Off-label Use of Dinutuximab Beta in Combination With Chemotherapy in Patients With Ewing Sarcoma: A Retrospective Single-center Case Series

Abstract

Surface expression of the disialoganglioside subtype GD2 has been observed on Ewing sarcoma (ES) cells, making it a suitable target for immunotherapy with the anti-GD2 antibody dinutuximab beta (DB). Here we report our experience of using DB in a cohort of 13 patients with GD2-positive, metastatic ES, in both the frontline (n=9) and relapsed/refractory (n=4) settings, when added to standard chemotherapeutic regimens. Outcomes were compared with 24 patients, primarily with localized ES, who were also treated at our center with standard therapy alone (without DB). Patients treated with DB had a median overall survival (OS) of 1877 days in the frontline setting and 810 days in the relapsed/refractory setting. Median time to progression was 1811 days and 782 days, respectively. In contrast, those treated with standard therapy alone in our center demonstrated a median OS of 1547 days and 210 days in the frontline and relapsed/refractory setting, respectively, with median times of progression of 1261 days and 113 days. DB treatment was well tolerated, with no new or unexpected adverse events reported. Anti-GD2 immunotherapy with DB represents a promising therapeutic option to improve outcomes in patients with metastatic ES, in both the frontline and relapsed/refractory settings.

Ewing sarcoma (ES) is an aggressive cancer thought to arise from mesenchymal stem cells that primarily affects bone and soft tissue, with a peak incidence in adolescents and young adults, affecting ~1.5 per million in that population.^1–3 It is often caused by translocation of the EWSR1 gene with one of the erythroblast transformation-specific (ETS) family of genes, most commonly EWSR1::FLI1. ^3,4 ES may occur in almost any bone or soft tissue (most commonly, the pelvis, axial skeleton, and femur) and patients typically present with pain and swelling in the affected site.⁵ Approximately 1 in 4 patients are diagnosed with metastatic disease, with metastasis most commonly affecting the lung, bone, and bone marrow.⁶ Current treatment for ES is multimodal, comprising combination chemotherapies, radiotherapy, and surgery, with event-free survival (EFS) rates for patients with localized disease approaching 75%.^3,7 However, such treatment is associated with acute and long-term toxicities and patients with metastatic or recurrent disease continue to have poor outcomes.³ There is therefore a need for new therapeutic approaches for ES, particularly for those with metastatic disease.

The disialoganglioside GD2 subtype, which is thought to be involved in cell signaling, is overexpressed in a wide range of tumors but has limited expression in normal tissues, making it a suitable target for cancer therapy.⁸ Tumors with high expression of GD2 include neuroblastoma, melanoma, osteosarcoma, soft tissue sarcoma, and ES.⁸ Indeed, GD2 surface expression has been observed on ES tumor cells, representing a suitable target antigen for immunotherapies.^9–11 Anti-GD2 antibodies, such as dinutuximab and dinutuximab beta (DB), bind to GD2 on the surface of tumor cells and target them for destruction by the body’s immune system via mechanisms thought to include antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and direct cytotoxicity by initiation of apoptosis.^8,12 On the basis of studies conducted by the International Society for Paediatric Oncology European Neuroblastoma (SIOPEN),^13,14 DB is approved in Europe for the treatment of high-risk neuroblastoma in patients ≥12 months old who have previously received induction chemotherapy and achieved at least a partial response, followed by myeloablative therapy and stem cell transplantation, as well as patients with a history of relapsed/refractory neuroblastoma, with or without residual disease.¹⁵ The addition of an anti-GD2 antibody, such as DB, to an existing cytotoxic regimen may also be effective in other tumors expressing high levels of GD2, such as osteosarcoma and ES.^8,16–18

We have previously reported 3 cases in which patients with newly diagnosed, metastatic, GD2-positive ES, or Ewing-like sarcoma were treated with DB in addition to standard chemotherapeutic regimens.¹⁷ In these cases, DB was well tolerated and all 3 patients achieved complete remission, without evidence of relapse.¹⁷ Here, we report our experience of using DB in a larger cohort of patients with ES, in both the frontline and relapsed/refractory settings.

