Possibilities and limitations of cytochemical methods in diagnosis of acute leukemia

144icron, Vol. 25, No. 4, pp. 317-329, 1994 Copyright 0 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0968-4328/94 526.00

Pergamon 09684328(94)EOOO3-8

REVIEW PAPERS

Possibilities and Limitations of Cytochemical Methods in Diagnosis of Acute Leukemia MARGITA KLOBUSICKA Department of Tumor Immunology, Cancer Research Institute, Slovak Academy of Sciences, Spit6lska 21, 82 2 32 Bratislava, Slovak Republic

Abstract-Accurate identification and classification of leukemic blast cells is a very important prerequisite of the precise diagnosis of acute leukemia and has a great impact on therapy and prognosis. The purpose of this review is to consider, in the broad sense of the word, the present possibilities and limitations of enzyme cytochemistry and to emphasize how cytochemistry may contribute, on integration with the other methods of study, to the final classification and differential diagnosis of acute leukemia, a highly variable hematological disorder. In this review, the role of conventional enzyme cytochemistry, either dominant or subsidiary, in the discrimination of acute leukemia subtypes is discussed. The survey confirms the absolute necessity of immunologic marker analysis in the accurate diagnosis of acute lymphoblastic leukemia, undifferentiated or minimally differentiated leukemia and mixed-lineage leukemia because in these cases, the cytochemical evaluation provides insufficiently relevant information regarding blast cell origin, specificity of leukemia subtypes and the discrete stages of leukemic cell maturation. On the other hand, cytochemical investigation is appreciated to be dominant over immunophenotyping in characterizing acute myeloid leukemia, because of the lack of specificity of the majority of immunological markers against myeloid antigens and, because of the availability of standardized and sufficiently specific cytochemical reactions. The cytogenetic, molecular biological and electron microscopic studies mentioned in this review supplement the important information for correct differential diagnosis of acute leukemia. The prognostic impact of enzyme cytochemistry in correlation to other methods is evaluated. Key words: Acute leukemia, acute myeloid leukemia, acute lymphoblastic leukemia, enzyme cytochemistry, immunophenotyping, prognosis.

CONTENTS I. Introduction ............................................................................................................................ II. Possibilities and limitations of cytochemical methods in the study of acute leukemia ................................................... A. Subclassification of acute leukemia .................................................................................................. B. Acute myeloid leukemia ............................................................................................................ C. Acute lymphoblastic leukemia ..................................................................................................... D. Acute undifferentiated leukemia .................................................................................................... E. Mixed-lineage acute leukemia ...................................................................................................... F. Prognostic implications ............................................................................................................ G. Conclusions.. .................................................................................................................... References ..............................................................................................................................

I. INTRODUCTION Acute leukemia (AL), a heterogeneous disorder, is believed to arise from a clonal expansion of normal hematopoietic cells arrested at a particular stage of differentiation that has either lymphoid or myeloid characteristics (Foon and Todd, 1986; Vellenga and Griffin, 1987; Drexler et al., 1988; Mirro, 1992; Pui et al., Abbreviations-AL, acute leukemia; ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; ANBE, a-naphthyl butyrate esterase; AP, acid phosphatase; APL, acute promyelocytic leukemia; BG, /I-glucuronidase; CAE, chloroacetate esterase; CD, cluster of differentiation; DPP IV, dipeptidylpeptidase IV; FAB, French-American-British; MAb, monoclonal antibody; MPO, myeloperoxidase; S’NT, S-nucleotidase; PCR, polymerase chain reaction; SBB, Sudan Black B; TCR, T cell receptor; TdT, Terminal deoxynucleotidyl transferase.

317

.318 318 319

.322 .324 .324 .324

..32 5 325

1993a). Because of the differences in therapy, it is important to distinguish between the myeloid and lymphoid type of leukemia (Bennett et al., 1976; Schumacher et al., 1991; Terstappen et al., 1991; Traweek, 1993). An accurate classification of acute leukemia has a major impact on diagnostic possibilities. In addition, recognition of acute leukemia subtypes is instrumental for appropriate prognosis (Basso et al., 1992; DuboscMarchenay et al., 1992; Pui et al., 1993a). The classification and diagnostic criteria based on standard morphologic and cytochemical analyses are at present supplemented by sophisticated approaches to reach information absolutely crucial to precise diagnosis of acute leukemia. This is important in cases revealing a degree of leukemic cell heterogeneity hidden by morphologic and cytochemical uncertainty (Bassan et al., 1989). 317

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M. KlobuSicka

Immunologic marker studies have greatly improved the accuracy of classification and diagnosis by providing specific information regarding the lineage assignment and discrete developmental stages of distinct leukemic blast cells (Foon and Todd, 1986; Pui et al., 1993a). The possibility of defining the immunologic phenotype of blast cells by using monoclonal antibodies (MAbs) reactive against various lymphoid or myeloid associated antigens allows one to distinguish acute lymphoblastic leukemia (ALL) from acute myeloid leukemia (AML) and to identify the subsets within each group (Foon and Todd, 1986; Neame et al., 1986; Drexler, 1987; Drexler et al., 1988; Ludwig and Thiel, 1990; Schumacher et al., 1991; van? Veer et al., 1992; Pui et al., 1993a). Cytogenetic analysis has also evolved as an important laboratory tool that influenced diagnosis of AL, especially in difficult cases in which the diagnosis could not be clearly established by morphologic, cytochemical or immunological methods (Second MIC Cooperative Study Group, 1988, Norton et al., 1988; Kalwinsky et al., 1990; Raimondi et al., 1993; Yunis and Tanzer, 1993). Cytogenetic abnormalities provide the focus for molecular studies designated to elucidate the mechanisms of cell transformation and aberrant regulation of leukemic cell growth (Pui et al., 1990). The gene rearrangement studies serve as clonal markers of leukemia (Campana et al., 1991). From the diagnostic standpoint, molecular biology studies have potential classification value in AL. The polymerase chain reaction (PCR), a new molecular technique used for the detection of genetic rearrangement, may offer advantages over cytogenetic techniques which are often unsatisfactory in cases of AL. Besides the timely diagnosis and correct therapeutic decision, the frontline use of extremely sensitive PCR has an important impact on the monitoring of high-risk patients (Izraeli et al., 1993), and furthermore, provides a useful tool for monitoring of minimal residual disease (Lee and Stass, 1993). Electron microscopic data are absolutely essential to establish the diagnosis of AL in cases in which light microscopic myeloperoxidase staining is negative (Kantarjian et al., 1990; Schumacher et al., 1991; Tauchi et al., 1991). The reclassification of cases of acute undifferentiated leukemia as acute myeloid leukemia following the use of ultrastructural myeloperoxidase staining has been possible (Le Maistre et al., 1988; Matutes et al., 1988). Recent investigations at the ultrastructural level have demonstrated some T-ALLs with blasts that contain this enzyme (Kantarjian et al., 1989; Tauchi et al., 1991). It should be stressed that it is very important to assess the degree to which these different laboratory approaches may influence the classification, definite diagnosis, therapy and prognosis of acute leukemia. In recent years, the enthusiasm aroused by the introduction of the new tools of laboratory investigation has led to the neglect and underestimation of the usefulness of cytochemistry in routine differential diagnosis and classification of hematologic disorders. However, the practical diagnostic value of enzyme cytochemistry still represents a helpful laboratory method. The signific-

ant contribution of standard cytochemistry to the understanding of pathogenesis of blood diseases, to the discrimination of leukemic cell functional heterogeneity and to the precise delineation of cell origin as well as to commitment to specific lineage has been documented in many reports (Crockard et al., 1984; Drexler et al., 1984; Browman et al., 1986; Quaglino and De Pasquale, 1987, 1988; Bassan et al., 1989; Schumacher et al., 1991; Boban et al., 1993). It is also relevant to consider in which cases AL cytochemistry has dominant importance and in which it plays only a supportive role to more prominent functions of other methods of study in classification, diagnosis and prognosis of AL (Quaglino and De Pasquale, 1988). Therefore, it has become obvious that criteria based on morphology, cytochemistry, immunology, cytogenetics and electron microscopy are of varying significance in the subclassification of AL subtypes. The main purpose of this review is to emphasize, especially with regard to its clinical, diagnostic and prognostic significance, the present possibilities and limitations of cytochemistry in the study of acute lymphoand myeloproliferative leukemia in correlation to other methods.

II. POSSIBILITIES AND LIMITATIONS OF CYTOCHEMICAL METHODS IN THE STUDY OF ACUTE LEUKEMIA A. Subclassijication of acute leukemia To obtain a rapid but reliable discrimination between ALL and AML is the major aim of a routine program for acute leukemia diagnosis. The classification of AL has traditionally been based on a combination of morphology and cytochemical staining. The French-American British (FAB) classification system is widely accepted (Bennett et al., 1976, 1985a,b, 1991) (Table 1). The application of FAB proposals have led to the identification of a predominant cell type within the whole leukemic cell population, either lymphoid or myeloid. The correct FAB classification is usually established without difficulties in the majority of AL cases. The precise cytochemical enzyme patterns are usually studied on cytocentrifuge preparation of isolated mononuclear cell suspension of Table 1. Groups

and subgroups

of acute leukemia

Leukemia AML Acute Acute Acute Acute

(reviewed)

FAB classification

myeloblastic leukemia promyelocytic leukemia myelomonocytic leukemia monocytic leukemia

ALL Acute lymphoblastic leukemia T phenotype Acute lymphoblastic leukemia B phenotype non T, non B acute lymphoblastic leukemia Acute undifferentiated leukemia Mixed-lineage acute leukemia

AML AML AML AML

Ml M3 M4 M5

and M2 (APL) (AMMoL) (AMoL)

T-ALL Ll and L2 B-ALL L3 non-T, non-B-ALL AUL

Cytochemistry of Acute Leukemia Table 2. List of enzymes reviewed in the present paper Enzymes

