Response to immunocytokine (IC) therapy is dependent on natural killer cells in murine neuroblastoma (NBL) models. Furthermore, killer immunoglobulin-like receptor (KIR)/KIR-ligand mismatch is associated with improved outcome to autologous stem cell transplant for NBL. Additionally, clinical antitumor response to monoclonal antibodies has been associated with specific polymorphic-FcγR alleles. Relapsed/refractory NBL patients received the hu14.18-IL2 IC (humanized anti-GD2 monoclonal antibody linked to human IL2) in a Children's Oncology Group phase II trial. In this report, these patients were genotyped for KIR, HLA, and FcR alleles to determine whether KIR receptor–ligand mismatch or specific FcγR alleles were associated with antitumor response. DNA samples were available for 38 of 39 patients enrolled: 24 were found to have autologous KIR/KIR-ligand mismatch; 14 were matched. Of the 24 mismatched patients, 7 experienced either complete response or improvement of their disease after IC therapy. There was no response or comparable improvement of disease in patients who were matched. Thus KIR/KIR-ligand mismatch was associated with response/improvement to IC (P = 0.03). There was a trend toward patients with the FcγR2A 131-H/H genotype showing a higher response rate than other FcγR2A genotypes (P = 0.06). These analyses indicate that response or improvement of relapsed/refractory NBL patients after IC treatment is associated with autologous KIR/KIR-ligand mismatch, consistent with a role for natural killer cells in this clinical response. Cancer Res; 70(23); 9554–61. ©2010 AACR.

The therapeutic role of natural killer (NK) cells in allogeneic hematopoietic stem cell transplant (HSCT) has been well described. Ruggeri et al. (1) first reported on the phenomenon of killer immunoglobulin-like receptor (KIR)/KIR-ligand incompatibility and response to HLA-haploidentical HSCT, primarily in adult patients with acute myeloid leukemia (AML). According to this analysis, a difference in HLA between the donor and recipient such that the donor KIR receptors lack their cognate ligand in the recipient results in improved leukemia control. Leung et al. defined the principle of missing KIR ligand (designated here as KIR/KIR-ligand mismatch), in which the HSCT recipient lacks 1 or more HLA class-I ligands for the HSCT donor's inhibitory KIRs (2). The KIR/KIR-ligand mismatch principle posits that a difference in HLA between the donor and recipient is not necessary for the benefit of KIR-HLA mismatching. They found that response of pediatric patients with AML and acute lymphoid leukemia (ALL) to haploidentical HSCT could be predicted by the presence of this KIR/KIR-ligand mismatch. An analysis of results of HLA-identical T cell–depleted sibling HSCT also revealed a benefit of KIR/KIR-ligand mismatch (3). KIR/KIR-ligand mismatch also extends to the autologous transplant setting. Because the genes encoding for KIR and HLA class I KIR ligands are inherited independently, it is possible for an individual to be KIR/KIR-ligand mismatched with himself. This scenario has been implicated as a favorable prognostic factor in pediatric solid tumor patients following autologous HSCT (ASCT) (4, 5). To date, there have been no reports relating KIR/KIR-ligand mismatch and response of cancer patients to immunotherapy or other treatments outside of the HSCT or allogeneic NK cell infusion settings (6).

Several groups have investigated the role of fragment c gamma receptor (FcγR) polymorphisms in the response of cancer patients to rituximab, the chimeric anti-CD20 immunoglobulin G1 (IgG1) monoclonal antibody (mAb). Some have found an association between FcγR genotypes for higher-affinity FcγR alleles (FcγR2A-131 H/H and FcγR3A-158 V/V genotypes) and response to rituximab-based therapy (7–9). Association between FcγR genotype and response, for both FcγR2A (found largely on neutrophils and monocytes/macrophages) and FcγR3A (found predominantly on NK cells) genotypes, has now been observed in cancer patients receiving other mAbs (i.e., cetuximab and trastuzumab) as well (10, 11). Cheung et al. (12) reported a correlation between FcγR2A polymorphism and response of neuroblastoma (NBL) patients to the anti-GD2 murine IgG3 mAb, 3F8, plus granulocyte macrophage colony-stimulating factor (GM-CSF). In that study, progression-free survival was associated with the FcγR2A 131-R/R alleles with the highest affinity for the 3F8 murine mAb.

GD2 is a disialoganglioside that is expressed on human melanoma and NBL cells. Our recent Children's Oncology Group (COG) phase III study has shown a significant improvement in overall survival and event-free survival for children receiving an immunotherapy regimen of a chimeric anti-GD2 mAb (ch14.18-IL2) in combination with interleukin 2 (IL2) and GM-CSF (13). The hu14.18-IL2 immunocytokine (IC) is a fusion protein linking a molecule of IL2 to the carboxyl terminus of each of the IgG1 heavy chains of the humanized anti-GD2 mAb hu14.18. Tumor response to this IC in mice is superior to the antitumor effects of comparable amounts of anti-GD2 mAb infused together with comparable amounts of IL2 (14). Antitumor effects in mice are primarily dependent on NK cells in a murine model of NBL (15), with better responses seen in the face of less-established disease (16). Furthermore, augmented major histocompatibility complex (MHC) class I expression on murine NBL was associated with escape from IC-mediated immunotherapy, implying a role for MHC-induced inhibition of NK cell function (17). Our recent phase II COG trial has demonstrated antitumor activity for hu14.18-IL2 in children with relapsed or refractory NBL (18).

In this study we evaluated the role of genotypes for KIR, KIR ligands (HLA class I), and FcγR polymorphisms, in the response of relapsed/refractory NBL patients to hu14.18-IL2 from our phase II trial (18).