MATERIALS AND METHODS

Patients

This was a case series of patients with ES treated with DB at the University Hospital “St. George,” Plovdiv, Bulgaria, between 2019 and 2024. Diagnosis of ES and Ewing-like sarcoma was confirmed by verifying the presence of ES transcripts using fluorescence in situ hybridization. Patients with metastatic ES expressing GD2 were treated with DB, which is not currently approved for the treatment of ES, in combination with chemotherapy in either the frontline or relapsed/refractory setting, over a follow-up duration of 2 to 5 years. The use of DB was approved by the Executive National Drug Agency and a steering committee of 3 pediatric onco-hematologists. Patients with metastatic ES who did not express GD2 and those with localized ES were treated according to standard of care without DB in the frontline and relapsed/refractory settings and included in the study for comparison. The comparator group also included GD2-positive patients with metastatic disease who chose not to be treated with DB. The presence of GD2 expression was assessed via immunohistochemistry in paraffin-embedded tumor specimens, using a murine anti-GD2 antibody, as previously described.¹⁷ All patients/their carers provided written informed consent.

Treatment

Chemotherapy Regimens

Patients treated in the frontline setting received the EURO EWING 2012 Protocol chemotherapy backbone (either Arm A or Arm B).¹⁹ DB was administered either during or following consolidation chemotherapy. Patients treated in the relapsed/refractory setting had previously received various standard first-line treatments and were treated with DB in several cycles in combination with a variety of chemotherapy reinduction regimens, including intrathecal chemotherapy, ifosfamide/carboplatin/etoposide (ICE regimen), and high-dose ifosfamide. Relapsed/refractory patients also received other treatments at or after relapse/progression, including radiotherapy, autologous stem cell transplantation, chemotherapy (ICE, ifosfamide, or irinotecan/temozolomide), and targeted therapy with pazopanib, regorafenib, or vorinostat.

Anti-GD2 Treatment With DB

All patients with GD2-positive, metastatic ES who were treated with DB in the frontline and relapsed/refractory settings received it under compassionate use as a long-term infusion (10 mg/m²/d over 10 days for 5 cycles or according to the clinician’s decision), in line with SIOPEN recommendations for the treatment of neuroblastoma.²⁰ DB was administered at the end of a chemotherapy cycle (not during it). Patients received granulocyte colony-stimulating factor when their absolute neutrophil count was <1000/mm³, but did not receive interleukin-2 during treatment. In addition, patients received gabapentin or intravenous tramadol (but not morphine) for pain prevention, and antihistamines (but not glucocorticoids), if needed.

Treatment Outcomes

Treatment outcomes included median overall survival (OS) and median time to progression. Adverse events were assessed using Common Terminology Criteria for Adverse Events (CTCAE; version 5.0).

RESULTS

Patients

A total of 37 patients with ES, including 1 patient with Ewing-like sarcoma (with CIC-DUX4 fusion), were treated at the University Hospital “St. George,” Plovdiv, Bulgaria, between 2019 and 2025. Immunohistochemistry revealed that 15/16 (93.8%) patients with metastatic ES who were tested for GD2 were GD2-positive, and one (6.3%) was GD2-negative. Of the 15 GD2-positive patients, 13 were treated with DB: 9 (7 males, 2 females; ages 5 to 17 y) were treated in the frontline setting (Table 1)²¹ and 4 (3 males, 1 female; ages 1 to 18 y) were treated in the relapsed/refractory setting (Table 2). Among the frontline patients, 6 previously underwent surgery (4 with no residual tumor [R0]; 2 with macroscopic residual tumor [R2]); 7 received the EURO EWING 2012 Protocol Arm A induction chemotherapy regimen, and 2 received Arm B. The relapsed/refractory patients had experienced 2 to 6 prior relapses and had received 1 to 6 previous lines of chemotherapy, with the most recent remission duration ranging from 6 to 45 months; all received the EURO EWING 2012 Protocol Arm A induction chemotherapy regimen.

TABLE 1.

