A subtype of childhood acute lymphoblastic leukaemia with poor treatment outcome: a genome-wide classification study

Abstract

Background: Genetic subtypes of acute lymphoblastic leukaemia (ALL) are used to determine risk and treatment in children. 25% of precursor B-ALL cases are genetically unclassified and have intermediate prognosis. We aimed to use a genome-wide study to improve prognostic classification of ALL in children.

Methods: We constructed a classifier based on gene expression in 190 children with newly diagnosed ALL (German Cooperative ALL [COALL] discovery cohort) by use of double-loop cross-validation and validated this in an independent cohort of 107 newly diagnosed patients (Dutch Childhood Oncology Group [DCOG] independent validation cohort). Hierarchical cluster analysis with classifying gene-probe sets revealed a new ALL subtype, the underlying genetic abnormalities of which were characterised by comparative genomic hybridisation-arrays and molecular cytogenetics.

Findings: Our classifier predicted ALL subtype with a median accuracy of 90.0% (IQR 88.3-91.7) in the discovery cohort and correctly identified 94 of 107 patients (accuracy 87.9%) in the independent validation cohort. Without our classifier, 44 children in the COALL cohort and 33 children in the DCOG cohort would have been classified as B-other. However, hierarchical clustering showed that many of these genetically unclassified cases clustered with BCR-ABL1-positive cases: 30 (19%) of 154 children with precursor B-ALL in the COALL cohort and 14 (15%) of 92 children with precursor B-ALL in the DCOG cohort had this BCR-ABL1-like disease. In the COALL cohort, these patients had unfavourable outcome (5-year disease-free survival 59.5%, 95% CI 37.1-81.9) compared with patients with other precursor B-ALL (84.4%, 76.8-92.1%; p=0.012), a prognosis similar to that of patients with BCR-ABL1-positive ALL (51.9%, 23.1-80.6%). In the DCOG cohort, the prognosis of BCR-ABL1-like disease (57.1%, 31.2-83.1%) was worse than that of other precursor B-ALL (79.2%, 70.2-88.3%; p=0.026), and similar to that of BCR-ABL1-positive ALL (32.5%, 2.3-62.7%). 36 (82%) of the patients with BCR-ABL1-like disease had deletions in genes involved in B-cell development, including IKZF1, TCF3, EBF1, PAX5, and VPREB1; only nine (36%) of 25 patients with B-other ALL had deletions in these genes (p=0.0002). Compared with other precursor B-ALL cells, BCR-ABL1-like cells were 73 times more resistant to L-asparaginase (p=0.001) and 1.6 times more resistant to daunorubicin (p=0.017), but toxicity of prednisolone and vincristine did not differ.

Interpretation: New treatment strategies are needed to improve outcome for this newly identified high-risk subtype of ALL.

Funding: Dutch Cancer Society, Sophia Foundation for Medical Research, Paediatric Oncology Foundation Rotterdam, Centre of Medical Systems Biology of the Netherlands Genomics Initiative/Netherlands Organisation for Scientific Research, American National Institute of Health, American National Cancer Institute, and American Lebanese Syrian Associated Charities.

Figure 1. Schematic view of the approach to determine a gene expression signature that enabled classification of pediatric ALL in known immunophenotypic and genetic subtypes and which resulted in the discovery of the poor-prognostic BCR-ABL-like ALL subtype

(A), Outline for construction and validation of a gene expression signature that enabled classification of pediatric ALL. A double-loop cross validation method was used to determine the number of gene probe sets that most optimally predicted cases assigned to the inner loop-test set (blue arrows) of the COALL cohort. Next, this number of probe sets was used to construct a classifier that becomes tested in the outer loop to estimate the prediction accuracy (red arrows). Upon construction of the final classifier in the total COALL cohort, the true accuracy of this classifier is determined using the independent DCOG validation cohort which is tested only once. (B), Flow diagram of the discovery of the BCR-ABL-like subtype in pediatric ALL. The different steps taken that lead to the identification of the novel BCR-ABL-like subtype have been summarized.

Figure 2. Clustering of ALL subtypes by gene expression profiles

Hierarchical clustering of 190 COALL (A) and 107 DCOG (B) study patients using 110 gene probe sets that were selected to classify pediatric ALL. Heat map shows which gene probe sets are relatively over-expressed (in red) and which gene probe sets are relatively under-expressed (in green) compared to the mean expression of all gene probe sets, see scale bar. *, cases with E2A-rearranged subclone (15–26% positive cells). T-ALL, red; MLL-rearranged, light blue; E2A-rearranged, dark blue; TEL-AML1 positive, light green; hyperdiploid, dark green; BCR-ABL positive, yellow; novel BCR-ABL-like; yellow/dotted; white, cases with unknown or other genetic abnormalities.

Figure 3. Kaplan-Meier estimates for the probability of disease-free survival (pDFS) in children with precursor B-ALL

(A), pDFS of COALL cohort precursor B-ALL cases; The COALL precursor B-ALL cohort consisted of 145 COALL-92/97 treated and 9 DCOG ALL-9 treated Sophia Children’s Hospital-patients. As a reference, data of 22 BCR-ABL positive cases enrolled in the COALL-92/97 protocol were included. Univariate analysis of pDFS comparing 30 BCR-ABL-like to 119 remaining precursor B-ALL cases (excluding 5 BCR-ABL positives): P=0.012. Kaplan-Meier curves were virtually the same when the 9 Sophia patients were excluded from this analysis. (B), pDFS of DCOG cohort precursor B-ALL cases. The DCOG precursor B-ALL cohort consisted of 92 DCOG-ALL8 treated children. As a reference, data of 25 BCR-ABL positive cases enrolled in DCOG ALL 7, 8 and 9 were included. Univariate analysis of pDFS comparing 14 BCR-ABL-like and 77 remaining precursor B-ALL cases (excluding BCR-ABL positive case): P=0.026. Numbers at the bottom indicate patients at risk.

Figure 4. In vitro cytotoxicity of 4 major drugs used in the treatment of pediatric ALL compared between BCR-ABL-like and other precursor B-ALL cases

LC50 left Y-axis: PRED, prednisolone; L-ASP, L-asparaginase. Right Y-axis: VCR, vincristine; DNR, daunorubicin. Box represents median LC50 value, whiskers indicate 25th and 75th percentiles. BCR-ABL-like data are depicted in red, those of other precursor B-ALL cases are depicted in green. Comparison between BCR-ABL-like and B-other group: p=0.001 for L-asparaginase (*) and p=0.017 for daunorubicin (**), other drugs p>0.05 (2-sided).

Figure 5. Genome-wide copy number alterations in pediatric precursor B-ALL

Genome-wide copy number data are visualized for BCR-ABL-like patients (n=44), BCR-ABL-positive (n=15) and other precursor B-ALL cases (B-other, n=25). Deletions are visualized in red, whereas amplifications are shown in blue. Centromere is indicated by grey line per chromosome.