Immunophenotyping of Acute Leukaemias Immunophänotypisierung Akuter Leukämien

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].

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 disease (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 available. 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 the diagnosis. More importantly, the leukaemic blast and cell size by leukaemic blast light scatter properties karyotyping can reveal important prognostic data. (i.e. forward and side scatter characteristics, FSC and There are various methods for the genetic characteri- SSC) [7, 18-22]. In light of these technical achieve- zation of leukaemic blasts [28]. Chromosome banding ments, immunophenotyping by multiparameter flow •analysis summarizes all chromosome abnormalities cytometry has emerged as an optimal method for im- detectable by light microscopy. Submicroscopical mu-

• JLab Med 2001; 25 (11/12): 512-532 513 Immunhämatologie tations can also be detected using fluorescence in situ • AML M2 with a higher number of megakaryocytes, hybridisation (FISH)» Southern blot, or polymerase usually with two small nuclei and in many cases chain reaction (PCR). Such techniques are only effec- normal or even elevated platelet counts, was seen in tive in selecting the right probes if prior knowledge of patients with inv(3)(q21q26) [38-40] the genetic aberrations is available and only provide • AML M3 [41] and its variant (AMLM3v) [15] were information concerning those chromosomes/genes for found to bear the same cytouenetic abnormality: which probes were used. t(15;17) • In AML M4 with abnormal eosinophils (M4eo). the inv(16) or t(16;16) was detected [42-45]. Unlike The FAB classification of AML normal eosinophils, abnormal eosinophils have large basophilic granules and abnormal granular The FAB classification of AML follows an algorithm positivity with CAE and can identify eleven different subtypes (Table 1) • Patients with AML M4 or M5 with erythrophagocy- 11-4, 14, 15]. These strict definitions of the FAB clas- tosis had detectable t(8; 16) sification for AML per se do not include primary bio- • Another correlation was observed in patients with logical characteristics. dysplastic granulopoiesis and pseudo-Pelger-Huet anomalies, sometimes with changes in the chromosome 17p involving the p53 gene [46] Correlations of morphological and cyto- • In patients with monocytic AML (M4. M5). the chemical features with immunopheno- Ilq23 chromosome area was often involved. typing, cytogenetics, and molecular The strongest correlations between cytomorphology genetics in AML and cytogenetics were clearly observed in AML M3(v) and AML M4eo. All other associations were much Since the FAB classification for acute leukaemias was weaker. It should be noted, however, that only cytoge- published in 1976, several papers from the FAB group netic and/or molecular genetic analyses could load to have been revised and supplemented. The observers the definition of a biological entity. Therefore, the agreed 65%-80% [I, 30-32] of the time, making it threshold of 5 % abnormal eosinophils for the diagno- possible to correlate specific and recurrent morpholog- sis of AML M4eo is arbitrary, because some eases ical features with cytogenetic and immunophenotypic show less than 1 % clearly abnormal eosinophils. results. In AML, the FAB system led to the description of more specific morphological details and correla- Immunophenotyping of AML tions with cytogenetic data: • AML M2 (or Ml) showing dysgranulopoiesis, in- Immunophenotyping by flow cytomctry has been in- crease of normal eosinophils, very mature blasts strumental in recognizing minimally differentiated (type II and so-called type III) and long, needle-like AML (AML-MO), acute megakaryoblastic leukaemia Auer rods were found to be cytogenetically associ- (AML-M7), and AML co-expressing lymphoid-ussoci- ated with t(8:21) [33-36] ated antigens [3, 4, 21, 24]. It has also been particu- • AML M2 baso showed a higher number of ba- larly helpful in distinguishing AML with monocytic sophils along with the typical M2 morphology and differentiation from AML-MO/M1 or AML subtypes was sometimes correlated with the t(6;9) [37] with granulocytic differentiation (i.e. AML-M2/M3)

I Table 1 FAB Classification of acute myeloid leukaemia AML MO No maturation, MPO < 3%, but Immunophenotyping: myeloid markers AML M1 Blasts must be 90 % or more of nonerythroid cells, MPO minimum 3 % AML M2 > 10% of myeloid cells show maturation from promyelocyte stage, monocytes < 20% AML M3 Most cells are highly abnormal promyelocytes AML M3v Most cells are bilobulated blasts with strong MPO AML M4 Myelomonocytic blast cells, with monocytic component of more than 20% but less than 80% AML M4eo Like M4, with abnormal eosinophils (usually over 5 %) AML M5a Monoblasts > 80% in the bone marrow AML M5b Monoblasts and monocytes > 80 % in the bone marrow AML M6 Erythroblasts > 50 % of total nucleated cells and at least 30 % of nonerythroid cells are blasts AML M7 Immunophenotyping: blasts demonstrate to be'megakaryoblasts (CD41+, CD61+)

514 J Lab Med 2001; 25 (11/12): 512-532 G. Rothe

[47]. The diagnostic sensitivity of a comprehensive niques may enable identification of leukaemic cells. panel of moAbs to myeloid and lymphoid lineage as One of these techniques is the leukocyte common anti- well as progenitor cell-associated antigens has been gen (CD45)/side scatter (SSC) gating procedure, demonstrated in both childhood and adult AML [21, which allows an efficient discrimination between the 48-51J. blast cell population and the normal cells and facili- Although none of the anti-myeloid moAbs used in tates the analysis of leukaemic blasts present in low these studies recognized the blast cells of all AML pa- proportions [53, 54]. The use of CD45/SSC gating, tients, nowadays nearly all AML cases can be detected primarily gated on blast cells identified according to using a combination of two or three pan-myeloid the low/intermediate CD45 density, correlates with reagents (i.e. CD 13, CD33, CD65) with moAbs to bone marrow differential and provides characteristic myeloperoxidase (MPO), which detect both the proen- flow-cytometric profiles for most subtypes of AML zymatic and the enzymatic forms of MPO, except for [21, 47, 54]. Moreover, this gating strategy has been MPO and megakaryocytic-associated antigens (e.g. demonstrated to give similar results in leukaemic spec- CD41a, CD61). However, expression of myelpid-asso- imens enriched for leukaemic blasts by density-gradi- ciated markers is not restricted to AML. Attempts to ent separation techniques and in lysed whole bone correlate immunophenotypic features with the various marrow or peripheral blood samples [54). On the basis AML subtypes (AML-M1 through AML-M6) accord- of these observations, several authors have suggested ing to the FAB classification have been largely unsuc- that CD45/SSC gating should replace FSC/SSC gat- cessful [18, 21, 52]. Although some AML subtypes " ing, and that this method could contribute to cost re- (e.g. AML-M3, see below) show a characteristic im- duction without affecting diagnostic quality [54-56]. munophenotypic profile, there are few entirely consis- Recently immunophenotyping studies using multi- tent relationships between morphology and im- parameter flow cytometry have shown that the anti- munophenotype (see Table 2). Therefore, cases with genie profiles of AML differ from the antigen expres- identical antigen expression may belong to different sion pattern of normal bone marrow. These phenotyp- FAB subtypes, and different immunophenotypic fea- ic aberrations of leukaemic blasts, otherwise known as tures can be observed in the same FAB subtypes. asynchronous or aberrant antigen expression, are prob- In AML, interpretation of immunophenotyping ably related to the underlying genetic alterations as studies may be confusing, because leukaemic blasts in well as to a disturbed regulatory control of certain pro- bone marrow and peripheral blood specimens are fre- teins [57-59] and may be of use when screening for quently admixed to normal haematopoietic cells. The genetic abnormalities and monitoring MRD. Several blast cell population can also be heterogeneous. There- antigen screening panels for immunophenotyping of fore, various multiparameter flow cytometry tech- AML have been recommended [21, 24, 60, 61]. These

Table 2 Relationships between morphology, immunophenotypic features and genetic aberrations in AML

Antigen MO M2 M3 M4Eo M5 M5 M7 t(8;21) t(15;17) inv(16) | MPO -*/- j CD2 ! CD7 -/+ j CD13 ' +/- CD14 ' CD15 CD19 CD33 +/- CD34 +/- CD56 CD41/CD61 CD64 CD65 -1+ CD117 +/- HLA-DR +/- - = Antigen not expressed -/+ = Antigen expressed in less than 50 % .of patients +/- = Antigen expressed in most patients + = Antigen expressed Open fields represent partial expression without specificity for diagnosis or lack of reliable data.

