Leukemia Research Vol. 15, No. 7, pp, 627-639, 1991. Printed in Great Britain.

0145-2126/91 $3.00 + 0.00 Pergamon Press plc

A B N O R M A L L O C A L I Z A T I O N OF I M M A T U R E P R E C U R S O R S (ALIP) IN T H E B O N E M A R R O W OF MYELODYSPLASTIC SYNDROMES: C U R R E N T STATE OF K N O W L E D G E A N D F U T U R E DIRECTIONS M. H. MANGI, J. R. SALISBURY* and G. J. MUFTIt Departments of Haematological Medicine and *Morbid Anatomy, King's College School of Medicine and Dentistry, Denmark Hill, London SE5 8RS, U.K.

(Received 21 January 1991. Accepted 27 January 1991) Abstract--It has been suggested that the occurrence of abnormal localization of immature precursors

(ALIP) in the bone marrow biopsy (BMB) may be of diagnostic and prognostic significance in myelodysplastic syndromes (MDS). The recognition of ALIP has been based exclusively on bone marrow histological appearances. During the last decade technical advances have led to the widespread use of various immunophenotypic markers for the diagnostic and prognostic purposes which has contributed enormously in understanding the development of haemopoietic cells and the cellular origin of various haematological malignancies. In addition proliferation antigens, growth factors, oncogenes, anti-oncogenes and other biological discoveries have opened new vistas to our knowledge of the normal and neoplastic growth processes. Despite this, the precise nature of ALIP and their significance in relation to the aetiopathogenesis and evolution of MDS remains unclear. Indeed the diagnostic value of ALIP in MDS is debatable. Furthermore, the precise cell lineages which comprise ALIP are not defined. The purpose of this review is to address these issues and to incorporate our new findings on the histological and immunophenotypic characterization of immature cell aggregates.

Key words: Abnormal localization of immature precursors (ALIP), bone marrow biopsy (BMB), immunohistochemistry, lectins, monoclonal antibodies, myelodysplastic syndromes (MDS).

N O R M A L L O C A L I Z A T I O N OF HAEMOPOIETIC PRECURSORS

mechanisms underlying these observations are poorly understood. However, work in murine bone marrow provides supporting evidence for the peritrabecular zone being a fostering area for the early developmental stages of granulopoiesis [8-12]. Secretion of various haemopoietic growth factors from the bone marrow haemopoietic and stromal cells is an area of ongoing research [13] and it is anticipated that future studies may provide insights into the mechanisms underlying the topography of haemopoietic cells within the marrow.

NORMAL human bone marrow biopsies reveal a spatial distribution pattern of haemopoietic cells. The erythropoietic islands and megakaryocytes are associated with marrow sinusoids in the central region of marrow cavities, early granulocytic precursors lie close to endosteal surfaces and the more mature forms are found in the central intertrabecular areas [1--4] (Fig. 1). In contrast to erythroblasts which show a focal pattern of distribution (erythron) during maturation, the maturing granulocytic cells are more or less randomly distributed [4-7]. An explanation for this absence of clustering of granulocytic precursors might be that with increasing maturation granulocytic cells exhibit increasing locomotive activity and so can leave their site of origin and move towards the sinusoids. The nests of erythroblasts and megakaryocytes are usually observed in close proximity of sinusoidal wall, thus obtaining an easy access to the intravascular space [1-7]. The biological

A B N O R M A L L O C A L I Z A T I O N OF I M M A T U R E P R E C U R S O R S (ALIP) Krause [14] observed a distinctive topographic distortion of the bone marrow cells in MDS. There was displacement of progenitor cells from peritrabecular areas towards intertrabecular areas, a bone marrow histological pattern termed clusters of immature cells in MDS. This finding of architectural distortion, in particular with centrally located immature precursors, [14] did not receive much attention until Tricot

t To whom correspondence should be addressed. 627

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and his colleagues [15-19] examined plastic embedded bone marrow biopsies (BMB) from 40 patients with primary myelodysplastic syndromes (P-MDS). The finding of abnormal localization of immature precursors was not only confirmed but was shown to be of prognostic importance [18-22] (Fig. 2). An attempt was made to define this abnormal localization of immature precursors (ALIP). Tricot et al. [18] defined ALIP as myeloblasts and promyelocytes clustering away from trabeculae in the marrow of myelodysplastic patients. They were further subdivided in two types:

phological grounds alone [31-39]. Therefore, immuno or enzyme labelling is necessary. Until now BMB studies have lacked the immuno- or enzyme labelling of various cell lineages involved in MDS. It is, therefore, important to complement the histomorphological findings with immunohistological labelling of BMB to demonstrate the cell lineages beyond reasonable doubt.

