Paediatric ambiguous lineage leukaemia with monocytic differentiation at diagnosis: case series and review of literature.

Abstract

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.1-4 MPAL accounts for 1–2% of paediatric leukaemia.3 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.1, 4 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.5, 6 The outcome for MPAL is inferior to other common paediatric acute leukaemia.7-11 The International Berlin-Frankfurt-Münster 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 AML-type or combined therapy.5 AML-type treatment was effective in those with CD19− or in the absence of lymphoid markers.5 Cytogenetic abnormalities typical for ALL or AML can be used to steer the type of chemotherapy.5 Treatment is largely guided by MRD and bone marrow transplant is reserved for those with inadequate response to therapy.5, 9 Previous studies have shown non-inferior outcomes in both CD19+ and CD19− cases compared to non-ALAL cases with MRD 0·1% or higher after induction treated on current intensified protocols.5, 12, 13 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 (HLA-DR; 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 blast-population 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. Clone 1: (20%) Precursor B cell CD10+, CD19+, CD34+, TdT+, CD79a, CD66c/CD123+, CD73/CD304+ and negative for MPO Clone 2: (65%)-monocytic cells CD13+, CD33+, CD34(wk)+, CD19+, CD64+, MPO+ and negative for CD117− and CD79a− Clone 1: Precursor B cell with CD10(weak), CD19+, CD34+, CD22+, HLA-DR+, TdT+ and negative for CD3, CD7 and MPO. Clone 2: Monocytic cells CD33+, CD34+, CD19+, CD14+, CD13+, CD4+, CD64+, HLA-DR+ and was negative for TdT. Immunophenotype at relapse: 9% myeloid blasts positive for CD45+, CD33+, CD34+, CD117+, CD14+, CD13+, CD64+, HLA-DR+, MPO+, cCD13+ UKALL2011 Regimen B, CR after induction, MRD positive, escalated to Regimen C week 5–9. On treatment relapse. Salvage with FLAD (fludarabine, cytarabine, liposomal daunorubicin), CR with MRD 0·01% Rising MRD to 0·1% pre-transplant. CR post-transplant, MRD negative Clone 1: CD10+, CD19+, CD34+, Tdt+ Clone 2: Myeloid/monocytic – CD13+, CD33+, CD34−, CD117−, CD11c+, CD14+, CD64+ Peripheral blood MFC showed a predominant monocytic population CD13+, CD33+, MPO+ and another small CD19+ population. A provisional diagnosis of AML with monocytic differentiation was considered and commenced on AML treatment as per UK national guidelines due to rising white blood cell counts (Table I). Bone marrow immunophenotyping confirmed MPAL B/Myeloid and switched to ALL-type protocol and remains in complete remission (CR) (Fig 1A–C; Table I). After induction he achieved CR; however, had risk MRD (0·5%) and therapy was escalated to Regimen C consolidation of the same protocol. Disease reassessment at week 9 showed a rising MRD (2%) with a rapid morphological relapse (Table I). He is currently on follow-up and remains disease-free at 4-years since diagnosis. He achieved CR after induction and MRD low risk and subsequently escalated to Regimen C in view of a diagnosis of MPAL. He is on maintenance chemotherapy with an uneventful follow-up (Table I). Bilineal and/or biphenotypic presentation is extremely rare with few case reports. Our patient population is unusual for various reasons. Firstly, all patients presented with monocytoid blasts at diagnosis, which has not been previously reported in the paediatric age group. Reported cases have been adults with T/myeloid phenotype,6 in contrast to our B/myeloid cohort. Recent evidence shows that myeloid potential may be retained even when lineage branches segregate towards B and T cells.14 Additionally, the blasts may arise before loss of myeloid differentiation potential. Secondly, all cases were biphenotypic and bilineal. This could be due to presence of two different clones or a distinct population arising from a single clone as reported previously.14 This rare subgroup of leukaemia poses both difficulties with diagnosis and treatment, as seen in Patient 1 (Table I). Most groups have treated with either ALL or AML protocols, or both. Lymphoid-directed treatment has shown better outcomes compared to AML or combined protocols.5, 7, 11, 15 Treatment is directed by MRD with bone marrow transplant for poor responders.5 Switching treatment based on the residual/relapse clone has been used by various groups as in our present Patient 2 (Table I). MFC MRD is used effectively in 90–95% cases of ALL and in 80–85% of AML; however, in paediatric MPAL data are limited to a recent large study that highlighted reliance on flow cytometry for diagnosis and MRD monitoring.5 The MFC technique identifies LAIP for tracking in follow up testing. MFC MRD identifies leukaemia cells down to 0·01% as a minimum in ALL and when >4 million cells are measured for ALL, MFC MRD is considered as sensitive as polymerase chain reaction-based methods. In AML, MFC MRD identifies leukaemia cells down to 0·1% and MFC MRD can be appropriately applied to MPAL cases.16, 17 Biphenotypic and bilineal ALAL is extremely rare in children with associated diagnostic and therapeutic challenges. Lymphoid-directed therapy with MFC MRD is an effective approach in children.