J Lab Med 2001; 25 (11/12): 512-532 515 Jmmunhämatologie include, for the most part, moAbs directed towards AML and indicate that AML-MO, similar to AML-M1, antigens expressed by earJy haematopoietic progeni- is not a unique subtype of leukaemia but probably in- tors and relatively lineage-restricted antigens. cludes distinct malignant myeloid processes with dif- We will now describe the immunophenotypic fea- ferent underlying cyto- and/or molecular genetic de- tures of AML-MO and AML-M7 and summarize the fects [64, 65]. The cytogenetic abnormalities and the antigenic profiles that may be linked with clinically rel- higher level of P-glycoprotein expression described in evant entities such as AML with t(8;21), AML with ab- most studies may be a contributing factors in the case normal bone marrow eosinophilia and inv(16) or of poor therapeutic outcome observed in adults with t(16;16). acute promyelocytic leukaemia with t(15;17), AML-MO [65, 66, 70]. and AML with Ilq23 abnormalities (Table 2). Despite the fact that the relevance of immunophenotyping for AML M2/t(8;21) the identification of AML subtypes carrying specific ge- Many studies have 'described the distinctive im- netic abnormality has been put in question, recent stud- munophenotypic features of AML-M2 cases harbour- ies based on multivariate phenotypic pattern and light ing the t(8;21) translocation that complement the char- scatter characteristics instead of individual antigen ex- acteristic morphological findings of this AML subtype pression have shown a great improvement in sensitivity [59, 71-74]. These features include expression of and specificity of immunophenotyping [57, 62]. CD 13, CD 15, CD33, CD34, CD65, HLA-DR with frequent co-expression of the antigen CD 19 associated AML minimally differentiated (AML MO) with the B-cell and the neural cell adhesion molecule Leukaemias of the MO subtype cannot be made on CD56. It should however be noted that recent case re- morphological grounds alone and these make up ports of adult patients with a so-called myeloid surface 3 %-6 % of paediatric and up to 10 % of adult AML. antigen-negative phenotype have been published In 1991, the FAB Cooperative Group listed cytochem- [75-77]. This indicates that low levels or absence of ical, morphological, and immunophenotypic diagnos- pan-myeloid antigens CD 13 and CD33 may occur par- tic criteria, as well as proposing the designation "MO" allel to expression of MPO as detected by cytochemi- for these leukaemias [6]. The criteria included: nega- cal staining or flow cytometry. tive cytochemical reactions of MPO and Sudan Black Findings regarding the frequency and intensity of B (SBB), no evidence of lymphoid differentiation by CD 19 and/or CD56 expression in AML-M2 with immunophenotyping, and expression of myeloid anti- t(8;21) [50, 78) are very controversial and have ques- gens (e.g. CD 13 or CD33) or the enzyme MPO shown tioned whether these aberrant phenotypic features by immunophenotyping and/or electron microscopy occur often enough to be able to select cases for mol- analysis. ecular screening on the basis of immunophenotyping More recently, stricter guidelines for excluding lym- [73).'The usually weak and variable expression of phoblastic and megakaryoblastic leukaemias have been CD 19 and CD56 on AML cells may be causing this proposed, based on the availability of more specific lin- discrepancy. Therefore, special gating strategies are re- eage-restricted moAbs, the use of multicolour flow cy- quired to separate blasts from whole mononuclear cell tometry, and the cytoplasmic detection of myeloid anti- fractions in flow-cytometric analysis by applying dif- gens in fixed cells (e.g. CD13, MPO) [21, 24, 63]. In ferent staining techniques or using other methods, in- accordance with these criteria, acute leukaemias with- cluding the use of different CD 19 moAbs. out detectable MPO can only be classified as AML MO In recent studies, an overexpression of CD34 in an when there are no lineage-restricted lymphoid (e.g. asynchronous combination with cytoplasmic MPO has CD3, CD22, CD79a, TCRß) and megakaryocytic anti- been present. It has also been suggested that quantifi- gens (e.g. CD41, CD61). Most MO cases express cation of CD34 expression could be useful for both CD13, CD33, CD65 as well as progenitor cell-associ- rapid diagnosis and remission assessment in AML with ated antigens, e.g. HLA-DR, CD7, CD34, and CD117 t(8;21)[59]. [64-67], whereas other myeloid lineage-associated antigens (e.g. CD14, CD 15) are uncommon. Acute promyelocytic leukaemia with t(15;17) It has been shown that anti-MPO antibodies are re- The characteristic immunophenotypic features of APL liable in detecting minimal myeloid differentiation in with t(15;17), which are by no means unique, include cases with negativity of CD13 and CD33 [65, 68, 69]. (a) absence or low expression of HLA-DR, CD7, It is possible that up to 80 % of MO cases have a com- CD 14, CD 15 (as detected with moAbs VIM-D5 or plex composite immunophenotype with expression of VIM-C6), and CD34; (b) variable expression of myeloid and nonlineage-restricted lymphoid markers, CD lib, CD65, and CD 117; (c) weak expression of including CD2, CD4, CD7, CDI9, and TdT, which CD64 and strong expression of CD9, CD 13, CD33, means it is difficult to classify them as AML, ALL, or CD68, and MPO [47, 50, 79-81]. In recent times, the biphenotypical acute leukaemia (see below) [63]. By sensitivity and specificity of immunophenotyping showing a variety of clonaJ abnormalities, such as studies for rapid screening of APL, especially in cases complex karyotypes, anomalies of chromosome 5 with. M3-variant (v) morphology or rare cases of and/or 7, trisomy 8, trisomy 13, cytogenetic studies re- t(15;17)-positive leukaemias resembling AML-M 1 and flect the heterogeneity of minimally differentiated AML-M2 (83, 84], have been improved by using a

