2 Immunophenotypic classification of acute lymphoblastic leukaemia

2 Immunophenotypic classification of acute lymphoblastic leukaemia W O L F - D I E T E R LUDWIG ANAND R A G H A V A C H A R ECKHARD THIEL

INTRODUCTION Recent advances in immunophenotyping of acute leukaemias have led to important insights into normal haematopoietic differentiation as well as into the cellular diversity and origins of leukaemic blasts, especially in acute lymphoblastic leukaemia (ALL). These advances include the production and standardization of monoclonal antibodies (mAbs) to a variety of lymphoid- and myeloid-lineage-associated antigens, the characterization of reactivity patterns of these reagents with leukaemic blasts, the establishment of an internationally accepted cluster of differentiation (CD) nomenclature and, finally, technical innovations in the evaluation of antigen expression on individual leukaemic cells. Immunophenotyping has become an important element in the modern diagnosis of acute leukaemia for several reasons. First, use of a standardized panel of mAbs to B-cell, T-cell and myeloid as well as non-lineage-restricted antigens permits allocation of more than 98% of acute leukaemias to their respective lineage (Janossy et at, 1989; Campana et al, 1990a). Secondly, in ALL, immunophenotyping has established a solid basis for precise and biologically oriented classification of the disease (Foon and Todd, 1986; Greaves, 1986); and, in acute myeloid leukaemia (AML), immunologic markers are particularly important for identifying acute leukaemia with minimal myeloid or megakaryoblastic differentiation (Bennett et al, 1985; Bennett et al, 1991; Buccheri et al, 1992). Thirdly, based on recent observations that leukaemic blasts frequently disclose aberrant or asynchronous antigen expression compared with normal haematopoietic cell differentiation, leukaemia-associated phenotypic features have been used to detect minimal residual disease (MRD) in both ALL and AML (reviewed in Campana et al, 1991a; van Dongen et al, •992). Finally, immunophenotyping alone or in conjunction with more recently developed cytogenetic and molecular-biological techniques has identified biologically and clinically distinct subsets within the major diagnostic groups and has been essential for Bailli~re' s Clinical Haematology--

Vol. 7, No. 2, June 1994 ISBN 0-7020-1825-2

235 Copyright © 1994, by Bailli~re Tindall All rights of reproduction in any form reserved

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monitoring risk groups in therapeutic studies (reviewed in Borowitz, 1990; Pui et al, 1990a; Secker-Walker, 1990). Several reviews have been published on the advances made during the late 1970s and early 1980s in the area of immunophenotyping of acute leukaemia (Crist et al, 1985; Thiel, 1985; Foon and Todd, 1986; Drexler et al, 1988; Borowitz, 1990; Bain, 1990) and more recent comprehensive articles have dealt in depth with technical aspects of immunofluorescence and flow cytometry methods, new fluorescent chromophores for labelling antibodies and immunoenzymatic staining procedures that can be applied directly to peripheral blood or bone-marrow smears for immunophenotyping of leukaemic blasts (Janossy et al, 1986; Mason et al, 1986; Campana et al, 1990b). This chapter discusses the contribution of mAbs to lymphoid- and myeloid-associated antigens towards improving diagnostic precision and establishing a reproducible classification of ALL. The incidence, the clinical and biological features and the prognostic relevance of immunophenotypic subgroups will be reviewed with emphasis on results obtained within the framework of the German ALL multicentre trials in both childhood and adult ALL. DIAGNOSIS AND CLASSIFICATION OF IMMUNOPHENOTYPIC SUBGROUPS

The last decade witnessed a marked increase in the diagnostic accuracy of immunophenotyping. While it was possible as early as 1980 to correctly classify approximately 90% of acute leukaemias as ALL or AML by using heterologous antisera to HLA-DR, terminal deoxynucleotidyl transferase (TdT), common ALL and T-cell-associated antigens (Janossy et al, 1980), the availability of mAbs has further improved diagnostic precision to the extent that 98-99% of acute leukaemias can now be reliably classified by immunological marker analysis (Chan et al, 1985; Janossy et al, 1989). In addition, ALL could be subdivided according to normal maturational stages of B- or T-lineage (reviewed in Thie11985; Foon and Todd, 1986; Borowitz, 1990). Current procedures for the diagnosis, lineage affiliation, and characterization of maturational stages of acute leukaemias are outlined in Figure 1. Table 1 specifies the most informative reagents for immunodiagnosis of ALL and describes their main cellular reactivity with normal haematopoietic cells as well as their diagnostic value for immunophenotyping leukaemic blasts in ALL. Acute leukaemia is diagnosed according to the criteria of the FrenchAmerican-British (FAB) classification system prior to immunophenotyping (Bennett et al, 1976). In the first step of phenotypic characterization, leukaemic blasts will be assigned to one of three main categories of acute leukaemias (i.e. B- or T-lineage ALL or AML) by combining mAbs to pan-B (CD19), pan-T (CD7), and pan-myeloid surface antigens (CD13, CD33, CDw65) with reagents to lineage-specific antigens that are already expressed very early in lymphoid or myeloid differentiation and are first

IMMUNOPHENOTYPIC CLASSIFICATION OF ALL

237

Acute Leukaenda: Diagnosis according to FAB criteria Assianment to Ivmohoid or mveloid lineage: CD19/cyCD22; CD7/cyCD3; CD13/CD33/CDw65/MPO B-cell precursor ALL, B-ALL,T-lineage ALL; AML

....

B-lineage ALL

Maturation stage/ immunoohenotvoic suboroup: CD20/CD24,cylgM,SIg

T-lineageALL

CD1/CD2/CD3/CD4/CD8/TCR proteins

AML

CD14/CD15/CD61/CD64/glyA

Figure 1. Flow chart indicating the antigens essential for lineage assignment and definition of maturational stage and/or immunophenotypic subgroup of acute leukaemia.

exhibited in the cytoplasm (cy) of B- (cyCD22), T-cell (cyCD3), and myeloid precursor cells (myeloperoxidase, MPO) (Bainton et al, 1971; D6rken et al, 1987; Campana et al, 1987; van Dongen et al, 1988). The latter are of utmost importance in cases lacking expression of any pan-B, -T or -myeloid surface antigens or disclosing equivocal phenotypic features (Janossy et al, 1989; Buccheri et al, 1992). Next, the maturational stage at which the majority of lymphoblasts are arrested is defined by analysing antigens more closely associated with distinct maturational stages of B- and T-cell lineage (i.e. B-lineage: CD20, cy immunoglobulin tx heavy-chain (cyIgM) and surface immunoglobulin (SIg); T-lineage: CD1, CD2, CD4, CD8, and membrane, mCD3) (Figure 1). It should be emphasized that both the lineage affiliation and the definition of maturational stage are based on the patterns of antigen expression demonstrated by an appropriate selection of CD mAbs rather than on the presence or absence of a single antigen. In addition, it is noteworthy that the dominant phenotype of a leukaemic cell population reflects the degree of maturation achieved by a leukaemic clone and may not correspond to the initial target cell of the disease, mostly a more immature progenitor cell. Criteria applied to define subgroups of B- and T-lineage ALL differ markedly between clinical studies, and a generally accepted classification system has not yet been established for immunophenotypic subgroups of ALL. Most terms used to designate ALL immunophenotypic subgroups have been based either on the presumptive B- and T-cell differentiation stages of normal tymphopoiesis (Reinherz et al, 1980; Nadler et al, 1984; Loken et al, 1987) defining four to six subgroups of B-lineage and three to