Leukemia

Myeloperoxidase (MPO) Sudan Black B (SBB) Chloroacetate esterase (CAE) a-Naphthylbutyrate e&erase (ANBE)

myeloid* myeloid* myeloid myeloid

Acid phosphatase (AP) Acid a-naphthyl acetate esterase (ANAE) /I-glucuronidase (BG) Dipeptidylpeptidase IV (DPP IV) 5’ Nucleotidase (SNT)

lymphoidt lymphoid lymphoidt lymphoid lymphoid

* Scarcely in lymphoid. t In some cases in myeloid.

blood and/or bone marrow by standard methods. Within AL, the practical usefulness of the following enzymes in the discrimination of leukemic cell types is assessed: myeloperoxidase (MPO), Sudan Black B (SBB), chloroacetate esterase (CAE), a-naphthylbutyrate esterase (ANBE), acid phosphatase (AP), acid a-naphthyl acetate esterase (ANAE), B-glucuronidase (BG), dipeptidylpeptidase IV (DPP IV) and S-nucleotidase @‘NT), (Table 2, Figs 14). These enzyme activities seem to reflect the degree of cell differentiation and maturation of leukemic as well as normal cells (Boesen et al., 1984; Crockard, 1984; Borgers and Verheyen, 1985; Quaglino and De Pasquale, 1988). However, the extreme heterogeneity in myeloid cell population and functionally distinct lymphoid cell subsets may largely limit or complicate the accurate recognition and classification of individual leukemic cells. To overcome difficulties, the addition of immunological, karyotypic, genotypic or electron microscopic refinements to the morphologic/cytochemical patterns has been recommended (Mirro, 1992). B. Acute myeloid leukemia AML, characterized by asynchronous proliferation and abnormal differentiation is formed by an extremely heterogeneous population (Vellenga and Griffin, 1987; Terstappen et al., 1991). The specific cytoplasmic parameter of myeloid cells is the cytochemically detected MPO (Hayhoe, 1984; Bennett et al., 1976,1985a; Quaglino and De Pasquale, 1988). According to FAB recommendations, the presence of more than 3% of MPO positive blasts in AL supports diagnosis of AML. Cytochemical MPO reaction discriminates satisfactorily between myeloid and nonmyeloid forms of acute leukemia (Tetteroo et al., 1987). The enzyme is localized in the primary granules of myeloid cells. The pattern of cytochemical reactivity with various available peroxidase substrates is not identical. Different methods of peroxidase staining can produce variable results (Hayhoe and Quaglino, 1988). The variables observed in different types of myeloid leukemic cells may depend on the presence of multiple molecular: forms of peroxidase (Lippi and Cappelletti, 1982). MPO ultracytochemistry is thought to be very useful in providing final diagnosis of some AML, particularly

319

when light microscopic cytochemistry is non-contributory (Nguyen et al., 1989). Recently, the usefulness of immunocytochemical detection of MPO in the diagnosis of undifferentiated or very poorly differentiated AML (with less than 10% of cytochemically MPO positive blasts) was recommended (Tetteroo et al., 1987; van der Schoot et al., 1990; Bennett et al., 1991; Lepelley et al., 1993). The presence of immunologically reactive, but enzymatically inactive MPO could be explained by the presence of either abnormal mature form of MPO or proMPO (Lepelley et al., 1993). van der Schoot et al. (1990) suggested that some myeloid blasts were capable of synthetizing MPO, but were unable to process the enzyme to the mature active form. Recent studies have shown that monoclonal anti-MPO is more sensitive than the cytochemical stains for MPO and may be of value if cytochemistry is negative (van der Schoot et al., 1990; Storr et al., 1990; Lepelley et al., 1993). Sudan Black B reactivity generally parallels MPO activity in myeloid cells; therefore for practical diagnostic purposes, SBB can be considered as myeloid series specific (Hayhoe, 1984; Bennett et al., 1985a). SBB staining may be more sensitive in detecting myeloid differentiation in some cases of AL (Davey et al., 1988). Hoyle et al. (1991) preferred the use of SBB to MPO since, in their experience, the SBB staining was often stronger than MPO in AML. The potential value of SBB staining in the classification of poorly differentiated leukemia was indicated by Quaglino and De Pasquale (1988). This finding is in perfect agreement with our own observations (Klobuiick6 et al., 1993). The high positivity of SBB in correlation with the expression of CD34 antigen (a marker of very immature hematopoietic cells (Civin et al., 1987)), as well as the absence of more mature myeloid cell antigens and lack of T and B cell markers helped us to diagnose very poorly differentiated AML in 11 patients. In these cases of AML, MPO staining was negative or hardly visible. Although the presence of SBB-positive leukemic cells is a strong evidence in support of diagnosis of AML, it is not invariable because rare cases of adult ALLs with blasts possessing SBB have been reported (Tricot et al., 1982; Charak et al., 1988). In addition to MPO and SBB a considerable practical value in the classification of myeloid leukemia has been ascribed to enzymes chloroacetate esterase (CAE) and anaphthyl-butyrate esterase (ANBE). The esterase reactions have been helpful in distinguishing granulocytic from monocytic cells (Lippi et al., 1983). Granulocytes (including promyelocytes, myelocytes as well as some myeloblasts) show a very strong activity for CAE, whereas monocytes, basophils and lymphocytes show low or no activity (Lippi et al., 1983; Tomonaga et al., 1985; Davey et al., 1989). a-Naphthyl-butyrate esterase, a sensitive and selective marker of monocytes, is strongly positive in monocytes, whereas neutrophils, eosinophils, basophils and lymphocytes are non-reactive (Lippi et al., 1983). The discriminative ability of CAE and ANBE may be stressed by employing both enzymes in combination

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M. KlobuSicka

Figs 14. Cytochemical staining of leukemic blast cells. (1) Cytoplasmic reactivity of AP in lymphoblasts of T-ALL. (2) Surface membrane localization of 5’NT activity in leukemic cells of non-T, B-precursor ALL (common ALL). (3) Combined staining for CAE (black) and ANBE (reddish-brown) in acute myelomonocytic leukemia. (4) ANBE positivity in leukemic blasts of acute monocytic leukemia. Scale bar indicates 10 nm/nm.

Cytochemistry of Acute Leukemia

(Tavassoli et al., 1979; Tavassoli, 1984). In AML, three subgroups, identifiable with the combi,n,e&esterqe reaction, have been found. Because CAE is considered to be granulocyte specific, the cells stained for CAE form the group of granulocytic leukemia. Since ANBE is considered a characteristic selective marker of monocytes, the leukemic blasts strongly positive for this enzyme (but not for CAE) represent cells of pure monocytic leukemia. The third group, where leukemic cells contain both esterases in the same cell in varied proportions, appeared to represent myelomonocytic leukemia. The presence of double esterase in a single cell supports the concept of a common origin for these subgroups of myeloid leukemia. In general, the combined method satisfactorily defines the degree of differentiation along the granulocytic and monocytic cell lineage (Tavassoli, 1984; Davey et al., 1989). Within the group of acute myelomonocytic leukemia, according to our own experience, marked enzymatic heterogeneity was noticed. The leukemia type with the prevalence of myeloid (granulocytes) cells had high MPO, SBB and CAE activity in contrast to the cases where the prevalence of monocytes was found, that displayed low degree of MPO, SBB and stronger ANBE as CAE. A large proportion of cells had cytochemical features of both granulocyte and monocyte on the same cell. The combined staining of promyelocytes with ANBE and CAE has not been reported (Davey et al., 1989), however Lemei (1988) observed a case of acute promyelocytic leukemia (APL, FAB M3) with strong ANBE. Being a monocytic differentiation marker, the high ANBE positivity might cause problems in differentiating promyelocyte (characteristic cytochemical features are SBB, MPO and CAE) and myelocytic leukemia. The combined method for MPO and ANBE in the same smear (MPO reaction is performed before ANBE reaction) provides the presence of granular MPO in neutrophils and eosinophils and poorly positive or negative reaction in monocytes (Lippi et al., 1983). It can be stated that the cytochemical methods may precisely identify the degree of granulocytic and monocytic differentiation in AML. The activity of MPO, SBB, CAE and ANBE varied according to the differentiation stage of myeloid cells. Although monoclonal antibodies are useful in distinguishing AML from ALL, they have less certain utility in the subclassification of AML. The discrete stages of myeloid cell differentiation are hard to recognize using surface membrane antigens (De Rossi et al., 1990). The expression of most myeloid antigens can be recognized at all stages of granulocytic and monocytic maturation (Ludwig and Thiel, 1990; Dubosc-Marchenay et al., 1992; Traweek, 1993). This fact limits the immunophenotypic discrimination among different AML subtypes. The practical diagnostic value of immunophenotyping in AML is a matter of controversy and discussion (Bassan et al., 1989; Terstappen et al., 1991). No absolute lineage and differentiation stage specificity of myeloid antigens has been reported (Drexler, 1987; Roberts et al., 1992;

321

Table 3. The basic panel of immunophenotyping htigen

_;_

,...

-

markers

-._

CD*

Reactivity

CD1 CD2 CD3 CD4 CD5 CD6 CD7 CD8 CD9 CD10 CD19 CD20 CD21 CD22 CD23 CD24

Cortical thymocytes, B cell subset T lymphocytes (associated with E-rosette receptor) T lymphocytes (> 95%), thymocytes T cell subset (helper/inducer) T lymphocytes (; 95%), thymocytes (70%), B-CLL T lymphocytes T lymphocytes (99%), thymocytes (70%) T cell subset (cytotoxic/suppressor), thymocytes Pre-B cells, monocytes, platelets Pre-B cells, activated B, lymphoid progenitors B lymphocytes B lymphocytes (60%) B lymphocytes (85%) B lymphocytes B lymphocytes (25%), activated B B lymphocytes (40%), granulocytes (60%)

CDllb CD13 CD14 CD15 CD33 CD34 CDw65

Granulocytes, monocytes Granulocytes, monocytes Monocytes Granulocytes, monocytes, activated T Myeloid progenitors, monocytes (55%) Myeloid/lymphoid stem cells Granulocytes, monocytes

HLA-DR

B lymphocytes (> 95%), monocytes (l(r90%)

* CD, clusters of differentiation antigens.

