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Aim: To evaluate top-ranking genes identified through genome-wide association studies for an association with corticosteroid-related osteonecrosis in children with acute lymphoblastic leukemia (ALL) who received Dana–Farber Cancer Institute treatment protocols. Patients & methods: Lead SNPs from these studies, as well as other variants in the same genes, pooled from whole exome sequencing data, were analyzed for an association with osteonecrosis in childhood ALL patients from Quebec cohort. Top-ranking variants were verified in the replication patient group. Results: The analyses of variants in the ACP1-SH3YL1 locus derived from whole exome sequencing data showed an association of several correlated SNPs (rs11553746, rs2290911, rs7595075, rs2306060 and rs79716074). The rs79716074 defines *B haplotype of the APC1 gene, which is well known for its functional role. Conclusion: This study confirms implication of the ACP1 gene in the treatment-related osteonecrosis in childhood ALL and identifies novel, potentially causal variant of this complication.

Aim: To evaluate the association between human leukocyte antigen (HLA) alleles and native Escherichia coli asparaginase hypersensitivity (AH) in children with acute lymphoblastic leukemia (ALL) who received Dana-Farber Cancer Institute treatment protocols. Patients & methods: HLA-DQA1, HLA-DRB1 and HLA-DQB1 alleles were retrieved from available whole exome sequencing data of a subset of childhood ALL patients from Quebec ALL cohort and analyzed for an association with AH. PCR assay was developed to analyze associated alleles in the entire discovery and replication cohorts. Results: Two alleles in linkage disequilibrium ( HLA-DRB1*07:01 and DQA1*02:01) were associated with AH. Additional analyses, performed to distinguish between HLA-DRB1*07:01 haplotypes with and without DQB1*02:02 allele, showed that the association was dependent on the presence of DQB1*02:02. Conclusion: This study confirms the implication of HLA-DRB1*07:01, DQA1*02:01 and DQB1*02:02 alleles in developing AH in childhood ALL.

Osteonecrosis (ON) is corticosteroid-related complication, reported in children with acute lymphoblastic leukemia (ALL). We have previously found that polymorphisms in BCL2L11 gene coding for pro-apoptotic Bim protein influence reduction of overall survival (OS) in a corticosteroid (CS) dose-dependent manner in childhood ALL patients. The same set of SNPs was here investigated for an association with CS-related ON assessed retrospectively in 304 children with ALL from Quebec (QcALL cohort) who received Dana-Farber Cancer Institute (DFCI) ALL treatment protocols. Two-year cumulative incidence of symptomatic ON was 10.6%. Two BCL2L11 polymorphisms, the 891T>G (rs2241843) in all QcALL patients and 29201C>T (rs724710) in high-risk group were significantly associated with ON, P = 0.009 and P = 0.003, respectively. The association remained significant in multivariate model (HR891TT = 2.4, 95% CI 1.2–4.8, P = 0.01 and HR29201CC = 5.7, 95% CI 1.6–20.9, P = 0.008). Both polymorphisms influenced viability of dexamethasone treated lymphoblastoid cell lines (P ≤ 0.03). The 891T>G influenced Bim gamma isoform levels (0.03) and its association with ON was also confirmed in replication DFCI cohort (N = 168, P = 0.03). QcALL children had a high incidence of ON during therapy, which was highly associated with BCL2L11 polymorphisms.

Aim: To identify genetic markers associated with vincristine-induced peripheral neuropathy (VIPN) in childhood acute lymphoblastic leukemia. Patients & methods: Whole-exome sequencing data were combined with exome-wide association study to identify predicted-functional germline variants associated with high-grade VIPN. Genotyping was then performed for top-ranked signals (n = 237), followed by validation in independent replication group (n = 405). Results: Minor alleles of rs2781377/SYNE2 (p = 0.01) and rs10513762/MRPL47 (p = 0.01) showed increased risk, whereas that of rs3803357/BAHD1 had a protective effect (p = 0.007). Using a genetic model based on weighted genetic risk scores, an additive effect of combining these loci was observed (p = 0.003). The addition of rs1135989/ACTG1 further enhanced model performance (p = 0.0001). Conclusion: Variants in SYNE2, MRPL47 and BAHD1 genes are putative new risk factors for VIPN in childhood acute lymphoblastic leukemia.

Aim: We have previously reported an association of dihydrofolate reductase promoter polymorphisms with reduced event-free survival in childhood acute lymphoblastic leukemia (ALL) patients treated with Dana Farber Cancer Institute protocol. Here, we assessed whether these associations are applicable to other protocol, based on different methotrexate doses. Methods: Genotypes for six tag polymorphisms and resulting haplotypes were analyzed for an association with ALL outcome. Results: The association was found with the polymorphisms A-680C, A-317G and C-35T in high-risk group patients. Carriers of haplotype *1 had a remarkably higher risk of events compared with noncarriers and a lower probability of event-free survival (21.4 vs 81.3%). Conclusion: The role of DHFR variants in predicting the outcome of childhood ALL extends beyond single-treatment protocol and can be useful biomarker in personalizing treatment.

