Stem cell transplantation.

Stem cell transplantation in pediatric patients with leukemia has been a matter of debate since the first successful transplantation in a child with aplastic anemia in Germany in 1975. Since then, there has been a long way to implement fully stem cell transplantation strategies into the treatment studies organized within BFM Groups for first line or second line treatment of acute and chronic leukemias and myelodysplastic syndrome. The role of different risk groups, different donors, stem cell sources and alternative approaches as haploidentical transplantation as well have been studied as the impact of chimerism and minimal residual disease. Finally, for all leukemias and myelodysplastic syndromes integrated treatment pathways have been developed, which integrate stem cell transplantation on an evidence base into the treatment of the patient.

Identification and selective depletion of alloreactive T-cells for adoptive immunotherapy.

BACKGROUND: T-cell-depleted allografts may exhibit delayed T-cell recovery, severe infections, and relapse after haploidentical hematopoietic stem cell transplantation (HSCT). Required donor lymphocyte infusions containing nonalloreactive cells may transfer immune function without causing graft-versus-host disease. METHODS: We developed an ex vivo approach for the immunomagnetic depletion of alloreactive CD25+, CD69+, and HLA-DR+ T-cells. To achieve highest rates of alloantigen expression, we cocultured peripheral blood mononuclear cells (PBMNCs) with PBMNCs (A/B*), dendritic cells (A/B* DCs), or cytokine-pretreated PBMNCs (A/B* cyt cells). Functional analyses were performed after depletion. RESULTS: After coculture with PBMNCs (A/B* cells), 29% of T-cells became CD25+, CD69+, and HLA-DR+. In modified mixed lymphocyte reactions (MLR) (A/B* cyt cells and A/B* DCs), 35% and 37% of T-cells became CD25+, CD69+, and HLA-DR+. Alloactivation was confirmed by interferon gamma release and proliferation. Immuno-magnetic depletion produced <1% alloactivated cells. Furthermore, this depletion strategy was allospecific and hardly impaired the immune function of the retained cells. DISCUSSION: The efficiency of immunomagnetic depletion depended on the stimulatory capacity of stimulator cells and was improved by using cytokine-pretreated PBMNCs for alloactivation. Overall, this approach might be a promising strategy for restoration of the immune system, particularly after haploidentical HSCT.

Haematopoietic stem cell transplantation trends in children over the last three decades: a survey by the paediatric diseases working party of the European Group for Blood and Marrow Transplantation.

This paper describes the trends in haematopoietic stem cell transplantation (HSCT) activity for children in Europe over the last three decades. We analysed 31,713 consecutive paediatric HSCTs reported by the European Group for Blood and Marrow Transplantation (EBMT) centres between 1970 and 2002. Data were taken from the EBMT registry and were compared according to period and centre category (paediatric or combined). Since 1996, there has been a significant increase in the number of HSCTs performed exclusively by paediatric centres, as well as in the number of alternative donor HSCTs, and in the use of peripheral blood stem cells (P<0.0001). The number of allogeneic HSCTs (allo-HSCTs) for acute lymphoblastic leukaemia, acute myeloblastic leukaemia and chronic myeloid leukaemia remained stable, whereas it increased for myelodysplastic syndromes and lymphomas, and decreased significantly for non-malignant diseases (P<0.0001). Multivariate analysis showed that younger age, human leukocyte antigen genoidentical donors, HSCT performed after 1996 and transplant centres performing more than 10 allo-HSCT/year were all associated with decreased transplant-related mortality (TRM) (P<0.0001). The number of autologus HSCTs (auto-HSCTs) for acute leukaemia decreased significantly, whereas it increased for solid tumours (P<0.0001). Multivariate analysis showed that both auto-HSCT performed before 1996 and paediatric solid tumours (P<0.0001) had higher TRM. Indications for paediatric HSCT have changed considerably during the last seven years. These changes provide tools for decision making in health-care planning and counselling.

Patterns of cross-resistance to the antifolate drugs trimetrexate, metoprine, homofolate, and CB3717 in human lymphoma and osteosarcoma cells resistant to methotrexate.

