CD19 Negative Relapse in B-ALL Treated with Blinatumomab Therapy: Avoiding the Trap

Acute lymphoblastic leukaemia of the B lineage (B-ALL) is an aggressive neoplasm of B lymphocyte precursors that expresses the pan B cell marker CD19 in the majority of cases. For this reason CD19 has played a pivotal role as a 'gating' antigen in the analysis of leukaemic cells by flow cytometry, both at diagnosis and during minimal residual disease (MRD) monitoring.

In recent years, novel therapeutic strategies have been developed for the treatment of B-ALL. Blinatumomab, a bispecific T cell engager (BiTE®) antibody and chimeric antigen receptor-modified T cells (CARTs) are treatments with targeted anti-CD19 activity that have shown promising efficacy in clinical trials.

Despite the burgeoning promise of targeted anti-CD19 strategies a concerning number of CD19 negative relapses have been reported in this setting - this argues strongly for down regulation of CD19 by the leukaemic clone or selection of pre-existing CD19 negative clones as mechanisms of therapy resistance.

The trials published to date have used PCR-based methods for MRD monitoring which has allowed for early detection of molecular relapse, with CD19 status being assessed by flow cytometry after morphological relapse.

Outside of the trial setting flow cytometry based MRD monitoring is regularly used. This poses a major practical issue for institutions that treat B-ALL patients with targeted anti-CD19 therapies as conventional CD19 gating based B-ALL MRD panels are insensitive to the presence of CD19 negative leukaemic cells.

We present the case of a 69 year old male with relapsed CD19 positive B-ALL who attained complete immunophenotypic remission after one cycle of single agent blinatumomab. He then relapsed with CD19 negative disease as detected by an alternative gating strategy within the Clinical Oncology Group-based antibody panel used routinely for B-ALL assessment at our institution.

B-ALL MRD analysis by flow cytometry at our institution involves the collection of whole bone marrow in sodium heparin, erythrocyte lysis and analysis on a Beckman Coulter Navios^TM flow cytometer within 24 hours using the following single tube antibody cocktail: CD45-KO, CD20-PacBlue, CD38-APC750, CD19-APC700, CD58-APC, CD13+33-PC7, CD56-PC5.5, CD34-ECD, CD10-PE and CD9-FITC. Kaluza® analysis software is used to define viable cells by excluding doublets, erythrocytes and cellular debris followed by the gating of CD19+ cells, excluding plasma cells and comparing the expression of other antigens to normal B-lineage maturation patterns. B-ALL is defined as a cluster of viable CD19+ cells that do not conform to normal B-lineage maturation patterns in multiple dimensions, typically: CD20vsCD10, CD38vsCD34, CD45vsCD34, CD45vsCD10, CD45vsCD38+/- aberrant CD9, CD13+33 or CD58 expression.

Following therapy in this case and in the absence of detectable CD19+ cells, gating was based on first identifying known major normal bone marrow cell populations using known antigen expression and location on the CD45/side scatter(SSC) plot: mature lymphocytes (CD45-high/SSC-low), granulocytes (CD10+/SSC-high), plasma cells (CD38++) and myeloblasts (CD34+/CD45+/13+33+/SSC-medium). In this case B-ALL cells were identified as CD34+/SSC-low, residing outside of expected regions for normal CD34+ B-lymphoid precursors in dimensions not dependant on CD19. This finding was followed by morphological relapse within 3 months.

This case highlights the potential limitations of CD19 gating for B-ALL MRD assessment by flow cytometry in patients treated with targeted anti-CD19 therapies. It demonstrates an alternative algorithm that may assist diagnostic laboratories with limited access to PCR-based methodologies by using an already widely available antibody panel. It is acknowledged that in this case the retained expression of CD34 by the leukaemic B-lymphoblasts was pivotal in tracking the leukaemia after therapy. In order to address the possibility of CD34/CD19 dual negative disease, the use of alternative B-lineage gating and/or maturation antigens such as CD22 and CD81 is being evaluated.

Awareness of this important issue and the need for an adapted approach to gating in this clinical setting will become increasingly important as targeted therapies become more widely adopted for the treatment of B-ALL.