IOB4 B1 HD6

B lineage CD19 CD20 CD22

My7 My9 V1M-2 CLB-MPO-1

OKlal

Various antisera

HLA-DR$

Td'I~

Immature lymphoid cells

Precursor/B/activated T-cells, monocytes

Precursor B-cells, cortical thymocytes Myeloid/lymphoid precursor cells

CFU-GM to granulocytes/monocytes CFU-GEMM to promyelocytes/monocytes Most immature and mature myeloid cells Myeloid cells

All B-cells Subset of B-cells Immature B-cells (cytoplasmic) Mature B-cells (membrane) Most B-cells, granulocytes Pre-B-cells Mature B-celUs

Cortical thymocytes Most T-cells, subset NK-cells Immature T-cells (cytoplasmic) Mature T-cells (membrane) Subset of thymocytes/mature T-cells Most T-cells, subset of B-cells All T-cells, subset of NK-cells Subset of thymocytes/mature T/NK-cells

Main cellular reactivity with haematopoietic cells

Defines common ALL, expressed by approx. 40% of T-ALL Expressed by 60-70% of BCP-ALL, < 10% of immature T-ALL (mostly adults), and 40-50% of AML Expressed by all BCP-/B-ALL, < 10% of immature T-ALL (mostly adults), and most AML Expressed in virtually all BCP-, T-lineage ALL, and 15-20% of AML

Co-expressed by approx. 5-20% of ALL Co-expressed by approx. 5-20% of ALL Co-expressed by most ALL with t(4;ll) Distinction between AML and ALL

Expressed by virtually all BCP-/B-ALL Expressed by 40% of BCP-ALL Cytoplasmic expression by virtually all BCP-ALL, membrane expression by more mature BCP-/B-ALL Expressed by > 90% of BCP-ALL and some AML Defines pre-B-ALL subset Defines B-ALL

Expressed by 90-95% of T-ALL Expressed by virtually all T-ALL and approx. 15% of AML

Defines intermediate T-ALL Expressed by 70-85% ofT-ALL and approx. 10% of AML Cytoplasmic expression by virtually all T-ALL, membrane expression by approx. 25% of T-ALL

Comments regarding diagnostic value in ALL

* CD, cluster of differentiation (according to the 4th International Workshop on Human Leukocyte Differentiation Antigens). From Knapp et al (1989). t Antibodies routinely applied to immunophenotyping of ALL in the German reference immunodiagnostic laboratory. :~ Unclustered.

J5 HPCA-1

CD10 CD34

Non-lineage-assoeiated

CD13 CD33 CDw65 MPO$

Myeioid lineage

OKB2 Various antisera Various antisera

Leu-3a Leu-1 Leu-9 Leu-2a

CD4 CD5 CD7 CD8

CD24 cylgM$ Slg$

Na 1/34 OKTll Leu-4

Antibodies'~

CDla CD2 CD3

T lineage

CD* design

Table 1. Antibodies useful in the diagnosis and classification of ALL.

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IMMUNOPHENOTYPIC CLASSIFICATION OF ALL

239

four subgroups of T-lineage ALL (Reinherz et al, 1980; Roper et al, 1983; Nadler et al, 1984; Foon and Todd, 1986; Garand et al, 1989); or on the expression of CD10, cy or SIg and different T-cell antigens, thus distinguishing broader categories of B-cell precursor and T-lineage ALL (First MIC Cooperative Study Group, 1986; Thiel et al, 1987; Crist et al, 1989; Janossy et al, 1989). We have recently proposed an obligatory panel of mAbs and definitions for immunological subclassification of childhood ALL in order to facilitate the comparability of clinical studies with respect to the clinical features and prognostic impact of immunologic subtypes (van der Does-van den Berg et al, 1992). Figure 2 depicts the antigenic profile characterizing the different B- and T-lineage ALL subgroups as well as the corresponding immunoglobulin (Ig) and T-cell antigen receptor (TCR) gene patterns. The subset previously defined as null or unclassified ALL (Greaves et al, 1981a; Hoelzer et al, 1984; Sobol et al, 1985; Thiel et al, 1987) comprised a very heterogeneous group of immunophenotypes such as CD 10 (CALLA)negative non-T-, non-B-ALL with expression of early B-cell antigens (First MIC Cooperative Study Group, 1986; Janossy et al, 1989) and acute leukaemias positive for TdT and HLA-DR but lacking B-, T- and myeloid-lineageassociated markers (Sobol et al, 1985). Although most of the null-ALL cases with unclassifiable or ALL morphology corresponded to immature pre-preB or pro-B subtypes, as shown later by the combined expression of early B-cell antigens (e.g. CD19, CD24) and rearrangement of Ig heavy-chain genes, it is probable that some immature AML and pre-T ALL were also previously included in this subset. Since lineage commitment of leukaemic blast cells can now be established in > 98% of cases, the imprecise and ill-defined term null-ALL should be abandoned.

B-cell precursor ALL

The analysis of CD10 (the common ALL antigen), cylgM and SIg allows the division of B-lineage ALL into three subgroups of B-cell precursor ALL (BCP-ALL) and more mature B-ALL. The most immature subtype of BCP-ALL (diversely designated as prepre-B ALL or pro-B, early B-precursor ALL or B-lymphoid null-ALL) (First MIC Cooperative Study Group, 1986; Thiel et al, 1987; van't Veer et al, 1992; Knapp, 1992; van der Does-van den Berg et al, 1992; Greaves et al, 1993) is characterized by the expression of CD19, cyCD22, and mostly CD24, while CD10, cyIgM and SIg are negative. More recently identified pan-B markers such as the CD72 antigen occur, like CD19 and cyCD22, at the earliest stages of B-cell differentiation and may be useful for the identification of immature BCP-ALL subsets (Pezzutto et al, 1989). The majority of childhood and adult ALL patients express CD10 in combination with CD19, cy or mCD22, CD24, and this subgroup is termed common ALL. A variable proportion of common ALL also expresses CD20. This antigen presumably occurs just before the expression of cyIgM

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Figure 2. Scheme illustrating the antigenic profile as well as Ig and T-cell receptor gene patterns in B-cell precursor ALL, B-ALL and T-lineage ALL. Antigens important for the definition of distinct B-cell-precursor or T-lineage ALL subgroups and B-ALL are framed in boxes; antigens in parentheses are not always expressed.