516 J Lab Med 2001; 25 (11/12): 512-532 G. Rothe combination of three phenotypic criteria (i.e. existence not show a specific immunophenotypic pattern in of a single blast cell population, heterogeneous reac- leukaemic blasts that might be used to differentiate tivity for CD 13, and the pattern of expression of acute myelomonocytic leukaemias with Ilq23 translo- CD34/CD15 as detected by the Leu-Mi mo Ab [57]) as cations from FAB M4 or M5 cases without 1 Iq23 in- well as the availability of antibody reagents directed volvement [93, 94]. However, our results in children against the PML protein [82]. Immunophenotyping has and adults with MLL rearrangements, usually due to also been helpful in distinguishing between acute the t(9;ll), showed characteristic features such as myelomonocytic or monocytic leukaemias and the mi- strong expression of HLA-DR, CD33, CD65, and crogranular variant, which, unlike AML-M4 or M5, CD4, whereas other myeloid lineage-associated anti- does not usually express CD4, CD 14, CD36, and gens (e.g. CD 13, CD 14) and CD34 were detected in HLA-DR. It also shows distinct light scatter character- less than 30% of cases [50, 95, 961. Moreover, we istics as a result of its high MPO content. [96] have observed a frequent co-expression of CD56 Recent studies have suggested a correlation be- in AML with monocytic differentiation and rearrange- tween immunophenotypic characteristics and morpho- ment of the MLL gene. This has also been confirmed logical, molecular genetic, and clinical features of by others [97]. childhood and adult APL that may be of use for deter- It is of interest that previous studies that have test- mining the biological and clinical heterogeneity of this ed the reactivity of moAb 7.1, which detects the subtype more easily. A strong association of the S- human homologue of the rat NG2 chondroitin sulfate (short) transcript, which results from a break at bcr3 of proteoglycan molecule, have discovered a strong asso- the PML gene, and M3v morphology with CD2 posi- ciation between blast cell expression of the NG2 mol- tivity has been described [81, 85, 86] as well as a more ecule, FAB M4/M5 morphology, and 1 Iq23 abnormal- frequent expression of CD56 in APL with the S-iso- ities in childhood AML [98, 99). Concurrent to these form subtype [87]. It is interesting to learn that both results, we were recently able to demonstrate in a large aberrant immunophenotypic features were significant series of patients, including children and adults with to the prognosis. CD2 positivity predicted a better CR AML, that the moAb 7.1 is a sensitive but not entire- rate and EPS in APL [81], whereas CD56 was linked ly specific marker for identifying 1 Iq23-associated with a poor therapeutic outcome in a small group of AML [96]. Furthermore, a frequent co-expression of adult patients with varying treatment protocols [87]. NG2 and CD56 in AML with monocylic differentia- Immunophenotypic features of rare AML cases, tion was observed. This raised the question whether which are morphologically similar to AML M3 and do these molecules, which are probably both involved in not express the PML/RARa fusion gene but show re- cell adhesion and migration mechanisms, have any arrangement of the RARa locus with genes other than pathophysiological impact on the clinical behaviour of the PML gene on chromosome 15, such as t(ll;17) this AML subset. and t(5;17), resemble the pattern seen in typical AML Recent studies suggested that a great improvement M3. The fact that expression of CD56. in conjunction in flow-cytometric detection of AML with monocytic with functional NK cell-mediated cytotoxicity was ob- differentiation [47] might be achieved by adding served in four cases with t(l 1;17) [88, 89] was of par- CD64 and CD45 intensity versus logarithmic side ticular interest. scatter analysis to CD 14, a highly specific but insensi- tive monocytic marker. Moreover, data relating to a AML-M4Eo large group of Japanese adult patients suggested that Multiparameter tlow-cytometric analysis, in detecting AML with myelomonocytic differentiation, which is small subpopulations with aberrant immunophenotyp- often associated with Ilq23 abnormalities or with ic features is of great diagnostic value. This has been inv(16), showed a typical surface antigen expression illustrated in AML-M4Eo by demonstrating expression pattern (i.e. CD34IOW, CD33hi^h, CDllbhieh, GM-CSF- of CD2 on leukaemic blasts with inv(16) or t(16;16) Rhi$h, CD4P°sitive) [100]. [90, 91]. Using multiparameter flow cytometry, two Future studies will have to demonstrate whether the major leukaemic cell populations are evident in AML- analysis of these phenotypic features by multiparame- M4Eo with expression of pan-myeloid and granulocyt- ter flow cytometry will aid in achieving a more rele- ic or monocytic antigens, including CD4, CD 13, vant subdivision of AML with monocytic differentia- CD 14, CD 15, CD33, and CD65. Other characteristic tion and whether they may have an impact on progno- features of AML-M4Eo, similar to AML-M2 with sis. t(8;21), include frequent positivity of CD34 and absence of CD7 [50]. The availability of moAbs to the Acute megakaryocytic leukaemia (AML M7) chimeric CBFß-MYHll protein can be used in flow- The differentiation of AML M7 from ALL, AML MO, cytometric analyses to screen for the inv(16) abnor- and sometimes small tumours in children, is not usual- mality [92J. ly possible by cytochemical and morphological studies. The diagnosis of AML M7 must therefore be con- AML-M5 with Ilq23 aberrations firmed by immunophenotypic detection of different Earlier studies using patients with acute myelomono- platelet glycoproteins indicating megakaryocytic dif- cytic leukaemias associated with 1 Iq23 aberrations did ferentiation (e.g. CD41a, CD61) or by ultrastructural