Patient Demographic, Clinical, and Treatment Characteristics and Outcomes in Frontline Patients Treated With DB (n=9)

Patient number	Age (y)	Sex	PS	Type of metastatic sites	GD2 expression	ES transcript status	Treatment before DB	Age at start of DB (y)	Timing of DB administration and number of cycles	Response before DB	Response after DB	Best response
1	10	M	Femur	Lung (multiple)	Positive	EWSR FLI	Surgery, R0 RT	11	Consolidation after chemotherapy 5 cycles	PR	CR	CR at the end of therapy
2	17	M	Pelvis	Lung	Positive	EWSR ETV4	RT	18	Consolidation after chemotherapy + ASCT 5 cycles	Partial metabolic	Partial morphologic+ full metabolic response	CR metabolically and PR morphologically
3	6	M	Temporal	Bone (single)	Positive	EWSR FLI1	Surgery, R2 RT	6	In the course of postoperative chemotherapy 5 cycles	CR	CR	CR after the induction chemotherapy + RT
4	15	F	Vertebrae T1-T4	Bone (skip), pleural effusion, lung	Positive	EWSR1 ERG	Surgery, R2 RT	16	In the course of postoperative chemotherapy 5 cycles	PR	CR	CR after the first DB cycle
5	5	M	Vertebrae C3-C7	Pulmonary	Positive	EWSR FLI1	Surgery, R0 RT	5	In the course of postoperative chemotherapy 5 cycles	CR	CR	CR at the end of induction
6	12	F	Femur	Lung, bone marrow	Positive	EWSR CIC DUX	Surgery, R0 RT	12	In the course of postoperative chemotherapy 5 cycles	Partial metabolic and morphologic	CR	CR after DB initiation
7	14	M	Pelvis	Lung, bone, bone marrow	Positive	EWSR FLI1	RT	15	Consolidation after chemotherapy 5 cycles	Partial metabolic and morphologic	Full metabolic and partial morphologic	CR metabolically and PR morphologically at the end of chemotherapy
8	17	M	Tibia	Lung, bone marrow	Positive	EWSR ETV1	Surgery, R0 RT	18	Consolidation after chemotherapy 5 cycles	Partial morphologic and full metabolic	Full metabolic and morphologic	CR in the course of consolidation
9	13	M	Mandible	Lung	Positive	EWSR FLI1	RT	14	In the course of postoperative chemotherapy 5 cycles	CR	CR	CR in the course of induction

ASCT indicates autologous stem cell transplantation; CR, complete response; DB, dinutuximab beta; ES, Ewing sarcoma; F, female; GD2, disialoganglioside subtype GD2; M, male; PR, partial response; PS, primary site; R0, no residual tumor; R2, macroscopic residual tumor; RT, radiotherapy.

TABLE 2.

Patient Demographic, Clinical, and Treatment Characteristics and Outcomes in Relapsed/Refractory Patients Treated With DB (n=4)

Patient number	Age (y)	Sex	PS	Type of metastatic sites	GD2 expression	ES transcript status	Metastatic stage at diagnosis/at relapse	Treatment lines before DB	Timing of DB administration and number of cycles	Response before DB	Response after DB	Other treatment at/after relapse
1	2	M	Scapula	Bone marrow	Positive	EWSR ERG	2A/4B	2	Consolidation after second-line chemotherapy, radiotherapy, and ASCT 5 cycles	Partial metabolic response	Full metabolic response	At relapse: I/T chemotherapy, RT After relapse: ASCT, targeted therapy (pazo) Targeted therapy after progression (rego)
2	1	F	Spine	Lung, bone, bone marrow	Positive	EWSR FLI1	2B/4B	5	In the course of seventh-line chemotherapy 4 cycles	PD	Further progression and a later fatal outcome	At relapse: I/T / ICE chemotherapy, RT After relapse: Targeted therapy (pazo) Targeted therapy after progression (rego)
3	18	M	Sternum	Bone (multiple), lung, lymph nodes	Positive	EWSR FLI1	4B/4B	1	In the course of second-line chemotherapy 7 cycles	PD	Full metabolic response, PR morphologically	At relapse: Ifos/ICE chemotherapy After relapse: surgery, ASCT, targeted therapy (rego) Targeted therapy after progression (vori)
4	11	M	Humerus	Bone, bone marrow, lungs	Positive	EWSR FLI1	3B/4B	1	Consolidation after second-line chemotherapy, radiotherapy, and ASCT 5 cycles	SD	CR at the end of treatment, full metabolic and morphologic response	At relapse: RT After relapse: surgery Amp, ASCT, targeted therapy (rego)

Amp indicates amputation; ASCT, autologous stem cell transplantation; CR, complete response; DB, dinutuximab beta; ES, Ewing sarcoma; F, female; GD2, disialoganglioside subtype GD2; H, humerus; I/T, irinotecan/temozolomide; ICE, ifosfamide/carboplatin/etoposide; Ifos, ifosfamide; M, male; pazo, pazopanib; PD, progressive disease; PR, partial response; PS, primary site; rego, regorafenib; RT, radiotherapy; SD, stable disease; vori, vorinostat.

