Evaluation for Myelodysplasia - Morphology and Cytogenetics
The ability of 4-color flow cytometry (FC) to help identify myelodysplastic syndromes (MDSs) was evaluated in 124 bone marrow aspirates from unselected patients with unexplained cytopenias and/or monocytosis. The morphologic features of bone marrow aspirate smears were correlated with FC and cytogenetic findings blindly, and patterns of antigen expression were compared with patterns seen in nonneoplastic and normal marrow specimens. Of 124 cases, 58 (46.7%) had definitive FC abnormalities ("flow-abnormal"), 19 cases (15.3%) had mild FC abnormalities of indeterminate significance, and 47 cases (37.9%) had essentially normal FC. Highly significant differences were identified between the flow-abnormal group and other groups in mean myeloid blast percentages and numbers of abnormal antigens expressed, even when the analysis was limited to cases with fewer than 5% myeloid blasts. Strikingly, flow-abnormal cases constituted 50 (89%) of the 56 morphologically abnormal cases and 31 (94%) of the 33 cytogenetically abnormal cases, demonstrating the strong concordance of FC-identified antigenic abnormalities with morphologic features and cytogenetics in the evaluation of patients with unexplained cytopenias.

The diagnosis of the myelodysplastic syndromes (MDSs) historically has relied on combining the clinical history, morphologic features of the peripheral blood and/or bone marrow sample, and cytogenetic information. However, because morphologic evaluation is inherently subjective, and cytogenetics, while objective, identifies abnormalities in only 30% to 40% of MDSs, additional objective correlates of MDS are needed.

Multiparametric flow cytometry (FC) represents a highly reproducible and objective way of assessing the expression of multiple antigens on a single cell. By comparing patterns of antigen expression on a given cell population with the patterns identified on normal cells of that type, one potentially can identify abnormalities that, if sufficiently great, might substitute for clonality studies in identifying malignancy. Patterns of expression of a number of antigens during normal myelopoiesis have been described in relatively great detail.

A number of studies during the past 15 years have applied FC to the study of MDSs. Most of the earlier studies investigated a relatively small number of surface antigens, and these studies are well reviewed and critiqued by Elghetany. Some of the described antigenic abnormalities have included the following: (1) loss of erythrocyte A, B, and H antigens in MDSs; (2) decreased expression of c-mpl, glycoprotein IIb/IIIa, and glycoprotein Ib on platelets from patients with refractory anemia (RA); (3) dyssynchronous expression of CD11b and CD16 in the developing neutrophils of patients with MDS; (4) aberrant coexpression of CD14 and CD66 on the myeloid cells in a subset of MDSs; (5) decreased CD10 on neutrophils in MDSs; (6) changes in a variety of leukocyte activation antigens, including FcR I, FcR II, and FcR III, in MDSs; (7) greater variability in the expression of CD38, CD71, CD13, and CD33 in RA vs normal marrow or marrow involved by aplastic anemia; and (7) aberrant coexpression of CD56 on myeloid blasts in MDSs. Two recent studies identified immunophenotypic abnormalities among relatively mature myeloid cells in the bone marrow and peripheral blood neutrophils of patients with MDSs, including decreased side light scatter, decreased CD10, and increased HLA-DR, CD11a, and CD66.

A variety of studies have used FC to document abnormalities in apoptosis and/or proliferation in MDSs. The latter studies generally have found abnormally increased apoptosis on bone marrow precursors in RA and RA with ringed sidero-blasts, with associated increases in caspase 3 activation and in expression of proapoptotic bcl-2 family members such as bad, bak, and bcl-xS. In contrast, in RA with excess blasts (RAEB), RA with excess blasts in transformation (RAEB-T), and chronic myelomonocytic leukemia (CMML), there tends to be a relative increase in proliferation.

Several studies have looked more generally at the role of 3-color FC in the diagnosis or classification of MDSs. Stetler-Stevenson and colleagues used 3-color FC to identify immunophenotypic abnormalities in the bone marrow of 45 patients with well-established MDSs and then applied FC to a series of 20 equivocal cases to demonstrate that FC is helpful in making the final diagnosis of MDS. Ogata and colleagues used 3-color FC to evaluate enriched populations of blasts from 95 patients with MDSs and 21 patients with acute myeloid leukemia arising from an MDS. They found that blasts from patients with a low-risk MDS (RA or RA with ringed sideroblasts) often expressed antigens associated with some degree of myeloid maturation, whereas blasts from patients with a high-risk MDS (RAEB, RAEB-T, and CMML) tended to have a less mature immunophenotype. Maynadie and coworkers used 3-color FC along with a hierarchical clustering algorithm to identify immunophenotypic features that could help distinguish different types of MDS as defined by the French-American-British classification. Additional immunophenotypic clusters were found to correlate with the International Prognosis Scoring System score. Del Canizo et al used 3-color FC to evaluate 7 myeloid-associated antigens and noted that 90% of samples from patients with an MDS showed aberrant immunophenotypes, compared with control subjects. Finally, Wells and coworkers used 3-color FC to develop a scoring system for quantifying the overall extent of immunophenotypic abnormalities. This group found that the FC scores correlated inversely with leukocyte and absolute neutrophil counts and directly with International Prognostic Scoring System risk categorization and the likelihood of relapse following stem cell transplantation for MDS.

In the University of Washington Hematopathology Laboratory (UWHL), Seattle, we have used 4-color FC since 1998 to evaluate bone marrow aspirates from patients suspected of having myeloid stem cell neoplasms and have accumulated a large data set of more than 800 normal and abnormal bone marrow samples. This data set has allowed us to identify reproducible patterns of antigen expression in normal granulocytic and monocytic maturation, including changes seen in benign and reactive settings such as marrow regeneration. This understanding of benign patterns of myeloid antigen expression, in turn, has allowed us to use 4-color FC to identify antigenic abnormalities that, if sufficiently extensive, can be used to support the possibility of a myeloid stem cell neoplasm, as previously demonstrated in the workup of myeloproliferative disorders of the nonchronic myelogenous leukemia type. We now validate our 4-color method for assisting in the diagnosis of MDS by showing strong concordance of the FC results with the morphologic and cytogenetic features of an unselected series of patients with unexplained cytopenias or monocytosis.