Patients

Patients included in this analysis were eligible for and enrolled on a COG phase II trial of hu14.18-IL2 (ANBL0322); the clinical findings from that study have been reported separately (18). In brief, children (ages 1–21 years) with refractory or recurrent NBL who met all study criteria were enrolled in 1 of 2 strata. Stratum 1 included 15 patients with measurable disease (using standard radiographic criteria). Stratum 2 included 24 patients with disease that was not measurable by standard imaging, but was evaluable by 123I-metaiodobenzylguanidine (MIBG) scan and/or bone marrow histology. DNA samples were available for 38 of the 39 enrolled patients. Informed consent was obtained from Institutional Review Board–approved clinical protocols for all patients and/or their families.

Hu14.18-IL2

The clinical grade hu14.18-IL2 IC (EMD 273063) was generously provided by Merck Serono (Darmstadt, Germany) via Dr. Toby Hecht, and the NCI-Biological Resources Branch (Frederick, MD).

KIR/KIR-ligand genotyping

KIR genotyping was performed on patient DNA samples by PCR sequence-specific primer technique (SSP Unitray assay; Invitrogen Corporation, Carlsbad, CA). KIR-ligand typing was performed at low resolution on the same samples for HLA-B and -C loci by reverse PCR-SSO methodology (LifeMatch assay; Gen-Probe, Inc., Stamford, CT) and for high-resolution HLA-C alleles by direct sequencing (AlleleSEQR assay; Abbott Labs, Des Plaines, IL).

FcγR genotyping

Genotyping of FcgR2A 131-H/R (rs1801274) and FcgR3A 158-F/V (rs396991) polymorphisms were detected using pyrosequencing and TaqMan (19). Forward and reverse primers are given in Table 1. PCR was performed in 25 μL reactions using 2X PCR Master Mix (Promega, Madison, WI) with 5 pmole forward, 0.5 pmole reverse, and 4.5 pmole of universal biotin primer. The biotinylated PCR products were isolated with Streptavidin-Sepharose HP (GE Healthcare, Piscataway, NJ) and Sepharose beads. Allele quantification was performed using a Pyrogold reagent kit (dATP, dCTP, dGTP, dTTP, enzyme, and substrate mixtures) on the PSQ HS96A system (Biotage AB, Uppsala, Sweden). Negative controls were included on each plate. Plates were read on a PSQ HS pyrosequencer using PSQ HS 96A SNP analysis software version 2.1 in AQ mode.

Table 1.

Forward and reverse primer sequences for FcγR2A 131-H/R & FcγR3A 158-V/F polymorphism genotyping

PrimersSequences

 
FcγR2A 131-H/R: Forward TTT TGC TGC TAT GGG CTT TC 
FcγR2A 131-H/R: Reverse /5Biosg/CCA GAA TGG AAA ATC CCA GAA A 
FcγR2A 131-H/R: Sequencing AAG GTG GGA TCC AAA 
FcγR3A 158-V/F: Forward /5Biosg/CAC TCA AAG ACA GCG GCT CCT 
FcγR3A 158-V/F: Reverse ATT CCA GGG TGG CAC ATG TC 
FcγR3A 158-V/F: Sequencing AGT CTC TGA AGA CAC ATT TTT 
PrimersSequences

 
FcγR2A 131-H/R: Forward TTT TGC TGC TAT GGG CTT TC 
FcγR2A 131-H/R: Reverse /5Biosg/CCA GAA TGG AAA ATC CCA GAA A 
FcγR2A 131-H/R: Sequencing AAG GTG GGA TCC AAA 
FcγR3A 158-V/F: Forward /5Biosg/CAC TCA AAG ACA GCG GCT CCT 
FcγR3A 158-V/F: Reverse ATT CCA GGG TGG CAC ATG TC 
FcγR3A 158-V/F: Sequencing AGT CTC TGA AGA CAC ATT TTT 

To account for variation in the accuracy of FcγR3A 158-V/F allele frequency determination, standard curves were generated by using synthesized biotin-labeled DNA oligonucleotides. Two oligonucleotides with either a “T” or “G” in the mutation position (representing F and V alleles, respectively) were combined in ratios (10:0, 9:1, …1:9, 0:10) and underwent pyrosequencing. The expected and observed allele frequencies were plotted. Backwards fitting was used to find the most parsimonious fit.

Genotyping of FcγR3A 158-V/F mutation polymorphisms was confirmed using TaqMan SNP genotyping assay (C_25815666–10, Chr-1, DIS484) in accordance with the manufacturer's instructions on a 7300 Real-Time PCR system (Applied Biosystems, Foster City, CA). This TaqMan Assay uses primers and probes that are predesigned and validated by the company. The probes are conjugated with VIC-MGB or FAM-MGB dyes, 1 to each allele. The amount of volume required for each 25 μL reaction was TaqMan Genotyping PCR Master Mix 12.5 μL and TaqMan SNP Genotyping Assay 1 μL. The reaction conditions were as follows: 95°C for 10 minutes, and 50 cycles of 92°C for 15 seconds and 60°C for 1 minute. Plates were read by the 7300 Real-Time PCR system according to manufacturer protocols.

Statistical methods

Fisher's exact test was used to examine the association between 2 categorical variables (i.e., KIR mismatch versus response/improvement, genotype versus response/improvement, and KIR mismatch versus stratum). Chi-square test of homogeneity was used to determine whether there was a difference in distributions of KIR mismatch/match and FcγR genotypes between our study population and others' (5, 12). For the analysis of the association between genotype and response/improvement, FcγR2A were dichotomized into H/H versus H/R or R/R, and FcγR3A into V/V versus V/F or F/F. Statistical significance was defined as a 2-tailed P < 0.05, and a P value from 0.05 to 0.1 was regarded as marginally statistically significant. Because of the exploratory nature of this study, no multiplicity adjustment was made for significance tests.

