Leukemia Research Vol. 16, No. 1, pp. 15-33, 1992. Printed in Great Britain.

0145-2126/92 $5.00 + .00 Pergamon Press pie

MORPHOLOGIC CLASSIFICATION OF THE MYELODYSPLASTIC SYNDROMES (MDS): COMBINED UTILIZATION OF BONE MARROW ASPIRATES AND TREPHINE BIOPSIES REINER BARTL,* BERTHA FRISCHt AND ROLF BAUMGART:~ *Department of Bone Marrow Diagnosis, University of Munich; tInstitute of Haematology, Ichilov Hospital, University of Tel-Aviv; SMedizinische Klinik III, Klinikum Grol3hadern, University of Munich, F.R.G. Abstract--In a retrospective and prospective follow-up study from 1975 to 1991, bone marrow biopsies, aspirates and clinical features of 495 patients with MDS were investigated. Sections of undecalcified plastic embedded biopsies and smears of bone marrow aspirates were stained according to Giemsa. Bone marrows with MDS were characterized by three main categories of morphologic alterations: (1) cellular abnormalities, (2) architectural disorganization in the bone marrow and (3) stromal changes; the combined use of aspirates and trephine biopsies enabled a more reliable and accurate diagnosis of MDS than either one alone. The bone marrow findings fell into one of 7 subtypes, with the frequencies and median survivals in brackets: (1) MDS sideroblastic (19%, 62 months), (2) MDS megaloblastoid (13%, 56 months), (3) MDS proliferative (22%, 31 months), (4) MDS blastic (15%, 9 months), (5) MDS hypoplastic (15%, 26 months), (6) MDS fibrotic (6%, 29 months), and (7) MDS inflammatory (10%, 42 months). In follow-up studies patients with secondary MDS were excluded and the prognosis and subsequent evolution for each of the morphologic subtypes were evaluated. The conclusion is drawn that aspirates and trephine biopsies are complementary procedures and both are required for diagnosis, classification and decisions on current treatment modalities of patients with MDS.

Key words: Bone marrow biopsy and aspirate, myelodysplastic syndromes, classification, prognosis, evolution, effects of therapy.

INTRODUCTION

differences between these conditions are only descriptive, as their clinical features overlap and all are characterized by an ineffective bone marrow [3]. During the past decade the term "myelodysplasia" has emerged in attempts to resolve the confusion regarding nomenclature. Further clarification was provided by the FAB group in 1982 who classified the MDS into 5 subtypes based on peripheral blood films and aspirates: RA, RARS, R A E B , RAEB-t and C M M L [81. But how to define "dysplasia" of the bone marrow? Strictly speaking, dysplasia is a histopathologic term and means "a loss in the uniformity of the individual cells, as well as a loss in their architectural orientation". Moreover, "Dysplasia is characteristically associated with protracted chronic irritation or inflammation" [9]. When it occurs in the bone marrow, "myelodysplasia" results and is characterized by a disturbed differentiation of haematopoiesis. This raises the question: are percentages, obtained from marrow aspirates and peripheral blood films, sufficient to characterize the complexity of myelodys-

THE term "myelodysplastic syndromes" encompasses a heterogeneous group of leukaemia-related disorders, characterized by cytopenia(s) and bone marrow dysfunction [1-4]. The various designations [5] used to describe these conditions fall into two main groups: terms to identify patients with "refractory anaemias" and terms to define preleukaemic states retrospectively [6, 7]. Nevertheless some Abbreviations: MDS, myelodysplastic syndromes; pMDS, primary MDS; CMPD, chronic myeloproliferative disorders; A L, acute leukaemia; SA L, subacute leukaemia; Ery. L, erythroleukaemia; MF/OMS, myelofibrosis/osteomyelosclerosis; Apl. A, aplastic anaemia; LPD, lymphoproliferative disorders; FAB, French-American-British; RA, refractory anaemia; RARS, RA with ring sideroblasts; RAEB, RA with excess of blasts; RAEB-t, RAEB in transformation; ALIP, abnormal localization of immature precursors; GM-CSF, granulocyte-macrophage colonystimulating factor; Ara-C, cytosine arabinoside; 1L-3, interleukin 3. Correspondence to: Prof. Dr R. Bartl, Ehrenfelsstr 5, 8000 Miinchen 70, F.R.G. 15

