James F. Beck

Increasing Mixed Chimerism Is an Important Prognostic Factor for Unfavorable Outcome in Children With Acute Lymphoblastic Leukemia After Allogeneic Stem-Cell Transplantation: Possible Role For Pre-Emptive Immunotherapy?

PURPOSE We recently reported that children with acute leukemias who show increasing mixed chimerism (MC) after allogeneic stem-cell transplantation have a significantly enhanced risk of relapse. Here we present the results of a prospective multicenter study to investigate (1) whether relapse of acute lymphoblastic leukemia (ALL) can be determined in advance by serial analysis of chimerism, and (2) if outcome can be influenced by withdrawal of immunosuppression and/or by low-dose donor lymphocyte infusion when increasing MC is detected. PATIENTS AND METHODS Serial and quantitative analysis of chimerism was performed using a fluorescent-based short-tandem-repeat-polymerase chain reaction in 163 children with ALL. RESULTS One hundred one patients revealed complete chimerism (CC) or low-level MC (CC/low-level MC); increasing MC was found in 46 patients; and decreasing MC, in 16 patients. Relapse was significantly more frequent in patients with increasing MC (26 of 46) than in patients with CC/low-level MC (eight of 101) or in patients with decreasing MC (0 of 16; P <.0001). The probability of 3-year event-free survival (EFS) was 54% for all patients, 66% for patients with CC/low-level MC (n = 101), 66% for patients with decreasing MC (n = 16), and 23% for patients with increasing MC (n = 46; P <.0001). Of the 46 patients with increasing MC, 31 received immunotherapy. This group had a significantly higher 3-year EFS estimate (37%) than the 15 patients who did not receive immunotherapy (0%; P <.001). CONCLUSION Serial analysis of chimerism reliably identifies patients at highest risk to relapse. The 3-year EFS of patients with increasing MC without immunotherapy was 0%, by which overt relapse could be prevented in a considerable group of patients.

Serial and quantitative analysis of mixed hematopoietic chimerism by PCR in patients with acute leukemias allows the prediction of relapse after allogeneic BMT

Within a prospective study we analyzed hematopoietic chimerism in serial peripheral blood samples taken from 55 patients with acute leukemias (ALL 21, AML 20, MDS 14) with a median age of 13.5 years at very short time intervals following allogeneic bone marrow transplantation (allo-BMT). The investigation was performed to determine the implications of mixed hematopoietic chimerism (MC) with regard to the clinical outcome in patients with acute leukemias after allo-BMT. Analysis of chimerism was performed by PCR of variable number of tandem repeat (VNTR) sequences with a maximum sensitivity of 0.8%. Thirteen male patients transplanted with the marrow of a female donor were also studied by amplification of a Y-chromosome- specific alphoid repeat (0.1–0.01% sensitivity). VNTR analysis in 55 patients revealed complete chimerism (CC) in 36 cases, MC in 18 follow-ups and autologous recovery in one patient. Quantitative analysis of MC identified 10/18 patients with increasing autologous patterns in whom 9/10 subsequently relapsed. The patient with autologous recovery relapsed as well. Eight of 18 patients with MC showed decreasing amounts of autologous DNA and became CC upon further follow-up. In contrast, only 7/36 patients with CC in the prior analysis of chimerism status relapsed. However, in 4/7 patients the interval between last CC confirmation and relapse was more than 4 months. In 2/7 patients autologous DNA was not detectable in peripheral blood but in bone marrow aspirates. One of these seven patients developed a fulminant relapse within 3 weeks. The probability of relapse-free survival for patients with CC is 0.67 (n = 36), for patients with decreasing MC 1.0 (n = 8) and for patients with increasing MC 0.1 (n = 10). In summary, the results demonstrate that serial and quantitative chimerism analysis at short time intervals by PCR provides a reliable and rapid screening method for the early detection of recurrence of underlying disease and is therefore a prognostic tool to identify patients at highest risk of relapse.

