Irinotecan and temozolomide in combination with dasatinib and rapamycin versus irinotecan and temozolomide for patients with relapsed or refractory neuroblastoma (RIST-rNB-2011): a multicentre, open-label, randomised, controlled, phase 2 trial.

BACKGROUND: Neuroblastoma is the most common extracranial solid tumour in children. Relapsed or refractory neuroblastoma is associated with a poor outcome. We assessed the combination of irinotecan-temozolomide and dasatinib-rapamycin (RIST) in patients with relapsed or refractory neuroblastoma. METHODS: The multicentre, open-label, randomised, controlled, phase 2, RIST-rNB-2011 trial recruited from 40 paediatric oncology centres in Germany and Austria. Patients aged 1-25 years with high-risk relapsed (defined as recurrence of all stage IV and MYCN amplification stages, after response to treatment) or refractory (progressive disease during primary treatment) neuroblastoma, with Lansky and Karnofsky performance status at least 50%, were assigned (1:1) to RIST (RIST group) or irinotecan-temozolomide (control group) by block randomisation, stratified by MYCN status. We compared RIST (oral rapamycin [loading 3 mg/m2 on day 1, maintenance 1 mg/m2 on days 2-4] and oral dasatinib [2 mg/kg per day] for 4 days with 3 days off, followed by intravenous irinotecan [50 mg/m2 per day] and oral temozolomide [150 mg/m2 per day] for 5 days with 2 days off; one course each of rapamycin-dasatinib and irinotecan-temozolomide for four cycles over 8 weeks, then two courses of rapamycin-dasatinib followed by one course of irinotecan-temozolomide for 12 weeks) with irinotecan-temozolomide alone (with identical dosing as experimental group). The primary endpoint of progression-free survival was analysed in all eligible patients who received at least one course of therapy. The safety population consisted of all patients who received at least one course of therapy and had at least one post-baseline safety assessment. This trial is registered at ClinicalTrials.gov, NCT01467986, and is closed to accrual. FINDINGS: Between Aug 26, 2013, and Sept 21, 2020, 129 patients were randomly assigned to the RIST group (n=63) or control group (n=66). Median age was 5·4 years (IQR 3·7-8·1). 124 patients (78 [63%] male and 46 [37%] female) were included in the efficacy analysis. At a median follow-up of 72 months (IQR 31-88), the median progression-free survival was 11 months (95% CI 7-17) in the RIST group and 5 months (2-8) in the control group (hazard ratio 0·62, one-sided 90% CI 0·81; p=0·019). Median progression-free survival in patients with amplified MYCN (n=48) was 6 months (95% CI 4-24) in the RIST group versus 2 months (2-5) in the control group (HR 0·45 [95% CI 0·24-0·84], p=0·012); median progression-free survival in patients without amplified MYCN (n=76) was 14 months (95% CI 9-7) in the RIST group versus 8 months (4-15) in the control group (HR 0·84 [95% CI 0·51-1·38], p=0·49). The most common grade 3 or worse adverse events were neutropenia (54 [81%] of 67 patients given RIST vs 49 [82%] of 60 patients given control), thrombocytopenia (45 [67%] vs 41 [68%]), and anaemia (39 [58%] vs 38 [63%]). Nine serious treatment-related adverse events were reported (five patients given control and four patients given RIST). There were no treatment-related deaths in the control group and one in the RIST group (multiorgan failure). INTERPRETATION: RIST-rNB-2011 demonstrated that targeting of MYCN-amplified relapsed or refractory neuroblastoma with a pathway-directed metronomic combination of a multkinase inhibitor and an mTOR inhibitor can improve progression-free survival and overall survival. This exclusive efficacy in MYCN-amplified, relapsed neuroblastoma warrants further investigation in the first-line setting. FUNDING: Deutsche Krebshilfe.

Long-term, continuous infusion of single-agent dinutuximab beta for relapsed/refractory neuroblastoma: an open-label, single-arm, Phase 2 study.