The 24 patients who were not treated with DB include 20 patients who were treated in the frontline setting and 4 in the relapsed/refractory setting. The 20 frontline patients (13 males, 7 females; ages 10 mo to 18 y) comprised 17 patients with localized disease, 1 GD2-negative patient with metastatic disease, and 2 GD2-positive patients with metastatic disease who did not want to be treated with DB (Table 3).²¹ Seventeen patients underwent surgery (15 with no residual tumor [R0]; 1 with no residual tumor/amputation [R0/Amp]; 1 with no residual macroscopic tumor but margins demonstrating presence of tumor microscopically [R1]). Eight received the EURO EWING 2012 Protocol Arm A induction chemotherapy regimen and 12 received Arm B. The 4 relapsed/refractory patients all had metastatic disease, had experienced 1 to 6 relapses, and received ≥2 lines of chemotherapy; all 4 patients received the EURO EWING 2012 Protocol Arm A induction chemotherapy regimen (Table 4).

TABLE 3.

Patient Demographic, Clinical, and Treatment Characteristics and Outcomes in Frontline Patients Not Treated With DB (Comparator Group; n=20)

Patient number	Age (y)	Sex	PS	Type of metastatic sites	GD2 expression	ES transcript status	Treatment	Best response
1	17	F	Femur	Bone, lymph nodes	Not evaluated	EWSR FLI1	Surgery, R0 RT	CR at the end of treatment
2	11	F	Femur	Localized disease	Not evaluated	EWSR ETV1	Surgery, R0 RT	CR after surgery
3	18	M	Pelvis	Bone marrow, multiple bones, lungs, lymph nodes	Negative	EWSR FLI1	RT	Full metabolic response, partial morphologic at the end of treatment
4	10	M	Femur	Localized disease	Not evaluated	EWSR FLI1	Surgery, R0 RT	CR after surgery
5	4	M	Vertebrae L3-S2	Lungs	Not evaluated	EWSR ERG	Surgery, R0 RT	CR at the end of treatment
6	12	F	Femur	Localized disease	Not evaluated	EWSR FLI1	Surgery, R0 RT	CR after the surgery
7	12	F	Femur	Bone Lymph nodes Lungs	Not evaluated	EWSR FLI1	Surgery, R0 RT	CR after the surgery
8	17	M	Tibia	Localized disease	Not evaluated	EWSR FLI1	Surgery, R0/Amp RT	After the Amp of the limb
9	10 mo	M	Right infratemporal fossa/mastoid	Locally aggressive/infiltration of the ear and brain	Not evaluated	EWSR FLI1	RT	CR after chemotherapy before RT
10	13	F	Scapula	Lungs, bone	Not evaluated	EWSR FLI1	Surgery, R0 RT	CR at the end of treatment
11	9	F	Scapula	Bone marrow, bones, lungs	Not evaluated	EWSR FLI1	Surgery, R0 RT	CR at the end of treatment
12	7	M	Femur	Localized disease	Not evaluated	EWSR FLI1	Surgery, R0 RT	CR at the end of treatment
13	15	F	Pelvis	Skip in pelvis	Not evaluated	EWSR ETV1	Surgery, R0 RT	CR after induction
14	13	M	Finger	Localized disease	Not evaluated	EWSR FLI1	Surgery, R0 RT	CR after surgery
15	8	M	Ulna	Localized disease	Not evaluated	EWSR FLI1	Surgery, R0 RT	CR after surgery
16	10	M	Fibula	Localized disease	Not evaluated	EWSR FLI1	Surgery, R1 RT	CR after surgery
17	15	M	Tibia	Lungs, lymph nodes	Not evaluated	EWSR FLI1	Surgery, R0 RT	No remission (<3 mo)
18	6	M	Vertebrae C5–C6	Localized disease	Positive	EWSR FLI1	Surgery, R0 RT	CR after surgery
19	16	M	Femur	Lungs	Not evaluated	EWSR FLI1	Surgery, R0 RT	PR at the end of treatment, followed by progression
20	18	M	Pelvis	Skip in the pelvis and proximal femur	Positive	EWSR FLI1	Surgery RT	Partial metabolic and full morphologic remission at the end of induction Early discontinuation of the treatment followed by progression