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IMMUNOPHENOTYPIC CLASSIFICATION OF ALL

241

in normal B-cell development but, at least in our experience, has shown no clear-cut association with the presence of cyIgM in BCP-ALL. The pre-B-ALL phenotype is characterized by the expression of cyIgM and, just as in common ALL, leukaemic cells are mostly positive for CD10, CD19, cy or mCD22, CD24, while CD20 is expressed in varying proportions. Since cyIgM has not been widely analysed in clinical studies, especially in adult ALL, pre-B cases have sometimes been included in the common ALL category. Virtually all BCP-ALL are positive for HLA-DR and TdT, whereas CD34, another progenitor-cell-associated antigen, is expressed in only about 60-70% of these cases (Borowitz et al, 1990; W.-D. Ludwig et al, unpublished observations). The distinction between these three subgroups of BCP-ALL has been somewhat arbitrary due to the different cut-off points applied (Janossy et al, 1989). Most studies defined common ALL by the surface antigen expression of CD10 on >I 20% of leukaemic cells, while the diagnosis of pre-B-ALL required detection of cyIgM in I> 10% of blasts. It is obvious that the incidence of immunophenotypic subgroups is strongly dependent on the cut-off points applied. The immunophenotypic features of BCP-ALL subgroups and the normal stages of B-lymphocyte development differ remarkably, one unique feature being that CD19 expression precedes CD10 acquisition in pre-pre-B-ALL, whereas both antigens are expressed concomitantly on normal B-cells (Loken et al, 1987; Uckun and Ledbetter, 1988). Furthermore, recent studies analysing in greater detail the immunophenotypic features of normal B-lymphocytes and their presumed malignant counterparts describe frequent asynchronous or aberrant expression of cell surface antigens by leukaemic blasts of BCP-ALL (Ryan et al, 1987; Hurwitz C.A. et al, 1988; Janossy et al, 1989; Ross et al, 1990; Hurwitz et al, 1992). Based on these observations, it was suggested that leukaemic blasts in B-cell precursor ALL may not originate from phenotypically identical normal cellular counterparts, or, alternatively, that these immunophenotypic subgroups could result from the malignant proliferation of rare normal lymphoid cells not detectable with methods presently available (Hurwitz C.A. et al, 1988). The B-lineage association of the different BCP-ALL subgroups has also been confirmed by the rearrangement of Ig heavy-chain genes (Korsmeyer et al, 1981; Nadler et al, 1984). B-cell ALL B-ALL occurs rarely in both childhood and adult ALL and accounts for approximately 2-3% of all ALL cases. Most B-ALLs can be identified morphologically because they fall into the L3 category according to the FAB classification (Bennett et al, 1981). More recent studies, however, have shown considerable morphological and immunological heterogeneity within childhood B-ALL, i.e. combination of Burkitt's cell morphology and BCPALL phenotype or Ll-subtype associated with monoclonal SIg-positive B-cell phenotype (Ganick and Finlay, 1980; Finlay and Borcherding, 1988;

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Sullivan et al, 1990), indicating that morphology alone does not adequately define B-ALL. Leukaemic blasts in B-ALL express SIg with K or h lightchain restriction, B-cell antigens including CD19, CD20, CD22, CD24 and usually CD10. In contrast to BCP-ALL, leukaemic cells in B-ALL are mostly TdT-negative despite descriptions of some typical TdT-positive cases with L3-morphology, monoclonal SIg expression, and t(8;14) (Drexler et al, 1986).

T-lineage ALL As in BCP-ALL, a consensus has not yet been reached as to the optimal basis for subclassification of T-lineage ALL. T-ALL was initially classified based on the concept that phenotypic features of T-leukaemic blasts correspond to those seen in normal thymic development (Reinherz et al, 1980), and a model has been proposed with three different subgroups representing early, common and mature T-cell differentiation stages derived by analysing a series of differentiation-related markers detectable with mAbs such as CD7, CD5, CD2, CD1, CD3, CD4, and CD8 (Foon and Todd, 1986). In paediatric studies, the criteria for subclassification of T-lineage ALL were slightly modified from those proposed for normal T-cell differentiation, and three subgroups were distinguished mainly on the basis of membrane CD1 and CD3 expression (Roper et al, 1983; Crist et al, 1988; van der Does-van den Berg et al, 1992). Children with an early thymocyte phenotype express cyCD3, CD7, CD5 and usually CD2 and are negative for CD1 and mCD3. Those with an intermediate or common phenotype are characterized by the expression of CD 1 in combination with CD7, CD5 and CD2 with variable expression of mCD3, CD4, and CD8. In the mature subtype, all cases are positive for mCD3, CD7, CD5 and CD2, while CD1 is negative. CD4 and CD8 are present in most cases of mature T-ALL, and usually only one of these antigens is expressed in the latter subtype. Others have divided T-lineage ALL on the basis of either E-rosetting or CD2 reactivity, with early T-precursor ALL (or pre-T ALL) being CD7+/ TdT+/E-rosettes (or CD2)- and T-cell ALL being CD7/TdT/E-rosettes (or CD2) + (Foa et al, 1985; First MIC Cooperative Study Group, 1986; Thiel et al, 1989). As in BCP-ALL, various deviations from the composite phenotype of normal thymic T-cells were observed in malignant T-cells, suggesting that T-ALL subtypes could not be easily reconciled with the model of normal T-cell differentiation (Greaves et al, 1981b; Koziner et al, 1982). More recently, the combined analysis of T-cell ontogeny by immunophenotyping and molecular biological studies of TCR gene patterns has stimulated remarkable progress in understanding the very earliest stages of T-cell development (reviewed in Haynes et al, 1989). These studies have revealed a hierarchy of assembly of the ~, % 13, and o~ TCR loci that precedes functional TCR protein expression in T-ALL and is generally coordinated with expression of T-cell-associated antigens (van Dongen et al, 1987). In a large series of adult pre-T-ALL diagnosed on the basis of CD7 and cyCD3 expression as well as TdT positivity and E-rosette negativity, the leukaemic

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cells expressed the haematopoietic progenitor cell marker CD34 in 17 of 28 cases and H L A - D R in five cases (Raghavachar et al, 1989; Thiel et al, 1989). This very primitive group of T-ALL turned out to be heterogeneous with respect to TCR gene rearrangements and the expression of CD2 and CD5 molecules. In a third of pre-T-ALL of this series, CD7 and cyCD3 expression already occurred at a very immature stage of T-cell differentiation prior to rearrangement of TCR 13, ~/, and 8 chain genes. The strongest criteria for T-cell commitment in the latter cases are (a) the expression of cyCD3 (van Dongen et al, 1988), (b) the absence of B-cell antigens (Thiel et al, 1989), and (c) the lack of Ig heavy- and light-chain rearrangements (Raghavachar et al, 1986a). The absence of any TCR or Ig gene rearrangements suggests that expression of cyCD3, CD7 and, in part, of CD2 and CD5 is acquired prior to entry of cells into the thymus, as has recently been demonstrated in bone-marrow cultures (Hurwitz J.C. et al, 1988). While the subgroups of pre-T and E-rosette-positive ALL differed significantly in some clinical features including outcome (see below), the different immunogenotypes did not correlate with a specific clinical course (Thiel et a!, 1989). More recently, mAbs to different TCR chains have been applied to investigate the appearance of TCR-related proteins during normal T-cell ontogeny as well as in discrete maturational stages of T-ALL (Campana et al, 1989; Campana et al, 1991b). These studies have shown that discrete stages of TCR development in T-lineage ALL reflect the maturational stages seen in the normal thymus, and it has been suggested that TCR protein analysis may describe the maturational stages of malignant cells more precisely than other T-cell differentiation antigens mentioned above (Campana et al, 1991b). A new T-ALL classification system of this kind, however, has to be examined in further studies with emphasis on the correlation between TCR expression, clinical features and treatment outcome.

