Long-Term Follow-up of a Phase III Study of ch14.18 (Dinutuximab) + Cytokine Immunotherapy in Children with High-Risk Neuroblastoma: COG Study ANBL0032

This article is featured in Highlights of This Issue, p. 2121

Survival from neuroblastoma, the most common extracranial solid tumor of childhood, has improved with multimodality therapy. However, relapse is common for patients with high-risk disease (1). Dinutuximab is a chimeric mAb that binds the disialoganglioside GD2 and activates complement (2). GD2 is a glycolipid that is strongly expressed on the surface of neuroblastoma cells with little intratumor or intertumor heterogeneity in expression, while normal tissue expression is restricted to neurons, melanocytes, and peripheral pain fibers. Dinutuximab mediates antibody-dependent cellular cytotoxicity (ADCC) by neutrophils and natural killer (NK) cells. These effector cells bind antibody via cell surface Fcγ receptors (FcγR), a family of cell surface glycoproteins mediating clearance and phagocytosis of immune complexes, ADCC and the release of inflammatory cytokines. The FCGR genes display polymorphisms that greatly influence the affinity of FcγR for IgG (3–5), which may contribute to the action of cytotoxic antibodies. GM-CSF enhances ADCC (6). IL2 augments ADCC of neuroblastoma cells by NK cells (7).

Phase I studies of dinutuximab and GM-CSF (8), and dinutuximab plus GM-CSF alternating with IL2 and isotretinoin (9), led to a randomized phase III trial of dinutuximab combined with alternating cycles of GM-CSF and IL2 followed by isotrentinoin in children with high-risk neuroblastoma [Children's Oncology Group (COG) study ANBL0032]. This trial tested the hypothesis that anti-GD2 immunotherapy would eradicate minimal residual neuroblastoma that persisted following multiagent induction chemotherapy, primary tumor resection, myeloablative chemotherapy, external beam radiotherapy, and isotretinoin. The initial results of this trial demonstrated that immunotherapy plus isotretinoin improved both EFS and OS (2), leading to FDA approval of ch14.18, renamed dinutuximab (Unituxin, United Therapeutics) in 2015. Here we report a long-term clinical update of this phase III trial and the results of immune correlative analyses.

This study (NCT00026312) opened in October 2001 and enrollment to the randomized portion ended in January 2009. All patients had high-risk neuroblastoma. Eligibility criteria included age <31 years, completion of intensive therapy including induction chemotherapy, high-dose chemotherapy followed by autologous stem cell transplant (ASCT), local radiotherapy, and at least partial remission without a history of progressive disease. Time from stem cell infusion to enrollment was recommended to be <100 days; time from start of induction therapy had to be ≤9 months. Patients were also required to have a total absolute phagocyte count [white blood cell × % (segs + bands + monos)] >1,000/μL following ASCT, performance score ≥50, life expectancy ≥2 months, and adequate organ function. Written informed consent was obtained from parents or guardians before the initiation of any study-related treatment or procedures, and the protocol was approved by the Ethics Committees from all participating institutions. The study was conducted in accordance with Good Clinical Practice principles, and the Declaration of Helsinki.

The trial involved a 1:1 randomization to six cycles of isotretinoin with or without five concomitant cycles of immunotherapy. Randomization occurred at the time of enrollment and was stratified on the basis of factors thought to potentially affect the posttransplantation outcome: response before ASCT, induction therapy protocol, number of ASCT cycles, and use of purged versus nonpurged stem cells (10). Patients with biopsy-proven persistent disease after autologous stem cell transplantation and radiotherapy were nonrandomly assigned to the immunotherapy group and were excluded from the primary outcome analyses. As reported previously (2), isotretinoin 160 mg/m²/day was given orally for 14 days every 28 days for six cycles for patients assigned to isotretinoin alone. The immunotherapy arm consisted of dinutuximab (25 mg/m²/day × 4 days) given every 28 days in combination with GM-CSF (Leukine, Berlex/Bayer; 250 mcg/m²/day × 14 days) in cycles 1, 3, and 5 or IL2 (Proleukin, Chiron) at 3 × 10⁶ IU/m²/24 hours × 96 hours during the week prior to dinutuximab and at 4.5 × 10⁶ IU/m²/24 hours × 96 hours during the dinutuximab. Dinutuximab (provided by the NCI) was infused over 5–20 hours each day based on tolerability. Note, 25 mg of dinutuximab provided as an experimental agent by the NCI was later determined to be equivalent to 17.5 mg of the FDA-approved formulation of dinutuximab (Unituxin, United Therapeutics; ref. 11).

