Most cases of acute leukemia (AL) are classified as either myeloid or lymphoid lineage based on the expression of a set of antigens. However, there are cases of patients with blasts expressing both lymphoid and myeloid features at diagnosis, known as biphenotypic acute leukemia (BAL) or mixed-phenotype acute leukemia (MPAL) [1]. These divergent morphologic and immunophenotypic features may be uniformly present in one blast population (biphenotypic leukemia) or may be seen on distinct blast populations in a single patient (bilineal leukemia) [2].

The European Group for Immunological Classification of Leukemias (EGIL) proposed a set of diagnostic criteria for BAL. This scoring system was based on the number and degree of specificity of certain makers for myeloid or T/B lymphoid blasts [3,4]. In 2008, the new World Health Organization (WHO) criteria were proposed for the classification of acute leukemia of ambiguous lineage (ALAL), which include significant modifications of the diagnostic criteria for AL of mixed phenotype. Cases with no lineage-specific markers were designated as acute undifferentiated leukemia (AUL), which often express CD34, HLA-DR, and/or CD38, and sometimes TdT, but lack specific myeloid or lymphoid antigens [5,6]. A type of leukemia with blasts that co-expresses certain antigens of more than one lineage on the same cells or that has separate populations of blasts of different lineages is referred to as MPAL. They can be further designated as B-myeloid or T-myeloid based on flowcytometric immunophenotyping, irrespective of whether one or more than one population of blasts was found. Thus, MPAL includes both biphenotypic and bilineal ALs. These WHO criteria were more stringent than those of the EGIL criteria and relied heavily on positivity for myeloperoxidase (MPO) [5]. In 2016, the WHO classification was updated but the criteria for ALAL remained unchanged [6].

The exact incidence of BAL or ALAL in childhood is unknown. The optimal therapy for this subtype of leukemia has not been established, especially in pediatric patients. Little is known about the prognostic implications of immunophenotypes of BAL [7]. The role of hematopoietic stem cell transplantation (HSCT) in first remission remains contentious [2,8,9,10].

The present study aimed to retrospectively investigate the incidence and clinicopathological characteristics of pediatric BAL or ALAL patients in Gwangju and Chonnam province from 2001 to 2017.

The incidence of BAL or ALAL varies according based on the criteria (EGIL or 2016 WHO) used for the diagnosis. BAL is reported to account for 2–5% of all ALs based on the EGIL criteria, while ALAL 1–2.5% based on the WHO criteria [2,9,14], which are similar to the current study of 2.9% and 1.9%, respectively. Additionally, the incidence of AUL and MPAL among ALAL was 0.5% and 1.3%, respectively, in this study. Although the incidence was not clearly defined, the incidence of AUL was reported to be 0.2–1% in some studies [15,16].

In contrast to the EGIL approach of scoring a detailed blast immunophenotype with numerous markers, the WHO criteria emphasize a few key lineage-defining markers with particular emphasis on CD19 for B lineage, CD3 for T lineage, and MPO for myeloid lineage. The WHO approach is simpler but relies heavily on the sensitivity and specificity of a few markers. Moreover, the WHO classification does not specify thresholds for positivity of these key markers, leaving it up to individual laboratories to decide on the definition of significant expression [13]. In addition, the WHO diagnostic criteria defined AUL as a type of leukemia that does not involve the expression of those specific antigens and hence set up the classification of ALAL, including MPAL and AUL. In the present study, the incidence might have been underestimated as some immunological markers such as cyCD79a were not tested in our institute.

The phenotype distribution in our patients was no different from those reported in previously published studies. The number of patients having the B-lymphoid/myeloid phenotype was more than the number of patients having the T-lymphoid/myeloid phenotype [2,17]. In adult studies, more than 70% of BAL patients had the B-lymphoid/myeloid phenotype, whereas only 23–33% had the T-lymphoid/myeloid phenotype. The B-lymphoid/T-lymphoid/myeloid phenotype and B-lymphoid/T-lymphoid phenotype were extremely rare [7,18,19,20].

In the WHO 2016 diagnostic criteria, t(9;22)/Ph+ positive and MLL rearrangement were classified separately. Although MPAL was not associated with a consistent cytogenetic/molecular abnormality, the Philadelphia chromosome was the most common abnormality, with 17–41% incidence in BAL and MPAL patients with almost exclusively B-lymphoid/myeloid phenotype [7,14,21]. BCR-ABL1 was more common among adult MPAL patients (15%) than among pediatric MPAL patients (3%) [14,15,22]. However, all Ph+ patients should be identified as quickly as possible because they may benefit from the addition of tyrosine kinase inhibitors (TKI) same as those with Ph+ ALL [12,23,24].

The second most common cytogenetic abnormality was MLL rearrangement as reported in other studies. Its incidence ranged from 10% to 15% [7,8,14] and were higher in the pediatric age group than in the adult group (15% vs. 3%) [14,15,25]. In the present study, the incidence of cytogenetic abnormality was also 16.7%. No single chromosomal abnormality is unique to ALAL. However, the present data and those of other studies showed that structural abnormalities are common.

MPALs may have worse prognosis than other types of leukemia. The proposed reasons were as follows: the mixed-phenotype leukemic stem cells are chemoresistant owing to slow replication, the blasts can adapt to therapy by switching phenotype, and some MPALs express high levels of multidrug resistance proteins [12,26].