R. BARI"Letal.

16

plasia? Furthermore, new therapeutic strategies designed to restore effective haematopoiesis require a broader based morphologic evaluation of patients with MDS than hitherto. This report, based on retrospective and prospective data, extends studies published in 1986 [5] and examines the practical value of the proposals made therein. The aims of this study are (1) to test our proposed histologic classification in a larger series of patients and include survival times, (2) to assess the prognostic impact of data derived from both bone marrow aspirates and trephine biopsies, and (3) to test our proposed morphologic classification based on both aspirates and biopsies with respect to its prognostic significance, to therapeutic decisions and evolution of disease. MATERIALS AND METHODS

Patients In the retrospective study, 450 patients with MDS were investigated from 1975 to 1991. No patient had received therapy prior to the initial bone marrow biopsy and aspirate/imprint. The diagnosis of MDS was based on the combined assessment of biopsy sections, marrow smears and peripheral blood values. There was a male preponderance (M/F ratio 1:5) and the median age was 64 years (range 14-86). Once the diagnosis was made, most of the patients were treated only with supportive measures and observation. In 45 patients, chemotherapy with low-dose Ara-C with or without GM-CSF was instituted. A total of 112 patients had sequential biopsies and smears taken in clinical phases of progression or of transformation. Survival times were available for 300 patients, measured from time of initial biopsy. In the prospective study, 48 patients with MDS were investigated from 1987 to 1991 at the Klinikum Grol3hadern, University of Munich. Initial bone marrow biopsies and aspirates together with peripheral blood values and films of all patients were assessed. The subsequent treatment modalities were: supportive care only (n=20), glucocorticoid therapy only (n=3), GM-CSF only (n=3), lowdose Ara-C and GM-CSF (n=14), interleukin 3 only (n=2), low-dose Ara-C and IL-3 (n=4) and bone marrow transplant (n=2). Sequential biopsies/aspirates were taken in all 48 patients, with a maximum of 6 follow-up biopsies. Biopsies In both studies a total of 1040 bone marrow biopsies were taken (after informed consent had been obtained) from the posterior or anterior iliac crest by means of a manual trephine (85% of the biopsies) or an electric drill; aspirates were taken from another spot on the anaesthesized area of the periosteum after complete withdrawal of the biopsy needle. All biopsy cores were embedded in plastic without decalcification, and 3 ~tm sections were stained with gallamine blue Giemsa for cytologic detail, Gomori's stain for fibres and Berlin blue stain for haemosiderin. Only sections with more than 30 sqmm bone marrow area were used for histologic and histomorphometric evaluations which were made with a Zeiss integration disc. Smears of the aspirates and imprints, and peripheral blood films were

stained according to Giemsa [10-12]. Electron microscopy: in some cases small pieces (1.5 × 1 mm) cut from the biopsies were processed by routine methods and embedded in Spurr's medium; sections were cut and viewed in a Zeiss EM 10 after staining with uranyl acetate and lead citrate. A computer-based analysis of clinical, laboratory, cytologic and histologic data was performed and selected BMDP computer programs (2D,1L) were utilized for statistical evaluation.

Morphological criteria for diagnosis of MDS The fundamental abnormality shared by all MDS is impairment of differentiation with retention of a greater or lesser proliferative capacity, i.e. "ineffective" haematopoiesis. In bone marrow smears this defect was sometimes manifest in only one cell line, but in sections the dysplasia was generally trilineal, though megakaryopoiesis proved to be the most sensitive parameter for its diagnosis (some striking cytologic abnormalities are illustrated in Figs 1--4). The various cellular abnormalities of MDS have been extensively described using bone marrow smears (for references see [13]). Though alterations in cellular and nuclear size and shape and in staining characteristics may be found in other conditions, some abnormalities are highly characteristic and almost pathognomonic of MDS: presence of hypo- and hypergranular neutrophils, neutrophils deficient in myeloperoxidase, Pelger-Huet anomaly, ringed sideroblasts, PAS-positive normoblasts, "micromegakaryoeytes" and megakaryocytes with multiple, separate, small round nuclei (Fig. 5a) [14, 15]. In some patients, however, the cellular abnormalities were not pronounced enough for a definite diagnosis at first presentation, and ancillary investigations including sequential bone marrows were necessary [3]. Bone marrow histology enabled a more precise evaluation of the complex haematopoietic defect than was possible from aspirate cytology alone. Histology revealed some of the most important diagnostic features of MDS: (1) cellularity (volume percentages), (2) topography of the different cell lines (Fig. 5b), (3) state of the microenvironment including extracellular matrix, and (4) inflammatory reactions including fibrosis and haemophagocytosis [12, 16-20]. RESULTS All biopsies were scored into three groups according to the cellularity: (a) hypercellular, more than 50 vol% haematopoiesis, 50% of the cases, (b) normocellular, 20-50 vol%, 30%, and (c) hypocellular, less than 20 vol%, 20% (Fig. 6). There was no correlation between the cellularity in the biopsy section and the blast percentages in the marrow smears. In some cases a hypocellular marrow was present even in R A E B - t , the high-risk group of the FAB classification. A more or less pronounced disorganization of marrow topography was evident in all biopsies of MDS (Fig. 7). Though all cell lines showed topographic abnormalities, it was most easily seen in megakaryopoiesis, with clusters of micromegakaryocytes, loss of sinusoidal orientation and para-

