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Disparity for HLA in unrelated donor bone marrow transplantation (BMT) increases the risk of graft rejection and graft-versus-host disease (GVHD) and may compromise transplant outcome. We have compared the outcome of matched and mismatched transplants from unrelated donors in 137 children with acute lymphoblastic leukemia (ALL). Their disease status was complete remission (CR)-1, 24 patients; CR-2, 88 patients; CR-3, 18 patients; CR-4, 2 patients; and relapse, 5 patients. CAMPATH monoclonal antibodies were used for T-cell depletion and cyclosporin A was given to 134 children together with short-course methotrexate in 43, mainly when there was HLA disparity. Fifty-two donor/recipient pairs were HLA-mismatched, 41 at HLA-A and -B and 11 at HLA-DR and -DQ loci. Overall graft failure was increased in recipients of marrow mismatched at either HLA-A, -B, -DR, or -DQ (15.7% v 4.8%; P = .057) mainly because there was a higher proportion of children with primary graft failure (11.8% v 1.2%; P = .012). The presence of an HLA-C locus mismatch did not independently increase the likelihood of graft failure. There was no significant difference in the incidence of acute GVHD ≥ grade 2 between the matched and mismatched groups (P = .849). For patients in CR-2, the risk of relapse post-BMT was significantly lower if leukemic relapse occurred off-treatment (P = .005). The Kaplan-Meier overall and leukemia-free survival (LFS) estimates for recipients of matched and mismatched BMT, respectively, at 36 months were 49% versus 42% (P = .380) and 45% versus 40% (P = .654). Although HLA mismatching results in an increased occurrence of primary graft failure with T-cell–depleted allografts, it allows more donors to be identified rapidly for children with ALL without compromising overall transplant outcome.

The efficacy of allografting in acute lymphoblastic leukemia (ALL) is heavily influenced by remission status at the time of transplant. Using polymerase chain reaction (PCR)-based minimal residual disease (MRD) analysis, we have investigated retrospectively the impact of submicroscopic leukemia on outcome in 64 patients receiving allogeneic bone marrow transplantation (BMT) for childhood ALL. Remission BM specimens were taken 6 to 81 days (median, 23) before transplant. All patients received similar conditioning therapy; 50 received grafts from unrelated donors and 14 from related donors. Nineteen patients were transplanted in first complete remission (CR1) and 45 in second or subsequent CR. MRD was analyzed by PCR of Ig or T-cell receptor δ or γ rearrangements, electrophoresis, and allele-specific oligoprobing. Samples were rated high-level positive (clonal band evident after electrophoresis; sensitivity 10−2 to 10−3), low-level positive (MRD detected only after oligoprobing; sensitivity 10−3 to 10−5), or negative. Excluding 8 patients transplanted in CR2 for isolated extramedullary relapse (all MRD−), MRD was detected at high level in 12 patients, low level in 11, and was undetectable in 33. Two-year event-free survival for these groups was 0%, 36%, and 73%, respectively (P < .001). Follow-up in patients remaining in continuing remission is 20 to 96 months (median, 35). These results suggest that MRD analysis could be used routinely in this setting. This would allow identification of patients with resistant leukemia (who may benefit from innovative BMT protocols) and of those with more responsive disease (who may be candidates for randomized trials of BMT versus modern intensive relapse chemotherapy).

Adequate viability after cryostorage is essential to allow engraftment of autologous haematopoietic progenitor cells (HPC). Information on the effect of prolonged cryopreservation on HPC viability in humans is limited although donations are routinely stored for over a year. Long-term storage is expensive and demonstration of clinical efficacy of HPC after long-term cryopreservation is important in order to justify the use of resources. Available reports describe only isolated cases in which old cells have been infused. We have conducted a retrospective survey of transplants using HPC stored >1 year within the National Blood Service in England. In total 75 patients received HPC that had been frozen for >1yr in NBS Laboratories. Patients had AML (21), NHL (21), myeloma (18), CML (5), Hodgkins disease (6) and other diseases (4). Fifty seven patients received PBSCs cryopreserved for >1 yr with median time in storage of 23 (12.1–65) months and median infused CD34 dose of 4.6 (1.2–85) ×106/kg. Neutrophil recovery to >0.5 × 109/l was 12 (4–49) days and platelets to > 20 × 109/l was 13 (3–43) days. Thirteen patients received bone marrow that had been cryopreserved for >1 year (median 32 (13–67) months) with infused TNC dose of 1.7 (0.91 – 3.64) × 108/kg. Time to neutrophils >0.5 × 109/l was 17 (12–95) days and platelets > 20 × 109/l 16 (12–66) days. A further 5 patients received a mixture of PBSCs and BM products all of which had been stored for > 1 year. Delayed neutrophil engraftment (neutrophils

