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Prognostic relevance of subclonal mutations in ALL
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.
pediatric ALL. This finding is particularly relevant for IKZF1 alterations, which are currently used or implement- ed for treatment stratification in multiple upfront treat- ment protocols.14,20
The selection of these genes was made based on enriched mutation frequencies in relapse found in previ- ous studies. Of all alterations identified in this study, 75% were subclonal at diagnosis, suggesting that these relapse- associated gene mutations accumulate during progression of the leukemia before the initial diagnosis, thereby increasing the clonal complexity. Whereas seven of the genes selected in our study showed this high mutational burden at diagnosis, both in terms of numbers and level of clonality, we identified only a single, not previously reported, subclonal NT5C2 mutation in a non-relapsed case (follow-up time 9.5 years). NT5C2 encodes the cytosolic nucleotidase, which is responsible for inactivat- ing cytotoxic thiopurine monophosphate nucleotides, and activating mutations in this gene are recurrently found in relapsed ALL, mainly T-cell ALL.4,9,36-38 One explanation for the low number of activating NT5C2 mutations at diagnosis is that these mutations decrease cell fitness, and only obtain their selective advantage during treatment with thiopurine.36 If already present at the time of initial diagnosis, these mutations are usually detectable in only a very small subset of cells, far below the detection level of our smMIP analysis.36
Hotspot RAS pathway mutations have been detected in nearly half of the cases, often of the hyperdiploid subtype, and their frequency and clonal burden varied between the different mutations. In our study, we used this variability to compare the potential of different hotspot mutations to drive clonal expansion under physiological conditions. Compared to diagnosis, we observed a less diverse spec- trum of KRAS and NRAS hotspot mutations in relapse, with G12D, G12V and G13D together accounting for two-thirds of KRAS and NRAS hotspot mutations found in relapse-fated clones. Studies in other cancers have demonstrated that the prevalence of different RAS path- way mutations varies depending on the type of cancer and tissue of origin, with KRAS mutations G12D, G12V, G13D and G12C being among the most common ones.39,40 Comparison of oncogenic capacities of different RAS
hotspots has also been performed using in vitro and in vivo modeling studies, focusing primarily on KRAS. These studies identified KRAS mutations G12D, G12V and G13D as having higher proliferative and transforming potential compared to other common hotspots in various tumors of epithelial origin.39,41,42 Our data indicate that in competition of multiple RAS hotspot mutations, some of these not only confer a proliferative advantage but can also more effectively sustain a treatment-induced selective sweep.4,10
The presence of IKZF1 deletions has been shown to be associated with relapse and survival in multiple clinical ALL studies,12,14-19 and these deletions have been described to play a role in resistance to tyrosine kinase inhibitors and glucocorticoids.43-46 Therefore, with the advance of more sensitive detection techniques, the question of whether subclonal alterations are also associated with relapse is very relevant, both from biological and from clinical perspectives. We here demonstrate that, in con- trast to major clone IKZF1 exon 4-7 deletions, cases that carry this deletion only in a subset of the cells do not show an association with relapse. Moreover, whereas all major clone exon 4-7 deletions were preserved in cases that relapsed, none of the relapses from cases with subclonal exon 4-7 deletions at diagnosis carried this deletion. Importantly, the majority of subclonal deletions had allele frequencies below 10% (Online Supplementary Table S7). Therefore, since a threshold to distinguish subclonal from major clonal deletions is difficult, deletions closer to our threshold of 25% should be evaluated with caution. Nevertheless, the difference between major and minor clone IKZF1 4-7 deletions is striking, and the reason behind this remains unclear. Possibly, the functional impact of full-clonal IKZF1 deletions, which arise early during leukemia development, is different from that of deletions that occur in later stages when the leukemia has already expanded. Other deletions in IKZF1 show much less clustering in their breakpoints and, therefore, screen- ing for these subclonal deletions in diagnostic samples is much less efficient. We did not, therefore, directly assess the stability and potential prognostic importance of whole gene and rare intragenic IKZF1 deletions. However, a pre- vious study showed that other IKZF1 deletion subtypes
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