HLA-DRB1*07:01 is associated with a higher risk of asparaginase allergies

Abstract

Asparaginase is a therapeutic enzyme used to treat leukemia and lymphoma, with immune responses resulting in suboptimal drug exposure and a greater risk of relapse. To elucidate whether there is a genetic component to the mechanism of asparaginase-induced immune responses, we imputed human leukocyte antigen (HLA) alleles in patients of European ancestry enrolled on leukemia trials at St. Jude Children's Research Hospital (n = 541) and the Children's Oncology Group (n = 1329). We identified a higher incidence of hypersensitivity and anti-asparaginase antibodies in patients with HLA-DRB1*07:01 alleles (P = 7.5 × 10(-5), odds ratio [OR] = 1.64; P = 1.4 × 10(-5), OR = 2.92, respectively). Structural analysis revealed that high-risk amino acids were located within the binding pocket of the HLA protein, possibly affecting the interaction between asparaginase epitopes and the HLA-DRB1 protein. Using a sequence-based consensus approach, we predicted the binding affinity of HLA-DRB1 alleles for asparaginase epitopes, and patients whose HLA genetics predicted high-affinity binding had more allergy (P = 3.3 × 10(-4), OR = 1.38). Our results suggest a mechanism of allergy whereby HLA-DRB1 alleles that confer high-affinity binding to asparaginase epitopes lead to a higher frequency of reactions. These trials were registered at www.clinicaltrials.gov as NCT00137111, NCT00549848, NCT00005603, and NCT00075725.

Figure 1

HLA-DRB1 and HLA-DQB1 alleles were imputed for asparaginase hypersensitivity studies. The HLA-DRB1 and HLA-DQB1 alleles of patients enrolled on either SJCRH or COG protocols were imputed to 4-digit resolution using SNP data from the patients and the T1DGC reference panel with both SNP data and 4-digit resolution HLA typing. (A) The discovery cohort consisting of SJCRH patients was used to identify associations between asparaginase hypersensitivity reactions and imputed HLA-DRB1 and/or HLA-DQB1 alleles. The validation cohort consisted of COG patients. (B) Using the imputed HLA-DRB1 alleles, the HLA-DRB1 amino acid sequence was inferred for every patient, and the association between polymorphic amino acids and asparaginase hypersensitivity reactions was determined. (C) Using the IEDB, imputed HLA alleles were scored for their binding affinity to E coli asparaginase peptide fragments. The imputed HLA alleles of patients were categorized into “high” or “low” binding categories for asparaginase, and the categories were tested for association with asparaginase hypersensitivity.

Figure 2

HLA class II allele imputations resulted in 54 unique class II HLA alleles in patients. The imputation of HLA-DRB1 alleles within the combined data sets (n = 1870) resulted in (A) 39 unique HLA-DRB1 alleles and (B) 15 unique HLA-DQB1 alleles of varying allele frequency.

Figure 3

Association of HLA-DRB1 and HLA-DQB1 alleles with asparaginase hypersensitivity. Using a general linear model adjusted for gender, age, treatment arm, and ALL immunophenotype, (A) HLA-DRB1*07:01 (P = .001 [P_adjusted = .023]) was associated with asparaginase hypersensitivities within the discovery cohort (n = 541). The dashed horizontal red line identifies a permuted significance threshold of P < .05 that is adjusted for multiple testing. Patients with the HLA-DRB1*07:01 allele had a higher incidence of hypersensitivity compared with patients that did not have the allele within the (B) discovery cohort (n = 541), (C) validation cohort (n = 1329), and (D) the combined cohort (n = 1870).

Figure 4

Polymorphic amino acid positions of HLA-DRB1 are associated with asparaginase hypersensitivity. (A) Amino acid position 73 had the strongest association with asparaginase hypersensitivity, and positions 11, 13, 14, 25, 30, 57, 60, 74, and 78 were associated with hypersensitivity to a lesser extent. The y-axis shows the permuted P value (P_adjusted) of each association tested and the x-axis represents the amino acid positions of the HLA-DRB1 protein, excluding the leader signal sequence. The dashed horizontal red line identifies a permuted significance threshold of P < .05 that is adjusted for multiple testing. (B) Patients with a glycine (Gly) residue at HLA-DRB1 amino acid position 73 had a higher incidence of hypersensitivity compared with patients with an alanine (Ala/Ala) (n = 1,870, P = 6.45 × 10⁻⁶ [P_adjusted < 1.00 × 10⁻⁴]).

Figure 5

The 3-dimensional structure of HLA-DR. The 3-dimensional ribbon model of HLA-DR is based on Protein Data Bank entry 3PDO with the DR α chain shown in violet and the DR β chain in gray. Amino acid positions identified within the HLA-DRB1 protein by the association analysis are shown for positions 11 (black), 13 (blue), 14 (cyan), 25 (orange), 30 (brown), 57 (yellow), 60 (pink), 73 (red), 74 (green), and 78 (purple).

Figure 6

High-affinity binding of HLA-DRB1 alleles was associated with asparaginase hypersensitivity reactions. (A) Patients with high-affinity binding alleles had a higher incidence of asparaginase hypersensitivity compared with patients with low-affinity binding alleles (n = 1870, P = 3.3 × 10⁻⁴). (B) Estimated ORs for association of HLA-DRB1*01:01, HLA-DRB1*04:02, HLA-DRB1*04:05, HLA-DRB1*04:08, HLA-DRB1*07:01, HLA-DRB1*14:01, and HLA-DRB1*15:01 alleles with asparaginase hypersensitivity, all at P < .1, are shown vs the predicted binding category (predicted low-binding alleles are shown in blue; predicted high-binding alleles are shown in red). All HLA-DRB1 alleles had predicted binding affinities consistent with the estimated odds ratios.

Figure 7

Asparaginase antibodies were associated with HLA-DRB1*07:01 genotype, amino acid composition, and binding affinity. Anti-asparaginase IgG antibody status was available for 502 ALL patients. A higher incidence of asparaginase antibodies was detected for patients with (A) HLA-DRB1*07:01 (P = 1.4 × 10⁻⁵), (B) a glycine amino acid within HLA-DRB1 amino acid position 73 (P = 5.2 × 10⁻⁵), or (C) HLA-DRB1 alleles with predicted high-affinity binding for asparaginase (P = 1.4 × 10⁻³). Ala, alanine; Gly, glycine; het, heterozygous; homo, homozygous.