KIR/KIR-ligand genotyping

The 4 inhibitory KIR genes (KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1) evaluated in this study and their corresponding KIR-ligands (HLA-C1, HLA-C2, or HLA-Bw4) are listed in Table 2. KIR/KIR-ligand mismatch was defined as absence of 1 or more HLA alleles known to be ligands for the inhibitory KIR genes present, using previously published criteria (2). The specific KIR and KIR-ligand genotypes for all 38 patients (with DNA available) are shown in Table 3. Two patients showed evidence for only 2 of the 3 inhibitory KIR gene specificities evaluated in this study; the other 36 patients had all 3 inhibitory KIR gene specificities present. KIR/KIR-ligand mismatch was observed in 24 of 38 patients whereas in 14 of 38 patients these were matched (Table 4). This ratio of matched to mismatched genotypes, when using this set of KIR genes and KIR ligand genes, is not significantly different (P = 0.93) from that reported for the set of NBL patients undergoing ASCT reported by Venstrom et al. (5).

Table 2.

KIR receptors and their MHC class I ligands that were determined by genotyping

ReceptorLigand

 
KIR2DL1 (CD158a) HLA-C2 (Cw2, Cw4, Cw5, Cw6, Cw15, Cw1602, Cw17, Cw18) 
KIR2DL2/KIR2DL3 (CD158b) HLA-C1 (Cw1, Cw3, Cw7, Cw8, Cw12, Cw13, Cw14, Cw1601) 
KIR3DL1 (CD158e) HLA-Bw4 
ReceptorLigand

 
KIR2DL1 (CD158a) HLA-C2 (Cw2, Cw4, Cw5, Cw6, Cw15, Cw1602, Cw17, Cw18) 
KIR2DL2/KIR2DL3 (CD158b) HLA-C1 (Cw1, Cw3, Cw7, Cw8, Cw12, Cw13, Cw14, Cw1601) 
KIR3DL1 (CD158e) HLA-Bw4 
Table 3.

KIR, KIR ligand, FcγR, and response data for each patient

Patient no.StratumKIR genotypeKIR ligandMatch vs mismatchFcγR2A-131FcγR3A -158Response

 
  1 2DL1, 2DL2/2DL3, 3DL1 Cw1, Cw5, Bw4 R/R F/V PD 
  2 2DL1, 2DL2/2DL3, 3DL1 Cw1, Cw3, Bw4 MM R/R F/V CR 
  3 2DL1, 2DL3, 3DL1 Cw4, Cw15 MM H/H F/V 
  4 2DL1, 2DL2/2DL3, 3DL1 Cw2, Cw8, Bw4 R/R F/V SD 
  5 2DL1, 2DL2/2DL3, 3DL1 Cw7, Cw16, Bw4 MM R/R F/V PD 
  6 2DL1, 2DL3, 3DL1 Cw6, Cw7 MM H/H F/V PD 
  7 2DL1, 2DL2/2DL3, 3DL1 Cw7 MM H/H F/V PD 
  8 2DL1, 2DL3, 3DL1 Cw12, Bw4 MM R/R b PD 
  9 2DL1, 2DL2/2DL3, 3DL1 Cw6, Cw7, Bw4 R/R F/V PD 
10 2DL1, 2DL3, 3DL1 Cw4, Cw7 MM H/H F/F CR 
11 2DL1, 2DL3, 3DL1 Cw5, Bw4 MM R/R F/F PD 
12a        
13 2DL1, 2DL3, 3DL1 Cw2, Cw7, Bw4 H/R F/V NE 
14 2DL1, 2DL2/2DL3, 3DL1 Cw3, Cw6, Bw4 H/R F/F PD 
15 2DL1, 2DL3, 3DL1 Cw7 MM H/H F/F PD 
16 2DL1, 2DL3, 3DL1 Cw12, Cw16, Bw4 MM R/R F/V PD 
17 2DL1, 2DL2/2DL3, 3DL1 Cw2, Cw16 MM H/R V/V SD 
18 2DL1, 2DL3, Cw4, Bw4 MM H/R F/F PD 
19 2DL1, 2DL2/2DL3, 3DL1 Cw2, Cw7, Bw4 H/H F/V SD 
20 2DL1, 2DL2/2DL3, 3DL1 Cw7, Bw4 MM R/R V/V PD 
21 2DL1, 2DL2/2DL3, 3DL1 Cw4, Cw6, Bw4 MM H/R F/V 
22 2DL1, 2DL2/2DL3, 3DL1 Cw5, Bw4 MM H/H F/F CR 
23 2DL1, 2DL3, 3DL1 Cw8, Cw15 MM H/R F/F PD 
24 2DL1, 2DL2/2DL3, 3DL1 Cw2, Cw3, Bw4 H/R F/F NR 
25 2DL1, 2DL3, 3DL1 Cw5, Bw4 MM R/R F/F SD 
26 2DL1, 2DL2/2DL3, 3DL1 Cw2, Cw6, Bw4 MM H/R F/V PD 
27 2DL1, 2DL3, 3DL1 Cw12, Cw16, Bw4 MM H/H F/V CR 
28 2DL1, 2DL2/2DL3, 3DL1 Cw6, Cw8, Bw4 H/R F/V PD 
29 2DL1, 2DL3, 3DL1 Cw5, Cw7 MM R/R F/V CR 
30 2DL1, 2DL2/2DL3, 3DL1 Cw12, Cw15, Bw4 H/H F/F PD 
31 2DL1, 2DL3, 3DL1 Cw4, Cw7 MM H/R F/F PD 
32 2DL1, 2DL3, 3DL1 Cw6, Cw7, Bw4 H/R F/V PD 
33 2DL1, 2DL3, 3DL1 Cw6, Cw7, Bw4 R/R F/F PD 
34 2DL2, 3DL1 Cw12, Cw17 MM H/R F/F PD 
35 2DL1, 2DL2, 3DL1 Cw6, Cw7 MM H/R b PD 
36 2DL1, 2DL2/2DL3, 3DL1 Cw3, Cw7, Bw4 H/H F/F PD 
37 2DL1, 2DL3, 3DL1 Cw3, Cw14, Bw4 MM H/R F/F PD 
38 2DL1, 2DL3, 3DL1 Cw4, Cw7, Bw4 H/R F/F PD 
39 2DL1, 2DL2/2DL3, 3DL1 Cw6, Cw7, Bw4 H/R F/F PD 