BACKGROUND: Short-term infusions of dinutuximab beta plus isotretinoin and cytokines administered in previous immunotherapy studies in neuroblastoma were associated with severe pain. Here, long-term, continuous infusion of single-agent dinutuximab beta was evaluated in patients with relapsed/refractory neuroblastoma. METHODS: In this open-label, single-arm, Phase 2 study, patients with either refractory or relapsed high-risk neuroblastoma received dinutuximab beta by continuous infusion over 10 days of each cycle, for up to five cycles. The primary endpoint was objective response rate 24 weeks after the end of cycle 5. Secondary endpoints included adverse events, intravenous morphine use, best response, duration of response, and three-year progression-free and overall survival. RESULTS: Of the 40 patients included, 38 had evaluable response. Objective response rate was 26% and best response rate 37%. Median duration of response was 238 days (IQR 108-290). Three-year progression-free and overall survival rates were 31% (95% CI 17-47) and 66% (95% CI 47-79), respectively. Prophylactic intravenous morphine use and duration of use decreased with increasing cycles. The most common grade 3 treatment-related adverse events were pain, diarrhea, and hypokalemia. CONCLUSION: Long-term continuous infusion of single-agent dinutuximab beta is tolerable and associated with clinically meaningful responses in patients with relapsed/refractory high-risk neuroblastoma. CLINICAL TRIAL REGISTRATION: The study is registered with ClinicalTrials.gov (NCT02743429) and EudraCT (2014-000588-42).

Endoscope-assisted microsurgical resection of a third ventricular immature teratoma.

BACKGROUND: Reaching a tumor within the third ventricle is challenging, and planning an accessible trajectory is crucial without injuring the surrounding structures. We report a 5-year-old boy presented with headache and a seizure where sequential MRI brain studies in a short time period revealed a rapid growing immature teratoma within the third ventricle with hydrocephalic changes. Several management procedures were performed for CSF diversion and medical treatment of the tumor with chemotherapy and stem cell therapy. The tumor was rapidly growing, and surgical excision was decided. Total resection was achieved via endoscope-assisted microsurgical transcallosal approach. Seven years after surgery, the patient experienced no recurrence of the tumor with a favorable clinical condition. CONCLUSION: We report a rare case of posterior third ventricular immature teratoma where the endoscope-assisted microsurgical technique was implemented with favorable long-term postoperative outcome.

Integrative medicine during the intensive phase of chemotherapy in pediatric oncology in Germany: a randomized controlled trial with 5-year follow up.

BACKGROUND: Integrative medicine is used frequently alongside chemotherapy treatment in pediatric oncology, but little is known about the influence on toxicity. This German, multi-center, open-label, randomized controlled trial assessed the effects of complementary treatments on toxicity related to intensive-phase chemotherapy treatment in children aged 1-18 with the primary outcome of the toxicity sum score. Secondary outcomes were chemotherapy-related toxicity, overall and event-free survival after 5 years in study patients. METHODS: Intervention and control were given standard chemotherapy according to malignancy & tumor type. The intervention arm was provided with anthroposophic supportive treatment (AST); given as anthroposophic base medication (AMP), as a base medication for all patients and additional on-demand treatment tailored to the intervention malignancy groups. The control was given no AMP. The toxicity sum score (TSS) was assessed using NCI-CTC scales. RESULTS: Data of 288 patients could be analyzed. Analysis did not reveal any statistically significant differences between the AST and the control group for the primary endpoint or the toxicity measures (secondary endpoints). Furthermore, groups did not differ significantly in the five-year overall and event-free survival follow up. DISCUSSION: In this trial findings showed that AST was able to be safely administered in a clinical setting, although no beneficial effects of AST between group toxicity scores, overall or event-free survival were shown.

First-line Anti-GD2 Therapy Combined With Consolidation Chemotherapy in 3 Patients With Newly Diagnosed Metastatic Ewing Sarcoma or Ewing-like Sarcoma.

Despite multimodal therapy, the prognosis of patients with metastatic Ewing sarcoma (ES) remains poor, with new treatments urgently needed. The disialoganglioside GD2, a well-established tumor-associated antigen, is expressed in 40% to 90% of ES cells, making it a suitable therapeutic target. Here we report 3 cases with newly diagnosed, metastatic, GD2-positive ES or Ewing-like sarcoma treated with the anti-GD2 antibody dinutuximab beta in addition to standard chemotherapeutic regimens. Treatment was well-tolerated, and all patients achieved complete remission, without evidence of relapse. First-line anti-GD2 immunotherapy in patients with metastatic, GD2-positive ES or Ewing-like sarcoma represents a promising therapeutic option that warrants further clinical evaluation.

