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 NaviosTM 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. Disclosures No relevant conflicts of interest to declare.

D3: A Gene Induced During Myeloid Cell Differentiation of Linlo c-Kit+ Sca-1+ Progenitor Cells

In an effort to characterize molecular events contributing to lineage commitment and terminal differentiation of stem/progenitor cells, we have used differential display reverse transcription polymerase chain reaction (DDRT-PCR) and cell lines blocked at two distinct stages of differentiation. The cell lines used were EML, which is representative of normal multipotential primitive progenitors (Sca-1+, CD34+, c-Kit+, Thy-1+) able to differentiate into erythroid, myeloid, and B-lymphoid cells in vitro, and MPRO, which is a more committed progenitor cell line, with characteristics of promyelocytes able to differentiate into granulocytes. One clone isolated by this approach was expressed in MPRO but not in EML cells and contained sequence identical to the 3′ untranslated region of D3, a gene cloned from activated peritoneal macrophages of unknown function. We have observed a novel pattern of D3 gene expression and found that D3 is induced in EML cells under conditions that promote myeloid cell differentiation (interleukin-3 [IL-3], stem cell factor [SCF], and all-trans-retinoic acid [atRA]) starting at 2 days, corresponding to the appearance of promyelocytes. D3 RNA expression reached a maximum after 5 days, corresponding to the appearance of neutrophilic granulocytes and macrophages, and decreased by day 6 with increased numbers of differentiated neutrophils and macrophages in vitro. Induction of D3 RNA in EML was dependent on IL-3 and was not induced in response to SCF or atRA alone or SCF in combination with 15 other hematopoietic growth factors (HGF) tested. Similarly, D3 was not expressed in the normal bone marrow cell (BMC) counterpart of EML cells, Linlo c-Kit+Sca-1+ progenitor cells. D3 RNA expression was induced in these cells when cultured for 7 days in IL-3 plus SCF. A comparison of the expression of D3 RNA in cell lines and normal BMC populations demonstrated that D3 is induced during macrophage and granulocyte differentiation and suggests a potential physiological role for D3 in normal myeloid differentiation.

D3: A Gene Induced During Myeloid Cell Differentiation of Linlo c-Kit+ Sca-1+ Progenitor Cells

In an effort to characterize molecular events contributing to lineage commitment and terminal differentiation of stem/progenitor cells, we have used differential display reverse transcription polymerase chain reaction (DDRT-PCR) and cell lines blocked at two distinct stages of differentiation. The cell lines used were EML, which is representative of normal multipotential primitive progenitors (Sca-1+, CD34+, c-Kit+, Thy-1+) able to differentiate into erythroid, myeloid, and B-lymphoid cells in vitro, and MPRO, which is a more committed progenitor cell line, with characteristics of promyelocytes able to differentiate into granulocytes. One clone isolated by this approach was expressed in MPRO but not in EML cells and contained sequence identical to the 3′ untranslated region of D3, a gene cloned from activated peritoneal macrophages of unknown function. We have observed a novel pattern of D3 gene expression and found that D3 is induced in EML cells under conditions that promote myeloid cell differentiation (interleukin-3 [IL-3], stem cell factor [SCF], and all-trans-retinoic acid [atRA]) starting at 2 days, corresponding to the appearance of promyelocytes. D3 RNA expression reached a maximum after 5 days, corresponding to the appearance of neutrophilic granulocytes and macrophages, and decreased by day 6 with increased numbers of differentiated neutrophils and macrophages in vitro. Induction of D3 RNA in EML was dependent on IL-3 and was not induced in response to SCF or atRA alone or SCF in combination with 15 other hematopoietic growth factors (HGF) tested. Similarly, D3 was not expressed in the normal bone marrow cell (BMC) counterpart of EML cells, Linlo c-Kit+Sca-1+ progenitor cells. D3 RNA expression was induced in these cells when cultured for 7 days in IL-3 plus SCF. A comparison of the expression of D3 RNA in cell lines and normal BMC populations demonstrated that D3 is induced during macrophage and granulocyte differentiation and suggests a potential physiological role for D3 in normal myeloid differentiation.

