Immunophenotyping of Acute Leukaemias Immunophänotypisierung Akuter Leukämien
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Immunhämatologie Redaktion: G. Rothe Immunophenotyping of Acute Leukaemias Immunophänotypisierung akuter Leukämien T. Benter, R. Rätei, W.-D. Ludwig Summary: For nearly 100 years the classification of menden Einfluß gewonnen. Der Grund liegt in den blood cells and the diagnosis of leukaemia have been Fortschritten der Laser- und Computertechnologie, based on cytomorphological features after staining. aber auch an der Verfügbarkeit von Hunderten ver- Even in the era of molecular biology this is still es- schiedener monoklonaler Antikörper (moAB). die sential. Therapy of acute myeloid leukaemia (AML) is gegen eine Vielfalt von Antigenen hämatopoetischer mostly dependent on the interpretation of the morpho- Zellen gerichtet sind. logical appearance of blasts under the microscope. Cy- Dieser Übersichtsartikel fokussiert auf die Immun- tomorphology should also lead to a rational use of phänotypisierung von Patienten mit akuten Leukämien techniques like immunophenotyping, cytogenetics, flu- und zeigt den Einfluß auf die Diagnostik und Thera- orescence / situ hybridisation (FISH), and poly- pie. merase chain reaction (PCR). In the past two decades, the impact of immunophe- Schlüsselwörter: akute Leukämie: Immunophänoty- notyping by flow cytometry in the diagnosis and man- pisierung: Klassifikation von Leukämien. agement of acute leukaemia has expanded rapidly. This has been mainly attributed to significant advances in laser and computer technologies and the production of several hundred monoclonal antibodies (moAbs) to he gold standard for classifying acute myeloid a variety of antinens expressed by haematopoietic Tleukaemia (AML) has been based on morphologi- cells. cal, cytochemical, and iinmunophenotypic criteria as This review concentrates on immunophenotyping of defined by the French—American—British (FAB) sys- cells from patients with acute leukaemia and shows the tem 11-4]. Eight subgroups of AML (AML MO-AiML clinical impact on diagnostics and treatment. M7) have now been identified by this classification and by using lineage commitment and the degree of Keywords: acute leukaemia; immunophenotyping; blast cell differentiation. Whilst original consideration classification of leukaemias. was given to the morphological, immunological. and cytogenetic (MIC) working classification of AML |5|, Zusammenfassung: Die Klassifikation von Blutzel- a more accurate classification system can be achieved len und die Diagnose von Leukämien beruht seit by the routine use of cellular and molecular genetics to annähernd 100 Jahren auf einer zytomorphologischen supplement the FAB system. Valuable insights have Beurteilung. Trotz der in diesen Bereichen erfolgrei- been gained into the pathogenesis of AML and treat- chen Molekularbiologie ist die Färbung von Präpara- ment strategies that target underlying specific molecu- ten des Blutes und des Knochemarks unerläßlich. Die lar abnormalities. Indikation, zur Therapie der akuten myeloischen However, the FAB classification of acute lym- Leukämie ist weitgehend von der Interpretation der phoblastic leukaemia (ALL) [1) has not been identi- blastären Zellen unter dem Mikroskop abhängig. Die fied as having significant immunophenotypic, genetic, Zytomorphologie sollte die Basis für den gezielten and clinical correlates, with the exception of the L3 Einsatz spezifischer Techniken wie Immunphänotypi- subtype. For this reason, it has been largely replaced sierung, Zytogenetik, Fluoreszenz in. situ Hybridisie- by immunophenotyping and genetic systems. Lineage- rung (FISH) und Polymerasekettenreaktion bilden. specific and/or maturation-specific monoclonal anti- In den letzten 20 Jahren hat die Immunphänotypi- bodies (moAbs) have enabled an accurate assignment sierung mittels Durchflußzytometrie bei der Diagnose of leukaemic lymphoblasts to specific lineages. Today, und der Behandlung von akuten Leukämien zuneh- the primary diagnosis and subclassification of ALL re- lies on immunophenotyping [6, 7). As with AML, a wide range of-novel genetic markers have been dis- Corresponding author: Wolf-Dieter Ludwig, M.D., Helios-Klinikum covered in the last few years that provide vital infor- Berlin, Department of Haemätology, Oncology and Tumor Im- mation for an understanding of the biological basis of munology. Robert-Roessle-Clinic, Charite. Campus Berlin-Buch, ALL. These markers can also be used for diagnosis ündenberger Weg 80, D-13122 Berlin, Germany. Tel.. +49 30 9417-1314. Fax: -1-49 30 9417-1314. and prognosis., revealing important clues for rational E-Mail: [email protected] therapeutic interventions [8-10]. 