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A. Agraz-Doblas et al.
primitive Lin-CD34+CD38-CD19- FL HSPC (hematopoietic stem cells, multipotent progenitors, and lymphoid-primed multipotent progenitors, which lie upstream of B progen- itors) very similar to t(4;11)+ iBCP-ALL, while FL-commit- ted B progenitors clustered as a transcriptionally different entity (Figure 7C).
Discussion
We set out to perform multi-layered sequencing on a large cohort iBCP-ALL patients, all enrolled in the interna- tional, collaborative Interfant treatment protocol. The fact that all patients were identically treated provides legitima- cy and confidence in potential correlations of clinical value. Our study revealed an average of 2.5 non-silent sin- gle nucleotide variants, a 2-fold higher number than that reported by Andersson et al.,20 likely reflecting the 3-fold larger sequencing coverage. This silent mutational land- scape, even in non-MLL iBCP-ALL, likely reflects the very young age of these patients, reinforcing the notion that infant cancer is a developmental disease with not enough time to develop somatic mutations. We also found the only recurrent, but subclonal, mutations occur in the KRAS and NRAS genes (gain-of-function mutations), although the frequency of subclonal NRAS mutations is
significantly higher in t(4;11)+ patients. In line with our previous work we found no recurrent mutations in the FLT3 gene.40
Analysis of clonal evolution of RAS-mutated clones from diagnosis to relapse revealed that one-third of the patients still carry RAS mutations at relapse, whereas the other two-thirds of patients who relapse have lost the diagnostic RAS mutation. This is in accordance with recently published data by Trentin et al.,36 and suggests that the therapy is able to eliminate the RAS-mutated clone in some patients, while in other patients the RAS mutation seems to confer chemoresistance, allowing these clones to evade treatment.44 Intriguingly, ~25% of the patients carry more than one RAS-mutated clone at diag- nosis, indicating a selection bias towards mutations in the RAS genes, or activated RAS pathways during leukemic transformation. From this perspective, the occurrence of patients carrying multiple distinct clones with activated RAS pathways may point to convergent evolution of clones capable of controlling the proliferation rate. However, arguing against this is the substantial represen- tation of patients not carrying RAS mutations at all. Hence, the role of RAS mutations in t(4;11)+ iBCP-ALL remains obscure, and the available data suggest that RAS pathway mutations are unlikely leukemia-initiating lesions. Indeed, Tamai et al.45 showed that leukemogenesis
Figure 6. Analysis of B-cell receptor repertoires suggest a hematopoietic stem cell/early pre-VDJ progenitor as the cell- of-origin for t(4;11)/MLL-AF4+ infant B-cell precursor acute lymphoblastic leukemia. (A) Cloud-plots of B-cell receptor (BCR) repertoires from two rep- resentative t(4;11)+ infant B-cell precursor acute lymphoblastic leukemia (iBCP-ALL) patients depicting the existence of many minor non-expanded B-cell clones either at diagnosis or relapse. Each vertex represents a unique BCR sequence, and the relative vertex size is propor- tional to the number of identical reads. (B) Largest BCR clone size in t(4;11)+ iBCP-ALL, healthy individuals and non- t(4;11)+ pediatric BCP-ALL. (C) Cloud-plots of BCR repertoires of representative t(1;19)/E2A- PBX1+, t(12;21)/TEL-AML1+ and t(9;22)/BCR-ABL+ patients showing high clonality of B-cell clones. The samples from the iBCP-ALL patients who were BCR-sequenced were four MLL- AF4+ diagnostic-relapse pairs, three E2A-PBX1+ samples, one TEL-AML1+ sample and one BCR-ABL+ sample.
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haematologica | 2019; 104(6)