• J Lab Med 2001; 25 (11/12): 512-532 517 Immunhämatologie demonstration of platelet peroxidase [3,114]. However, tor-, myeloid- or lymphoid-associated antigens and the immunophenotyping studies are more readily per- therapeutic outcome, especially in childhood AML [50, formed than ultrastructural studies,, which have largely 111, 112], others suggested a significant influence of been replaced by the former [101, 102]. specific antigens or combined phenotypic features on Leukaemic blasts in AML M7 express CD61, the complete remission (CR) rate and/or CR duration CD41a, and less frequently CD42b. Moreover, most and survival. Antigens that appear to have an adverse cases express CD4, CD33, CD34, CD36, HLA-DR, prognostic effect include CD7, CD9, CD lib. CD 13, and, less frequently, CD 13. Co-expression of lym- CD14, HLA-DR, CD34, and TdT [69, 113-116). On phoid antigens, especially CD7 or CD2, has been de- the other hand, a better therapeutic outcome has been scribed. Cytoplasmic expression of platelet glycopro- associated with the presence of CD 15, CD65, and CD2 teins may precede the cell-surface expression of these [117, 118|. These findings (e.g. CD2, CD7, CD34, markers. This should be tested in cases with undiffer- TdT) [50, 111, 112. 118-120] could not be confirmed entiated morphology and negative or inconclusive cy- by other authors. The comparability of most of these tochemistry in order to differentiate AML M7 from results, however, is made difficult due to the use of dif- AML MO and ALL [103]. Since platelet adherence to ferent methods such as choice of moAbs and tech- leukaemic blasts and nonspecific binding of glycopro- niques applied in order to detect antigen expression, in- teins lib/Ilia to AML-M5 may result in false-positive consistencies in criteria for defining antigen positivity, CD41 a and CD61 staining results, caution must be ex- " or variation in the group of patients studied (i.e. chil- ercised. Therefore, in all cases with equivocal im- dren and/or adults) and in treatment given. munophenotyping and morphological findings, confir- In addition, the prognostic value of relating clinical mation of flow-cytometric results by cytospin im- outcome to specific antigens instead of evaluating the munofluorescence should be performed. composite immunophenotype has to be questioned in Characteristic immunophenotypic features have re- the light of recent findings demonstrating that expres- cently been described within the CD34+ stem cell sion of certain antigens can be associated with compartment in patients with AML M7 [104]. Com- favourable as well as poor prognostic genetic aberra- pared to CD34+ cells in AML-MO through AML M6 tions. For example, t(8:21), inv(16), chromosome 5 subtypes, the CD34+ megakaryoblasts expressed and 7 aberrations, as well as complex karyotypes, were CD61, glycophorin A but were CD38-. These results observed more frequently in CD34+ AML [49, 121), correspond to the hypothesis of a common immature and CD 19 co-expression may occur in both AML with progenitor cell for the megakaryocytic and erythroid t(8;2l) and with t(9;22) [50, 122. 123). These results cell lineage [105] and the expression of megakary- suggest that CD34+ and/or CD 19+ AML comprise a ocytic antigens sometimes observed in acute ery- heterogeneous group of patients with good as well as throleukaemia as well as the positivity of glycophorin poor risk factors. Recent data confirm this statement A in some cases of AML M7. by suggesting a prognostic role of CD56 expression in The chromosomal abnormalities linked to AML M7 AML with t(8;2l) [74) and acute promyelocytic include t(l;22)(pl3;q!3) [106], constitutional or ac- leukaemia [87] but not in AML cases with Ilq23 quired trisomy of chromosome 21 [107-110], and translocations [93]. sometimes a rearrangement of 3q21 and 3q26. In con- Moreover, future studies should interpret expression trast to t(l;22), which is only observed in infants and of surface antigens in the context of other cell-biolog- not associated with any dysplastic features, rearrange- ical features. These include differentiation stage and ments of 3q21 and 3q26 are not specific for AML M7. functional characteristics, which reflect cellular resis- This has been demonstrated in all subtypes of AML, tance mechanisms to cytotoxic drugs (e.g. multidrug- with the exception of AML M3, and occurs for the resistance phenotype, expression of apoptosis-regulat- most part in older patients whose leukaemic blasts ing proteins) [120, 121, 124-126]. Our personal re- may display dysplastic features [40]. sults in a large series of untreated de novo children and Infants with Down's syndrome, in particular, tend to adults with AML enrolled in the German AML-BFM present with a transient myeloproliferative disorder, and AMLCG studies do not indicate any influence of which commonly shows evidence of megakaryocytic the expression of individual myeloid, lymphoid, and and erythroid differentiation by immunophenotyping progenitor cell-associated antigens on prognosis [110, studies that is indistinguishable from AML by light 119, 127, 128]. They therefore do not show that im- microscopy or immunophenotyping [109, 110]. munophenotyping alone can be applied for risk strati- Leukaemic blasts in children with Down's syndrome fication in AML^at diagnosis. These findings are con- and AML M7 also often show erythroid differentiation current with other recent studies in children [111, 112] and co-express CD7 [109, 110]. and adults [48] with AML. Prognostic impact of immunophenotyping in AML Implication of chromosome abnormali- It is still controversial how significant surface antigen expression in AML is to the prognosis. Despite the fact ties on prognosis that some investigators were not able to show any cor- The most important, independent prognostic parameter relation between the expression of individual progeni- in AML is the karyotype of the leukaemic blasts. For

518 J Lab Med 2001; 25 (11/12): 512-532 G. Rothe clinical purposes, 'karyotype analysis allows discrimi- taken into consideration for a definite diagnosis of L3- nation between three major prognostic groups. A type Burkitt cell leukaemia. The morphological ap- favourable outcome using current treatment regimens pearance of L3 can be copied by AML MO, M1, Ì5 or was observed in several studies in patients with even by many undifferentiated solid tumours. Also, t(8;21)(q22;q22), inv(16)(p!3q22), or t(15;17) vacuolation can be seen in rare cases of LI and L2- (q22;qll-12). Chromosome aberrations with an un- ALL [13]. favourable clinical course are -5/del(5q), -7/del(7q), In some cases of ALL, over 40 % of lymphoblasts inv(3)/t(3;3), and complex aberrant karyotype. The are hand-mirror shaped, but this can also be a feature rest are assigned to an intermediate prognostic group, in rare cases of AML. At the moment, this morpholog- which is very heterogeneous because it includes pa- ical feature appears to be merely a morphological vari- tients with a normal karyotype and rare chromosome ant without distinguishing clinical correlations [13]. aberrations with yet unknown prognostic impact. In It has been suggested that a number of BCR/ABL- future, this group will need to be subdivided further. positive ALL have a unique morphological appear- There is still no general agreement concerning the ance: There are not only the dominant lymphoid blast last detail between the large clinical study groups as to cell population but also larger blasts with myeloid the classification of AML patients according to kary- characteristics, some of the latter even showing a pos- otype and prognosis. According to their experience itive MPO reaction (mostly < 3 %). In some cases, this [129-133], different clinical study groups assign cyto- may falsely lead to the diagnosis of AML MO or even genetic categories to different prognostic subgroups. It AML Ml with Philadelphia translocation [13]. Con- is important to remember that treatment itself influ- versely, · some AML may prove to be Philadelphia ences the impact of prognostic parameters. In the fu- chromosome positive [135]. ture, the major objective will be to find the best therapy for each biological entity. In order to reach this objective, the biological entities must be defined very Immunophenotyping of ALL clearly and large, well-designed prospective trials are Immunophenotyping has become essential in the diag- required to allow a randomized comparison of various nosis of ALL since the demonstration by Borella and treatment strategies even in small subgroups. For APL, Sen [136] that in some children with ALL, leukaemic this goal has already been achieved. There is world- lymphoblasts were of thymic origin. It has also made wide consensus that this subgroup of patients should a substantial contribution to a more precise and bio- be treated in separate trials and ATRA should be im- logically oriented classification of the disease (re- plemented in the treatment. viewed in [6, 18. 19, 26, 137-139]). Patients with t(8;21) or inv(16) benefit from treat- Over the last two decades, immunophenotyping in ment with high-dose cytarabine according to data from ALL, performed at first with polyclonal antisera and Bloomfield et al. Patients with t(8:21) or inv(lo) had tfien using a rapidly expanding panel of moAbs, has the best outcome overall compared to other cytogenet- mainly been applied to distinguishing ALL from AML, ic risk groups and they also benefited most from in- lineage assignment of leukaemic blasts, phenotyping creasing doses of cytarabine [131]. These data also of pathological cell subsets, and examining the signif- stress that the prognosis of distinct cytogenetic sub- icance of membrane antigen expression in predicting groups can be influenced by different treatment strate- treatment response (reviewed in [6, 7, 19, 21, 138]). In gies. addition, leukaemia-associated phenotypic features One important discovery relating to cytogenetic ab- have been routinely used to detect MRD in ALL (re- normalities and prognosis was that although the inci- viewed in [23]) based on observations that leukaemic dence of distinct chromosome abnormalities varies blasts often reveal aberrant or asynchronous antigen with age,'the prognosis of defined cytogenetic aberra- expression compared to normal haematopoietic cell tions is age independent. differentiation. In recent times, immunophenotyping in conjunction with cytogenetic and molecular genetic studies has The FAB-classification of ALL identified clinically and biologically distinct subsets The domain of immunophenotyping and cyto-/molec- within the major diagnostic subgroups of precursor B- ular genetic analyses is the lineage assignment, sub- and T-cell ALL. It has also become decisive in moni- classification of precursor B- or T-cell leukaemia, and toring risk groups in therapeutic studies (reviewed in stratification of treatment according to cell-biological [6, 138-140]). risk groups in ALL. The morphological categories Rather than being based on the presence or absence L1-L3 - originally suggested by the FAB group - are of a single antigen, it should be emphasized that both no longer of clinical importance except for the L3 sub- the lineage affiliation and the definition of matura- type [1,13, 134]. Even in cases displaying the L3 mor- tional stage in ALL are based on patterns of antigen phology with relatively uniform blasts having inten- expression shown by an appropriate selection of clus- sively basophilic cytoplasm and sharply defined, fat- ter of differentiation (CD) moAbs (Table 3). More- containing vacuoles, the cytogenetic result, the t(8;14) over, it is worth noting that the dominant phenotype of or its variants, and the immunophenotyping should be a leukaemic cell population reflects the degree of mat-