 
Patient no.StratumKIR genotypeKIR ligandMatch vs mismatchFcγR2A-131FcγR3A -158Response

 
  1 2DL1, 2DL2/2DL3, 3DL1 Cw1, Cw5, Bw4 R/R F/V PD 
  2 2DL1, 2DL2/2DL3, 3DL1 Cw1, Cw3, Bw4 MM R/R F/V CR 
  3 2DL1, 2DL3, 3DL1 Cw4, Cw15 MM H/H F/V 
  4 2DL1, 2DL2/2DL3, 3DL1 Cw2, Cw8, Bw4 R/R F/V SD 
  5 2DL1, 2DL2/2DL3, 3DL1 Cw7, Cw16, Bw4 MM R/R F/V PD 
  6 2DL1, 2DL3, 3DL1 Cw6, Cw7 MM H/H F/V PD 
  7 2DL1, 2DL2/2DL3, 3DL1 Cw7 MM H/H F/V PD 
  8 2DL1, 2DL3, 3DL1 Cw12, Bw4 MM R/R b PD 
  9 2DL1, 2DL2/2DL3, 3DL1 Cw6, Cw7, Bw4 R/R F/V PD 
10 2DL1, 2DL3, 3DL1 Cw4, Cw7 MM H/H F/F CR 
11 2DL1, 2DL3, 3DL1 Cw5, Bw4 MM R/R F/F PD 
12a        
13 2DL1, 2DL3, 3DL1 Cw2, Cw7, Bw4 H/R F/V NE 
14 2DL1, 2DL2/2DL3, 3DL1 Cw3, Cw6, Bw4 H/R F/F PD 
15 2DL1, 2DL3, 3DL1 Cw7 MM H/H F/F PD 
16 2DL1, 2DL3, 3DL1 Cw12, Cw16, Bw4 MM R/R F/V PD 
17 2DL1, 2DL2/2DL3, 3DL1 Cw2, Cw16 MM H/R V/V SD 
18 2DL1, 2DL3, Cw4, Bw4 MM H/R F/F PD 
19 2DL1, 2DL2/2DL3, 3DL1 Cw2, Cw7, Bw4 H/H F/V SD 
20 2DL1, 2DL2/2DL3, 3DL1 Cw7, Bw4 MM R/R V/V PD 
21 2DL1, 2DL2/2DL3, 3DL1 Cw4, Cw6, Bw4 MM H/R F/V 
22 2DL1, 2DL2/2DL3, 3DL1 Cw5, Bw4 MM H/H F/F CR 
23 2DL1, 2DL3, 3DL1 Cw8, Cw15 MM H/R F/F PD 
24 2DL1, 2DL2/2DL3, 3DL1 Cw2, Cw3, Bw4 H/R F/F NR 
25 2DL1, 2DL3, 3DL1 Cw5, Bw4 MM R/R F/F SD 
26 2DL1, 2DL2/2DL3, 3DL1 Cw2, Cw6, Bw4 MM H/R F/V PD 
27 2DL1, 2DL3, 3DL1 Cw12, Cw16, Bw4 MM H/H F/V CR 
28 2DL1, 2DL2/2DL3, 3DL1 Cw6, Cw8, Bw4 H/R F/V PD 
29 2DL1, 2DL3, 3DL1 Cw5, Cw7 MM R/R F/V CR 
30 2DL1, 2DL2/2DL3, 3DL1 Cw12, Cw15, Bw4 H/H F/F PD 
31 2DL1, 2DL3, 3DL1 Cw4, Cw7 MM H/R F/F PD 
32 2DL1, 2DL3, 3DL1 Cw6, Cw7, Bw4 H/R F/V PD 
33 2DL1, 2DL3, 3DL1 Cw6, Cw7, Bw4 R/R F/F PD 
34 2DL2, 3DL1 Cw12, Cw17 MM H/R F/F PD 
35 2DL1, 2DL2, 3DL1 Cw6, Cw7 MM H/R b PD 
36 2DL1, 2DL2/2DL3, 3DL1 Cw3, Cw7, Bw4 H/H F/F PD 
37 2DL1, 2DL3, 3DL1 Cw3, Cw14, Bw4 MM H/R F/F PD 
38 2DL1, 2DL3, 3DL1 Cw4, Cw7, Bw4 H/R F/F PD 
39 2DL1, 2DL2/2DL3, 3DL1 Cw6, Cw7, Bw4 H/R F/F PD 

 

aPatient 12 not included, DNA was not available.

bFcγR3A genotyping results from patients 8 and 35 excluded from analysis due to discrepancy in results of pyrosequencing and TaqMan.

Abbreviations: CR, complete response; I, (clinically) improved; M, match; MM, mismatch; PD, progressive disease; SD, stable disease.

Table 4.

