Path. Res. Pract. 186, 589-596 (1990)
Pro-Megakaryoblasts in Bone Marrow Tissue from Patients with Primary (Idiopathic) Osteo-Myelofibrosis (Agnogenic Myeloid Metaplasia)! An Immunomorphometric Study on Trephine Biopsies J. Thiele, S. Wagner, H.-P. Bertsch, S. Wienhold and R. Fischer Institutes of Pathology, University of Cologne
D. Dienemann and H. Stein Free University Berlin Steglitz, FRG
R. Zankovich First Clinic of Medicine, University of Cologne, FRG
SUMMARY An immunomorphometric study was performed on bone marrow biopsies from 40 patients with primary osteomyelofibrosis - OMF, (agnogenic myeloid metaplasia) by employment ofa monoclonal antibody against glycoprotein IlIa (Y2151) to determine the number of pro-megakaryoblasts. Specimens from 15 individuals without any hematological disorder served as controls. With reference to the pertinent literature on megakaryocyte precursors and following a pilot study on corresponding smears, in tissue sections pro-megakaryoblasts were characterized by a size of 42.1 ± 2.6 ftm 2 (diameter 7.5 ± 0.3 ftm). In comparison with controls, in OMF no relevant increase in the number of pro-megakaryoblasts per square and cubic millimeter bone marrow was evaluable. The relative frequency of these precursors was significantly reduced due to an increase in the total amount of conspicuously large and abnormal megakaryocytes. Statistical analysis failed to reveal any correlations between counts for pro-megakaryoblasts or the total number of Y2151 - positive megakaryocytic elements with the density of argyrophilic fibers (determined by morphometry) or the platelet values. Our findings imply that in OMF the marked increase in circulating progenitor cells ofthe megakaryocyte lineage may be generated by extramedullary, probably splenic hematopoiesis. Moreover, the evolution of medullary fibrosis is thought to be associated with the striking predominance of large atypical, possibly overaged and hyperpolyploid megakaryocytes and not with an increase in precursor cells. Introduction Primary (idiopathic) osteo-myelofibrosis with myeloid metaplasia (OMF) is a chronic myeloproliferative disorder (CMPD) due to a defect occurring at the level of the 1 Supported in part by a grant from the Deutsche Forschungsgemeinschaft (DFG-Th 390/1-1).
© 1990 by Gustav Fischer Verlag, Stuttgart
pluripotent hematopoietic stem cell which confers on it a growth advantage over its normal counterparts 1,33. The condition has various names among which are agnogenic myeloid metaplasia (AMM). The latter term is frequently used synonymously24, although it may also indicate a myelofibrosis secondary to polycythemia vera (P. vera), primary (essential) thrombocythemia (PTH) or chronic granulocytic leukemia (CGL). The clonal expansion of 0344-0338/90/0186-0589$3.50/0
590 . Thiele,]. et al.
single stem cells in OMF has been implied by appearance of increased numbers of pluripotent and lineage-restricted circulating progenitor cells9,12, 14, 17,27,29,30,32,33,50,60. Invitro results demonstrate that the hematopoietic stem cell involved in this disorder retains the potential for differentiation towards the megakaryocyte lineage and is capable of an increased and spontaneous formation of colony forming unit megakaryocytes - CFU-MK13,29,30,33,35.1t is now widely assumed that abnormal megakaryocyte proliferation in the bone marrow of patients with OMF plays an important role in fibrillogenesis 10,44. Atypical megakaryopoiesis, which is always a conspicuous feature in this disorder6,8,21,25,57, stimulates fibroblasts to proliferate and secrete collagen by release of a specific growth factor and platelet factor 4, which inhibits the collagenase activity7, 10, 11,31,38,53. An imbalance of increased formation and decreased degradation of collagen is therefore proposed as the basic mechanism leading to the development of medullary fibrosis 1o . To obtain further insights into the abnormalities of megakaryopoiesis in OMF we focused in this study on the determination of promegakaryoblasts in bone marrow tissue and possible correlations with the content of argyrophilic (reticulin and collagen) fibers and the platelet count. Recently, studies involving the identification of megakaryocyte precursor cells, even in routinely processed paraffin embedded trephine biopsies of the bone marrow became feasible by the employment of a monoclonal antibody against platelet glycoprotein IlIa, termed Table 1. Clinical and hematological features in primary osteomyelofibrosis (agnogenic myeloid metaplasia arising "de novo") at time of diagnosis respectively bone marrow biopsy in 40 patients Mean
S.D.