Methotrexate (MTX)-resistant sublines of malignant human cells were selected in vitro by stepwise increase in drug concentration in the medium. By this procedure a subline of Burkitt's lymphoma cells (RAJI) was made 290-fold resistant (RAJI/MTX-R), T-cell leukemia cells (CCRF-CEM) were obtained 210-fold resistant (CEM/MTX-R), and 3 MTX-resistant human osteosarcoma lines were selected: TE-85/MTX-R (19-fold resistant; relative to wild-type); MG-63/MTX-R (8-fold resistant); and SAOS-2/MTX-R (200-fold resistant). We also studied a B-cell lymphoblastoid line, WI-L2/m4, that was 13,000-fold resistant. Assay of cellular dihydrofolate reductase (DHFR) showed the following pattern of activity in resistant cell lines, relative to parental cell activity: RAJI/MTX-R, 550-fold increased; CEM/MTX-R, unchanged; TE-85/MTX-R, 4-fold increased; MG-63/MTX-R, 6-fold increased; SAOS-2/MTX-R, unchanged; and WI-L2/m4, 110-fold increased. Measurement of MTX membrane transport showed decreased uptake in CEM/MTX-R and SAOS-2/MTX-R, relative to parental cell lines. The other DHFR-overproducing cells all gave normal initial MTX uptake rates but increased total uptake. The DHFR-overproducing lines all had significant cross-resistance to both metoprine and trimetrexate; the two lines with defective MTX transport were not cross-resistant, and the CEM/MTX-R cells showed collateral sensitivity to these agents. Only minor cross-resistance to homofolic acid was found in all MTX-resistant lines. The highly MTX-resistant RAJI/MTX-R and WI-L2/m4 cells showed minor cross-resistance to the dual inhibitor of thymidylate synthetase and DHFR, CB3717 (5- and 15-fold, respectively). These studies demonstrated that, depending upon the mechanism of resistance, MTX-resistant human tumor cells may be effectively killed by antifolates with different routes of uptake into cells, or with a different enzyme target. Thus, there are at least three functionally distinct classes of folate antagonist with antitumor activity.

Ascorbic acid enhances the effects of 6-hydroxydopamine and H2O2 on iron-dependent DNA strand breaks and related processes in the neuroblastoma cell line SK-N-SH.

Neuroblastoma cells accumulate ascorbic acid and iron. It was hypothesized that these features could be exploited for sensitizing neuroblastoma cells for therapy in combination with reactive oxygen intermediates. In the present study the effects of 6-hydroxydopamine (6-OHDA) and H2O2 on metabolic parameters critical for cell survival were investigated in cells with low and high ferritin content in the presence and absence of ascorbate. Human neuroblastoma SK-N-SH cells were pretreated with 100 microM FeSO4 and 10 microM desferrioxamine, respectively, for 24 h yielding cells with different ferritin contents. The effects of 6-OHDA and H2O2 (25 microM-250 microM) in the absence and presence of 1 mM ascorbic acid on DNA strand break formation, activation of poly(ADP-ribose) polymerase, and finally decrease in NAD+ and ATP concentration were investigated. All these parameters were influenced by 6-OHDA and H2O2 in a concentration-dependent manner in a similar way. The effects were most pronounced in ferritin-rich cells and in the presence of ascorbic acid. Using isolated CCC PM2 DNA, 6-OHDA and ascorbic acid caused strand breaks that were prevented in the presence of mannitol or desferrithiocine. H2O2-mediated strand breaks were observed only in the presence of ascorbic acid. Based on these data and data published by others a model explaining the deleterious effects of ascorbic acid on neuroblastoma cells is presented. It is suggested that continuous application of a high dosage of ascorbic acid might be a useful approach in neuroblastoma therapy.

Cytotoxic effects of m-[131I]- and m-[125I]iodobenzylguanidine on the human neuroblastoma cell lines SK-N-SH and SK-N-LO.

