How to think about acute leukemia of ambiguous lineage

Visual Abstract

Abstract

Classification of acute leukemia involves assigning lineage by resemblance of blasts to normal progenitor cells. This approach provides descriptive information that is useful for disease monitoring, provides clues to pathogenesis, and can help to select effective chemotherapeutic regimens. Acute leukemias of ambiguous lineage (ALAL) are those leukemias that either fail to show evidence of myeloid, B-lymphoid, or T-lymphoid lineage commitment or show evidence of commitment to more than 1 lineage, including mixed-phenotype acute leukemia (MPAL). The different treatment regimens for acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) make ALAL a challenge both diagnostically and therapeutically. Current classification criteria have reduced the reported incidence of mixed lineage leukemias by emphasizing fewer markers and categorizing some biphenotypic leukemias with recurrent cytogenetic abnormalities as other entities. Several recent studies have explored the genomic and epigenetic landscape of MPAL and emphasize the genomic heterogeneity of MPAL. Two classification proposals of myeloid malignancies recently been published and include International Consensus Classification and fifth edition of the World Health Organization Classification of Haematolymphoid Tumours. Our review aims to discuss the diagnostic challenges in the setting of classification updates, recent genomic studies, and therapeutic strategies in this poorly understood disease.

Learning Objectives

• Understand how mixed-phenotype acute leukemia classification has changed over the years and current criteria have reduced the number of markers and included genomic findings

• Recognize the genomic data for mixed-phenotype acute leukemia reveal mutations commonly seen in both acute myeloid leukemia and acute lymphoblastic leukemia

CLINICAL CASE

A 56-year-old male with a history of diabetes, coronary artery disease, and heart failure presented with 4 months of fatigue and pancytopenia (white cell count 2.7  ×  10⁹/L, hemoglobin 9.0  g/dL, platelets 89  ×  10⁹/L) with 25% circulating blasts. The blasts were medium to large and demonstrated scant cytoplasm, large nuclei, open chromatin, and prominent nucleoli. Flow immunophenotypic analysis revealed a 49% population of immature cells that were positive for CD34, CD19, CD22 (partial), CD79a, and terminal deoxynucleotidyl transferase (TdT), but were negative for CD10, CD20, CD33, CD123, and myeloperoxidase (MPO); the analysis also revealed a 13% myeloblast population that was positive for CD34, CD13, CD15, CD64 (partial), and MPO but negative for TdT, CD19, CD20, and CD22 (Figure 1). By immunohistochemistry, blasts were mostly positive for CD34 and negative for lysozyme and E-cadherin. Subsets of blasts were positive for CD117, TdT, and PAX5. Cytogenetic studies showed an abnormal complex male karyotype with 3 related clones demonstrating multiple structural and numerical abnormalities, as follows: 46 - 71,XY,add(1)(p22),add(4)(q21),der(4)t(4;9)(q31.1;q22),add(5)(q11.2), -7,der(9)t(1;9)(p22;q13), +10, +ider(11)(q10)del(11)(q11q21)x3, t(12;15)(p13;q22),del(14)(q22),add(17)(p11.2), +22/46 - 53,idem,add(2 (q37)/49,idem,der(2;11)(q10;q10),add(18)(q23)/46,XY. Next generation sequencing studies revealed mutation and amplification of KMT2A; mutation and loss of TP53; mutation of BCORL1; amplification of IDH1; loss of ASXL1, CDKNB1, ETV6, and TET2; losses of chromosomes 16, 5q, and 7q; and loss of heterozygosity of FBXW7.

Figure 1.

Clinical case.

Introduction and historical overview

Acute leukemias of ambiguous lineage (ALALs) include biologically diverse leukemias that fail to show commitment to either the myeloid or lymphoid lineages or that do display evidence of commitment to more than 1 lineage. Cases in the former group are typically referred to as acute undifferentiated leukemias (AULs), while those in the latter group are identified as mixed-phenotype acute leukemias (MPALs).