Fresh heparinized blood was obtained from patients receiving immunotherapy at the following timepoints: pretreatment (day 1); day 6 (prior to fourth dinutuximab dose during cycle 1, corresponding to a “peak” dinutuximab level); day 80 (prior to first dose of IL2 during cycle 4, corresponding to a “trough” dinutuximab level); day 90 (prior to fourth daily dinutuximab dose during cycle 4, corresponding to a “peak” dinutuximab level); day 111 (prior to initial dose of GM-CSF during cycle 5, corresponding to a “trough” dinutuximab level); day 118 (prior to fourth daily dinutuximab dose during cycle 5, corresponding to a “peak” dinutuximab level). These timepoints and their relationship to protocol therapy are summarized in Supplementary Table S1. Blood samples were collected from patients randomized to isotrentinoin alone prior to the fourth cycle and end of therapy. Sample size varies depending on the number of specimens available at each timepoint.

Dinutuximab levels were measured in plasma samples by ELISA as described previously (8, 9).

Human anti-chimeric antibody (HACA) levels were estimated in plasma samples by the ELISA “bridging assay” as published previously (8, 9, 12) for all patients entered into this trial that received dinutuximab and provided plasma samples. Briefly, for these assays optical density (OD) values <0.3 were considered negative; OD values ≥0.3 but <1.0 were considered intermediate and OD values ≥1.0 were considered strong.

Soluble IL2 receptor (sIL2R) levels in plasma were measured as reported previously (8, 9, 13).

The region surrounding the polymorphic codon 158 of FCGR3A (rs396991; ref. 5) and codon 131 of FCGR2A (rs1801274; ref. 14) were selectively amplified, purified, and genotyped by direct sequence analysis, as shown below:

FCGR3A was selectively amplified over the highly homologous FCGR3B (AH003573) using a FCGR3A-specific antisense primer in combination with a common sense primer (4983F). The genotype of codon 158 was assessed by direct sequence analysis with primers 5128F and 5390R. Specific amplification of FCGR3A over FCGR3B is validated by assessing the sequence of polymorphic codons 129, 140, 148, which differ between the two genes.

Primers for FCGR3A

3A-X5R-C 5′-CCT TCC AGT CTC TTG TTG AGC TTC G-3′

4983F 5′-CTC AGG ATC TGG GTG GTA CG-3′

5128F 5′-TGA GGT GTC ACA GCT GGA AG-3′

5390R 5′-AGT GTG ATT GCA GGT TCC ACT-3′

FCGR2A was selectively amplified over the relatively dissimilar isoforms FCGR2B (AH005422.2) or FCGR2C (AH002832.2) using FCGR2A selective primers Fc2A-JF and FC2A-JR. The genotype of codon 131 was assessed by direct sequence analysis with FCGR2A selective primer FC2A-JiR. Specific amplification of FCGR2A was validated by assessing the sequence of the multiple polymorphic codons unique to FCGR2A.