Traweek, 1993) (Table 3). For example, CD13, CD33 and CD15 antigens appeared to be pan-myeloid cell surface markers. As the CD13 marker is expressed by blast cells from all AML subtypes, it does not permit one to distinguish the different AML subgroups (DuboscMarchenay et al., 1992). Similarly, the CD33 antigen commonly covers the whole granulocyte/monocyte differentiation pathway (Drexler, 1987, Traweek, 1993). Nevertheless, the CD15 .antigen expression was found to be helpful for distinguishing between the less and more differentiated AML subtypes (Neame et al., 1986). The immunological and cytochemical findings differ profoundly in some cases of AML. The CD14 antigen considered to be monocyte lineage specific (Drexler, 1987; Drexler et al., 1988; Dubosc-Marchenay et al., 1992) is not absolutely restricted to all maturation stages of monocytes. The lack of this antigen was demonstrated in acute monoblastic leukemia (San Miguel et al., 1986). Monoblasts and promonocytes were CD14 marker insensitive, but according to our experience, strongly ANBE positive. Thus no practical diagnostic usefulness of CD14 antigen for the detection of early stages of monocyte differentiation was found in some patients with monocytic leukemia. Acute promyelocytic leukemia (APL) is easily recognized by its peculiar morphologic and cytochemical features (De Rossi et al., 1990; Frankel, 1993). The identification of a specific myeloid phenotype for all APL would be a helpful diagnostic tool. The study of De Rossi et al. (1990) showed that APL has a characteristic phenotype: HLA-DR negative, CD7 negative, CD9 positive, CD13 positive, CD33 positive, TdT negative. This phenotype is identical for FAB M3 and M3 v, and was never related to other myeloid or lymphoproliferative

322

M. KlobuSickSl

disorders. On the other hand, the presence of Auer rods in leukemic cells is useful in alerting the clinician to the possibility of diagnosis for APL, because it is an unusual finding in other forms of AML (Davey et al., 1989). The very immature types of AML cannot be diagnosed on morphologic/cytochemical grounds alone as the blast cells are usually agranular, SBB and light microscopy MPO cytochemistry negative (Matutes et al., 1988; Bennett et al., 1991). The demonstration of small granules positive for MPO at the ultrastructural level is a very important diagnostic characteristic of these minimally differentiated AMLs FAB MO (Bennett et al., 1991). In the immature FAB MO/Ml subtypes of AML it is only possible to detect one of the myeloid differentiation antigens, either CD13 or CD33 (Lee et al., 1987). The FAB M 1 subtype may be differentiated from FAB M2 on the basis of CD1 5 marker expression being negative in M 1 and positive in M2 (Neame et al., 1986). Some diagnostic discrepancies appeared between FAB Ml vs M2 and Ml/M2 vs M4 classification (Castoldi et al., 1993). Approximately one third of AMLs have been found to have abnormal chromosomal translocation with chromosomal breakpoints (Yunis, 1989; Yunis and Tanzer, 1993). The t(8; 21) (q22; q22) translocation is a characteristic chromosomal aberration in common AML subtype (FAB M2 category) (Kalwinsky et al., 1990; Hurwitz et al., 1992). In acute promyelocytic leukemia the disease shows a highly specific t(l5; 17) (q22;q21) translocation in both granulocytic and microgranulocytic variants (Yunis and Tanzer, 1993). The t(9;ll) translocation is found mostly in acute monocytic leukemia FAB M5 (Kalwinsky et al., 1990). Myeloid-associated antigens CD15 and CDw65 are preferentially expressed in cases with t(4; 11) translocation (Pui et al., 1991a). In conclusion, the diagnosis of the majority of AML subtypes can be readily established by morphology and well standardized and specific cytochemical reactions. The immunologic markers do not have sufficient discriminative capacity because of the lack of specificity for the great majority of monoclonal antibodies against myeloid antigens. MPO staining at the ultrastructural level has influenced the diagnosis of AML in cases in which light microscope cytochemistry was not contributory. Cytogenetic analysis has revealed supportive data. C. Acute Zymphoblustic leukemia ALL is an immunophenotypically heterogeneous disease with clinically important subtypes representing clonal expansion of lymphoblasts at different stages of maturation (Foon and Todd, 1986; Pui et al., 1993a; van? Veer et al., 1993). It should be emphasized that immunological marker analysis has proven to be of dominant relevance in the diagnosis of ALL to confirm the origin of blasts, to distinguish them from undifferentiated cells or megakaryocytes as well as to specify the subtype and maturation stage ofblast cells (Bennett et al., 1985b, 1991; Drexler et al., 1988; Traweek, 1993). The individual discrete stages of T and B cell differentiation are well characterized by the expression of specific surface mem-

brane antigens. The early thymocytes express CD2, CD5 and CD7 antigen without CDl, CD4, CD8 and CD3 expression. The lymphocytes considered to be in intermediate developmental stage are characterized by expression of CDl, CD2, CD5, CD7 and with variable expression of CD4 and CD8. Mature lymphocytes express CD3, CD5, CD7, CD2, CD4 and CD8 (Pui et al., 1993a). The most immature differentiation stage of TALL is characterized by the expression of cytoplasmic CD3 marker as well as the presence of CD7 and TdT (Crist et al., 1988; van Dongen et al., 1988; Mori et al., 1988; Thiel et al., 1989). CD3 surface membrane antigen expression is rarely found in T-ALL (Ludwig and Thiel, 1990). The antigens CD19 and CD22 appear earliest during B cell ontogeny and are expressed in virtually all cases of Blineage ALL (Janossy et al., 1989). The CD22 antigen, has been shown to occur intracytoplasmically at the same early stage of differentiation as the CD19 marker (Ling et al., 1987). According to Pui et al. (1993a), the temporal sequence of B-lineage antigen expression can be summarized as: CD24-+CDlO-+CD20-CD22 (cytoplasmic)+ CD%l+sIg (surface immunoglobulin)+CD23. Despite lineage specificity of lymphocyte surface antigens documented in many reports, only a few antigenic determinants are entirely restricted to a particular cell lineage (Mirro and Kitchingman, 1989). On the basis of this statement, CD2, CD4, CD7, CD10 and CD19 antigens are misnamed when referred entirely to as lymphoid-associated antigens (Campos et al., 1992; Drexler et al., 1993). The CD2 marker is expressed in some cases of acute promyelocytic leukemia, especially in the microgranular variant (Claxton et al., 1992). CD4 is expressed in many cases of myelomonocytic and monocytic leukemias (Szabo et al., 1990; Lombard and Mansvelt, 1992). Due to its broad spectrum of reactivity, the CD7 antigen is no longer considered to be solely a Tlineage marker, but appears to identify the less differentiated subtypes of AML (FAB Ml) (Kondo et al., 1992). Recent studies have shown CD10 antigen expression not only on leukemic cells of B-lineage (Foon and Todd, 1986; Pieters et al., 1992), but also on T cells (Shuster et al., 1990; Pui et al., 1993b) and terminally differentiated granulocytes (Braun et al., 1983), as well as on some nonhematopoietic cells (Mechtersheimer and Moller, 1989). CD19, a B-lineage-associated antigen, can be found on blasts of AML with the t(8;21) chromosome aberration (Kita et al., 1992) whereas CD15, a myeloid cell restricted antigen, is very often expressed in ALL cases with the t(4; 11) translocation (Pui et al., 1991a). Besides the dominance of immunophenotyping in the classification and diagnosis of ALL, cytochemical studies are particularly helpful in the characterization of this disorder by describing typical enzyme patterns of distinct lymphoid leukemic cells. The group of enzymes such as acid phosphatase (AP), acid a-naphthyl-acetate esterase (ANAE), /I-glucuronidase (BG), dipeptidylpeptidase IV (DPP IV) and 5’ nucleotidase (5’NT) has been cytochemically investigated in lymphoid leukemias in an attempt to define the possible

Cytochemistry of Acute Leukemia

lymphocyte subsets or different stages of lymphocyte maturation (Kulenkampff et al., 1977; Boesen et al., 1984; Crockard, 1984). Much controversy still exists concerning the cell specificity of these enzymes when considered in relation to their normal and pathologic cell behavior (Quaglino and De Pasquale, 1988). Their possible diagnostic usefulness is assumed in the characterization of three subgroups of ALL (T-ALL, B-ALL, non T, Bprecursor ALL), where the predominant cell types identify the relevant pathologic entities (Quaglino and De Pasquale, 1987). The enzyme AP granular pattern reaction was reported to be a characteristic feature of lymphoblasts in T-ALL (Wirthmtiller, 1980; Greaves, 1981; Boesen et al., 1984). Its diagnostic usefulness is undoubted. Although immunophenotyping is documented to be absolutely necessary in all cases of ALL (Foon and Todd, 1986; Drexler et al., 1988; Schumacher et al., 1991),.diagnostitally important correlation was found in T-ALL between T cell antigen markers and a strong positivity of AP in the majority of our patients (Babu8ikovH et al., 1991). Staining for ANAE has shown a typical ‘dot like’ pattern in normal circulating T lymphocytes (Knowles et al., 1978; Boesen et al., 1984). Discrepancies exist regarding ANAE activity in malignant T lymphoblasts as a weak or null reaction has been noted by some authors (Kulenkampff et al., 1977; Wirthmtiller, 1980; Knowles and Halper, 1982; Veerman et al., 1983) and a moderate one by others (Basso et al., 1980b). The possible explanation of these variations may be the fact that the different maturation stages of lymphocytes are characterized by different enzymes. The simultaneous determination and comparative evaluation of ANAE and AP can be of great value in the discrimination of the particular maturation stages of lymphocytes (Yang et al., 1982; Veerman et al., 1983). The most immature thymocytes were negative for ANAE (Kulenkampff et al., 1977; Basso et al., 1980a), but strongly positive for AP activity (Wirthmtiller, 1980). This may suggest that ANAE positivity is established at a later stage of T cell development than AP activity. Veerman et al. (1983) mentioned that both lysosomal enzymes, ANAE and AP, are not expressed at the same stage of lymphocyte differentiation and that they may be expressed differently in distinct cell lineages. Basso et al. (1980a) confirmed the ANAE and AP presence together only in mature circulating peripheral T lymphocytes. A rather uncertain significance in relation to normal as well as to lymphoid leukemia cell behavior have bglucuronidase (BG) and dipeptidylpeptidase IV (DPP IV). The presence of BG was demonstrated in correlation with its presence in the lymphocytes during the later phase of cell maturation (Basso et al., 1980a). Although BG is not absolutely restricted to T cell lineage of ALL, it has been reported to be useful in the characterization of T cell malignancies. Abnormally expressed BG was noticed in some cases of T-ALL, common acute lymphoblastic leukemia antigen (CALLA, CDlO) positive ALL and acute undifferentiated leukemia (Broadhead et al., 1981). An apparent restriction of DPP IV (lymphocyte protease)

