3 Morphology and cytochemistry of acute lymphoblastic leukaemia

3 Morphology and cytochemistry of acute lymphoblastic leukaemia HELMUT LOFFLER WINFRIED GASSMANN

Morphologic examination and cytochemical staining of air-dried bone marrow and peripheral blood smears continue to form the basis for diagnosis and classification of acute leukaemias. Acute lymphoblastic leukaemia (ALL) is usually characterized by small to medium sized leukaemic blasts with a rather low grade of cell-to-cell variability. The nucleocytoplasmic ratio is high with just a small cytoplasmic rim in many cases. The cytoplasm tends to be moderately basophilic, is usually agranular and is free of vacuoles. The presence of azurophilic granules, inclusions or vacuoles in the blasts, however, does not exclude the diagnosis of ALL as long as the cytochemical tests are compatible with the diagnosis. Auer rods are never present. Compared with AML, the chromatin is more condensed. The nucleoli tend to be indistinct; they are smaller in size and in number than in blasts of myelogenous leukaemias. Cases with more than one prominent nucleolus are the exception to the rule. There are no dysplastic features in the granulocytic lineage. Morphology of the neutrophils of the peripheral blood is normal. There are no pseudoPelger-HuEt forms and no agranular or hypogranular polymorphs. FAB CLASSIFICATION OF ALL

The French-American-British (FAB) classification of haematological malignancies, proposed in 1976 (Bennett et al, 1976, 1981), has gained widespread acceptance and can be regarded as the standard classification system of acute leukaemias. A L L is separated into three categories: ALL L1, L2, and L3. Separation of L1 and I~ is based on four criteria: ALL Lt is characterized by (i) high nucleocytoplasmic ratio with the cytoplasm representing less than 10% of the cell surface area, (ii) one single small or even no clearly visible nucleolus in the majority of the cells, (iii) regular nuclear outline, and (iv) a rather small size of the blasts. In some cases of A L L Lt, it may even be difficult to discriminate the leukaemic blasts from lymphocytes. The Bailli&e's Clinical Haematology--

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features of ALL L2 are complementary: (i) cytoplasm over 20%, (ii) prominent or multiple nucleoli, (iii) irregular nuclear outline, and (iv) a cell size twice the diameter of small lymphocytes or larger. There is no correlation between Lt or L2 morphology and immunological phenotype of the cells. Moreover, it must be kept in mind that morphology often differs from peripheral blood to bone marrow where cells appear to be more regular. ALL WITH L3 MORPHOLOGYDIDENTIFICATION OF B.ALL

Rapid and reliable identification of B-ALL is of outstanding importance, since this type of leukaemia cannot be cured by the treatment approaches which are successful for all other types of ALL. The cells of L3 ALL are medium sized to large and have a characteristically homogeneous appearance. In this context, homogeneity means homogeneity of structure and not of cell size and shape. The blasts have dense but finely stippled nuclear chromatin. The nucleus is round to oval and may have more or less prominent nucleoli. The cytoplasm is intensely basophilic and moderately abundant. Usually, it completely surrounds the nucleus. The most characteristic feature of this type of leukaemia is the prominent cytoplasmic vacuolation in the majority of the cells (Gill et al, 1986; Fenaux et al, 1989). It is important to note that this vacuolation may be missing in the blasts of peripheral blood. In case no bone marrow smears are available, basophilia of the cytoplasm may be the only indicator of the mature B-cell phenotype of the leukaemia. According to our experience, there is a high correlation of the classical L3 phenotype as defined by morphology with B-ALL defined immunologically with surface expression of immunoglobulins. Twenty-six patients of the German ALL-Study Group with L3 morphology were analysed immunologically by Dr W.-D. Ludwig and Prof. Dr E. Thiel (Berlin). In 18 of 22 evaluable cases the diagnosis of B-ALL could be confirmed immunologically. Three cases had common ALL (c-ALL) and one other had pre-pre-B-ALL. Similar data have been reported by other groups (Mangan et al, 1985). The reverse, however, has also been observed. We and others have observed cases classified as B-ALL immunologically missing the characteristic morphological L3 phenotype (Finlay and Borcherding, 1988; Michiels et al, 1988). The question remains, however, of whether it is the immunologically determined B-nature or the L3 phenotype as seen under the microscope that is associated with the very poor prognosis under conventional ALL therapy. CYTOCHEMISTRY The use of cytochemistry has greatly facilitated the discrimination of ALL from the various types of acute myeloblastic leukaemia (AML). The following cytochemical reactions are of special practical value: peroxidase,