Primers for FCGR2A

Fc2A-JF 5′-GGA AAA TCC cAG AAA TTc TCG C-3′

Fc2A-JR 5′-CAA CAG CCT GAC TAC CTA TTA CGC GGG-3′

Fc2A-JiR 5′-AGC TCT GGC CCC TAC TTG TT-3

Amplifications were performed using 50 ng genomic DNA extracted from mononuclear cells or granulocytes using the Gentra DNA isolation kit (Qiagen). Fc receptors were amplified in a 50 μL reaction mixture consisting of 2 mmol/L MgSO4, 100 μmol/L 4dNTP, 10 pmol each sense and antisense primer, 1.25 units Platinum Taq DNA Polymerase High Fidelity (Invitrogen) in 1× Platinum Taq DNA Polymerase High Fidelity buffer. Reactions were denatured at 94°C for 2 minutes, then cycled 35× at 94°C (30 seconds), [64°C (15 seconds) for FCGR3A, 55°C (60 seconds) FCGR2A], and 68°C (120 seconds).

PCR products were purified using Qiagen QiaQuick or Zymo DNA Clean and Concentrate DNA purification columns and sequenced either at the Moores-UCSD Cancer Center shared sequencing resource or at Retrogen Inc.

FCGR genotypes were correlated with EFS/OS of patients based on actual treatment received using a log-rank test.

The primary analysis was an intention-to-treat comparison of EFS between the two randomized treatment groups (2). The study was designed to enroll 386 randomized patients, with 80% power using a two-sided log-rank test with alpha = 0.05 (or one-sided test with alpha = 0.025) to detect an absolute difference of 15% in 3-year EFS between the two randomized groups.

Sequential monitoring of the intention-to-treat population was performed as reported previously. The study met criteria for early stopping owing to efficacy, and randomization was halted (2). Patients on the isotretinoin-alone arm were permitted to cross-over to receive antibody after randomization was stopped. Patients with biopsy-proven persistent disease after ASCT and radiotherapy were nonrandomly assigned to the immunotherapy group and were excluded from the primary outcome analyses.

EFS time was defined as the time from study enrollment until first occurrence of relapse, progressive disease, secondary cancer, or death or, if none occurred, until last contact. OS time (secondary endpoint) was defined as the time from study enrollment until death, or last contact if death did not occur. Kaplan–Meier survival curves (ref. 15; reported as point estimates ± SE) were compared by treatment group using a one-sided log-rank test; all other survival comparisons were two sided. Cross-over patients from the isotretinoin-alone arm who received antibody were censored at the start of antibody therapy in survival analyses. Patient characteristics at baseline were compared between randomized groups using a χ² test. P < 0.05 were considered statistically significant.

Wilcoxon signed-rank tests were performed to test for differences in median levels of dinutuximab and sIL2R between timepoints. The data tables for these plasma assays indicate the number of patients with evaluable samples at the specified timepoints. Cox proportional hazard models were used to test for association between EFS and dinutuximab levels at each timepoint. Wilcoxon rank-sum tests compared levels of dinutuximab or sIL2R at each timepoint between (i) patients in the randomized cohort that received dinutuximab, with versus without occurrence of a dose-limiting toxicity (DLT) and, (ii) between HACA-positive versus HACA-negative patients. DLT was defined as ≥grade 4 allergic reaction, vascular leak syndrome, or persistent neuropathic pain ≥4 days after completing ch14.18 infusion, or ≥grade 3 motor neuropathy for ≥2 weeks. Log-rank tests were used to compare EFS and OS between HACA-positive versus HACA-negative patients. Associations between occurrence of a DLT at any time during treatment and HACA positivity were tested with a Fisher exact test. Comparisons were two sided, except for tests of HACA versus dinutuximab levels which were one sided based on findings (12) that strong antidrug antibody responses (like HACA) can neutralize detection of plasma levels of the therapeutic drug, resulting in lower dinutuximab levels in HACA-positive patients.

Except as otherwise stated, analyses were performed using SAS version 9.4.

The authors have not been paid to write this article. The corresponding author had full access to all of the data and had final responsibility for the decision to submit for publication. The full protocol for this trial entitled “Isotretinoin With or Without Dinutuximab, Aldesleukin, and Sargramostim Following Stem Cell Transplant in Treating Patients With Neuroblastoma.” can be accessed by COG members at www.cogmembers.org.