GD2 targeting by dinutuximab beta is a promising immunotherapeutic approach against malignant glioma.

PURPOSE: Disialoganglioside GD2 is expressed by glioblastoma multiforme (GBM) cells representing a promising target for anti-GD2 immunotherapeutic approaches. The aim of the present study was to investigate anti-tumor efficacy of the chimeric anti-GD2 antibody (Ab) dinutuximab beta against GBM. METHODS: Expression levels of GD2 and complement regulatory proteins (CRP; CD46, CD55 and CD59) on well-known and newly established primary tumor originated GBM cell lines were analyzed by flow cytometry. Ab-dependent cellular (ADCC) and complement-dependent cytotoxicity (CDC) mediated by dinutuximab beta against GBM cells were determined by a non-radioactive calcein-AM-based assay. RESULTS: Analysis of primary GBM cells revealed a heterogeneous GD2 expression that varied between the cell lines analyzed with higher expression levels in the tumor surface compared to the core originated cells. Both GD2-positive and -negative tumor cells were detected in every cell line analyzed. In contrast to CDC, ADCC mediated by dinutuximab beta was observed against the majority of GBM cells. Importantly, CDC-resistant cells showed high expression of the CRP CD46, CD55 and CD59. CONCLUSION: Our present data show anti-tumor effects mediated by dinutuximab beta against GBM cells providing a rationale for a GD2-directed immunotherapy against GBM. Due to high CRP expression, a combining of GD2-targeting with CRP blockade might be a further treatment option for GBM.

EZH2 Inhibition in Ewing Sarcoma Upregulates GD2 Expression for Targeting with Gene-Modified T Cells.

Chimeric antigen receptor (CAR) engineering of T cells allows one to specifically target tumor cells via cell surface antigens. A candidate target in Ewing sarcoma is the ganglioside GD2, but heterogeneic expression limits its value. Here we report that pharmacological inhibition of Enhancer of Zeste Homolog 2 (EZH2) at doses reducing H3K27 trimethylation, but not cell viability, selectively and reversibly induces GD2 surface expression in Ewing sarcoma cells. EZH2 in Ewing sarcoma cells directly binds to the promoter regions of genes encoding for two key enzymes of GD2 biosynthesis, and EZH2 inhibition enhances expression of these genes. GD2 surface expression in Ewing sarcoma cells is not associated with distinct in vitro proliferation, colony formation, chemosensitivity, or in vivo tumorigenicity. Moreover, disruption of GD2 synthesis by gene editing does not affect its in vitro behavior. EZH2 inhibitor treatment sensitizes Ewing sarcoma cells to effective cytolysis by GD2-specific CAR gene-modified T cells. In conclusion, we report a clinically applicable pharmacological approach for enhancing efficacy of adoptively transferred GD2-redirected T cells against Ewing sarcoma, by enabling recognition of tumor cells with low or negative target expression.

Social inequalities in the participation and activity of children and adolescents with leukemia, brain tumors, and sarcomas (SUPATEEN): a protocol for a multicenter longitudinal prospective observational study.

BACKGROUND: About 2000 children and adolescents under the age of 18 are diagnosed with cancer each year in Germany. Because of current medical treatment methods, a high survival rate can be reached for many types of the disease. Nevertheless, patients face a number of long-term effects related to the treatment. As a result, physical and psychological consequences have increasingly become the focus of research in recent years. Social dimensions of health have received little attention in health services research in oncology so far. Yet, there are no robust results that allow an estimation of whether and to what extent the disease and treatment impair the participation of children and adolescents and which factors mediate this effect. Social participation is of great importance especially because interactions with peers and experiences in different areas of life are essential for the development of children and adolescents. METHODS: Data are collected in a longitudinal, prospective, observational multicenter study. For this purpose, all patients and their parents who are being treated for cancer in one of the participating clinics throughout Germany will be interviewed within the first month after diagnosis (t1), after completion of intensive treatment (t2) and half a year after the end of intensive treatment (t3) using standardized questionnaires. Analysis will be done by descriptive and multivariate methods. DISCUSSION: The results can be used to identify children and adolescents in high-risk situations at an early stage in order to be able to initiate interventions tailored to the needs. Such tailored interventions will finally reduce the risk of impairments in the participation of children and adolescents and increase quality of life. TRIAL REGISTRATION: ClinicalTrials.gov: NCT04101123.