Because selection of an anti-leukemic chemotherapy regimen for AL is largely based on whether a case is classified as myeloid or lymphoid, the presence of markers for both lineages may have important implications for treatment. Thus, there are no agreed chemotherapy protocols for patients with ALAL as yet [12,27]. In the current study, the induction regimen was selected based on the morphology of the blasts and cytochemical stains. All 6 (100.0%) patients who underwent ALL induction therapy reached a CR. On the contrary, among six patients who received AML induction therapy along, 1 (16.7%) achieved a CR after one cycle, while 2 achieved a CR after FLAG-IDA salvage chemotherapy. The remaining three patients reached a CR after switching to ALL induction therapy. Thus, the current study demonstrated that an ALL-directed induction chemotherapy is more effective in achieving CR than AML-type therapy, although patient numbers are small and follow-ups are not long. Rubnitz et al. [2] reported that ALL-type therapy was associated with higher CR rate than AML-type induction therapy (83% vs. 52%, respectively). Similar results were documented from studies by Matutes et al. [14] and by Al-Seraihy et al. [17]. In a Korean pediatric study, a significantly lower CR rate of 52% was reported even predominantly with ALL-type therapy. In this study, eight of ten patients who failed to achieve a CR with AML-type therapy subsequently entered a CR with ALL-type induction therapy [8]. Gerr et al. [15] reported significantly better survival in patients who received ALL-type induction therapy than those with AML-type induction therapy (81% vs. 41%, P=0.0009). In this study, same tendency was observed but they were not significantly different.

The issue of HSCT remains contentious. In this study, among 9 (75% of total cohort) patients who received an allogeneic HSCT, 5 (55.5%) survived. Among three patients who did not receive HSCT, only one survived. Our decision to proceed with transplant was primarily based on the availability of HLA-matched stem cell donor. The current study demonstrated a favorable outcome for patients transplanted in CR1. The post-allogeneic-HSCT outcome was much better than with post remission chemotherapy in a single institution (N=32, 3-year OS 77% vs. 16%) [28]. However, a Korean pediatric study reported no benefit from HSCT over chemotherapy alone in patients with BAL [8]. In general, similar outcomes have been reported between transplanted vs. non-transplanted cases, but the issues need to be clarified because of the lack of randomized study, due the small number and heterogeneity of patients, stem cell donor, and transplantation procedures. Thus, routine use of HSCT may not be required in patients with ALAL, especially in those who attain a molecular remission after induction therapy. However, still a significant portion of the patients may need a prompt HSCT. Some recommend that an HSCT should be reserved for the case of Philadelphia chromosome-positive MPAL, infants particularly those with 11q23 abnormalities and those who have a poor response to early therapy [28,29]. However, whether older children with 11q23 abnormalities should also be transplanted remains unclear and require further evaluation using larger, possibly multi-institutional studies. Other risk-determining variables need to be included in this decision process [10,12,29].

So far, the survival rate reported in both pediatric and adult studies ranged from 8.1% to 60% [2,17,30]. In the current study, the 5-year OS of the total cohort was 51.1±15.8%. Most recently, the survival rate of MPAL based on the WHO classification was about 80% in children [16]. Killick et al. showed a better OS for the younger patients than older counterparts (75% vs. 17% at 2 yr; P=0.01) [7]. Gerr et al. [15] demonstrated that the 5-year EFS probability of ALAL patients (62±5%) was lower than those of ALL patients (80±1%, P<0.001), but better than those of AML patients (49±2%, P=0.027). A few studies demonstrated the survival of AUL or its comparison with MPAL, because of the extreme rarity of the cases. In the current study, the survival of MPAL (75.0±21.7%) was better than that of AUL (0.0%) (P=0.008) [30].

The prognostic implication of high leukocyte counts at diagnosis, and immunophenotypic subtype, expression of the specific antigen, as well as the selection of induction regimen and the use of HSCT in BAL or ALAL need to be further established in prospective, multicenter fashion. Pediatric ALAL may be distinct from its adult counterpart in terms of clinical and cytogenetic characteristics, and response to chemotherapy, even though they are extremely rare. With better understanding of novel mechanism and pathogenesis of these rare situations, the best therapeutic approaches including target therapy will be available in the future.

Flow diagram of the classification, treatment and outcome.

Abbreviations: CR, complete remission; MPAL, mixed-phenotype acute leukemia; NR, non-responders; PR, partial remission; SCT, stem cell transplantation.

The 5-year Kaplan-Meier plot for (A) OS and (B) EFS for all biphenotypic acute leukemia patients.

Abbreviations: BAL, biphenotypic acute leukemia; EFS, event-free survival; OS, overall survival.

The 5-year Kaplan-Meier plot of (A) OS and (B) EFS of AUL and MPAL by 2016 WHO classification.

Abbreviations: AUL, acute undifferentiated leukemia; EFS, event-free survival; MPAL, mix-phenotype acute leukemia; OS, overall survival.

The 5-year Kaplan-Meier plot of (A) OS and (B) EFS according to initial leukocyte count.

Abbreviations: EFS, event-free survival; OS, overall survival.

The 5-year Kaplan-Meier plot of (A) OS and (B) EFS for patients with T/myeloid and B/myeloid biphenotypic leukemia.

Abbreviations: EFS, event-free survival; OS, overall survival.

The 5-year Kaplan-Meier plot for (A) OS and (B) EFS for biphenotypic acute leukemia patients treated with ALL-directed induction therapy and AML-directed induction therapy.

Abbreviations: EFS, event-free survival; OS, overall survival.

The 5-year Kaplan-Meier plot for (A) OS and (B) EFS for biphenotypic acute leukemia patients treated with chemotherapy alone and those treated with HSCT in first remission.

Abbreviations: EFS, event-free survival; HSCT, hematopoietic stem cell transplantation; OS, overall survival.

The 5-year Kaplan-Meier plot for (A) OS and (B) EFS for biphenotypic acute leukemia patients treated with HSCT in first remission and those treated with HSCT in the second remission after relapse.

Abbreviations: EFS, event-free survival; HSCT, hematopoietic stem cell transplantation; OS, overall survival.