DUX4r, ZNF384r and PAX5-P80R mutated B-cell precursor acute lymphoblastic leukemia frequently undergo monocytic switch

Abstract

Recently, we described B-cell precursor acute lymphoblastic leukemia (BCP-ALL) subtype with early switch to the monocytic lineage and loss of the B-cell immunophenotype, including CD19 expression. Thus far, the genetic background has remained unknown. Among 726 children consecutively diagnosed with BCP-ALL, 8% patients experienced switch detectable by flow cytometry (FC). Using exome and RNA sequencing, switch was found to positively correlate with three different genetic subtypes: PAX5-P80R mutation (5 cases with switch out of 5), rearranged DUX4 (DUX4r; 30 cases of 41) and rearranged ZNF384 (ZNF384r; 4 cases of 10). Expression profiles or phenotypic patterns correlated with genotypes, but within each genotype they could not identify cases who subsequently switched. If switching was not taken into account, the B-cell-oriented FC assessment underestimated the minimal residual disease level. For patients with PAX5-P80R, a discordance between FC-determined and PCR-determined MRD was found on day 15, resulting from a rapid loss of the B-cell phenotype. Discordance on day 33 was observed in all the DUX4r, PAX5-P80R and ZNF384r subtypes. Importantly, despite the substantial phenotypic changes, possibly even challenging the appropriateness of BCP-ALL therapy, the monocytic switch was not associated with a higher incidence of relapse and poorer prognosis in patients undergoing standard ALL treatment.

Figure 1.

Monocytic switch appearance. In cytometric plots, mononuclear cells after exclusion of doublets and nonmalignant T cells and B cells (if available) are shown. Green, orange and red rectangles highlight the preswitched B-precursor blasts, B-monocytoid intermediate cells and fully switched monocytoids, respectively. Examples of patients with the respective genotypes are shown at day zero (d0) (bone marrow [BM]), d+8 (peripheral blood [PB]) and d+15 (BM). The percentage of each population is shown (of all nuclear cells) in the corresponding color. Polymerase chain reaction (PCR)-determined minimal residual disease (MRD) values in percentages are shown in black. We observed DUX4r monocytic switch pattern in 43 of 45 patients, PAX5-P80R pattern in seven of seven patients and ZNF384r pattern in three of five patients within the respective genotypes in all patients analyzed (n=745).

Figure 2.

Multidimensional analysis of RNA sequencing and flow cytometry data. (A) Overview of the gene expression found for all patient (n=197) using UMAP as the dimensionality reduction algorithm for RNA sequencing data. Only the genes with the most variability (genes with an SD 0.4-fold higher than the maximum SD, n=271) were used. Patients with switch are shown as full circles, and patients without switching are shown as empty circles. Relevant genetic subtypes are shown in color. (B) Overview of immunophenotypes of all cases (n=745) using UMAP as dimensionality reduction algorithm. Blasts were gated using common backbone (forward scatter, side scatter, CD45). The data (expression of markers on blast population in percentage) were prepared from multiple tubes and merged together. Missing values were replaced with marker median values. Open circles represent cases without monocytic switch, full circles represent cases which developed switching. Relevant genetic subtypes are shown in color.

Figure 3.

Correlation of the mimal reidual disease results obtained by flow cytometry and polymerase chain reaction in selected patient groups. Only samples with appropriate measured sensitivity are shown (the flow cytometry [FC] sensitivity is 0.0001 if the polymerase chain reaction [PCR]-determined minimal residual disease [MRD] <0.01; for samples with PCR-determined MRD ≥0.01, the FC measurement sensitivity is at least one log value lower than the actual PCR-determined MRD log value). In the upper part of each graph, the number of patients with MRD values FC^pos/PCR^neg, FC^pos/PCR^{nq pos}, and FC^pos/PCR^pos (upper lane); and FC^neg/PCR^neg, FC^neg/PCR^{nq pos}, and FC^neg/PCR^pos (bottom lane) are indicated. Spearman's rank correlation coefficient indicated if the P-value was <0.05. Nq pos: nonquantifiable positivepositive; BM: bone marrow.

Figure 4.

Event-free survival of B-cell precursor acute lymphoblastic leukemia patients with and without switch. The prospective cohort (n=725) is shown. One patient was lost to follow-up.