ALALs are uncommon, and the true incidence of these leukemias is likely to be lower because of the prior inclusion of otherwise typical acute lymphoid or myeloid leukemia with aberrant cross-lineage antigen expression that does not meet current lineage definitions, as well as cases with differentiation along more unusual lineages. Previous reports suggest that MPAL accounts for 2% to 3% of acute leukemia cases and 0.35 cases per 1 million person-years with a male predominance (~1:1.6).^1-3 Among cases of MPAL, it has been estimated that the B-myeloid subtype accounts for 59% of cases, whereas the T-myeloid, B/T, and more rare trilineage subtypes account for 35%, 4%, and 2% of cases, respectively. According to Surveillance, Epidemiology, and End Results database records between 2000 and 2016, a total of 1888 cases of AUL were diagnosed (1.34 per 1 million person-years).^4,5

The markedly different treatment regimens for acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) make ALAL a challenge both diagnostically and therapeutically.³ One of the major challenges in the subclassification of ALAL in general and MPAL in particular is that classification based primarily on driver mutations does not explain the phenotypic diversity seen in these leukemias.⁶ Reported survival outcomes for MPAL have ranged between 36% and 80%; however, since patients with an MPAL phenotype were excluded from frontline clinical trials until recently, all available treatment and outcome data for MPAL is retrospective. While most patients with MPAL respond to ALL- directed therapy, there is no clear consensus on how to treat this heterogeneous disease.⁷

Diagnostic criteria and challenges

To create a standardized diagnostic approach, different classification systems have been proposed over the years. The European Group for the Immunological Characterization of Leukemias (EGIL) algorithm was developed between 1995 and 1998 and was based on a point system with a requirement of more than 2 points in 2 separate lineages for a diagnosis of MPAL.⁸ In 2008, the World Health Organization (WHO) classification system grouped bilineal and biphenotypic acute leukemias into a unified heading of MPAL and further provided more stringent criteria using fewer but more lineage-specific antibodies.^9,10 The 2016/2017 WHO revisions essentially reaffirmed this decision.¹⁰ Most recently, 2 classification proposals of myeloid malignancies have been published and are hereby named as International Consensus Classification (ICC) and 5th edition of the WHO Classification of Haematolymphoid Tumours. (WHO-HAEM5).^11,12

The total sum of MPAL blasts in blood or bone marrow must be greater than or equal to 20%, with the individual populations of bilineal MPAL classified according to standard AML and ALL criteria. In contrast, biphenotypic MPAL requires the use of lineage-assignment criteria. Some retrospective clinical reviews have suggested that patients with bilineal MPAL have a worse prognosis, although classification challenges in biphenotypic leukemia complicate this issue.¹³ In practice, the accurate identification of minor blast populations of divergent lineage presents a challenge in diagnosing bilineal acute leukemia. Identification of immunophenotypic aberrancies can be essential to differentiating a small bilineal blast population from residual normal myeloid blasts or hematogones (physiologic B-cell precursors). In addition, it is important to consider the possibility of a monocytic blast population coexisting with ALL, which most often occurs in the context of KMT2A translocations, because monocytic blasts often resemble normal monocytes in flow cytometry analysis.¹⁴

When only a small percentage of aberrant clones of divergent lineage are identified (<5%), a diagnosis of MPAL can be rendered if clear immunophenotypic aberrancies are identified.⁶ Identification of such aberrancies is essential to differentiate a small bilineal blast population from normal myeloid or B-cell precursors. For cases in which aberrant clones represent less than 5% of all cells, ICC classification recommends that a diagnosis should be based on the major leukemic population with a descriptive modifier; an example would be “Predominantly ALL with a small leukemic population of myeloid lineage of uncertain significance detected.” Additional details should then be provided in the body of the report.