FIG. 1. Erythroid series: (a) dyserythropoiesis, note bi- and multinuclearity, Giemsa × 600, (b) PAS-positive erythroblasts, PAS × 400, (c) megaloblastoid erythropoiesis, Giemsa x 600, (d) erythropoietic "hot spots" with maturation arrest, biopsy section, Giemsa x 250.

FIG. 2. Megakaryocyticseries: (a) binucleated "micromegakaryocyte", Giemsa x 600, (b) megakaryocyte with multiple small round nuclei, Giemsa × 600, (c) dysplastic megakaryocytes, biopsy section, Giemsa × 50, (d) dysplastic megakaryocytes with fine fibrosis, biopsy section, Gomori × 250. 17

(a)

FIG. 3. Granulocytic series: (a) defective myeloperoxidase in granulocyte (upper right), peripheral blood film, myeloper-

oxidase, x 600, (b) variable granularity in myeloid precursors, Giemsa x 600, (c) Auer rods in case of RAEB-t, Giemsa x 600, (d) ALIP surrounded by fat cells, biopsy section, Giemsa x 250.

FIG. 4. Monocytic series: (a) iron accumulation in macrophages, biopsy section, Berlin blue x 250, (b) large macrophages engorged with PAS-positive inclusions, PAS x 600, (c) haemophagocytosis, Giemsa x 600, (d) atypical monocytes in CMML, peripheral blood film, Giemsa x 600.

18

(a)

(b)

FIG. 5. Electron micrographs in case of MDS proliferative type: (a) megakaryocyte with multiple, separate, small round nuclei, M=megakaryocyte, × 25 000, (b) architectural disorganization showing dysplastic erythroblasts in paratrabecular region, TB =trabecular bone, x 20000. 19

y,,

FIG. 6. Bone marrow cellularity in MDS: (a) hypercellular, (b) normocellular, (c) hypocellular, all Giemsa x 100.

20

FIG. 8. Topographic distortion in MDS: (a) groups of immature erythroblasts and megakaryocytes without orientation to the sinusoid in the centre, (b) intravascular localization of a micromegakaryocyte, (c) "abnormal localization of immature precursors" (ALIP), (d) perivascular plasmacytosis between two dysplastic megakaryocytes with multiple small nuclei, all Giemsa × 100. 21

FIG. 10. Morphologic classification of MDS: (a) MDS sideroblastic, with immature erythroid islands and a paratrabecular megakaryocyte, Giemsa x 250, Insert: >15% sideroblasts in Berlin blue stain, (b) MDS megaloblastoid, note hypercellular marrow with erythroid hyperplasia and maturation arrest, hyperplastic sinusoidal system, Giemsa × 250, (c) MDS proliferative, hypercellular marrow predominantly involving the granulocytic lineage, paratrabecular cluster of atypical megakaryocytes, Giemsa x 100, (d) MDS blastic, note the high percentage of various blast cells (myeloblasts, erythroblasts, megakaryoblasts and undifferentiated blasts as shown in cytochemical 22

Y

4

(Fig. 10 continued) and immunocytologic reactions not illustrated), Giemsa x 250, (e) MDS hypoplastic, with increased fatty tissue, prominent sinusoids and extravasation of erythrocytes, Giemsa x 100, (f) MDS fibrotic, with coarse fibrosis and marked reduction of haematopoiesis. Insert: fibres composed of collagen type III, immunohistology, FITC, (g) MDS inflammatory, with edema, extravasation of erythrocytes and vasculitis, Giemsa x 100, (h) CMML, for comparison, with myelomouocytic infiltration and emperipolesis in a megakaryocyte, Giemsa x 250. 23

FIG. 11. Variable cellularity of the bone marrow, evident in biopsy sections: (a) subcortical zone and (b) central zone of hypoplasia, Gomori × 10.