512 J Lab Med 2001: 25 (11/12): 512-532 © 2001 Blackwell Wissenschafts-Verlag, Berlin G. Rothe A gene-based classification system is obviously munodiagnosis of haematopoietic malignancies, large- more effective than pne relying mainly on indirect ly replacing immunocytochemical microscopic analy- measures of blast ceil diversity such as morphology sis. Indeed, flow cytometry provides an objective, sen- and immunophenotype. It is fairly certain that new ap- sitive, and rapid multivariate analysis of a large num- proaches to acute leukaemia classification, such as ber of cells. It is now generally accepted that multipa- gene expression profiling using DNA microarrays, will rameter flow cytometry is a powerful diagnostic tool give important, information in identifying acute for immunophenotyping acute leukaemias and chronic leukaemia subtypes with distinct clinical phenotypes lymphoproliferative disorders, defining immunophe- and a variable clinical course [9-12]. notypic subsets, and detecting minimal residual dis- ease (MRD). More recently, multiparameter flow cy- Morphological classifications of acute tometry has aided in the development and monitoring of antibody-based treatment strategies [7. 18, 19, 22^ leukaemias 23]. The FAB classifications of acute leukaemias [1-3, 14, Most previous studies that investigated the diagnos- 15] follow an algorithm and are based on several tic impact of immunophenotyping and the association thresholds. In the era of biological description of enti- between antigen expression and therapeutic outcome ties, some rules seem to be arbitrary, as considered for in acute leukaemias used 20 % of cells stained with the new proposals such as the WHO classification [16].· moAb for surface markers and 10 % for more specific However, the FAB system still forms the basis for the markers with cytoplasm expression (e.g. myeloperoxi- cytomorphological classification of AML and MDS, dase, CD79a, cytoplasmic CD3) as the general cut-off but not of ALL, The definition of acute leukaemia and point for marker positivity [24]. These cut-off points the distinction between AML and ALL according to were chosen randomly and have been criticized [25] FAB is based on two criteria: for not being based on physiologic knowledge but • The percentage of blasts in the bone marrow is > 30 merely serving as a convenient method for collecting % of all nucleated cells data. Moreover, many clinical studies that describe im- • ± 3 % of blasts show a positive reaction for MPO or munophenotypic features of acute leukaemias and cor- SBB in the bone marrow relating prognoses by immunophenotyping for ALL and AML were carried out as single-colour analyses. The definition of complete remission in acute Evidently, these studies were not always able to dis- leukaemias has been published by the Cancer and tinguish malignant from normal haematopoietic cells, Leukaemia Group B (CALGB) and includes the fol- and, more importantly, did not consider multiparame- lowing criteria [17]: ter flow cytometry data [20]. Bone marrow blasts < 5 % Several studies have convincingly demonstrated Neutrophils > 1500/ìÉ that three- or four-colour immunophenotyping can re- Platelet count > 150,000/ìÉ liably resolve unique subsets of malignant cells within a complex population. This application has substan- Some other definitions use different thresholds for tially expanded our knowledge of normal and malig- neutrophils (i.e. whole white blood cell count) and nant subsets of haematopoietic cells. Nevertheless, the platelets (> 100,000/ìÉ). Most study groups, however, clinical relevance of multiparameter flow cytometry in accept the CALGB criteria, which should remain the acute leukaemias has only yet been demonstrated for standard until other criteria are available. flow-cytometric detection of MRD (reviewed in [23]). Future studies on acute leukaemias shall have to prove whether additional diagnostically and clinically rele- Immunophenotyping vant information can be provided by multivariate During the past two decades, flow cytometry-based analysis of phenotypic patterns of leukaemic blasts, in- immunophenotyping has impacted the diagnosis and cluding the density of antigen expression [26, 27] and management of acute leukaemia immensely. Mainly its pattern of reactivity (e.g. homogeneous versus het- due to significant advances in laser and computer erogeneous) by using well-established and by then technologies, several hundred moAbs to a variety of hopefully standardized flow-cytometric procedures. antigens expressed by haematopoietic cells can be pro- duced cost effectively. Moreover, distinct fluo- Genetic characterization rochromes conjugated with moAbs have become avail- able. Now, at least three to four cellular antigens can In acute leukaemia, genetic analysis is an obligatory be measured simultaneously in combination with two diagnostic tool that not only contributes to confirming intrinsic parameters, such as cytoplasmic complexity