Page 101 - 2019_06-Haematologica-web

Cellular origin and clinical prognostic markers of infant MLLr B-ALL
provides a detailed analysis of B-cell population dynamics and clone tracking.42,43 BCR sequencing was therefore per- formed to address whether t(4;11)+ iBCP-ALL cells expressed fully rearranged BCR from which increased lev- els of B-cell clonal expansion may be observed and to determine whether there are detectable levels of B-cell clonal persistence over time indicative of B-cell clonal sur- vival. BCR sequencing was performed on t(4;11)/MLL- AF4+ iBCP-ALL peripheral blood samples (blasts >98%) using a polymerase chain reaction-based method37 with additional incorporation of unique molecular barcodes, allowing for accurate quantitation of relative B-cell clone frequency. After BCR sequence filtering, each sample yielded between 1,583-46,863 BCR (1,213-38,426 unique BCR) (Online Supplementary Table S5).
We first delineated the relative clonality in these patients, and found that the BCR repertoires from t(4;11)+ patients did not exhibit significantly expanded VDJ- rearranged B-cell clones (Figure 6A) either at diagnosis or relapse compared to healthy peripheral blood samples
(Figure 6B). This is in contrast to non-MLL BCP-ALL patients (n=5) including three patients with t(1;19)/TCF3- PBX1 (EF2-PBX1), one with t(12;21)/ETV6-RUNX1 (TEL- AML1) and one with t(9;22)/BCR-ABL1, which were all found to be significantly clonal, with large B-cell clones comprising ~3-40% of total BCR (Figure 6B,C).37 Given the persistence of both t(4;11) and RAS mutations in MLL- AF4+ iBCP-ALL, the lack of B-cell clonal expansion or per- sistence supports the model that t(4;11)/MLL-AF4+ iBCP- ALL malignant cells are developmentally stalled at the pro- B stage, and that the cellular origin of such genomic driv- ers has to be pre-VDJ stem/progenitor cells.
Finally, in order to understand whether the fetal cell-of- origin in iBCP-ALL lies upstream of committed B progen- itors, we compared the transcriptome of iBCP-ALL blasts (n=42) with that of highly purified human FL HSPC pop- ulations (3-7 for each population) (Figure 7A,B and Online Supplementary Table S6) by RNA-sequencing. In keeping with the results of the BCR analysis, our principal compo- nent analysis revealed a gene expression signature for
Figure 5. Specific transcriptional differences between MLL-AF4+ and MLL-AF9+ or MLLwt infant B- cell precursor acute lymphoblas- tic leukemia patients. Here, FL- derived CD34+CD19+ progenitors were not included as normalizers in the analysis in order to avoid potential bias. Gene set enrich- ment analysis (GSEA) was per- formed with the genes differen- tially expressed between MLL- AF4+ patients and MLL-AF9+ or MLLwt patients. MLL-AF4+ infant B-cell precursor acute lym- phoblastic leukemia (iBCP-ALL) patients showed a significant overexpression of genes associ- ated with cellular catabolism, coupled to a significant downreg- ulation of negative regulators of the PI3-MAPK pathway, as well as of genes involved in lymphoid differentiation and RNApol II transcriptional regulation as compared to both MLL-AF9+ and MLLwt iBCP-ALL patients. The bottom panels represent positive pathway enrichment called by
GSEA software. Total patients: 27 t(4;11)+, 5 t(9;11)+ and 10 MLLwt.
42
haematologica | 2019; 104(6)
1183