Range
Erythrocytes X 10 12/1
4.2
0.8
2.6-
Hemoglobin gldl
11.5
2.3
7.9- 17.1
Leukocytes x 109/1
12.6
9.8
1.0- 47.6
polymorphonuclear %
60.3
18.2
18.0- 95.0
myeloblasts %
1.4
2.0
0-
erythro-normoblasts %
2.6
2.8
0.0- 10.0
5.8
6.0
Thrombocytes x 109/1
652
427
15-1600
LDH
537
391
116-1700
LApl
94
87
0- 304
Spleen size2
6
6
0- 20
Liver size2
3
3
0- 12
1 Normal range 10-80. S.D. standard deviation.
2
Centimeter below costal margin
Y2/51 19 ,23. Because it has been shown that glycoprotein IlIa antibodies stain very immature stem cell derived megakaryocytes (pro- and megakaryoblasts) at about day 5 of culture58 ,59, this seems to be an appropriate method to characterize pro-megakaryoblasts in addition to more mature megakaryocytic elements.
Material and Methods Patients A total number of 40 patients (19 male/21 female; median age 66 years) were studied. These presented with OMF at all stages of the disease according to hematological and particularly bone marrow findings 43 • Since, with informed consent biopsies were performed on admission, a relatively high number (26 of 40 cases) revealed so-called early hyperplastic stages 6,8,21,24,25,43,56 frequently associated with thrombocytosis 6,24,56, but with an only minimal to slight degree of bone marrow fibrosis 6,8,25,43,56. In 9 of these cases repeated biopsies were performed at a minimal interval of 6 months, and evolution into advanced OMF could be observed. The pertinent data regarding our patients are given in Table 1. No patient had a history of a preceding CMPD, i.e. P. vera, PTH, or CGL and no other identifiable explanation for bone marrow fibrosis. Moreover, no patient had received any therapy, particularly no cytostatics, splenectomy or transfusion of packed cells before the study, and exclusively all cases had been seen the first time for a hematological disorder. Bone marrow specimens from 15 individuals (6 male/9 female, median age 50 years) without any hematological disorder and a thrombocyte count within the normal range (140-345 x 109/1) served as controls.
Bone Marrow Biopsies Representative trephine biopsies of the bone marrow (mean size 21 x 2 mm) were performed from the posterior iliac crest34 of patients as initial examination. Fixation was done in an aldehyde solution for 12 to 48 hours (2 ml 25% glutardialdehyde, 3 ml 37% formaldehyde, 1.58 g calcium acetate and distilled water per 100 ml) and further processing included decalcification for 3-4 days in 10% buffered EDTA, pH 7.4, paraffin embedding and employment of several staining techniques54 • These included Giemsa, PAS (periodic acid Schiff) reaction and the silver impregnation method after Gomori.
Immunostaining with Y2/S1 The monoclonal antibody Y2/51, which is directed against platelet glycoprotein IlIa 19,23, was obtained by courtesy of D. Y. Mason, Oxford. Before staining procedures all slides of the paraffin embedded marrow specimens were predigested with pronase (1 mg per 1 ml tris buffered saline) for approximately 30 min at 37.5 0c. Thereafter samples were stained according to the alkaline phosphataseanti-alkaline phosphatase (APAAP) method 13 using neo-fuchsin as the alkaline phosphate substrate55 .
Immunomorphometry Following immunostaining with Y2/51 morphometric evaluation was performed by a manual optic planimeter (MOPA-M01-Kontron) with a standard program set (Kontron software) on large trephine biopsies with an artifact-free marrow area of 7.53 ± 3.8 mm2 • Frequency of megakaryocytes: Count for this cell line per square millimeter was done at 500x magnification by calculation
Pro-Megakaryoblasts in Primary Myelofibrosis . 591 of the evaluable marrow area of the trephine biopsy and the total number of the corresponding glycoprotein IlIa (Y2/51) - positive elements. By this method exclusively nucleated megakaryocytes were regarded, i.e. no pyknotic - degenerative forms or naked (bare) nuclei and no (a-nuclear) cytoplasmic fragments, in order to avoid confusion with giant platelets. In bone marrow specimens of our 40 patients with OMF the total number of counted megakaryocytes was 7,424. Identification of pro-megakaryoblasts: Following previous studies on the normal megakaryopoiesis and its precursor cells, pro-megakaryoblasts were characterized as mono-nuclear lymphoid-like cells5,42,46,52,58,59. These showed a striking variety of reported diameters ranging from 7-14 f.lm on smear preparations of human bone marrow, stem cell derived cultures or enriched suspensions of this cell fraction 37,42,46. For recognition of these very immature megakaryocytic elements we performed a pilot study on smears of sternal aspirates from several control patients stained with Y2/51. This preliminary investigation disclosed that the smallest elements clearly identifiable as members of the megakaryocyte series, i.e. pro-megakaryoblasts5 revealed a diameter of 9.3 ± 1.8 f.lm (size 61.7 ± 14.8 f.lm 2) and a nearly round cell perimeter and nucleus (size 26.2 ± 15.4 f.lm 2, diameter 5.7 ± 1.3 f.lm). Noteworthy was that these measurements concurred with the size (diameter 8-10 f.lm) of stem cell derived very early megakaryocyte precursors described after employment of the glycoprotein IlIa staining