐1 (anti‐CD52) monoclonal antibodies in 50 consecutively referred patients with relapsed ALL in second remission. All were previously treated according to MRC protocols UKALL X and XI, and then given chemotherapy on MRC R1 from relapse until UD‐BMT. 19 patients had relapsed on and 31 off therapy.

Minimal residual disease status as a predictor of relapse after allogeneic bone marrow transplantation for children with acute lymphoblastic leukaemia.

We have analysed the behaviour of minimal residual disease (MRD) after allogeneic bone marrow transplantation (allo-BMT) in 71 children with acute lymphoblastic leukaemia (ALL). The method relied on PCR of IgH, TCRdelta and/or TCRgamma gene rearrangements followed by electrophoretic size resolution and allele-specific oligoprobing. Patients were similarly conditioned; 55 received marrow from unrelated donors and 16 from related donors. MRD was assessed at various time-points up to 24 months after BMT. Three children were not evaluable due to transplant-related mortality. MRD was detected in 28/32 patients (88%) who relapsed post-BMT; 16 were positive at all times and 12 were initially negative but became positive at a median of 3 months (range 1.5-11) prior to relapse. In contrast, only eight of 36 (22%) patients who remained in continuing complete remission (CCR) (median follow-up 43 months, range 20-94) showed MRD at any time after BMT (P<0.0001). In these eight patients MRD was found up to 9 months after transplant and at low levels (0.01-0.001%). All eight (median follow-up 39 months, range 24-87) had at least two MRD-negative samples tested subsequently and five of the eight had evidence of grade I-II acute graft-versus-host disease (GvHD), raising the possibility of a graft-versus-leukaemia effect. In general, any evidence of MRD after allo-BMT is a poor prognostic sign. However, if immunotherapy were to be targeted towards patients with evidence of persisting MRD after BMT, the method described would expose only a small proportion of patients to unnecessary additional toxicity.

PCR assessment of bone marrow status in 'isolated' extramedullary relapse of childhood B-precursor acute lymphoblastic leukaemia.

SUMMARY. Approximately one‐third of first relapses of childhood ALL occur at an extramedullary site without morphological evidence of bone marrow disease. However, the high incidence of subsequent medullary relapse in these cases strongly suggests that leukaemia is present at submicroscopic levels at the time of ‘isolated’relapse. PCR analysis of immunoglobulin heavy chain (IgH) and T‐cell receptor (TCR) gene rearrangements now allows detection of leukaemia at levels as low as 0.001%. We have therefore used this technique to reassess bone marrow status at morphologically isolated relapse in 13 children with B‐lineage ALL (11 with off‐treatment relapses, two on treatment). In 12 of these 13 patients marrow disease was detectable by PCR at the time of this relapse—in all cases at levels below the threshold of light microscopy. Where relapse occurred off‐therapy this indicated re‐emergence of disease, since MRD has never been detected by PCR at this stage in patients remaining in long‐term remission. In both patients who relapsed on‐therapy the level of MRD at the time of relapse represented an increase on that seen in their previous marrow sample. We conclude that re‐emerging bone marrow disease can be detected in most cases of ‘isolated’relapse when investigated by this highly sensitive technique. Our findings at a molecular level confirm a long‐held clinical suspicion and indicate that full systemic re‐induction as well as local therapy is obligatory for these children.

The significance of detection of minimal residual disease in childhood acute lymphoblastic leukaemia

Summary. In acute lymphoblastic leukaemia (ALL), minimal residual disease (MRD) can be defined as disease occurring at a subclinical level and beyond detection by conventional methods of assessment. Application of the polymerase chain reaction (PCR) to the hypervariable segment of the immunoglobulin heavy chain (IgH) gene, allows detection of MRD at a level of one leukaemic cell in 104‐105 normal marrow cells. We have performed a retrospective study using this technique in the assessment of children with precursor B‐cell ALL in whom the clinical outcome is known. In the early treatment period MRD is commonly detected in children who remain in complete remission on subsequent follow up. Thus, the detection of MRD at this time may have little value in the prediction of future relapse. However, at the end of treatment, children who remain in complete remission have no evidence of MRD. Conversely, detectable MRD at this time would seem to predict for future relapse, though this can be a delayed event. Remarkably, in two children who suffered a bone marrow relapse 8.5 and 9 years after completing therapy for their initial disease, MRD was detected, in their end of initial treatment marrow samples. Clearly PCR technology is changing the definition of the remission state in childhood ALL. and may have predictive value in the assessment of children who are at a high risk of future relapse. Large prospective studies of molecular monitoring are now required to confirm these preliminary results.

Second allogeneic bone marrow transplants from unrelated donors for graft failure following initial unrelated donor bone marrow transplantation

Graft failure is a common and severe complication of unrelated donor bone marrow transplantation (UD-BMT). However, there are few reports of a second UD-BMT in this setting. We describe 12 patients with graft failure (five primary, seven secondary) who had a second transplant, five from their original donor and seven from a different donor. Their median age was 9 years. Two patients died before day 10 of regimen-related toxicity. Nine of 10 evaluable patients engrafted in a median of 17 days. Secondary graft failure was seen in one patient. Transplant-related morbidity was significant. Six of nine developed acute GHVD, there were five severe infections and five patients developed Bearman grade 3 or 4 extramedullary toxicity. Overall, five patients survive at a median of 38 months after the second BMT and two are in continuous complete remission. Second transplants from unrelated donors for graft failure can result in prolonged survival.