Interleukin 2 with anti-GD2 antibody ch14.18/CHO (dinutuximab beta) in patients with high-risk neuroblastoma (HR-NBL1/SIOPEN): a multicentre, randomised, phase 3 trial.

BACKGROUND: Immunotherapy with the chimeric anti-GD2 monoclonal antibody dinutuximab, combined with alternating granulocyte-macrophage colony-stimulating factor and intravenous interleukin-2 (IL-2), improves survival in patients with high-risk neuroblastoma. We aimed to assess event-free survival after treatment with ch14.18/CHO (dinutuximab beta) and subcutaneous IL-2, compared with dinutuximab beta alone in children and young people with high-risk neuroblastoma. METHODS: We did an international, open-label, phase 3, randomised, controlled trial in patients with high-risk neuroblastoma at 104 institutions in 12 countries. Eligible patients were aged 1-20 years and had MYCN-amplified neuroblastoma with stages 2, 3, or 4S, or stage 4 neuroblastoma of any MYCN status, according to the International Neuroblastoma Staging System. Patients were eligible if they had been enrolled at diagnosis in the HR-NBL1/SIOPEN trial, had completed the multidrug induction regimen (cisplatin, carboplatin, cyclophosphamide, vincristine, and etoposide, with or without topotecan, vincristine, and doxorubicin), had achieved a disease response that fulfilled prespecified criteria, had received high-dose therapy (busulfan and melphalan or carboplatin, etoposide, and melphalan) and had received radiotherapy to the primary tumour site. In this component of the trial, patients were randomly assigned (1:1) to receive dinutuximab beta (20 mg/m2 per day as an 8 h infusion for 5 consecutive days) or dinutuximab beta plus subcutaneous IL-2 (6 × 106 IU/m2 per day on days 1-5 and days 8-12 of each cycle) with the minimisation method to balance randomisation for national groups and type of high-dose therapy. All participants received oral isotretinoin (160 mg/m2 per day for 2 weeks) before the first immunotherapy cycle and after each immunotherapy cycle, for six cycles. The primary endpoint was 3-year event-free survival, analysed by intention to treat. This trial was registered with ClinicalTrials.gov, number NCT01704716, and EudraCT, number 2006-001489-17, and recruitment to this randomisation is closed. FINDINGS: Between Oct 22, 2009, and Aug 12, 2013, 422 patients were eligible to participate in the immunotherapy randomisation, of whom 406 (96%) were randomly assigned to a treatment group (n=200 to dinutuximab beta and n=206 to dinutuximab beta with subcutaneous IL-2). Median follow-up was 4·7 years (IQR 3·9-5·3). Because of toxicity, 117 (62%) of 188 patients assigned to dinutuximab beta and subcutaneous IL-2 received their allocated treatment, by contrast with 160 (87%) of 183 patients who received dinutuximab beta alone (p<0·0001). 3-year event-free survival was 56% (95% CI 49-63) with dinutuximab beta (83 patients had an event) and 60% (53-66) with dinutuximab beta and subcutaneous IL-2 (80 patients had an event; p=0·76). Four patients died of toxicity (n=2 in each group); one patient in each group while receiving immunotherapy (n=1 congestive heart failure and pulmonary hypertension due to capillary leak syndrome; n=1 infection-related acute respiratory distress syndrome), and one patient in each group after five cycles of immunotherapy (n=1 fungal infection and multi-organ failure; n=1 pulmonary fibrosis). The most common grade 3-4 adverse events were hypersensitivity reactions (19 [10%] of 185 patients in the dinutuximab beta group vs 39 [20%] of 191 patients in the dinutuximab plus subcutaneous IL-2 group), capillary leak (five [4%] of 119 vs 19 [15%] of 125), fever (25 [14%] of 185 vs 76 [40%] of 190), infection (47 [25%] of 185 vs 64 [33%] of 191), immunotherapy-related pain (19 [16%] of 122 vs 32 [26%] of 124), and impaired general condition (30 [16%] of 185 vs 78 [41%] of 192). INTERPRETATION: There is no evidence that addition of subcutaneous IL-2 to immunotherapy with dinutuximab beta, given as an 8 h infusion, improved outcomes in patients with high-risk neuroblastoma who had responded to standard induction and consolidation treatment. Subcutaneous IL-2 with dinutuximab beta was associated with greater toxicity than dinutuximab beta alone. Dinutuximab beta and isotretinoin without subcutaneous IL-2 should thus be considered the standard of care until results of ongoing randomised trials using a modified schedule of dinutuximab beta and subcutaneous IL-2 are available. FUNDING: European Commission 5th Frame Work Grant, St. Anna Kinderkrebsforschung, Fondation ARC pour la recherche sur le Cancer.

