Relative expansion of CD19‐negative very‐early normal B‐cell precursors in children with acute lymphoblastic leukaemia after CD19 targeting by blinatumomab and CAR‐T cell therapy: implications for flow cytometric detection of minimal residual disease

Abstract

CD19-directed treatment in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) frequently leads to the downmodulation of targeted antigens. As multicolour flow cytometry (MFC) application for minimal/measurable residual disease (MRD) assessment in BCP-ALL is based on B-cell compartment study, CD19 loss could hamper MFC-MRD monitoring after blinatumomab or chimeric antigen receptor T-cell (CAR-T) therapy. The use of other antigens (CD22, CD10, CD79a, etc.) as B-lineage gating markers allows the identification of CD19-negative leukaemia, but it could also lead to misidentification of normal very-early CD19-negative BCPs as tumour blasts. In the current study, we summarized the results of the investigation of CD19-negative normal BCPs in 106 children with BCP-ALL who underwent CD19 targeting (blinatumomab, n = 64; CAR-T, n = 25; or both, n = 17). It was found that normal CD19-negative BCPs could be found in bone marrow after CD19-directed treatment more frequently than in healthy donors and children with BCP-ALL during chemotherapy or after stem cell transplantation. Analysis of the antigen expression profile revealed that normal CD19-negative BCPs could be mixed up with residual leukaemic blasts, even in bioinformatic analyses of MFC data. The results of our study should help to investigate MFC-MRD more accurately in patients who have undergone CD19-targeted therapy, even in cases with normal CD19-negative BCP expansion.

A Mathematical Description of the Bone Marrow Dynamics during CAR T-Cell Therapy in B-Cell Childhood Acute Lymphoblastic Leukemia

“…The model predicted relative proportions of B-cell subsets and CAR T subsets directly attainable from the flow cytometry data of long-term remission patients. In particular, the reorganization of the B-cell subsets, with a predominant CD19 − compartment, has recently been reported in patients who respond to anti-CD19 therapy [54]. This otherwise unnoticeable precursor could reach up to 100% of all the B-cell precursors, as shown here.…”

Strong expansion of normal CD19‐negative B‐cell precursors after the use of blinatumomab in the first‐line therapy of acute lymphoblastic leukaemia in children

“…On the other hand, the use of other early B‐line antigens [CD22, CD24, CD10, intracellular (i) CD79a] instead of CD19 for primary B‐cell gating, such as to improve the MFC‐MRD technique in the case of CD19‐targeted treatment, 7,10,11 could lead to errors in the interpretation of the immunophenotype of normal haematopoietic cells. Recently we have shown that after CD19 targeting in children with R/R BCP‐ALL by either blinatumomab or chimaeric antigen receptor (CAR)‐T cells, a relative expansion of normal very early CD19‐negative BCPs can be observed 12 . Due to the specific immunophenotype, which is very different from that of mature CD19‐positive BCPs, these normal cells could be misinterpreted during the MFC‐MRD assessment as developing a CD19‐negative relapse 12 .…”

“…Recently we have shown that after CD19 targeting in children with R/R BCP‐ALL by either blinatumomab or chimaeric antigen receptor (CAR)‐T cells, a relative expansion of normal very early CD19‐negative BCPs can be observed 12 . Due to the specific immunophenotype, which is very different from that of mature CD19‐positive BCPs, these normal cells could be misinterpreted during the MFC‐MRD assessment as developing a CD19‐negative relapse 12 . MRD studies are central to evaluating the effectiveness of targeted treatment 5,13 .…”

“…MRD studies are central to evaluating the effectiveness of targeted treatment 5,13 . Therefore, the possible detection of such CD19‐negative normal precursors in the majority (almost 60–70%) of children after CD19‐directed immunotherapy should be viewed as one of the cornerstones of MFC‐MRD monitoring after blinatumomab therapy 12 . At the same time, the inclusion of blinatumomab in treatment protocols for primary ALL requires additional assessment of the normal antigen expression pattern of bone marrow (BM) cells.…”

“…The MRD‐targeted combinations contained fluorochrome‐conjugated antibodies against CD45, CD19, CD10, CD38, CD34, CD58, CD20, CD22, CD24 and iCD79a (Table SII). CD19‐negative B‐cell precursors were gated as described previously 12 …”