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WHO Classification of Tumours
Overview: Acute leukaemias of ambiguous lineage


Definition
Acute leukaemias of ambiguous lineage encompass those leukaemias that show no clear evidence of differentiation along a single lineage. They include leukaemias with no lineage-specific antigens (acute undifferentiated leukaemia (AUL) and those with blasts that express antigens of more than one lineage to such a degree that it is not possible to assign the leukaemia to any one lineage with certainty (mixed phenotype acute leukaemias, MPAL). The latter can either contain distinct blast populations, each of a different lineage, or one population with multiple antigens of different lineages on the same cells, or a combination.

Terminology
Historically, there has been confusion both in the terminology and definition of MPAL. The term acute bilineal (or bilineage) leukaemia has been applied to leukaemias containing separate populations of blasts of more than one lineage, and the term biphenotypic leukaemia to those containing a single population of blasts coexpressing antigens of more than one lineage

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Hanson CA, Abaza M, Sheldon S, Ross CW, Schnitzer B, Stoolman LM (1993)
Acute biphenotypic leukaemia: immunophenotypic and cytogenetic analysis.
Br J Haematol 84: 49-60




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Legrand O, Perrot JY, Simonin G, Baudard M, Cadiou M, Blanc C, Ramond S, Viguié F, Marie JP, Zittoun R (1998)
Adult biphenotypic acute leukaemia: an entity with poor prognosis which is related to unfavourable cytogenetics and P-glycoprotein over-expression.
Br J Haematol 100: 147-55




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Matutes E, Morilla R, Farahat N, Carbonell F, Swansbury J, Dyer M, Catovsky D (1997)
Definition of acute biphenotypic leukemia.
Haematologica 82: 64-6




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Sulak LE, Clare CN, Morale BA, Hansen KL, Montiel MM (1990)
Biphenotypic acute leukemia in adults.
Am J Clin Pathol 94: 54-8



, although sometimes the latter term also encompassed bilineal leukaemia. Here the term mixed phenotype acute leukaemia applies to this group of lesions in general, and, as defined below, the more specific terms B/myeloid (B/MY) and T/myeloid (T/MY) leukaemia to refer to leukaemias containing the two lineages specified, ­irrespective of whether one or more than one population of blasts is seen.

Some well-defined myeloid leukaemic entities may have immunophenotypic features that might suggest that they be classified as B/myeloid (B/MY) or T/myeloid (T/MY) leukaemias. However, MPAL, as defined here, excludes cases that can be classified in another category, either by genetic or clinical features. These specifically include cases with the recurrent acute myeloid leukaemia (AML)-associated translocations t(8;21), t(15;17) or inv(16); the first of these especially frequently expresses multiple B-cell markers

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Tiacci E, Pileri S, Orleth A, Pacini R, Tabarrini A, Frenguelli F, Liso A, Diverio D, Lo-Coco F, Falini B (2004)
PAX5 expression in acute leukemias: higher B-lineage specificity than CD79a and selective association with t(8;21)-acute myelogenous leukemia.
Cancer Res 64: 7399-404



. In addition, cases of leukaemia with FGFR1 mutations are not considered T/MY leukaemias. Cases of chronic mye­­logenous leukaemia (CML) in blast crisis, MDS-related AML and therapy-­related AML should be classified primarily as such, even if they have a mixed phenotype, with a secondary notation that they have a mixed phenotype.

The diagnosis of ambiguous lineage leukaemias rests on immunophenotyping. Flow cytometry is the preferred method for establishing the diagnosis, especially when a diagnosis of MPAL is dependent upon demonstrating coexpression of lymphoid and myeloid differentiation antigens on the same cell. Cases in which the dia­gnosis rests on demonstration of two distinct leukaemic populations with a different phenotype may also be established by immunohistochemistry in tissue sections, or with cytochemical stains for myeloperoxidase on smears coupled with flow cyto­metry to detect a leukaemic B or T lymphoid population.

Myeloid component of MPAL
The myeloid component of an MPAL can be recognized in one of three ways (Table 7.01): 1) When there are two or more distinct populations of leukaemic cells, one of which would meet immunophenotypic criteria for acute myeloid leukaemia (with the exception that this population need not comprise 20% of all nucleated cells) 2) When there is a single population of blasts that by itself would meet criteria for B acute lymphoblastic leukaemia (B-ALL) or T acute lymphoblastic leukaemia (T-ALL) and the blasts also express myeloperoxidase, most frequently shown by flow cyto­metric positivity, on blast cells coexpressing lymphoid markers. The myeloid lineage antigens CD13, CD33 and CD117 are not specific enough to allow identification of a mixed phenotype leukaemia. 3) When there is a single population of cells that by itself would meet criteria for B or T-ALL in which the blasts also show unequivocal evidence of monoblastic differentiation: either diffuse positivity for non-specific esterase or expression of more than one monocytic marker such as CD11c, CD14, CD36, CD64 or lysozyme. The first of these three instances would previously have been considered "bilineage leukaemia" while alternatives 2 and 3 represent what would have been termed "biphenotypic leukaemia".

T-cell component of MPAL
The T-cell component of an MPAL is re-cognized by strong expression of cytoplasmic CD3, either on the entire blast population, or on a separate subpopulation of leukaemic cells (Table 7.01). Surface CD3, though rare, also indicates T-cell lineage. Expression of cCD3 is best determined by flow cytometry using relatively bright fluorophores such as phycoerythrin or allophycocyanin, and should be as bright or nearly as bright as that of normal resi­dual T cells present in the sample. T-cell lineage can also be demonstrated by CD3 expression on blasts by immunohisto­chemistry on bone marrow biopsies, though it should be noted that polyvalent T-cell antibodies used in immunohistochemistry also react with the zeta (ζ) chain of the T-cell receptor present in the cytoplasm of NK-cells, and are thus not absolutely T-cell specific.