Haploidentical Stem Cell Transplantation for Refractory/Relapsed Neuroblastoma.

Pediatric patients with refractory or relapsed metastatic neuroblastoma (NBL) have a poor prognosis despite autologous stem cell transplantation (SCT). Allogeneic SCT from a haploidentical donor has a remarkable alloreactive effect in patients with leukemia; thus, we evaluated this approach in children with very high-risk NBL. We analyzed data from 2 prospective phase I/II trials. A total of 26 patients with refractory (n = 5), metastatic relapsed (n = 20), or locally relapsed MYCN-positive (n = 1) NBL received a median of 17 × 106/kg T/B cell-depleted CD34+ stem cells with 68 × 103/kg residual T cells and 107 × 106/kg natural killer cells. The conditioning regimen comprised melphalan, fludarabine, thiotepa, OKT3, and a short course of mycophenolate mofetil post-transplantation. Engraftment occurred in 96% of the patients. Event-free survival and overall survival at 5 years were 19% and 23%, respectively. No transplantation-related mortality was observed, and the single death was due to progression/subsequent relapse. The median duration of follow-up was 8.1 years. Patients in complete remission before SCT had a significantly better prognosis than those with residual tumor load (P < .01). All patients with progressive disease before SCT relapsed within 1 year. Grade II and grade III acute graft-versus-host disease (GVHD) occurred in 31% and 12% of the patients, respectively. Chronic limited and extensive GVHD occurred in 28% and 10%, respectively. Our data indicate that haploidentical SCT is a feasible treatment option that can induce long-term remission in some patients with NBL with tolerable side effects, and may enable the development of further post-transplantation therapeutic strategies based on the donor-derived immune system.

Inflammatory response and treatment tolerance of long-term infusion of the anti-GD2 antibody ch14.18/CHO in combination with interleukin-2 in patients with high-risk neuroblastoma.

BACKGROUND: The monoclonal anti-GD2 antibody ch14.18/CHO in combination with IL-2 is active and effective in high-risk neuroblastoma (NB) patients. Here, we investigated the inflammatory response and treatment tolerance of long-term infusion (LTI) of ch14.18/CHO (10 × 10 mg/m2 ; 24 hr) in combination with subcutaneous (s.c.) IL-2 in a single center program. METHODS: Fifty-three NB patients received up to six cycles of 100 mg/m2 ch14.18/CHO (d8-18, where d represents day(s)) as LTI combined with 6 × 106 IU/m2 s.c. IL-2 (d1-5; 8-12) and 160 mg/m2 oral 13-cis retinoic acid (RA) (d19-32). Side effects of ch14.18/CHO and IL-2 treatment require hospitalization of patients on d8. Treatment tolerance was evaluated daily with clinical parameters (body temperature, vital signs, Lansky performance status, requirement of i.v. concomitant medication) to define an outpatient candidate status. sIL-2-R and C-reactive protein values were determined to assess the inflammatory response. RESULTS: LTI of ch14.18/CHO (d8-18) in combination with s.c.IL-2 (d8-12) showed an acceptable treatment tolerance that allowed all patients to receive part of the treatment as an outpatient (median time point of discharge: d15 for all cycles). The treatment tolerance improved from cycle to cycle and the time to become an outpatient candidate decreased from d15 to d13 in subsequent cycles. Clinical and laboratory parameters indicate a maximum inflammatory response at d11 of each cycle. Interestingly, the soluble IL-2 receptor remained increased at baseline of the next cycle indicating immune activation over the entire treatment period of 6 months. CONCLUSIONS: LTI of ch14.18/CHO combined with s.c.IL-2 shows an improved tolerance in subsequent cycles allowing outpatient treatment.