24

L

11,

J .J

e

r

FlG. 13. Sequential biopsies in patients with MDS: (a) massive acute inflammatory reaction of the bone marrow under treatment with IL-3 0 4 0 ~tg/d), several micromegakaryocytes indicate the basic MDS, Giemsa x 400, (b) evolution of MDS proliferative to megakaryocytic myelosis, with a cluster of highly atypical megakaryocytes, Giemsa × 400, (c) gradual evolution of MDS hypoplastic to a hypocellular variant of smouldering AL, with small blasts loosely distributed between the fat cells, Giemsa x 100, (d) abrupt evolution of MDS blastic to a proliferative variant of A M L after treatment with low-dose Ara-C, some residual micromegakaryocytes. On attempts at aspiration, a dry tap caused by a concomitant fine fibrosis, Gomori x 400. 25

Classification of MDS

27 TABLE 1. BONE MARROW REPORT IN M D S

Aspirate cytology

NORMAL

DYSPLASTIC

FIG. 7. Schematic representation of architectural disorganization in MDS: on the left normal architecture of the bone marrow, with granulopoietic precursors at endosteal surface, erythrons and megakaryocytes around the central sinusoids. In MDS there is topographic distortion, with precursors of the three cell lines found in all marrow regions. There is no stromal organization.

--Erythrocytic lineage Erythroid hyperplasia (1), megaloblastoid appearance (2), multinuclearity, cytoplasmicvacuolization, nuclear budding and fragmentation, internuclear bridges, mitotic figures, sideroblasts, PAS-positive normoblasts (3), ring sideroblasts (>15%) (4). --Megakaryocytic lineage Micromegakaryocytes, large megakaryocytes with single or multiple small round nuclei (5), megakaryoblasts, pyknosis, mitotic figures, giant platelets. --Granulocytic lineage Granulopoietic hyperplasia (6), increased blast cells (7), paramyeloid cells (8), Auer rods (9), hypo- and hypergranularity (10), Pelger-Huet anomaly, persistence of cytoplasmic basophilia in mature cells, eosinophils with ring-shaped non-lobated nuclei and variable granularity. --Monocytic lineage Monoblasts and monocytes, sometimeswith multiple elongated lobes and azurophil granules, haemophagocytosis, iron-loaded macrophages.

Biopsy histology trabecular localization of dysplastic and pyknotic megakaryocytes. Groups of myeloid precursors in central marrow areas were a further important sign of dysplasia, the so-called ALIP: abnormal localization of immature precursors [18]. Erythroid islands with maturation arrest in both inter- and paratrabecular regions were further characteristic features of myelodysplasia (Fig. 8a-c). In addition, alterations of the microenvironment were consistently found together with ineffective haematopoiesis: these included areas of edema and extravasation of erythrocytes caused by disrupted sinusoids, vasculitic changes, fibrosis, often patchy, lymphoid nodules, increases in plasma cells, mast cells, lymphocytes, macrophages, and even haemophagocytosis (Figs 8 and 9). In some cases of MDS, inflammatory changes were the most prominent morphologic feature, with high percentages of plasma cells, mast cells and lymphocytes in smears of the corresponding bone marrow aspirates.

Morphologic classification of MDS All patients were grouped according to the FAB classification, using the findings in peripheral blood and bone marrow aspirates (Table 1). Chronic myelomonocytic leukaemia (CMML), being by definition a leukaemia, does not belong in the group of MDS regarded as pre-leukaemic conditions. Patients with CMML were therefore excluded. On the basis of results of both aspirate cytology and trephine histology (criteria listed in Table 1), the patients were also assigned to one of 7 morphologic types (the frequencies and median survivals in brackets) (Fig. 10).

--Cellularity Hypercellular (>50 vol% haematopoiesis) (11), normocellular (20-50 vol%) (12), hypocellular (