Two hundred and fifty-seven patients were enrolled; enrollment was stopped prior to enrolling the planned 386 patients due to statistical evidence of efficacy (2). Four patients were deemed ineligible and 27 with persistent disease were nonrandomly assigned to immunotherapy. These 31 patients were excluded from all analyses, resulting in an analytic cohort of 226 patients randomized to receive isotrentinoin and immunotherapy (n = 114) or isotrentinoin alone (n = 112). There were no significant differences in baseline characteristics between randomized cohorts (Table 1). Four patients were permitted to cross-over to the immunotherapy arm after the randomization was stopped. Six patients randomized to immunotherapy refused protocol therapy and received isotrentinoin only; 7 patients randomized to isotrentinoin alone received immunotherapy.

For 226 eligible randomized patients, the 5-year EFS and OS estimates were 51.4 ± 3.5% and 65.2 ± 3.3%, respectively. The median follow-up time in the 113 patients alive without an event was 9.97 (range, 0.7–15.3) years. Among baseline characteristics, only age at diagnosis, disease stage, and pre-ASCT response had significant impact on the clinical outcomes (Table 1). The EFS for 183 stage 4 patients was significantly worse than for non-stage 4 patients (5-year EFS: 46.7 ± 3.8% vs. 83.4 ± 7.1%; P = 0.0009). For 174 patients in complete remission (CR)/very good partial remission (VGPR) pre-ASCT, EFS was superior to those in PR (5-year EFS: 56.1 ± 3.9% vs. 35.6 ± 6.7%; P = 0.0055). For the randomized comparison, the 5-year EFS was 56.6 ± 4.7% for immunotherapy versus 46.1 ± 5.1% for isotretinoin only (P = 0.042; Fig. 1A). The 5-year OS was 73.2 ± 4.2% for immunotherapy versus 56.6 ± 5.1% for isotretinoin only (P = 0.045; Fig. 1B). As shown in Fig. 1A, early relapses occurred more frequently in the isotretinoin group than in the immunotherapy group, with 93.3% versus 69.8% of events, respectively, occurring within the initial 2 years. After 2 years, there were 4 and 16 events in the isotretinoin and immunotherapy arms, respectively. All events were due to relapse/progression, except for 1 patient in each group with a secondary malignancy. For 158 stage 4, ≥18 months old patients, EFS (5-year: 48.3 ± 5.6% vs. 36.7 ± 6.1%; P = 0.022) and OS (70.1 ± 5.2% vs. 49.1 ± 6.3%; P = 0.025) were statistically significantly higher in patients assigned to immunotherapy compared with those assigned to isotretinoin (Fig. 2A and B).

Table 2 shows trough (days 80 and 111) and peak (days 6, 90, and 118) dinutuximab levels obtained from the six timepoints (Supplementary Table S1). Formal pharmacokinetic parameters could not be assessed from these available peak and trough values. Dinutuximab trough levels were higher than the pretreatment level (P < 0.0001), indicating the persistence of dinutuximab for 2.5 weeks following the prior dinutuximab dose and consistent with previously published detailed pharmacokinetic evaluations (16). Peak levels of dinutuximab during GM-CSF–containing cycles (1 and 5) were similar, but the peak value on D90 during the IL2-containing cycle (cycle 4) was lower (P < 0.005). There was no significant correlation of either peak or trough dinutuximab levels with DLT or EFS (Supplementary Table S2).

sIL2R is released by IL2-responsive cells when activated by IL2. The sIL2R levels were significantly greater during IL2-containing cycles (day 90) compared with the three GM-CSF–containing cycles and a pre-IL2 level (day 80; Table 2). The sIL2R values for day 6 (GM-CSF containing) were significantly greater than pretreatment levels, consistent with the ability of GM-CSF to induce endogenous IL2 (13). The trough levels following either a GM-CSF– or IL2-containing cycle (day 80 and 111) also remained greater than the pretreatment levels (P < 0.0001), indicating sustained and sufficient activation of the endogenous IL2 system to cause continued release of sIL2R. There was no correlation between sIL2R level and DLT status at any time.