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.

Prevention of relapse in pediatric patients with acute leukemias and MDS after allogeneic SCT by early immunotherapy initiated on the basis of increasing mixed chimerism: a single center experience of 12 children

The success of allogeneic stem cell transplantation (allo-SCT) in children is mainly affected by relapse or graft rejection. We have recently shown in a study of 55 patients with acute leukemias (ALL 21, AML 20 and MDS 14), that patients who demonstrate increase amounts of autologous marrow repopulation (increasing mixed chimerism) have a significantly enhanced risk of relapse (P < 0.0001). based on these findings, we asked whether post-transplant relapse can be prevented by withdrawal of immunosuppression and/or by donor lymphocyte infusion (dli). we describe the results of a pilot study where adoptive immunotherapy was used to treat 12 patients (five all, three aml, four mds) who showed increasing mixed chimerism (mc) post-transplant. a response to immunotherapy, defined as the re-establishment of complete chimerism (cc) and continuous complete remission (ccr), was achieved in four patients (two all, two aml) following withdrawal of csa and in a further six patients (three all, three mds) after additional dli. one all patient, who initially responded to dli, developed severe gvhd that required further immunosuppression. gvhd was controlled but this patient subsequently relapsed. another patient with all became a cc but developed an isolated relapse in the bone marrow 260 days later. one patient with mds developed severe gvhd after dli and died. two children (one aml and one mds) did not show any response to interventional treatment and died due to relapse. of the 12 patients treated, seven remain in ccr at a median follow-up of 747 days (range 351–1109 days). in summary, these results provide evidence that increasing mc can be used to guide adoptive immunotherapy strategies and that these treatment modalities can be used to prevent relapse in children with acute leukemias or mds after allo-sct.

Mixed hematopoietic chimerism after allogeneic bone marrow transplantation: the impact of quantitative PCR analysis for prediction of relapse and graft rejection in children.

It still remains unclear whether patients with mixed hematopoietic chimerism (MC) after allogeneic bone marrow transplantation (allo-BMT) have an increased risk of developing relapse or graft failure. To address this question, we monitored the individual dynamics of chimerism after allo-BMT in pediatric patients within a prospective case control study. The individual ratio of donor to recipient peripheral white cells was determined by quantification of genomic variable number of tandem repeats (VNTRs) with a polymerase chain reaction (PCR) approach. Within the study period from 1 January 1994 until 1 July 1996 we investigated 50 sequences of 46 pediatric patients after allo-BMT (32 with malignant, 18 with nonmalignant diseases). We found complete chimerism (CC) in 34/50 cases, MC in 12/50 follow-ups and 4/50 patients revealed autologous recovery (AC). Eight of 12 patients with MC showed increasing autologous patterns and subsequently relapsed or rejected their graft, 3/12 showed decreasing amounts of recipient DNA and turned to CC upon further follow-up. One patient of 12 who had severe combined immunodeficiency (SCID), attained engraftment with a stable MC pattern. Three patients of 34 with CC relapsed lacking a transitional MC interval. However, the time span between last CC confirmation and relapse in each of these three patients was 6 months or longer. We suggest that these patients also developed a stage of transitional MC but that the critical timepoint of molecular confirmation by PCR was missed as time intervals in the individual follow-up of these three patients were too long (⩾6 months). In summary, the results demonstrate that the individual risk of developing relapse or graft failure is significantly enhanced in the MC situation (P < 0.0005). therefore the quantitative analysis of mc at short time intervals might be of great value to identify high risk patients which will have a significantly enhanced risk for relapse or graft rejection.

