The recent active use of immunotherapy based on CD19/CD3 bispecific T-cell engager (blinatumomab), brings significant problems on monitoring Minimal Residual Disease (MRD) based on Multiparameter Flow Cytometry (MFC), due to possible CD19 downregulation by tumor cells. This phenomenon breaks the well-established approach of MFC strategy analysis, based gating on CD19 positivity. Also, the selective pressure of the CD19-directed immunotherapy increases the incidence of the so-called lineage switch, which has become one of the possible mechanisms of BCP-ALL resistance to immunotherapy. At present, there is no consensus approach to MFC-MRD monitoring after blinatumomab therapy for BCP-ALL. There are several described changes in MRD methodology, including the addition of other early B-lineage antigens (e.g., CD22, CD24 and CD79a) or only the modification of the gating strategy using conventional sets of antibodies.

Evaluated two different antibody panels to monitor MFC-MRD in patients Relapsed/Refractory (R/R) B-cell ALL after blinatumomab therapy.

All Bone Marrow (BM) samples the MRD detection was performed with panel EuroFlow BCPALL MRD protocol standardized and added two more tubes (8 colors) with early B-lineage antigens according to previously described in literature (e.g., Tube 1: CD66b/CD22/CD34/CD19/CD24/CD38/CD10/CD45; Tube 2: CD20/CD22/CD34/CD19/CD79a/CD38/CD10/CD45). All BM samples were studied following the EuroFlow SOPs with the acquisition at least ≥5×106 events per samples and in both panels. The cytometer FACSCanto 8-color and Infinicyt software was used for acquisition and analyzed data files. The study enrolled real-world children in public health care systems and was approved by the local ethics committees.

A total of 54 BM samples obtained from 11 children -48% M and 52% F with a median age of 10-years (0‒19) with R/R B-cell ALL were studied at different time points after treatment with blinatumomab, including: pré-transplant (n = 24) and post-transplant (n = 30). As controls without use of blinatumomab were include 04 BM from children with B-cell ALL at D15 of treatment with standard chemotherapy protocol. MRD were detected in 35% (19/54) of BM studied and of these 2/11 relapsed. We found 100% agreement between the two panels used. Only one sample was discordant at first time, but upon review it was noted that this CD19 negative population were very early CD19- normal B cell progenitors.

The wide implementation of CD19-directed therapy in the treatment in protocol for R/R BCP-ALL significantly complicated the rather routine procedure of MRD monitoring. Despite the subjective impressions by these investigators that other B-cell antigens such as CD22, CD24 and cyCD79a brings more confidence to analyze B-cell ALL MRD samples from patients treated with anti-CD19 therapy, for this studied cohort, we did not find difference on the results of MRD with the standard EuroFlow panel and the alternative panel.

No difference was found on the MRD detection with the standard EUFOLOW panel and the alternative panel. Although introduction of CD22, and CD24 markers, with 8 colors strategy, it was not advantageous. Using 8 colors strategy, it may be recommended to investigate the comparative financial impact of the EuroFlow, and the alternative panel in the context of Blinatumomab therapy.