Multiclonal complexity of pediatric acute lymphoblastic leukemia and the prognostic relevance of subclonal mutations

Abstract

Genomic studies of pediatric acute lymphoblastic leukemia (ALL) have shown remarkable heterogeneity in initial diagnosis, with multiple (sub)clones harboring lesions in relapse-associated genes. However, the clinical relevance of these subclonal alterations remains unclear. We assessed the clinical relevance and prognostic value of subclonal alterations in the relapse-associated genes IKZF1, CREBBP, KRAS, NRAS, PTPN11, TP53, NT5C2, and WHSC1 in 503 ALL cases. Using Molecular Inversion Probe sequencing and breakpoint-spanning PCR we reliably detected alterations below 1% allele frequency. We identified 660 genomic alterations in 285 diagnosis samples of which 495 (75%) were subclonal. RAS pathway mutations were common, particularly in minor subclones, and comparisons between RAS hotspot mutations revealed differences in their capacity to drive clonal expansion in ALL. We did not find an association of subclonal alterations with unfavorable outcome. Particularly for IKZF1, an established prognostic marker in ALL, all clonal but none of the subclonal alterations were preserved at relapse. We conclude that, for the genes tested, there is no basis to consider subclonal alterations detected at diagnosis for risk group stratification of ALL treatment.

Figure 1.

Schematic representation of the study design. Single-molecule molecular inversion probe-based sequencing approach and real-time quantitative polymerase chain reaction were used in order to detect alterations in known relapse-associated genes in a large cohort of diagnostic samples from patients with acute lymphocytic leukemia. Detected alterations were correlated with outcome and Sanger sequencing was performed on available relapse samples in order to confirm that exactly the same alteration was present in the major clone in relapse. smMIP: single-molecule molecular inversion probe; MLPA: multiplex ligation-dependent probe amplification assay; PCR: polymerase chain reaction; qPCR: real-time quantitative polymerase chain reaction.

Figure 2.

Prevalence and distribution of alterations in eight relapse-associated genes. (A) Violin plot showing the variability in mutation allele frequency at diagnosis in the genes studied. The color of the dots indicates whether the mutation was detected in a case without relapse (blue) or with relapse (red). (B) Bar plot showing the frequencies of major clone (high-clonal) and subclonal alterations per case in the genes studied. RAS pathway genes (NRAS, KRAS, PTPN11) were the most frequently mutated. Subclonal mutations (yellow bar) were highly prevalent in all genes tested. A subset of cases had both clonal and subclonal alterations in the same gene (gray bar).

Figure 3.

Potency of RAS pathway mutations to drive clonal expansion. (A-C) Schematic representation of KRAS (A), NRAS (B) and PTPN11 (C) indicating the prevalence of common hotspot mutations. (D) Violin plot showing allele frequency in hotspot mutations of three investigated RAS pathway genes. The median allele frequency was significantly higher in KRAS, indicating a high potential of KRAS hotspot mutations to drive clonal expansion. (E) Violin plot showing allele frequencies in frequent KRAS and NRAS hotspot mutations. The median allele frequency was significantly higher in KRAS G13D, suggesting their higher potential to drive clonal expansion compared to other RAS hotspot mutations.

Figure 4.

Prevalence of relapse associated genomic alterations at diagnosis. Bar plot showing the percentage of relapses in cases with high-clonal (blue) or subclonal (yellow) mutations in seven relapse-associated genes, and in cases that were wild-type (black) for these genes. Only cases with high-clonal IKZF1 4-7 deletions showed a significantly higher percentage of relapse development compared to wild-type cases (Fisher exact test, P<0.01) (Online Supplementary Table S9).

Figure 5.

Cumulative incidence of relapse for high-clonal and subclonal IKZF1 deletions. The cumulative incidence of relapse (CIR) was estimated using a competing- risk model with death as a competing event. CIR plots are presented for the representative ALL9 (left) and ALL10 (right) cohorts. Lines represent the IKZF1 deletion status and include wild-type (black line), subclonal exon 4-7 deletion (yellow), other high-clonal deletion (purple), and high-clonal exon 4-7 deletion (blue). Straight lines depict relapses and dotted lines death. P-values shown are obtained by employing the Gray test to compare CIR curves. The 5-year CIR was higher in cases with high-clonal IKZF1 deletions, compared to wild-type cases in both representative cohorts.

Figure 6.

Preservation of clonal and subclonal mutations at the time of relapse. Tracing of major clone (top) and subclonal (bottom) alterations detected in initial diagnosis samples from relapsed patients in the matched relapse samples. The pie charts depict the fractions of preserved (blue) and lost (orange) alterations at the time of relapse.