323

to a particular T cell subset was documented in several reports (Lojda, 1981; Crockard et al., 1984; Ansorge and Schiin, 1987). The number of DPP IV positive cells correlates well with those showing a focal reaction of ANAE. The lack of cytochemically demonstrable DPP IV was found in B lymphocytes (Crockard et al., 1984). The highest level of DPP IV reactivity was noticed in the more mature thymocytes (Chilosi et al., 1982), when tracing DPP IV expression during T cell development. DPP IV activity never parallels the presence of AP, thus T-ALL that represents cells at early thymocyte stage of maturation does not display significant DPP IV activity (Chilosi et al., 1982; Feller et al., 1984). AP and DPP IV are present in T-ALL, but not at the same stage of maturation. Recently, DPP IV was classified as an activation marker of human lymphocytes (Schiin and Ansorge, 1990). The enzymes AP, ANAE, BG and DPP IV appear to be useful markers of lymphoid cell differentiation steps. Cytochemical stains for lysosomal enzymes (AP, ANAE, BG) are therefore helpful in discriminating T and B cells (Prasthofer et al., 1988). On the other hand, the AP and BG activities are not strictly lymphoid cell associated. The diffuse pattern of AP (in contrast to granular in ALL) was seen in some myeloid blasts, especially in the AML FAB Ml and M2 subgroups (Bennett et al., 1976; Drexler et al., 1984) as well as in AML FAB M4 and M5 subtypes (Besley et al., 1983). Similarly, normal or high activity of a diffuse BG reaction was observed in some cases of acute myelomonocytic and monocytic leukemia (Besley et al., 1983; Drexler et al., 1984). A high percentage of BG positive cells was expressed in six of our cases of acute undifferentiated leukemia, where leukemic blasts lacked the expression of surface membrane antigens either lymphoid or myeloid as well as cytochemical markers except for BG activity. The strong activity of BG and the presence of HLA-DR molecules were thought to suggest the possible assignment of these leukemic cells to a myelo/monocyte lineage (KlobuSicki et al., 1988). Much interest has been focused on enzyme Snucleotidase (SNT). Although ubiquitous, this enzyme lacks specificity, but is of great diagnostic value in AL (Veerman et al., 1985; Gutensohn and Thiel, 1990; Pieters et al., 1992). It is worthwhile to include 5’NT in the battery of markers suitable for subclassification of ALL. The strong correlation between 5’NT positivity and expression of CD10 (CALLA) marker in non-T, non-BALL was noteworthy (Foon and Todd, 1986; Gutensohn and Thiel, 1990; Pieters et al., 1992). The differences between enzymic activity of 5’NT in the CD10 positive group of non-T-ALL and the CD10 negative subgroups were statistically significant (Gutensohn and Thiel, 1990). T-ALL cells display very low or no activity of 5’NT (Drexler et al., 1984; Veerman et al., 1985; Gutensohn and Thiel, 1990). The differential diagnostic value of 5’NT was appreciated in the distinction between the myeloid and lymphoid phenotype in blast crisis of chronic myeloid leukemia (Gutensohn and Thiel, 1981; Koya et al., 1981). In mixed CD10 antigen positive T-ALL in children, a simultaneously expressed activity of both 5’NT (charac-

M. KlobuSicki

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teristic marker of non-T, B-precursor ALL) and AP (characteristic feature of T-ALL) has also been noted (BabuSikova et al., 1991). A noteworthy finding was reported by Koya et al. (198 1 ), who observed that peroxidase-negative AML had much higher 5’NT activity than MPO-positive counterparts. This observation is in agreement with our experience gained by cytochemical and immunological analysis of a very immature variant of AL, positive for CD34 antigen. It was rather unexpected to find this surface membrane-associated 5’NT positivity in a high proportion of CD3Cpositive, MPO-negative, but strongly SBBpositive myeloid blasts. The CD34 antigen is the immunologic marker of very early, immature stages of both lymphoid and myeloid cells (Civin et al., 1987). Thus, 5’NT in correlation to CD34 marker expression and SBBpositivity appeared to be a useful characteristic of poorly differentiated myeloid leukemia (KlobuSicki et al., 1993). Recent investigations have shown ultrastructural evidence of MPO in lymphoblasts of T-ALL (Kantarjian et al., 1989, 1990; Tauchi et al., 1991). The MPO-positivity in ALL may represent leukemogenesis from a common myeloid-lymphoid progenitor. Further studies regarding the expression and significance of MPO presence in otherwise typical ALL are necessary. These studies should incorporate molecular genetic assessment for MPO mRNA and immunoglobulin light chain gene rearrangement (Hammer et al., 1992). A large proportion of ALLs have been found to have genetic defects involving a specific chromosome breakpoint (Izraeli et al., 1993; Yunis and Tanzer, 1993). The chromosomal abnormalities in T cell ALL usually include genes encoding either the 1 or /I chain of the T cell receptor (TCR) (Yunis and Tanzer, 1993). Campana et al. (1991) recently investigated TCR protein expression that reacted exclusively with CD3 positive T cells and did not label in B-lineage or myeloid blast cells. This approach suggested a new tool for the classification of T-ALL based on the type and pattern of TCR protein expression. The t( 11; 14) gene translocation is the most common anomaly among childhood T-ALLs (Ribeiro et al., 1991). Several subgroups of B cell ALL are also associated with specific chromosomal translocations. Pre-B ALL is usually combined with t(5; 14) or t(8; 14) gene rearrangements (Yunis and Tanzer 1993). B-ALL is invariably associated with the common t(8; 14) or one of the t(2;8) or t(8;22) translocation variants (Yunis, 1989). Finally, immunophenotyping appears to be more instrumental in the final differential diagnosis of ALL because morphologic and cytochemical analyses provide insufficient information regarding the lineage and distinct steps of leukemic cell maturation. Clearly cytochemical investigation exerts a subsidiary diagnostic supplement in the majority of ALLs. D. Acute undifferentiated leukemia In a small fraction of ALs, commitment to specific lineage is not possible because of the lack of expression of lineage-associated antigens on the cell surface and the

lack of distinguishing morphologic and cytochemical characteristics (Shende et al., 1992; Traweek, 1993). The differentiation between ALL and AML in morphologically and cytochemically undifferentiated leukemias is of clinical relevance. The expression of CD13 and/or CD33 antigens as well as ultrastructural MPO activity proved to be helpful for the recognition of poorly differentiated AML which otherwise would remain unclassifiable or could be misdiagnosed as ALL (Matutes et al., 1988; Le Maistre et al., 1988). Recently, a definition of acute undifferentiated leukemia and a system for its classification on the basis of the current knowledge of phenotypic features of these cells and their clonal counterparts that exist at early stages of normal hematopoiesis was proposed by Shende et al. (1992). Cytochemical studies on acute undifferentiated leukemia have been limited. E. Mixed-lineage acute leukemia This group of AL (Mirro and Kitchingman, 1989; Traweek, 1993) is very heterogeneous. As mentioned above, the coexpression of one or more markers associated with other lineages (aberrant expression) was described in some studies (Childs et al., 1989; Drexler et al., 1991; Wiersma et al., 1991). The increased utility of immunophenotyping analysis is required for this type of AL (Traweek, 1993). Clinically, the cases of mixed leukemia can be divided into lymphoid-positive AML or myeloid antigen-positive ALL (Pui et al., 1991b). The Ly+ AML cases are usually characterized by FAB Ml or M2 morphology, a low level of MPO activity and a combined population of MPO-positive large and small blasts (Pui et al., 1987; Ferrara et al., 1990). The My+ ALL cases have an increased frequency of FAB L2 morphology (Pui et al., 1991b; Wiersma et al., 1991). The most common examples of mixed-lineage leukemia include AML expressing some of the lymphoid markers. CD2-positive AML has low MPO and SBB activity (Pui et al., 1987; Cross et al., 1988). CD10 antigen expression was found in coexpression with myeloid-associated antigen CDw65 (Boban et al., 1993). This type of coexpression is hardly associated with other myeloid markers (Bradstock et al., 1989). CD7 antigen-positive AML represents a particular subset of immature AML with a worse prognosis than standard AML (Kondo et al., 1992; Drexler et al., 1993). CD19 and CD20 antigen expression in myeloid cells was reported by Campos et al. (1987) and Gerhartz and Schmetzer (1990). The myeloid cell-associated antigens CDllb, CD13, CD33 and CD14 expression were noticed in some cases of ALL (Hanson et al., 1993). F. Prognostic implications The prognostic importance of immunophenotyping in AL has been documented in a large number of studies (Callea et al., 1991; Dubosc-Marchenay et al., 1992; Hoelzer, 1992; Drexler et al., 1993; Traweek, 1993). Although the FAB classification system appears to be helpful in differential diagnosis, so far only minimum