method 59 . Accordingly, in sections of normal marrow tissue (control specimens) pro-megakaryoblasts were defined as Y2/51-positive nucleated elements with a size less than 50 f.lm 2 (diameter < 8 f.lm), a round nucleus (size < 20 f.lm 2 and diameter < 5.5 f.lm) displaying a dispersed chromatin pattern and frequently one or two prominent nucleoli and a small rim of cytoplasm (see also Fig. 2c-e). To avoid an erroneous counting of peripheral sections of more mature megakaryocytes or hypoploid micromegakaryocytes, no elements were regarded showing irregular shapes of cells or nuclei (form perimeter> 0.84 respectively 0.87 - see below) and a clumping or margination of chromatin. Calculation of the number of pro-megakaryoblasts and the content of argyrophilic fibers: To determine the immature small as well as medium-sized to large megakaryocytes, i.e. stages I to IV5 within the bone marrow tissue, the total area of the trephine biopsy was divided into five segments of the same size. More than 20 randomly selected Y2/51-positive megakaryocytic elements were measured in each field at a magnification of 1250x with calculation of morphometric variables. The total number of 100 megakaryocytic elements per biopsy proved to be high enough for the characterization of pro-megakaryoblasts (see above). Quantification of pro-megakaryoblasts per unit of bone marrow tissue volume (mm 3 ) was performed by employment of a previously described algorithm 15. The form perimeter or circular deviation (CD) of the megakaryocyte precursors and their nuclei was defined as CD = 4n NO (C = circumference and A = area), giving the value 1.00 for a circular shape and a lower factor indicating an ellipsoid outline or increased irregularity. The reticulin and collagen fiber content was determined following silver impregnation (Gomori's stain) by counting the number of intersections (i) with the lines of a grid ocular at a magnification of 500x in 20 randomly selected trabeculae-free fields (equalling 1.14 mm 2). The area covered by fat cells was subtracted and the density of argyrophilic fibers expressed as number of intersections per square millimeter fat cell-free hematopoietic tissue (i/mm 2).
Statistical Evaluation The statistical analysis included the computation of correlation coefficients between the number of pro-megakaryoblasts and the
total count for megakaryocytes with the density of argyrophilic (reticulin plus collagen) fibers and the platelet values. For calculating the correlation coefficients we used the method of Pearson 20.
Results An overview on bone marrow tissue stained by antiglycoprotein lIla revealed a distinctive reaction of all megakaryocytic elements and thrombocytes (Figs. 1a-d; 2a-e). In comparison with control specimens (Fig. 1a) the number of positively reacting megakaryocytes was significantly increased in OMF, particularly in those specimens displaying an early hyperplastic stage (Fig. 1b). Advanced stages of OMF were characterized by conspicuous fibroosteosclerotic changes and a prominent clustering of megakaryocytes along sinusoidal structures (Fig. 1c) or adjacent to the newly formed endophytic bone (Fig. 1d). In addition to groupings of pleomorphic and often bizarre appearing large megakaryocytes (Fig. 2a, b), infrequently very small Y2/51-positive cells could be observed intermingled with other hematopoietic elements (Fig. 2c-e). These cells were identified as pro-megakaryoblasts and showed a size of 42.1 ± 2.6 ftm 2 (diameter 7.5 ± 0.3 ftm) with a relatively large nucleus (size 16.6 ± 3.2 ftm 2, diameter 4.9 ± 0.5 ftm) and a nuclear cytoplasmic ratio of 0.45 ± 0.04. The overall size distribution of all Y2/51-positive megakaryocytic elements in the bone marrow tissue of the control group and our cohort of 40 patients with OMF are given in Fig. 3. In OMF large to giant megakaryocytes with a size exceeding 500 ftm 2 occurred in more than 20% in contrast to less than 10% in the normal marrow. Due to this predominance of large atypical, probably overaged and hyperpolyploid megakaryocytes (Fig. 2a, b), a shift to the right could be encountered in specimens showing OMF. A detailed morphometric evaluation failed to disclose significantly increased numbers of precursor cells in OMF, when compared with samples from control patients (Table 2). Only 3 of 40 patients with OMF had a pro-megakaryoblast count in excess of the upper limit of the normal value. In congruence with the marked increase in the total megakaryocyte count, the relative frequency of pro-megakaryoblasts was also reduced. As could be expected, the density of argyrophilic (reticulin and collagen) fibers proved to be conspicuously greater in OMF cases (Table 2). Statistical analysis could not reveal any correlations between number of pro-megakaryoblasts and total megakaryocyte count with the density of fibers or the corresponding value for platelets. Discussion Our immunomorphometric study on marrow tissue from patients with OMF extends results which were derived from in-vitro cultures of megakaryocyte progenitor cells from peripheral blood 9 , 12, 14, 26,29, 30, 33 and more important, also from the bone marrow 12 ,29. In these
592 . Thiele, J. et al.