INCIDENCE AND CLINICAL FEATURES OF IMMUNOPHENOTYPIC SUBGROUPS Comparison of the relative frequencies of immunological subgroups between childhood and adult ALL is hampered by the differences in criteria applied in assigning leukaemic blasts to the major immunophenotypic subgroups of ALL and by the lack of detailed information, mainly in adult ALL (Sobol et al, 1985; Hussein et al, 1989; Ellison et al, 1991; Linker et al, 1991), regarding the distribution of the different subsets of BCP- and T-lineage ALL. Moreover, some recent studies classified patients as ALL despite the fact that their leukaemic blasts expressed exclusively myeloid antigens without B- or T-cell-associated markers (Sobol et al, 1985; Sobol et al, 1987; Hussein et al, 1989; Ellison et al, 1991). These cases probably represented minimally differentiated acute myeloid leukaemias (Bennett et al, 1991) and should not have been included among the immunophenotypic subgroups of ALL.

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W.-D. LUDW1G ET AL

Childhood/Adult ALL

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63 Y

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pre-B

Adults N=946

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pre-T

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T 0

10

20

30

40

50

60

70

% Figure 3. Incidence of immunophenotypic subgroups in childhood (open bars) and adult ALL (hatched bars) as determined in the German multicentre trials ALL-BFM 86/90 and ALL/ AUL-BMFT 03/87 and 04/89.

Figure 3 illustrates the incidence of immunophenotypic subgroups in children and adults with ALL as determined in the German prospective multicentre trials. The immunological analyses were performed in one central laboratory, and patients were assigned to the subgroups described above according to the reactivity patterns of leukaemic cells with mAbs and heterologous antisera (e.g. TdT, anti-Ig). These data reveal that children differ markedly from adults regarding the incidence of immunophenotypic subgroups. Thus, immature subtypes of BCP- (i.e. CD10-negative pre-preB phenotype) or T-lineage ALL (i.e. pre-T phenotype) occur more frequently in adult than in childhood ALL, indicating a higher proportion of cases being arrested at a very immature stage of lymphoid differentiation. Moreover, as reported previously (Foa et al, 1985; Hoelzer et al, 1988; Thiel et al, 1989; Ludwig et al, 1990), a higher incidence of T-lineage ALL was observed in adults than in children. It should be noted that the standard application of mAbs detecting pan-B, pan-T and pan-myeloid antigens as well as lineage-specific intracytoplasmic markers (see above) permitted the unequivocal assignment of most (> 99%) childhood and adult ALL to BCPor T-lineage ALL subgroups, thus strongly decreasing the incidence of the phenotypically unclassifiable or null-ALL subgroup. Our findings based on prospective studies in a large number of patients underline the diagnostic precision of immunophenotyping in ALL and are in agreement with other recent investigations suggesting that phenotypic characterization with appropriate reagents identifies features of lineage commitment in the vast majority of acute leukaemias (Janossy et al, 1989; Campana et al, 1990a).

IMMUNOPHENOTYPIC CLASSIFICATION OF A L L

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Interestingly, a recent update of a collaborative study group on childhood ALL subtypes in different geographic and ethnic settings revealed a striking variation in the incidence rates of common ALL between 'developed' and 'underdeveloped' countries that might be attributed to differences in socioeconomic factors and patterns of infection in infancy (Greaves et al, 1993). Numerous studies have analysed the correlations between ALL immunophenotypic subgroups and clinical/laboratory features at presentation in childhood or adult ALL (Greaves et al, 1981a; Pui et al, 1986; Hoelzer et al, 1988; Crist et al, 1989; Garand et al, 1989; Ludwig et al, 1989a; Thiel et al, 1989; Sullivan et al, 1990; Reiter et al, 1992) and have described pronounced differences between BCP- and T-lineage ALL subgroups and B-ALL with respect to the main clinical risk factors (e.g. age and leukocyte counts) as well as other clinical characteristics. While an immature pre-pre-B phenotype is often associated with unfavourable clinical features such as age < 1 year, high leukocyte counts, an increased incidence of massive hepatosptenomegaly, and central nervous system (CNS) disease at diagnosis (Pui et al, 1986; Garand et al, 1989; Ludwig et al, 1989b; Pui et al, 1993), no significant differences have as yet been observed between common and pre-B immunophenotypic subgroups with the exception of slightly increased leukocyte counts and serum lacticdehydrogenase concentrations within the pre-B subgroup (Lilleyman and Hinchliffe, 1989). The most striking features of B-ALL in both childhood and adult ALL are the frequent presence of abdominal lymphomatous tumours, an early involvement of the CNS, a high incidence of kidney involvement, the low leukocyte counts, and a male prevalence (Garand et al, 1989; Sullivan et al, 1990; Reiter et al, 1992). Patients with T-lineage ALL, usually male adolescents or young male adults, are characterized by a mediastinal mass, a higher incidence of extramedullary manifestations including hepatosplenomegaly and CNS disease, and increased leukocyte counts (Crist et al, 1988; Hoelzer et al, 1988; Crist et al, 1989; Garand et al, 1989; Ludwig et al, 1989a; Thiel et al, 1989; Garand et al, 1990; Ludwig et al, 1990; Pui et al, 1990b). In our experience, adults with pre-T-ALL were less likely to present with a mediastinal mass, lymphadenopathy or thrombocytopenia, suggesting a lower incidence of extramedullary involvement that may result from a more pronounced bone-marrow homing of prethymic-T leukaemia cells (Thiel et al, 1989; Ludwig et al, 1990). In contrast, childhood T-lineage A L L subgroups classified according to their maturational stage showed no significant differences with respect to the main clinical features, although some controversial results have been published regarding the incidence of a mediastinal mass in mature T-ALL (Crist et al, 1988; Ludwig et al, 1989a; Ludwig et al, 1993). IMMUNOPHENOTYPIC-GENOTYPIC ASSOCIATIONS IN ALL Advances in immunophenotyping, cytogenetics and molecular genetics, and their combined application in characterizing leukaemic blasts have not only

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considerably advanced our knowledge of the pathophysiology of acute leukaemias but have also contributed towards establishing a refined ALL classification and identifying distinct clinicopathologic entities. Improved banding methods have now identified clonal chromosome abnormalities in 75-90% of ALL cases (Secker-Walker, 1990), and several studies, mainly in childhood ALL, have analysed the relationship of karyotypic findings to different immunophenotypic subgroups of ALL (Williams et al, 1984; Pui et al, 1988; Uckun et al, 1989). These studies have identified distinct ploidy groups as well as significant associations between numerical chromosome abnormalities, especially leukemic cell hyperdiploidy, and immunophenotypic features (reviewed in Pui et al, 1990a; Raimondi, 1993). Recent studies in childhood ALL have shown that patients whose leukaemic cells have a modal chromosome number > 50 (or a D N A index > 1.16) fare much better than those with lower chromosome numbers and have also indicated that, among children with BCPALL, a D N A index >1.16 was the strongest predictor of a favourable response to chemotherapy (Look et al, 1985; Bloomfield et al, 1986; Trueworthy et al, 1992). Thus, much interest has focused on the biological and clinical features of this subgroup. Hyperdiploidy (>50 chromosomes) accounts for about 25% of childhood and 10% of adult ALL cases and occurs much more frequently in common and pre-B ALL than in pre-pre-B, B- or T-lineage ALL (Bloomfield et al, 1986; Pui et al, 1988; Trueworthy et al, 1992; Harbott et al, 1993; Hiddemann et al, 1993). This ploidy group is also associated with other favourable prognostic features--lower leukocyte counts, lower serum lactic-dehydrogenase levels, white race, and age between 2 and 10 years (reviewed in Pui et al, 1990a; Raimondi, 1993)--and can now be reliably identified by flow cytometric analysis of leukaemic cell D N A content. A clear-cut correlation between other numerical chromosomal abnormalities and immunophenotypic features has not yet been established, the only exceptions being a higher incidence of T-lineage ALL in the normal diploid and near-tetraploid subgroups (reviewed in Pui et al, 1990a; Raimondi, 1993). In addition, strong associations have been recognized between established structural chromosomal abnormalities and immunophenotypic subgroups (see below), especially in B-lineage ALL. These correlations of karyotype and immunophenotype form the basis for the 'Morphologic, Immunologic, and Cytogenetic' (MIC) working classification of ALL, the first to combine the three major diagnostic approaches (First MIC Cooperative Study Group, 1986). Six immunophenotypic categories of B- or T-lineage ALL with corresponding chromosomal abnormalities have thus far been defined. The classification is open-ended, thus allowing new categories to be added as recognized. The following briefly discusses the immunophenotypic characteristics of the most prominent specific chromosomal rearrangements. B-cell precursor ALL