(1) ALIP aggregates: more than five immature precursors. (2) ALIP clusters: three to five immature precursors.

Recently Mangi et al. [40-41] analysed material from 63 cases with primary myelodysplastic syndromes (P-MDS) for bone marrow immunohistochemical patterns. An indirect immunoperoxidase technique on formalin-fixed and EDTA decalcified bone marrow biopsy [42] employed mouse monoclonal antibodies recognising CD15 (Leu M1), CD68 (KP1), HLA-DR, polyclonal CD3; lectin UEA-1 [36] and enzyme histochemistry for chloroacetate esterases (CAE) [42]. On the basis of immunohistochemical assessment three types of immature cell aggregates have been described which were difficult to distinguish on histological appearances alone [40-41]. These were erythroid aggregates (pseudo ALIP), megakaryocytic aggregates (pseudo ALIP) and myeloblastic and monoblastic aggregates (true ALIP) (Figs 3-7). Table 1 shows various descriptions used by different authors to describe clusters of immature haemopoietic cells in MDS.

A case was classified as A L I P + if at least three aggregates or clusters were easily detectable in one section. This classification has been modified by Verhoef et al. [23] who reported three types of ALIP on histological grounds. These were: (1) ALIP without a proliferation of large blastic cells. (2) ALIP accompanied by proliferation of myeloblasts and promyelocytes. (3) ALIP surrounded by a mixture of granulocytic and monocytic cells. When BMB from MDS patients, were divided into ALIP positive and negative groups, this grouping was found to have clinical relevance, irrespective of French-American-British (FAB) subtypes [18--25]. Although the occurrence of ALIP in cases of MDS has been confirmed by several authors, there is continuing debate surrounding their prognostic importance [26-30]. This could be due to inter-observer errors and the inherent pitfalls in the analysis of bone marrow histology. PITFALLS OF ALIP R E C O G N I T I O N (1) When the three-dimensional structure of bone marrow is considered; it is possible that these immature blasts are overlying bone trabeculae which are not in the field of view. (2) Immature erythroid cells are conventionally recognized by the company they keep; but in poor BMB preparations, as well as in cases with predominant erythroid hyperplasia [28], erythroid precursors can be mis-diagnosed as ALIP. (3) Like BM smears, plastic embedded BMB allow a detailed analysis of morphology, nevertheless it is often difficult and sometimes impossible to differentiate between various cell lineages on mor-

I M M U N O P H E N O T Y P I N G AND TYPES OF I M M A T U R E CELL A G G R E G A T E S

FAB SUBTYPES VS PRESENCE OF ALIP ALIP is predominantly a feature of the more malignant type of MDS, i.e. refractory anaemia with excess of blasts (RAEB), refractory anaemia with excess of blasts in transformation (RAEBt) and some cases of chronic myelomonocytic leukaemia (CMML) which contain more than 5% blasts in the bone marrow [24-25]. However, some R A and RARS cases which by definition contain less than 5% bone marrow blasts and some cases of CMML with less than 5% bone marrow blasts also demonstrate presence of ALIP [18-23, 26-30; 40-41]. This shows the heterogenous nature of cases with MDS and demonstrates a limitation of bone marrow aspiration alone in the diagnosis of these disorders. There are several explanations for the discrepancy between BM cytology and BM histology. (1) The number of the blast cells in BM cytological preparations also depends on the ease of the aspir-

FIG. 1. Staining for chloroacetate esterase (CAE) showing intimate relationship between bone marrow trabeculae and CAE-positive granulocytic precursors. T = T r a b e c u l a (magnification 1000x). FIG. 2. Clusters of abnormal localization of immature precursors (arrow) as seen in the bone marrow biopsy (BMB) of MDS with Giemsa stain (magnification 400×). m = Mast cells. 629

FIG. 3. Clusters of abnormal localization of immature granulocytic cells in the centre of the marrow stained for CAE without counterstain. (Magnification 1000x). FIG. 4. True ALIP: clusters of myeloblasts stained for HLA-DR (arrow). An immunoperoxidase technique, magnification 1000×. Note morphologcially similar pseudo ALIP (open arrow). 630

