Early emergence of PNH-like T cells after allogeneic stem cell transplants utilising CAMPATH-1H for T cell depletion.

CAMPATH-1H (C-1H) is widely used in vivo and / or in vitro for T cell depletion in hematopoietic SCT. This humanised monoclonal antibody is specific for CD52, a marker coexpressed on the majority of human lymphocytes with CD48 and other glycosylphosphatidyl-inositol (GPI) anchored proteins. We detected CD52 / CD48 dual expression on >99% of CD3(+) lymphocytes from normal individuals and all 15 post-SCT patients whose transplants did not utilise C-1H. By contrast, 23 / 26 patients with transplants involving C-1H (in vivo, in vitro or both) exhibited populations lacking CD52 expression that accounted for 49.7% (4.2-86.2%) of the CD3+ lymphocytes (median and range) in samples evaluated at a median of 2 months post-SCT. Most CD52- cells also lacked CD48 expression. These GPI- T cells were of either donor or mixed donor / recipient origin. They were predominant in the early months after SCT at times of profound lymphopenia and inversely correlated with the recovery of the absolute lymphocyte count (r= - 0.663, P<0.0001). The presence of CD52- cells has been correlated previously with clinical outcome after CAMPATH therapy for both malignant and nonmalignant diseases.

Factors affecting the outcome of stem cell transplantation from unrelated donors for childhood acute lymphoblastic leukemia in third remission.

Between July 1990 and March 2002, 35 consecutive children with ALL in third complete remission (CR3) underwent stem cell transplantation (SCT) from unrelated donors (UD). All patients received CAMPATH-1M 5-20 mg daily for 5 days. Grafts were T-cell depleted in 30 patients, 29 by CAMPATH antibodies and one by CD34 selection. Median follow-up was 3.8 years (0.3-9.3). Event-free survival (EFS) at 3 years was 35% (SE 8%); relapse rate and transplant-related mortality (TRM) at 3 years was 42 and 23%. Short first complete remission (CR1) <2.5 years was associated with lower EFS (P=0.001), higher TRM (P=0.019) and higher relapse rate (P=0.023). Short second complete remission (CR2) <2.5 years was associated with lower EFS (P=0.003) and higher TRM (0.009). Higher relapse rate and lower EFS were associated with isolated first extramedullary relapse (P=0.019, 0.012). There was no significant difference in outcome between mismatched unrelated donor stem cell transplantation (MMUD-SCT) and matched unrelated donor stem cell transplantation (UD-SCT). We conclude that UD-SCT is an effective treatment of ALL in CR3. The outcome remains limited by TRM and a high relapse rate. Short duration of CR1 and of CR2 and extramedullary site at first relapse are particularly adverse. MMUD should also be considered in high-risk patients, since the outcome of MMUD appears similar to MUD.

Gram-negative bacteraemia (GNB) after 428 unrelated donor bone marrow transplants (UD-BMT): risk factors, prophylaxis, therapy and outcome.

Gram-negative infection is an important cause of morbidity and mortality after unrelated donor-bone marrow transplantation (UD-BMT). We performed a retrospective case-control study to examine the risk factors, prophylaxis, therapy and outcome of Gram-negative bacteraemia (GNB) in 428 patients undergoing UD-BMT. The incidence of GNB was 3.6% in children and 19% in adults. Of the adults, 11% developed GNB >60 days post UD-BMT. Predisposing risk factors for GNB included 'high-risk' disease status, chronic graft-versus-host disease and use of systemic steroids. Fever, a raised C-reactive protein (CRP) and hypotension were common findings at presentation. Patients were routinely given prophylactic ciprofloxacin: resistance to this antibiotic was seen in 33% of isolates. We identified an age-matched control group undergoing UD-BMT over the same time period as the study group. Gram-positive bacteraemia was significantly more common in cases than controls. Mortality from GNB was 17% in children and 24% in adults. We conclude that GNB is a common complication of UD-BMT with a high associated mortality. Patients should be educated further to present rapidly with symptoms suggestive of infection.

Sacrococcygeal teratomas: the UK Children's Cancer Study Group's experience. I. Neonatal.