J-Lab Med 2001; 25 (11/12): 512-532 519 Immunhämatologie uration achieved by a leukaemic clone and may not translocations in precursor B-cell ALL by simply clas- correspond to the initial target cell of the disease, sifying antigen expression as either positive or nega- which is usually a more immature progenitor cell. tive. More complex descriptions of patterns of expres- We "Will now discuss significant associations be- sion or combinations of antigens are required for this tween immunophenotypic features and numerical purpose. and/or structural chromosomal abnormalities that have recently helped, to establish a refined ALL classification, particularly in precursor B-cell ALL B-cell precursor ALL (Table 4). It should be noted that it is not possible to The t(4;l I)(q21;q23) chromosomal abnormality is pre- obtain accurate phenotypic predictions of specific sent in about 2 %-6 % of both adults and children with

Table 3a Clusters of differentiation (CD) antigens useful in the diagnosis and classification of AML and ALL (myeloid lineage)

CD Molecular and functional Cellular reactivity within the Comments on diagnostic value in group characteristics lymphohaematopoietic system leukaemia diagnosis Myeloid lineage CD 13 150-kD type II membrane glyco- Early committed progenitors of Expressed in most AML, protein, homodimer, amino- granulocytes and monocytes co-expressed in 20 %-35 % of ALL peptidase N (CFU-GM) and maturing cells of these lineages CD14 55-kD glycosylphosphatidylin Mature monocytes (strong), Expressed predominantly in mature ositol-linked glycoprotein; macrophages, granulocytes myelomonocytic leukaemias LPS receptor (weak) (AML M4, M5b) CD15 Carbohydrate,. 3-FAL, X-hapten, Mature granulocytes and mono- Expressed in 50 % of AML, Lewis-X (Lex); adhesion cytes, myeloid and monocytic aberrantly expressed in 5%-10% molecule, ligand for E-, P-, cells, Langerhans cells of ALL, predominantly in L-selectin (CD15s) pro-B-ALL with t(4;11) CD33 67-kDa transmembrane protein, Myeloid and monocytic cells, Expressed in most AML, sialoadhesin early erythroblasts, co-expressed in 20 %-35 % of ALL megakaryoblasts CD36 88-kDa jlycoprotein, Megakaryocytes, platelets, Expressed mainly in AML M5-M7 platelet 3p Illb, GpIV mature monocytes and macrophages, erythroid precursors CD41 Platelet glycoprotein lib, allb Megakaryocytes and platelets Expressed in AML M7 integrin chain,' forms complex with CD61/ß3 integrin chain CD42b Platelet glypoprotein Ib·, forms Megakaryoctes and platelets, Expressed in AML M7 complex with CD41c (disulfide absence of CD42 complex leads bond) and CD41a, CD41d; to Bernard-Soulier syndrome CD41a-d: receptor for vWf (von Willebrand factor) CD61 Platelet glycoprotein Ilia, Megakaryocytes and platelets Expressed in AML M7 ß3 integrin chain, forms complex with CD41/allb integrin chain CD64 72-kDa glycoprotein, high affinity Monocytes and macrophages, Expressed in monoblastic/ IgG Fc receptor (FcyRI), immature granulomonocytic pro- monocytic leukaemia and in sub- receptor-mediated endocytpsis of genitors, subset of dendritic cells, sets of immature AML IgG-antigen complexes, antibody- early myeloid lineages dependent cellular cytotoxicity CD65/ Carbohydrate, ceramide-dodeca- Mature granulocytes, myeloid Expressed in most AML, aberrantly CD65s saccharide/sialylated-CD65 cells, monocytes expressed in 5%-10% of ALL, predominantly in pro-B-ALL with t(4;11) CD66c 90-kDa GPI-linked glycoprotein, Mature granulocytes, myeloid Expressed by distinct subsets of member of the carcinoembryonic cells, monocytes prec-B-cell ALL (e.g. Ph+, ETV6- antigen family AML1", and hyperdiploid cases)

520 J Lab Med 2001; 25 (11/12): 512-532 G. Rothe

ALL, and has been associated with characteristic im- the early B-cell commitment of blast cells with this cy- munophenotypic and clinical features (e.g. high leuko- togenetic abnormality. cyte counts, predominance, of females in infants, fre- On the basis of our experience in a large series of quent organ enlargement, and increased incidence Of childhood and adult ALL patients with Ilq23 re- CNS leukaemia at diagnosis) (reviewed in [141, 142]). arrangements [145, 152, 155, 156], these features, par- Earlier reports, mainly in infant ALL, have suggested' ticularly the missing or weak expression of CD24 as that t(4;ll)-associated acute leukaemias originate for compared with CD 19, and the co-expression of the most part in multipotent or very early CDlO-nega- CD65s, usually associated with negativity of other tive B progenitor cells with a high frequency of pan-myeloid antigens (e.g. CD 13, CD33), are highly myeloid-antigen positivity [143, 144]". predictive for the cytogenetic and/or molecular Recent studies have made an analysis of the im- demonstration of MLL rearrangements, mostly due to munophenotypic and genotypic features of this sub- a t(4;ll) or, less frequently, other Ilq23 aberrations. group in greater detail. In the vast majority of ALL More recently, the use of moAb 7.1, recognizing a spe- with t(4;ll), leukaemic blasts reveal a typical anti- cific antigen of the chondroitin sulfate proteoglycan genie profile (e.g. CD 19+, CD 10-, CD24- or weakly family, has been shown to detect with a high degree of +, cylgM- or +, CD 15 and/or CD65s+) indicative of sensitivity childhood ALL with MLL rearrangements, an immature pro-B phenotype with frequent co-ex- but does not distinguish between the different translo- pression of particular myeloid antigens (i.e. CD 15, cation partners involved in MLL rearrangements [96, CD65s). This clear-cut association of immunopheno- 157]. The need for molecular genetic analyses cannot, typic features with t(4;ll), which was initially de- therefore, be dismissed. Our personal results in a large scribed in infant ALL [145-147], has recently also number of childhood and adult ALL patients suggest been detected in adult patients [148-154]. Southern that carefully constructed antibody panels, including blot analysis revealed Ig heavy-chain gene rearrange- 7.1, may be helpful for identifying ALL carrying MLL ments in virtually all cases as well as oligoclonal dis- rearrangements and for detecting MRD by flow cy- ease in some of them [145]. These results underline tometry [96].