Increasing mixed chimerism defines a high-risk group of childhood acute myelogenous leukemia patients after allogeneic stem cell transplantation where pre-emptive immunotherapy may be effective

Summary:Children with leukemias and increasing mixed chimerism (increasing MC) after allogeneic stem cell transplantation have the highest risk to relapse. Early immunological intervention was found to be effective in these cases. To substantiate this on a defined group of pediatric acute myelogenous leukemia (AML) patients, we performed serial analysis of post transplant chimerism and pre-emptive immunotherapy in patients with increasing MC. In total, 81 children were monitored, 62 patients revealed complete chimerism (CC), low-level MC or decreasing MC. Increasing MC was detected in 19 cases. Despite early immunological intervention relapse was still significantly more frequent in patients with increasing MC (9/19) than in patients with CC, low-level or decreasing MC (8/62, P<0.005). The probability of 3-year event-free survival (EFS) was 52% for all patients (n=81), 59% for patients with CC, low-level MC, 60% for patients with decreasing MC (n=62), and 28% for patients with increasing MC (n=19, P<0.005). Patients with increasing MC who received early immunological intervention showed a significantly enhanced probability for event-free survival (pEFS 36%, n=15) compared to patients with increasing MC without intervention (pEFS 0%, n=4, P<0.05). These results prove that pediatric AML patients with increasing MC are at highest risk for relapse and that early immunological intervention can prevent relapse in these patients.

Treatment of neuroblastoma stage 4 with 131I-meta-iodo-benzylguanidine, high-dose chemotherapy and immunotherapy. A pilot study

Disseminated neuroblastoma after infancy has a prognosis of approximately 10-20% with conventional therapy. We investigated the role of high-dose chemotherapy (HDCT) with peripheral blood stem cell (PBSC) rescue in combination with 131I-metaiodobenzylguanidine ([131I-m]IBG). 11 children with neuroblastoma stage 4 were pretreated within the German Neuroblastoma Trial NB90 and included in a high-dose concept for consolidation. Remission was documented by ultrasound, CT, NMR, or [123I-m]IBG scanning. HDCT was a combination of melphalan (180 mg/m2), carboplatin (1,500 mg/m2) and etoposide (40 mg/kg). All children were treated by [131I-m]IBG (0.58 GBq/kg) prior to high-dose treatment. All 11 children were additionally treated with antiGD2 murine- or chimeric-antibody (ch14.18). 4 children had no change to their remission status but three achieved a complete response (from a partial response to first line) and one a partial response (from no response to first line). The other 3 children progressed, 2 dying of their disease. Using Kaplan-Meier analysis, the probability of progression-free survival was 0.70 +/- 0.15 with a median observation time of 19 months. 9/11 children are alive, 8 without progression or relapse, whilst 2 have died of their disease. The combination of mIBG plus high-dose chemotherapy with PBSC support supplemented by immunotherapy with antiGD2 antibody appears to be a feasible and effective treatment regimen for disseminated neuroblastoma in this limited series. Larger numbers of patients should be treated to confirm these results.

Detection of neuroblastoma cells in CD34+ selected peripheral stem cells using a combination of tyrosine hydroxylase nested RT-PCR and anti-ganglioside GD2 immunocytochemistry.

A sensitive assay was developed for the detection of neuroblastoma cell contamination in CD34+ selected and unseparated peripheral blood stem cells (PBSC) used for autologous transplantation in stage 4 neuroblastoma patients. Specifically, we established a non-radioactive nested cDNA-PCR (nPCR) for detection of tyrosine hydroxylase (TH) gene expression combined with anti-disialoganglioside GD2 immunocytochemistry with the murine monoclonal antibody (MAb) 14G2a. Sensitivities of TH nPCR determined with a number of neuroblastoma cell lines and PBSCs correlated to cell line dependent basal TH gene expression levels and ranged from 1:10(4) to 1:10(6). The sensitivity obtained by immunocytochemistry was 1:10(5). We observed the highest PBSC contamination rate of 47% (18/38) among 38 PBSC specimens exclusively obtained from stage 4 neuroblastoma patients by using TH nPCR and GD2 immunocytochemistry in combination. Furthermore, a clinically applied purging method, CD34+ selection by immunoabsorption (CD34+ purity 42.4%), was used on 16 PBSCs. 10/16 (63%) preparations were contaminated prior to CD34+ selection and 56% (9/16) remained contaminated. A significant reduction of neuroblastoma cell contamination by CD34+ selection was not detectable, but the absolute amount of re-infused tumour cells was decreased due to 100-fold smaller cell counts of CD34+ selected grafts used for transplantation. 22 PBSC preparations were used for transplantation. A Kaplan-Meier analysis showed an event-free survival probability of 0.56 +/- 0.22 (n = 9) in the group with contaminated PBSCs versus 0.88 +/- 0.12 (n = 8) with no detectable neuroblastoma-cell contamination. Our data suggest that the combined use of TH nPCR and GD2 immunocytochemistry is optimal to detect contamination and monitor purging strategies.

Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of monoamine transporters in neuroblastoma cell lines: Correlations to meta-iodobenzylguanidine (MIBG) uptake and tyrosine hydroxylase gene expression

Radiolabelled meta-iodobenzylguanidine (MIBG) has been widely used in scintigraphy and targeted radiotherapy in patients with neuroblastoma. Recently, it has been demonstrated that MIBG is incorporated into neuroblastoma cells by the noradrenaline transporter. In vitro experiments on SK-N-SH human neuroblastoma cells performed in the present study showed that uptake of MIBG is inhibited by noradrenaline, more so by dopamine and to a lesser extent, by serotonin, indicating that the respective transporters may also contribute to MIBG uptake. However, neither dopamine nor serotonin transporter gene expression was detected. Noradrenaline transporter gene expression was found in 4 of 6 investigated cell lines, which correlated with specific MIBG uptake. Furthermore, an inverse correlation of noradrenaline transporter and tyrosine hydroxylase gene expression, the key regulatory enzyme of catecholamine synthesis, was observed. These data show that MIBG is specifically incorporated only in neuroblastoma cells in which there is noradrenaline transporter gene expression. Furthermore, the catecholamine status in neuroblastoma cells is regulated by a coordinate expression of the key elements of catecholamine synthesis and reuptake systems.

Histone deacetylase inhibitors induce cell death and enhance the apoptosis-inducing activity of TRAIL in Ewing’s sarcoma cells

PurposeThe present in vitro study was conducted to evaluate the effects of the histone deacetylase inhibitors (HDIs) suberoyl anilide hydroxamic acid (SAHA), sodium butyrate (NaB) and MS-275 applied as single agents or in combination with TRAIL in Ewing’s sarcoma.MethodsCytotoxic activities were assessed by cytofluorometric analysis of propidium iodide uptake, DNA fragmentation and mitochondrial depolarisation as well as by measuring caspase-9 and -3 activities. Cell-surface expression of TRAIL receptors was determined by cytofluorometry, and histone H4 acetylation was assessed by western blot.ResultsAll three HDIs potently induced cell death in the two cell lines explored, SK-ES-1 and WE-68. However, they seemed to differ in their modes of action. SAHA and NaB induced mitochondrial depolarisation as well as caspase-9 and -3 activities, and their cytotoxic effects could be significantly reduced by the pan-caspase inhibitor z-VAD-fmk. MS-275 was a much weaker inducer of caspase-9 and -3 activities as well as mitochondrial injury; consistently, z-VAD-fmk had little effect on MS-275-mediated activities. Combined treatment of HDIs and TRAIL led to an additive effect in SK-ES-1 cells and a supra-additive effect in WE-68 cells. Yet, HDIs did not increase cell-surface expression of TRAIL receptor 2, but rather decreased it. Selective inhibition of caspase-8 in WE-68 cells and HDI treatment of CADO-ES-1 cells, a Ewings sarcoma cell line deficient in caspase-8 expression, revealed that caspase-8 was not required for HDI-mediated apoptosis.ConclusionsThese results suggest that HDIs may be considered as a novel treatment strategy for Ewing’s sarcoma either applied as monotherapy or in combination with TRAIL.