The aim of this study was to review the United Kingdom Children's Cancer Study Group (UKCCSG) experience of sacrococcygeal teratomas (SCT) including histological presentation, response to surgery and chemotherapy, and long term effects of the tumour and treatment. This paper presents the results for those children diagnosed during the neonatal period. Children aged up to 4 weeks with biopsy proven localised or metastatic sacrococcygeal germ cell tumours were eligible. From 1st January 1989 to 31st December 1997 (9 years), 15 UKCCSG centres registered 51 neonates with SCT into GC 8901. Surgery alone was performed in all and the prognosis was good - except for 1 baby who died from massive haemorrhage at the initial operation and 1 who died from the complications of prematurity. Seven of the 51 children (14%) who had teratomas in the neonatal period (5 mature, two immature) had yolk sac tumour (YST) recurrence at: 4, 12, 15, 20, 20, 28 and 32 months of age. These children received chemotherapy in the form of etoposide/bleomycin/carboplatin (JEB) and are alive and well at review. These results emphasise the need for oncological follow-up of SCT and the good response to JEB chemotherapy of malignant teratomas and YST.

The clinical relevance of detection of minimal residual disease in childhood acute lymphoblastic leukaemia.

Risk directed treatment forms a central component of modern protocols for childhood acute lymphoblastic leukaemia (ALL). A review of recent studies of minimal residual disease (MRD) analysis shows that it is a powerful prognostic factor in both first line and relapse treatment. However, the value of MRD analysis is both time point and protocol specific, and the threshold for MRD detection of the technique used impacts upon the results obtained. MRD analysis does have a useful role to play in the risk directed treatment of childhood ALL, and this is currently being investigated in large prospective studies.

Minimal residual disease (MRD) status prior to allogeneic stem cell transplantation is a powerful predictor for post-transplant outcome in children with ALL.

We have retrospectively investigated the relationship between the level of minimal residual disease (MRD) detected in bone marrow taken prior to conditioning therapy and outcome following stem cell transplantation for high risk childhood ALL. Forty-one patients, in whom both a molecular marker of MRD and sufficient archival material was available, were included in the study. All were in remission at BMT: eight in CR1, 32 in CR2 and five in greater than CR2. MRD was measured by PCR amplification of antigen receptor gene rearrangements and clone-specific oligoprobing, the median sensitivity of detection being one leukaemic cell in 10000 normals. Results were classified as high-level positive (if a clonal band was evident after electrophoresis), low-level positive (if MRD was detected only after oligoprobing) and negative. MRD was detected at high levels in 17 patients, at low levels in 10 patients and 14 patients were MRD negative at the time of transplant. The 5-year event-free survival for these groups was 23%, 48% and 78%, respectively (P = 0.022). Limited multivariate analysis confirmed the significance of MRD (P = 0.0095) vs CR status, donor type, sex, immunophenotype and acute GvHD. This study confirms the strong relationship between MRD level and outcome following allogeneic transplantation. In contrast to a previous study we observed that a minority of children with high-level pre-BMT MRD can enter long lasting remission. The possible role for acute GVHD coupled with a graft-versus-leukaemia effect in the clearance of high level MRD in patients with ALL is discussed.

The use of zanamivir to treat influenza A and B infection after allogeneic stem cell transplantation.

The use of zanamivir in seven patients with influenza (three A and four B) post allograft is described. Inhaled zanamivir (10 mg twice daily) was continued from the diagnosis of influenza until excretion of virus ceased (median duration 15 days, range 5 to 44 days). There was no toxicity attributable to zanamivir and rapid resolution of influenza symptoms was seen. There was no mortality due to influenza in the seven patients. The good outcome of 30 previous patients with influenza post transplant is described. A randomised multicentre study would be required to demonstrate efficacy.

An optimised biphasic culture system for the generation of functional dendritic cells from patients with acute lymphoblastic leukaemia at presentation and in clinical remission.