The t(4;ll)(q21;q23) chromosomal abnormality occurs in about 2-6% of

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both children and adults with ALL and has been associated with characteristic immunophenotypic and clinical features (e.g. high leukocyte counts, predominance of females, frequent organ enlargment, and increased incidence of CNS leukaemia at diagnosis) (reviewed in L6glise et al, 1990). Previous reports, mainly in infant ALL, have suggested that t(4;11)associated acute leukaemias mostly originate in multipotent or very early CD10- B-progenitor cells with a high frequency of myeloid-antigen positivity (Mirro et al, 1986; Raghavachar et al, 1986a; Hagemeijer et al, 1987; Lampert et al, 1987; Katz et al, 1988). Recent studies have analysed their immunophenotypic and genotypic features in greater detail. In the vast majority of ALL with t(4;11), leukaemic blasts disclose a typical antigenic profile (e.g. CD19 +, CD10-, CD24- or weakly +, cyIgM - or +, CD15 and/or CDw65 ÷) indicative of an immature pre-pre-B phenotype coexpressing two myeloid-associated antigens (CD15, CDw65) with similar biochemical characteristics. This clear-cut association of immunophenotypic features with t(4;11) was established by a study we performed in a large number of infants aged < 1 year (Ludwig et al, 1989b) and has been confirmed by others (Pui et al, 1991; Borowitz et al, 1993). Southern blot analysis revealed Ig heavy-chain gene rearrangements in virtually all cases as well as oligoclonal disease in some of them (Raghavachar et al, 1986a; Hagemeijer et al, 1987; Katz et al, 1988; Ludwig et al, 1989b), thus underlining the early B-cell commitment of blast cells with this cytogenetic abnormality. Meanwhile, the characteristic correlation of immunophenotype and karyotype has also been observed in adult ALL with t(4,11) (Secker-Walker et al, 1991; Schardt et al, 1992; Ludwig et al, 1994). These unusual phenotypic features can be used to predict the t(4;11) with great precision and may additionally provide a means of monitoring these patients for MRD. It should be noted, however, that other 11q23 abnormalities such as t(1; 11) or t(11;19) occasionally display differing immunophenotypes, and t(9;11) is closely associated with acute monoblastic leukaemia, indicating that these leukaemias may segregate into distinct biological and molecular subsets (Katz et al, 1988, K611er et al, 1989; Ludwig et al, 1989b; Ludwig et al, 1994). Most interestingly, recent molecular investigations have identified a large gene (MLL or ALL-l) that spans the 11q23 breakpoints in t(4;11), t(9;11), and t(11;19) and may be of fundamental importance in the pathogenesis of 11q23-associated acute leukaemias (reviewed in Raimondi, 1993). The Philadelphia (Ph) chromosome, or t(9;22)(q34;q11), found in 15-30% of adults and 3-5% of children with ALL, is usually associated with a common or pre-B-ALL phenotype (Crist et al, 1990a; Maurer et al, 1991; Secker-Walker et al, 1991; Tien et al, 1992; Harbott et al, 1993; Rieder et al, 1993). Some studies have identified a low proportion of immature CD10B-precursor, mature B- or pure T-lineage features in Ph ÷ ALL (McKinley et al, 1986; Pui et al, 1988; Uckun et al, 1989; Crist et al, 1990a; Maurer et al, 1991; Harbott et al, 1993). Interestingly, our data as well as the findings of others suggest that myeloid antigens are not co-expressed more frequently on Ph ÷ ALL than on Ph- ALL (Tien et al, 1992; Westbrook et al, 1992; Ludwig et al, 1994),

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whereas 'mixed-lineage' features with co-expression of myeloid and lymphoid antigens were observed in most cases of Ph + AML (Chen et al, 1988; Tien et al, 1992). Given the heterogeneity of Ph + ALL in its breakpoint location and lineage involvement with either a pluripotent-stem or lymphoid-progenitor target cell, it might be interesting to determine whether these subsets differ in their immunophenotypic features (Secker-Walker and Craig, 1993). No extensive analyses have as yet been performed to investigate the correlation between surface antigen expression and, for instance, the two different breakpoint cluster regions (bcr) detected on chromosome 22 (i.e. major- or minor-bcr) in Ph + ALL. Previous studies indicated that t(1;19)(q23;p13) is found in about 25% of childhood ALL with a cyIgM + pre-B phenotype (reviewed in Crist et al, 1990b). Although subsequent studies confirmed the close association between t(1;19) and pre-B phenotype in both childhood and adult ALL (Raimondi et al, 1990; Secker-Walker et al, 1992), this abnormality was also detected in some cyIgM- common ALL (Raimondi et al, 1990; Crist et al, 1990c; Secker-Walker et al, 1992; Harbott et al, 1993) and altogether accounts for approximately 5-6% of childhood ALL cases (reviewed in Pui et al, 1990a; Raimondi, 1993). More recently, the consequences of this chromosomal rearrangement have been characterized molecularly (Mellentin et al, 1989). Interestingly, t(1 ;19)-induced expression of the E2A-PBX1 fusion gene was reliably detected in cyIgM + but not in cyIgM- BCP-ALL harbouring t(1;19) (Privitera et al, 1992). These data indicate that the molecular breakpoints differ between cyIgM- and cyIgM + BCP-ALL cases with t(1;19) and suggest that a systematic characterization of molecular abnormalities within the broad category of BCP-ALL may provide sharper distinctions than those achieved by immunophenotyping and cytogenetics. B-ALL

While, in BCP-ALL and T-lineage ALL, specific cytogenetic abnormalities do not show a close relationship to the FAB subtype, leukaemic blasts in mature SIg + B-ALL are usually characterized by FAB L3 morphology and invariably exhibit t(8;14)(q24;q32) (in 75-85% of the patients) or one of the variant translocations t(2;8)(pll-12;q24) and t(8;22)(q24;q11). The high predictive value of these B-ALL-associated chromosomal abnormalities for L3-morphology has recently been demonstrated (Mitelman and Heim, 1992). It should be noted, however, that exceptions to these associations have been described in some cases of B-ALL with L3 morphology lacking t(8;14), B-ALL with t(1;19), Ph chromosome or t(1;22), and pre-B ALL with t(8;14) and t(14;18) (Mufti et al, 1983; Mangan et al, 1985; McKinley et al, 1986; Sullivan et al, 1990; Reiter et al, 1992). T-lineage ALL

A large number of different chromosomal abnormalities have been described in T-lineage ALL (reviewed in Pui et al, 1990a; Raimondi, 1993).