Table 3b Clusters of differentiation (CD) antigens useful in the diagnosis and classification of AML and ALL (T lineage)

CD Molecular and functional Cellular reactivity within the Comments on diagnostic value in group characteristics lymphohaematopoietic system leukaemia diagnosis T lineage CD1a 49-kDa type I transmembrane Cortical thymocytes, Defines cortical prec-T-cell ALL glycoprotein, MHC Mike; binds Langerhans cells to a-microglobulin; nonpeptide antigen-presenting molecule CD2 50-kDa type I.transmembrane Thymic and mature T cells, Expressed in 70-85 % of prec-T-cell glycoprotein, LFA-1; receptor for most NK cells ALL and approx. 10 % of AML CD58 (LFA-3); adhesion and (especially M3 and M4Eo signal transducing molecule subtypes) CD3 Complex of 6 polypeptide Thymic and mature T cells Cytoplasmic expression defines chains, component of the TCR prec-T-cell ALL, membrane ex- (associated with TCRa or pression in 25 % of T-ALL defines TCRyo) mature prec-T-cell ALL CD4 55-kDa transmembrane glyco- Subset of thymocytes and mature Variably expressed by pre-T, protein, receptor for MHC class II T cells (helper/inducer), mono- cortical, mature prec-T-cell ALL, molecules, receptor for HIV cytes, macrophages and AML (especially of monocytic envelope glycoprotein (gp120) origin) CDS 67-kDa glycoprotein, scavenger Thymic (weak expression) and Expressed by 90 %-95 % of receptor cysteine-rich (SRCR) . mature (strong expression) prec-T-cell ALL family, costimulatory molecule and T cells, subset of mature B cells receptor for CD72 j CD7 40-kDa glycoprotein T cells, NK cells, haematopoietic Expressed in virtually all prec-T-cell i stem cells ALL and approx. 15 % of AML j CD8 32-kDa, áá homodimer or áâ Subset of thymocytes and.mature Variably expressed by pre-T, j heterodimer, co-receptor with TCR T cells (suppressor/cytotoxic), cortical, and mature ! for MHC class I molecules NK cell subset prec-T-cell ALL

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Table 3c Clusters of differentiation (CD) antigens useful in the diagnosis and classification of AML and ALL (B lineage, non-lineage accociated)

CD Molecular and functional Cellular reactivity within the Comments on diagnostic value in group - characteristics lymphohaematopoietic system leukaemia diagnosis B lineage CD19 95-kDa glycqprotein, associated Expression from the earliest Expressed in virtually all with CÖ21; signal transduction recognizable B-lineage cells to prec-B-cell ALL and a subset of mature B cells, follicular dendritic AML (especially, AML M2 with cells t(8;21)) CD20 33- to 37-kDa phosphoprotein, B cells Expressed in 40 % of B cell activation .prec-B-cell ALL CD22 135-kDa type I glycoprotein, Precursor and mature B cells Cytoplasmic expression in virtually adhesion and signalling all prec-B-cell ALL, membrane expression in most prec-B-cell ALL and B-ALL CD24 35- to 45-kpa glycosylphosphati- Precursor and mature B cells, Expressed in > 90 % of prec-B-cell dylinositol-linked glycoprotein neutrophilic granulocytes ALL and some AML CD79a 40- to 65-kDa glycoprotein, Precursor and mature B cells, Expressed in virtually all associated with CD79ß; com- plasma cells prec-B-cell ALL and aberrantly in ponent of B cell antigen receptor some AML NK CD56 175-220-kDa glycoprotein, neural NK cells, subset of T cells Expressed in some AML with cell adhesion molecule (NCAM), t(8;21), t(15;17), acute monocytic homotypic and heterotypic cell leukaemia and NK cell neoplasms adhesion (in neural development) Non-lineage associated CD10 100-kDa glycoprotein, zinc metallo- Lymphoid precursors, germinal Defines common-ALL, expressed protease, neutral endopeptidase, centre B cells, mature neutrophilic in approx. 40 % of prec-T-cell ALL common ALL antigen (CALLA) granulocytes CD34 105- to 120-kDa type I trans- Early lymphohaematopoietic stem Expressed in 60 %-70 % of membrane glycoprotein, cell and progenitor cells prec-B-cell ALL, < 10% of prec-T- adhesion cell ALL, and 40 %-50 % of AML CD45 180- to 220-kDa glycoprotein, Expressed, typically at high Expressed in 90 % of all leukocyte common antigen (LCA), levels, on all haematopoietic cells prec-B-cell ALL, almost all tyrosine pnosphatase, T and prec-T-cell ALL, and nearly all AML B cell antigen-receptor-mediated activation CD117 145-kDa glycoprotein,.tyrosine Haemopoietic stem and Expressed in approx. 85 %-90 % of kinase receptor type 3, c-kit, stem progenitor cells prec-B-cell ALL and practically all cell factor (SCF) receptor other subtypes of acute leukaemia

The Philadelphia (Ph) translocation, or myeloid antigen expression [151, 166], positivity of t(9;22)(q34;ql 1), which occurs in 15 %-30 % of adults the KOR-SA3544 antigen [170], co-expression of and 3 %-5 % of children With ALL, is usually associ- CD34 and CD 10 [154], and expression of CD25 [164], ated with a common or pre-B ALL phenotype [148, are associated with Ph+ ALL, there does not seem to 158-167]. Some studies have been able to identify a be a definite correlation between this translocation and low proportion of immature CDlO-negative precursor immunophenotype. Not only our data in childhood and B-cell, B- or pure T-lineage features in Ph+ ALL [158, adult ALL [163, 171] but also the findings of others 168]. Recent data have shown that, similar to chronic [172, 173] suggest that myeloid antigens are not co- myelogenous leukaemia, a primitive haematopoietic expressed more-often on Ph+ ALL than on Ph- ALL. cell is the target for the leukaemia transformation in As yet, no extensive analyses have been carried out to Philadelphia-positive ALL [169]. · investigate «the relation between surface antigen ex- Although it has been suggested that several im- pression and the two different breakpoint cluster re- munophenotypic features, for example increased gions (bcr) detected on chromosome 22 (i.e. minor or

