Faculty Bibliography

Glucocorticoids are important therapy for acute lymphoblastic leukemia (ALL) and their major adverse effect is osteonecrosis. Our goal was to identify genetic and nongenetic risk factors for osteonecrosis. We performed a genome-wide association study of single nucleotide polymorphisms (SNPs) in a discovery cohort comprising 2285 children with ALL treated on the Children's Oncology Group AALL0232 protocol (NCT00075725 https://clinicaltrials.gov/ct2/show/NCT00075725), adjusting for covariates. The minor allele at SNP rs10989692 (near the glutamate receptor GRIN3A locus) was associated with osteonecrosis (hazard ratio = 2.03, P=3.59x10-7). The association was supported by two replication cohorts, including 361 children with ALL on St. Jude's Total XV protocol (NCT00137111 https://clinicaltrials.gov/ct2/show/NCT00137111) and 309 non-ALL patients from Vanderbilt University's BioVU repository treated with glucocorticoids (odds ratio = 1.87 and 2.26, P = 0.063 and 0.0074 respectively). In a meta-analysis, rs10989692 was also highest ranked (P = 2.68x10-8), and the glutamate pathway was the top ranked pathway (P = 9.8x10-4). Osteonecrosis-associated glutamate receptor variants were also associated with other vascular phenotypes including cerebral ischemia (OR = 1.64, P = 2.5x10-3) and arterial embolism and thrombosis (OR = 1.88, P = 4.2x10-3). In conclusion, osteonecrosis was associated with inherited variations near glutamate receptor genes. Further understanding this association may allow interventions to decrease osteonecrosis.

Abnormal gene expression in cancer represents an under-explored source of cancer markers and therapeutic targets. In order to identify gene expression signatures associated with survival in acute lymphoblastic leukemia (ALL), a strategy was designed to search for aberrant gene activity, which consists of applying several filters to transcriptomic datasets from two pediatric ALL studies. Six genes whose expression in leukemic blasts was associated with prognosis were identified:three genes predicting poor prognosis (AK022211, FASTKD1 and STARD4) and three genes associated with a favorable outcome (CAMSAP1, PCGF6 and SH3RF3). Combining the expression of these 6 genes could successfully predict prognosis not only in the two discovery pediatric ALL studies, but also in two independent validation cohorts of adult patients, one from a publicly available study and one consisting of 62 newly recruited Chinese patients. Moreover, our data demonstrate that our six gene based test is particularly efficient in stratifying MLL or BCR.ABL negative patients. Finally, common biological traits characterizing aggressive forms of ALL in both children and adults were found, including features of dormant hematopoietic stem cells, suggesting new therapeutic strategies.

Minimal residual disease (MRD) is highly prognostic in pediatric B-precursor acute lymphoblastic leukemia (ALL). In COG High Risk B-ALL study AALL0232 we investigated MRD in subjects randomized in a 2X2 factorial design to receive either High-Dose (HD-MTX) or Capizzi Methotrexate (C-MTX) during interim maintenance (IM), or Prednisone or Dexamethasone during induction. Subjects with end induction MRD>=0.1% or those with morphologic slow early response were non-randomly assigned to receive a second IM and delayed intensification phase. MRD was measured by 6-color flow cytometry in one of two reference labs, with excellent agreement between the two. Subjects with end induction MRD<.01% had a 5y EFS of 87+/-1% vs 74+/-4% for those with MRD 0.01%-0.1%; increasing MRD amounts was associated with progressively worse outcome. Subjects converting from MRD positive to negative by end consolidation had a relatively favorable 79+/-5% 5 y DFS vs 39+/-7% for those with MRD>=0.01%. Although HD-MTX was superior to C-MTX, MRD retained prognostic significance in both groups (86+/-2% vs 58+/-4% for MRD negative vs positive C-MTX subjects; 88+/-2% vs 68+/-4% for HD-MTX subjects). Intensified therapy given to subjects with MRD>0.1% did not improve either 5y EFS or OS. However, these subjects showed an early relapse rate similar to that seen in MRD negative ones, with EFS/OS curves for patients with 0.1%-1% MRD crossing those with 0.01%-0.1% MRD at 3 and 4 years, thus suggesting that the intensified therapy altered the disease course of MRD positive subjects. Additional interventions targeted at the MRD positive group may further improve outcome. NCT00075725 at www.clinicaltrials.gov.

