Dirk Schwabe

Localized infant neuroblastomas often show spontaneous regression: results of the prospective trials NB95-S and NB97.

PURPOSE The excellent prognosis of localized neuroblastoma in infants, the overdiagnosis observed in neuroblastoma screening studies, and several case reports of regression of localized neuroblastoma prompted us to initiate a prospective cooperative trial on observation of localized neuroblastoma without cytotoxic treatment. PATIENTS AND METHODS For infants with localized neuroblastoma without MYCN amplification, chemotherapy was scheduled only in cases with threatening symptoms; otherwise, the tumor was either resected or observed by ultrasound and magnetic resonance imaging (MRI). RESULTS Of 340 eligible participants, 190 underwent resection, 57 were treated with chemotherapy, and 93 were observed with gross residual tumor. Of those 93 patients with unresected tumors, spontaneous regression was seen in 44, local progression in 28, progression to stage 4S in seven, and progression to stage 4 in four. Time to regression was quite variable, with first signs of regression noted 1 to 18 months after diagnosis and in 15 of 44 patients even after the first year of life. So far, complete regression was observed in 17 of 44 patients 4 to 20 months after diagnosis. Known clinical risk factors were not able to differentiate between patients with regression and regional or metastatic progression. Overall survival (OS; 3-year OS, 0.99 +/- 0.01) and metastases-free survival (rate at 3 years, 0.94 +/- 0.03) for patients with unresected tumors was excellent and was not different from patients treated with surgery or chemotherapy. CONCLUSION Spontaneous regression is regularly seen in infants with localized neuroblastoma and is not limited to the first year of life. A wait-and-see strategy is justified in those patients.

Cellular immunotherapy of malignancies using the clonal natural killer cell line NK-92.

For years activated natural killer (A-NK) cells have been explored with respect to their efficacy in anticancer therapy, but, except for some anectdotal reports, no clear clinical benefit has been shown. However, as the understanding about the interactions of NK cells and tumor cells advances, the use of A-NK cells might be revisited with more sophisticated approaches that pay tribute to mechanisms which allow tumor cells to escape immune surveillance. Here the highly cytotoxic NK cell line NK-92 seems to be an attractive alternative for use in adoptive immunotherapy, because it was shown to exhibit substantial antitumor activity against a wide range of malignancies in vitro as well as in xenografted SCID mice. NK-92 cells are characterized by an almost complete lack of killer cell immunglobulin-like receptors (KIRs) yet conserved ability to perforin and granzyme B-mediated cytolytic activity, which make them unique among the few established NK and T cell-like cell lines. NK-92 is the only natural killer cell line that has entered clinical trials. Here we discuss the current status of development of this cell line for adoptive immunotherapy (AIT) of malignancies and review our first clinical experience in patients with advanced cancer who have received repeated transfusions of irradiated NK-92 in a phase I/II trial. Also we discuss issues that address safety aspects of immunotherapy with clonal cell lines and describe further manipulations, which hold the potential of significantly improving the clinical outcome of AIT with NK-92.

Five-year-survey of invasive aspergillosis in a paediatric cancer centre. Epidemiology, management and long-term survival

The epidemiology, management, and long‐term survival of invasive aspergillosis was assessed in a prospective, 5‐year observational study in 346 unselected paediatric cancer patients receiving dose‐intensive chemotherapy for newly diagnosed or recurrent malignancies. Invasive aspergillosis occurred exclusively in the context of haematological malignancies, where it accounted for an incidence of 6.8% (n=13 of 189). The lung was the primary site in 12 cases, and dissemination was present in three of those. Prior to diagnosis, the overwhelming majority of patients had been profoundly neutropenic for at least 14 days (n=11 of 13) and were receiving systemic antifungal agents (n=10 of 13). Clinical signs and symptoms were nonspecific but always included fever. All 11 patients who were diagnosed and treated during lifetime for a minimum of 10 days responded to either medical or combined medical and surgical treatment, and seven were cured (64%). Nevertheless, the overall long‐term survival was merely 31% after a median follow‐up of 5.68 years after diagnosis. Apart from refractory or recurrent cancer, the main obstacles to successful outcome were failure to diagnose IA during lifetime and bleeding complications in patients with established diagnosis. The frequency of invasive aspergillosis of greater than 15% in paediatric patients with acute myeloblastic leukaemia and recurrent leukaemias warrants the systematic investigation of preventive strategies in these highly vulnerable subgroups.