For biphenotypic MPAL, T-lineage can be assigned when blasts express cytoplasmic or surface CD3 and WHO-HAEM5 recommends that at least a portion of leukemic cells should have a level of CD3 expression exceeding 50% of that seen on normal mature T cells in the same sample. It is suggested that the best way to make such assessments is through flow cytometry using a bright fluorochrome because that method provides an increased dynamic range.¹² T lineage can also be demonstrated by immunohistochemistry for CD3 withjing the caveat that the polyclonal antibodies used may react with the CD3 ζ chain also present in normal NK cells and so may not be entirely T-lineage specific.¹²

Assignment of B lineage requires multiple lines of evidence: strong expression of CD19 plus 1 additional B-cell marker (CD79a, CD22, or CD10) or weak CD19 expression and 2 additional markers. WHO-HEAM5 recommends that expression of CD19 approaching the level seen on normal B progenitors (exceeding 50% of the level seen on normal B progenitors on at least a portion of the leukemia cells) has considerable specificity for B lineage, but it still should occur with expression of 1 or more other B-lineage antigens to define B lineage.¹² PAX5 by immunohistochemistry is considered to have a high specificity for B lineage by ICC, but, as WHO-HEAM5 points out, there is relatively little literature describing its lineage specificity in ALAL or MPAL; therefore, its use for this purpose should be considered tentative.

Table 1.

Acute leukemia of ambiguous lineage entities as defined by WHO-HAEM5 and ICC classification

Acute leukemia of ambiguous lineage
MPAL with defining genetic alterations	ICC	WHO-HAEM5
MPAL with BCR::ABL1
	MPAL, with t(v;11q23.3); KMT2A rearranged
	MPAL with ZNF384 rearrangement
	MPAL with BCL11B activation	ALAL with BCL11B rearrangement
MPAL with defining immunophenotypic changes	B/Myeloid MPAL T/Myeloid MPAL B/T/Myeloid MPAL B/T MPAL	MPAL, rare types
Acute undifferentiated leukemia (AUL)*
ALAL*, NOS

^* AUL is defined by a lack of lineage-specific markers.

^** Rare cases may exist in which the criteria for MPAL are not fulfilled, the criteria for acute leukemia of a single lineage are not fulfilled, and the patient has at least 1 of the lineage-specific markers in Table 2. Such cases are best referred to as acute leukemia of ambiguous lineage, not otherwise specified (ALAL, NOS).

Expression of MPO or at least 2 monocytic markers (nonspecific esterase, CD11c, CD14, CD64, or lysozyme) denotes myeloid lineage. This can be investigated using flow cytometry, immunohistochemical stains, or cytochemical stains. WHO-HEAM5 recommends that the tests should show that the MPO level exceeds 50% of that seen on mature neutrophils or show a variable pattern reminiscent of that seen on normal CD34- positive myeloid progenitors. The role of MPO in defining myeloid lineage or mixed lineage remains a topic of active discussion.² The WHO classification does not set a threshold for MPO positivity, and cases with an otherwise B-ALL immunophenotype with MPO as the sole aberrancy present a unique challenge, particularly as some reagents show nonspecific binding to MPO, and MPO mRNA can frequently be detected in B-ALL.¹⁵ ICC states that no specific threshold is set for MPO positivity, but care should be taken in instances when fewer than 10% MPO-positive cells are seen in flow cytometry. The presence or absence of MPO is also a discriminating factor between the diagnosis of early T-cell precursor acute lymphoblastic leukemia and MPAL, T-myeloid subtype. ETP-ALL is defined by an immature hematopoietic phenotype with nonspecific myeloid features, such as CD13 or CD33.¹⁶ The most prominent distinction between T-myeloid MPAL and ETP-ALL is the presence of MPO (or, rarely, monocytic markers) in T-myeloid MPAL.

Historically, criteria for AUL have remained consistent in that a diagnosis of AUL requires the lack of expression of lineage-specific markers. AUL lacks cCD3, MPO, CD19, cCD22, and CD79a and usually expresses only 1 surface lineage marker (ie, CD13, CD33, or CD7). As such, CD34 and HLA-DR expression with or without CD38 or TdT is common. Based on a recent multi-institutional study, cases with a partial or single full myeloid marker can be considered as AUL.¹⁷ ICC classification clarified that cases with an immunophenotype of AUL but cytogenetic abnormalities and molecular findings diagnostic of AML with myelodysplasia-related cytogenetic abnormalities or AML with myelodysplasia-related mutation should be diagnosed as AML.

