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Ž. Antić et al.
Figure 1. Schematic representation of the study design. Single-molecule molecular inversion probe-based sequencing approach and real-time quantitative poly- merase 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.
tions previously identified using a standard MLPA method, as well as 28 additional cases carrying deletions that were missed with the MLPA technique. All break- points were sequenced to determine their unique break- point-spanning sequences (Online Supplementary Table S6). Using a dilution series of a control sample with a full-clon- al IKZF1 exon 4-7 deletion, we determined the level of clonality of the deletions, which ranged from 100% down to 0.32% (Figures 1 and 2, Online Supplementary Figure S1C). All but one of the subclonal IKZF1 exon 4-7 dele- tions had allele frequencies below 10% (Online Supplementary Table S7).
Subclonal alterations in relapse-associated genes are common at diagnosis
Combining sequence mutations and IKZF1 exon 4-7 deletions, we detected 660 genomic alterations in 285 diagnostic samples, of which 165 (25%) were present in the major fraction of cells (allele frequency ≥25%), which were referred to as high-clonal. The remaining 495 muta- tions (75%), most of which had an allele frequency <10% were referred to as subclonal (Online Supplementary Figure S2, Online Supplementary Table S7). A total of 147/285 patients carried at least one alteration in a major clone, while 138/285 (48%) patients carried exclusively subclon- al alterations. NRAS and KRAS were the most frequently affected genes, showing major clone mutations in 6% and 8% of the cases and subclonal mutations in 20% and 15% of the cases, respectively (Figure 2A, B).
The proportion of subclonal alterations, relative to major clone alterations, was variable among different genes, ranging from 59% for IKZF1 exon 4-7 deletions to 86% for PTPN11 mutations (Figure 2B). Only one thus far unknown (subclonal) NT5C2 mutation (p.Arg507Trp) was identified in a leukemia sample from a patient who did not relapse (Figure 2A, B). Subclonal mutations were rela- tively common in hyperdiploid ALL (184 cases), particu- larly for mutations in RAS pathway genes (190/256; 74%), WHSC1 (22/26; 85%) and CREBBP (13/27; 48%) (Online Supplementary Table S8). Major clone WHSC1 mutations
were mostly identified in ETV6-RUNX1-positive cases (4/10, 40%).
Potency of RAS pathway genes as drivers of clonal expansion
We identified 473 RAS pathway mutations in 225/503 (45%) cases, of which 78% were subclonal (median allele frequency = 3.5%). Over half of the RAS-affected cases were hyperdiploid (>47 chromosomes), in line with previ- ous studies indicating that RAS mutations are associated with hyperdiploidy at diagnosis.10,33 The abundance of these mutations in major and minor clones suggests that these mutations drive clonal expansion during the devel- opment of leukemia. Major clone RAS pathway mutations (n=102; all being known hotspots) were found to be mutu- ally exclusive, and 52/102 (51%) of these RAS-mutated cases had at least one additional subclonal mutation in one of the three RAS pathway genes. The mutations mostly affected codons 12 and 13 of KRAS and NRAS (Figure 3A- C), and considerable variability in the level of clonality was observed between the different RAS hotspot muta- tions at the time of diagnosis. For example, NRAS G12A (10 cases), NRAS G12V (7 cases), and PTPN11 E76K (7 cases) were never found to be present in a major clone, whereas 55% (n=11) of the KRAS G13D and 27% (n=9) of the KRAS G12D mutations were found in major clones. With these high numbers of RAS mutations, the variabili- ty in clonal burden between hotspot mutations may pro- vide an opportunity to compare the capacity of different hotspots to drive clonal expansion of ALL. In order to test this hypothesis we compared allele frequencies and per- formed statistical analyses. We found that KRAS hotspot mutations had a significantly higher allele frequency com- pared to both NRAS and PTPN11 mutations (Wilcoxon signed-rank test, P<0.01) (Figure 3D). When comparing the different hotspot mutations within KRAS, A146V showed the lowest allele frequency, indicating a weaker potential of this hotspot to drive clonal expansion com- pared to the other KRAS hotspots. Furthermore, the allele frequency of KRAS G13D was significantly higher than
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haematologica | 2021; 106(12)