Cytochemistry

of Acute Leukemia

information has been obtained regarding prognosis. A few reports are designated to the prognostic implication of the leukemic blast cell’s enzymatic features (Basso et al., 1984; Veerman et al., 1985; Pieters and Veerman, 1987; Gutensohn and Thiel, 1990; Hoyle et al., 1991; Pieters et al., 1992; Valdts and Torres-Valdivieso, 1992). Moreover, in general the literature data regarding the clinical significance of these enzymes are contradictory. It was confirmed that the evaluation of an enzyme presence (SNT, AP, BG, ANAE in ALL and SBB and MPO in AML) in blast cells might be of value for the monitoring of the complete remission, effectiveness of therapy and for the detection of impending relapse. From the clinical standpoint, the expression of high 5’NT activity in leukemic cells showed a lower probability of complete remission and higher relapse rate than those with a low or negative 5’NT in CDlO-positive non-T, nonB-ALL (Veerman et al., 1985). Of note is the fact that CD10 expression has been linked to a more favorable prognosis (Pui et al., 1993b). Contradictory results on the adverse prognostic factor of a high AP activity in childhood ALL have been published (Basso et al., 1984; Pieters and Veerman, 1987; Pieters et al., 1992). Within the CDlO-positive non-T, non-B-ALL, ANAE-positive cases had a significantly shorter complete remission than ANAE-negative cases (Pieters and Veerman, 1987). BG presence in leukemic cells was reported to be a negative prognostic factor in non-T, non-B-ALL (Basso et al., 1984). In our series of 42 childhood ALL cases the presence of BG in blast cells was noticed to be an unfavorable prognostic indicator as compared to the complete remission probability of BG-positive and BGnegative cases. Telek et al. (1983) and Kuriyama et al. (1985) observed that a dense granular pattern of BG activity in blast cells was associated with significantly shorter survival time than the scattered granular pattern of BG activity in T-ALL patients. The reason why the pattern of BG activity in lymphoblasts reflected the time of survival of adult ALL cases, was not explained. The phenotype of T-ALL and BG-positivity in non-T, non-BALL, according to Basso et al. (1984), had a similar prognostic significance. This author confirmed a more aggressive clinical course and poorer response to treatment in children with ALL whose leukemic blasts have a more complete enzyme profile. Immunophenotyping of AML has not been prognostically useful except for the expression of a few specific antigens (Foon and Todd, 1986; Koehler et al., 1991). Moreover, little information is available for prognostic impact of enzyme cytochemistry in AML. The percentage of SBB-positive blasts, a simple and reproducible test, was shown by Hoyle et al. (1991) to be the most important prognostic factor in AML. ValdCs and Torres-Valdivieso (1992) proposed MPO as a good marker for maturity of myeloid cells as well as for the prognostic evaluation of AML. The clinicians pay particular attention to the relationship of specific chromosomal abnormalities to the therapeutic response and prognosis of AL (Rivera et al., 1991; Mirro, 1992). In both, AML and ALL, cytogenetic

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abnormalities represent an independent prognostic factor (Kalwinsky et al., 1990; Pui et al., 1990). Patients with aberrant chromosomal translocation are usually assigned to a higher risk group (Izraeli et al., 1993). It appears that all the chromosomal subtypes of T-ALL have a similar phenotype and poor prognosis (Rabbitts, 1991). The clinical course of the patients with ALL FAB L2 associated with the presence of the t(14; 18) chromosomal translocation suggests an unfavorable prognosis (Borrego et al., 1992). The prognostic importance of mixedlineage leukemia was published in some studies (Ferrara et al., 1990; Ball et al., 1991; Drexler et al., 1993).

G. Conclusions

The cytochemical investigation of leukemic blast cells, despite some difficulties and limitations, is exploitable for the detection and classification of AL. The practical differential diagnostic, therapeutic and prognostic usefulness of cytochemistry has been appreciated in many studies. In our opinion, although none of the enzymatic reactions appears to be absolutely specific, the cytochemical analysis allows the clinician to assess the diagnosis in the vast majority of AL cases. We admit that other methods, e.g. immunology, cytogenetic techniques and molecular biology, are necessary and dominant in the cases revealing a degree of cellular heterogeneity hidden by morphological and cytochemical uncertainty. According to our experience, immunophenotyping in AML permits only an inadequate assignment to the subtypes and the majority of AMLs may be recognized accurately and diagnosed correctly by cytochemistry. However, enzyme cytochemistry is limited in distinguishing subtypes of acute lymphoblastic leukemia, undifferentiated acute leukemia and minimally differentiated acute myeloid leukemia. The advances in cytogenetic and molecular biological technologies have been helpful for confirming the diagnosis of AL. The classification and diagnosis of AL based solely on enzyme cytochemistry are rarely found in literature. However, when cytochemistry is used in close conjunction with immunophenotyping, cytogenetic analysis, electron microscopy investigation and molecular biological technologies, it can still make a significant contribution to the precise diagnosis of acute leukemia.

REFERENCES Ansorge, S. and Schijn, E., 1987. Dipeptidyl peptidase IV (DPPIV), a functional marker of the T lymphocyte system. Acta Histochem., 82, 41-46. BabuBikovi, O., &p, J., HrivfigkovB, A., KlobuSickL, M., MesBroiovB, A. and KonikovP, E., 1991. Purine metabolism enzyme pattern, cytochemical characteristics and clinicopathologic features of dDlO-positive childhood T-cell leukemia. &‘eopZas&z, 38, 595-602. Ball. E. D., Davis. R. B.. Griffin. J. D.. Maver. R. J.. Davev. F.. Arthur. fi. C., Wurstei-Hill,‘D., Neil, W.,EI de&any, t., All&, S: L., Rail K., Lee, E. J., Schiffer, C. A. and Bloomfield, C. D., 1991. Prognostic value of lymphocyte surface markers in acute myeloid leukemia. Blood, 17,2242-2250.

326

M. KlobuSick&

Bassan, R., Rambaldi, A., Viero, P., Borleri, G. and Barbui, T., 1989. Integrated use of morphology, cytochemistry and immune marker analysis to identify acute leukemia subtypes. Haematologica, 74, 487490. Basso, G., Cocito, M. G., Semenzato, G., Pezzuto, A. and Zanesco, L., 1980a. Cytochemical study of thymocytes and T lymphocytes. Br. J. Haemat., 44, 577-582. Basso, G., Cocito, M. G., Poletti, A., Colleselli, P. and Zanesco, L., 1980b. Study ofcytochemical markers ACP and ANAE in childhood lymphoma and lkukemia. Br. J. Cancer, 41, 835-838. Basso. G.. Agostini, C.. Cocito, M. G., Pezzutto, A., Destro, R.. Capuio, %., Gazzola, M. V., Raimondi, R., Zanesco, L. and Semenzato, G., 1984. Non T, non B childhood acute lymphoblastic leukemia. Correlation between cytochemical markers and first remission. Cancer, 54, 981-985. Basso, G., Putti, M. C., Cant&Rajnoldi, A., Saitta, M., Santostasi, T., Santoro, N., Lippi, A., Comelli, A., Felici, L., Favre, C., Russo Mancuso, G., Guglielmi, C., Paolucci, P., Biondi, A., Rondelli, R. and Pession, A., 1992. The immunophenotype in infant acute lymphoblastic leukaemia: correlation with clinical outcome. Br. J. Haemat., 81, 184-191. Bennett, J. M., Catovsky, D., Daniel, M. T., Anta, J. P., Orfao, A. and Lopez-Barrasca, A., 1976. Proposals for the classification of the acute leukemias: French&American-British Cooperative group. Br. J. Haemat., 33, 451458. Bennett, J. M., Catovsky, D., Daniel, M. T., Flandrin, G., Galton, D. A. G., Gralnick, H. R. and Sultan, C., 1985a. Proposed revised criteria for the classification of acute myeloid leukemia: A report of the French-American-British Cooperative group. Ann. Intern. Med., 103, 621624. Bennett, J. M., Catovsky, D., Daniel, M. T., Flandrin, G., Galton, D. A. G., Gralnick, H. R. and Sultan, C., 1985b. Criteria for the diagnosis of acute leukemia of megakaryocytic lineage (M7): a report of the French-American-British Cooperative group. Ann. Intern. Med., 103,46%462. Bennett, J. M., Catovsky, D., Daniel, M. T., Flandrin, G., Galton, D. A. G., Gralnick. H. R. and Sultan. C., 1991. Proposal for the recognition of minimally differentiated acute myeloid leukemia (AML-MO). Br. J. Haemat., 78, 325-329. Besley, G. T., Moss, S. E., Bain, A. D. and Dewar, A. E., 1983. Correlation of lysosomal enzyme abnormalities in various forms of adult leukemias. J. c/in. Path., 36, 1000-1004. Boban, D., SuSiC, M., Markovib-GlamoEak, M., Uiarevii-, B., Batinik, D., MaruSiC, M., Nemet, D., Labar, B. and Hitrec, V., 1993. Correlation of morphological FAB classification and immunophenotyping: value in recognition of morphological, cytochemical and immunological characteristics of mixed leukaemias. Eur. J. Cancer, 29A, 1167-l 172. Boesen, A. M., Hokland, P. and Jensen, 0. M., 1984. Acid phosphatase and acid esterase activity in neoplastic and non-neoplastic lymphoid cells. &and. J. Haemat., 32, 313-322. Borgers, M. and Verheyen, A., 1985. Enzyme cytochemistry. Intern. Rev. Cytol., 95, 163-225. Borrego, S., Antifiolo, G., Parody, R. and Jimenez, F., 1993. Translocation t(14;18) in a patient with common acute lymphoblastic leukemia (FAB L2). Cancer Genet Cytogenet., 65, 177-178. Bradstock. K. F.. Kirk.. Grimslev. P. G.. Kabral. A. and Hughes, W. G.. 1989. Unusual immunophen&ype in acute leukemias: incidence and clinical correlation. Br. J. Haemat., 72, 512-518. Braun, M. P., Martin, P. J., Ledbetter, J. A. and Hansen, J. A., 1983. Granulocytes and cultured human fibroblasts express common acute lymphoblastic leukemia antigens. Blood, 61, 718-725. Broadhead, D. M., Besley, G. T., Moss, S. E., Bain, A. D., Eden, 0. E. and Sainsbury, C. P., 1981. Recognition of abnormal lysosomal enzyme patterns in childhood leukemia by isoelectric focusing with special reference to some properties of abnormally expressed components. Leuk. Res., 5, 29-40. Browman, G. P., Neame, P. B. and Soamboonsrup, P., 1986. The contribution of cytochemistry and immunophenotyping to the reoroducibilitv of the FAB classification in acute leukemia. Blood, 68: 90&905. Callea, V., Morabito, F., Martino, B., Stelitano, C., Oliva, B. and Nobile, T., 1991. Diagnostic and prognostic relevance of the immunophenotype in acute myelocytic leukemia. Tumori, 77,28-3 1. Campana, D., van Dongen, J. J. M., Mehta, A., Coustan-Smith, E., Wolvers-Tettero, I. L. M., Ganeshaguru, K. and Janossy, G., 1991. Stages of T-cell receptor protein expression in T-cell acute lymphoblastic leukemia. Blood, 77, 1546-1554. Campos, L., Guyotat, D., Gentilhomme, O., Treille, D., Fiere, D. and Germain, D., 1987. Expression of a B-lymphoid differentiation