522 J Lab Med 2001; 25 (11/12): 512-532 G. Rothe

CD66c [176] and complete or partial lack of both CD9 Table 4 Classification of acute lymphoblastic and CD20 expression [179, 180]. leukaemia (ALL) . fc Previous studies have indicated that t(I;19) a (q23;p!3), observed in 5 %-6% of childhood and less 1. B lineage ALL (CD19+ and/or CD79 + and/or CD22+): than 5 % of adult ALL, is strongly linked to cytoplas- Pro-B-ALL (B-l) (no expression of other mic 'ì+ pre-B ALL [181]. This abnormality was also differentiation B-cell detected in some cytoplasmic ì- common ALL [158, antigens) 182, 183], although subsequent studies confirmed the Common ALL (B-ll) CD10+ close association between t(l;19) and pre-B phenotype Pre-B-ALL (B-ll!) cytoplasmic lgM+ in both childhood and adult ALL [182]. Mature B-ALL (B-IV) cytoplasmic or surface kappa or lambda* More recently, a pattern of surface antisen expression (i.e. CD9+, CD19+, CD22+, CD20+, CD34-, b 2. T lineage ALL (cytoplasmic/membrane CD45hlgh) has been described that is characteristic of CD3+): ALL with t(l;19) but lacks specific details [184, 185]. Pro-T-ALL (T-l) CD7+ Pre-T-ALL (T-lI) CD2+ and/or CD5+ and/or Polyclonal and monoclonal antibodies can now be CD8+ used for the flow-cytometric detection of the E2A- Cortical T-ALL (T-lll) CD1a+ PBX1 protein in the nucleus of t(l;19)+ leukaemic Mature-T-ALL (T-IV) membrane CD3+, CD1a- blasts [186]. á/â + T-ALL (group a) anti-TCR á/â+ ã/ä + T-ALL (group b) anti-TCR ã/ï+ B-ALL 3. ALL with myeloid antigen expression Although certain cytogenetic abnormalities in precur- (My + ALL) sor B- and T-cell ALL do not enjoy a close relationship to the FAB subtype, leukaemic blasts in mature sur- a Positive at least with two of the three markers; most face immunoglobulin (slg)+ B-ALL are normally cases are TdT+, HLA-DR+ with the exception of B-IV, characterized by FAB L3 morphology and usually ex- which is frequently TdT- b hibit t(8;14)(q24;q32) (in 75 %-85 % of the patients) The majority of cases are TdT+, HLA-DR- CD34-, but these marker's are not taken into consideration for or one of the variant translocations t(2:8)(pl l-12;q24) diagnosis or disease classification and t(8;22)(q24;ql 1). Recently, a high predictive value of these B-ALL-associated chromosomal anomalies was demonstrated [29] for L3-morphology. Do note major -bcr) in Ph+ ALL. Preliminary results in adult the exceptions, however, such as paediatric ALL pa- ALL have suggested that M-BCR rearrangements are tients with the (8:14) translocation. FAB L3 morphol- more common in My+ than in My- ALL 1163]. ogy, and laboratory and clinical features consistent It was previously reported that the monoclonal anti- with B cell ALL whose leukaemic blasts showed a less body KOR-SA3544 could recognize Ph+ ALL with high differentiated B-precursor immunophenotype [187] or sensitivity [170]. However, more recent studies have lacked surface and cytoplasmic Ig [188], and slg light- shown that this antibody specifically recognizes the non- chain positive adult ALL of LI or L2 morphology specific cross-reacting antigen (NCA)-50/90 (CD66c), without t(8;14) or its variants [189]. one of the carcinoembryonic antigen (CEA)-related gly- These unusual findings demonstrate the importance coproteins [174], and reacts with certain subsets of pre- of evaluating patients with a combination of diagnos- cursor B-cell ALL,.lncluding Ph-r ALL, ETV6-AML1 tic tools in order to assign them to or to exclude them negative ALL, and hyperdiploid ALL [175, 176]. from recognized disease subgroups. Moreover, out- The most common translocation in childhood ALL is come data and clinical courses in these patients sug- the ETV6-AML1 fusion, created by the t(12;21). which gest that laboratory tests with cytogenetic evidence of occurs in about 20 %-25 % of cases but is only present t(8;14) are dictated by a hierarchy of clinical relevance in less than 3 % of adult ALL cases. The ETV6-AML1 or of variant translocations in assigning patients to B- translocation is restricted to patients with nonhyper- cell ALL-specific protocols [187]. On the other hand, diploid precursor B-cell.ALL, and most cases show a therapeutic response in other subtypes without one of common ALL or, more rarely, a pre-B ALL phenotype. these three translocations resembles the precursor B- The high frequency of myeloid antigen expression (i.e. cell ALL. CD 13 ^nd/or CD33) that was reported originally in a large series of children with ETV6-AML1 rearrange- T-lineage ALL ment enrolled in the German and Italian miilticentre trials was confirmed by other reports [177-179]. It is in- Precursor T-cell ALL, like precursor B-cell ALL, is teresting to note that other characteristic immunophe- heralded by a strong interpatient biological hetero- notypic features have been described recently that are geneity. Leukaemic transformation may occur at dis- highly predictive of ETV6-AML1 rearrangement and tinct stages of T-cell ontogeny, resulting in lynv may be implemented as a screening test for this genet- phoblasts^with immunophenotypic features that corre- ic 'abnormality [296]. These include negativity of *spond to immature or more mature T-cell progenitors.

J Lab Med 2001; 25 (11/12): 512-532 523 Immunhämatologie

In marked contrast to precursor B- or mature B-cell (e.g. Ilq23 rearrangements) and clinical features (e.g. ALL, clear-cut relationships -have not yet been estab- high tumour burden, age < 1 year). lished between the specific chromosomal changes oc- Cytogenetic and molecular genetic studies provide curring in approximately 44%-6l % of paediatric T- conclusive evidence that children and adults with com- lineage ALL patients [190, 191] and the maturational mon and pre-B ALL differ significantly as far as the stage of T-ALL blasts or a particular pattern of surface incidence of the known favourable or unfavourable antigen expression [165, 190-192]. Characteristic phe- chromosomal translocations is concerned. For exam- notypic features within groups showing common chro- ple, the t(9;22) accounts for up to 55 % of adult but mosomal abnormalities in T-ALL [190, 192] have only less than 5 % of children with CD 10+ precursor B-cell been identified in children with t(ll;14) whose ALL. The reported frequency for the t(12;21), associ- leukaemic blasts expressed a profile of membrane sur- ated with a good prognosis in most recent studies, face antigens (i.e. CD4+, CD8+, and CD3±) associat- ranges between 12%. and 36% in childhood common ed with more mature thymocytes [192. 193]. Most data or pre-B ALL, and rarely occurs in adult patients [148, were derived from cytogenetic analyses in childhood 159, 173] (reviewed in [206]). These results may part- ALL. Convincing evidence on the correlation of im- ly explain the major differences observed in therapeu- munophenotype and karyotype in adult patients with tic success rates between children and adults with precursor T-cell ALL is not yet available. common or pre-B ALL. Recent data show that the most common nonran- . Confirmation of the prognostic importance of the dom genetic defect associated with precursor T-cell pre-B ALL immunophenotype has been limited to se- ALL, occurring in about 10%-25% of patients quential studies of the Paediatric Oncology Group [194-196], is represented by alterations of the TALI (POG). No wonder, until recently, this was the only proto-oncogene on chromosome Ip32, either by group performing cytoplasmic testing in the context translocation or other rearrangements. Many reports of large prospective clinical trials. Earlier POG studies demonstrated unanimously that TALI alterations in T- suggested that the pre-B phenotype might be an inde- ALL exclusively occurred" in CD3- or CD3+ T-ALL of pendent prognostic marker for reduced event-free sur- the -B lineage, although a clear association of TALI vival (EPS) [181]. More recent data, however, have re- gene rearrangements with a distinct stage of thymo- vealed that the subgroup of children with pre-B ALL cyte maturation has not yet been established and t(l;l9) has a worse therapeutic outcome [207]. By [194-197]. contrast, the German ALL-BFM trials, the analysis by the Medical Research Council (MRC) UKALL trial XI, and a single-centre study did not show any major Prognostic impact of immunopheno- differences in the duration of remission between com- typing in ALL mon and pre-B ALL [202, 208]. The paucity of standardized criteria for the classifica- In children with precursor B-cell ALL, the progno- tion of immunophenotypic subgroups in the past and a sis has been associated with other immunophenotypic lack of controlled prospective studies on the therapeu- features such as CD20, CD34, and CD45 expression. tic response in precursor B- and T-cell ALL subsets It has also been suggested that the lack of CD20 and and the different treatment strategies administered CD45 antigens or the presence of CD34 on leukaemic have all encumbered the assessment of the prognostic blasts may be linked with a longer EPS [209-212]. In impact of immunophenotyping studies in ALL. By view of the relationship of these immunophenotypic virtue of the strong_cprrelation between certain im- features (e.g. absent CD45) to other biologically munophenotypic subgroups and cytogenetic or clinical favourable characteristics [180, 211, 213], however, features, the merit of immunophenotyping in indepen- their significance to the prognosis has to be evaluated dently predicting therapeutic outcome has come into in further studies by adapting results based on multiple question. Many studies have shown that the prognostic risk factors. impact of immunophenotypic subgroups and chromo- The prognosis in B-ALL can be remarkably im- somal abnormalities is lower as a result of improved proved by "novel and intensive treatment strategies, efficacy of chemotherapy. Obviously, prognostic fac- adapted to the clinical and biological features of this tors have to be evaluated in the context of therapy ad- disease [200, 201]. This has been shown by several ministered [154, 182, 198-201]. studies in childhood and adult ALL. The data have im- No significant differences in remission rates were pressively illustrated that the negative prognostic im- recorded for immunophenotypic subgroups in precur- pact of biological characteristics, such as the im- sor B-cell ALL, although several studies did show a munophenotype or chromosomal translocations, can correlation between the maturational stage of B lym- be offset by more effective treatment. phoblasts and remission time. Most studies in both In T-lineage ALL, various immunophenotypic fea- childhood and adult ALL reported a worse prognosis tures,, including an immature pro-/pre-T-ALL pheno- for patients whose leukaemic blasts expressed an im- type, membrane expression of CD3 or MHC class II mature CDJO-negative pro-B phenotype, also called antigen, and negativity of CD2, CD5, THY antigen early pre-B or null-ALL [138, 153, 154, 202-205], (similar to CD1), or CD 10 [138, 202, 209, 214-218], which was often connected with adverse biological seem to be connected with an increased risk of nonre-