As we have reported recently, the human neuroblastoma cell line SK-N-SH is able to take up and store m-iodobenzylguanidine (mIBG). This is in contrast to several other neuroblastoma cell lines, among which are SK-N-LO cells. Both cell lines were used in cell killing experiments with unlabeled and radioactive-labeled mIBG. Using 1-200 microCi m-[131I]IBG (1 h incubation time), only SK-N-SH cells could to a large extent be destroyed in a dose-dependent manner. This effect is completely caused by the radioactive labeling of the molecule, because unlabeled mIBG proved not to be toxic in the concentration range used in experiments with radiolabeled mIBG (30 nM-3 microM). The killing effect was strongly reduced when m-[131I]IBG with low specific activity (0.2-0.3 mCi/mg) was used instead of 20-30 mCi/mg. Similar effects in both cell lines were obtained using m-[131I]-and m-[125I]IBG. SK-N-SH cells that survived a first treatment with m-[131I]IBG were less sensitive to a second treatment. SK-N-LO cells were more sensitive against m-[131I]- and m-[125I]IBG than SK-N-SH cells if both cell lines are exposed to these radioactive compounds over a long period of time (24 h). The reason that only SK-N-SH cells could be destroyed in short-term incubation experiments is that mIBG is stored for approximately 7 days in these cells only. SK-N-LO cells could only be destroyed to a significant degree if m-[131I]IBG was permanently present in the test system. Bone marrow stem cells (CFU-c) also proved to be sensitive against m-[131I]IBG, although the effects were less pronounced than on SK-N-SH cells.

Specific uptake of m-[125I]iodobenzylguanidine in the human neuroblastoma cell line SK-N-SH.

The uptake of m-[125I]iodobenzylguanidine (mIBG), a compound structurally analogous to the antihypertensive drug guanethidine, was examined in various human cell lines. Of three neuroblastoma lines, SK-N-LO, IMR-32, and SK-N-SH, only the last showed specific uptake of the compound. In contrast, only a nonspecific uptake could be demonstrated for the other neuroblastoma lines, as well as for an osteogenic sarcoma line (SAOS-2) and a melanoma line (IgR 3). Based on analyses of uptake characteristics from Lineweaver-Burk plots it is evident that two different transport mechanisms are responsible for mIBG uptake into SK-N-SH cells: a nonspecific diffusion mechanism, and a specific, active uptake system. The latter was dramatically reduced at 4 degrees compared to 37 degrees, as well as in the presence of ouabain or the absence of oxygen. A competitive inhibition of the transport of mIBG by norepinephrine was observed. When drug-treated SK-N-SH cells were incubated in fresh medium, 20 to 30% of mIBG was still retained in the SK-N-SH cells 24 h after the end of incubation with mIBG, whereas no mIBG was detectable in SK-N-LO cells already after 1 h.

Antibody-targeted superantigens induce lysis of major histocompatibility complex class II-negative T-cell leukemia lines.

CTLs bearing certain T-cell receptor V beta-regions are directed by the bacterial superantigen Staphylococcus enterotoxin A (SEA) to lyse MHC class II-positive cells. In order to extend superantigen-dependent cytotoxicity to MHC class II-negative carcinoma cells, covalent conjugates of superantigen and mAbs against surface markers of these cells have been used. We now describe a novel strategy which allows rapid selection of mAb suitable for superantigen targeting against MHC class II-negative tumor cells. A recombinant fusion protein of protein A and SEA binding to the mAbs CD7 or CD38 was able to mediate T cell-dependent lysis of MHC class II-negative Molt-4 and CCRF-CEM acute lymphatic leukemia cell lines. Lysis was dose dependent and correlated with E:T cell ratio. In contrast, SEA alone did not induce any significant lysis. In order to decrease the MHC class II affinity of the protein A-SEA complex, a point mutation was introduced into SEA (protein A-SEA mu9). The mutated fusion protein had similar potency as protein A-SEA against Molt-4 cells but was 100-fold less active against MHC class II-positive cells. Considering the efficiency and specificity of the mutated SEA protein interacting with mAb in targeting T lymphocytes against MHC class II-negative leukemia cells while only marginally affecting normal MHC class II-positive cells, we suggest the development of SEA-mAb fusion proteins as a potential adjuvant therapy of leukemias.

Susceptibility of multidrug-resistant human leukemia cell lines to human interleukin 2-activated killer cells.

Considering the possibility to overcome drug resistance by other treatment strategies than chemotherapy we investigated the susceptibility of three independently selected multidrug-resistant sublines of the T-lymphoblastoid leukemic cell line CCRF-CEM to lymphokine-activated killer (LAK) cells. We found that two of the multidrug-resistant sublines were significantly less susceptible targets to LAK cells. A third one, however, was as susceptible as the parental CCRF-CEM cell line. Moreover, a multidrug-resistant subline that reverted to an almost drug-sensitive phenotype was observed to be also revertant for resistance against LAK cells. We found an inverse relationship between the expression of the mdr1 gene (P-glycoprotein) and the susceptibility to LAK cells. Verapamil, a calcium channel blocker, while increasing the drug sensitivity of a multidrug-resistant subline, did not induce a reversal of the suppression of LAK susceptibility. The possibility of enhanced resistance to LAK cells of multidrug-resistant cells should be taken into account when one is looking for therapy strategies to overcome multidrug resistance.