Asparaginase is used to treat acute lymphoblastic leukemia (ALL); however, hypersensitivity reactions can lead to suboptimal asparaginase exposure. Our objective was to use a genome-wide approach to identify loci associated with asparaginase hypersensitivity in children with ALL enrolled on St. Jude Children's Research Hospital (SJCRH) protocols Total XIIIA (n = 154), Total XV (n = 498) and Total XVI (n = 271) or Children's Oncology Group protocols POG 9906 (n = 222) and AALL0232 (n = 2,163). Germline DNA was genotyped using the Affymetrix 500K, Affymetrix 6.0, or the Illumina Exome Beadchip array. In multivariate logistic regression, the intronic rs6021191 variant in NFATC2 had the strongest association with hypersensitivity (P = 4.1x10-8, OR = 3.11). RNA-seq data available from 65 SJCRH ALL tumor samples and 52 Yoruban HapMap samples showed that samples carrying the rs6021191 variant had higher NFATC2 expression compared to non-carriers (P = 1.1x10-3 and 0.03, respectively). The top ranked non-synonymous polymorphism was rs17885382 in HLA-DRB1 (P = 3.2x10-6, OR = 1.63), which is in near complete linkage disequilibrium with the HLA-DRB1*07:01 allele we previously observed in a candidate gene study. The strongest risk factors for asparaginase allergy are variants within genes regulating the immune response.

Glucocorticoids are universally used in the treatment of acute lymphoblastic leukemia (ALL), and resistance to glucocorticoids in leukemia cells confers poor prognosis. To elucidate mechanisms of glucocorticoid resistance, we determined the prednisolone sensitivity of primary leukemia cells from 444 patients newly diagnosed with ALL and found significantly higher expression of CASP1 (encoding caspase 1) and its activator NLRP3 in glucocorticoid-resistant leukemia cells, resulting from significantly lower somatic methylation of the CASP1 and NLRP3 promoters. Overexpression of CASP1 resulted in cleavage of the glucocorticoid receptor, diminished the glucocorticoid-induced transcriptional response and increased glucocorticoid resistance. Knockdown or inhibition of CASP1 significantly increased glucocorticoid receptor levels and mitigated glucocorticoid resistance in CASP1-overexpressing ALL. Our findings establish a new mechanism by which the NLRP3-CASP1 inflammasome modulates cellular levels of the glucocorticoid receptor and diminishes cell sensitivity to glucocorticoids. The broad impact on the glucocorticoid transcriptional response suggests that this mechanism could also modify glucocorticoid effects in other diseases.

There is incomplete understanding of genetic heterogeneity and clonal evolution during cancer progression. Here we use deep whole-exome sequencing to describe the clonal architecture and evolution of 20 pediatric B-acute lymphoblastic leukaemias from diagnosis to relapse. We show that clonal diversity is comparable at diagnosis and relapse and clonal survival from diagnosis to relapse is not associated with mutation burden. Six pathways were frequently mutated, with NT5C2, CREBBP, WHSC1, TP53, USH2A, NRAS and IKZF1 mutations enriched at relapse. Half of the leukaemias had multiple subclonal mutations in a pathway or gene at diagnosis, but mostly with only one, usually minor clone, surviving therapy to acquire additional mutations and become the relapse founder clone. Relapse-specific mutations in NT5C2 were found in nine cases, with mutations in four cases being in descendants of the relapse founder clone. These results provide important insights into the genetic basis of treatment failure in ALL and have implications for the early detection of mutations driving relapse.