Detectible HACA responses were rare; 8 and 5 patients developed strong and intermediate responses, respectively (Supplementary Table S3). No HACA reactivity was detected during cycle 1 (day 6) while HACA was detected in 6 of 105 (5.7%) by cycle 3 (day 80) and 9 of 91 (9.9%) by cycle 5 (day 118). The presence of HACA was associated with significantly lower trough and peak levels of dinutuximab during cycle 4 (days 80 and 90) and 5 (days 111 and 118; Supplementary Table S4). There was no association of HACA reactivity with EFS or OS (P ≥ 0.41; Supplementary Fig. S1), or with occurrence of DLT (Supplementary Table S5). When the characteristics shown in Table 1 were compared in the patients who did or did not develop HACA, no statistically significant differences were found. Following the detection of HACA, the peak plasma levels of dinutuximab in cycle 4 (day 90) and cycle 5 (day 118) of 6 HACA-positive patients were <10% of the expected value; namely the peak dinutuximab level in cycle 1 (day 6) for the same patient (Supplementary Table S6) demonstrated that the HACA response, once developed, could neutralize detectible dinutuximab in vivo. Detection of a HACA response at any time after starting treatment was associated with higher levels of sIL2R on day 6 (P = 0.0022) and day 90 (P = 0.010; Supplementary Table S7).

Polymorphisms of the FCGR2A at codon 131 and FCGR3A at codon 158 are summarized in Table 3. The frequency of the FCGR2A and FCGR3A genotypes was similar in the immunotherapy and isotrentinoin only groups, consistent with that reported in the literature (17). No association between FCGR2A or FCGR3A genotype with survival was identified in either treatment group. Similarly, categorization of patients with heterozygous genotypes as high or low affinity had no association with outcome (data not shown). Patients were also categorized as having a high-affinity profile if they did not harbor a homozygous low-affinity genotype of either receptor (18) or if they had a homozygous high-affinity FCGR2A or FCGR3A genotype, regardless of the low allele genotype (Tables 4 and 5). While slightly different distribution profiles were observed using the two criteria, neither profile was associated with outcome (data not shown).

The results of our randomized phase III trial of immunotherapy with dinutuximab, IL2, and GM-CSF have changed the standard of care for patients with high-risk neuroblastoma (2) and led to FDA approval of dinutuximab. Our long-term follow-up data confirm superior EFS and OS for newly diagnosed patients with high-risk neuroblastoma randomized to immunotherapy compared with those randomized to isotretinoin alone, including the cohort of stage 4 patients. It is noteworthy that 13 of 226 (6%) randomized patients did not adhere to the randomized treatment they were assigned. This should be considered when evaluating the intent-to-treat results reported here. Nontheless, our findings attest to the important role of dinutuximab-based immunotherapy in high-risk neuroblastoma.

With longer follow-up, the improved EFS and OS associated with immunotherapy reported are decreased compared with our original 2010 report, due to late relapses in the immunotherapy group. Delayed relapses following immunotherapy with dinutuximab were also observed in a German study (19) in which the EFS declined from 50% at 2 years to 41% at 9 years. Downregulation of GD2 in bone marrow reported in a minority of patients may account for the relpases. Strategies to reduce late relapse following dinutuximab-based immunotherapy include increasing the number of immunotherapy cycles or delivering higher doses of an anti-GD2 antibody, but this could expose patients to more dinutuximab-associated toxicities (2, 11). However, hu14.18K322A, a humanized ch14.18 with a point mutation (K322A) that reduces complement-dependent lysis and allodynia (20), is dosed approximately 3-fold higher than the dose of dinutuximab that was administered in our trial and promising preliminary efficacy data in patients with relapsed or refractory neuroblastoma have been reported previously (20). Alternatively, addition of agents that may act synergistically with anti-GD2 immunotherapy should be considered. A recent COG study combining dinutuximab with chemotherapy led to an impressive 41.5% response rate in 53 patients with recurrent/refractory neuroblastoma (21), a finding consistent with the response rate observed when chemotherapy was combined with hu14.18K322A (22). High levels of polyamines and ornithine decarboxylase (ODC) activity are found in many human cancers including neuroblastoma. Eflornithine α-difluoromethylornithine (DFMO), an inhibitor of ODC, was shown to inhibit neuroblastoma initiation and progression in mice (23). DFMO also ameliorates anti-GD2 antibody–induced allodynia (24), making combination of dinutuximab and DFMO an attractive strategy. This approach is being studied in an ongoing COG trial (NCT03794349). Separate preclinical studies have also shown synergism between anti-GD2 antibodies in combination with anti-PD1 checkpoint blockade (25) further supporting future combinatorial therapy trials designed to enhance the efficacy of GD2-directed immunotherapy and reduce toxicities.