Amp indicates amputation; CR, complete response; DB, dinutuximab beta; ES, Ewing sarcoma; F, female; GD2, disialoganglioside subtype GD2; M, male; mo, months; PR, partial response; PS, primary site; R0, no residual tumor; R1, no residual macroscopic tumor but margins still demonstrate presence of tumor microscopically; RT, radiotherapy.

TABLE 4.

Patient Demographic, Clinical, and Treatment Characteristics and Outcomes in Relapsed/Refractory Patients Not Treated With DB (Comparator Group; n=4)

Patient number	Age (y)	Sex	PS	Type of metastatic sites	GD2 expression	ES transcript status	Metastatic stage at diagnosis/relapse	Other Treatment at/after relapse
1	15	M	Pelvis	Bone, bone marrow, lungs	Not evaluated	EWSR 1FLI	4В/4B	At relapse: Cyclophosphamide/topotecan chemotherapy, RT Progression 1: Gemcitabine/ docetaxel/bevacizumab Progression 2: Gemcitabine/ carboplatin/liposomal doxorubicin
2	9	F	Right tibia	Bone, bone marrow, liver, lungs, lymph nodes	Not evaluated	EWSR1 ERG	2B/4B	At relapse: Oral cyclophosphamide/ celecoxib + I/T
3	18	M	Rib	Pleural effusion, unilateral lymphadenomegaly, bone	Not evaluated	EWSR1 FLI1	3B/4B	At relapse: I/T + pazo in alternation HD Ifos After progression: Gemcitabine/ docetaxel/bevacizumab
4	15	M	Femur	Bone, localized disease	Not evaluated	EWSR FLI1	4A/4B	At relapse: I/T + ASCT + HD Ifos + zoledronic acid At progression: Nivolumab + ipilimumab + gemcitabine/ docetaxel/bevacizumab

ASCT indicates autologous stem cell transplantation; DB, dinutuximab beta; ES, Ewing sarcoma; F, female; GD2, disialoganglioside subtype GD2; HD, high dose; I/T, irinotecan/temozolomide; Ifos, ifosfamide; M, male; pazo, pazopanib; RT, radiotherapy.

Survival Outcomes

Patients treated with DB in the frontline setting demonstrated a median OS of 1877 days and a median time to progression of 1811 days. In contrast, those frontline patients who did not receive DB, but were treated with standard therapy, had a median OS of 1547 days and a median time to progression of 1261 days. In the relapsed/refractory setting, our 4 patients treated with DB demonstrated a median OS of 810 days, with a median time to progression of 782 days, whereas those treated with standard therapy alone in our center had a median OS of 210 days and a median time to progression of 113 days.

Adverse Events in Patients Treated With DB

All patients treated with DB experienced Grade 1 or 2 pain during the first course of DB infusion; there were no cases of Grade 3 or 4 pain. Three patients (1 frontline; 2 relapsed/refractory) experienced Grade 1 or 2 peripheral neuropathy; again, during the first course of DB. One patient with relapsed/refractory ES experienced Grade 3 capillary leakage syndrome after the sixth course of DB, which resolved following treatment discontinuation for 48 hours and supportive care (human albumin, diuretics, and dopamine infusion). Three patients (1 frontline; 2 relapsed/refractory) experienced Grade 1 or 2 optic nerve disorders; 2 patients had total resolution after the end of the treatment, and the other had a persisting disorder up until the time of their death. One patient, treated in the frontline setting, experienced transverse myelitis during the last day of their final (fifth) course of DB. The DB infusion was discontinued 2 hours earlier than scheduled, and the transverse myelitis totally resolved after steroid treatment for 3 months; there was no relapse of transverse myelitis or ES over the subsequent 15 months of follow-up. All patients experienced pancytopenia, as expected with their chemotherapy regimens. Febrile neutropenia occurred in 20% of patients treated with DB and in 25% not treated with DB. The infection rates in patients treated with DB were as follows: 4% gastrointestinal infections, 10% mucositis, 1% sepsis, 5% urinary tract infections, and 12% respiratory infections. In patients not treated with DB, the infection rates were 2% gastrointestinal infections, 5% mucositis, 2% sepsis, 6% urinary tract infections, and 10% respiratory infections. There were no hypersensitivity reactions or central nervous system side effects in either group.