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Sudan Black B staining (SBB), non-specific esterase with ~-naphthyl acetate as substrate, periodic acid Schiff reaction (PAS), acid phosphatase, and dipeptidyl aminopeptidaseIV (DAP IV). Peroxidase and SBB

Peroxidase activity or SBB staining of leukaemic cells are indicators for the myeloid nature of blast cells and negativity of these tests is a prerequisite for the diagnosis of ALL. However, SBB staining of blasts has been demonstrated in rare cases of ALL (Tricot et al, 1982). Stass et al (1983) found SBB positivity in six out of 350 patients with immunologically proven ALL. Consequently, we prefer the peroxidase reaction and use SBB just in case of peroxidase deficiency. Non-specific esterase

The non-specific esterase with oL-naphthyl acetate as substrate shows dotlike positivity in some cases of T-lineage ALL. The main intention of using this cytochemical test is to exclude poorly differentiated monoblastic leukaemia which shows diffuse esterase activity. PAS

For evaluation of PAS cytochemistry it is necessary to evaluate the pattern of the reaction. When present, PAS positivity of ALL is seen as granules or blocks against a negative cytoplasmic background. In ALL the PAS reaction never shows a diffuse pattern. On the contrary, diffuse PAS positivity is an indicator of the myeloid nature of the leukaemia. It should be noted that PAS reactivity is an indicator of low priority which is 'overruled' by positive results of any other of the cytochemical tests mentioned. Blasts being positive for peroxidase as well as for PAS belong to the myeloid lineage independent of the PAS-reaction product. Acid phosphatase

Focal acid-phosphatase positivity is highly correlated with ALL of a Tophenotype and to a lesser degree with ALL of pre-T-phenotype; 70 versus 40% of the respective cases are focally positive. However, this type of acid-phosphatase positivity can also be found in 15% of the cases of Blineage ALL (L6ffler et al, 1987). DAP IV

In contrast to acid-phosphatase, DAP IV has a very high specificity of near 100% but lower sensitivity for uncovering T-lineage of leukaemic blasts. Twenty percent of T-ALL and 5% of pre-T-ALL are positive for this enzyme. Until now, we have found only one exception to the rule, a patient suffering from c-ALL showing DAP IV positive blasts (L6ffler et al, 1987).

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T H E MORPHOLOGICAL/CYTOCHEMICAL DIAGNOSIS OF A C U T E U N D I F F E R E N T I A T E D LEUKAEMIA

Until recently, acute leukaemias proving negative for all cytochemical tests especially for the PAS reaction and for the focal type of acid-phosphatase, were termed acute undifferentiated leukaemia (AUL). However, this morphological/cytochemical diagnosis can easily be confused with the immunological diagnosis of unclassifiable leukaemia. According to our experience with over 2000 leukaemias classified by morphology and immunology, A U L (morphological/cytochemical) can be classified as ALL in 95% and as AML in 5% by immunological analysis. For cases with PAS or acid-phosphatase reaction typical for ALL, the respective percentages are 97 and 3%. Accordingly, the term 'acute undifferentiated leukaemia' should be reserved for cases which can be classified neither by morphology/cytochemistry nor by immunology nor by electron microscopy (Matutes et al, 1988; Heil et al, 1991).

D I S C R I M I N A T I O N OF ALL F R O M P O O R L Y D I F F E R E N T I A T E D AML

The morphological and cytochemical distinction of ALL from poorly differentiated AML remains a problem (Lee et al, 1987; Campana et al, 1990; Van't Veer, 1992). According to the FAB classification of acute leukaemias, AML M0 and AML M1 are defined as acute myelogenous leukaemias with less than 10% of the leukaemic cells showing maturation to or beyond the promyelocytic stage. Distinction of cases without clear-cut myelogenous morphology from ALL is provided by peroxidase cytochemistry in AML M1 (Bennett et al, 1976) whereas AML M0 is characterized by negativity for peroxidase and immunological proof of the myeloid nature of the leukaemia (Bennett et al, 1991; Catovsky et al, 1991). The peroxidase reaction is considered positive by the FAB group, when the enzyme can be demonstrated in more than 3% of the blasts. The threshold of 3% had been chosen although lymphatic cells are uniformly negative for this enzyme. The rationale behind it is that a small number of residual non-leukaemic neutrophil blasts being positive for peroxidase may be mistaken as leukaemic cells and may lead to an erroneous classification of the disease. Recently, we analysed the discriminative power of this FAB cut-off limit of peroxidase cytochemistry in prospective studies of the AML Cooperative Group (AMLCG) and the German ALL Study Group (Gassmann et al, 1992). Cytological analysis was done by the authors, immunological analyses were performed by Dr W.-D. Ludwig and Prof. Dr E. Thiel (Berlin). Patients were recruited from February 1988 to December 1991. Immunological analysis established the diagnosis of AML in 347 patients. On