We have tested the hypothesis that functional dendritic cells (DC) may be generated from patients with acute lymphoblastic leukaemia (ALL). We evaluated the production of DC from blast cells taken at presentation from nine children with ALL. Blast cells were expanded in serum-free medium supplemented with Flt3L, G-CSF, GM-CSF, IL-3, IL-6 and SCF for 7 days and subsequently stimulated with Flt3L, GM-CSF and TGF-beta for a further 14 days, with the addition of TNF-alpha for the final 48 h of culture. Cultured cells had the morphological appearance of DC and expressed the DC-associated antigens CD1A (range 2-87%) and CD83 (15-44%). Expression of the co-stimulatory molecules CD80 and CD86 was increased and the majority of these cells retained their expression of CD34 (73+/-4%) and HLA-DR (79+/-5%). Seven of the nine ALL had a leukaemia-specific abnormality and DC generated from five of these seven cases were derived from the leukaemic clone. Leukaemic DC derived from four HLA-A*02-positive ALL pulsed with CMV-associated peptides could induce significant proliferation of peptide-specific CD8+ T cells. This specificity was verified using tetrameric complexes of HLA class l/antigenic peptide. DC could also be generated from cells taken at times of complete remission of ALL and from normal controls using these culture conditions. These findings show that functional DC can be generated both from ALL blasts and from patients in remission; these might be utilised in future for immunotherapeutic strategies in the treatment of ALL.

Second malignancies in children: the usual suspects?

The aim of this article is to provide an up to date review of second malignant neoplasms (SMN's) following treatment for childhood cancer, referring to their incidence, the role of genetic factors, and how the primary malignancy and treatment received influence the type, site and prognosis of SMN's. The role of genetic factors will be discussed as far as they impact upon a predisposition to later development of SMN's. The primary malignancies that have important associations with SMN's will then be discussed, in particular Hodgkin's disease, retinoblastoma and acute lymphoblastic leukaemia. The important second malignancies will be highlighted, including tumours of the CNS and thyroid, osteosarcoma, secondary acute myeloid leukaemia and melanoma. Emphasis will be put upon identifying which patients are most likely to suffer from these tumours. An important part of the article are case histories. These are provided in combination with illustrations as a useful adjunct to the text, with a particular emphasis on radiological features, diagnosis and screening. Finally, the important but different roles of causal agents, in particular chemotherapy and radiotherapy are highlighted.

The clinical features, risk factors and outcome of thrombotic thrombocytopenic purpura occurring after bone marrow transplantation.

In this study, we retrospectively analysed the clinical features, risk factors and outcome of 22 patients with thrombotic thrombocytopenic purpura (TTP) occurring after allogeneic stem cell transplantation. All but two of these patients received stem cells from unrelated donors (UDs), two-thirds were female, three-quarters were adults and leukaemia was the major reason for transplant. The incidence of TTP was 20 out of 332 patients (6%) with UD transplants and two out of 104 recipients (2%) of matched sibling allografts (P = 0.16). In order to ascertain basic demographic risk factors for the development of TTP, we compared the 22 patients with 434 patients who did not develop TTP. Compared with patients who did not develop TTP, patients with TTP were nearly three times older (P < 0.001) and were more than twice as likely to be female (P = 0.001). Because > 90% of patients were recipients of UD marrow, we then compared the 20 UD-bone marrow transplantation (BMT) patients with 60 randomly selected UD-BMT patients who did not develop TTP. On univariate analysis, age and female gender were again significant risk factors, as was grade II-IV acute graft-versus-host disease (GvHD) (P = 0.002), and there was a trend towards an association with chronic GvHD (P = 0.083). However, after logistic regression analysis, only age and sex remained significant (P < 0.001 and 0.009 respectively). We report an 86% mortality with only three survivors out of 22 patients, and one of these remains thrombocytopenic and red cell transfusion dependent, possibly in part because of graft hypoplasia. Six out of 17 patients responded to plasmapheresis, but the majority of them ultimately succumbed because of TTP, often in association with GvHD or fungal infection.

Second malignancies in children: the usual suspects?