FIG. 5. Pseudo ALIP: erythroid aggregates stained for UEA-1 (arrow). An immunoperoxidase technique, magnification 1000x. (A)Proerythroblasts showing membranous and dot-like cytoplasmic staining. (B) Binuclear megakaryocytes showing deep granular cytoplasmic staining. FIG. 6. Pseudo ALIP: megakaryocytic aggregates stained for UEA-1. In contrast to erythroblasts, megakaryoblasts show deep granular cytoplasmic staining for UEA-1 (arrow) (magnification 1000 x). Note morphologically similar unstained myeloblasts (open arrow). 631

FIG. 7. True ALIP: CD68 (KP1) showing immature granulocytic aggregates (an immunoperoxidase technique, magnification 1000x). Note morphologically similar pseudo ALIP (open arrow). FIG. 8. BMB frozen section from a case of MDS showing staining for CD34. Note the difficulty in interpretation due to cross-labelling of endothelium and immature erythroid cells (the APAAP technique, magnification 400x). 632

FIG. 9. PC10 showing nuclear staining for proliferative cells. PC10-positive erythroid clusters (arrow) (an immunoperoxidase technique, magnification 1000×). FIG. 10. Recombinant human granulocyte colony stimulating factor (rh-GCSF) antibody: positive ceils in MDS (perinuclear staining for some of the monocytic/ macrophage cells). An immunoperoxidase technique, magnification 1000×. Note absence of rhGCSF in myeloblasts (open arrow). 633

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ALIP in MDS TABLE 1. Authors

Method

Term

Krause, 1981 [14].

Giemsa/H&E staining of paraffin-embedded BMB. Giemsa/H&E staining of plastic embedded BMB. Giemsa/H&E staining of plastic embedded BMB.

Clusters of immature cells. (1) ALIP aggregates. (2) ALIP clusters. (1) ALIP without proliferation of large blast cells. (2) ALIP with proliferation of large blast cells. (3) ALIP surrounded by myeloblasts and monocytic cells. 1. Erythroid aggregates (pseudo ALIP). 2. Megakaryocytic aggregates (pseudo ALIP). 3. Myeloid aggregates (true ALIP) (granulocytic and monocytic aggregates).

Tricot et al., 1984 [17]. Verhoef et al., 1990 [23].

Mangi et al., 1990 [40--41].

Immunohistochemical assessment of routinely processed (formalin-fixed and paraffin-embedded BMB.

ation process and amount of suction applied, which will vary from case to case [42, 43]. (2) The problem of BM aspiration may be further compounded by an increased amount of reticulin. This has been found in up to 70% of cases with MDS [15-23, 40, 41] and these reticulin fibres may preferentially retain the blasts and in some cases there may be markedly increased reticulin fibrosis with MDS as described by various authors [43-45]. (3) In BM cytological preparations, although the morphology of individual cells is preserved, the architecture is completely disrupted. In contrast, BM trephine sections yield a larger volume of tissue in its natural architecture, where blasts aggregates are not disrupted and they are easier to detect with the aid of immunohistochemical stains. Hence, BM histological preparations reduce the chances of error which are inherent in BM aspirate preparations [46]. PROGNOSTIC I M P O R T A N C E OF ALIP An additional value of BMB investigation in MDS lay in pinpointing those smaller number of R A / RARS cases with ALIP, which are otherwise misrepresented on the basis of the BMA blast count. According to our experience [41, 42] and others [18-23] the clinical behaviour of R A / R A R S cases with ALIP was different from R A / R A R S cases without ALIP. However, it has to be stressed that for precise identification of ALIP and their prognostic role in MDS immunohistochemical was necessary [41, 42].

U N U S U A L P H E N O T Y P E AND ALIP Early haemopoietic cell markers are of considerable interest for both diagnostic and prognostic purposes in myeloproliferative disorders [47, 48]. Twenty-three cases of P-MDS have been examined (Mangi, 1990 an unpublished observation] using fresh frozen BMB sections and an alkaline phosphatase and anti-alkaline phosphatase technique [49,50] employing a mouse monoclonal antibody to CD34 and a rabbit polyclonal antibody to terminal deoxynucleotidyl transferase (TdT). Anti-CD34 labelled myeloid and erythroid progenitors as well as endothelium which hampered the interpretation of BMB (Fig. 8). Due to technical reasons this study failed to confirm or refute the expression of CD34 by ALIP [51]. Occasional TdT-positive cells (

Abnormal localization of immature precursors (ALIP) in the bone marrow of myelodysplastic syndromes: current state of knowledge and future directions.

It has been suggested that the occurrence of abnormal localization of immature precursors (ALIP) in the bone marrow biopsy (BMB) may be of diagnostic ...
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