Thymic Medulla Epithelial Cells Acquire Specific Markers by Post-Mitotic Maturation

The development of thymocyte subsets and of the thymic epithelium in SCID and RAG-2-/– mice was monitored after normal bone-marrow-cell transfer. The kinetics of thymic reconstitution and their relationships with cell proliferation were investigated by using bromodeoxyuridine to detect DNA-synthesizing cells among lymphoid cells by 3-color flow cytometry, and in epithelial compartments by staining frozen sections. Thymocytes started to express CD8 and CD4 10 days after transfer, simultaneously with extensive proliferation. The first mature CD4+single-positive cells were generated, from resting CD4+CD8+cells after day 15. During this day 10–15 period, many epithelial cells positive for cortexspecific or panepithelial markers were labeled with BrdUrd after pulse-injection. Organized medullary epithelium also developed after day,15, that is, synchronously with the appearance of mature thymocytes, but medullary cells were never found BrdUrd+. These results suggest that, in these models, the reconstitution of the thymic epithelial network proceeds through expansion of preexisting cortical or undifferentiated cells and by later maturation (acquisition of specific markers) of medullary cells. This last process is dependent of the presence of mature thymocytes.

A rabbit bone marrow model system for evaluation of cytotoxicity: characterization of normal bone marrow cell cycle parameters by flow cytometry.

Characterization of a rabbit model system for the study of cell cycle effects of myelotoxic agents in normal bone marrow is described. Cell cycle phase distributions are obtained by computer analysis of flow cytometric single cell DNA histograms. Comparison of marrow aspirates with marrow samples from sacrificed animals indicates that dilution of aspirates with peripheral blood is not significant. Aspiration of marrow from one bone does not affect the cell cycle distribution of unsampled bones. Hence, sequential aspirates of different bones in a single animal may be used as representative samples for further study of effects of myelotoxins on marrow proliferation and differentiation.

Diagnosis of Neonatal Bacterial Infection: Hematologic and Pathologic Findings in Fatal and Nonfatal Cases

Consecutive newborn autopsy cases were divided into infected and noninfected groups on the basis of pathologic findings and cultures, and were compared to a concomitant consecutive group of neonatal survivors with proven bacterial sepsis. Newborns dying with bacterial infection often demonstrated leukopenia, neutropenia, and thrombocytopenia, usually associated with normal bone marrow cell production. Those with nonfatal sepsis frequently had neutrophiia with an increase in absolute band counts. Of infected newborns 80% showed one or more hematologic abnormalities as did 43% of newborns dying without bacterial infection. Of newborns dying with bacterial infection 13% had no hematologic abnormality. Blood cultures were negative in 18% (seven) of the infants dying with bacterial infection. Abnormalities of the white blood cell, differential and platelet counts are not invariably specific for bacterial infection nor do normal values adequately exclude it. Blood cultures may be negative in newborns dying with significant foci of bacterial infection.

Effect of Erythropoietin on RNA Synthesis by Normal and Leukemic Bone Marrow and Spleen Cell Suspensions In Vitro

Erythropoietin (EPO) induced a 42% increase in 3H-uridine incorporation into RNA after a 5-hr culture of normal bone marrow cell suspensions. Bone marrow cells obtained from rats 3-5 days after the initiation of a myelogenous leukemia exhibited a decreased responsivity to EPO. At this time incorporation of the isotope into RNA in the presence of EPO was approximately 50% of controls. Rats rendered leukemic 8-10 days prior to culture showed no bone marrow response to EPO even in those instances where leukemic cells comprised a relatively small percentage of the marrow compartment. EPO had little or no effect on RNA synthesis by spleen cells obtained from normal and leukemic rats. This was noted even in those leukemic spleens in which erythropoiesis was observed. The data suggest that the anemia associated with myelogenous leukemia may, in part, be due to a loss of EPO-responsive cells and/or a loss of sensitivity of these elements to normal humoral control.