Hematopoietic Cell Lineagespecific Markers

Most of the antigens expressed by leukemic cells are lineage-associated, but few are lineage-specific. As a general rule, the most immature and the most mature markers of a given cell lineage are specific for that lineage. Figure 15-1 illustrates that the three antigens specific for the T- and B-lymphoid and the myeloid lineages are intracytoplasmic CD3, intracytoplasmic CD22, and intracytoplasmic myeloperoxidase protein, respectively. This conclusion is based on experience in leukemia

Figure 15—1. Antigenic overlap between the major cell lineages. Lineage-specific antigens are in bold letters. This figure also represents the minimal panel of antibodies and antigens that needs to be tested in a new case of acute leukemia.

immunophenotyping rather than on the fact that these antigens are the first to appear in normal immature cells committed to their cell lineage. In fact, there do exist immature AML cases that lack myeloperoxidase by antibody staining but do express surface CD33 and/or CD13 and lack any evidence of lymphoid, megakaryocytic, or erythroid differentiation. The intracellular localization of these crucial antigens is unfortunate because their detection by flow cytometry requires technical skill and experience. In fact, routine clinical laboratories may shy away from testing intracellular antigens by flow cytometry. Aside from choosing optimal fixation and permeabilization media,66-68 it is essential that data reflect gated blast cells. Following fixation and permeabilization, scattergram characteristics (size and granularity) of cells change, which makes cell populations collapse. Without the surface marker staining the leukemic cell population, blast cells can often no longer be separated from residual normal bone marrow or peripheral blood cell components. This may lead to the occasional misleading report of myeloperoxidase protein expression in ALL. The other pitfall in the interpretation of immunophenotypic myeloperox-idase detection in a case of presumed ALL results from the use of morphology as the gold standard in assigning lineage affiliation to a leukemic blast cell population. It cannot be emphasized enough that, although viewed together, neither morphology nor immunophenotyping forms the basis of the other's interpretation.

The proposed specificity of cytoplasmic CD79a for B-cell-related ALL requires some discussion. The CD79a (mb-1) polypeptide appears in the cytoplasm early during B-cell differentiation. Together with the later-appearing CD79b, it eventually associates in the B-cell membrane with immunoglobulin and has been assumed to be restricted to B cells, whether normal or neoplastic.69,70 Given its primary usefulness as an intracellular marker in characterizing B-lin-eage ALL, antibodies to CD79a were initially produced to intracellular epitopes of the molecule (clones HM47, JCB117). Recently, the diagnostic value of CD79a received an interesting boost when a new antibody to a cell surface epi-tope of CD79a, clone ZL7.4, was found to distinguish between B-cell chronic lymphocytic leukemia (surface CD79a-negative) and mantle cell lymphoma,71 as well as B-cell prolymphocytic leukemia and hairy cell leukemia (all surface CD79a-positive). Surprisingly, clone ZL7.4 reacts with intracellular structures in selected cases of AML, particularly of the immunophenotypically undifferentiated subtype (Paietta E, unpublished observation). Further studies are needed to explain this puzzling observation. The occasional observation of CD79a in the cytoplasm of T-ALLs72,73 and AML,74,75 particularly APL,75 even when using antibodies to intracellular CD79a epitopes, further confuses the lineage specificity of this antigen.

The more immature a leukemia subtype, the more the laboratory investigator or pathologist will have to rely on the detection of lineage-specific antigens, because other lineage-associated antigens, which usually assist in establishing the immunophenotypic diagnosis, are absent or sparse. The more mature a leukemia immunophenotype, the more easily the affiliation with a certain cell lineage is established. Therefore, lineage-specific mature antigens are less important in establishing lineage affiliation in the mature subtypes of acute leukemia, where they allow, however, for determining the level of blast cell differentiation. For instance, a leukemic blast cell mature enough to express B-cell-specific surface immunoglobulins will certainly express a series of B-lineage-associated cell surface markers (e.g., CD19, CD22, CD24), which will leave no doubt about lineage affiliation. However, the finding of monoclonal surface immunoglobulin will confirm the diagnosis of mature B-ALL, which has prognostic implications. In the case of T-ALL, the detection of surface T-cell receptor proteins will establish a mature subtype of the disease, and the lineage affiliation will be supported by the presence of additional T-cell markers. As a word of caution, only the expression of immunoglobulins and T-cell receptor proteins, but not immunoglobulin or T-cell receptor gene rearrangements, should be considered specific for their respective cell lineages. For the myeloid lineage, the most mature antigen is lactoferrin, which however is too mature to be found in a leukemic myeloblasts. All cell surface myeloid antigens have been seen on leukemic lymphoblasts and therefore cannot be considered specific for myeloid lineage.

The presence of one of the lineage-specific antigens determines lineage affiliation. It is astonishing and concerning that these antigens were not considered essential in a recent consensus meeting on optimal reagents for acute leukemia immunophenotyping.76 A blast cell population negative for any of the discussed lineage-specific antigens may fall into one of four categories: true lineage-negative acute leukemia, which is extremely rare; acute monocytic leukemia (AMOL), which is usually myeloperoxidase-negative; acute megakaryocytic leukemia (AMegL); and acute erythroleukemia (AEL). In each of these instances, comprehensive testing will yield the appropriate immunophenotypic diagnosis.

Antigens not established as lineage-specific are considered lineage-associated, and they represent the majority. A selection of antibodies and antigens tested routinely in acute leukemias is compiled in Table 15-1. Calling them lineage-associated means that although they occur predominantly in their respective cell lineage during normal hematopoiesis, they may be found on other cell lineages. They may appear transiently outside their predominant cell lineage during normal development (e.g., CD10 during normal T-cell differentiation) or on leukemic blast cells of another cell lineage (e.g., CD10 on T lymphoblasts). Another example would be CD24. The presence of this antigen supports the diagnosis of B-line-age disease if this diagnosis is suggested by other immunophe-notypic parameters. However, CD24 is also extremely valuable in determining the immature state of a given myeloid population because it is expressed by cells at and beyond the promye-locyte stage but not by more immature myeloid cells. In summary, lineage-associated antigens must be considered supportive but not determinant. Along the same line, their expression across lineages is common, as will be discussed in the next section.

Table 15-1. Antibodies (CDs) and Antigens to be Included in Testing Panels for Leukemia Immunophenotyping Arranged According to Lineage Affiliation

Myeloid/ Erythroid/ Uncommitted Monocytic Lymphoid Megakaryocyte

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