Table 2.

Markers used in lineage assignment in WHO-HAEM4 classification

Myeloid	MPO
	Or
	Evidence of monocytic differentiation: 2 of NSE, CD11c, CD14, and CD64
B lineage	Strong CD19 and 1 strongly expressed marker: CD79a, cCD22, or CD10
	Or
	Weak CD19 and 2 strongly expressed markers: CD79a, cCD22, or CD10
T lineage	cCD3 (at level of expression of background T cells) or surface CD3

NSE, nonspecific esterase.

Table 3.

Classification updates to the lineage markers

			WHO-HAEM5	ICC classification
B lineage	Strong CD19 and	1 or more marker expressed: CD10, CD22 or CD79a	CD19 intensity in part exceeds 50% of normal B-cell progenitor by flow cytometry	CD19 expression should be at least similar to that seen in stage I B-cell precursors or mature B-cells
	Weak CD19 and	2 more strongly expressed: CD10, CD22, CD79a
				Consider immunohistochemical stains for B-lineage PAX5, OCT2, BOB1
T lineage	CD3 (surface or cytoplasmic)		Intensity in part exceeds 50% of mature T-cell level by flow cytometry
Myeloid lineage	MPO or		MPO intensity in part exceeds 50% of mature neutrophil level Immunocytochemistry positive with non-zeta chain reagent
	Monocytic differentiation	NSE, CD64, CD11c, CD14 or lysozyme

^* The immunophenotypic criteria described here are for cases of suspected MPAL and are not required for straightforward cases of AML or ALL.

Genetic analysis

Studies have shown that distinct subsets of MPALs are classified based on recurring genetic alterations. Two distinct classes of MPALs are classified based on cytogenetics: MPAL with BCR::ABL1 and MPAL with KMT2A rearrangements. MPAL with KMT2A are more common in the pediatric age group (and especially infants) and account for about 10% of MPALs, and BCR::ABL1 in adults accounts for about 20% to 30% of MPALs.¹⁸ These conditions are commonly associated with the B-myeloid phenotype. B-myeloid MPALs also frequently show del(1)(p32), trisomy 4, del(6q), 12p11.2 aberrancies, and near tetraploidy.¹⁹

In addition to MPAL with BCR::ABL1 and MPAL with KMT2A gene rearrangements, 2 new molecularly defined entities have been recognized in both classifications, respectively: B-myeloid MPAL with ZNF384 rearrangements and T-myeloid MPAL with BCL11B-activation. A recent study reported ZNF384 rearrangements (with TCF3, EP300, TAF15, and CREBBP) in as many as 48% of the pediatric B-myeloid MPALs, although this aberration has not been identified in adult patients.²⁰ The genomic landscape of B-ALL with ZNF384-r and MPALs with ZNF384-r was similar, with exception of KDM6A, which was observed only in MPALs, and these cases showed higher FLT3 expression.²⁰ ALAL with rearrangements leading to deregulation of BCL11B (BCL11B activated) comprise 10% to 15% of MPALs and are further enriched in patients with MPAL T-myeloid cases, representing up to one-third of this population.²¹

Genetic studies have shown that mutations seen in MPAL cases appear to be a mixture of those commonly seen in both AML and ALL. AML-associated epigenetic modifiers TET2, EZH2, and ASXL1 and ALL-associated deletions of IKZF1 have been frequently reported in B-myeloid leukemias.⁴ An extensive study of the genetics of childhood MPAL on 115 cases of ALAL identified 158 recurrently mutated genes, including the common AML-associated genes FLT3, RUNX1, CUX1, and CEBPA as well as the ALL-associated genes CDKN2 and ETV6.¹⁹ Interestingly, most of the MPAL cases harbored mutually exclusive mutations of WT1, ETV6, RUNX1, or CEBPA. Whole-exome sequencing identified frequent mutations of epigenetic modifiers, including DNMT3A, and mutations of activated signaling pathways, tumor suppressors, and transcription factors that were also frequent in MPAL patients.²² Xiao et al reported similar mutations, including PHF6, DNMT3A, TET2, WT1, RUNX1, FLT3, ETV6, and ASXL1.²³ Takahashi et al described frequent occurrence of NOTCH1, RUNX1, DNMT3A, and IDH2 in 38 adult patients with MPAL.²⁰ A series of 9 B/T MPAL cases revealed that the genomic landscape that strongly resembles that of T-ALL subgroups is associated with early developmental arrest, while genetic alterations that are common in B-ALL are rarely seen.²⁴