SODIUM VALPROATE INHIBITS IN VIVO GROWTH OF HUMAN NEUROBLASTOMA CELLS

Sodium valproate (VPA) belongs to the group of simple branched-chain fatty acids and due its anticonvulsive activity is broadly applied in the treatment of epilepsy. We previously showed that VPA is able to induce cellular differentiation, to enhance immunogenicity and to inhibit proliferation of human neuroblastoma (NB) cells in vitro. Furthermore, we demonstrated that VPA inhibits proliferation, enhances neural cell adhesion molecule expression and decreases CD44 expression of human and rat glioma cells in vitro. In the present study we investigated the antitumoral effects of VPA on established human NB xenografts from UKF-NB-3 human NB cells in athymic (nude) mice. When the animals developed s.c. tumors of about 100 mm3 volume they were treated with 400 or 200 mg/kg/day VPA i.p. At the end of the treatment period (40 days) tumor volumes in animals treated with 400 and 200 mg/kg VPA were about 4- (p < 0.0001) and 2-fold (p < 0.0005) smaller than in the saline-treated control group, respectively. Histological examination of the remnant tumors of treated animals revealed induction of differentiation by induction of stroma-rich tumors and nodules that contained elongated NB cells. Pyknotic nuclei and apoptotic bodies indicated induction of apoptosis. We conclude that VPA is able to abrogate NB growth in vivo and may therefore be useful in the treatment of NB patients.

Treatment with sirolimus results in complete responses in patients with autoimmune lymphoproliferative syndrome

We hypothesized that sirolimus, an mTOR inhibitor, may be effective in patients with autoimmune lymphoproliferative syndrome (ALPS) and treated patients who were intolerant to or failed other therapies. Four patients were treated for autoimmune cytopenias; all had a rapid complete or near complete response. Two patients were treated for autoimmune arthritis and colitis, demonstrating marked improvement. Three patients had complete resolution of lymphadenopathy and splenomegaly and all patients had a reduction in double negative T cells, a population hallmark of the disease. Based on these significant responses, we recommend that sirolimus be considered as second‐line therapy for patients with steroid‐refractory disease.

Pre-emptive immunotherapy with purified natural killer cells after haploidentical SCT: a prospective phase II study in two centers

Adoptive immunotherapy with allogeneic purified natural killer (NK) cell products might exert graft-versus-tumor alloreactivity with little risk of GVHD. In a prospective phase II study in two centers, we administered purified NK cell products to high-risk patients treated with haploidentical T-cell-depleted SCT. Sixteen patients received a total of 29 NK cell infusions on days +3, +40 and +100 after transplantation. Median doses (and ranges) of infused NK- and T-cells per product were 1.21 (0.3–3.8) × 107/kg and 0.03 (0.004–0.72) × 105/kg, respectively. With a median follow-up of 5.8 years 4/16 patients are alive. Cause of death was relapse in five, GVHD in three, graft failure in three, and transplant related neurotoxicity in one patient. Four patients developed acute GVHD⩾grade II, all receiving a total of ⩾0.5 × 105 T cells/kg. Compared with historical controls, NK cell infusions had no apparent effect on the rates of graft failure or relapse. Adoptive transfer of allogeneic NK cells is safe and feasible, but further studies are needed to determine the optimal dose and timing of NK cell therapy. Moreover, NK cell activation/expansion may be required to attain clinical benefit, while careful consideration must be given to the number of T cells infused.

Pegylated asparaginase (OncasparTM) in children with ALL: drug monitoring in reinduction according to the ALL/NHL‐BFM 95 protocols

Hypersensitivity reactions are relevant adverse effects of asparaginase therapy. Therefore, children treated with native Escherichia coli asparaginase in induction therapy of acute lymphoblastic leukaemia (ALL) or non‐Hodgkins lymphoma (NHL) were switched to the pegylated enzyme for reinduction under drug monitoring. Seventy children, including four patients with allergic reactions during induction, were given one dose of OncasparTM 1000 U/m2 intravenously. Activity was determined every third or fourth day until it dropped below the limit of quantification. In current reinduction protocols [ALL/NHL‐Berlin–Frankfurt–Münster (BFM) 95 trials], four doses of 10 000 U/m2E. coli asparaginase deplete asparagine for about 2–3 weeks, therefore activities of ≥ 100 U/l up to day 14 and ≥ 50 U/l up to day 21 were targeted. In 66 patients without an allergic reaction during induction, the mean activity was 606 ± 313 U/l, 232 ± 211 U/l and 44 ± 50 U/l after 1, 2 and 3 weeks respectively. In 44/66 patients, activity was ≥ 100 U/l after 14 d. A rapid decline in activity was seen in the remaining 22 patients, including 8/22 patients who showed no activity after 1 week. Toxicity was low and comparable to the native enzymes but, in contrast to about 30% of hypersensitivity reactions with conventional reinduction therapy, no allergic reaction was seen. Substituting 4 × 10 000 U/m2 asparaginase medac for one dose of 1000 U/m2 OncasparTM was safe and well tolerated. Comparable pharmacokinetic treatment intensity was achieved in about two‐thirds of patients.