524 J Lab Med 2001; 25 (11/12): 512-532 G. Rothe sponse to therapy. The prognostic impact of these fac- cursor T-cell ALL with distinctive clinical and patho- tors, however, depends on the treatment strategies logical features and prognoses [226-228]. Further used, and immunophenotyping is still controversial prospective studies are needed to more thoroughly and has not been used for routine risk classification or characterize the cytobiological features of TCR-ãä + assignment to new treatment strategies in high-risk lymphoblasts and to confirm the better prognosis of precursor T-celi ALL patients. this subgroup compared to TCR-a + precursor T-cell In line with our observations, other studies have ALL [228]. shown that children and adults with pre-T ALL differ greatly with respect to their phenotypic and genotypic Acknowledgement features. This suggests an arrest of adult pro-/pre-T We would like to thank P.D. Dr.Dr. T. Haferlach and ALL at a less mature differentiation stage than in Dr. C. Schock for helpful discussions. childhood and may be closely related to the worse response observed in these patients [209, 214, 216, 219, 220]. Similar results have been published with refer- Impact of chromosome abnormalities on ence to adolescent and adult patients whose leukaemic prognosis blasts showed CD7 antigen expression in the absence The impact of each prognostic factor on clinical out- of myeloid, B-, or more mature T-cell differentiation come has to be analysed and viewed in light of thera- antigens [221]. Interestingly, the leukaemic blasts in · peutic strategies. There is no doubt that therapy signif- these patients were capable of multilineage differenti- icantly impacts prognosis. Success can be achieved by ation in vitro both spontaneously and after stimulation considering modern standard protocols t(12;21), with appropriate cytokines, suggesting that the acute dic(9;12), hyperdiploidy with a chromosome number leukaemia evolved from in vivo transformation of im- between 51 and 55, and t(10;14) in T-lineage ALL. An mature pluripotent haematopoietic cells showing a intermediate prognosis can be shown in patients with a poor response to conventional chemotherapy. In sup- normal karyotype, a deletion of the long arm of chro- port of this theory, patients with immature precursor T- mosome 6, a 9p- or 12p-deletion. The outcome is not cell ALL had a higher frequency of co-expression of particularly optimistic for patients with t(9:22), even CD34, CD 117, and/or myeloid antigens [209, 220, with very intensive treatment strategies including allo- 2221. geneic stem cell transplantation. By intensifying and Most other attempts to identify additional sub- risk-adapting treatment strategies, patients with groups of precursor T-cell ALL of prognostic rele- t(l:19), t(4;ll), or t(8;14) and variants might have a vance'have been unsuccessful in both childhood and better prognosis [152, 200, 230-232]. adult ALL [214, 223]. Using similar maturational stag- Improvements in therapy could change or diminish ing systems, at least three recently conducted multi- the importance of various cytogenetic abnormalities in centre trials in childhood ALL have yielded more evi- acute leukaemia. In childhood ALL, it has been proven dence that children with cortical (CDla+) precursor T- that with more intensive chemotherapy all patients fall cell ALL respond better and earlier to treatment, as il- into one positive prognosis group, regardless of ploidy lustrated by their in vivo response to corticosteroids. A or specific karyotypic abnormalities. Changes in treat- significantly longer event-free survival than those with ment strategies could even improve outcome in the an immature or mature precursor T-cell phenotype poor prognostic subgroup of Ph-positive patients. In [202, 224] was also observed. Similar data were re- recent studies in Ph-positive childhood ALL, it has cently published byJJhe Cancer and Leukaemia Group been shown that intensive chemotherapy improves the [154], who cited a major improvement in survival for outcome in a subgroup of patients [167, 229]. adult patients with CD1, CD2, CD4, and CD5 expression in comparison to patients not expressing these antigens. Despite a lack of explanations as to why patients with a cortical immunophenotype respond better References to treatment, recent investigations of apoptosis-related 1. Bennett JM, Catovsky D. Daniel MT, Flandrin G, Galton DA. Gralnick HR. Sultan C. Proposals for the classification of the acute parameters, including spontaneous apoptosis in vitro leukaemias. French-American-British (FAB) co-operative group. Br and modulation of apoptosis by IL-7, suggest that mat- J Haematol 1976;33:451-8. urational stages of precursor T-cell ALL differ with re- 2. Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA. Gralnick HR, Sultan C. Proposal for the classification of myelodys- gard to their accessibility to apoptotic mechanisms, plaslic syndromes. Br J Haematol 1982:51:189-99. with lymphoblasts expressing CD la or showing a se- 3. Bennett JM, Catovsky D, Daniel MT, Flandrin G, Gallon DA. lection-related phenotype that is more susceptible to Gralnick HR, Sultan C. Criteria for the diagnosis of acute leukemia of megakaryocyte lineage (M7). A report of the French-American- apoptosis than leukaemic lymphoblasts with an imma- Brilish Cooperative'Group. Ann Intern Med 1985; 103:460-2. ture phenotype [224, 225]. 4. Bennett JM. Catovsky D, Daniel MT, Flandrin G. Gallon DA, Many studies hint that a subclassification of mem- Gralnick HR, Sultan C. Proposal for the recognition of minimally brane CD3+ precursor T-cell ALL in accordance with differentiated acute myeloid leukemia (AML-MO). Br J Haematol 1991:78:325-9. T-cell antigen receptor (TCR) áâ or ãä expression can 5. Second MIC Cooperative Study Group. Morphologic, iminuno- give valuable clinical information. Indeed, TCR-ãä + logic and cytogenetic (MIC) working classification of the acute cases represent an important but rare subgroup of pre- myeloid leukaemias. Br J Haematol 1988:68:487-94.

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