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The overall frequency of translocations is about 44% with 30-40% of the abnormal karyotypes having their breakpoints within the 14ql 1, 7q34-q36, or 7p15 regions containing the TCR a/g, TCR [3, and TCR ~ genes, respectively (Raimondi et al, 1989). In contrast to BCP- or mature B-ALL, no obvious relationships have yet been established between particular structural chromosomal changes and the maturational stage of T-ALL blasts (Lampert et al, 1988; Raimondi et al, 1989). Moreover, it has not been possible to identify characteristic phenotypic features within groups of frequent chromosomal abnormalities in T-ALL such as aberrations involving band q l l of chromosome 14 (Lampert et al, 1988; Raimondi et al, 1989). Most of these data are derived from cytogenetic analyses in childhood ALL. Detailed results are not yet available on the correlation of immunophenotype and karyotype in adult T-lineage ALL. More recently, alterations of the T A L I proto-oncogene on chromosome lp32, either by translocation or other rearrangements, have been shown to represent the most common non-random genetic defect associated with T-lineage ALL, occurring in about 10-25% of patients with this immunophenotype (Bash et al, 1993; Breit et al, 1993; Janssen et al, 1993). Consistent with other recent reports, our own data indicate that T A L l alteration in T-ALL correlates with commitment of leukaemic blasts to TCRa[3 lineage, whereas no clear association of T A L l gene rearrangements with a distinct stage of thymocyte maturation could yet be detected (Macintyre et al, 1992; Bash et al, 1993; Breit et al, 1993; Janssen et al, 1993). PROGNOSTIC IMPACT OF IMMUNOPHENOTYPING IN ALL The lack of standardized criteria in the past for the classification of immunophenotypic subgroups, the paucity of controlled prospective studies on the treatment outcome of BCP- and T-lineage ALL subsets and the different treatment strategies applied complicate the assessment of the prognostic impact of immunophenotypes in ALL. In addition, the strong correlation observed between certain immunophenotypic subgroups (e.g. CD10- prepre-B-ALL, pre-B-ALL) and unfavourable cytogenetic or clinical features (see above) has called into question the value of immunophenotyping as an independent predictor of treatment outcome. Finally, several recent studies have shown that the prognostic impact of immunophenotypic subgroups as well as chromosomal abnormalities is diminished by improved efficacy of chemotherapy, and prognostic factors must therefore be evaluated in the context of therapy delivered (Fletcher et al, 1989; Raimondi et al, 1990; Reiter et al, 1992). In B-cell precursor ALL, no substantial differences in remission rates were recorded for immunophenotypic subgroups, but several studies revealed an association between the maturational stage of B-tymphoblasts and the duration of remission. Most studies in both childhood and adult ALL have reported a worse prognosis for patients whose leukaemic blasts express an immature CD10- pre-pre-B or null-ALL phenotype (Kersey et

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al, 1982; Pui et al, 1986; Thiel et al, 1987; Hoelzer et al, 1988; Ludwig et al, 1993; Pui et al, 1993) which, at least in childhood ALL, was associated with adverse biological (e.g. 11q23 rearrangements) and clinical features (e.g. high tumour burden, age < 1 year) occurring in up to 50% of patients. As pointed out above, the complexity of the historical null-ALL in adult series makes it difficult to assess the prognostic significance of the immature CD10- pre-pre-B immunophenotype separately. In the German multicentre trials, previous data indicated that adult patients had a significantly shorter remission duration with B-lymphoid null-ALL than with common and T-lineage ALL (Hoelzer et al, 1988). The prognostic impact of the pre-pre-B phenotype is currently being investigated. Cytogenetic and molecular studies have provided conclusive evidence that children and adults with common and pre-B-ALL differ significantly with respect to the incidence of the known poor-risk translocation, t(9;22), with up to 55% of adults and about 5% of children being Ph + and/or exhibiting BCR-ABL rearrangements (Maurer et al, 1991; Secker-Walker et al, 1991; Westbrook et al, 1992; reviewed in Hoelzer, 1993). These findings largely explain the striking differences observed in treatment outcome between children and adults with common or pre-B-ALL. Confirmation of the prognostic importance of the pre-B-ALL immunophenotype has been limited to sequential studies of the Pediatric Oncology Group (POG), since, until recently, this was the only group performing cyIgM testing in the context of large prospective clinical trials. Previous studies of the POG suggested that the pre-B phenotype might be an independent prognostic marker for reduced event-flee survival (EFS) (Crist et al, 1984; Crist et al, 1989). However, more recent data show that only the subgroup of children with pre-B-ALL and t(1;19) have a worse treatment outcome (Crist et al, 1990c). The ALL-BFM 86 as well as a recently published single-centre study revealed no significant differences in remission duration between common and pre-B-ALL (Lilleyman and Hinchcliffe, 1989; Ludwig et al, 1993). In children with BCP-ALL, the prognosis has been linked with other immunophenotypic features such as CD20, CD34 and CD45 expression, and it has been suggested that the lack of CD20 and CD45 antigens or the presence of CD34 on leukaemic blasts may be associated with a longer EFS (Ludwig et al, 1989a; Borowitz et al, 1990; Behm et al, 1992). However, in view of the relationship of these immunophenotypic features to other biologically favourable characteristics, their prognostic significance has to be evaluated in further studies by adjusting results for the presence of other risk factors. Several studies in childhood and adult ALL have shown that a remarkable prognostic improvement of B-ALL is achieved by the development of intensive treatment strategies especially adapted to the biological and clinical features of this disease (Reiter et al, 1992; Hoelzer et al, 1992; reviewed in Hoelzer, 1993). These data impressively illustrate that more effective treatment can offset the negative prognostic impact of biological characteristics such as the immunophenotype or chromosomal translocations.