Pro-Megakaryoblasts in Primary Myelofibrosis· 593
reports an expansion of the precursor pool of this lineage has been found in the majority of cases investigated. Similar findings have been described in P. vera and particularly in PTH14,26,29,32,33,36,37,39,4o,47. In these disorders spontaneous megakaryocyte colony formation was observed under suboptimal culture conditions or in the absence of any added stimulator9,29,3o,33,36,39,4o,47. However, patients with OMF having undergone splenectomy did not reveal an increased number of CFU-Mk in circulation30,32,3s. On the other hand, following removal of the spleen a spontaneous recovery in the amount of committed progenitor cells of the granulocyte - monocyte and erythroid lineages has been noticed in the peripheral blood after a short post-operative reduction s1 . For this
reason it was not entirely clear whether the enhancement of circulating megakaryocyte precursors was generated by the extramedullary hematopoiesis in the spleen as a site of colony forming cell production in OMF16. In this context our finding of a normal amount of pro-megakaryoblasts in the bone marrow implied that the increase in precursor cells in the peripheral blood may probably originate from an enhanced egress of these elements from the splenic pool. In several patients with OMF a considerable increase in the spontaneous growth of megakaryocyte colonies from bone marrow derived stem cells was reported 12,29, but there was no correlation detectable between the number of CFU-MK in the marrow and peripheral blood. One explanation for the increased colony formation of medullary progenitor
Fig. 2a-e. Mature megakaryocytes and pro-megakaryoblasts in bone marrow tissue of patients with primary osteo-myelofibrosis (agnogenic myeloid metaplasia arising "de novo") following immunostaining with an antibody against platelet glycoprotein lIla. Particularly in the early hyperplastic stages there are groupings of large and pleomorphic megakaryocytes containing a multi-lobulated nucleus surrounded by an extended veil-like portion of cytoplasm (a, b). For comparison with a large mature megakaryocyte (b), small (diameter 6.3 ~m - c), large (diameter 8.0 ~m - d) and medium sized (diameter 7.2 ~m - e) pro-megakaryoblasts (arrow heads) are shown. a - x 280; b - x 650. ... Fig. 1a-d. Survey of bone marrow tissue following immunostaining with an antibody against platelet glycoprotein lIla. Normal control specimen (a) in comparison with primary osteo-myelofibrosis (agnogenic myeloid metaplasia arising "de novo") in an initial hyperplastic (b), an early fibro-osteosclerotic (c) and a terminal, grossly osteosclerotic (d) stage. There are numerous large, often bizarre looking megakaryocytes in the early stage (b) frequently lying adjacent to dilated vascular structures (arrows in c) or clustered at the peritrabecular area of the the osteosclerotic bone tissue (d). a-d x 150.
594 . Thiele, J. et al.
Table 2. Immunomorphometric features of bone marrow tissue in controls and patients with primary osteo-myelofibrosis (agnogenic myeloid metaplasia) following anti-glycoprotein IlIa staining for the identification of megakaryocyte precursor cells Primary osteomyelofibrosis 40
Controls 15
n
± 29.9
Megakaryocytes total 24.4 ± 5.3 per mm 2
79.9
Pro-megakaryoblasts 1.6 ± 1.2 per mm 2 (range 0.3-5.0)
2.6 ± 3.3 (range 0.2-9.2) 3.1 ± 2.8
% of megakaryocytes 6.2 ± 4.5 per mm 3
140 (range 40-230)
170 (range 10-1200)
Density of fibers (i/mm 2 x 102)
19.6 ± 7.9
86.3 ± 23.5
Adipose tissue %
50.8 ± 8.7
21.0 ± 14.6
900
n
CONTROLS O/'o1F
... ...
600
... ,
,,
300
o
400
" ...
......
... ...
' ................