Purpose: Asparaginase (ASNase) is a standard and critical component in the therapy of childhood acute lymphoblastic leukemia (ALL), but it is also associated with several toxicities. Experimental design: We recently reported the results of an association study between ASNase pathway genes and event-free survival (EFS) in childhood patients with ALL. The same polymorphisms were interrogated here in relation to allergies, pancreatitis, and thrombotic events following treatment with E. coli ASNase. Results: Among patients of the discovery group, allergies, and pancreatitis were more frequent in individuals who are homozygous for the triple-repeat allele (3R) of the asparagine synthetase (ASNS) gene, resulting in remarkably higher risk of these toxicities associated with 3R3R genotype [OR for allergies, 14.6; 95% confidence interval (CI), 3.6–58.7; P < 0.0005 and OR for pancreatitis, 8.6; 95% CI, 2.0–37.3; P = 0.01]. In contrast, the ASNS haplotype *1 harboring double-repeat (2R) allele had protective effect against these adverse reactions (P ≤ 0.01). The same haplotype was previously reported to confer reduction in EFS. The risk effect of 3R3R genotype was not replicated in the validation cohort, whereas the protective effect of haplotype *1 against allergies was maintained (P ≤ 0.002). Analysis with additional polymorphisms in ASNS locus in lymphoblastoid cell lines showed that haplotype *1 is diversified in several subtypes of which one was associated with reduced in vitro sensitivity to ASNase (rs10486009, P = 0.01) possibly explaining an association seen in clinical setting. Conclusions: This finding might have implication for treatment individualization in ALL and other cancers using asparagine depletion strategies. Clin Cancer Res; 21(2); 329–34. ©2014 AACR. See related commentary by Avramis, p. 230

Background: Vincristine (VCR) is a standard component in the treatment of childhood acute lymphoblastic leukemia (ALL). VCR cytotoxicity is primarily due to its ability to disrupt the formation of microtubules of the mitotic spindle. Patients & methods: Seventeen polymorphisms in regulatory and coding regions of genes controlling VCR targets (TUBB1, MAP4, ACTG1 and CAPG) or potentially influencing VCR levels (ABCB1 and CYP3A5) were investigated for an association with peripheral neuropathy and outcome in childhood ALL patients. Results: High-grade neurotoxicity was more frequent in carriers of the A allele of synonymous (Ala310) G to A (rs1135989) variation in the ACTG1 gene. Substitution (rs4728709) in the promoter of the ABCB1 gene had a protective effect against lower grade neurotoxicity and C to A variation (rs3770102) located 17 nucleotides upstream from the transcription start site had a protective effect against high-grade neurotoxicity. Patients with the ABCB1 3435TT genotype had lower event-free survival; the association with event-free survival was not supported by the analysis in the replication patient set. Conclusion: The polymorphisms in the ACTG1, CAPG and ABCB1 genes may modulate VCR-related neurotoxicity, whereas the risk of relapse seems not to be affected by the genes of the VCR pathway. Original submitted 19 June 2013; Revision submitted 31 March 2014

The introduction of multiagent treatment protocols has led to a remarkable increase in survival rates for children diagnosed with acute lymphoblastic leukemia, yet for a subpopulation of patients, resistance to chemotherapeutics remains an obstacle to successful treatment. Here we investigate the role of the mitochondrial (or intrinsic) apoptosis pathway in modulating the onset and outcomes of childhood acute lymphoblastic leukemia. Cell death is a highly regulated process that plays an essential role in regulating cell homeostasis, particularly in tissues with high intrinsic proliferating capacity such as the hematopoietic system. Following the underlying paradigm that cis-acting genetic variation can influence disease risk and outcomes by modulating gene expression, we performed a systematic analysis of the proximal promoter regions of 21 genes involved in apoptosis. Using gene reporter assays, we show that promoter variations in 11 intrinsic apoptosis genes, including ADPRT, APAF1, BCL2, BAD, BID, MCL1, BIRC4, BCL2L1, ENDOG, YWHAB, and YWHAQ, influence promoter activity in an allele-specific manner. We also show that correlated promoter variation and increased expression of MCL1 is associated with reduced overall survival among high-risk patients receiving higher doses of corticosteroid, suggesting that increased expression of this anti-apoptosis gene could lead to reduced cell death and influence treatment response in a disease- and dose-responsive manner.

Purpose: Corticosteroids induce apoptosis in the malignant lymphoid cells and are critical component of combination therapy for acute lymphoblastic leukemia (ALL). Several genome-wide microarray studies showed major implication of proapoptotic Bim in mediating corticosteroid-related resistance in leukemia cells. Experimental Design: We investigated Bim gene polymorphisms and their association with childhood ALL outcome, and the mechanism underlying the observed finding. Results: Lower overall survival (OS) was associated with BimC29201T located in Bcl-2 homology 3 (BH3) domain (P = 0.01). An association remained significant in multivariate model (P = 0.007), was more apparent in high-risk patients (P = 0.004) and patients treated with dexamethasone (P = 0.009), and was subsequently confirmed in the replication patient cohort (P = 0.03). RNA analysis revealed that C29201T affects generation of γ isoforms (γ1) that lack proapoptotic BH3 domain. The phenotypic effect was minor suggesting the influence of additional factors that may act in conjunction with Bim genotype. Combined analysis with Mcl gene polymorphism (G-486T) revealed profound reduction in OS in individuals with both risk genotypes (P < 0.0005 in discovery and P = 0.002 in replication cohort) and particularly in high-risk patients (P ≤ 0.008). Conclusions: Increased expression of prosurvival Mcl1 and presence of Bim isoforms lacking proapoptotic function might explain marked reduction of OS in a disease and dose-dependent manner in ALL patients carrying Bim- and Mcl1-risk genotypes. Clin Cancer Res; 19(18); 5240–9. ©2013 AACR.

