The International Consensus Classification of acute leukemias of ambiguous lineage

All ALAL categories require that the total number of blasts in blood or bone marrow must be >20%. MPAL cases can contain 2 or more completely separable single-lineage leukemia populations or more than 1 lineage-defining marker in a single blast population.^4,5 In cases with separate blast populations, each population needs to be classified according to acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) criteria.¹ In cases in which small aberrant clones of divergent lineage are identified (>5%), a diagnosis of MPAL could be rendered if clear immunophenotypic aberrancies are identified. Identification of such aberrancies is essential to differentiate a small bilineal blast population from normal myeloid or B-cell precursors. For cases with aberrant clones, which represent <5% of all cells, a diagnosis should be based on the major leukemic population with a descriptive modifier, for example, “predominantly ALL with a small leukemic population of myeloid lineage detected of uncertain significance.” Additional details should be provided in the body of the report.

Similar to the criteria published in the revised fourth edition of the World Health Organization classification of these disorders, B-lineage designation in MPAL requires strong expression of CD19, along with at least 1 other B-cell marker.⁶ If CD19 expression is weak, 2 other B-lineage markers are needed (Table 2). T-lineage assignment requires a fraction of blasts to show strong cytoplasmic expression of CD3 (cCD3), with the brightest expression comparable to that of background mature T cells (Table 2). The negative predictive value of dim, rather than strong, cCD3 expression for T lineage is still unknown, and it often disappears during treatment or at relapse.^7,8 MPO has been considered the most specific marker for myeloid lineage in prior classification systems. At this time, no specific threshold is set for MPO positivity, but care should be taken in instances when <10% MPO⁺ cells are seen in flow cytometry.⁹ Additionally, extra care should be taken to discriminate small MPO populations from background nonneoplastic myeloid progenitors.¹⁰ Isolated MPO expression can also be seen in B-ALL cases with homogeneous expression of typical B lineage markers, and this alone should not change the diagnosis. AUL is defined by lack of lineage-specific markers (Table 2). A recent multiinstitutional study showed that only a small subset of AUL cases (25%) lacked all myeloid markers, whereas 62% showed partial or full expression of 1 myeloid marker.¹¹ 

Studies have shown that distinct subsets of MPALs are classified based on recurring genetic alterations. In addition to MPAL with BCR::ABL1 and MPAL with KMT2A gene rearrangements, 2 new molecularly defined entities have been described, B/myeloid MPAL with ZNF384 rearrangements and T/myeloid MPAL with BCL11B activation (Table 1). As with BCR::ABL1 and KMT2A, these 2 newer entities can manifest as ALAL or as single-lineage leukemias. In childhood B/myeloid MPAL, fusion genes involving the ZNF384 transcription factor gene were identified in nearly 50% of cases.¹² ALAL with rearrangements leading to deregulation of BCL11B (BCL11B-activated) comprise 10% to 15% of MPAL and are further enriched in patients with MPAL, T/myeloid cases, representing up to one-third of this population.^13-15 Unlike the BCL11B alterations described in typical T-ALL, including loss-of-function sequence mutations, deletions, and rearrangements deregulating oncogenes such as TLX3, the 14q32 structural variants identified in ambiguous-lineage leukemias result in deregulation of BCL11B expression through juxtaposition of hematopoietic stem cell enhancers or formation of neo-enhancers that also deregulated BCL11B expression.¹⁵ 

The majority (64% to 87%) of MPAL cases have an abnormal karyotype. Cytogenetic abnormalities and molecular findings diagnostic of AML with myelodysplasia-related cytogenetic abnormalities or AML with myelodysplasia-related mutations¹ are currently classified as AML, even if there is also evidence of concurrent lymphoid antigen expression or AUL immunophenotype, although such a feature could be annotated to highlight the immunophenotype.

Gene mutations found within MPAL are a mixture of those commonly seen in both AML and ALL and as such cannot be used to define a lineage.¹² A study of 115 pediatric MPAL/ALAL cases showed 158 recurrently mutated genes, including the AML-associated genes FLT3, RUNX1, and CEBPA and the ALL-associated genes CDKN2A and ETV6. Most of the MPAL cases harbored mutually exclusive mutations of either WT1, ETV6, RUNX1, or CEBPA.^16,ZNF384 fusions are also commonly seen in B-ALL and have an overlapping gene-expression profile with ZNF384-rearranged B/myeloid MPALs.¹² AUL cases are characterized by frequent PHF6 mutations and trisomy 13.¹¹ 

T/myeloid MPAL cases possess a more heterogeneous group of driver mutations, with fusions that overlap with T-ALL such as ZEB2::BCL11B, NUP214::ABL1 and fusions with ETV6 involving multiple different partners.¹⁶ Early T-cell precursor (ETP) ALL, characterized by expression of CD7, weak or absent CD5, and 1 or more myeloid/stem cell markers, has been shown to have a gene-expression profile that is intermediate between T/myeloid MPALs and T-ALL.¹⁷ The coexpression of myeloid markers and CD3 makes distinguishing ETP-ALL from T/myeloid MPAL problematic; molecular data blur the increasingly artificial distinction between these entities. The BCL11B cases present with absence of CD5 and cCD3 or MPO expression, resulting in variable classification. Moreover, emerging work is showing that the biology of immature leukemias is explained by diverse genomic alterations converging on a conserved set of stem cell genes showing phenotypic ambiguity: ETP, MPAL, or neither. This further suggests that genetic alterations rather than phenotypic expression may, in the future, be a better way of classifying these leukemias.¹⁶ 

The currently recommended treatment approach for MPAL in both pediatric and adult patients is induction therapy with an ALL regimen.^17-19 Patients with MPAL with BCR::ABL1 benefit from addition of a tyrosine kinase inhibitor.¹⁹ Immunophenotypic properties of MPAL are not stable over time: both variable proportions of different lineages and phenotypic alterations are known as lineage switch. Most cases of lineage transformation involve a switch from ALL to AML, and a retrospective review can often reveal the presence of an unrecognized or underappreciated malignant subclonal population present in the initial specimen, which displayed more chemoresistance and emerged after eradication of the chemosensitive dominant clone.²⁰ The selective pressure brought on by specific therapies can influence these transformations, especially in MPAL (eg, resultant AML after the use of B-cell–specific therapy such as blinatumomab). However, frank ALL to AML lineage switch must be distinguished from the B-monocytic transition commonly seen with ALL with DUX4-rearrangements and other alterations early in therapy,²¹ in which such changes do not affect prognosis with continued ALL therapy.

Contribution: O.K.W. drafted the first version of the manuscript based on discussions of a clinical advisory committee meeting; and all other authors participated in the classification draft and reviewed and edited the final manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Olga K. Weinberg, UT Southwestern Medical Center BioCenter, 2230 Inwood Rd, EB03.220G, Dallas, TX 75235; e-mail: olga.weinberg@utsouthwestern.edu.