Distribution of genotypes for KIR mismatch/match and FcγR genotypes among the 38 patients evaluated


 
KIR Mismatch: 24/38 (63%) KIR Match: 14/38 (37%)  P = 0.93a
FcγR2A 131-H/H: 10/38 (26%) FcγR2A 131-H/R: 16/38 (42%) FcγR2A 131-R/R: 12/38 (32%) P = 0.5b 
FcγR3A 158-F/F: 17/36 (47%) FcγR3A 158-F/V: 17/36 (47%) FcγR3A 158-V/V: 2/36 (6%) Pc = 0.92b 

 

 
KIR Mismatch: 24/38 (63%) KIR Match: 14/38 (37%)  P = 0.93a
FcγR2A 131-H/H: 10/38 (26%) FcγR2A 131-H/R: 16/38 (42%) FcγR2A 131-R/R: 12/38 (32%) P = 0.5b 
FcγR3A 158-F/F: 17/36 (47%) FcγR3A 158-F/V: 17/36 (47%) FcγR3A 158-V/V: 2/36 (6%) Pc = 0.92b 

 

aP value for the difference in distribution of KIR match/mismatch for this population versus comparable population of NBL patients evaluated for KIR match/mismatch by Venstrom et al. (5).

bP value for the difference in distribution of FcγR genotypes for this population versus that of comparable NBL patients described by Cheung et al. (12).

cP value remains 0.79 to 1.0 regardless of whether patients 8 and 35 are FF or FV and included.

FcγR genotyping

FcγR genotyping data for all patients tested are also included in Table 3, and is summarized in Table 4. The genotype distributions for both FcγR2A and FcγR3A were in Hardy–Weinberg Equilibrium (HWE). The FcγR2A 131-H/R genotype was more prevalent (16 of 38 patients) than the H/H genotype (10 of 38) and R/R genotype (12 of 38) (P value of HWE is 0.34). Two of 38 patients (patients 8 and 35) were excluded from the analysis of FcγR3A genotyping (and relevant analyses of associations) due to discrepant results of pyrosequencing and TaqMan; both patients were F/V by pyrosequencing and F/F by TaqMan. If patients 8 and 35 are included in the analyses as either F/F or F/V the relevant statistical analyses, as do their conclusions, remain virtually unchanged. The FcγR3A 158-F/F and F/V genotypes were equally prevalent (both present in 17 of 36 patients) in this patient population, whereas the V/V genotype was present in only 2 patients (P of HWE = 0.39). These distributions of the FcγR2A (P = 0.5) and FcγR3A (P = 0.92) genotypes were not significantly different from those reported for the population of NBL patients reported by Cheung et al. (12).

Clinical response to hu14.18-IL2

The clinical details for this phase II trial have been reported separately (18). Individual response and stratum data are included in Table 3. For the purposes of the associations examined in this report, 5 patients (2, 10, 22, 27, and 29) showed a complete response (CR). These CRs lasted 13, 9, 20, 30, and >35 months, respectively. Two additional patients (3 and 22) were scored as showing stable disease, but showed clear clinical signs of improvement (clearing of all marrow disease and MIBG improvement not quite meeting partial response criteria for patient 3, and resolution of all MIBG-detectable disease with near clearing of marrow disease for patient 22). All 5 patients with CR and the 2 with “improved” status were in stratum 2.

Associations between clinical response and genotype data

KIR. The analyses of KIR/KIR-ligand mismatch and FcγR genotyping data for clinical associations are summarized in Tables 57. All 7 patients who responded (5 of 38) or showed improvement (2 of 38) following IC therapy were mismatched for their KIR/KIR-ligand genotypes (i.e., lacked HLA ligand for at least 1 of their KIR genes; Table 5, Mismatch versus response/improvement of all patients). In contrast, none of the 14 KIR/KIR-ligand matched patients demonstrated any response or improvement. Thus, KIR/KIR-ligand mismatch was associated with clinical response/improvement following IC treatment (P = 0.03). When this same analysis was done for the 24 stratum 2 patients, there was a trend (that did not meet criteria for statistical significance) toward a similar association of mismatch and response/improvement (7 of 7) versus nonimprovement (11 of 17) (P = 0.13, Table 5, Mismatch versus response/improvement for patients in stratum 2 only). In addition, there appeared to be a greater fraction of KIR/KIR-ligand mismatched patients enrolled in stratum 2 (18 of 24, 75%) than in stratum 1 (6 of 14, 43%) with marginal statistical significance (P = 0.08, Table 5, Mismatch versus both strata 1 and 2).

Table 5.

Tests of association of mismatch versus response/improvement of all patients, response/improvement for patients in stratum 2 only, and both stratum 1 and stratum 2

KIR-mismatchKIR-matchTotal

 
Mismatch vs response/improvement (strata 1 and 2)a    
 Response/improvement   7 (29%)   0 (0%)   7 
 No response/no improvement 17 (71%) 14 (100%) 31 
 Total 24 14 38 
Mismatch vs response (stratum 2 only)b    
 Response/improvement   7 (39%) 0 (0%)   7 
 No response/no improvement 11 (61%) 6 (100%) 17 
 Total 18 24 
 Stratum 1 Stratum 2 Total 

 
Mismatch vs stratum (strata 1 and 2)c    
 KIR-mismatch   6 (43%) 18 (75%) 24 
 KIR-match   8 (57%)   6 (25%) 14 
 Total 14 24 38 

 
KIR-mismatchKIR-matchTotal

 
Mismatch vs response/improvement (strata 1 and 2)a    
 Response/improvement   7 (29%)   0 (0%)   7 
 No response/no improvement 17 (71%) 14 (100%) 31 
 Total 24 14 38 
Mismatch vs response (stratum 2 only)b    
 Response/improvement   7 (39%) 0 (0%)   7 
 No response/no improvement 11 (61%) 6 (100%) 17 
 Total 18 24 
 Stratum 1 Stratum 2 Total 

 
Mismatch vs stratum (strata 1 and 2)c    
 KIR-mismatch   6 (43%) 18 (75%) 24 
 KIR-match   8 (57%)   6 (25%) 14 
 Total 14 24 38 

 

aP = 0.03.

bP = 0.13.

cP = 0.08.

FcγR. The FcγR2A genotype data analysis for association with response suggests, with marginal statistical significance, that patients with the H/H genotype showed a higher likelihood of response/improvement (Table 6, P = 0.06). In contrast, there was no suggestion of association between FcγR3A genotype and response/improvement to IC (P = 1.0, Table 7).