antigen (CD19) on an acute non-lymphoblastic leukemia cells. Eur. J. Haemat., 38, 22&224. Castoldi, G. L., Liso., Fenu, S., Vegna, M. L. and Mandelli, F., 1993. Reproducibility of the morphological diagnostic criteria for acute myeloid leukemia: the GIMEMA group experience. Ann. Hemat., 66, 171-174. Charak, B. S., Advani, S. D., Karandikar, S. M., Parikh, P. M., Nair, C. N., Das Gupta, A., Gopal, R., Tapan, K. S., Nadkarni, K. S., Kurkure, P. A., Pai, S. K. and Pai, V. R., 1988. Sudan Black B positivity in acute lymphoblastic leukemia. Acta Haemat., 80, 199-202. Childs, C., Hirsch-Ginsberg, C., Walters, R. D., Andersson, B. S., Reuben, J., Trujillo, J. M., Cork, A., Stass, S. A., Freireich, E. J. and Zipf, T. J., 1989. Myeloid surface antigen positive acute lymphoblastic leukemia (My + ALL): immunophenotyping, ultrastructural, cytogenetic and molecular characteristics. Leukemia, 3, 777-783. Chilosi, M., Pizzolo, G., Menestrina, F., Iannucci, A. M., Bonetti, F. and Fiore-Donati, L., 1982. Dipeptidyl(amino)peptidaseIV (DAPIV) histochemistry on normal and pathological lymphoid tissues. Am. J. clin. Path., 77, 714719. Civin, C., Banquergo, M. L., Strauss, L. C. and Loken, M. R., 1987. Antigen analysis of hematopoiesis. VI. Flow cytometric characterization of MY-10 positive progenitor cells in normal human bone marrow. Expl Hairnat., 15,1&17. Claxton. D. F.. Reading. C. L.. Nagaraian. L.. Tsuiimoto. Y.. Andersson, i. S., Este;; E., Co;k, A.TH;h, i. d., Trljillo, J: and Deisseroth, A. B., 1992. Correlation of CD2 expression with PML gene breakpoints in patients with acute promyelocytic leukemia. Blood, 80, 582-586. Crist, W. M., Shuster, J. J., Falletta, J., Pullen, D. J., Berard, C. W., Vietti, T. J., Alvarado, C. S., Roper, M. A., Prasthofer, E. and Grossi, C. E., 1988. Clinical features and outcome in childhood Tcell leukemia-lymphoma according to stage of thymocyte differentiation: a Pediatric Oncology group study. Blood, 72, 1891-1897. Crockard, A. D., 1984. Cytochemistry of lymphoid cells: a review of findings in the normal and leukemic state. Histochem. J., 16, 107-150. Crockard, A. D., MacFarlane, E., Andrews, C., Bridges, J. M. and Catovsky, D., 1984. Dipeptidylaminopeptidase IV activity in normal and leukemic T cell subpopulations. Am. J. clin. Path., 82, 294. Cross, A. H., Goorha, R. M., Nuss, R., Behm, F. G., Murphy, S. B., Kalwinsky, D. K., Raimondi, S., Kitchingman, G. R. and Mirro, J. Jr, 1988. -Acute myeloid leukemia with T-lymphoid features: A distinct biologic and clinical entitv. Blood. 72, 579-587. Davey, F., Mick: R., Nelson, D. A:, MacCallum, J., Sobol, R. E., Royston, I., Cuttner, J., Ellison, R. R. and Bloomfield, C. D., 1988. Morphologic and cytochemical characterization of adult lymphoid leukemias which express myeloid antigens. Leukemia, 2, 420-426. Davey, F. R., Davis, R. B., MacCallum, J. M., Nelson, D. A., Mayer, R. J., Ball, E. D., Griffin, J. D., Schiffer, C. A. and Blomfield, C. D., 1989. Morphologic and cytochemical characteristics of acute promyelocytic leukemia. Am. J. Hemat., 30, 221-227. De Rossi, G., Avvisati, G., Coluzzi, S., Fenu, S., LoCoco, F., Lopez, M., Nanni, M., Pasqualetti, D. and Mandelli, F., 1990. Immunological definition of acute promyelocytic leukemia (FAB M3): A study of 39 cases. Eur. J. Haemat., 45, 168-171. Drexler, H. G., Gaedicke, G. and Minowada, J., 1984. Enzyme markers in acute leukemias: Advances during the last decade. J. natn. Cancer Inst., 72, 1283-1298. Drexler, H. G., 1987. Classification of acute myeloid leukemias-A comparison of FAB and immunophenotyping. Leukemia, 1, 697-705. Drexler, H. G., Thiel, E. and Ludwig, W.-D., 1991. Review of the incidence and clinical relevance of myeloid antigen-positive acute lymphoblastic leukemia. Blut, 5, 637-645. Drexler. H. G.. Thiel. E. and Ludwig. W.-D., 1993. Acute mveloid leukemias expressing lymphoid-associated antigens: Diagnostic evidence and prognostic significance. Leukemia, 7, 489498. Dubosc-Marchenay, N., Lacombe, F., Dumain, P., Marit, G., Montastrue, M., Belloc, F. and Reiffers, J., 1992. Role of blast cell immunophenotyping for the diagnosis and prognosis of acute myeloid leukemia. Hemat. Oncol., 10, 235-249. Feller, A. Ch., Parwaresch, M. R. and Lennert, K., 1984. Cytochemical distribution of dipeptidylaminopeptidase IV (DAP IV); (EC 3.4.14.5) in T-lyipiobl&tic lymphomafleukemia characterized with monoclonal antibodies. Leuk. Res., 8. 397-406. Ferrara, F., De Rosa, C., Fasanaro, A., Mele, 6., Finizio, O., Schiavone, E. M., Spada, 0. A., Rametta, V. and Del Vecchio, L., 1990. Myeloid antigen expression in adult acute lymphoblastic leukemia: Clinico-

Cytochemistry of Acute Leukemia hematological correlations and prognostic relevance. Hemat. Path., 4,93-98. Foon, K. A. and Todd, R. F., III., 1986. Immunologic classification of leukemia and lymphoma. Blood, 68, l-3 1. Frankel, S. R., 1993. Acute promyelocytic leukemia. New insight into diagnosis and therapy. Management Acute Leuk., 7, 109-138. Gerhartz, H. H. and Schmetzer, H., 1990. Detection of minimal residual disease in acute myeloid leukemia. Leukemia, 4, 508-516. Greaves. M. F.. 1981. Analvsis of the clinical and bioloaical significance of lymphoid phenotypes in acute leukemia. Cincer -Res., 41, 47524766. Gutensohn, W. and Thiel, E., 1981. High level of S’nucleotidase activity in blastic chronic myelogeneous leukemia with common ALLantigen. Leuk. Res., 5, 505-510. Gutensohn, W. and Thiel, E., 1990. Prognostic implication of ecto-5’nucleotidase activity in acute lymphoblastic leukemia. Cancer, 66, 1755-1758. Hammer, R. D., Collins, R. D., Ebrahimi, S. and Casey, T. T., 1992. Rapid immunocytochemical analysis of acute leukemias. Am. J. c/in. Path., 97, 876883. Hanson, C. A., Abaza, M., Sheldon, S., Ross, Ch. W., Schnitzer, B. and Stoolman, L. M., 1993. Acute biphenotypic leukaemia: immunophenotypic and cytogenetic analysis. Br. J. Haemat., 84, 4960. Hayhoe, F. G. J., 1984. Cytochemistry of acute leukaemias. Histochem. J., 16, 1051-1059. Hayhoe, F. G. J. and Quaglino, D., 1988. Haematological Cytochemistrv, (2nd edn). Churchil Livingston, Edinburgh. Hoelzer, D.; iG92. Prognostic factors in &ute.lymphobi&tic leukemia. Leukemia, 6 (Suppl. 4), 49-51. Hoyle, C. F., Gray, R. G., Wheatley, K., Swirsky, D., De Bastos, M., Sherrington, P., Rees, J. K. H. and Hayhoe, F. G. J., 1991. Prognostic importance of Sudan Black positivity: A study of bone marrow slides from 1386 patients with de novo acute myeloid leukaemia. Br. J. Haemat., 79, 398407. Hurwitz, C., Raimondi, S., Krance, R., Mirro, J., Ayers, D., Head, D. and Behm, F., 1992. Distinctive immunophenotypic features of t(8;21) (q22;q22) acute myeloblastic leukemia in children. Proc. Am. Sot. clin. Oncol., 11 (Abstr. 923), 278. Izraeli, S., Janssen, J. W. G., Haas, 0. A., Harbott, J., Brok-Simoni, F., Walther, J. U., Kovar, H., Henn, T., Ludwig, W.-D., Reiter, A., Rechavi, G., Bartram, C. R., Gadner, H. and Lion, T., 1993. Detection and clinical relevance of genetic abnormalities in pediatric acute lymphoblastic leukemia: a comparison between cytogenetic and polymerase chain reaction analyses. Leukemia, 7, 671-687. Janossy, G., Coustan-Smith, E. and Campana, D., 1989. The reliability of cytoplasmic CD3 and CD22 antigen expression in the immunodiagnosis of acute leukemia: A study of 500 cases. Leukemia, 3, 17(1-181. Kalwinsky, D. K., Raimondi, S. C., Schell, M. J., Mirro, J., Jr, Santana, V. M., Behm, F., Dahl, G. V. and Williams, D., 1990. Prognostic importance of cytogenetic subgroups in de novo pediatric acute nonlymphocytic leukemia. J. c/in. Oncol., 8, 75-83. Kantarjian, H., Freireich, E. J. and Stass, S. A., 1989. Lineage switch revisited: myeloperoxidase positive blasts by electron microscopy (EM) in patients with acute lymphoblastic leukemia. Blood, 74 (Suppl. l), 196a. Kantarjian, H., Hirsch-Ginsberg, C., Yee, G., Huh, Y., Freireich, E. and Stass, S. A., 1990. Mixed-lineage leukemia revisited: acute lymphocytic leukemia with myeloperoxidase-positive blasts by electron microscopy. Blood, 76, 808-813. Kita, K., Nakase, K., Miwa, H., Masuya, M., Mishii, K., Morita, N., Tatakura, N., Otsuju, A., Shirakawa, S., Ueda, T., Nasu, K., Kyo, T., Dohy, H. and Kamada, N., 1992. Phenotypic characteristics of acute myelocytic leukemia associated with the t(8;21) (q22;q22) chromosomal abnormality: Frequent expression of immature B-cell antigen CD19 together with stem cell antigen CD34. Blood, 80, 47w77. KlobuBickB, M. and BabuHikovB, O., 1988. The contribution of cytochemistry to the classification of diagnostically uncertain leukemias. Histochem. J., 20, 729-730 (Abstr.). KlobuSickL, M., BabuSikovl, 0. and HrivdLkovC, A., 1993. Relation of some cytochemical activities to the expression of CD34 marker in acute leukemia. Neoplasma, 40, 309-314. Knowles, D. M., II., Ho&an, T., Ferrarini, M. and Kunkel, H. C., 1978. The demonstration of acid a-naphthyl acetate esterase activity in human lymphocytes: usefulness as a T-cell marker. Cell Immunol., 35, 112-123. Knowles, D. M., II and Halper, J. P., 1982. Human T-cell malignancies. Correlative clinical, histopathologic, immunologic and cytochemical analysis of 23 cases. Am. J. Path., 106, 187-203.

