Genome-Wide Association Study (GWAS) of High-Risk Minimal Residual Disease and Relapse in B-Cell Acute Lymphoblastic Leukemia on Trial AIEOP-BFM ALL 2000

A significant proportion of patients with pediatric acute lymphoblastic leukemia (ALL) still suffers from relapse and therapy-related toxicities. Thus, the focus of further improvement of therapy for them is not only cure, but minimizing both short-term and long-term therapy-associated toxicity. This can be achieved by better personalized adjustment of therapy to the risk of relapse. Continuing research has identified numerous features with prognostic potential, including clinical and genetic variables as well as early response to chemotherapy being associated with treatment outcome. While most research activities on genetic prognostic factors have focused on somatic features, relatively little is known on hereditary contributions to treatment response and outcome in pediatric ALL. Therefore, we conducted a genome-wide association study (GWAS) of high-risk minimal residual disease (MRD) and relapse in B cell precursor (BCP) ALL patients treated on trial AIEOP-BFM ALL 2000.

On this trial, patients were stratified into three risk groups, mainly by MRD analyses. MRD standard-risk (MRD-SR) patients were MRD-negative on treatment days 33 and 78, MRD high-risk (MRD-HR) patients had residual disease of ≥5x10^-4 on treatment day 78. All the remaining MRD results were considered MRD intermediate-risk (MRD-IR). Besides MRD, established high-risk parameters were also retained for stratification: prednisone poor-response, induction failure, positivity for t(4;11)/ MLL-AF4 . Treatment used standard drugs and, in some of the patients, cranial irradiation and/or hematopoietic stem cell transplantation. DNA isolated from remission samples was genotyped using Illumina Human1M-Duo BeadChips containing 1.048.711 single SNV markers. For GWAS analyses gPLINK v2.050, PLINK v.1.09 and RStatistics v3.3.1 were used. The analysis on MRD was conducted in 69 MRD-HR patients (cases) and in 1034 patients of the MRD-SR and MRD-IR groups (controls). The analysis on relapse employed 145 relapsed (cases) and 1007 non-relapsed patients (controls). After quality control assessment, the final analyses included 749.262 SNPs among 62 MRD-HR patients compared to 927 controls and 129 relapsed patients compared to 925 controls.

In the analysis on MRD-HR five SNVs reached genome-wide significance at P ≤5×10⁻⁸. An intronic SNV (rs1570765) in STARD13 demonstrated the strongest association ( P =1.73x10^-10; odds ratio, OR=5.06). Two additional SNVs with P ≤5×10⁻⁸ in STARD13 were in linkage disequilibrium with rs1570765. The two remaining SNVs with P ≤5×10⁻⁸ were located in FEZ2 and NRIP1. Five additional SNVs demonstrated significant levels of ≤1×10⁻⁶ and were associatedwith the following genes: STARD13 , VIT and FICD . STARD13 is located on chromosome 13, belongs to the family of RhoGap proteins and is frequently lost in hepatocellular and breast carcinomas. Its RhoGAP domain activates members of the Ras superfamily which leads to a suppression of cell growth and differentiation (Lin et al. Oncogene 2010). In the GWAS approach on relapse three SNVs reached significant levels of ≤1×10⁻⁶ and were associatedwith the genes ASB3 (rs1549749, P =3.51x10^-7; rs12465316, P =4.26x10^-7) on chromosome 2 and NDN (rs9972595, P =1.04x10^-6) on chromosome 15. Twenty additional SNVs demonstrated significance levels of ≤1×10⁻⁵. ASB3 codes for a member of the ankyrin repeat and SOCS box-containing (ASB) family of proteins containing ankyrin repeat sequence and a SOCS box domain. The latter serves to couple suppressor of cytokine signalling (SOCS) proteins and their binding partners with the elongin B and C complex, possibly targeting them for degradation. NDN , a p53 target gene, codes for Necdin which was previously shown to regulate the quiescence and response to genotoxic stress of hematopoietic stem/progenitor cells (Asai et al., Blood 2012). Further replication results, an analysis of additive effects and multivariate analyses will be presented.

In conclusion, our results demonstrate that germline genetic variation is associated with high levels of MRD after induction/consolidation treatment for ALL and relapse on an AIEOP-BFM ALL protocol. In line with previous studies (Yang et al., JAMA 2009), our data suggest that genome-wide approaches may be useful to identify novel and clinically relevant SNVs for further refinement of risk-adapted treatment strategies in pediatric ALL.