Individualized risk assessment in neuroblastoma: does the tumoral metabolic activity on 123I-MIBG SPECT predict the outcome?

PURPOSE: Risk-adapted treatment in children with neuroblastoma (NB) is based on clinical and genetic factors. This study evaluated the metabolic tumour volume (MTV) and its asphericity (ASP) in pretherapeutic 123I-MIBG SPECT for individualized image-based prediction of outcome. METHODS: This retrospective study included 23 children (11 girls, 12 boys; median age 1.8 years, range 0.3-6.8 years) with newly diagnosed NB consecutively examined with pretherapeutic 123I-MIBG SPECT. Primary tumour MTV and ASP were defined using semiautomatic thresholds. Cox regression analysis, receiver operating characteristic analysis (cut-off determination) and Kaplan-Meier analysis with the log-rank test for event-free survival (EFS) were performed for ASP, MTV, laboratory parameters (including urinary homovanillic acid-to-creatinine ratio, HVA/C), and clinical (age, stage) and genetic factors. Predictive accuracy of the optimal multifactorial model was determined in terms of Harrell's C and likelihood ratio χ 2. RESULTS: Median follow-up was 36 months (range 7-107 months; eight patients showed disease progression/relapse, four patients died). The only significant predictors of EFS in the univariate Cox regression analysis were ASP (p = 0.029; hazard ratio, HR, 1.032 for a one unit increase), MTV (p = 0.038; HR 1.012) and MYCN amplification status (p = 0.047; HR 4.67). The mean EFS in patients with high ASP (>32.0%) and low ASP were 21 and 88 months, respectively (p = 0.013), and in those with high MTV (>46.7 ml) and low MTV were 22 and 87 months, respectively (p = 0.023). A combined risk model of either high ASP and high HVA/C or high MTV and high HVA/C best predicted EFS. CONCLUSIONS: In this exploratory study, pretherapeutic image-derived and laboratory markers of tumoral metabolic activity in NB (ASP, MTV, urinary HVA/C) allowed the identification of children with a high and low risk of progression/relapse under current therapy.

2017 GPOH Guidelines for Diagnosis and Treatment of Patients with Neuroblastic Tumors.

The clinical course of neuroblastoma is more heterogeneous than any other malignant disease. Most low-risk patients experience regression after limited or even no chemotherapy. However, more than half of high-risk patients die from disease despite intensive multimodal treatment. Precise patient characterization at diagnosis is key for risk-adapted treatment. The guidelines presented here incorporate results from national and international clinical trials to produce recommendations for diagnosing and treating neuroblastoma patients in German hospitals outside of clinical trials.

Targeting of heme oxygenase-1 as a novel immune regulator of neuroblastoma.

Heme oxygenase (HO)-1 catalyzes the degradation of cytotoxic heme into biliverdin and blocks antitumor immune responses, thus protecting cancer against host defense. Whether this scenario also applies to neuroblastoma (NB), the most common extracranial solid childhood tumor, is not known. Here, we demonstrate for the first time a prognostic relevance of HO-1 expression in samples from NB patients and show that targeting of HO-1 prevents both cancer resistance against cellular stress and immune escape in the syngeneic NXS2 A/J mouse model of NB. High HO-1 RNA expression in NB tissues emerged as unfavorable prognostic marker, in particular for patients older than 18 months as indicated by univariate as well as multivariate survival probability analyses including disease stage and MYCN status. On the basis of this observation we aimed to target HO-1 by systemic as well as tumor-specific zinc protoporphyrin-mediated HO-1 suppression in a syngeneic immunocompetent NB mouse model. This resulted in 50% reduction of primary tumor growth and a suppression of spontaneous liver metastases. Importantly, HO-1 inhibition abrogated immune cell paralysis affecting CD4 and CD8 T-effector cells. This in turn reverted HO-1-dependent immune escape mechanisms in NB by increasing NB apoptosis and improved DC maturation. In summary, HO-1 emerges as a novel immune regulator in NB and emerges as a promising target for the development of therapeutic approaches.