327

Koehler, M., Schell, M. J., Behm, F. G., Raimondi, S. C., Woodruff, L. S. and Mirro, J., 1991. Expression of a novel surface antigen MKW in childhood acute leukemia has prognostic significance. Leukemia, 5,41-48. Kondo, S., Okamura, S., Harada, N., Ikematsu, W., Kawasaki, C., Fukuda, T., Kubota, A., Shimoda, K., Harrada, M., Shibuya, T., Okamura, T., Gondo, H., Hayashi, S., Yamaga, S., Omori, F. and Niho, Y., 1992. CD7-positive acute myeloid leukemia: Further evidence of cellular immaturity. J. Cancer Res. c2in. Oncol., 118, 386388.

Koya, M., Kanoh, T., Sawada, H., Uchino, H. and Ueda, K., 1981. Adenosine deaminase and ecto-5’-nucleotidase activities in various leukemias with special reference to blast crisis: Significance of ecto5’-nucleotidase in lymphoid blast crisis of chronic myeloid leukemia. Blood, 58, 1107-l 111. Kulenkampff, J., Janossy, G. and Greaves, M. F., 1977. Acid esterase in human lymphoid cells and leukemic blasts. A marker for T lymphocytes. Br. J. Haemat., 36, 231-240. Kuriyama, K., Tomonaga, M., Nonaka, H., Jinnai, I., Matsuo, T., Yoshida, Y., Amenomori, T., Tagawa, M., Ichimaru, M., Ohmiya, A. and Tsuji, Y., 1985. A comparative study of immunological and cytochemical profiles between adult and childhood acute lymphoblastic leukemias (ALLs): Heterogeneity in adult common ALL. Leuk. Res., 9, 1237-1247. Lee, E. J., Pollak, A., Leavitt, R. D., Testa, J. R. and Schiffer, C. A., 1987. Minimally differentiated acute nonlymphocytic leukemia: a distinct entity. Blood, 70, 14OS1406. Lee, M.-Sh. and Stass, S. A., 1993.3. Uses ofpolymerase chain reaction in leukemia. Detection of minimal residual disease and identification of novel genetic mutation. In: Leukemia: Advances Research and Treatment, Freireich, F. J. and Kantarjian, H., (eds.). Kluwer Academic, pp. 3544. LeMaistre, A., Childs, C. C., Hirsch-Ginsberg, C., Reuben, J., Cork, A., Trujillo, J. M., Andersson, B., McCredie, K. B., Freireich, E. and Stass, S. A., 1988. Heterogeneity in acute undifferentiated leukemia. Hemat. Path., 2, 79-90. Lemei, P., 1988. A case of acute promyelocytic leukemia with “faggot cells” exhibiting alpha-naphthyl-butyrate esterase activity. Br. J. Haemat., 68, 138-139. Lepelley, P., Preudhomme, C., Sartiaux, C., Ghevaert, C., Lai, J. L., Iaru, T., Fenaux, P. and Cosson, A., 1993. Immunological detection of myeloperoxidase in poorly differentiated acute leukemias. Eur. J. Haemat., SO, 155-159. Ling, N. R., Lennan, Mac, I. C. M. and Mason, D. Y., 1987. B-cell and plasma cell antigens: new and previously defined clusters. In: Leucocyte Typing III., McMichael, A. J., (ed.). Oxford University Press, Oxford, pp. 302-335. Lippi, U. and Cappelletti, P., 1982. Cytochemical evidence of multiple molecular forms of myeloperoxidase (MPO). Huematologica, Pavia, 67,807-808. Lippi, U., Cappelletti, P. and Schinella, M., 1983. a-Naphthyl butyrate esterase (a selective cytochemical monocyte marker). La Ricerca c/in. Lab., 13,467-471. Lojda, Z., 1981. Proteinases in pathology. Usefulness of histochemical methods. J. Histochem. Cytochem., 29,481-493. Lombard, E. H. and Mansvelt, E. P. G., 1992. CD4+, CD33-, CD13-, CDlrl-acute monoblastic leukaemia. Acta Haemat., 87, 151-152. Ludwig, W.-D. and Thiel, E., 1990. Routine immunophenotyping of acute leukemias. Blut, 60, 48-50. Matutes, E., Pombo De Oliveira, M., Foroni, L., Morilla, R. and Catovsky, D., 1988. The role of ultrastructural cytochemistry and monoclonal antibodies in clarifying the nature of undifferentiated cells in acute leukemia. Br. J. Haemat., 69, 205-211. Mechtersheimer, G. and Miiller, P., 1989. Rapid communication: Expression of the common acute lymphoblastic leukemia antigen (CDlO) in mesenchymal tumor. Am. J. Path., 134,961-966. Mirro. J., Jr and Kitchingman. G. R., 1989. The mornholoev. cytochemistry, molecular-char&teristics and clinical significance% acute mixed-lineage leukemia. In: Leukemia Cytochemistry, Scott, S. C., (ed.). Chister, Ellis Horwood, pp. 115-179. Mirro, J., Jr, 1992. Pathology and immunology of acute leukemia. Leukemia, 6 (Suppl. 4), 1>15. Mori, N., Oka, K., Yoda, Y., Abe, T. and Kojima, M., 1988. Leu-4 (CD3) antigen expression in the neoplastic cells from T-ALL and Tlymphoblastic lymphoma. Am. J. clin. Path., 90, 244-249. Neame, P. B., Soamboonsrup, P., Browman, G. P., Meyer, R. M., Benger, A., Wilson, W. E. C., Walker, I. R., Saeed, N. and McBride, J. M., 1986. Classifying acute leukemia by immunophenotyping: A combined FAB-immunologic classification of AML. Blood, 68, 1355-1362.