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morphological/cytochemical analysis, 96 of these were classified as poorly differentiated leukaemias fulfilling the FAB criteria of AML M1, AML Mo or ALL, respectively. The leukaemic blasts of these 96 cases were analysed for peroxidase activity. In 33 of 96 cases no peroxidase activity could be demonstrated cytochemically. Fourteen patients had 0.2-1% of the blasts being positive for this enzyme and five patients had 1.1-2.9% positive blasts. Forty-four patients fulfilled the 3% threshold of the FAB group for AML and had 3-100% of the blasts positive for peroxidase. Thus, a total of 52 out of 96 patients with poorly differentiated AML with the myeloid nature proven by immunology could not be classified as AML on morphological and cytochemical grounds provided the FAB criteria were applied rigidly. In all 19 patients with peroxidase positive blasts in the range 0.2-3%, the myeloid nature of the leukaemia had been considered certain on morphological grounds. These data show that acute leukaemias with less than 3% of blasts being positive for peroxidase in which the positive blasts are morphologically identical to the negative ones, can be recognized as AML morphologically. Special attention has to be paid to morphology of the blasts that are counted as positive leukaemic cells. In addition, the absence of blasts being positive for peroxidase is no reliable indicator for the lymphatic nature of a leukaemia, even if the PAS reaction is typical for ALL. The morphological diagnosis of ALL needs confirmation by immunology in any instance.

SPECIAL VARIANTS OF ALL ALL with vacuoles (other than B-ALL)

Vacuolation is not a feature restricted to B-ALL. In cooperative British ALL studies with more than 2000 children, 29% of the patients had blast cells with cytoplasmic vacuoles (Figure la) (Lilleyman et al, 1992). In rare L1/L2 cases with vacuolation being so prominent that cytoplasm is difficult to evaluate and cell surface disrupted in many cells, the vacuoles contain PAS-positive material (Figure lb and c). ALL with granules

Granular ALL is characterized by the presence of more than 5% lymphoblasts with at least three clearly defined azurophilic granules (Figures 2 and 3). It is a rare variant of this disease. In a large study of the Pediatric Oncology Group, granular ALL accounted for 4.5% of 2096 cases of ALL (Cerezo et al, 1991). The authors did not observe an association of the granulation with the immunological sub-type with the exception of the 12 cases of B-ALL not showing granules. In adults the percentage of granular ALL is lower, accounting for 1.5% according to our experience.

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(b)

(c)

Figure 1. ALL with cytoplasmic vacuoles. (a) Many small distinct vacuoles in a moderately basophilic cytoplasm. They are PAS negative. Immunophenotype: cALL. May-Grt~nwaldGiemsa. (b) Very large vacuoles partially inflating the cytoplasm. May-Griinwald-Giemsa. (c) The vacuoles of the lymphoblasts shown in (b) are filled with PAS-positive material. Immunophenotype: cALL. PAS reaction.

(a)

(b)

(c)

Figure2. Granular ALL. (a) Granular ALL with many, sometimeslarge azurophilicgranulesin the cytoplasm. May-Griinwald-Giemsa. (b) Same case as (a) with rather strong acid phosphatase reaction in most granules. (c) Another case withsmall to mediumsizedazurophilic granules. May-Griinwald-Giemsa. The granules of A L L can be confused with the primary granules of granulopoiesis, with granules of immature basophils and with small A u e r rods. T h e y can be distinguished by their characteristic cytochemical staining reactions and by electron microscopy. The blasts are negative for peroxidase and SBB and the granules usually show positivity for esterase and acid-

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Figure 3. Electron microscopy of a lymphoblast with large membrane bound inclusions corresponding to the azurophilic granules (arrow).

phosphatase. Together with the electron microscopic appearance these are arguments in favour of a lysosomal nature of these granules. This variant of ALL with occasional co-expression of myeloid markers can be observed in any age group. Most cases belong to the immunological sub-group c-ALL. Hand-mirror cell leukaemia

'Hand-mirror' morphology of more than 40% of the leukaemic cells is seen in some cases of ALL but it has also been described in rare instances of AML. The immunologic phenotype usually is cALL or pre-pre-B-ALL. At present, hand-mirror cell leukaemia seems to be merely a morphologic variant without distinguishing clinical features (Mazur et al, 1986). Philadelphia-chromosome-positive ALL and acute leukaemia of mixedlineage type

The Philadelphia chromosome (Ph I) or its molecular equivalent, the fusion product BCR/ABL, is associated with a very poor prognosis and can be detected in a high percentage of c-ALL and pre-B-ALL depending on the age of the patients (Crist et al, 1990; Pui et al, 1990; Seeker-Walker, 1990; Maurer et al, 1991; Janssen et al, 1992; Westbrook et al, 1992).

