Differential Immunodiagnosis Of The Acute Leukemias

In ALL, the markers with the most reliable specificity are cytoplasmic CD3 (cCD3) for early T-cell ALL and cytoplasmic CD22 (cCD22) for early B-cell ALL, whereas antigens such as TdT, HLA-DR, CD2, CD7, CD19 and CD10, although informative and complementary for both diagnosis and prognosis, are not restricted to ALL. Somatic antigen receptor proteins also provide specific lineage assignment in that TCR protein expression is restricted to T-lineage ALL and ^H chains and LC are confined to B-lineage ALL and should be included in any primary acute leukemia immunophenotyping panel. Whereas pro tein expression is lineage-specific, TCR and Ig chain gene rearrangements occur across lineage borders286-289 and can be observed in AML, although rarely.290

In AML, intracytoplasmic myeloperoxidase (MPO) protein can be detected by antibody staining in leukemic cells that are negative for MPO by cytochemistry, which suggests the presence of a precursor MPO form lacking enzyme activity.291,292 Myeloperoxidase activity can be detected ultrastructurally in blasts lacking the enzyme by light microscopic cytochem-istry,293-295 which has prognostic significance inasmuch as such patients do poorly when treated with ALL treatment regi-mens.295 This observation is consistent with the proposed role of MPO as a mediator of the resistance of MPO-positive AML to vincristine by catalyzing the oxidative degradation of the drug.296 Reports on MPO protein detection in ALL297,298 must be viewed with caution and should only be considered if optimal fixation-permeabilization media were used and, most importantly, if data refer unequivocally and exclusively to gated lymphoblasts. Expression of MPO protein is specific for AML, whereas MPO gene expression, albeit at low levels, is observed in some cases of ALL, frequently in association with the Philadelphia chromosome.299,300 Myeloperoxidase gene expression has been detected in rare cases of CD7+CD5┬▒CD2- TALL, together with the finding of CD3e or CD3S mRNA in these lymphoblasts.301 Lack of or decreased levels of immuno-logically detectable MPO protein in cases of immature AML or acute monocytic leukemia (AMOL)292,294 are due to differential regulation of MPO gene transcription.302

It is mandatory to test leukemic cells for at least three myeloid surface antigens, CD33, CD13, and CD65 or CD65s as this trio will identify more than 90 percent of AMLs.303-305 In combination, these antigens are not expressed in ALL199,306 (Paietta E, unpublished observation) and cells with lymphoid morphology that express all three myeloid antigens should be tested carefully to rule out AML. In fact, reports of CD33+CD13+CD65s+ triple-positive ALL usually base the diagnosis of ALL on FAB criteria, lack MPO determination by flow, and/or use a 20 percent or lower arbitrary cutoff point to establish myeloid antigen positivity, although they neglect to gate strictly on blast cells, thereby allowing for the contamination of their observations with normal myeloid cells.

Provided that sufficient material is available, there should not be a difference in the composition and the size of antibody panels used to test AML versus ALL. The risk of rendering an immunodiagnosis from limited marker panels lies in the commonly observed coexpression of two lymphoid- or myeloid-associated antigens by blast cells from the opposite cell lineage and the finding that several antigens, particularly those expressed early in hematopoiesis, may be expressed by leukemias of all cell lineages (e.g., TdT, CD33, CD13, CD7, CD10).

An ongoing problem in clinically interfaced laboratory studies in leukemia is that investigators first define leukemia subtypes by strict FAB rules (for instance, more than 3 percent MPO-positive blast cells define AML) and subsequently study their immunophenotype. In other words, a classification system based on imprecise morphologic criteria subject to interpreta tion is correlated with the expression of cell characteristics that at the time the FAB system was designed were not taken into consideration and in many instances were not even known yet. As a result, the literature is replete with morphologically defined leukemia subtypes, which, when examined carefully, are immunophenotypically unequivocally committed to a cell lineage that contradicts the morphologic diagnosis. In extreme cases, even the diagnostic power of proven lineage-specific markers, such as cCD3, is ignored in favor of FAB criteria.307,308 The error in this approach is that a classification system with known prognostic limitations distorts valuable immunopheno-typic and clinically relevant information that would be provided by those cases if they were identified by their antigenic features. That this is in fact the case is best demonstrated by clinical results in patients who fail induction treatment that would be appropriate for the diagnosis suggested by morphology but who subsequently respond to therapy for the opposite cell lineage in agreement with the immunophenotypic informa-

tion.307

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