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In T-lineage ALL, various immunophenotypic features seem to be associated with an increased risk of treatment failure: an immature pre-T-ALL phenotype, membrane expression of CD3 or MHC class II antigen, and negativity of CD5, THY antigen (similar to CD1), or CD10 (Dowell et al, 1987; Thiel et al, 1989; Garand et al, 1990; Pui et al, 1990b; Shuster et al, 1990; Pui et al, 1993). The prognostic impact of these factors, however, has differed according to the treatment strategies used, and immunophenotypic criteria have not been generally accepted as the basis for risk assignment in T-lineage ALL. In our experience, children and adults with pre-T-ALL differed markedly with respect to their phenotypic and genotypic features, suggesting an arrest of adult pre-T-ALL at a less mature differentiation stage than in childhood, which may be closely related to the poor prognosis observed in these patients (Thiel et al, 1989; Raghavachar et al, 1989; Ludwig et al, 1990). Similar results have been published in a series of nine adolescent and elderly male patients whose leukaemic blasts disclosed CD7 antigen expression with absence of myeloid, B-, or more mature T-cell differentiation antigens (Kurtzberg et al, 1989). Most interestingly, these leukaemic blasts were capable of multilineage differentiation in vitro both spontaneously and after stimulation with appropriate cytokines, suggesting that acute leukaemia in these patients evolved from in vivo transformation of immature pluripotent haematopoietic cells that respond poorly to conventional chemotherapy. Further attempts to identify additional prognostically relevant subgroups of T-lineage ALL have been disappointing in both childhood and adult ALL (Crist et al, 1988; Thiel et al, 1989). Interestingly, in the ALL-BFM 86 study, life-table analyses of a large number of children with maturationally classified T-lineage ALL provided strong evidence for differences in treatment outcome between early, intermediate, and mature T-ALL (Ludwig et al, 1993; unpublished observations). Children with a CD1 + intermediate T-cell phenotype disclosed a significantly better in vivo response to corticosteroids and a longer duration of EFS than those with an early (including pre-T) or more mature T-cell phenotype. Further investigations towards better defining the correlation between these immunophenotypic subgroups and biological features (e.g. chromosomal abnormalities, expression of transcription factors) are in progress in the German multicentre trials. MYELOID-ANTIGEN-POSITIVE ALL Detailed analyses using immunological and molecular-genetic studies have recently documented typical ALL cases inappropriately expressing myeloid antigens as well as morphologically/cytochemically defined AML with lymphoid-associated markers (reviewed in: Greaves et al, 1986; Stass and Mirro, 1986; Hurwitz and Mirro, 1990). Different hypotheses have been postulated to explain the origin of these acute leukaemias with 'mixed-lineage' features (Mirro et al, 1985), including

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the concept of 'lineage infidelity' (Smith et al, 1983) and the theory of 'lineage promiscuity' (Greaves et al, 1986). Unfortunately, much controversy has surrounded both the terminology and classification criteria, and, more recently, several scoring systems were proposed based on different diagnostic weight given to myeloid- or lymphoid-lineageassociated markers in order to classify acute leukaemias with 'mixedlineage' features more precisely (Hurwitz and Mirro, 1990; Catovsky et al, 1991) and to evaluate the clinical significance of acute leukaemias in which individual blast cells simultaneously express markers normally restricted to a single lineage (reviewed in: Drexler et al, 1991; Drexler et al, 1993). In view of previous studies pointing to myeloid-antigen expression as a predictor of poor prognosis in both childhood and adult ALL (Sobol et al, 1987; Wiersma et al, 1991), considerable interest has focused on the biological features and clinical significance of this subgroup of acute leukaemias. Though reported My + ALL incidences have varied depending on the different criteria or immunologic techniques applied and myeloid markers tested, a critical review of data published in the literature revealed that about 5-10% of children and 10-20% of adults express myeloid-lineageassociated antigens on ALL blasts (Drexler et al, t991). My + ALL should be distinguished from rare cases of acute leukaemias undifferentiated by conventional morphological and cytochemical criteria but clearly expressing markers of different lineages and from acute leukaemias in which morphology, cytochemistry and/or immunological markers suggest the existence of two separate blast cell populations (Ludwig et al, 1988; Catovsky et al, 1991). In the German multicentre ALL trials, we have attempted to better define the incidence, biological features, and clinical relevance of My + ALL by prospectively analysing more than 1000 children and adults with respect to the expression of myeloid antigens (CD13, CD33, CDw65) on leukaemic blasts (Ludwig et al, 1994). Our preliminary evaluation confirmed a striking difference in My + ALL incidence between children and adults. About 7% of children and 19% of adults disclosed simultaneous expression of lymphoid markers and at least one myeloid-lineage-associated antigen, as demonstrated by flow cytometric analysis using double colour immunofluorescence assays (Ludwig et al, 1994; W.-D. Ludwig et al, unpublished results). In contrast to other recent studies (Pui et al, 1990c; Wiersma et al, 1991), we were able to demonstrate a clear association between the maturational stage of BCP-ALL and the presence of myeloid antigens. Almost 50% of cases with a CD10- pre-pre-B phenotype expressed myeloid antigens (usually CDw65) as opposed to less than 20% (adults) or 10% (children) in the other BCP-ALL and most T-lineage subgroups. It should be noted that a markedly increased incidence of myeloid antigen co-expression was also observed in adult patients with an immature pre-T-ALL phenotype. These findings are in line with our previous observations suggesting that adult pre-T-ALL is arrested at a less mature differentiation stage than in childhood (Thiel et al, 1989; Ludwig et al, 1990). Cytogenetic examinations, available in almost 60% of children and 50%

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of adults with My + ALL, have shown that an immature My + CD 10- pre-preB or, in exceptional cases, a pre-B phenotype with typical surface antigen expression as discussed above correlated strongly with rearrangements involving chromosome 11 band q23, mostly t(4;11). Clear-cut associations between the other My + ALL subgroups and specific chromosomal aberrations have not yet been observed. In keeping with other recent studies (Pui et al, 1990c), our results regarding the treatment outcome of children with My + ALL have thus far failed to demonstrate any prognostic value for myeloid-antigen expression in childhood ALL, suggesting that highly effective therapy will abolish the prognostic impact of My + ALL. Due to a relatively short follow-up of adult My + ALL patients, no definite conclusions can be drawn as to their treatment outcome. Further prospective studies consistently based on clearly defined diagnostic criteria such as those mentioned above (Catovsky et al, 1991) are clearly needed to elucidate more accurately the biological heterogeneity of My + ALL and to establish its clinical relevance.

PHENOTYPIC CONVERSION IN ACUTE LEUKAEMIA Different mechanisms have been suggested to account for phenotypic conversion (lineage switch) in acute leukaemia, usually from ALL to AML (Stass and Mirro, 1986; Hurwitz and Mirro, 1990). Leukaemic transformation may arise by a haematopoietic progenitor cell retaining the capacity to differentiate along either lymphoid or myeloid pathways, or, alternatively, by a new leukaemogenic event (e.g. therapy-induced) causing an apparent lineage switch in acute leukaemia. We have described three cases of in vivo conversion of phenotypes in CD10- BCP-ALL (Raghavachar et al, 1986b; Ludwig et al, 1988). In two cases, within a few days of starting treatment, the leukaemic phenotype changed from BCP-ALL to acute myelomonocytic lineage (FAB-L1, TdT + to FAB-M4 or -M5, TdT-). The initial antigenic profile of these leukaemias was characterized by CD 10- pre-pre-B features or by co-expression of both B-lymphoid and myeloid markers on the same cell. Examination of Ig heavy-chain gene rearrangements initially and after conversion revealed an identical monoclonal configuration of Ig heavy-chain sequences in all samples suggesting that in these patients leukaemia might have developed at a very early stage of haematopoiesis prior to full or irreversible commitment to a single lineage. Very similar findings have been described in 11@3associated acute leukaemias (see above) and, consistent with these data, one of our patients had leukaemic blasts disclosing a t(11;19) (Ludwig et al, 1988). Changes in phenotypic or genotypic characteristics of leukaemic blasts between diagnosis and relapse are another important issue, because they may provide clues to the pathogenesis of leukaemia relapse and possibly to the prognosis after retreatment. Most studies have observed only subtle