Table 6.

Response/Improvement for patients with higher-affinity receptor genotype (H/H) for FcγR2A-131 versus other 2 genotypes (H/R and R/R)

FcγR2AHHHR + RRTotal

 
Response/improvement   4 (40%) 3 (11%)   7 
Nonresponse/nonimprovement   6 (60%) 25 (89%) 31 
Total 10 28 38 

 
FcγR2AHHHR + RRTotal

 
Response/improvement   4 (40%) 3 (11%)   7 
Nonresponse/nonimprovement   6 (60%) 25 (89%) 31 
Total 10 28 38 

 

P = 0.06.

Table 7.

Response/Improvement for patients with higher-affinity receptor genotype (V/V) for FcγR3A-158 veersus other 2 genotypes (V/F and F/F)

FcγR3AVVVF + FFTotal

 
Response/improvement 0 (0%)   7 (21%)   7 
Non-Response/Non-improvement 2 (100%) 27 (79%) 29 
Total 34a 36 

 
FcγR3AVVVF + FFTotal

 
Response/improvement 0 (0%)   7 (21%)   7 
Non-Response/Non-improvement 2 (100%) 27 (79%) 29 
Total 34a 36 

 

P = 1.0.

aP value remains 1.0 regardless of whether patients 8 and 35 (both FF or FV) are included.

NK-mediated lysis of NBL cell lines in vitro is augmented by the addition of IL2 (20), especially when using NK cells from patients receiving IL2 (21). Furthermore, NK-mediated antibody dependent cell-mediated cytotoxicity (ADCC) is augmented when the NK cells are obtained following in vivo administration of IL2 (22). Initial studies with chimeric anti-GD2 antibody, ch14.18, fused with human IL-2 (ch14.18-IL2), demonstrated NK-mediated regression of local and disseminated murine NBL (15). Similar results were later seen in murine NBL models with administration of hu14.18-IL2 (16). As escape from this NK-mediated response to hu14.18-IL2 was associated with upregulation of MHC class I on NBL cells (known to induce inhibitory responses via Ly-49 receptors on murine NK cells) (17), we hypothesized that similar relationships may influence the clinical response to hu14.18-IL2. To test this hypothesis, it was first necessary to identify a population that shows some clinical response to hu14.18-IL2.

Our recently reported phase II study of hu14.18-IL2 in patients with relapsed or refractory NBL demonstrated CR or improved disease in 7 of 38 treated patients (18). All 7 of these responding/improved patients were in stratum 2 (evaluable but not radiographically measurable disease), consistent with our preclinical data showing greater detection of antitumor activity in animals with less tumor burden (16). Although the role of KIRs has been evaluated in the setting of autologous and allogeneic stem cell transplantation and infusions of allogeneic NK cells following lymphodepletive chemotherapy (1–6), it has not been studied for association with antitumor response in patients receiving only cytokines or mAbs for immunotherapy. We hypothesized that children with recurrent/refractory NBL, who received the hu14.18-IL2 in our phase II COG study, would demonstrate greater response to IC in the presence of KIR/KIR-ligand mismatch. When the data were analyzed for all 38 patients who had provided DNA samples, 24 of them were found to be KIR/KIR-ligand mismatched, and all 7 of the responding or improved patients were found in this group (P = 0.03, Table 5, Mismatch versus response/improvement of all patients). Because the KIR/KIR-ligand interaction is primarily a mechanism controlling NK cell activity, this result is consistent with the murine data showing that the anti-NBL effect of ch14.18-IL2 and hu14.18-IL2 is primarily mediated by NK cells (15–17). Even prior to the administration of hu14.18-IL2, there is a trend toward greater KIR/KIR-ligand mismatch in the patients who enter stratum 2 than in stratum 1 (P = 0.08, Table 5, Mismatch versus both strata 1 and 2). If additional data validate this trend, it would suggest that a child's endogenous KIR/KIR-ligand status might play a role in the clinical pattern of relapse; namely, the patients who are KIR-mismatched may be less likely to relapse with “bulky” (measurable/stratum 1) disease. Similarly, if additional data validate the trend (P = 0.13) that KIR/KIR-ligand mismatch is associated with response within Stratum 2 patients (Table 5, Mismatch versus response/improvement for patients in stratum 2 only); the presence of Stratum 2 status and KIR/KIR-ligand mismatch might be considered as eligibility criteria for future treatment with hu14.18-IL2 for children with relapsed or refractory NBL.

The roles of the activating Fc receptors involved in ADCC, FcγR2A ,and FcγR3A, have been demonstrated in response to rituximab, cetuximab, and trastuzumab (7–11). Cheung et al. (12) have found an association between FcγR2A polymorphism and outcome of NBL patients receiving the murine anti-GD2 IgG3 antibody, 3F8, but only when given in combination with GM-CSF. This result suggests that when neutrophils or monocytes/macrophages are activated with GM-CSF, they facilitate clinically meaningful ADCC via the high-affinity alleles of FcγR2A for 3F8 (12). This response was unlikely to be NK-mediated, as FcγR2A is not present on NK cells. As preclinical data and our current results (Table 5, Mismatch versus response/improvement of all patients) suggest that NK cells are playing a role in the clinical response to hu14.18-IL2 in NBL patients, we initially hypothesized that the response/improvement of NBL patients to the IgG1-containing IC might be associated with the presence of the high-affinity FcγR3A 158-V/V genotype (influencing NK function), and not the high-affinity FcγR2A 131-H/H genotype reflecting neutrophil and macrophage ADCC. However, we found that patients with the H/H genotype for FcγR2A did show a trend toward higher response rate, which was of marginal statistical significance (P = 0.06). Clearly these comparisons are underpowered. Even so, the near significant association of improvement with the FcγR2A 131-H/H genotype would be consistent with the activation of some neutrophil or macrophage-mediated ADCC. IL2 treatment is known to induce release of GM-CSF by IL2 responsive cells (23), and it is possible that activation of ADCC by these FcγR2A-bearing cells is resulting from GM-CSF production stimulated by the IL2 component of the IC.