We found that sIL2R levels were significantly elevated over baseline levels during GM-CSF–containing cycles, suggesting that dinutuximab + GM-CSF treatment induced an endogenous IL2 response. This was reported previously with administration of GM-CSF alone (13) in patients with refractory cancers and in other clinical settings (26). As IL2 is known to activate NK cells to mediate ADCC in vitro (7), the induction of sIL2R in the absence of IL2 administration suggests that some of the desired effects from IL2 administration might also be induced in vivo by the dinutuximab + GM-CSF combination. Furthermore, toxicity associated with IL2-containing cycles is significantly worse than toxicity associated with GM-CSF–containing cycles (2). In addition, clinical data from two randomized European trials (27, 28) showed no added benefit when IL2 was added to dinutuximab beta therapy. Although dinutuximab beta is produced in Chinese hamster ovary (CHO) cells and differs slightly in glycosylation compared with dinutuximab used in our trial (made with the same antibody plasmid construct but in murine hybridoma cells), the data still suggest that the addition of exogenous IL2 to dinutuximab in this setting may not be necessary. Data from a series of nonrandomized single-arm trials of a murine anti-GD2 antibody, 3F8, suggest that GM-CSF may augment the activity of immunotherapy in children with high-risk neuroblastoma (29). Taken together, our trial results and those of other investigators have prompted COG to eliminate IL2 from dinutuximab-based post-consolidation therapy, but retain GM-CSF as a way to augment the activity of the GD2-directed antibody.

Although large increases in anti-GD2 antibody–based dosing can slightly augment the response to in vivo ADCC in preclinical mouse models, clinical studies have not consistently shown a dose effect in anti-GD2 antibody trials (20). In our trial, neither peak nor trough dinutuximab levels correlated with progression or toxicity in this cohort. Trough dinutuximab levels remained detectable (∼100-fold the concentration needed to induce ADCC in vitro) nearly 2.5 weeks after the last dinutuximab infusion in the prior cycle (Table 2). It is possible that the variations in dinutuximab levels between patients may not be large enough to detectably influence outcome. Alternatively, greater plasma dinutuximab levels may be associated with improved outcome in other settings. For example, we have recently shown that in patients with relapsed/refractory neuroblastoma receiving dinutuximab in combination with irinotecan/temozolomide, those with complete or partial responses had significantly higher trough dinutuximab levels than nonresponders (21).

In this study, neither trough nor peak concentrations of dinutuximab correlated with the incidence of antibody-related toxicities. The etiology of dinutuximab-related toxicity is likely multifactorial. While pain is related to complement activation upon binding of dinutuximab to GD2 on nerve fibers (30), interpatient variability in the level of GD2 expression on nerve fibers, differences in downstream pain signaling pathways and variation in levels of complement components may also affect the magnitude of pain that is experienced. In addition, some of the immunotherapy-related toxicities are attributable to the effects of IL2 and GM-CSF. Polymorphisms in the genes encoding receptors for these cytokines or components of their downstream signaling pathways may influence the frequency and severity of the toxicities experienced by different patients. Taken together, these factors may impact toxicity to a greater degree than does the level of dinutuximab, resulting in the absence of a detectable association between dinutuximab level and regimen-related toxicity. Despite the known toxicities of dinutuximab, long-term follow-up analyses indicate that all toxicities appear to resolve with time (30, 31).