328

M. KlobuSickh

Norton, J. D., Campana, D., Hotlbrand, A. V., Janossy, G., CoustonSmith. E.. Jani. H.. Yaxlev. J. C. and Prentice. H. G.. 1988. Correiatioi of jmm;nophe&type with rearrangement of T cell antigen receptor beta and gamma genes in acute lymphoblastic leukemia in adults. Leukemia, 2, 27-34. Nguyen, D., Brynes, R. K., Macanlay, L., Kaplan, B. and Pinter-Brown, L. C., 1989. Acute myeloid leukemia FAB Ml microgranular variant: a multiparameter study. Hemat. Path., 3, 11-22. Pieters, R. and Veerman, A. J. P., 1987. Prognostic significance of acid alpha-naphthyl acetate esterase and acid phosphatase in childhood acute lymphoblastic leukemia. Leuk. Res., 11, 995-999. Pieters, R., Huismans, D. R., Loonen, A. H., Peters, G. J., Hahlen, K., van der Does-van den Berg, A., van Werng, E. R. and Veerman, A. J. P., 1992. Relation of S-nucleotidase and phosphatase activities with immunophenotype, drug resistance and clinical prognosis in childhood leukemia. Leuk. Res., 16, 873-880. Prasthofer, F., Zarcone, D. and Grossi, C. E., 1988. Distinctive morphological features of human peripheral blood lymphocytes. J. Immun. Immunopharmac., 8, 8495. Pui, C. H., Behm, F. G., Kalwinsky, D. K., Murphy, S. B., Butler, D. L., Dahl, G. V. and Mirro, J., 1987. Clinical significance of low level myeloperoxidase positivity in childhood acute nonlymphoblastic leukemia. Blood, 70, 51-54. Pui, C. H., Crist, W. M. and Look, A. T., 1990. Biology and clinical significance of cytogenetic abnormalities in childhood acute lymphoblastic leukemia. Blood, 76, 1449-1463. Puii C. H., Frankel, L. S., Carroll, A. J., Raimondi, S. C., Shuster, J. J., Head. D. R.. Crist. W. M.. Land. V. J.. Pullen. D. J.. Steuber. C. P.. Behi, F. G.‘and Bbrowitz; M. J.; 19919. Clinical characteristics and treatment outcome of childhood acute lymphoblastic leukemia with the t(4; 11) (q21; q23): a collaborative study of 40 cases. Blood, 77, 440-447. Pui, C. H., Raimondi, S. C., Head, D. R., Schell, M. J., Rivera, G. K., Mirro. J. Jr. Crist. W. M. and Behm. F. G.. 1991b. Characterization of chilhhooh acu;e leukemia with multiple myeloid and lymphoid markers at diagnosis and relapse. Blood; 78, 1327-1337. Pui. C. H.. Behm. F. G. and Crist. W. M.. 1993a. Clinical and biologic ;elevar& of ‘immunologic marker studies in childhood a&te lymphoblastic leukemia. Blood, 82, 343-362. Pui, C. H., Rivera, G. K., Hancock, M. L., Raimondi, S. C., Sandlund, J. T., Mahmoud, H. H.,Ribeiro, R. C., Furman, W. L., Hurwitz,C. A., Crist, W. M. and Behm, F. G., 1993b. Clinical significance of CD10 expression in childhood acute lymphoblastic leukemia. Leukemia, 7, 3540. Quaglino, D. and De Pasquale, A., 1987. Relationship between different cytobiological aspects of myeloid leukaemias. Acta Haemat., 78 (Suppl. l), 2631. Quaglino, D. and De Pasquale, A., 1988. Conventional cytochemistry, immunocytochemistry and their relationship to other cytobiological techniques in the clinical study of haematological diseases. Haematologica, 73, 143-152. Rabbitts, T. H., 1991. Translocations, master genes and differences between the origins of acute and chronic leukemias. Cell, 67, 641-644. Raimondi, S. C., Pui, C. H., Head, D. R., Rivers, G. K. and Behm, F. G., 1993. Cytogenetically different leukemic clones at relapse of childhood acute Ivmphoblastic leukemia. Blood, 82, 576-580. Ribeiro, R. C., Raimondi, S. C., Behm, F. G., Cherrie, j., Crist, W. M. and Pui, C. H., 1991. Clinical and biologic features of childhood Tcell leukemia with the t(11; 14). Blood, 78,466-470. Rivera, G. K., Raimondi, S. C., Hancock, M. L., Behm, F. G., ChingHon, P., Abromowitch, M., Mirro, J., Jr, Ochs, J. S., Look, A. T., Williams, D. L., Murphy, S. B., Dahl., Kalwinsky, D. K., Evans, W. E., Kun, L. E., Simone, J. V. and Crist, W. M., 1991. Improved outcome in childhood acute lymphoblastic leukemia with reinforced early treatment and rotational combination chemotherapy. Lancet, 337, 666. Roberts, G. T., Spence, D. G., Padmos, M. A., Sheth, K. V., Clink, H. and Ernst, P., 1992. Morphologic, immunophenotypic and cytogenetic patterns of adult acute myeloid leukemia in Saudi Arabia. Leuk. Res., 16, 181-190. San Miguel, J. F., Gonzalez, M., Canizo, M. C., Anta, J. P., Zola, H. and Borrasca, A., 1986. Surface marker analysis in acute myeloid leukemia and correlation with FAB classification. Br. J. Haemat., 64, 547-560. Schan, E. and Ansorge, S., 1990. Dipeptidyl peptidase IV in the immune system. Cytofluorometric evidence for induction of the enzyme on activated T lymphocytes. Biol. Chem., Hoppe-Seyler, 371, 699-705. Schoot, van der C. E., Daams, G. M., Pinkster, J., Vet, R. and von dem Borne, A. E. G., 1990. Monoclonal antibodies against myeloperoxi-

dase are valuable immunological reagents for the diagnosis of acute myeloid leukemia. Br. J. Haemat., 74, 173-178. Schumacher, H. R., Shrit, M. A., Kowal-Vern, A., Dizikes,G., Radvany, R. M. and Le Beau, M., 1991. Acute leukemia and related entities. Arch. Path. Lab. Med., 115, 331-337. Second MIC Cooperative Study Group, 1988. Morphologic, immunologic and cytogenetic (MIC) working classification of the acute myeloid leukemias. Br. J. Haem., 68, 487494. Shende, A. C., Bonagura, V. R., Cheah, M. S. C. and Lanzkowsky, P., 1992. Acute undifferentiated leukemia (AUL): A case report and a proposed system of classification. Am. J. Hemat., 40, 23&237. Shuster, J. J., Falletta, J. M., Pullen, D. J., Crist, W. M., Humphrey, G. B., Dowell, B. L., Wharam, M. D. and Borowitz, M., 1990. Prognostic factors in childhood T-cell acute lymphoblastic leukemia: a Pediatric Oncology Group study. Blood, 75, 166-173. Storr, J., Dolan, G., Counstan-Smith, E., Barnett, D. and Reilly, J. T., 1990. Value of monoclonal anti-myeloperoxidase (MP07) for diagnosing acute leukemia. J. clin. Path., 43, 847-849. Szabo, G., Miller, C. L. and Kodys, K., 1990. Antigen presentation by the CD4 positive monocyte subsets. /. Leuc. Biol., 47, 11 l-120. Tavassoli, M., Shaklai, M. and Crosby, W. H., 1979. Cytochemical diagnosis of acute myelomonocytic leukemia. Am. J. clin. Path., 72, 59-62. Tavassoli, M., 1984. Combined esterase in acute myelomonocytic leukaemia. Br. J. Haem., 57, 187. Tauchi, T., Ohyashiki, T., Ohyashiki, K., Ohyashiki, J. M., Saito, M., Nakazawa, Y., Enomoto, Y., Hosoda, Y. and Toyama, K., 1991. Acute lymphoblastic leukemia with azurophilic granules that contain ultrastructural myelopcroxidase activity. Am. J. Hemat., 36, 288-290. Telek, B., Kiss, D., Pecze, K. and Pak, K., 1983. Differential diagnostic value of acid phosphatase and beta-glucuronidase in acute leukemia. Blur, 46, 67. Terstappen, L. W. M. M., Safford, M., Koneman, S., Loken, M. R., Zurlutter, K., Buchner, T., Hiddemann, W. and Wormann, B., 1991. Flow cytometric characterization of acute myeloid leukemia. Part II. Phenotypic heterogeneity at diagnosis. Leukemia, 5, 757-767. Tetteroo, P. A. T., van der Schoot, C. E. and van dem Borne, A. E. G., 1987. New developments in the phenotyping analysis of (im)mature myeloid leukemia: Clinical relevance. (Abstract). Intern. Meeting on genotypic, phenotypic and functional aspects of haematopoiesis, Assisi, p. 13. Thiel, E., Kranz, B. R., Raghavachar, A., Bartram, C. R., Loffler, H., Messerer, D., Ganser, A., Ludwig, W.-D., Buchner, T. and Hoelzer, I)., 1989. Prethymic phenotype and genotype of pre-T (CD7+/ER-)-cell leukemia and its clinical significance within adult acute lymphoblastic leukemia. Blood, 73, 1247-1258. Tomonaga, M., Yoshida, Y., Tagawa, M., Jinnai, I., Kuriyama, K., Amenomori, T.. Yoshioka, A., Matsuo, T., Nonaka, H. and Ichimaru, M., 1985. Cytochemistry of acute promyelocytic leukemia (M3): Leukemic promyelocytes exhibit heterogeneous patterns in cellular differentiation. Blood, 66, 35&357. Traweek, S. T., 1993. Immunophenotypic analysis of acute leukemia. Am. J. clin. Path., 99, 504-512. Tricot, G., Broeckaert-Van Orshoven, A., Van Hoof, A. and Verwilghen, R. L.. 1982. Sudan Black B positivitv in acute lvmnhoblastic _ leukaemia. Br. J. Haemat., 51, 615621. _ ValdBs, M. D. and Torres-Valdivieso, M. J., 1992. Prognostic importance of myeloperoxidase positivity in de nouo acute myeloid leukaemia. Br. J. Haemat., 81,449-450. van Dongen, J. J. M., Krissansen, G. W., Wolvers-Tettero, I. L. M., Comans-Bitter, W. M., Adriaansen, H. J., Hooijkaas, H., Wering, van, E. Rand Terhorst, C., 1988. Cytoplasmic expression of the CD3 antigen as a diagnostic marker for immature T-cell malignancies. Blood, 71, 603-612. van? Veer, M. B., 1992. The diagnosis of acute leukemia with undifferentiated or minimally differentiated blasts. Ann. Hemat., 64, 161-165. van? Veer, M. B., van Putten, W. L. J., Verdonck, L. F., Ossenkoppele, G. J., Lovenberg, B., Kluin-Nelemans, J. C., Wijermans, P. W., Schouten, H. C., Sizoo, W. and Dekker, A. W., 1993. Acute lymphoblastic leukaemia in adults: immunological subtypes and clinical features at presentation. Ann. Hemat., 66, 277-282. Veerman, A. J. P., Huismans, D. R. and van Zantwijk, C. H., 1983. Immunologic phenotype related to acid alpha-naphthyl acetate esterase and acid phosphatase in childhood acute lymphoblastic leukaemia. Acta Haematol., Basel, 69, 32-35. Veerman, A. J. P., Hogeman, P. H. G., van Zantwijk, C. H. and

Cytochemistry of Acute Leukemia Bezemer, P. D., 1985. Prognostic value of 5’nucleotidase in acute lymphoblastic leukemia with common-ALL phenotype. Leuk. Res., 9, 1227-1229. Vellenga, E. and Griffin, J. D., 1987. The biology of acute myeloblastic leukemia. Sent. Oncol., 14,365-371. Wiersma, S. R., Ortega, J., Sobel, E. and Weinberg, K. J., 1991. Clinical importance of myeloid-antigen expression in acute lymphoblastic leukemia in childhood. N. Engl. J. Med., 324, 800-808. Wirthmiiller, R., 1980. Immunological and cytochemical characterization of human T-derived lymphoproliferative disorders. Biology of the Cancer Cells. Proceedings of the Fifth Meeting of the

329

European Association for Cancer Research, Vienna, 9912 September, pp. 83-89. Yang, K., Bearman, R. M., Pangalis, G. A., Zelman, R. J. and Rappaport, H., 1982. Acid phosphatase and alpha-naphthyl acetate esterase in neoplastic and non-neoplastic lymphocytes. A statistical analysis. Am. J. clin. Path., 78, 141-149. Yunis, J. J., 1989. Genes and chromosomes in the pathogenesis and prognosis of human cancer. In: Advances in Pathology, FenoglioPreiser, C. (ed.). Year Book Publishers, Chicago, pp. 147-188. Yunis, J. J. and Tanzer, J., 1993. Molecular mechanisms of hematologic malignancies. Crit. Rev. Oncogen., 4, 161-190.