Given the infrequency of the FcγR3A 158-V/V genotype in the general population, with only 2 of our 36 genotyped patients identified with this genotype, it is no surprise that a statistically significant association was not seen between this genotype and response. However, this lack of statistical significance cannot be equated with the lack of proof in this study due to inadequate sample size with a highly unbalanced design. Even so, it may be surprising that none of the 7 patients who showed response or improvement had this V/V genotype. This very preliminary result would suggest that there may not be an association between the V/V genotype and improvement in this population when treated with hu14.18-IL2. The lack of an association with FcγR3A alleles and response (even if confirmed in a larger sample) does not necessarily indicate that NK cells are uninvolved in mediating ADCC. Rather, it could suggest that there is no advantage for high versus lower affinity FcγR3A alleles in this setting of IC-mediated ADCC. We have recently shown that some NK cells can use their IL2 receptors to recognize the membrane-bound IL2 on tumor cells coated with tumor-reactive IC (Gubbels et al., submitted manuscript). This results in NK adhesion to the IC-coated tumor cells, activation of an immune synapse, and subsequent tumor cell destruction, apparently without requiring Fc receptors (Buhtoiarov et al., submitted manuscript). In the face of this IL2 receptor–facilitated IC-mediated killing, high-affinity FcγRs on the NK cells might not be needed. In other words, although high-affinity FcγRs play an important role in the clinical effects of FcR-mediated ADCC using conventional mAbs, they might not be as important for NK mediated antitumor effects of ICs. This might enable NK cells with the F/V or F/F FcγR3A alleles to mediate comparable in vivo destruction to that mediated by cells with V/V alleles. Clearly, these are speculative hypotheses as the results were obtained from a limited number of subjects and must be extended to a larger population to draw firmer conclusions.

The COG plans to perform a follow-up study of hu14.18-IL2 in stratum 2 patients, to further characterize these responses. That follow-up study should provide additional data to better test the associations analyzed in this report. Furthermore, the KIR/KIR-ligand mismatch might also be further tested by genotyping of subjects participating in larger trials of clinically effective mAbs that mediate ADCC, such as ch14.18, rituximab, cetuximab, and trastuzumab (7–11).

In summary, we conclude that response or improvement of relapsed or refractory NBL patients following treatment with hu14.18-IL2 is associated with KIR receptor–ligand mismatch, consistent with a role for NK cells in this clinical response. Although there appears to be no statistical significance of FcγR genotype in response of NBL patients to IC in this COG trial, further clinical and in vitro data are needed to better clarify the potential roles of FcγR2A and FcγR3A in these clinical responses.

No potential conflict of interest was disclosed.

We thank Dr. Toby Hecht and the NCI Biological Resources Branch for their provision, through a MCRADA with Merck Serono KGAa of Darmstadt, Germany, of the clinical grade hu14.18-IL2 used for this clinical trial, Bill Clements, Stuart McMillan, and Drs. Claudia Herrman, Jens Oliver Funk, and Jean Henslee Downey of Merck Serono/EMD Serono for their input and help in this clinical study, the physicians and researchers from the COG institutions that entered patients into this trial, the participating patients and their families, and Dr. Bartosz Grzywacz for helpful discussion and editing.

Grant Support This work was supported by U10CA98543 COG Group Chair Grant, U10 CA98413 COG Statistics and Data Center, R01-CA-32685–25, P30-CA14520, CA87025, CA81403, RR03186, K12 CA087718, and grants from the Midwest Athletes for Childhood Cancer Fund, The Crawdaddy Foundation, The St. Baldrick's Foundation, The Evan Dunbar Foundation, and Abbie's Fund.

