Flow cytometry:ETP-ALL

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Blastic plasmacytoid dendritic cell neoplasm (BPDCN)

This is a rare diagnosis but one that presents some practical diagnostic difficulties since BPDCN is derived from myeloid cells and therefore may have morphological and immunophenotypic characteristics which overlap with typical AML or acute undifferentiated leukaemia. Morphologically the primitive cells may resemble AML or have lymphoid morphology, and a skin rash is present in most cases. In view of the diagnostic overlap, correlation with morphology, histopathology and clinical information is important when making this diagnosis.

Using standard diagnostic panels, cases of BPDCN generally have the following features:

• BPCDN typically have features of primitive phenotype: the cells typically lie in the "blast area" - with dim to moderate CD45 expression with low side scatter. CD7, TdT, CD38 and HLA-DR are frequently expressed. CD34 is characteristically absent
• Lineage-defining markers of myeloid, T or B cell are not expressed: i.e. there is no expression of MPO, CD14 or lysozyme; cCD3 is not expressed, and CD19 is not expressed (as well as CD20, cCD22, CD79a)
• One or more myeloid lineage-associated features may be detected: expression of CD33 is relatively common (>50%), CD117 may also be expressed (<20%), CD13 may be found although is infrequent (<5%). Therefore cases may fit the criteria to assign myeloid lineage (two myeloid-associated markers).

Diagnosis is then based on specific criteria:

For cases meeting the criteria above, then the following are required:
Cells of BPDCN will express: bright CD56 and/or CD4
Then will also meet either Criteria set 1 OR Criteria set 2
Criteria for BPCN (set 1)	CD123 expressed together with one of: CD303, CD304, TCF4, TCL1
Criteria for BPCN (set 2)	CD123 not expressed but at least three of CD303, CD304, TCF4, TCL1 are expressed

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1. The immunophenotype of the blast cells should be consistent with their "primitive" nature

The immunophenotype of ETP-ALL requires careful consideration – this reflects that the condition was identified using gene expression profiling rather than by immunophenotype. Using immunophenotype to establish the diagnosis is therefore challenging and may underestimate the number of true cases.* and may not be easily separated from ALAL T/my or T-ALL

To diagnose ETP-ALL the cells need to meet specific criteria. These are given in the table below:

Requirement 1: Expressed antigens
1. T-lineage should be demonstrated: (may be heterogenous but should be expressed by ≥25% of blasts)Assignment of T-Lymphoid lineage in ETP-ALL 2. One or more myeloid antigen (CD11b, CD13, CD33, CD65, CD117) and/or stem cell antigens (CD34, HLA-DR) should be present Note CD7 is consistently positive in ETP-ALL and does not count as a stem cell antigen in this context
Requirement 2: Non-expressed antigens
*Required absence: CD3, CD1a and CD8 (<5% of blasts) Required absent/dim CD5 expression (<75% positive blasts)
NOTES
CD4 may have the same pattern

The ETP-ALL immunophenotype may also be established by immunohistochemistry.

Distinction from mixed-phenotype acute leukaemia (MPAL) requires that MPO is not expressed - in this context of ETP-ALL the WHO advocate a threshold of <3% to define negative myeloperoxidase expression (by cytochemistry or flow cytometry). This threshold is different from that of T/myeloid MPAL so may not be optimal but is retained at present.

• T-ALL cases that have an immunophenotype similar to ETP-ALL but where CD5 is expressed (≥75% of blasts) may be designated as “near-ETP-ALL”, but the clinical implications of such a designation remain unclear.