Modern classification systems for acute and chronic leukaemias are based on cytomorphology, cytochemistry, immunophenotyping, immunogenetics and molecular cytogenetics. Morphology forms the initial ...diagnosis of leukaemia, but generally is not sufficient to identify biologically and clinically relevant subsets within the main categories of leukaemia. Immunophenotyping precisely defines the lineage and stage of differentiation of malignantly transformed haematopoietic cells. This is usually sufficient for precise classification of mature lymphoid malignancies, although immunogenetic and (molecular) cytogenetic studies might be helpful to confirm the diagnosis of disseminated non-Hodgkin's lymphomas. However, certain categories of disease that are clearly defined by cytomorphology and immunophenotyping, particularly acute leukaemias, are still heterogeneous, mainly owing to different underlying leukaemogenic events. Immunophenotyping can reveal subgroups highly suggestive of certain chromosome aberrations but reliable identification of such aberrations requires cytogenetic or molecular studies. Such combined diagnostic information forms the basis of current WHO classification of tumours of haematopoietic and lymphoid tissues. This will be complemented in the near future with novel criteria revealed by microarray gene expression profiling.
This chapter summarizes and comments on the currently used immunophenotypic classification systems of acute and chronic leukaemias and on the added value of molecular diagnostics.
Classification systems for acute and chronic leukaemias SZCZEPANSKI, Tomasz; VAN DER VELDEN, Vincent H. J; VAN DONGEN, Jacques J. M
Best practice & research. Clinical haematology,
2003, Letnik:
16, Številka:
4
Journal Article
The WHO classification of myeloid disorders contribute to a more refined classification and prognostication of myelodysplastic syndromes (MDS). The considerable differences in clinical behaviour of ...pure refractory anemia (RA) versus refractory cytopenia with multilineage dysplasia (RCMD) with or without ringsideroblasts are of importance in the management of patients with MDS. Flow cytometry may add additional diagnostic criteria to adequately discriminate RA from RCMD (+/− ringsideroblasts; (RS)) and may contribute in identifying Idiopathic Cytopenia of Undetermined Significance. We developed a 4-colour flow-cytometric procedure that comprises all differentiation stages of granulocytic, monocytic and erythroid lineages, instrumental for the recognition of various subpopulations within all three lineages in normal bone marrow samples. In 43 evaluable patients with MDS (RA, RARS, RCMD, RCMD-RS, MDS-U, RAEB-1 and 2), aberrant expression of differentiation antigens were demonstrated in 1 or more lineages. Flow-cytometry identified aberrancies in granulopoiesis and monocytopoiesis in 93% and 74% of the cases, respectively. In the majority of cases abnormal relations between CD13, CD16, CD11b, CD15 and HLA-DR were prominent in the granulopoiesis. In 34% of the cases a striking monocytopenia was detected, whereas in 59% abnormal surface expression of CD14, CD36 and CD33 indicating aberrant differentiation of monocytes. We defined aberrant myeloid blasts by a leukaemia associated phenotype (LAP) according to the definitions used in acute myeloid leukaemia. In 47% of the patients a LAP was detectable by demonstrating co-expression of CD5, CD7, CD19 and CD56 on CD34+ myeloid blasts. In all patients diagnosed as RA/RARS and MDS-U (n=12) according to WHO criteria, additional flow aberrancies were identified including a leukaemia associated phenotype of myeloid blasts in 41% of the cases. Only in 3 out of 28 cases with RCMD/RCMD-RS no erythroid aberrancies were detectable by flow-cytometry. In 9 normal control BM samples, no flow-cytometric abnormalities were present. It is concluded that flow-cytometry in MDS identifies aberrancies in the granulocytic and monocytic lineages and may classify patients with multi-lineage aberrancies not otherwise determined by cytology (WHO). Flow-cytometry may discriminate pure RA or MDS-U from RCMD. Since new drugs are emerging in low-risk MDS, the value of flow-cytometry might be of importance to further refine the classification in MDS. The exact role of these aberrant differentiation patterns on IPSS, clinical behaviour, impact on treatment decisions and as tool in disease monitoring have to be determined in future prospective studies.
During the past two decades, flow-cytometric immunophenotyping of lymphocytes evolved from a research technique into routine laboratory diagnostics. Extensive studies in healthy individuals resulted ...in detailed age-related reference values for different lymphocyte subpopulations in peripheral blood. This is an important tool for the diagnosis of hematological and immunological disorders. Similar albeit less detailed information is now available for other lymphoid organs, e.g., normal bone marrow, lymph nodes, tonsils, thymus and spleen. Flow-cytometric immunophenotyping forms the basis of modern classification of acute and chronic leukemias and is increasingly applied for initial diagnostic work-up of non-Hodgkin's lymphomas. Finally, with multiparameter flow cytometry it is now possible to identify routinely and reliably low numbers of leukemia and lymphoma cells (minimal residual disease).
The quantitative analysis of minimal residual disease (MRD) with the use of junctional regions of rearranged immunoglobulin (Ig) and T-cell (TCR) receptor genes as MRD target sequences is a complex, ...multi-step procedure. It includes: the processing of biological material, the detection, clonality assessment, identification, and selection of optimal MRD target sequences, and MRD analysis with the use of real-time quantitative polymerase chain reaction (RQ-PCR), which includes: probe and primer selection, standard curve preparation, testing the sensitivity of primers, DNA quality and quantity testing, quantitative MRD analysis, and data interpretation. Although this approach is laborious, it requires experienced and qualified personnel, and extensive standardization, it is applicable in more than 95 % of acute lymphoblastic leukemia (ALL) patients and enables MRD assessment with high sensitivity (10−4 – 10−5), which makes it a valuable tool for the management of ALL treatment.
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