Disialoganglioside-specific human natural killer cells are effective against drug-resistant neuroblastoma.

The disialoganglioside GD2 is a well-established target antigen for passive immunotherapy in neuroblastoma (NB). Despite the recent success of passive immunotherapy with the anti-GD2 antibody ch14.18 and cytokines, treatment of high-risk NB remains challenging. We expanded the approach of GD2-specific, antibody-based immunotherapy to an application of a GD2-specific natural killer (NK) cell line, NK-92-scFv(ch14.18)-zeta. NK-92-scFv(ch14.18)-zeta is genetically engineered to express a GD2-specific chimeric antigen receptor generated from ch14.18. Here, we show that chimeric receptor expression enables NK-92-scFv(ch14.18)-zeta to effectively lyse GD2(+) NB cells also including partially or multidrug-resistant lines. Our data suggest that recognition of GD2 by the chimeric receptor is the primary mechanism involved in NK-92-scFv(ch14.18)-zeta-mediated lysis and is independent of activating NK cell receptor/ligand interactions. Furthermore, we demonstrate that NK-92-scFv(ch14.18)-zeta is able to mediate a significant anti-tumor response in vivo in a drug-resistant GD2(+) NB xenograft mouse model. NK-92-scFv(ch14.18)-zeta is an NB-specific NK cell line that has potential for future clinical development due to its high stability and activity toward GD2(+) NB cell lines.

Targeting of MYCN by means of DNA vaccination is effective against neuroblastoma in mice.

The MYCN oncogene is a strong genetic marker associated with poor prognosis in neuroblastoma (NB). Therefore, MYCN gene amplification and subsequent overexpression provide a possible target for new treatment approaches in NB. We first identified an inverse correlation of MYCN expression with CD45 mRNA in 101 NB tumor samples. KEGG mapping further revealed that MYCN expression was associated with immune-suppressive pathways characterized by a down-regulation of T cell activation and up-regulation of T cell inhibitory gene transcripts. We then aimed to investigate whether DNA vaccination against MYCN is effective to induce an antigen-specific and T cell-mediated immune response. For this purpose, we generated a MYCN-expressing syngeneic mouse model by MYCN gene transfer to NXS2 cells. MYCN-DNA vaccines were engineered based on the pCMV-F3Ub plasmid backbone to drive ubiquitinated full-length MYCN-cDNA and minigene expression. Vaccines were delivered orally with attenuated S. typhimurium strain SL7207 as a carrier. Immunization with both MYCN-DNA vaccines significantly reduced primary tumor growth of MYCN-expressing NB cells in contrast to negative controls. The immune response was mediated by tumor-infiltrating T cells in vivo, which revealed MYCN-specific and MHC class I-restricted lysis of inducible MYCN-expressing NB target cells in vitro. Finally, these antigen-specific T cells also killed MYCN-negative mammary carcinoma cells pulsed with MYCN peptides in contrast to controls. In summary, we demonstrate proof of concept that MYCN can be targeted by DNA vaccination, which may provide an approach to overcoming MYCN immune-suppressive activities in patients with MYCN-amplified disease.

Development of immunomonitoring of antibody­dependent cellular cytotoxicity against neuroblastoma cells using whole blood.

Neuroblastoma, a childhood tumour of neuroectodermal origin, accounts for 15 % of paediatric cancer deaths, which is often metastatic at diagnosis and despite aggressive therapies, it has poor long-term prognosis with high risk of recurrence. Monoclonal antibody (mAb) therapy targeting GD2, a disialoganglioside expressed on neuroblastoma, has shown promise in recent trials with natural killer cell (NK)-mediated antibody-dependent cellular cytotoxicity (ADCC) thought to be central to efficacy, although other immune effectors may be important. To further enhance therapy, immunomonitoring of patients is essential to elucidate the in vivo mechanisms of action and provides surrogate end points of efficacy for future clinical trials. Our aim was to establish a 'real-time' ex vivo wholeblood (WB) immunomonitoring strategy to perform within the logistical constraints such as limited sample volumes, anticoagulant effects, sample stability and shipping time. A fluorescent dye release assay measuring target cell lysis was coupled with flow cytometry to monitor specific effector response. Significant target cell lysis with anti-GD2 antibody (p < 0.05) was abrogated following NK depletion. NK up-regulation of CD107a and CD69 positively correlated with target cell lysis (r > 0.6). The ADCC activity of WB correlated with peripheral blood mononuclear cells (r > 0.95), although WB showed overall greater target cell lysis attributed to the combination of NK-mediated ADCC, CD16+ granulocyte degranulation and complement- dependent cytotoxicity. Response was maintained in heparinised samples stored for 24 h at room temperature, but not 4 °C. Critically, the assay showed good reproducibility (mean % CV < 6.4) and was successfully applied to primary neuroblastoma samples. As such, WB provides a resourceful analysis of multiple mechanisms for efficient end point monitoring to correlate immune modulation with clinical outcome.