1.
Ruggeri
L
,
Capanni
M
,
Casucci
M
,
Volpi
I
,
Tosti
A
,
Perruccio
K
, et al
Role of natural killer cell alloreactivity in HLA-mismatched hematopoietic stem cell transplantation
.
Blood
1999
;
94
:
333
9
.
2.
Leung
W
,
Iyengar
R
,
Turner
V
,
Lang
P
,
Bader
P
,
Conn
P
, et al
Determinants of antileukemia effects of allogeneic NK cells
.
J Immunol
2004
;
172
:
644
50
.
3.
Hsu
KC
,
Keever-Taylor
CA
,
Wilton
A
,
Pinto
C
,
Heller
G
,
Arkun
K
, et al
Improved outcome in HLA-identical sibling hematopoietic stem-cell transplantation for acute myelogenous leukemia predicted by KIR and HLA genotypes
.
Blood
2005
;
105
:
4878
84
.
4.
Leung
W
,
Handgretinger
R
,
Iyengar
R
,
Turner
V
,
Holladay
MS
,
Hale
GA
. 
Inhibitory KIR-HLA receptor-ligand mismatch in autologous haematopoietic stem cell transplantation for solid tumour and lymphoma
.
Br J Cancer
2007
;
97
:
539
42
.
5.
Venstrom
JM
,
Zheng
J
,
Noor
N
,
Danis
KE
,
Yeh
AW
,
Cheung
IY
, et al
KIR and HLA genotypes are associated with disease progression and survival following autologous hematopoietic stem cell transplantation for high-risk neuroblastoma
.
Clin Cancer Res.
2009
;
15
:
7330
4
.
6.
Miller
JS
,
Soignier
Y
,
Panoskaltsis-Mortari
A
,
McNearney
SA
,
Yun
GH
,
Fautsch
SK
, et al
Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer
.
Blood
2005
;
105
:
3051
7
.
7.
Cartron
G
,
Dacheux
L
,
Salles
G
,
Solal-Celigny
P
,
Bardos
P
,
Colombat
P
, et al
Therapeutic activity of humanized anti-CD20 monoclonal antibody and polymorphism in IgG Fc receptor FcgammaRIIIa gene
.
Blood
2002
;
99
:
754
8
.
8.
Weng
WK
,
Levy
R
. 
Two immunoglobulin G fragment C receptor polymorphisms independently predict response to rituximab in patients with follicular lymphoma
.
J Clin Oncol
2003
;
21
:
3940
7
.
9.
Paiva
M
,
Marques
H
,
Martins
A
,
Ferreira
P
,
Catarino
R
,
Medeiros
R
. 
FcgammaRIIa polymorphism and clinical response to rituximab in non-Hodgkin lymphoma patients
.
Cancer Genet Cytogenet
2008
;
183
:
35
40
.
10.
Zhang
W
,
Gordon
M
,
Schultheis
AM
,
Yang
DY
,
Nagashima
F
,
Azuma
M
, et al
FCGR2A and FCGR3A polymorphisms associated with clinical outcome of epidermal growth factor receptor expressing metastatic colorectal cancer patients treated with single-agent cetuximab
.
J Clin Oncol
2007
;
25
:
3712
8
.
11.
Musolino
A
,
Naldi
N
,
Bortesi
B
,
Pezzuolo
D
,
Capelletti
M
,
Missale
G
, et al
Immunoglobulin G fragment C receptor polymorphisms and clinical efficacy of trastuzumab-based therapy in patients with HER-2/neu-positive metastatic breast cancer
.
J Clin Oncol
2008
;
26
:
1789
96
.
12.
Cheung
NK
,
Sowers
R
,
Vickers
AJ
,
Cheung
IY
,
Kushner
BH
,
Gorlick
R
. 
FCGR2A polymorphism is correlated with clinical outcome after immunotherapy of neuroblastoma with anti-GD2 antibody and granulocyte macrophage colony-stimulating factor
.
J Clin Oncol
2006
;
24
:
2885
90
.
13.
Yu
AL
,
Gilman
AL
,
Ozkaynak
MF
,
London
WB
,
Kreissman
S
,
Chen
H
, et al
. 
Chimeric anti-GD2 antibody with GM-CSF, IL2 and 13-cis retinoic acid for high-risk neuroblastoma: a Children's Oncology Group (COG) phase 3 study
.
New Engl J Med
2010
;
363
:
1324
34
.
14.
Lode
HN
,
Xiang
R
,
Varki
NM
,
Dolman
CS
,
Gillies
SD
,
Reisfeld
RA
. 
Targeted interleukin-2 therapy for spontaneous neuroblastoma metastases to bone marrow
.
J Natl Cancer Inst
1997
;
89
:
1586
94
.
15.
Lode
HN
,
Xiang
R
,
Dreier
T
,
Varki
NM
,
Gillies
SD
,
Reisfeld
RA
. 
Natural killer cell-mediated eradication of neuroblastoma metastases to bone marrow by targeted interleukin-2 therapy
.
Blood
1998
;
91
:
1706
15
.
16.
Neal
ZC
,
Yang
JC
,
Rakhmilevich
AL
,
Buhtoiarov
IN
,
Lum
HE
,
Imboden
M
, et al
Enhanced activity of hu14.18-IL2 immunocytokine against murine NXS2 neuroblastoma when combined with interleukin 2 therapy
.
Clin Cancer Res
2004
;
10
:
4839
47
.
17.
Neal
ZC
,
Imboden
M
,
Rakhmilevich
AL
,
Kim
KM
,
Hank
JA
,
Surfus
J
, et al
NXS2 murine neuroblastomas express increased levels of MHC class I antigens upon recurrence following NK-dependent immunotherapy
.
Cancer Immunol Immunother
2004
;
53
:
41
52
.
18.
Shusterman
S
,
London
WB
,
Gillies
SD
,
Hank
JA
,
Voss
S
,
Seeger
RC
, et al
Anti-tumor activity of hu14.18-IL2 in relapsed/refractory neuroblastoma patients: a Children's Oncology Group (COG) phase II study
.
J Clin Oncology
. Epub
2010 Oct 4
.
19.
Ronaghi
M
,
Elahi
E
. 
Discovery of single nucleotide polymorphisms and mutations by pyrosequencing
.
Comp Funct Genomics
2002
;
3
:
51
6
.
20.
Rossi
AR
,
Pericle
F
,
Rashleigh
S
,
Janiec
J
,
Djeu
JY
. 
Lysis of neuroblastoma cell lines by human natural killer cells activated by interleukin-2 and interleukin-12
.
Blood
1994
;
83
:
1323
8
.
21.
Hank
JA
,
Weil-Hillman
G
,
Surfus
JE
,
Sosman
JA
,
Sondel
PM
. 
Addition of interleukin-2 in vitro augments detection of lymphokine-activated killer activity generated in vivo
.
Cancer Immunol Immunother
1990
;
31
:
53
9
.
22.
Hank
JA
,
Robinson
RR
,
Surfus
J
,
Mueller
BM
,
Reisfeld
RA
,
Cheung
NK
, et al
Augmentation of antibody dependent cell mediated cytotoxicity following in vivo therapy with recombinant interleukin 2
.
Cancer Res
1990
;
50
:
5234
9
.
23.
Heslop
HE
,
Duncombe
AS
,
Reittie
JE
,
Bello-Fernandez
C
,
Gottlieb
DJ
,
Prentice
HG
, et al
Interleukin 2 infusion induces haemopoietic growth factors and modifies marrow regeneration after chemotherapy or autologous marrow transplantation
.
Br J Haematol
1991
;
77
:
237
44
.