600
--800
Fig. 3. Size distribution of nucleated megakaryocytic elements in bone marrow tissue calculated from normal control specimens and patients with primary osteo-myelofibrosis (agnogenic myeloid metaplasia arising "de novo") following immunostaining with an antibody against platelet glycoprotein IlIa. In comparison with the controls there is a significant tendency for larger cell forms of this lineage (shift to the right) in primary osteomyelofibrosis.
cells may be an interaction of accessory cells from the bone marrow microenvironment, which may be present in the culture dish. Accessory cells have been assumed to playa role in hematopoietic proliferation, mediated by release of either stimulating or conversely inhibitory factors 29 . However, it should be mentioned that several patients involved in these cell culture studies on circulating megakaryocyte progenitors in CMPDs had previously received chemotherapy30,32,37 which is known to inhibit spontaneous colony formation 35 . In congruence with our finding of a normal amount of pro-megakaryoblasts in the bone marrow of patients presenting with various stages of OMF (Table 1, Fig. 1 b-d), the relative frequency was conspicuously smaller than in the control specimens (Table 2). This result
was certainly related to the absolute increase in megakaryocytes (counts per square or cubic millimeter) and the obvious predominance of larger, apparently more mature and atypical elements of this series (Fig. 2a, b; Fig. 3). The exact amount of medullary megakaryocyte precursors should be of a special interest, because of the role of this cell line in the development of fibrosis. According to a model proposed by several authors 10,28,44 abnormal megakaryocytes and platelets die within the marrow and release certain factors into the intracellular space. There are two principal factors involved in this process: a growth factor discharged by degraded megakaryocytes and platelet factor 4, which in a combined action conversely generate marrow fibrosis 7,1O,1l,31,38,53. This hypothesis on medullary fibrosis was supported by morphometric measurements in patients with OMF. As could be shown, a two-fold increase in fiber density was significantly associated with features consistent with an augmented pleomorphism of megakaryocytes and a higher amount of bare (pyknotic-naked) nuclei, i.e. degenerative elements of this celllineage57 . In consideration of this model, the failure to detect any relevant correlations between density of fibers and numbers of pro-megakaryoblasts was remarkable. In order to explain this finding one has to take into account that probably only overaged-degenerated and mature platelet shedding megakaryocytes release these mediators into the myeloid stroma, which is an essential requirement for the evolution of marrow fibrosis1o,28,44. It is tempting to speculate that precursors or immature cells of the megakaryopoiesis apparently were not able to synthesize these factors. Consequently a predominance of the largesized, possibly hypcrpolyploid subpopulation of this cell line was observahle, 111 addition to many degenerated forms as pyknotic (naked) nuclei57 . However, these denuded nuclei could not be assessed in the present study for their obvious lack of immunoreactivity. In-vitro studies have shown that interferon alpha has an antiproliferative capacity on myeloid precursor cells 18 ,22, 48. In patients with OMF, growth of circulating hematopoietic progenitors was suppressed in a dose-dependent manner by this reagent9,48. Noticeable was that no resolution of the bone marrow fibrosis could be achieved by this therapy2,49. On the other hand, chemotherapy with busulfan and 6thioguanin did not result in a significant reduction of megakaryocyte proliferation in OMF, but in a decrease of medullary fibrosis 45 . The assumed mechanism of action accomplished by cytotoxic therapy in the"se patients was therefore suggested to include a reduction in the synthesis and/or release of platelet - derived factors by the mature megakaryocytes, but particularly platelet factor 4 which inhibits collagenase activity28, 44. Following a quantitation of bone marrow fiber production in OMF no significant relationships between density of fibers and platelet count could be assessed 3,57. Because it is known that an enlarged spleen can pool a vast amount of the marrow platelets, this finding was not surprising. Consequently in P. vera and PTH there were no interactions calculable between counts for megakaryocytes and the corresponding platelet values 4,41. Determination of the thrombocyte production rates, degree of megakaryocyte
Pro-Megakaryoblasts in Primary Myelofibrosis . 595
