Immunophenotyping of B-Acute Lymphoblastic Leukaemia
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IMMUNOPHENOTYPING OF B-ACUTE LYMPHOBLASTIC LEUKAEMIA Dr Lee Shir Ying Sr Consultant Haematologist Department of Laboratory Medicine National University Hospital Singapore Introduction • Case study illustrating the principles of immunophenotyping for B-ALL • MRD detection in B-ALL Cell characterization • Means of characterization: – Morphology, cytochemistry, immunohistochemistry, flow cytometry immunophenotyping – Characterization into: –Lineage, maturity, abnormality Flow Cytometry Technology • Simultaneous measurement of multiple physical characteristics of a single cell as the cell flows in suspension through a measuring device Definition • B-ALL: Neoplasm of precursors (lymphoblasts) committed to B-cell lineage • When blood and bone marrow are extensively involved, acute lymphoblastic leukemia (B-ALL) is appropriate • When disease is confined to mass with absent or minimal blood and marrow involvement, term lymphoblastic lymphoma (B-LBL) is used • If patient presents with mass lesion and blood and bone marrow involvement, 25% blasts in marrow defines leukemia (B-ALL) Case Study • 58-year-old man. No past medical history of note. • Referred for investigation of pancytopaenia and constitutional symptoms. • Also a paravertebral mass, which was biopsied (also sent for flow cytometry) • Full blood count – Total white cell count - 2.9 x 109/L – Haemoglobin - 7.7 g/dL – Platelet count - 23 x 109/L ➢ Bone marrow aspirate MGG stained ➢ Infiltrated by small uniform blast-like cells ➢ Fine chromatin, high N/C ratio, nucleoli present, irregular nuclear contours Bone marrow trephine: ➢ Hypercellular for age ➢ Marked decrease in trilineage haematopoiesis Sheets of darkly-stained cells with high N/C • Round to irregular nuclei • Evenly dispersed chromatin • Inconspicuous to small nucleoli PAX5 CD79a CD20 Morphology appears to be a straightforward B-cell acute lymphoblastic leukaemia, but..... Markers of immaturity, CD34, nuTDT TdT CD34 CD117CD117 Blasts without CD34 and nuTDT…. could they be erythroblasts or megakaryoblasts? • Erythroid lineage : CD71 (transferrin receptor) • Megakaryocytic lineage : CD61 (GP-IIIa) CD71 CD61 Other markers of immaturity / lymphoblasts, CD10 and CD99…. CD10 CD99 Immunophenotype of B-ALL • B-cell markers: CD19, CD22, CD79-α, PAX-5 • Immature lymphoid cell markers: TdT, CD34, CD99 • Degree of differentiation determines immunophenotype: – Earliest stage blasts express cytoplasmic CD22 and TdT and CD34 (Pro-B ALL), CD10- – Intermediate stage blasts express CD10 (Common Early Pre- B-ALL) – Most mature stage blasts express cytoplasmic μ chains, CD34 often negative (Pre-B ALL) • CD20 expressed in 30-40% of cases • Ki-67 high (50-95%) Differential diagnosis • Burkitt Lymphoma – Burkitt lymphoma (BL) is mature, high-grade B-cell lymphoma – In smears: Deeply basophilic cytoplasm with cytoplasmic vacuoles – Surface IgM(+), CD20(+), CD10(+), Ki-67(+), TdT(-), Bcl-2(-) – Translocations involving MYC • Other Leukemias – Immunophenotyping is helpful to distinguish B-ALL from other leukemias • Haematogones – Can be numerous in children, especially during recovery after BM insults – Show a continuum of expression of markers of B-cell maturation – Demonstrate gradual gain of CD20 expression – Gradual loss of CD10, TdT and CD34 – Lack of aberrant antigen expression CD34 negative, TDT negative B-ALL…. need to exclude Burkitt’s lymphoma C-myc Ki-67 Cytogenetics: 46 XY FLOW CYTOMETRY RESULTS First Things First…. Orientation of Bone Marrow Haematolymphoid Populations using CD45 (LCA) vs SSC RED CELLS = 85% of total nucleated cells FSC-A vs SSC-A (cell size and internal complexity in comparison with lymphocytes) RED CELLs Are small to intermediate in size Low internal cytoplasmic complexity Determine Lineage of Abnormal Population Other B-lineage markers What we have so far for B-lineage markers…. • Positive: CD19, CD24, cyCD79a, (PAX5) • Dim or Partial: CD22, CD10 • Negative: CD20, CD79b, smIgM, surface light chain & cytoplasmic light chain • Negative: cyMPO, cyCD3, sCD3, CD7, CD5 Why are some B-markers present and others not? Relationship to B-cell maturation Pro-B Pre-BI Pre-BII Mature B (Pre-Pre-B) (Pre-B) Ref: Prof A Orfao Phenotype of Normal B-cells in Relation to B-Cell Maturation Precursors (Immature B-cells) Mature B-cells Van Lochem et al. Clinical Cytometry 60B: 1-13 (2004) CD34 and nuTDT are expressed only in the earliest Pre-B cell stage and parallel each other CD34+ TDT+ Summary of B-cell antigens • Precursor (Immature) B-cells are characterized by: • Positive CD19, CD10, CD24, cyCD79a • Dim CD22 • Negative CD20, CD79b, sIgM, surface light chains (usually) and cytoplasmic light chains • CD34 and nuTDT