Patients treated with murine mAbs often generate neutralizing human anti-mouse antibodies (HAMA) against the therapeutic antibody. Chimeric or humanized mAbs have been used to decrease the likelihood of developing a neutralizing antibody and mitigate the effects of antidrug antibodies by minimizing the number of xenogeneic antigenic epitopes recognizable on the mAb. The incidence of detectible HACA response in this study was relatively low, which may be due to the chimeric nature of dinutuximab. It may also reflect the effects of immunosuppressive therapy as part of induction and consolidation therapy received prior to dinutuximab administration. The rarity of HACA detected here is in contrast to the 33% rate of HACA development in adult patients with melanoma following treatment with dinutuximab without prior chemotherapy exposure (12), and the 19% HACA rate in patients with relapsed neuroblastoma receiving a genetically similar anti-GD2 antibody (dinutuximab beta) that was produced in CHO cells (32). While the rate of HACA was low in our study, it was associated with diminished dinutuximab plasma levels, suggesting that the HACA response can neutralize dinutuximab in vivo. In analyses of patients with neuroblastoma receiving the murine 3F8 mAb, a substantial fraction (75%) developed a HAMA response (33). Interestingly, even though strong HAMA responses lead to discontinuation of murine anti-GD2 therapy, patients that develop a HAMA response have better survival outcomes than those who do not. This may reflect induction of an anti-idiotype network response with resultant generation of endogenous “antibody-3,” which is cross-reactively able to recognize GD2 (33). In this study, we saw no difference in outcome based on HACA status; however, the number of HACA-positive patients may be too small (Supplementary Fig. S1) to detect a correlation. Separate analyses suggest the HACA response may be blunted when anti-GD2 is given concurrently with chemotherapy (21, 22).

Polymorphisms of Fc receptors can influence antibody-binding affinity and ADCC mediated by NK cells and granulocytes, which can in turn impact therapeutic efficacy of antibody therapy (34). In patients with high-risk neuroblastoma treated with the murine 3F8, those with homozygous high-affinity FCGR2A genotypes had significantly longer PFS than those with the homozygous low-affinity genotypes, but no such association was identified with respect to FCGR3A genotype (35). A more recent trial demonstrated that patients with high-risk neuroblastoma harboring a high-affinity FCGR2A + FCGR3A genotype “profile” had a better EFS compared with patients with low FCGR2A + FCGR3A genotype profile when treated with dinutuximab beta (18). However, we did not find any relationship between Fc receptor genotypes and outcome in this study. This is consistent with previous data showing that Fc receptor genotype had little consistent association with patient outcome even when the same antibody was used in different studies (34).

Genotype analyses from this trial did not show an association of inherited Killer-cell Immunoglobulin-like Receptor (KIR) genes and their KIR ligands with outcome among patients that received immunotherapy. However, these association analyses did suggest that this immunotherapy regimen may provide greater benefit for individuals with a certain combination of KIR/KIR ligands than those without the potentially “favorable” genotype (36); these KIR and their ligands are known to influence NK function and ADCC. Work is ongoing to validate whether the association of KIR/KIR-ligand genotype and benefit from this immunotherapy regimen may extend to a separate nonrandomized cohort of patients with neuroblastoma treated with this same dinutuximab-based regimen on the same protocol, following cessation of randomization in 2009. Separately, analyses of additional factors/biomarkers (including evaluation of the tumor microenvironment, and of gene expression) that impact these immunotherapeutic processes are also in progress.