Optical genome mapping (OGM) is a groundbreaking DNA-based nonsequencing technology that offers a more comprehensive and precise assessment of cytogenomic architecture and allows whole genome examination with remarkably high resolution that will be greatly needed to study ALAL in more depth.²⁵

CLINICAL CASE (continued)

The diagnosis in the described case is favored to be MPAL, B-myeloid based on immunophenotype, but the complex karyotype suggests AML. MPAL with a complex karyotype is a rare, somewhat controversial entity with limited clinical and genetic data.²⁶ Both the WHO-HEAM5 classification and ICC classification exclude MPAL with complex karyotype from the MPAL category to include them in the myelodysplasia-related categories of AML. TP53 is one of the most frequently mutated genes across all cancer types. AML patients with TP53 mutations have a nearly uniformly poor prognosis, suggesting a biologically homogeneous single group.^27,28 In the updated ICC classification, AML with mutated TP53 is now recognized as a separate single entity¹¹ and this case would be considered as AML with mutated TP53.

Table 4.

Comparative mutational analysis of MPAL in recently published large series

Mutation	MPAL B-myeloid			MPAL T-myeloid		B/T MPAL
Reference	MDACC21	SKACC23	SJCCC20	SKACC23	SJCCC20	BCH24
Number of patients	N  =  13	N  =  12	n  =  35	n  =  11	n  =  48	n  =  9
ASXL1	3 (23%)	1 (8%)	-	1 (9%)	-	1 (11%)
SRSF2	3 (23%)	-	-	-	-	0
TET2	2 (15.4%)	2 (16%)	-	2 (18%)	-	0
FLT3	3 (23%)	0	6 (17%)	2 (18%)	21 (44%)	0
CEBPA	0	1 (8%)	0	1 (9%)	5 (10.4%)	0
RUNX1	6 (46%)	1 (8%)	8 (23%)	1 (9%)	3 (6%)	1 (11%)
PTNP11	1 (7.7%)	0	6 (17%)	0	4 (8.3%)	2 (22%)
ZNF384	0	0	15 (43%)	0	0
NOTCH1	0	1 (8%)	0	0	8 (17%)	1 (11%)
WT1	1 (7.7%)	1 (8%)	2 (6%)	3 (27%)	20 (41%)	1 (11%)
PHF6	0	1 (8%)	1 (3%)	2 (18%) + 3 (27%)	0	5 (55%)
DNMT3A	1 (7.7%)	0	-	4 (36%)	-	1 (11%)
ETV6	0	0	8 (23%)	2 (18%)	12 (25%)	2 (22%)
NRAS	3 (23%)	1 (8%)	11 (31%)	2 (18%)	4 (8.3%)	0
KRAS	0	2 (16%)	3 (8.5%)	2 (16%)	3 (6%)	0
TP53	1 (7.7%)	0	2 (6%)	2 (18%)	0	2 (22%)

Most studies describe frequency of an abnormal karyotype in more than 50% of MPAL cases and fewer cases with normal karyotype. The WHO classification of MPAL explicitly excludes cases with genomic lesions pathognomonic for AML, including those that consist of t(8;21), inv(16), or t(15;17). The current WHO-HEAM5 and ICC classifications are not clear on how ALAL- and myelodysplastic syndrome (MDS) related mutations should be classified, but both suggest excluding cases that qualify for a diagnosis of AML-myelodysplasia related from classification as ALAL. However, the clinical and genetic features of ALAL with secondary mutations remain poorly characterized and should be addressed in future studies.