are positive only in the earliest pre-B-cells and negative in intermediate pre-B- cells Case example – CD34 Partial CD34 Partial CD34 Partial CD34 expression expression expression BM Tube 1 BM Tube 2 Mass 8% of blasts express CD34 Case example – TDT, CD10 Equivocal TDT expression 5% of blasts express CD10 Are there any other clues to the precursor nature of the B-cells? CXCR5 is negative to weakly expressed CD81 is brightly expressed Chemokine receptors CXCR4 CXCR5 CCR5 CCR6 in association with developmental stages of B-cells Expression of tetraspanin molecules during normal B-cell development Barrena. Leukemia (2005), 1376–1383 Identify Aberrancies – are these pathological B- lymphoblasts or normal B-cell precursors? • Different from normal approach • Leukaemia associated phenotype (LAP) approach • Combination of both Normal Precursor B-cell maturation • 1) simplified to 3 19+/34-/Tdt-/10-/ broad maturation stages 22++/45++/38dim/20++ • 2) earliest precursor 19+/34-/Tdt-/10+/ B-cell is CD19-/Tdt+/45- 22dim/45+/38++/20dim /34+/10+/22dim • 3) Useful mAb: 19dim/34+/Tdt+/10++/ 45, 34, 10, 19, 20, 22 22dim/45dim/38++/20- Search for different from normal aberrancies Old and New LAP Antigens Gaipa et al. Cyt B Case example – Aberrancies of LAP markers CD13, CD45, CD38 Cross-lineage CD13+ expression Dim/heterogenous CD38 Case example – Aberrancies of LAP markers CD66c, CD123, CD73, CD304 CD73 and/or CD304 aberrantly expressed CD66c and CD123 not expressed Could this be Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN)? • Great mimicker • The classic phenotype is described as CD4+, CD56+, CD123+ • BPCA4+ (CD304), BPCA2+ (CD303), TCL1+, CD68+ CD4, CD123, CD56 are Negative 5 out of 186 (2.7%) B-cell ALL cases are negative for TdT MINIMAL RESIDUAL DISEASE (MRD) DETECTION IN B-ALL Do Clinicians think MRD Testing is Useful? Minimal residual disease MRD strongly correlates with poor outcome in childhood and adult ALL MRD strongest predictor of treatment outcome and efficacy • Paediatric ALL Which method? • • Adult ALL Immunophenotyping • PCR based • AML Which time point? • Myeloma • Early Day 14 • After induction • Chronic lymphocytic • End of consolidation leukaemia Which level? • Hairy cell leukaemia • 0.1% (1 in 103) • 0.01% (1 in 104) • 0.001% (1 in 105) B-ALL flow cytometry MRD - example Post Induction 0.1% Post-#2 0.04% 3 months later 0.07% 2 months later 1.0% 1 month later 18% Frank relapse Persistent MRD > 0.01% (1 in 104) Increasing MRD heralds disease relapse Euroflow B-ALL MRD Panel EuroFlow erythrocyte bulk-lysis procedure: sufficiently large volumes of BM, ie, containing >10 million cells, are lysed, allowing staining of 10 million cells in 100mL of cell suspension per tube. Sensitivity == Limit of Quantification How many cases will be detected by the antibody panel combination Limit of Quantification (LOQ) of MRD enumeration LOQ of the assay is a function of the total number of nucleated cell events acquired • E.g. for a target LOQ of 0.01% or 1 in 104 and 100 leukaemia cells (CV<10%), a minimum of 1 million events should be acquired • E.g. for a target LOQ of 0.001% or 1 in 105 and 100 leukaemia cells (CV <10%), a minimum of 10 million Arroz et al events should be acquired How many events to call a population? Coefficient of variation reflects the precision and repeatability of the assay Caveats for successful MRD • Good sample quality • Avoid haemodilution • Stringent quality control and instrument calibration and setup • Acquisition of sufficient events for desired LOQ • Gating and population identification. Pitfalls: – Failure to gate on the correct population – Mistaking normal variants for abnormal – Misinterpreting abnormal cells for normal – Phenotypic shift – either treatment related or clonal evolution Haematogone compartment reconstitution after ALL treatment Lack of normal haematogones at early time points Theunissen et al BJH 2017 Lost of immature markers during induction treatment of B-ALL CD10 low CD34 low CD45 high CD20 high Era of Immunotherapy CD19 Chimeric Antigen Bi-specific T-cell Engager Receptor T-cells (CAR-T) (BITE) Blinatumumab Autologous T-cells engineered by retroviral transduction to express Allows T-cells to recognize tumor anti-CD19 fragment linked to T-cell associated surface antigen activation domains. B-ALL leukaemia cell At diagnosis CD19+ CD45- CD10- CD20- CD34 Partial 2% Post Anti-CD19 CD19- CD45- CD10- CD20- CD24+ CD22+ 0.013% Take home message • Rarely, B-lymphoblastic leukaemia/lymphoma can be negative for CD34 and TdT • Normal B-cell maturation provides a frame of reference for B-ALL immunophenotyping • All B-ALL patients with morphologic CR should have an assessment for MRD • New therapies require application of different approaches to identify B-ALL cells THANK YOU.