The aim of this article is to provide an up to date review of second malignant neoplasms (SMN's) following treatment for childhood cancer, referring to their incidence, the role of genetic factors, and how the primary malignancy and treatment received influence the type, site and prognosis of SMN's. The role of genetic factors will be discussed as far as they impact upon a predisposition to later development of SMN's. The primary malignancies that have important associations with SMN's will then be discussed, in particular Hodgkin's disease, retinoblastoma and acute lymphoblastic leukaemia. The important second malignancies will be highlighted, including tumours of the CNS and thyroid, osteosarcoma, secondary acute myeloid leukaemia and melanoma. Emphasis will be put upon identifying which patients are most likely to suffer from these tumours. An important part of the article are case histories. These are provided in combination with illustrations as a useful adjunct to the text, with a particular emphasis on radiological features, diagnosis and screening. Finally, the important but different roles of causal agents, in particular chemotherapy and radiotherapy are highlighted.

T cell-depleted unrelated donor bone marrow transplantation for acute myeloid leukemia.

The outcome for 39 patients with acute myeloid leukemia (AML) in remission who had CAMPATH 1M T cell-depleted unrelated donor bone marrow transplantations (BMTs) is described. Conditioning was mainly with cyclophosphamide (120 mg/kg) and total body irradiation (TBI) (14.4 Gy), but 5 patients received busulfan in place of TBI and 200 mg/kg cyclophosphamide. All patients received cyclosporin, and short-course methotrexate was given to recipients of mismatched grafts. The patient population was predominantly pediatric (median age, 10 years), but one third of the patients was aged 15 years or above. Twenty-five patients were in second complete remission (CR2), and 14 had high-risk CR1 disease (primarily failed remission induction or antecedent myelodysplastic syndrome, often with complex cytogenetic abnormalities). Both recipient and donor were cytomegalovirus seronegative in 15 of 37 cases (38%); 51% of patients were matched for HLA class I and II. Grade II to IV acute graft-versus-host disease (GVHD) occurred in 24% of patients; chronic GVHD occurred in 5 of 31 evaluable patients (16%), 4 extensive and 1 limited. Relapse occurred in 5 cases (13%); 1 of these 5 patients survives, 24 months after a second unrelated donor transplantation. Two of these relapses were associated with secondary graft failure (incidence rate, 5%). All patients engrafted primarily. Severe viral infection was the major transplant-associated complication, with 12 episodes in 9 patients, 5 of them lethal. Twenty-five patients survive at a median follow-up of 44 months (range, 2-102 months), with estimated actuarial overall and disease-free survival rates at 44 months of 61% (SE 8%) and 57% (SE 8%), respectively. Nineteen patients are more than 2 years post-BMT and may be cured. The functional status of long-term survivors is excellent, with 19 of 21 patients who survive 6 months or more in full-time employment or full-time students. These encouraging results suggest that in patients lacking a sibling donor, unrelated donor BMT for AML in remission achieves survival figures as good as or better than those reported on patients with autologous stem cell transplantation, and that T-cell depletion of grafts is associated with a low relapse rate and an excellent functional status. However, only a randomized study comparing unrelated donor BMT and auto-grafting will resolve which of these treatment strategies is better for patients with AML.

The United Kingdom Children's Cancer Study Group's second germ cell tumor study: carboplatin, etoposide, and bleomycin are effective treatment for children with malignant extracranial germ cell tumors, with acceptable toxicity.

PURPOSE: To evaluate carboplatin, etoposide, and bleomycin (JEB) in children with malignant extracranial germ cell tumors (GCTs). PATIENTS AND METHODS: Malignant GCTs in children aged 0 to 16 years were excised without major morbidity or otherwise biopsied. Stage I testicular and some ovarian GCTs were resected and monitored with alpha-fetoprotein (AFP) ("watch-and-wait" approach). Patients with recurrent stage I disease and all other patients received JEB (etoposide 120 mg/m(2) on days 1 through 3, carboplatin 600 mg/m(2) on day 2, and bleomycin 15 mg/m(2) on day 3). Courses were administered every 3 to 4 weeks until remission, and then two more courses were given. Chemotherapy toxicities were assessed using World Health Organization or Brock grading. RESULTS: Between January 1989 and December 1997, 192 patients were registered. Eight were excluded because either there was no histologic diagnosis (n = 3) or chemotherapy was given off-study (n = 5). The remaining 184 patients had germinoma (n = 20), malignant teratoma (n = 55), embryonal carcinoma (n = 1), yolk sac tumor (n = 107), or choriocarcinoma (n = 1). Forty-seven patients were treated with surgery alone, and 137 patients received JEB. The 5-year survival rate in March 1999 for all 184 patients was 93.2% (95% confidence interval [CI], 87.9% to 96.3%); for the 137 JEB-treated patients, it was 90.9% (95% CI, 83.9% to 95.0%), with an event-free survival rate of 87.8% (95% CI, 81.1% to 92.4%). The median follow-up after JEB treatment was 53 months (range, 0 to 109 months); the median number of courses was five (range, three to eight). Site, stage, and AFP level had prognostic significance. Nonfatal hematologic toxicity was common, but deafness and pulmonary and renal toxicities were rare. One child died of a thoracic tumor and bronchopulmonary dysplasia, and another died of acute myeloid leukemia. CONCLUSION: Conservative surgery, a watch-and-wait approach after complete excision, and JEB for those requiring chemotherapy produced high cure rates and few serious complications.

