Neuroblastoma (NB) is the most frequent, solid malignancy in childhood outside the central nervous system. More than 90% of children with NB are diagnosed until the age of 6 years. The presence of MYCN amplification and/or metastatic disease (stage M) are considered high-risk features in patients with NB. In this group, 5-year event-free survival is still below 50% despite multimodal therapy including chemotherapy, surgery, radiotherapy, high-dose chemotherapy with autologous stem cell rescue and maintenance therapy.1 Therefore, identifying new treatment strategies for these patients is of major importance.

Disialoganglioside (GD₂) is a glycolipid of the cell membrane. It is found on all NB cells with limited expression on normal tissue,2 and is an established target for immunotherapy in patients with NB.

In the ANBL 0032 study of the Children’s Oncology Group, administration of the human/mouse chimeric anti-GD₂ antibody ch14.18 produced in SP2/0 cells (dinutuximab) in combination with granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin 2 (IL-2) resulted in an improved survival of patients with high-risk NB.3 Similarly, two trials of the International Society of Paediatric Oncology European Neuroblastoma (SIOPEN) group showed a benefit for patients with high-risk NB treated with dinutuximab beta (DB). DB is different from dinutuximab as this variant was produced in Chinese hamster ovary cells. This introduced variations in the glycosylation pattern followed by enhanced antibody effector functions.4 Improved survival was found in first-line maintenance treatment (HR-NBL-SIOPEN/1 study5) as well as in patients with relapsed and refractory NB.6

DB was approved by the European Medicines Agency in 2017 for the treatment of patients with relapsed or refractory NB. The primary mechanism of action of DB is the induction of an antibody-dependent cell-mediated cytotoxicity, mediated mainly by natural killer (NK) cells.7 The contribution of macrophages, monocytes and neutrophils to the clinical effect of DB is not clear to date. The cytotoxic response of effector cells is activated by immunoglobulin receptors (FCGR) on the cell surface on recognition of DB bound to NB cells.8 FCGR3A is expressed on the surface of NK cells and FCGR2A is expressed on macrophages, monocytes and neutrophils. Frequent clinical adverse effects of DB include the induction of neuropathic pain and capillary leak syndrome.6

Whereas passive immune therapy with DB has evolved as a treatment option for pediatric patients with high-risk NB, active immune therapy approaches such as checkpoint inhibitors have been developed and approved for adult patients with cancer.9 The first checkpoint inhibitor ipilimumab targeting the CTLA-4 molecule was approved for patients with melanoma in 2011.10 Programmed cell death protein 1 (PD-1) is another checkpoint mainly expressed on activated T cells and NK cells.11 PD-1 inhibits immune responses after binding to its programmed death ligands, PD-L1 and PD-L2. PD-L1 is expressed on hematopoietic and epithelial cells, PD-L2 on macrophages and dendritic cells. An upregulation of both ligands can be observed in malignant diseases, PD-L1 predominantly in solid tumors, PD-L2 in B cell lymphoma.9 In NB, PD-L1 expression is low. However, a constitutive and inducible PD-L1 expression was shown in several cell lines.12 13

Nivolumab is a monoclonal antibody that inhibits the PD-1/PD-L1 checkpoint by specifically binding to PD-1 and is approved for the treatment of patients with malignant diseases including melanoma, non-small cell lung cancer and Hodgkin lymphoma.

In a preclinical NB model it was demonstrated that low PD-L1 expression was upregulated by the treatment with DB, and a combined treatment with a murine anti-PD-1 antibody and DB induced a synergistic anti-NB immune response in a syngeneic mouse model.14 Therefore, this combination might be a reasonable approach for patients with NB. As both DB and nivolumab lead to immune system activation, autoimmune phenomena are possible adverse effects particularly to be considered with this approach. Here, we report the results of combined treatment with nivolumab and DB in two patients with relapsed/refractory NB.

First-line treatment for patients with high-risk NB involves a multimodal approach combining chemotherapy, surgery, high-dose chemotherapy with autologous stem cell rescue with or without mIBG therapy and external beam radiotherapy. In addition, maintenance therapy with DB is standard of care in high-risk NB.15

Patients with refractory disease or multiple relapses of NB have a dismal prognosis when standard treatment approaches have failed.16 Immune therapy with DB either alone or in combination with a haploidentical stem cell transplant procedure is an option currently explored for individual patients.17

In the past decade, checkpoint inhibitors have gained particular interest in various malignant diseases of adult patients including melanoma, non-small cell lung cancer and Hodgkin lymphoma.18–20 Since 2011, seven checkpoint inhibitors have been approved including nivolumab. Based on the observation that DB leads to an upregulation of the PD-1/PD-L1 checkpoint14 in a preclinical model with a synergistic therapeutic effect when combined with an anti-PD-1 antibody, we applied this combination in two heavily pretreated patients with relapsed/refractory NB. The outcome was very promising as both patients showed an excellent response to this treatment. In patient 1, progressive disease was suspected at staging examinations after 3 months of therapy. It is well known, that checkpoint inhibitors may result in pseudoprogression after the first few weeks of treatment due to immunotherapy-induced inflammatory reaction.21 Considering pseudoprogression and the excellent general condition of the patient, we decided to continue her treatment as planned. Four months later, all PET-positive lesions had disappeared. The complete response was confirmed by MRI scans. The CR was stable as further PET/CTs at the end of therapy and in the patient’s 3-month and 6-month follow-up revealed no evidence of disease. In patient 2, the response to treatment was documented by MRI and mIBG scans. Regression of tumor tissue was clearly seen in the patient’s soft tissue lesions and in the bone metastases. As both patients had previous, unsuccessful therapies with DB alone or in combination with IL-2 or chemotherapy, we consider the addition of immune therapy with nivolumab as the decisive element for the favorable outcome documented in our two patients.

We report the first results of a combined treatment with nivolumab and DB in two patients with relapsed/refractory NB. The response to treatment was excellent. The first patient is now 6 months after end of therapy in ongoing complete remission. The second patient has reached a partial remission and is still receiving treatment. Treatment-related toxicities were mild and well manageable; in particular, we did not observe any autoimmune phenomena. Both patients have an excellent quality of life and are fully active. These results suggest that combined immune therapy with nivolumab and DB may be a promising strategy to be explored in a prospective clinical trial.