Cytomegalovirus (CMV) represents a major cause of morbidity in patients undergoing allogeneic stem cell transplantation (SCT). Although antiviral prophylaxis has contributed to the reduction of early CMV disease, recovery of CMV-specific cytotoxic lymphocytes is still necessary for the long-term control of CMV reactivation after SCT. It has been demonstrated that CMV-specific CD4+ and CD8+ cytotoxic T lymphocytes from the donor are important in resolving CMV infection and protecting recipients from developing CMV disease post SCT. Recently CMV recombinant protein pp65, an immunodominant target for CD4+ and CD8+ T cell responses, has been developed for the stimulation and isolation of donor CMV-specific T cells. The aim of this study was to investigate the potential of pp65 to generate CMV-specific CD8+ and CD4+ T cells from seropositive subjects for use as adoptive immunotherapy after SCT. Peripheral blood mononuclear cells (5x108 cells) were isolated from single unit CMV-positive buffy-coats and were stimulated with pp65 for 16 hours. CMV-specific T cells that produced IFNγ were captured using the IFNγ-secretion assay and were enriched using the CliniMACS system. The phenotype of the cells, both pre and post enrichment was evaluated by flow cytometry using monoclonal antibodies against CD3, CD4 and IFNγ. Enriched IFNγ producing T cells were cultured with irradiated feeder cells in the presence of IL-2 for 18 days. Alloreactivity experiments were performed by mixing 105 enriched T cells at day 0 and day 18 of culture with 105 third party cells for seven days. Proliferation was assessed by the 3H-thymidine incorporation assay. Experiments to date have shown that pp65 is capable of stimulating both CD4+ and CD8+ T cells to secrete IFNγ. Flow cytometric analyses revealed that prior to enrichment, IFNγ+ cells represented 0.13% of the CD3+/CD4+ cell population and 2.1% of the CD3+/CD4− cell population. Whereas, following magnetic enrichment and an 18-day expansion phase, the proportion of IFNγ+ cells increased significantly (554-fold and 6-fold) to 72% and 13.3% in the CD3+/CD4+ and CD3+/CD4− subfractions, respectively. The alloreactivity of the expanded positive cells had decreased by an average 95.5% (SD=5.7, n=3) compared to prior to enrichment and cell expansion. The proliferation of the expanded cells was still high (Stimulation Index >3), but considerably reduced compared to the cells at day 0. The cytotoxicity of these cells will be assessed to determine their suitability for immunotherapeutic use post-SCT. Large scale CMV-specific T cell capture experiments, performed under GMP conditions, are ongoing.

Cytomegalovirus (CMV) infection is associated with increased transplant related mortality and decreased overall survival after stem cell transplantation (SCT) despite major advances in early detection, prophylaxis and treatment. This effect is most marked in CMV seropositive patients who have a statistically significant decrement in overall or disease free survival of 20–46% when compared to low risk (−/−) transplants (Boeckh Blood 2004 103: 2003–8). Transfer of cellular immunity from a seropositive donor results in reconstitution of T cells to CMV and protection from CMV disease post-SCT, and this underlies the recent development of cellular immunotherapeutic manoeuvres. As a centre looking to develop such therapy, we have carried out a retrospective analysis of a patient cohort in order to estimate how many patients would require this intervention. We used twice weekly quantitative PCR surveillance to analyse the CMV reactivation profiles of 104 recipients of 106 allogeneic SCT, transplanted between April 2002 and April 2004. Hence follow up was 5–27 months. The cohort comprised 41 adults and 65 children. Transplants were from related (42), unrelated (52) and haploidentical donors (12). T cell depletion was performed either (i) in vivo using either CAMPATH-1H (44 transplants) or ATG (5), (ii) ex vivo by either CD34 (8) or CAMPATH-1H in vitro (1), or (iii) by combinations of (i) and (ii) (26). SCTs were for malignancies in 92 cases and non-neoplastic disorders in 14. Many patients were considered high risk, necessitating short search to transplant times. A hierarchy of donor selection factors was considered: HLA matching was the primary determinant, with other factors including stem cell dose, age, gender and CMV status. Seventy-one patients were at risk of CMV reactivation. There were 28 episodes of CMV reactivation in 27 of these patients, with the following Recipient/Donor serostatus combinations: 0 (of 16) −/+, 22 (of 38) +/− and 6 (of 17) +/+. Seven patients (Group A) resolved their infections without intervention. Nine patients (Group B) resolved infections after antiviral drug treatment, whilst the remaining 12 reactivators (Group C) did not clear their CMV DNA load despite antiviral treatment (of whom 3 died, 1 relapsed and the remainder have ongoing CMV PCR positivity). As a minimum a CMV immunotherapy programme should allow prophylaxis or early treatment of all patients in groups B and C. However, using our current selection criteria only 5/21 cases had a CMV seropositive donor. Logistical problems e.g. CTL precursor frequency or availability of an immunodominant tetramer might have rendered some donors inappropriate providers of anti-CMV CTL. Thus a maximum of 5 out of 106 transplanted patients in our unit could have benefited, although this figure could be slightly improved by deliberate selection of CMV positive donors for CMV positive patients. Such numbers should be borne in mind by any centre contemplating the development of antiviral immunotherapy programmes.