DISCUSSION

Standard therapies for patients presenting with metastatic ES have been associated with low survival rates.²² Treatment failure rates range from 50% to 80% depending on the site of metastasis.²³ There is, therefore, an urgent need for innovative treatments to improve outcomes for patients with metastatic ES. In the current case series, the survival outcomes of 13 patients with GD2-positive metastatic ES who were treated at our center with DB in either the frontline (n=9) or relapsed/refractory (n=4) setting were substantially better than those of the frontline (n=20, with mainly localized disease) or relapsed/refractory (n=4, with metastatic disease) patients who were treated only with standard therapy. The safety findings from the study were consistent with DB’s safety profile in patients with neuroblastoma, although we observed no hypersensitivity reactions (a very common adverse reaction in neuroblastoma clinical trials),¹⁵ likely due to the administration of DB as a long-term infusion (10 mg/m²/d over 10 d), which has been shown to minimize the occurrence of adverse events. This case series builds on our previous experience, having initially reported promising outcomes for frontline treatment with DB in 3 patients with metastatic, GD2-positive ES, or Ewing-like sarcoma.¹⁷ Together, our findings indicate that DB seems to be effective in treating metastatic ES, in the frontline and the relapsed/refractory setting. However, prospective randomized clinical trials are needed to confirm these observations and to better assess the efficacy and safety of DB in combination with standard treatment. In the frontline setting, DB was effective when administered either during postoperative chemotherapy or as consolidation after autologous stem cell transplantation.

In Europe, the targeting of GD2 with DB is the standard of care maintenance treatment for high-risk neuroblastoma, and DB is also approved for use in patients with relapsed/refractory neuroblastoma.^15,24 In addition to expressing high levels of GD2,⁸ ES and neuroblastoma are similar in being “small round blue cell tumors,” these comprising a group that also includes other childhood tumors, such as non-Hodgkin’s lymphoma, rhabdomyosarcoma, and primitive neuroectodermal tumor.²⁵ These tumors appear similar using light microscopy, but differential diagnosis is possible using genetic analysis (eg, MYCN for neuroblastoma, EWSR1 for EWING sarcoma, and PAX3/7-FOXO1 for embryonal rhabdomyosarcoma^26–28) and immunohistochemistry with antibodies against specific tumor-related antigens, including neuron-specific enolase, CD99, NB84, SATB2, and GD2.^25,29,30 It is therefore possible that DB may be effective in other small round blue cell tumors associated with high GD2 expression, although this remains to be determined in future studies. Others have also previously suggested that anti-GD2 antibodies may be a suitable treatment option for some small round blue cell tumors.³¹ Immunohistochemistry for GD2 expression is best conducted using paraffin blocks rather than fresh frozen material (in the authors’ experience). Although we were able to confirm GD2 expression using fully automated immunohistochemistry of paraffin-embedded tumor specimens before DB treatment, a standardized immunohistochemistry protocol is required to routinely categorize tumors based on GD2 expression.¹⁷ A prospective randomized trial is also required to directly compare outcomes in patients with metastatic and nonmetastatic GD2-positive ES treated with conventional treatment plus DB versus patients with metastatic GD2-negative ES treated with conventional treatment alone.

Our study is limited by its small sample size and retrospective nature, which might have resulted in selection bias. It is also limited by the fact that the frontline comparator group was not case-controlled and comprised patients who predominantly (85%) had localized rather than metastatic disease. Nevertheless, since outcomes are considerably better for ES patients with localized versus metastatic disease,^3,21 it is all the more notable that DB treatment improved outcomes in our patients with metastatic disease to such an extent when compared with the group not treated with DB.

In summary, our study provides evidence that anti-GD2 immunotherapy with DB, when combined with chemotherapy, represents a promising therapeutic option to improve outcomes in patients with metastatic ES, in both the frontline and relapsed/refractory settings.