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36 Juvonen E, Partanen S, Ruutu T (1986) Colony formation by megakaryocytic progenitors in essential thrombocythaemia. Br J Haematol 66: 161-164 37 Juvonen E, Partanen S, lkkala E, Ruutu T (1988) Megakaryocytic colony formation in polycythaemia vera and secondary erythrocytosis. Br J Haematol 69: 441-444 38 Kaplan KL, Chao FC, Stiles CD, Antoniades HN, Scher CD (1979) Platelet alpha-granules contain a growth factor for fibroblasts. Blood 53: 1043-1052 39 Kimura H, Ishibashi T, Sato T, Matsuda S, Uchida T, Kariyone S (1987) Megakaryocytic colony formation (CFU-Meg) in essential thrombocythaemia: quantitative and qualitative abnormalities of bone marrow CFU-Meg. Am J Hematol 24: 23-30 40 Komatsu N, Suda T, Sakata Y, Eguchi M, Kaji K, Saito M, Miura Y (1986) Megakaryocytopoiesis in-vitro of patients with essential thrombocythaemia: effect of plasma and serum on megakaryocytic colony formation. Br J Haematol 64: 241-248 41 Kutti J, Ridell B, Weinfeld A (1973) The relation of thrombokinetics to bone marrow megakaryocytes and to the size of the spleen in polycythaemia vera. Scand J Haematol 10: 88-95 42 Levene RB, Williams NT, Lamaziere JMD, Rabellino EM (1987) Human megakaryocytes: IV. Growth and characterization of clonable megakaryocyte progenitors in agar. Exp Hematol 15: 181-189 43 Lewis SM (1985) Myelofibrosis: Historical perspective. In: Lewis SM (Ed.) Myelofibrosis. Pathophysiology and Clinical Management, pp. 1-13. Marcel Dekker, New York· 44 Mac Carthy DM (1985) Fibrosis in the bone marrow: content and causes. Br J Haematol 59: 1-7 45 Manoharan A, Pitney WR (1984) Chemotherapy resolves symptoms and reverses marrow fibrosis in myelofibrosis. Scand J Haematol 33: 453-459 46 Mazur EM, Hoffman R, Chasis J, Marchesi S, Bruno E (1981) Immunofluorescent identification of human megakaryocyte colonies using an antiplatelet glycoprotein antiserum. Blood 57: 277-286 47 Mazur EM, Cohen JL, Bogart L (1988) Growth characteristics of circulating hematopoietic progenitor cells from patients with essential thrombocythemia. Blood 71: 1544-1550 48 Neumann HA, Fauser AA (1982) Effect of interferon on pluripotential hematopoietic progenitors (CFU-GEMM) derived from human bone marrow. Exp Hematoll0: 587-590 49 Parmeggiani L, Ferrant A, Rodhain J, Michaux JL, Sokal G (1987) Alpha interferon in the treatment of symptomatic myelofibrosis with myeloid metaplasia. Eur J Haematol 39: 228-232
50 Partanen S, Ruutu T, Vuopio P (1982) Circulating haematopoietic progenitors in myelofibrosis. Scand J Haematol 29: 325-330 51 Partanen S, Ruutu T, Juvonen E, Pantzar P (1986) Effect of splenectomy on circulating haematopoietic progenitors in myelofibrosis. Scand J Haematol 37: 87-90 52 Rabellino EM, Levene RB, Leung LLK, Nachman RL (1981) Human megakaryocytes: II. Expression of platelet proteins in early marrow megakaryocytes. J Exp Med 154: 88-100 53 Ross R, Vogel A (1978) The platelet derived-growth factor. Cell 14: 203-210 54 Schaefer HE (1984) How to fix, decalcify and stain paraffin embedded bone marrow biopsies. In: Lennert K, Hiibner K (Eds.) Pathology of the Bone Marrow, pp. 6-9. Gustav Fischer, Stuttgart-New York 55 Stein H, Gatter KC, Asbahr H, Mason DY (1985) Methods in laboratory investigation. Use of freeze-dried paraffin-embedded sections for immunohistologic staining with monoclonal antibodies. Lab Invest 52: 676-683 56 Thiele J, Zankovich R, Steinberg T, Fischer R, Diehl V (1989) Agnogenic myeloid metaplasia (AMM) - correlation of bone marrow lesions with laboratory data: a longitudinal clinico-pathological study on 114 patients. Hematol Oncol 7: 1-17 57 Thiele J, Hoeppner B, Zankovich R, Fischer R (1989) Histomorphometry of bone marrow biopsies in primary osteomyelofibrosis/-sclerosis (so-called agnogenic myeloid metaplasia) - correlations between laboratory and morphological features. Virchows Archiv A (Pathol Anat) 415: 191-202 58 Vainchenker W, Deschamps JF, Bastian JM, Guichard J, Titeux M, Breton-Gorius J, McMichael AJ (1982) Two monoclonal antibodies as markers of human megakaryocyte maturation: immunofluorescent staining and peroxidase detection in megakaryocyte colonies and in vivo cells from normal and leukemic patients. Blood 59: 514-521 59 Vinci G, Tabilio A, Deschamps JF, Van Haeke D, Henri A, Guichard J, Tetteroo P, Lansdorp PM, Hercend T, Vainchenker W, Breton-Gorius J (1984) Immunological study of in-vitro maturation of human megakaryocytes. Br J Haematol 56: 589-605 60 WangJC, Cheung CP, Ahmed F, Steier W, Tobin MS (1983) Circulating granulocyte and macrophage progenitor cells in primary and secondary myelofibrosis. Br J Haematol 54: 301-307
Received November 24, 1989 . Accepted December 5, 1989