This long-term follow-up study demonstrates that immunotherapy with dinutuximab in combination with cytokines is associated with an improvement in survival in children with high-risk neuroblastoma. However, the magnitude of the survival benefit for patients randomized to immunotherapy versus isotretinoin has decreased over time due to late relapses. Further correlative biological studies may help to identify subsets of patients most likely to benefit from treatment with immunotherapy. Newer approaches to increase the antitumor efficacy and decrease the toxicity of anti-GD2–based immunotherapy have been identified with clinical trials underway.

A.L. Yu reports grants from NCI, St. Baldrick's Foundation, Alex's Lemonade Stand Foundation, Padres Foundation, and Bridgeman Memorial Fund and personal fees from Eusa Pharma during the conduct of the study. A.L. Gilman reports grants from NCI during the conduct of the study. A. Naranjo reports grants from NIH/NCI during the conduct of the study and personal fees from Novartis outside the submitted work. M.B. Diccianni reports grants from NIH and ALSF during the conduct of the study. A. Batova reports grants from NIH during the conduct of the study. W.B. London reports grants from NCI/NIH during the conduct of the study and personal fees from Merck and from Jubilant Draximage outside the submitted work. S.L. Cohn reports other from Children's Oncology Group during the conduct of the study and other from United Therapeutics outside the submitted work. No disclosures were reported by the other authors.

The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

A.L. Yu: Conceptualization, resources, data curation, formal analysis, supervision, funding acquisition, validation, investigation, methodology, writing–original draft, project administration, writing–review and editing. A.L. Gilman: Conceptualization, supervision, investigation, writing–original draft, project administration, writing–review and editing. M.F. Ozkaynak: Conceptualization, investigation, project administration, writing–review and editing. A. Naranjo: Resources, data curation, software, formal analysis, supervision, funding acquisition, validation, investigation, methodology, writing–original draft, writing–review and editing. M.B. Diccianni: Data curation, formal analysis, validation, investigation, methodology, writing–original draft, writing–review and editing. J. Gan: Data curation, formal analysis, investigation, methodology, writing–review and editing. J.A. Hank: Data curation, formal analysis, investigation, methodology, writing–review and editing. A. Batova: Data curation, investigation, methodology, writing–review and editing. W.B. London: Data curation, validation, methodology, writing–original draft, writing–review and editing. S.C. Tenney: Data curation, formal analysis, writing–review and editing. M. Smith: Resources, supervision, project administration, writing–review and editing. B.L. Shulkin: Investigation, writing–review and editing. M. Parisi: Investigation, writing–review and editing. K.K. Matthay: Conceptualization, supervision, investigation, writing–review and editing. S.L. Cohn: Conceptualization, supervision, investigation, project administration, writing–review and editing. J.M. Maris: Supervision, investigation, writing–review and editing. R. Bagatell: Data curation, supervision, investigation, writing–original draft, writing–review and editing. J.R. Park: Data curation, supervision, investigation, writing–original draft, writing–review and editing. P.M. Sondel: Conceptualization, resources, data curation, supervision, funding acquisition, investigation, methodology, writing–original draft, project administration, writing–review and editing.

The authors thank Ralph Reisfeld and Stephen Gillies for their pioneering work in the development of ch14.18, facilitating its translation to clinical testing and regulatory approval as dinutuximab. We thank Biopharmaceutical Development Program of the NCI for producing ch14.18 for this clinical trial and Bayer for providing the GM-CSF. Thanks also to the COG operations office, COG treatment sites involved in this trial, and patients and families that participated in this trial. This work was supported by NIH/NCI grants U10CA180899 (NCTN Statistics and Data Center), U10CA98543 (COG Chair's Grant), U10CA180886 (NCTN Operations Center Grant) and the St. Baldrick's Foundation, Alex's Lemonade Stand Foundation, Padres Foundation, the Lou Bridgeman Memorial Fund (to A.L. Yu and M.B. Diccianni), grants R35 CA197078 and support from the Midwest Athletes against Childhood Cancer to the University of Wisconsin (to P.M. Sondel), NCI R35 CA220500 (to J.M. Maris).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.