How and when should we monitor chimerism after allogeneic stem cell transplantation?

SUMMARY: Chimerism analysis has become an important tool for the peri-transplant surveillance of engraftment. It offers the possibility to realize impending graft rejection and can serve as an indicator for the recurrence of the underlying malignant or nonmalignant disease. Most recently, these investigations have become the basis for treatment intervention, for example, to avoid graft rejection, to maintain engraftment and to treat imminent relapse by pre-emptive immunotherapy. This invited review focuses on the clinical implications of characterization of hematopoietic chimerism in stem cell transplantation.

Late effects after stem cell transplantation (SCT) in children--growth and hormones.

Stem cell transplantation (SCT) has established itself as a very successful therapy in often otherwise unbeatable disorders. In a subset of children and adolescents there are, however, late effects, often as a combination of the underlying disorder, its primary treatment and subsequent SCT. In children and adolescents, disorders of growth and the endocrine system have been observed to occur frequently. The assurance of normal growth, puberty, fertility and thyroid function--including the prevention of secondary malignancies--is of utmost importance for the overall success of treatment and the maintenance of quality of life. This, however, requires a systematic and structured follow-up programme for patients after SCT. Patients and their families need to be made familiar with this concept early and physicians need to understand that such a system must be implemented as part of a comprehensive care.

Rituximab mediates in vitro antileukemic activity in pediatric patients after allogeneic transplantation.

Relapse is a major problem after allogeneic transplantation in children with acute B-lineage lymphoblastic leukemias (ALL) and lymphomas and additional therapeutic strategies are needed to increase graft versus leukemia effects without inducing graft versus host disease (GvHD). Several studies have shown the efficacy of a humanized CD20 antibody (rituximab) for treatment of CD20+ malignancies together with conventional chemotherapy but less is known about its post transplant usefulness. We studied the ability of rituximab to mediate antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) with effector cells and complement from patients who were transplanted with T-cell-depleted grafts from unrelated or mismatched related donors. Highest lytic activity (ADCC) was observed against leukemia-derived MHH4 cells and Burkitt's lymphoma-derived Raji cells in the first months after transplantation, corresponding to the high percentage of regenerating CD56+ CD16+ cells. Moreover, primary cryopreserved ALL-blasts from a pediatric patient were also efficiently lysed. Increased lysis was obtained after stimulation with interleukin-2. Combination of ADCC and CDC had additive effects. These findings encourage clinical trials on the use of rituximab for improving minimal residual disease control and relapse prevention after allogeneic high-risk transplantation in the small group of pediatric patients with CD20+ leukemias/lymphomas.

Survey on haematopoietic stem cell transplantation for children in Europe.

A recent report, prepared in March 2003, regarding the paediatric transplantation activity registered between 1970 and 2002 in the European Bone Marrow Transplantation (EBMT) database showed a decrease in the number of registrations in 2001 and in 2002. In order to validate this observation, the Paediatric Diseases Working Party (PDsWP) secretariat distributed a questionnaire to 395 institutions participating in the EBMT Registry. Each institution was requested to check the number of transplants they reported and to confirm or to correct the figures. As of 15 March 2004, replies had been received from 135 centres reporting a median of 48 transplants per centre over the study period, total 17 891 (58% of the total number). Among them, 55 confirmed their original figures, while 80 corrected the numbers. The overall number of autologous and allogeneic SCTs performed and not reported were 461 and 692, respectively. Most of the teams that corrected their figures stated that their data managers could provide missing data to the EBMT; 260 other teams, each reporting a median of 15 transplants during the study period, total 12 866 (42% of the total number) chose not to reply. A report prepared in March 2004, following the PDsWP survey, showed an increasing number of transplants performed on patients below 18 years of age between 1973 and 2002 and reported to the EBMT Registry (328 autologous and 628 allogeneic) as compared to the 2003 report. This first PDsWP survey, reaching more than 50% of activity in the field, illustrates that the decrease in activity we observed in the 2003 report does not correspond to a decrease in the number of transplants that were actually performed. It demonstrates the compliance of most major paediatric institutions and confirms the important role of cooperation between National Registries and EBMT Registries.

