PMC9760757_01
Immunophenotyping analysis of the bone marrow (BM) aspirate (t0, t1, and t2) and peripheral blood (PB) (t3 and t4) carried out at diagnosis (t0 - panel A), at first evaluation after cladribine therapy (t1 - panel B), and after rituximab treatment (t2 - panel C, t3 - panel D, and t4 - panel E).Any debris, dead cells, and clumps or doublets were excluded using forward scatter (FSC)-height (FSC-H) by FSC-area (FSC-A) parameters (black gate, "Not Doublets"). CD45+ lymphocytes (red gate) were gated on CD45 vs. the side scatter (SSC) dot plot on the Not Doublets gate. Hairy cell leukemia (HCL), monoclonal B-cell lymphocytosis (MBL)/B-cell chronic lymphocytic leukemia (B-CLL), and P3 populations (fuchsia, blue, and green gates, respectively) were identified from the lymphocyte gate. The expression of some specific markers (CD19, CD20, and CD25) and the physical parameter of SSCs were exploited to identify the three clonal populations (see the dot plots marked with *). At t0 (A), the HCL and MBL/B-CLL clones were identified by the different expression of CD19 vs. SSC. At t1 (B), t3 (D), and t4 (E), the three B-cell clones were identified by coexpression at the variable intensity of the CD19 and CD20 markers. At t2 (C), following treatment with rituximab, the gating strategy was based on the different intensities of the expression of CD19 vs. CD25 and/or CD19 vs. SSC.
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Table 2 ,  Figure 1A ). Table 1 ). The immunophenotyping of BM aspirate revealed an HCL population reduction to 0.9%, while the CLL population was 16% among WBCs ( Table 2 ,  Figure 1B ). Table 2 ,  Figure 1C ). The BRAF V600E mutation was still detectable ( Table 2 ). At +8 months (t3), the flow cytometric examination of the PB sample again evidenced the presence of both HCL and MBL populations, both positive for CD20 ( Table 2 ,  Figure 1D ). At the last follow-up (+27 months: t4), the percentage of the BRAF V600E mutation found on the PB was 0.65% and the immunophenotyping analysis of the PB confirmed the presence of both HCL and CLL populations ( Table 2 ,  Figure 1E ). Table 2 ,  Figure 1E ). The P3 population showed an MBL/B-CLL phenotype with a characteristic intensity of CD20 and CD19 markers that was used for the gating strategy. In particular, P3 expressed CD19+intermediate, CD20+intermediate, CD23+, CD5+high, CD25-, CD11c-, CD43+, CD103-, CD79b+low, FMC7+low, sIgk-, and sIglambda+intermediate immunophenotype ( Figure 1E ). P3 analyzing the previous flow cytometric files.  Figure 1  shows the gating strategy and the immunophenotype of the three populations at different time points. P3 was absent at diagnosis (t0) and appeared in post-therapy samples ( Figure 1 ). In particular, we found a P3 clone (0.94% of WBCs) in t1 at first evaluation after cladribrine treatment ( Figure 1B ,  Table 2 ). Figure 1B ). For this reason, we hypothesized that the P3 population may have originated from the HCL clone. Figure 1C ). P3 was also identified in t3 (8 months after the end of rituximab treatment) representing 0.23% of WBCs and showing the same immunophenotype of t1 ( Table 2 ,  Figure 1D ). Finally, we established to monitor the patient with a watch-and-wait approach until disease progression (HCL or CLL diseases).