Asparaginase is a standard and critical component in the therapy of childhood acute lymphoblastic leukemia. Asparagine synthetase (ASNS) and the basic region leucine zipper activating transcription factor 5 (ATF5) and arginosuccinate synthase 1 (ASS1) have been shown to mediate the antileukemic effect of asparaginase and to display variable expression between leukemia cells that are resistant and sensitive to treatment. Fourteen polymorphisms in the regulatory and coding regions of these genes were investigated for an association with acute lymphoblastic leukemia outcome. Lower event-free survival (EFS) was associated with ATF5 T1562C, tandem-repeat ASNS polymorphism, derived haplotype, and ASS1 G1343T and G34T substitutions (P ≤ .03). Associations were limited to patients who received Escherichia coli asparaginase. Variations that sustained correction for multiple testing (ATF5 T1562C, P = .005; ASNS tandem-repeat and related haplotype, P ≤ .01) were subsequently analyzed in the replication cohort. The E coli–dependent association of the ATF5 T1562 allele with reduced EFS was confirmed (P = .01). A gene-reporter assay showed that the haplotype tagged by T1562 had higher promoter activity (P ≤ .01). The remaining regulatory polymorphisms also appeared to affect ATF5 function; 2 additional high-activity haplotypes were identified (P ≤ .02) and were further corroborated by quantitative mRNA analysis in lymphoblastoid cell lines. The ATF5-regulated increase in ASNS expression in response to more efficacious E coli–induced asparagine depletion may explain our observed results.

Childhood acute lymphoblastic leukemia patients ( n = 310) were analyzed for four SNPs in the NR3C1 gene. Polymorphisms −627A/G, intron 2 +646C/G and 9bT/C were all associated with reduced event-free survival. Haplotypes composed of AGT alleles at these loci and tagged by the intron 2 +646G variant also associated with lower event-free survival ( p = 0.03). The progressive impact of this haplotype on outcome was seen with two copies associated with reduced overall survival ( p = 0.05). Quantitative mRNA analysis in lymphoblastoid cell lines showed that carriers of the AGT haplotype had a higher ratio of GR γ/α isoforms ( p = 0.04), which possibly explains its association with reduced event-free survival and overall survival.

Methotrexate and 6-mercaptopurine, important components of acute lymphoblastic leukemia treatment, are substrates for multidrug resistance-associated protein MRP4. Eight single nucleotide polymorphisms were analyzed in MRP4 gene, and 4 variants were identified as tagSNPs with frequency more than or equal to 5%. They were investigated for association with treatment responses in 275 children with acute lymphoblastic leukemia. The TC genotype of the regulatory T-1393C polymorphism was associated with better event-free survival (P = .02) and lower methotrexate plasma levels (P = .01). The CA genotype of A934C (Lys304Asn) substitution correlated in contrast with lower event-free survival (P = .02) and higher frequency of high-grade thrombocytopenia (P = .01). Gene reporter assay showed that the promoter haplotype uniquely tagged by the C-1393 allele conferred higher promoter activity compared with remaining haplotypes (P < .001). Further analyses are needed to replicate this pilot study and get closer insight into the functional effect of these polymorphisms.

Dihydrofolate reductase (DHFR) is the major target of methotrexate (MTX), a key component in childhood acute lymphoblastic leukemia (ALL) treatment. A total of 15 polymorphisms in DHFR promoter were analyzed, and 3 sites (C−1610G/T, C−680A, and A−317G) were identified as sufficient to define observed haplotypes (tag single nucleotide polymorphisms [tagSNPs]). These polymorphisms were investigated for association with treatment response in 277 children with ALL. Lower event-free survival (EFS) was associated with homozygosity for the allele A−317 and C−1610 (P = .03 and .02), and with the haplotype *1, defined by both C−1610 and A−317 alleles (P = .03). The haplotype *1 conferred higher transcriptional activity (P < .01 compared with haplotypes generating minimal luciferase expression). Quantitative mRNA analysis showed higher DHFR levels for particular haplotype *1 carriers (P < .01). The analysis combining haplotype *1 with thymidylate synthase (TS) and cyclin D1 (CCND1) genotypes previously shown to affect ALL outcome showed that the number of event-predisposing genotypes was associated with increasingly lower EFS (P < .001). In conclusion, DHFR promoter polymorphisms are associated with worse ALL outcome, likely due to a higher DHFR expression. Combined effects among genes of the folate cycle can further accentuate differences in the response to the treatment.