MDR1, MRP, topoisomerase IIalpha/beta, and cyclin A gene expression in acute and chronic leukemias.

Gene expression was analyzed by cDNA-PCR at the mRNA level in bone marrow samples (>80% blasts) from ALL (28 primary, 22 first relapses, 10 recurrent relapses), from AML (14 primary, 23 relapses), In peripheral blood lymphocytes from CLL (five untreated, 10 treated), in one CML in blast crisis in the course of the disease (four samples), and in bone marrow samples from healthy donors (12 specimens). We found low mean MDR1 expression in primary ALL, first relapses of ALL, and primary AML. Significantly higher mean relative MDR1 expression levels were seen in recurrent relapses of ALL, and in the group of relapsed state AML. MDR1 expression measured intermediate in bone marrow samples from healthy donors. The CLL lymphocytes showed generally relatively high MDR1 expression levels. MRP gene expression measured very similar in primary ALL, first relapses of ALL, primary AML, and normal bone marrow. Significantly increased MRP mRNA levels were observed in the groups of recurrent ALL and relapsed state AML. CLL lymphocytes also showed high MRP expression levels. A combined increase of MDRI (about 20-fold) and MRP (about four-fold) was monitored in samples obtained from the CML in blast crisis after chemotherapy. While no significant differences of the mean topoisomerase IIbeta mRNA levels were found throughout, a significantly decreased topoisomerase IIalpha gene expression was measured in first and recurrent relapses of ALL. In CLL lymphocytes either the expression of the topoisomerase IIalpha gene was not detectable by cDNA-PCR, or it measured very low. Topoisomerase IIalpha gene expression was correlated to cyclin A gene expression in the samples of acute leukemias, Indicating the link of topoisomerase IIalpha expression to the proliferative activity of these leukemic blast cells. Our results point to a potentially multifactorial emergence of multidrug resistance in particular states and types of leukemias.

Expression of PKC isozyme and MDR-associated genes in primary and relapsed state AML.

For investigation of relative differences in mRNA expression levels and of correlations in the expression of genes possibly involved in multidrug resistance (MDR) of acute myelogenous leukemias (AML), a complementary DNA polymerase chain reaction (cDNA-PCR) analysis was established for the genes encoding MDR1/P-glycoprotein, the multidrug resistance-associated protein (MRP), topoisomerase II alpha, topoisomerase II beta, topoisomerase I, glutathione S-transferase pi, protein kinase C (PKC) isozymes alpha, beta 1, beta 2, epsilon, eta, theta and cyclin A. In a first descriptive study comprising samples of childhood or adult AML we calculated the mean values from primary (n=14) or relapsed (n=23) states of the diseases, respectively. We found in the latter significant increases of MDR1, MRP, gst pi, and PKC theta gene expression. MDR1 and MRP gene expression levels were generally correlated (rs= +0.4128, P<0.02, n=37), as well as topoisomerase II alpha and cyclin A gene expression levels (rs= +0.8727, P<0.0001, n=35). Within the group of relapsed state AML a significant negative correlation between the gene expression levels of MDR1 and topoisomerase II alpha (rs= -0.5500, P<0.01, n=22) was observed. Remarkably, highly significant positive correlations were found for MDR1/PKC eta (rs= +0.5560, P<0.001, n=32), MRP/PKC theta (rs= +0.6573, P<0.0001, n=34) and MRP/PKC eta (rs= +0.5241, P<0.005, n=32).

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.

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.

Clinical strategies for the treatment of neuroblastoma.

Neuroblastoma is the most common solid extracranial tumour in childhood. In spite of intensive efforts of clinicians and scientists the prognosis for advanced disease is still poor. This paper presents a short review of the state-of-the-art in conventional treatment including surgery, chemotherapy, and radiation. This is followed by a review of the treatment attempts with high dose chemotherapy followed by autologous bone marrow or stem cell transplantation. One of the main problems with this approach is the contaminating tumour cells. Finally the various immunotherapeutic strategies are summarised which are used to remove minimal residual disease. Later, our new approach, combining various treatment modalities, is described.

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.

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.