Flow cytometric characterization of acute myeloid leukemia: IV. Comparison to the differentiation pathway of normal hematopoietic progenitor cells.

Gradual increase of CD38 on cells expressing CD34 characterizes the early cell differentiation pathway of normal human hematopoietic progenitors. In this study the coordinated expression pattern of CD34 and CD38 was assessed on leukemic blasts from bone marrow aspirates of 95 patients with newly diagnosed acute myeloid leukemia (AML). Expression was divided into six categories analogous to the differentiation pathway of normal bone marrow. The CD38 antigen was expressed on the leukemic cells of all patients and CD34+ leukemic cells were found in 79 patients (83%). In 93 patients, the leukemic cells were found along the differentiation pathway defined by CD34 and CD38. In 33 of the 93 patients, a part of the CD34+ cells did not express the CD38 antigen (categories 1 and 2). In another 33 patients, all CD34+ cells expressed CD38 (categories 3 and 4). In the remaining 27 patients, only cells were found which dimly expressed CD34 or did not express CD34 (categories 5 and 6). Of the 93 patients, 88 were treated with intensive chemotherapy according to the protocol of the German AML Cooperative Group. Of these, 21 died early and were not evaluable for treatment response. Complete remission was achieved in 14 of 22 patients (64%) in categories 1 and 2, in 19 of 26 patients (73%) in categories 3 and 4, and in 18 of 19 patients (95%) in categories 5 and 6. The event-free survival was significantly longer in patients of categories 5 and 6 compared to patients in categories 1 and 2 (p less than 0.01) and categories 3 and 4 (p less than 0.05), respectively. We conclude that in the majority of AML patients the immunophenotype of leukemic cells follows the early cell differentiation pathways defined by coordinated expression of CD34 and CD38 similar to that of normal hematopoietic progenitors. The presence of cells in the late cell differentiation stages (CD34+/-, CD38 /+) identifies patients with a higher complete remission rate and longer complete remission duration.

Flow cytometric characterization of acute myeloid leukemia. Part II. Phenotypic heterogeneity at diagnosis.

The frequency and distribution of aberrant antigen expression are analyzed on bone marrow aspirates from 80 patients with newly diagnosed acute myeloid leukemia (AML) by multidimensional flow cytometry. Parameters examined are the light scatter profile of the leukemic cells and the correlative expression of different combinations of the CD2, 4, 5, 7, 11b, 11c, 13, 14, 15, 16, 33, 34, 38, and HLA-DR antigens. Antigen expression on leukemic cells in bone marrow is described by characteristic antigen expression patterns describing: (i) the percentage of cells expressing the antigen; (ii) the antigen density; and (iii) the distribution of the antigen on the leukemic cells. Typically the non-myeloid antigens are homogeneously expressed by the leukemic cells, whereas the myeloid associated antigen CD11b, CD11c, CD14, and CD15 are heterogeneously expressed. Comparison of the antigenic profiles of 80 bone marrow aspirates revealed an extreme interclonal heterogeneity. Comparison of the antigen expression patterns found in AML patients with the antigen expression in normal bone marrow revealed four patterns of aberrant antigen expression in AML: (i) expression of nonmyeloid antigens (i.e. CD2, CD5, and CD7 were present in 57, 60, and 37% of the patients, respectively); (ii) asynchronous expression of myeloid associated antigens (i.e. co-expression of CD34 and CD15 in 25% of the patients and expression of CD16 on immature myeloid cells in 15% of the cases); (iii) over-expression of myeloid associated antigens (e.g. CD34 in 16% of the cases and CD14 on neutrophilic cells in 19% of all patients); and (iv) absence of expression of myeloid associated antigens (e.g. lack of CD33 in 21% of the cases and lack of both CD11b and CD15 in 6% of all patients. Multidimensional flow cytometric analysis of bone marrow aspirates of AML patients disclosed that the leukemic cells of each AML patient had a unique antigenic profile and could be discriminated from their normal counterparts based on aberrant antigen expression and typical light scatter profiles. The ability to distinguish leukemic cells from normal cells allows the detection of residual leukemic cells during and after chemotherapy.

Flow cytometric characterization of acute myeloid leukemia. Part 1. Significance of light scattering properties.

Acute leukemias are classified using the morphological and cytochemical criteria set forward by the French, American and British (FAB) group. Immunophenotyping is helpful for the differential diagnosis but is secondary to the morphological criteria. Immunophenotyping performed by flow cytometry, however, can yield valuable information on cell morphology in addition to cell surface antigen expression. To provide a basis of a combined evaluation of both morphology, i.e. light scattering, and immunophenotype by flow cytometry we have compared the light scattering profiles of 70 patients newly diagnosed with acute leukemia with normal bone marrow and related the findings to the FAB classification. Three main light scattering profiles were observed in the bone marrow aspirates of the 70 patients (A1,2; B1,2,3; C1,2,3,4). A1,2, characterized by a predominant cell cluster with low forward and orthogonal light scattering, contained only and all patients diagnosed as acute lymphoblastic leukemia, acute undifferentiated leukemia, and acute non-lymphocytic leukemia M6 and M1. B1,2,3 is characterized by a predominant cell cluster with large forward and low to high orthogonal light scattering. Category B1 contained the majority of patients classified as M5; the M3 leukemias were categorized as B2. C1,2,3,4 is characterized by a predominant cell cluster with low forward and orthogonal light scattering that branches towards regions with larger light scattering. Categories C1 and C2 contained the majority of the patients classified as M2. Category C3 was specific for M4 and M4eo leukemias. The patients diagnosed as M4 were heterogeneous and equally distributed over the B and C categories. The clear relationship found between the FAB classification and classification by the light scattering profile of the acute leukemias enhances the importance of the flow cytometric classification of leukemias. In contrast with light microscopy, flow cytometry can now provide the hematologist with an objective technique to classify leukemias by the simultaneous assessment of cell surface antigen expression and cell morphology, i.e. light scattering.