Comparison of allogeneic transplant versus chemotherapy for relapsed childhood acute lymphoblastic leukaemia in the MRC UKALL R1 trial. MRC Childhood Leukaemia Working Party.

BACKGROUND: Although reinduction rates are good for children with relapsed acute lymphoblastic leukaemia there is no consensus on whether bone marrow transplantation (BMT) is the most effective treatment to prolong second remission. PATIENTS AND METHODS: Analyses comparing the outcome of related donor allogeneic BMT (related allograft) with chemotherapy are unreliable because of selection biases. To avoid these biases, the MRC UKALL R1 trial was analysed by HLA-matched donor availability. RESULTS: No significant difference in outcome was found between the donor and no donor groups. The donor group had a non-significant eight-year event-free survival (EFS) advantage of 8%, (95% confidence interval -9%-24%) over the no donor group. Patients with a first remission less than two years appeared to benefit most from having a donor, although the effect was only marginally significantly different from patients with longer first remission. Analysis by treatment received gave similar results, with BMT patients having a 5% (P = 0.8) eight-year EFS advantage over patients who received chemotherapy. CONCLUSIONS: Related allograft was not found to be significantly better than chemotherapy, but there was the possibility of a moderate EFS benefit with related allograft. especially in patients with a short first remission.

Functional analysis of the CD8+CD57+ cell population in normal healthy individuals and matched unrelated T-cell-depleted bone marrow transplant recipients.

The biological activities of CD8+ that co-express CD57 remain poorly defined. It is unclear whether all CD8+ cells have the potential to become CD57+ or whether they represent a unique subset with distinct functions. Several studies have reported the association between elevated numbers of CD8+CD57+ and a wide range of clinical disorders such as viral reactivation of human cytomegalovirus (HCMV). In this study, we have investigated the relationship between viral reactivation and the effect of diminished interleukin (IL)-2 production. Using CD8+ cells isolated from patients at various times after allogeneic transplants and in vitro models of HCMV infection, we determined their combined effect on CD8+CD57+. Our results show that high numbers of CD8+CD57+ correlated with diminished killing of HCMV-infected targets. In addition, we showed a synergistic effect between IL-2 and HCMV in the expansion of CD8+CD57+ cells. Furthermore, these cells after anti-CD3 stimulation did not produce tumour necrosis factor (TNF)-alpha or interferon (IFN)-gamma. Interestingly, IL-10 production was elevated in several patients which appeared to be associated with the time from transplant.

Malignant infantile osteopetrosis presenting with neonatal hypocalcaemia.

Presentation characteristics were reviewed in 14 children from 12 families with malignant infantile osteopetrosis seen at two large referral centres for bone marrow transplantation. Children from six of these families presented initially with symptoms of hypocalcaemia. These comprised early or late neonatal convulsions in six cases (corrected serum calcium < 1.5 mmol/l), and vomiting and irritability (serum calcium 1.68 mmol/l) in another. One other related child had severe and persistent jittering episodes almost certainly attributable to hypocalcaemia. In seven of eight cases, these symptoms developed during the first 14 days of life. Although occasionally reported previously, malignant infantile osteopetrosis remains essentially unrecognised as a cause of neonatal hypocalcaemia, often resulting in diagnostic confusion and delay. This is important in the context of curative haemopoietic stem cell transplantation where preservation of sight may depend on early intervention.