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Morphology of these cases usually is unremarkable with a few but important exceptions (Tachibana et al, 1987; Hirsch-Ginsberg et al, 1988; Pui et al, 1991; Jackson et al, 1992). We have seen a considerable number of PhLpositive acute leukaemias with a dominant lymphatic and a minor myeloid leukaemic blast cell population with the latter being positive for peroxidase. These leukaemias of mixed lineage type do represent a large portion of cases with discrepancies between morphology and immunology in the studies of the German ALL Study Group and the AMLCG. Since 1988, in 14 patients with an immunological diagnosis of c-ALL or pre-B-ALL established by Dr W.-D. Ludwig and Prof. Dr E. Thiel (Berlin), an additional myeloid blast population has been identified or suspected by morphology and cytochemistry. In seven of these cases a chromosome or polymerase chain reaction analysis had been performed. In six of these, a Ph 1 could be identified. An analogous mixed-lineage acute leukaemia of the T-cell series has been observed consistently in the German registries of the ALL Study Group and the A M L C G and by others (Kantarjian et al, 1990; Pui et al, 1991). In contrast to the B-lineage cases, we, as well as Pui et al (1991), have not seen any case of co-existence of myetoid and T-lymphatic blasts which proved positive for the Ph 1 or the BCR/ABL fusion product. ALL with eosinophilia Few patients with either c-ALL or ALL of the T-lineage show a marked eosinophilia (Catovsky et al, 1980). Some of these are characterized by a recently identified translocation between chromosomes 5 and 14 (Hogan et al, 1987). They must be distinguished from other hypereosinophilic syndromes and from FAB MeEo which is characterized by another cytogenetic abnormality. SUMMARY ALL is characterized by small to medium sized leukaemic blasts with a rather low grade of cell-to-cell variability. The nucleocytoplasmic ratio is high with just a small cytoplasmic rim in many cases. The cytoplasm tends to be moderately basophilic. Usually, though not in each instance, it is agranular and free of vacuoles. The chromatin is more condensed than in AML and the nucleoli tend to be indistinct. The FAB classification of haematological malignancies separates ALL into three categories: ALL L1, L2, and L3. However, just the identification of the L3 variant is of major importance. The L3 cells are medium sized to large and are characterized by intensely basophilic and moderately abundant cytoplasm with prominent cytoplasmic vacuolation in the bone marrow but not necessarily in the peripheral blood. According to our experience there is a high but not universal correlation of the L3 phenotype as defined by morphology with the immunotogically defined B-ALL with surface expression of immunoglobulins.

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Until recently, acute leukaemias proving negative for all c y t o c h e m i c a l tests especially for the P A S reaction and for the focal type of acid p h o s p h a t a s e , were t e r m e d 'acute undifferentiated l e u k a e m i a ' ( A U L ) . H o w e v e r , this morphological/cytochemical diagnosis m a y be c o n f u s e d with the i m m u n o l o g i c a l diagnosis o f unclassifiable leukaemia. Since a l m o s t any o f these cases can be recognized as A L L or A M L by i m m u n o l o g y , the term A U L should be reserved for cases which can be classified neither by m o r p h o l o g y / c y t o c h e m i s t r y nor by i m m u n o l o g y . T h e m o r p h o l o g i c a l and cytochemical distinction of A L L f r o m p o o r l y differentiated A M L remains a p r o b l e m , especially if the F A B criteria for distinguishing A L L f r o m A M L by cytochemistry ( 3 % of the blasts positive for peroxidase) are applied rigidly. A small but significant p e r c e n t a g e of p o o r l y differentiated leukaemias have less than 3 % of the blasts positive for peroxidase and the m y e l o i d n a t u r e of the l e u k a e m i a can be identified by cytochemistry. T h e absence of blasts being positive for peroxidase is no reliable indicator for the lymphatic n a t u r e of a leukaemia, even if the P A S reaction is typical for A L L . T h e m o r p h o l o g i c a l diagnosis of A L L n e e d s confirmation by i m m u n o l o g y in each instance.

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