Cerebrovascular complications of L-asparaginase in the therapy of acute lymphoblastic leukemia.

l-asparaginase is frequently used in combination therapy for the treatment of lymphoid malignancies. We report 5 children aged between 8 and 14 years with neurologic complications presenting with headache and seizures during the first three weeks of l-asparaginase treatment. Three patients had venous thrombosis, one presented a parenchymal hemorrhage, and one showed a peculiar encephalopathy with extended cortical and subcortical lesions suggesting a neurotoxic reaction. Decreased fibrinogen and antithrombin III levels were found. Early MRI is critical even in cases with mild neurologic symptoms. Diagnosis should be followed by early cessation of l-asparaginase application.

IL-2-driven regulation of NK cell receptors with regard to the distribution of CD16+ and CD16- subpopulations and in vivo influence after haploidentical NK cell infusion

To characterize natural killer (NK) cell subpopulations during activation, we analyzed the NK cell receptor repertoire and functionality of purified clinical scale CD56+CD3− donor NK cells during stimulation with 1000 U/mL interleukin (IL)-2 for up to 14 days. In a phase I/II trial, we investigated the efficacy and feasibility of nonidentical NK cell infusion in patients with neuroblastoma after haploidentical stem cell transplantation. After IL-2 stimulation, large differences in the distribution of CD16negative and CD16positive subpopulations were found in 12 donors. Thereby, surface expression for all natural cytotoxicity receptors (NCRs) and NKG2D increased. In addition, killer cell immunoglobulin-like receptor (KIR)+ NK cells were overgrown by KIR− proportion and the homing receptor CD62L was lost during stimulation. NK cell cytotoxicity against K562 and neuroblastoma cells increased and significantly higher cytokine secretion (eg, interferon-γ, tumor necrosis factor-β, macrophage inflammatory protein-1α, macrophage inflammatory protein-1β) was observed after IL-2 stimulation compared with freshly isolated NK cells. However, NK cells of donors showing an initially enhanced cytotoxicity combined with NCRbright and CD69 expression, seemed to be exhausted and did not favor a stimulation period over 9 days. When IL-2–stimulated NK cells were given to transplant recipients, they induced a decrease of peripheral blood NK, in particular of CD56bright-NK cells. Our data indicate that IL-2 stimulation increases the expression of activating receptors and emphasizes mechanisms beside KIR/human leukocyte antigen. Furthermore, the results suggest that the expansion period of purified NK cells has to be individualized to optimize NK cell immunotherapy.

IL-2-activated haploidentical NK cells restore NKG2D-mediated NK-cell cytotoxicity in neuroblastoma patients by scavenging of plasma MICA

NK group 2D (NKG2D)‐expressing NK cells exhibit cytolytic activity against various tumors after recognition of the cellular ligand MHC class I chain‐related gene A (MICA). However, release of soluble MICA (sMICA) compromises NKG2D‐dependent NK‐cell cytotoxicity leading to tumor escape from immunosurveillance. Although some molecular details of the NKG2D‐MICA interaction have been elucidated, its impact for donor NK (dNK) cell‐based therapy of solid tumors has not been studied. Within an ongoing phase I/II trial, we used allogeneic IL‐2 activated dNK cells after haploidentical stem cell transplantation for immunotherapy of patients with high‐risk stage IV neuroblastoma. NKG2D levels on activated dNK cells increased strongly when compared with freshly isolated dNK cells and correlated with enhanced NK‐cell cytotoxicity. Most importantly, elevated sMICA levels in patients plasma correlated significantly with impaired dNK‐cell‐mediated cytotoxicity. This effect could be reversed by high‐dose infusion of activated dNK cells, which display high levels of surface NKG2D. Our data suggest that the provided excess of NKG2D leads to clearance of sMICA and preserves cytotoxicity of dNK cells via non‐occupied NKG2D. In conclusion, our results identify this tumor immune escape mechanism as a target to improve immunotherapy of neuroblastoma and presumably other tumors.