Comparison of fluorescent consensus IgH PCR and allele‐specific oligonucleotide probing in the detection of minimal residual disease in childhood ALL

The sensitivity of detection of residual disease by two IgH PCR strategies, fluorescent framework 3 (Ffr3) and allele‐specific oligonucleotide probing (ASOP), was compared in 57 ‘remission’ BM samples obtained from 19 children with B‐lineage acute lymphoblastic leukaemia (ALL). Oligonucleotide probing was more sensitive than FFr3 PCR in 10/16 cases, achieving a sensitivity of 0.01% or greater in 15/16 cases. Comparable sensitivities were obtained in the six remaining cases; the FFr3 PCR achieving a sensitivity of 0.1% or greater in 14/16 cases. 39/57 ‘remission’ BM samples analysed showed no evidence of MRD by either technique although 18 were positive by ASOP and 14 positive by FFr3 PCR. The level of disease was estimated to be 0.01% or less in the four false negative samples.

Unrelated donor bone marrow transplantation in children and young adults with acute myeloid leukaemia in remission

The role of unrelated donor bone marrow transplantation (UD‐BMT) in the management of patients with acute myeloid leukaemia (AML) is uncertain. We describe 18 patients with a median age of 13 years (range 4–31) who received an ex vivo T‐cell‐depleted UD‐BMT for AML (13 in second complete remission (CR2) and five in first complete remission (CR1) with high‐risk features). Nine donor recipient pairs were fully matched; eight of these donor–recipient pairs had a single class I HLA mismatch; one patient had both single class I and class II HLA mismatches. Grade II GVHD of the skin occurred in four patients (22%) and limited chronic GVHD in two patients (11%). There have been four deaths: one from relapse and three from infection. With a median follow‐up of 27 months, 14 patients survive and the actuarial event‐free survival at 2 years is 70 ± 20% (95% confidence interval). We conclude that unrelated donor BMT can result in prolonged disease‐free survival in children and young adults with AML.

THE CLINICAL VALUE OF DETECTING GENE REARRANGEMENT IN ACUTE LEUKAMIAS

Introduction The last 2 years have seen the cloning and characterization of the majority of the common recurring chromosomal translocations in human acute leukaemias. This has resulted in the discovery of numerous novel genes of great scientific interest, the function of which remains to be elucidated. The clinical impact of this information in improving patient management has yet to be fully realized. In this annotation we define the translocations which can be detected, suggest how these may improve diagnosis, and describe how they can be used in the subsequent management of patients. We will also discuss how these techniques compare with immunoglobulin heavy chain (IgH) or T-cell receptor (TCR) gene rearrangements when used for the detection of residual disease in lymphoid malignancies.

Matched and mismatched unrelated donor bone marrow transplantation for juvenile chronic myeloid leukaemia

Juvenile chronic myeloid leukaemia (JCML) is a rare haematological condition of childhood curable only by bone marrow transplantation (BMT). We report our experience using matched and mismatched unrelated donor BMT for JCML in five patients. Although the procedure is hazardous in terms of toxicity and relapse, two patients are alive and disease‐free 28 and 49 months post BMT.

A laboratory comparison of T cell depletion by CD34+ cell immunoaffinity selection and in vitro Campath-1M treatment: clinical implications for bone marrow transplantation and donor leukocyte therapy.

Donor leukocyte infusions (DLI) have been used effectively to induce remission in patients who relapse after BMT. Using CD34+ cell immunoaffinity enrichment, donor T cells may be captured in the unadsorbed (residual) fraction and we assessed this as a potential source of functional T cells for post-BMT immunotherapy. We extended our study to compare CD34+ cell selection and antibody-mediated cell lysis using Campath-1M and measured T cell-depletion, CD34+ cell recovery and relative progenitor proliferative potential. The recovery of CD3+ cells (responsive to IL-2 or PHA) in the unadsorbed fraction was 84 ± 12% (mean ± s.d.) using a laboratory scale CD34+ cell selection process (CEPRATE LC). The immunoselected (CD34+ cell enriched) product contained 55 ± 12% of the starting CD34+ cells (purity, 75 ± 6%) with recoveries of 44 ± 12% and 42 ± 13% for CFU-GM and BFU-E respectively. T cell depletion was 99.8 ± 0.2% (FACS) and the frequency of clonable T cells estimated at 1:640 (limiting dilution assay). In comparison, Campath-1M-treated marrow samples gave recoveries of CD34+ cells, CFU-GM and BFU-E of 50 ± 7%, 78 ± 20% and 79 ± 18%, respectively. The frequency of clonable T cells was 1:2700 despite an estimated T cell depletion of 98.4 ± 1.9%. Data obtained from four BM harvests processed on the clinical grade CEPRATE SC system was comparable in every respect to the laboratory scale system. The yield of 1259 ± 222 × 106 CD3+ cells in the unadsorbed fraction would allow for multiple graded incremental T cell aliquots for DLI for patients with acute leukaemia.