LAUSR

Ambiguous lineage leukaemia (ALAL) is a rare sub-type of paediatric acute leukaemia. There are four main subgroups according to the lineage of differentiation, namely: (i) mixed phenotype acute leukaemia (MPAL), (ii) bilineal acute leukaemia, (iii) undifferentiated acute leukaemia, and (iv) acute leukaemia with early switch to a different lineage. MPAL accounts for 1–2% of paediatric leukaemia. The World Health Organization (WHO) classifications (2008 and 2016) have established strict criteria for diagnosis of MPAL, emphasising myeloperoxidase for myeloid lineage, cytoplasmic cluster of differentiation 3 (CD3) for T lineage, and CD19 and other B markers for B lineage. Flow cytometry has been pivotal in diagnosis, treatment response and minimal residual disease (MRD) assessment in MPAL. The diagnosis of MPAL is unlikely to be suspected by morphology except in cases with distinct dual-blast population with either lymphoid or myeloid features. Therefore, the diagnosis is reliant on immunophenotyping and exclusion of cytogenetics suggestive of acute myeloid leukaemia (AML), whereas bone marrow morphology is useful to rule out any dysplasia. The outcome for MPAL is inferior to other common paediatric acute leukaemia. The International Berlin-Frankfurt-M€ unster Study of Leukemias of Ambiguous Lineage (iBFM-AMBI2012) study showed a superior 5-year event-free survival (80 4% vs. 36 7 2%) in those treated with acute lymphoblastic leukaemia (ALL)-type than with AMLtype or combined therapy. AML-type treatment was effective in those with CD19 or in the absence of lymphoid markers. Cytogenetic abnormalities typical for ALL or AML can be used to steer the type of chemotherapy. Treatment is largely guided by MRD and bone marrow transplant is reserved for those with inadequate response to therapy. Previous studies have shown non-inferior outcomes in both CD19 and CD19 cases compared to nonALAL cases with MRD 0 1% or higher after induction treated on current intensified protocols. We conducted a multicentre retrospective study with chart and pathology review of all cases of MPAL/ALAL diagnosed and treated at University Hospital Southampton (UHS) and Sheffield Children’s Hospital (SCH), UK from the year 2015 until 2020. A total of three children with bilineal and biphenotypic acute leukaemia and a predominant monocytic component (UHS, one; SCH, two) were enrolled. Immunophenotyping by multiparameter flow cytometry (MFC) was performed on bone marrow samples following bulk lysis. Initial screening performed using two tubes for both myeloid and lymphoid lineages; eight surface markers: CD33 (Becton Dickinson [BD], Franklin Lakes, NJ, USA; 345800), CD34 (BD; 345803), CD117 (BD; 339217), CD45 (BD; 641417), human leucocyte antigen-DR isotype (HLADR; BD; 655874), CD10 (BD; 341112), CD19 (BD; 345791) and CD7 (BD; 642916) and five cytoplasmic markers: immunoglobulin M (Dako, Glostrup, Denmark; R511101), terminal deoxynucleotidyl transferase (Bio-Rad, Pleasanton, CA, USA; OBT0012), CD3 (BD; 345766), CD79a (Dako; R7159) and myeloperoxidase (MPO; BD; 333138). Post blastpopulation identification, two standardised MRD tubes were used for flow MRD monitoring in B-lineage ALL using a lyophilised cocktail mix by Cytognos (Salamanca, Spain; CD81 FITC, CD34 PerCP 5.5, CD45 OC515, CD20 PB, CD38 APC AF750, CD19 PC7) with four drop ins on PE – CD123 (BD; 554529), CD66c (Beckman Coulter, Brea, CA, USA; IM2357U), CD304 (BioLegend, San Diego, CA, USA; 354504) and CD73 (BD 550527). The identified leukaemia associated immunophenotype (LAIP) was used for downstream analyses. Clinical and treatment details are described in Table I.