Copy number alterations in B-cell development genes, drug resistance, and clinical outcome in pediatric B-cell precursor acute lymphoblastic leukemia

Abstract

Pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is associated with a high frequency of copy number alterations (CNAs) in IKZF1, EBF1, PAX5, CDKN2A/B, RB1, BTG1, ETV6, and/or the PAR1 region (henceforth: B-cell development genes). We aimed to gain insight in the association between CNAs in these genes, clinical outcome parameters, and cellular drug resistance. 71% of newly diagnosed pediatric BCP-ALL cases harbored one or more CNAs in these B-cell development genes. The distribution and clinical relevance of these CNAs was highly subtype-dependent. In the DCOG-ALL10 cohort, only loss of IKZF1 associated as single marker with unfavorable outcome parameters and cellular drug resistance. Prednisolone resistance was observed in IKZF1-deleted primary high hyperdiploid cells (~1500-fold), while thiopurine resistance was detected in IKZF1-deleted primary BCR-ABL1-like and non-BCR-ABL1-like B-other cells (~2.7-fold). The previously described risk stratification classifiers, i.e. IKZF1^plus and integrated cytogenetic and CNA classification, both predicted unfavorable outcome in the DCOG-ALL10 cohort, and associated with ex vivo drug cellular resistance to thiopurines, or L-asparaginase and thiopurines, respectively. This resistance could be attributed to overrepresentation of BCR-ABL1-like cases in these risk groups. Taken together, our data indicate that the prognostic value of CNAs in B-cell development genes is linked to subtype-related drug responses.

Figure 1

CNA landscape of B-cell development genes in the different subtypes of pediatric BCP-ALL. CNA profile of 515 newly diagnosed pediatric BCP-ALL patients, representing all major BCP-ALL subtypes, was determined using MLPA. Association between CNAs and subtypes was studied using the Fisher Exact test. The proportion of patients per subtype with a specific CNAs is shown. CNAs tested included IKZF1 (A), EBF1 (B), PAX5 (C), ETV6 (D), CDKN2A/B (E), RB1 (F), BTG1 (G), PAR1 (H). **p ≤ 0.01, *p ≤ 0.05. del = deletion.

Figure 2

Co-occurence of CNAs in B-cell development genes in the different BCP-ALL subtypes. Heatmap of CNA profile of 515 newly diagnosed pediatric BCP-ALL patients, representing the major BCP-ALL subtypes. CNAs are shown per subtype. Colors indicate presence of a CNA and absence of CNAs is shown in white. The heatmap is sorted on IKZF1 deletions followed by CNAs in PAX5. Each column represents an individual patient. The co-occurrence between the different CNAs in all BCP-ALL cases was calculated using the Fisher Exact test. Odds ratios and p-values of significant associations are shown.

Figure 3

The association between CNAs and MRD levels after induction therapy and the first consolidation course in newly diagnosed BCP-ALL. MRD levels of DCOG-ALL treated BCP-ALL cases (all risk groups) after induction (TP1; n = 183) and first consolidation course (TP2; n = 183). The percentage of cases with high (≥10⁻³), medium (10⁻⁴ ≤ MRD < 10⁻³), and undetectable MRD levels (<10⁻⁴) is depicted per CNA. The Fisher’s Exact test was applied to study associations between CNAs and MRD levels. **p ≤ 0.01, *p ≤ 0.05. del = deletion.

Figure 4

Prognostic value of CNAs in DCOG-ALL10 treated cases. (A) The association between CNAs in all risk groups and cumulative incidence of relapse (CIR) and event-free-survival (EFS) was examined. BCP-ALL patients (n = 210) were treated according to DCOG-ALL10 protocol. CIR was estimated using a competing risk model. Relapse and non-response were considered as event, and death as competing event. To test equality of the CIRs, the Gray’s test was applied. Non-response, relapse, and death were considered as events for EFS. EFS rates were determined using Cox regression, and compared using the Wald test. For reliable test results, groups should contain at least 5 cases. (B) CIR and EFS curves of cases without or with an IKZF1 deletion. Curves contain either all risk groups, or the medium risk arm only.

Figure 5

The association between CNAs and the ex vivo cellular drug response. (A) Leukemic cells were incubated for four days with a concentration range of prednisolone (µg/ml), vincristine (µg/ml), L-asparaginase (IU/ml), daunorubicin (µg/ml), 6-mercaptopurine (µg/ml), and 6-thioguanine (µg/ml), after which cell viability was measured using an MTT assay. The Mann-Whitney U test was applied to compare LC50-values. No association is depicted in grey, resistance in blue (p < 0.05, fold induction (FI) > 1), sensitive in green (p < 0.05, FI < 1), and not determined in white. The number of cases that were tested for prednisolone is depicted, and represent the maximum number of cases. For reliable test results, groups should contain at least 5 cases (groups ≤ 5 are depiced as ND). Results of single CNAs are depicted for all risk groups and for BCR-ABL1-like/B-other cells, high hyperdiploid cells, and ETV6-RUNX1 cells. In addition, associations between the risk classifiers IKZF1^plus and integrated cytogenetic and CNA classification (poor risk) and cellular drug resistance are shown^,. (B) LC50 values for prednisolone (µg/ml), 6-thiogunanine (µg/ml), and 6-mercaptopurine (µg/ml) of cases without or with IKZF1 deletion. Columns include all BCP-ALL subtypes (grey), BCR-ABL1-like/B-other cells (blue), and high hyperdiploid cells (green). The red line represent the median LC50 value in the each group. (C) LC50 values for prednisolone (µg/ml) of cases without or with PAX5 CNAs. Columns include all BCP-ALL subtypes (grey), BCR-ABL1-like/B-other cells (blue), high hyperdiploid cells (green), and ETV6-RUNX1 cells (yellow). The red line represent the median LC50 value in the each group. (D) LC50 values for prednisolone (µg/ml), vincristine (µg/ml), and daunorubicin (µg/ml) of primary leukemic cells without or with ETV6 deletion. Columns include all BCP-ALL subtypes (grey), BCR-ABL1-like/B-other cells (blue), high hyperdiploid cells (green), and ETV6-RUNX1 cells (yellow). The red line represent the median LC50 value in the each group. *p < 0.05, **p < 0.01; Mann-Whitney U test.