MPAL

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Important Note

Flow cytometry is an important element that may alert clinicians to a diagnosis of MPAL and has an advantage of rapidity. However conclusions may subsequently be modified (e.g. by results of immunohistochemistry, cytogenetics, or genetics). In some cases, findings will re-assign with significant treatment implications. It is important that this is acknowledged when assigning an initial diagnosis of MPAL.

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Frequency of MPAL types

• MPAL with features of Myeloid and B-lineage
• MPAL with features of Myeloid and T-lineage
• MPAL with features of T- and B-lineage
• acute Undifferentiated leukaemia

Following further analysis cases may be assigned additionally as: MPAL with t(9;22) (q34.1;q11.2); BCR-ABL1) MPAL with t(v;11q23.3); with KMT2A re-arrangement

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Requirements to assign myeloid lineage in MPAL The assignment of myeloid lineage recognises MPO expression to be a defining marker of myeloid lineage. However MPO is expressed only by around 80% of AML cases. The assignment of myeloid lineage can therefore also be made if monocytic lineage can be established, requiring at least 2 of 5 possible lineage markers to be detected (although only 3 of these can be established by flow cytometry).
Marker option 1
Demonstrate expression of Myeloperoxidase (MPO)	Myeloperoxidase expression alone may be sufficient to establish myeloid lineage, but be aware of the limitations: (1) intensity should be at least half of that of mature neutrophils in at least of proportion of cells measured by the same method; (2) there are circumstances where judgement is required (see notes).
Marker option 2
Demonstrate clear evidence of monocytic lineage	If MPO is not demonstrated then myeloid lineage may still be assigned through demonstration of monocytic features. This can be assigned by the detection of at least two of the following features: By flow cytometry: CD11c, CD14, CD64; by other approaches: lysozyme or non-specific esterase in malignant cells (enzyme cytochemistry)

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NOTE Interpretation of MPO positivity in MPAL The WHO classification advises that MPO expression is assessed by its intensity of expression. The is based on two considerations: 1. The techniques used to detect MPO do not have the same sensitivity: immunohistochemistry > flow cytometry > enzyme-cytochemistry. This means diagnosis of MPAL may be method dependent – expressing MPO as intensity allows expression to be compared with that of normal cells detected using the same method. 2. MPO is not entirely specific for myeloid lineage: this is particularly the case when expression is dim, so an element of subjectivity is allowed when interpreting (particularly when detected by more sensitive techniques such as immunocytochemistry). Some suggestions have been made that may aid interpretation (weak evidence base): Applying a threshold of >10% cells being positive for MPO may improve specificity. *Variability of expression level by blast cells may suggest partial maturation and support myeloid lineage origin. *The expression of other myeloid markers may allow greater confidence that MPO is lineage specific. Be aware of common patterns of aberrancy that are associated with specific alternative diagnoses: *Dim (weak) expression of MPO may be a feature of otherwise typical B-LL/LBL Burkitt-like entities may have strong MPO staining however other investigations will confirm their nature and other myeloid markers will be absent.

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Requirements to assign B-lymphoid lineage in MPAL For B-lymphoid lineage assignment the key lineage-marker is CD19. However, this marker has recognised aberrant expression in AML cases so additional criteria of expression intensity it is required that other markers must be expressed in addition to allow B-lineage assignment.
Marker option 1
Required Strong expression of CD19	To meet the definition of "strong" the expression intensity must exceed that of 50% of normal B lymphocytes
In addition 1 of: CD10, CD22, or CD79a (surface or cytoplasmic)	If CD19 is strong then B-lineage can be assigned if there is at least ONE of these additional markers
Marker option 2
Required Weak expression of CD19	To meet the definition of "weak" the expression intensity must be less than that of 50% of normal B lymphocytes
In addition 2 of: CD10, CD22, or CD79a (surface or cytoplasmic)	If CD19 is weak then B-lineage can be assigned only if there are at least TWO of these additional markers

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Requirements to assign T-lymphoid lineage in MPAL For T-lymphoid lineage assignment the key lineage-marker is CD3. Provided that this marker meets the intensity criteria then it is T-lineage defining in MPAL. This expression can be determined either by CD3 detection by flow cytometry OR by immunocytochemistry on trephine.
Marker requirement
Either CD3 surface or cytoplasm (strong)	To meet the definition of "strong" the expression intensity must exceed that of 50% of normal T lymphocytes
Or CD3 by immunocytochemistry (strong)	For immunohistochemistry it is important a non-zeta chain reagent is used