B-cell component of MPAL
In contrast to what is described above with myeloid and T-cell lineages, no single marker is sufficiently specific to indicate B-cell differentiation with certainty, so that a constellation of findings is needed (Table 7.01). B-cell differentiation can be recognized when there is a distinct subpopulation of cells that by itself meets criteria for B-ALL. When only one population of cells is present, then B-lineage assignment requires either 1) strong CD19 expression coupled with strong expression of at least one of the following antigens: CD10, CD79a or cCD22; or 2) weak CD19 expression coupled with strong expression of at least two of the following: CD10, CD79a and cCD22. Rarely, a case may be assigned as B lineage even if CD19 is negative, though care must be taken when doing this because of the relative lack of specificity of CD10 and CD79a.

Diagnostic considerations
Cases of MPAL based on one criterion at diagnosis (e.g. "biphenotypic leuk­aemia") may change over time or at relapse to the other ("bilineage leukaemia"), or vice versa. Also, following therapy, persistent disease or relapse may occur as either pure ALL or AML. Some cases of what has been termed "lineage switch"

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Pane F, Frigeri F, Camera A, Sindona M, Brighel F, Martinelli V, Luciano L, Selleri C, Del Vecchio L, Rotoli B, Salvatore F (1996)
Complete phenotypic and genotypic lineage switch in a Philadelphia chromosome-positive acute lymphoblastic leukemia.
Leukemia 10: 741-5




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Reardon DA, Hanson CA, Roth MS, Castle VP (1994)
Lineage switch in Philadelphia chromosome-positive acute lymphoblastic leukemia.
Cancer 73: 1526-32



may reflect this phenomenon.

Ambiguous lineage leukaemias are rare and account for less than 4% of all cases of acute leukaemia. Many cases of what have been reported as undifferentiated leukaemia can be demonstrated to be leukaemias of unusual lineages, and many cases of what have been reported as biphenotypic acute leukaemias may in fact represent acute lymphoid or myeloid leukaemias with cross-lineage antigen expression, so that the actual frequency may even be lower. These leukaemias occur both in children and adults but more frequently in the latter, although some subtypes of MPAL may be more common in children

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Killick S, Matutes E, Powles RL, Hamblin M, Swansbury J, Treleaven JG, Zomas A, Atra A, Catovsky D (1999)
Outcome of biphenotypic acute leukemia.
Haematologica 84: 699-706




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Owaidah TM, Al Beihany A, Iqbal MA, Elkum N, Roberts GT (2006)
Cytogenetics, molecular and ultrastructural characteristics of biphenotypic acute leukemia identified by the EGIL scoring system.
Leukemia 20: 620-6



.

A variety of genetic lesions have been reported in ambiguous lineage leukaemias, especially MPAL. Two of these, the t(9;22) (q34;q11) BCR-ABL1 translocation, and translocations associated with the MLL gene occur frequently enough and are associated with distinctive features that they are considered as separate entities.


> Mixed pnenotype acute leukaemia, NOS - rare types
Some cases of leukaemia have been seen in which leukaemic blasts show clear-cut evidence of both T and B lineage commitment as defined above. This is a very rare phenomenon, with a frequency that is likely lower than what has been reported in the literature. As strictly applied, the most recent EGIL criteria for biphenotypic leukaemia (scores higher than 2 in more than one lineage), which assigned 2 points to CD79a expression {53, 187} would likely overestimate the incidence of B/T leukaemia because CD79a can be detected in T-ALL {1750}. In assigning B lineage to a case of T-cell leukaemia, CD79a and CD10 should not be considered as evidence of B-cell differentiation. There have also been a few cases with evidence of trilineage (B, T and myeloid lineage) assignment. Overall there are too few cases of either of these to make any specific statements about clinical features, genetic lesions or prognosis.

To date, there have been no reports of B or T/megakaryocytic or B or T/erythro - leukaemias. Because it has been suggested that erythroid and megakaryocytic lineages are the earliest to branch off from the pluripotent haematopoietic stem cell, leaving progenitor cells with T, B and myeloid potential {11}, neoplasms of these combinations of lineages may not occur. If they do occur, it is possible that the definitions used here might not detect all cases, as these leukaemias would not be expected to express MPO.


> Other ambiguous lineage leukaemias
Under some circumstances leukaemias may express combinations of markers that do not allow classification as either AUL or MPAL as defined above, yet definitive classification along a single lineage may be difficult. Examples of such cases might include cases that express T-cell-associated but not T-cell-specific markers such as CD7 and CD5 without cytoplasmic CD3, along with myeloid-associated antigens such as CD33 and CD13 without myeloperoxidase. Such cases are best considered acute unclassifiable leukae mias. With more extended panels containing newer, less commonly used markers, such leukae mias might be able to be classified.







Michael J. Borowitz
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Michael J. Borowitz
Department of Pathology and Oncology
The John Hopkins Medical Institutions
Baltimore
USA




Marie-Christine Bene
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Marie-Christine Bene
Laboratory of Immunology
Nancy University UHP & Nancy CHU
Vandoeuvre-Les-Nancy
France




Nancy Lee Harris
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Nancy Lee Harris
Pathology-Warren 2
Massachusetts General Hospital
Boston
USA




Anna Porwit
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Anna Porwit
Department of Pathology
Karolinska University Hospital
Stockholm
Sweden




Estella Matutes
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Estella Matutes
Section of Haemato-Oncology
Institute of Cancer Research/Royal Marsden Hospital
London
UK