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Chronic natural killer large granular lymphocyte (NK-LGL) leukemia, also referred to as chronic lymphoproliferative disorder of NK cells, is a rare disorder defined by prolonged expansion of clonal NK cells. Similar prevalence of STAT3 mutations in chronic T-LGL and NK-LGL leukemia is suggestive of common pathogenesis. We undertook whole-genome sequencing to identify mutations unique to NK-LGL leukemia. The results were analyzed to develop a resequencing panel that was applied to 58 patients. Phosphatidylinositol 3-kinase pathway gene mutations (PIK3CD/PIK3AP1) and TNFAIP3 mutations were seen in 5% and 10% of patients, respectively. TET2 was exceptional in that mutations were present in 16 (28%) of 58 patient samples, with evidence that TET2 mutations can be dominant and exclusive to the NK compartment. Reduced-representation bisulfite sequencing revealed that methylation patterns were significantly altered in TET2 mutant samples. The promoter of TET2 and that of PTPRD, a negative regulator of STAT3, were found to be methylated in additional cohort samples, largely confined to the TET2 mutant group. Mutations in STAT3 were observed in 19 (33%) of 58 patient samples, 7 of which had concurrent TET2 mutations. Thrombocytopenia and resistance to immunosuppressive agents were uniquely observed in those patients with only TET2 mutation (Games-Howell post hoc test, P = .0074; Fisher’s exact test, P = .00466). Patients with STAT3 mutation, inclusive of those with TET2 comutation, had lower hematocrit, hemoglobin, and absolute neutrophil count compared with STAT3 wild-type patients (Welch’s t test, P ≤ .015). We present the discovery of TET2 mutations in chronic NK-LGL leukemia and evidence that it identifies a unique molecular subtype.

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.

Asparaginase therapy is an important component of treatment for childhood acute lymphoblastic leukemia (ALL). However, immune responses to asparaginase during treatment can lead to suboptimal asparaginase exposure and possibly to worse outcome. Potential factors that can affect the incidence of asparaginase allergies include the pharmaceutical formulation of asparaginase, the number of asparaginase doses, treatment schedule, concurrent medications, ALL lineage, and racial ancestry. While genetic variations in the Human Leukocyte Antigen (HLA) genes have been implicated in allergic reactions to several drugs, no investigation has determined the influence of HLA on asparaginase allergies. Asparaginase allergy data from children with ALL who were enrolled on either SJCRH protocols [Total XIIIA (n =91), Total XV (n =320) and Total XVI (n =130)] or Children’s Oncology Group (COG) protocols [POG-9906 (n= 125) and AALL0232 (n= 1,204)] were used to investigate the influence of HLA class II genes on asparaginase allergies. Germline DNA was interrogated using the Affymetrix 500K, Affymetrix 6.0, or Illumina Exome Beadchip arrays. The T1DGC reference panel that contains both SNP data (Illumina Immunochip) and typed HLA class II alleles of unrelated European individuals (n= 4,938) was used to impute the HLA alleles of ALL patients with confirmed European ancestry. BEAGLE was used to impute HLA-DRB1 and HLA-DQB1 alleles at four-digit resolution. In a subset of 61 patients whose HLA type had been determined clinically, there was 94% concordance between imputed and typed HLA status. Using data on the occurrence of allergic reactions in patients enrolled in SJCRH protocols and adjusting for treatment arm, gender, age, and ALL lineage, HLA-DRB1*07:01 was identified as an asparaginase allergy risk allele (n = 541, p = 1.4 x 10-3, OR = 1.92), and the association was validated in the cohort of COG patients (n = 1,329, p = 0.01, OR = 1.49). Overall, patients with the HLA-DRB1*07:01 allele had a higher incidence of allergies compared to those without the allele (26% versus 18%, n = 1,870, p = 7.5 x 10-5, OR = 1.64). To determine the amino acid features of HLA-DRB1*07:01 that confer a greater risk of allergy, polymorphic amino acid positions associated with the imputed HLA-DRB1 alleles were inferred. Association analysis identified a glycine residue in HLA-DRB1 amino acid position 73 as the most significant residue associated with asparaginase allergies (n = 1,870, p = 4.5 x 10-5, OR = 1.50). This variant and other significantly associated variants (p < 5.5 x 10-4) were determined to be localized within the antigen binding pocket of the HLA-DRB1 protein, suggesting that binding of asparaginase peptide fragments to the HLA protein is an important determinant of the immune response to asparaginase. To investigate if HLA-DRB1 alleles with higher affinity for asparaginase peptide fragments were associated with a higher incidence of asparaginase allergy, the Immune Epitope Database (IEDB) was used to computationally predict the affinity of various HLA alleles for asparaginase. Using the IEDB binding scores, alleles were assigned to either a high or low binding category. Using the imputed HLA-DRB1 alleles of ALL patients, we found that patients whose alleles predicted high affinity binding to asparaginase had a higher incidence of allergies compared to those predicted to carry low-binding alleles (n = 1,870, p = 3.3 x 10-4, OR = 1.38). HLA-DRB1*07:01 was predicted to be a high-binding allele, consistent with its association with allergy. We conclude that HLA-DRB1*07:01 is associated with asparaginase allergy due to amino acid variants within the binding pocket of HLA-DRB1 that confer high binding affinity of HLA-DRB1 to asparaginase. Disclosures: Relling: Sigma-Tau Pharmaceuticals: I receive funding for investigator-initiated research on the pharmacology of asparaginase from Sigma-Tau Pharmaceuticals., I receive funding for investigator-initiated research on the pharmacology of asparaginase from Sigma-Tau Pharmaceuticals. Other; St. Jude Children’s Research Hospital : I have in the past received a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms as clinical diagnostics., I have in the past received a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms as clinical diagnostics. Patents & Royalties. Hunger:Jazz Pharmaceuticals: I receive funding for investigator-initiated research of asparaginase from Jazz Pharmaceuticals., I receive funding for investigator-initiated research of asparaginase from Jazz Pharmaceuticals. Other; Sigma-Tau Pharmaceuticals: I receive funding for investigator-initiated research of asparaginase from Sigma-Tau Pharmaceuticals. Other. Evans:St. Jude: In accordance with institutional policy, St. Jude allocates a portion of the income it receives from licensing inventions and tangible research materials to those researchers responsible for creating this intellectual property. Patents & Royalties, Under this policy, I and/or my spouse have in the past received a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms as clinical diagnostics., Under this policy, I and/or my spouse have in the past received a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms as clinical diagnostics. Other.