Mimics of ALAL

ALAL with rearrangements leading to deregulation of BCL11B (BCL11B-activated) comprise 10%-15% of MPALs and are further enriched in patients with MPAL T-myeloid, representing up to one-third of this population.⁶ 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 neoenhancers that also deregulate BCL11B expression.

Myeloid/lymphoid neoplasms with eosinophilia with FGFR1 rearrangement was initially described as 8p11 myeloproliferative syndrome due to the frequent involvement of the short arm of chromosome 8p11.²⁹ The disease is known for its complex presentations, including myeloproliferative neoplasm (MPN), MDS/MPN, AML, T- or B-ALL/lymphoblastic leukemia/lymphoma (LBL), but also includes MPAL, frequently associated with peripheral blood and/or bone marrow eosinophilia. In such instances, the background features of MPN may be obscured by the blasts and become more obvious after myeloablative treatment. Hematologic malignancies with gene fusions involving FLT3 have also recently been included among myeloid/ lymphoid neoplasms with eosinophilia and tyrosine kinase gene fusions in the WHO-HAEM5 and ICC and often present as MPN with eosinophilia or as MDS/MPN, resembling chronic myelomonocytic leukemia, atypical chronic myeloid leukemia, juvenile myelomonocytic lukemia, or systemic mastocytosis associated with hematologic malignancy.²⁹ Extramedullary involvement is frequent, including T-ALL, MPAL, myeloid sarcoma and, rarely, B-ALL/LBL. Conventional cytogenetic analysis is reliable in demonstrating translocations at 8p11. The t(8;13) is the most common translocation, and, thus, ZMYM2 is the most common fusion-partner of FGFR1, but there are 14 additional fusion partners.

MLL-r leukemias make up approximately 10% of acute leukemias in all age groups. For the most part, however, leukemias arising from rearrangements of the MLL gene manifest as either ALL or AML, and only a minority of MPAL carry MLL rearrangements. An MPAL with a KMT2A rearrangement usually has a B-myeloid immunophenotype.³⁰ The PICALM:MLLT10 fusion is observed in approximately 7% of T-lymphoblastic leukemia/lymphoma (T-ALL), 0.3%-2% of AML, and, occasionally, MPAL and is notable for its association with a poor prognosis.³¹

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematologic malignancy that can involve the bone marrow, peripheral blood, skin, lymph nodes, and the central nervous system.^32,33 Immunophenotypically, BPDCN cells are positive for pDC-associated markers, including CD123, BDCA2 (CD303), and TCF4 and frequently express CD4, CD56, HLA-DR, and TCL1. The neoplasm may also express various other lymphoid- or myeloid- related markers, including CD2, CD5, CD7, CD33, CD38, CD68, CD117, HLA-DR, and TdT and can resemble MPAL in rare cases. Nevertheless, by definition, BPDCN cells are negative for lineage-specific antigens for B cells (CD19), T cells (CD3), and myeloid cells (MPO), and these markers should be thoroughly investigated. Atypical presentation of schwannoma, other solid tumors (including metastatic, carcinoma of unknown primary, and germ cell tumors), and infections should be considered, and biopsy and imaging studies should be performed.

In summary, MPAL comprises a heterogenous group of leukemias that are genetically, immunophenotypically, and clinically diverse. Advancements have been made in redefining the classification and discovering the genomic underpinnings of MPAL in the past decade. Addressing the knowledge gap and clinical therapies for acute leukemias of ambiguous lineage will require future prospective clinical trials.

Contributions

Olga K. Weinberg designed research, performed research, contributed vital new reagents or analytical tools, analyzed data, and wrote the paper.

Conflict-of-interest disclosure

Olga K. Weinberg: no competing financial interests to declare.

Off-label drug use

Olga K. Weinberg: There are no off label drug use discussion and nothing to disclose.