Flow cell sorting followed by PCR-based clonality testing may assist in questionable diagnosis and monitoring of acute lymphoblastic leukemia.

INTRODUCTION: Multicolor flow cytometry (MFC) has highly reliable and flexible algorithms for diagnosis and monitoring of acute lymphoblastic leukemia (ALL). However, MFC analysis can be affected by poor sample quality or novel therapeutic options (e.g., targeted therapies and immunotherapy). Therefore, an additional confirmation of MFC data may be needed. We propose a simple approach for validation of MFC findings in ALL by sorting questionable cells and analyzing immunoglobulin/T-cell receptor (IG/TR) gene rearrangements via EuroClonality-based multiplex PCR. PATIENTS AND METHODS: We obtained questionable MFC results for 38 biological samples from 37 patients. In total, 42 cell populations were isolated by flow cell sorting for downstream multiplex PCR. Most of the patients (n = 29) had B-cell precursor ALL and were investigated for measurable residual disease (MRD); 79% of them received CD19-directed therapy (blinatumomab or CAR-T). RESULTS: We established the clonal nature of 40 cell populations (95.2%). By using this technique, we confirmed very low MRD levels (<0.01% MFC-MRD). We also applied it to several ambiguous findings for diagnostic samples, including those with mixed-phenotype acute leukemia, and the results obtained impacted the final diagnosis. CONCLUSION: We have demonstrated possibilities of a combined approach (cell sorting and PCR-based clonality assessment) to validate MFC findings in ALL. The technique is easy to implement in diagnostic and monitoring workflows, as it does not require isolation of a large number of cells and knowledge of individual clonal rearrangements. We believe it provides important information for further treatment.

Reliable Flow-Cytometric Approach for Minimal Residual Disease Monitoring in Patients with B-Cell Precursor Acute Lymphoblastic Leukemia after CD19-Targeted Therapy.

We aimed to develop an antibody panel and data analysis algorithm for multicolor flow cytometry (MFC), which is a reliable method for minimal residual disease (MRD) detection in patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) treated with CD19-directed therapy. The development of the approach, which was adapted for the case of possible CD19 loss, was based on the additional B-lineage marker expression data obtained from a study of primary BCP-ALL patients, an analysis of the immunophenotypic changes that occur during blinatumomab or CAR-T therapy, and an analysis of very early CD19-negative normal BCPs. We have developed a single-tube 11-color panel for MFC-MRD detection. CD22- and iCD79a-based primary B-lineage gating (preferably consecutive) was recommended. Based on patterns of antigen expression changes and the relative expansion of normal CD19-negative BCPs, guidelines for MFC data analysis and interpretation were established. The suggested approach was tested in comparison with the molecular techniques: IG/TR gene rearrangement detection by next-generation sequencing (NGS) and RQ-PCR for fusion-gene transcripts (FGTs). Qualitative concordance rates of 82.8% and 89.8% were obtained for NGS-MRD and FGT-MRD results, respectively. We have developed a sensitive and reliable approach that allows MFC-MRD monitoring after CD19-directed treatment, even in the case of possible CD19 loss.

Lineage Conversion in Pediatric B-Cell Precursor Acute Leukemia under Blinatumomab Therapy.

We report incidence and deep molecular characteristics of lineage switch in 182 pediatric patients affected by B-cell precursor acute lymphoblastic leukemia (BCP-ALL), who were treated with blinatumomab. We documented six cases of lineage switch that occurred after or during blinatumomab exposure. Therefore, lineage conversion was found in 17.4% of all resistance cases (4/27) and 3.2% of relapses (2/63). Half of patients switched completely from BCP-ALL to CD19-negative acute myeloid leukemia, others retained CD19-positive B-blasts and acquired an additional CD19-negative blast population: myeloid or unclassifiable. Five patients had KMT2A gene rearrangements; one had TCF3::ZNF384 translocation. The presented cases showed consistency of gene rearrangements and fusion transcripts across initially diagnosed leukemia and lineage switch. In two of six patients, the clonal architecture assessed by IG/TR gene rearrangements was stable, while in others, loss of clones or gain of new clones was noted. KMT2A-r patients demonstrated very few additional mutations, while in the TCF3::ZNF384 case, lineage switch was accompanied by a large set of additional mutations. The immunophenotype of an existing leukemia sometimes changes via different mechanisms and with different additional molecular changes. Careful investigation of all BM compartments together with all molecular -minimal residual disease studies can lead to reliable identification of lineage switch.

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.

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.

Immunophenotypic changes of leukemic blasts in children with relapsed/refractory B-cell precursor acute lymphoblastic leukemia, who have been treated with Blinatumomab.

Not available.

Chimerism evaluation in measurable residual disease-suspected cells isolated by flow cell sorting as a reliable tool for measurable residual disease verification in acute leukemia patients after allogeneic hematopoietic stem cell transplantation.

BACKGROUND: The presence of minimal/measurable residual disease (MRD) before or after hematopoietic stem cell transplantation (HSCT) is known as a predictor of poor outcome in patients with acute myeloid (AML) or lymphoblastic (ALL) leukemia. When performed with multiparameter flow cytometry (MFC), assessment of residual leukemic cells after HSCT may be limited by therapy-induced shifts in the immunophenotype (e.g., loss of surface molecules used for therapeutic targeting). However, in such cases, questionable cells can be isolated and tested for hematopoietic chimerism to clarify their origin. METHODS: Questionable cell populations were detected during the MFC-based MRD monitoring of 52 follow-up bone marrow samples from 37 patients diagnosed with T cell neoplasms (n =14), B cell precursor ALL (n = 16), AML (n = 7). These cells (suspected leukemic or normal) were isolated by flow cell sorting and tested for hematopoietic chimerism by RTQ-PCR. RESULTS: The origin of cells was successfully identified in 96.15% of cases (n = 50), which helped to validate the results of MFC-based MRD monitoring. CONCLUSIONS: We believe that a combination of MFC, cell sorting, and chimerism testing may help confirm or disprove MRD presence in complicated cases after HSCT.