Key words: Megakaryocyte precursors - Osteomyelofibrosis - Morphometry - Anti-glycoprotein IlIa - Bone marrow biopsies Juergen Thiele, M.D., Institute of Pathology, University of Cologne, Joseph-Stelzmann-Str. 9, D-5000 Cologne 41, FRG
Pro-Megakaryoblasts in Bone Marrow Tissue from Patients with Primary (Idiopathic) Osteo-Myelofibrosis (Agnogenic Myeloid Metaplasia)! An Immunomorphometric Study on Trephine Biopsies J. Thiele, S. Wagner, H.-P. Bertsch, S. Wienhold and R. Fischer Institutes of Pathology, University of Cologne
D. Dienemann and H. Stein Free University Berlin Steglitz, FRG
R. Zankovich First Clinic of Medicine, University of Cologne, FRG
SUMMARY An immunomorphometric study was performed on bone marrow biopsies from 40 patients with primary osteomyelofibrosis - OMF, (agnogenic myeloid metaplasia) by employment ofa monoclonal antibody against glycoprotein IlIa (Y2151) to determine the number of pro-megakaryoblasts. Specimens from 15 individuals without any hematological disorder served as controls. With reference to the pertinent literature on megakaryocyte precursors and following a pilot study on corresponding smears, in tissue sections pro-megakaryoblasts were characterized by a size of 42.1 ± 2.6 ftm 2 (diameter 7.5 ± 0.3 ftm). In comparison with controls, in OMF no relevant increase in the number of pro-megakaryoblasts per square and cubic millimeter bone marrow was evaluable. The relative frequency of these precursors was significantly reduced due to an increase in the total amount of conspicuously large and abnormal megakaryocytes. Statistical analysis failed to reveal any correlations between counts for pro-megakaryoblasts or the total number of Y2151 - positive megakaryocytic elements with the density of argyrophilic fibers (determined by morphometry) or the platelet values. Our findings imply that in OMF the marked increase in circulating progenitor cells ofthe megakaryocyte lineage may be generated by extramedullary, probably splenic hematopoiesis. Moreover, the evolution of medullary fibrosis is thought to be associated with the striking predominance of large atypical, possibly overaged and hyperpolyploid megakaryocytes and not with an increase in precursor cells. Introduction Primary (idiopathic) osteo-myelofibrosis with myeloid metaplasia (OMF) is a chronic myeloproliferative disorder (CMPD) due to a defect occurring at the level of the 1 Supported in part by a grant from the Deutsche Forschungsgemeinschaft (DFG-Th 390/1-1).
© 1990 by Gustav Fischer Verlag, Stuttgart
pluripotent hematopoietic stem cell which confers on it a growth advantage over its normal counterparts 1,33. The condition has various names among which are agnogenic myeloid metaplasia (AMM). The latter term is frequently used synonymously24, although it may also indicate a myelofibrosis secondary to polycythemia vera (P. vera), primary (essential) thrombocythemia (PTH) or chronic granulocytic leukemia (CGL). The clonal expansion of 0344-0338/90/0186-0589$3.50/0
590 . Thiele,]. et al.
single stem cells in OMF has been implied by appearance of increased numbers of pluripotent and lineage-restricted circulating progenitor cells9,12, 14, 17,27,29,30,32,33,50,60. Invitro results demonstrate that the hematopoietic stem cell involved in this disorder retains the potential for differentiation towards the megakaryocyte lineage and is capable of an increased and spontaneous formation of colony forming unit megakaryocytes - CFU-MK13,29,30,33,35.1t is now widely assumed that abnormal megakaryocyte proliferation in the bone marrow of patients with OMF plays an important role in fibrillogenesis 10,44. Atypical megakaryopoiesis, which is always a conspicuous feature in this disorder6,8,21,25,57, stimulates fibroblasts to proliferate and secrete collagen by release of a specific growth factor and platelet factor 4, which inhibits the collagenase activity7, 10, 11,31,38,53. An imbalance of increased formation and decreased degradation of collagen is therefore proposed as the basic mechanism leading to the development of medullary fibrosis 1o . To obtain further insights into the abnormalities of megakaryopoiesis in OMF we focused in this study on the determination of promegakaryoblasts in bone marrow tissue and possible correlations with the content of argyrophilic (reticulin and collagen) fibers and the platelet count. Recently, studies involving the identification of megakaryocyte precursor cells, even in routinely processed paraffin embedded trephine biopsies of the bone marrow became feasible by the employment of a monoclonal antibody against platelet glycoprotein IlIa, termed Table 1. Clinical and hematological features in primary osteomyelofibrosis (agnogenic myeloid metaplasia arising "de novo") at time of diagnosis respectively bone marrow biopsy in 40 patients Mean
S.D.