Chronic diseases in pregnant women: prevalence and birth outcomes based on the SNiP-study.

BACKGROUND: The subject of "pregnancy and disease" is of particular importance for maternal well-being and neonatal outcomes. The international literature has focused on acute diseases during pregnancy; however, there are only a few studies investigating chronic diseases in pregnant women. The focus of this study is on diseases of women in childbearing age that are not related to the pregnancy. The objective of the paper is to deliver population based prevalences of chronic dieases in childbearing women and compare the two groups of chronically ill women and healthy women in detail regarding sociodemography, peri- and prenatal parameters and birth outcomes. METHODS: Data of n = 5320 childbearing women were evaluated in the context of the population-based Survey of Neonates in Pomerania (SNiP). Data were obtained via face-to-face interviews, self-applied questionnaires, and abstraction from medical records at the time of giving birth. Sociodemographic and health status data were assessed, including chronic diseases that were taken out of medical records. A comprehensive set of pre- and perinatal varaiables were assessed. RESULTS: In the SNiP, every fifth pregnant woman suffers from at least one chronic disease, and higher prevalence rates have been reported in the literature. There was a significant difference between chronically ill women and healthy women in age, education and income. Prenatal complications were more frequent in the healthy group than in the chronic disease group. Women with chronic diseases delivered by Cesarean section more frequently than women in the healthy group. Every tenth woman with at least one chronic disease gave birth to a premature infant, while only one in every 13 woman in the healthy control group gave birth to a premature infant. CONCLUSIONS: This analysis is the first population-based study in which all chronic diseases could be taken into consideration. The population-based prevalences rates in the SNiP data are consistently lower than those found in the literature. There are differences between chronically ill women and healthy women in peri- and prenatal variables as well as birth outcome on the population level. However, they are less frequent than expected and further analyses are need focusing on specific diseases.

Preclinical Development of CAR T Cells with Antigen-Inducible IL18 Enforcement to Treat GD2-Positive Solid Cancers.

PURPOSE: Cytokine-engineering of chimeric antigen receptor-redirected T cells (CAR T cells) is a promising principle to overcome the limited activity of canonical CAR T cells against solid cancers. EXPERIMENTAL DESIGN: We developed an investigational medicinal product, GD2IL18CART, consisting of CAR T cells directed against ganglioside GD2 with CAR-inducible IL18 to enhance their activation response and cytolytic effector functions in the tumor microenvironment. To allow stratification of patients according to tumor GD2 expression, we established and validated immunofluorescence detection of GD2 on paraffin-embedded tumor tissues. RESULTS: Lentiviral all-in-one vector engineering of human T cells with the GD2-specific CAR with and without inducible IL18 resulted in cell products with comparable proportions of CAR-expressing central memory T cells. Production of IL18 strictly depends on GD2 antigen engagement. GD2IL18CART respond to interaction with GD2-positive tumor cells with higher IFNγ and TNFα cytokine release and more effective target cytolysis compared with CAR T cells without inducible IL18. GD2IL18CART further have superior in vivo antitumor activity, with eradication of GD2-positive tumor xenografts. Finally, we established GMP-compliant manufacturing of GD2IL18CART and found it to be feasible and efficient at clinical scale. CONCLUSIONS: These results pave the way for clinical investigation of GD2IL18CART in pediatric and adult patients with neuroblastoma and other GD2-positive cancers (EU CT 2022-501725-21-00).