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alterations in immunophenotypic features between lymphoblasts at diagnosis and at relapse (reviewed in Borowitz, 1990). In accordance with these findings, our investigations in a series of both childhood and adult ALL cases studied at first presentation and at relapse did not reveal significant changes in the immunological subtype (Raghavachar et al, 1987; Raghavachar et al, 1988). However, examination of Ig and TCR-[3 gene rearrangements to determine the ultimate clonality of lymphoid neoplasms revealed marked heterogeneity of leukaemic cell clones at diagnosis and relapse. These clonal variations occurred at both early and late relapses and are not restricted to late recurrences. With respect to immunophenotype stability, they may reflect the evolution of subclones within a single malignant cell population rather than the occurrence of two independent clones. In agreement with these observations, cytogenetic studies in a large number of children at initial diagnosis and at relapse did not provide compelling evidence for the emergence of a new clone, since the incidence was only one in 116 cases analysed (Abshire et al, 1992). S U M M A R Y AND FUTURE DIRECTIONS

During the last two decades, immunophenotyping has yielded significant new information regarding the biological heterogeneity of ALL and has provided a solid basis for a biologically oriented and reliable classification of this disease. At present, lineage commitment of acute leukaemias can be achieved in more than 98% of cases by applying a standardized panel of mAbs to pan-B-cell (CD19, cyCD22), pan-T-cell (cyCD3, CD7) and pan-myeloid antigens (CD13, CD33, MPO) that are expressed either on the surface or in the cytoplasm of the earliest progenitors of the respective cell lineage. Further subclassification of ALL based on the analysis of antigens more closely associated with different maturational stages of B- and T-cell lineage has proven useful for the identification of biologically and clinically distinct entities in both B-cell precursor and T-lineage ALL. Immunophenotyping in more than 2000 patients recruited for the German multicentre trials has shown that children and adults differ markedly in frequency distribution of immunologic subgroups with a higher adult incidence of immature B-cell precursor (i.e. pre-pre-B-ALL) and T-lineage ALL immunophenotypes (i.e. pre-T-ALL). Several immunophenotypic features in both childhood and adult ALL have been associated with a poor prognosis (e.g. pre-pre-B-ALL or nullALL, pre-B-ALL, B-ALL, pre-T-ALL; myeloid-antigen-positive ALL). At least in B-cell precursor ALL, however, the worse prognosis of the pre-pre-B or pre-B phenotype could be attributed mainly to the distinct biological and clinical features of these subgroups, and the independent value of immunophenotyping in predicting outcome has not yet been established. In addition, a number of studies have shown that more effective treatment may lessen the negative prognostic impact of immunophenotypic features (e.g. in B-ALL).

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T h e c o m b i n a t i o n of i m m u n o p h e n o t y p i n g a n d c y t o g e n e t i c t e c h n i q u e s m o r e r e c e n t l y u s e d to c h a r a c t e r i z e a c u t e l e u k a e m i a s h a s m a d e it p o s s i b l e to e s t a b l i s h a r e f i n e d ( M o r p h o l o g i c , I m m u n o l o g i c , C y t o g e n e t i c ; M I C ) classific a t i o n o f A L L a n d to i d e n t i f y distinct c l i n i c o p a t h o l o g i c e n t i t i e s w i t h a g o o d ( e . g . , h y p e r d i p l o i d c o m m o n o r p r e - B - A L L in c h i l d h o o d ) o r p o o r p r o g n o s i s (e.g. m y e l o i d - a n t i g e n - p o s i t i v e p r e - p r e - B - A L L with r e a r r a n g e m e n t s o f c h r o m o s o m e 11 b a n d q23; P h - p o s i t i v e c o m m o n or p r e - B - A L L ; p r e - B - A L L w i t h t(1;19)). A s t h e m o l e c u l a r c o n s e q u e n c e s o f t h e s e c h r o m o s o m a l t r a n s l o c a t i o n s h a v e b e e n c h a r a c t e r i z e d (e.g. B C R - A B L or E2A-PBX r e a r r a n g e m e n t s ) a n d t h e r e b y r e n d e r e d a m e n a b l e to e a s y t e s t i n g b y , f o r e x a m p l e , a R N A - b a s e d p o l y m e r a s e c h a i n r e a c t i o n , i n f o r m a t i o n f r o m such a n a l y s e s s h o u l d b e a d d e d to t h a t o b t a i n e d f r o m i m m u n o p h e n o t y p i n g a n d c y t o g e n e t i c s t u d i e s in o r d e r to a c h i e v e a s h a r p e r d i s t i n c t i o n o f b i o l o g i c a l l y a n d clinically r e l e v a n t s u b g r o u p s w i t h i n B- a n d T - l i n e a g e A L L . F u t u r e s t u d i e s will d e m o n s t r a t e w h e t h e r t h e p r o g n o s i s o f such s u b g r o u p s c a n b e i m p r o v e d b y i n d i v i d u a l l y a d j u s t i n g t r e a t m e n t p r o t o c o l s to t h e b i o l o g i c a l f e a t u r e s o f t h e l e u k a e m i c cells i n v o l v e d .

Acknowledgements The authors wish to thank all participants in the German multicentre trials ALL/AUL-BMFT (Chairman: Prof. Dr D. Hoelzer) and ALL-BFM (Chairman: Prof. Dr H. Riehm) for continuous support. We are indebted to Prof. Dr C. Fonatsch, Prof. Dr C. R. Bartram, Prof. Dr W. Gassmann, Dr J. Harbott, Prof. Dr W. Hiddemann, Prof. Dr F. Lampert, Prof. Dr H. L6ffler, Dr J. Maurer and Dr H. Rieder for their fruitful co-operation. We thank Mrs S. B6ttcher, B. Komischke, M. Martin, and A. Sindram for skillfnl technical assistance and J. Weirowski, PhD, for editing the manuscript. These studies were supported in part by the Bundesministerium ftir Forschung und Technologie, Deutsche Leuk~mie-Forschungshilfe and Deutsche Krebshilfe.

REFERENCES Abshire TC, Buchanan GR, Jackson JF et al (1992) Morphologic, immunologic and cytogenetic studies in children with acute lymphoblasticleukemia at diagnosis and relapse: a Pediatric Oncology Group study. Leukemia 6: 357-362. Bain BJ (1990) Immunological, cytogenetic and other markers. In Bain BJ (ed.) Leukaemia Diagnosis, pp 61-88. London: Gower Medical Publishing. Bainton DF, Ullyot DF & Farquhar MG (1971) ]'he development of neutrophilic polymorphonuclear leukocytes in human bone marrow: origin and content of azurophil and specific granules. Journal of Experimental Medicine 134: 907-934. Bash RO, Crist WM, Shuster JJ et al (1993) Clinical features and outcome of T-cell acute lymphoblastic leukemia in childhood with respect to alterations at the TALl locus: A Pediatric Oncology Group study. Blood 81: 2t10-2117. Behm FG, Raimondi SC, Schell MJ et al (1992) Lack of CD45 antigen on blast cells in childhood acute lymphoblastic leukemia is associated with chromosomal hyperdiploidy and other favorable prognostic features. Blood 79: 1011-1016. Bennett JM, Catovsky D, Daniel MT et al (1976) Proposals for the classification of the acute leukemias. French-American-British (FAB) Co-operative Group. British Journal of Haematology 33: 451-458. Bennett JM, Catovsky D, Daniel MT et al (1985) Criteria for the diagnosis of acute leukemia of megakaryocytic lineage (M7). Annals of Internal Medicine 103: 460-462.

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