Quantification of NG2-positivity for the precise prediction of KMT2A gene rearrangements in childhood acute leukemia.

It has long been known that there is a link between neuron glial antigen 2 (NG2) surface expression and KMT2A gene rearrangements in acute leukemia (AL). However, the exact levels of NG2 positivity that predict the presence of KMT2A rearrangement are not known. The current study focuses on a cohort of 505 pediatric AL patients who showed any level of positive NG2 expression (greater than 1% of cells) for whom comprehensive genetic data were available. NG2 expression was measured as either the percentage of positive cells or the number of molecules on the cell surface. KMT2A gene rearrangements were identified by FISH. The fusion partner was detected with RT-PCR, LDI-PCR or anchored multiplex PCR followed by high-throughput sequencing. KMT2A-positive samples comprised a substantial proportion of the NG2-positive cohort (180 of 505, 36%), with a total of 19 different types of translocation. Despite its occurrence in other AL genetic subgroups, NG2 expression was significantly increased in AL patients with KMT2A rearrangements in terms of both the cell percentage and number of molecules per cell. The threshold levels (TL) for NG2-positivity were established by ROC analysis of the whole cohort and separately for children less than 1 years old and older with lymphoblastic (ALL) and myeloid (AML) leukemia. The lowest TL was defined in infants with ALL (7%), while in older children, the threshold was higher (12%). In AML patients, the situation was reversed, with 28% NG2-positivity in infants and 14% in patients >1 year old. The defined TLs resulted in improved diagnostic performance compared to the conventional thresholds of 10% and 20% for all patient groups.

FLAER-negative CD15+ neutrophils can be used for the simplified screening of suspected PNH cases.

BACKGROUND: The flow cytometry analysis of GPI-linked proteins on red blood cells and leukocytes is crucial for paroxysmal nocturnal hemoglobinuria (PNH) diagnostics. However, the commonly used multicolor panels cannot be implemented in low-resourced hematology laboratories. In order to develop a simple prediagnostic test for PNH screening, we analyzed the diagnostic accuracy of the two-color (FLAER/CD15) detection of GPI-deficient neutrophils. METHODS: We reanalyzed multicolor data set of 1594 peripheral blood samples of patients screened for PNH applying only two markers (FLAER/CD15). The quantitative positivity/negativity was reported. Then, these results were compared in a blinded manner with previously obtained multicolor data from the same samples. RESULTS: Among the 1594 samples included in the study, 507 samples were PNH-positive by the multicolor assay. The two-color method revealed 510 PNH-positive samples. The detailed examination of this discrepancy revealed 12 false-positives and 9 false-negatives. Therefore, FLAER/CD15 screening method displayed 98.90% of the diagnostic specificity and 98.22% of the sensitivity. CONCLUSION: This simple two-color evaluation of FLAER-negative neutrophils is a highly effective screening test for PNH. Although this approach is not intended to replace the multicolor diagnostic procedure, it could minimize the number of patients requiring a conventional multicolor flow cytometric assay.

Heterogeneity of childhood acute leukemia with mature B-cell immunophenotype.

BACKGROUND: Flow cytometry (FCM) plays a crucial role in the differential diagnosis of Burkitt lymphoma/leukemia (BL) and B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The presence of surface IgM (sIgM) alone or with light chain restriction indicates a mature blast phenotype (BIV by EGIL) and is usually observed in BL. However, sIgM expression could also be detected in transitional BCP-ALL cases. These similarities in immunophenotype and ambiguous correspondence with other laboratory findings may challenge the correct BL diagnostics. METHODS: We retrospectively reviewed the available data from immunophenotypic, morphological, cytogenetic, and molecular genetic studies of 146 children (85 boys and 61 girls) with a median age of 10 years (range 0-18 years) who were diagnosed with BL and BCP-ALL. The blasts' immunophenotype was studied by multicolor FCM. The conventional cytogenetic analysis included G-banded karyotyping and fluorescence in situ hybridization (FISH). RESULTS: In 54 children classified as BIV-ALL according to the EGIL, it was demonstrated that sIgM in a minority of cases can be associated with various types of BCP-ALL. Analysis of the antigen expression profile of 105 patients with verified BL (n = 21) and BCP-ALL (n = 84) showed significant differences in BL and the sIgM(+) vs BCP-ALL immunophenotype. Thus, even in cases of ambiguous sIgM expression, these two diseases could be reliably discriminated by complex immunophenotyping. Moreover, 10 patients (7 boys and 3 girls) with BL leukemic cells did not express sIgM, and they were diagnosed with BL on the basis of other laboratory and clinical signs. CONCLUSIONS: In conclusion, our study shows that BIV subtype is heterogeneous group of leukemia including not only the BL, but also BCP-ALL. In ambiguous cases, only a combination of multiple immunophenotypic, cytomorphologic, and genetic diagnostic technologies can allow the precise discrimination of BL and BCP-ALL and selection of the appropriate treatment scheme.