Range
Erythrocytes X 10 12/1
4.2
0.8
2.6-
Hemoglobin gldl
11.5
2.3
7.9- 17.1
Leukocytes x 109/1
12.6
9.8
1.0- 47.6
polymorphonuclear %
60.3
18.2
18.0- 95.0
myeloblasts %
1.4
2.0
0-
erythro-normoblasts %
2.6
2.8
0.0- 10.0
5.8
6.0
Thrombocytes x 109/1
652
427
15-1600
LDH
537
391
116-1700
LApl
94
87
0- 304
Spleen size2
6
6
0- 20
Liver size2
3
3
0- 12
1 Normal range 10-80. S.D. standard deviation.
2
Centimeter below costal margin
Y2/51 19 ,23. Because it has been shown that glycoprotein IlIa antibodies stain very immature stem cell derived megakaryocytes (pro- and megakaryoblasts) at about day 5 of culture58 ,59, this seems to be an appropriate method to characterize pro-megakaryoblasts in addition to more mature megakaryocytic elements.
Material and Methods Patients A total number of 40 patients (19 male/21 female; median age 66 years) were studied. These presented with OMF at all stages of the disease according to hematological and particularly bone marrow findings 43 • Since, with informed consent biopsies were performed on admission, a relatively high number (26 of 40 cases) revealed so-called early hyperplastic stages 6,8,21,24,25,43,56 frequently associated with thrombocytosis 6,24,56, but with an only minimal to slight degree of bone marrow fibrosis 6,8,25,43,56. In 9 of these cases repeated biopsies were performed at a minimal interval of 6 months, and evolution into advanced OMF could be observed. The pertinent data regarding our patients are given in Table 1. No patient had a history of a preceding CMPD, i.e. P. vera, PTH, or CGL and no other identifiable explanation for bone marrow fibrosis. Moreover, no patient had received any therapy, particularly no cytostatics, splenectomy or transfusion of packed cells before the study, and exclusively all cases had been seen the first time for a hematological disorder. Bone marrow specimens from 15 individuals (6 male/9 female, median age 50 years) without any hematological disorder and a thrombocyte count within the normal range (140-345 x 109/1) served as controls.
Bone Marrow Biopsies Representative trephine biopsies of the bone marrow (mean size 21 x 2 mm) were performed from the posterior iliac crest34 of patients as initial examination. Fixation was done in an aldehyde solution for 12 to 48 hours (2 ml 25% glutardialdehyde, 3 ml 37% formaldehyde, 1.58 g calcium acetate and distilled water per 100 ml) and further processing included decalcification for 3-4 days in 10% buffered EDTA, pH 7.4, paraffin embedding and employment of several staining techniques54 • These included Giemsa, PAS (periodic acid Schiff) reaction and the silver impregnation method after Gomori.
Immunostaining with Y2/S1 The monoclonal antibody Y2/51, which is directed against platelet glycoprotein IlIa 19,23, was obtained by courtesy of D. Y. Mason, Oxford. Before staining procedures all slides of the paraffin embedded marrow specimens were predigested with pronase (1 mg per 1 ml tris buffered saline) for approximately 30 min at 37.5 0c. Thereafter samples were stained according to the alkaline phosphataseanti-alkaline phosphatase (APAAP) method 13 using neo-fuchsin as the alkaline phosphate substrate55 .
Immunomorphometry Following immunostaining with Y2/51 morphometric evaluation was performed by a manual optic planimeter (MOPA-M01-Kontron) with a standard program set (Kontron software) on large trephine biopsies with an artifact-free marrow area of 7.53 ± 3.8 mm2 • Frequency of megakaryocytes: Count for this cell line per square millimeter was done at 500x magnification by calculation
Pro-Megakaryoblasts in Primary Myelofibrosis . 591 of the evaluable marrow area of the trephine biopsy and the total number of the corresponding glycoprotein IlIa (Y2/51) - positive elements. By this method exclusively nucleated megakaryocytes were regarded, i.e. no pyknotic - degenerative forms or naked (bare) nuclei and no (a-nuclear) cytoplasmic fragments, in order to avoid confusion with giant platelets. In bone marrow specimens of our 40 patients with OMF the total number of counted megakaryocytes was 7,424. Identification of pro-megakaryoblasts: Following previous studies on the normal megakaryopoiesis and its precursor cells, pro-megakaryoblasts were characterized as mono-nuclear lymphoid-like cells5,42,46,52,58,59. These showed a striking variety of reported diameters ranging from 7-14 f.lm on smear preparations of human bone marrow, stem cell derived cultures or enriched suspensions of this cell fraction 37,42,46. For recognition of these very immature megakaryocytic elements we performed a pilot study on smears of sternal aspirates from several control patients stained with Y2/51. This preliminary investigation disclosed that the smallest elements clearly identifiable as members of the megakaryocyte series, i.e. pro-megakaryoblasts5 revealed a diameter of 9.3 ± 1.8 f.lm (size 61.7 ± 14.8 f.lm 2) and a nearly round cell perimeter and nucleus (size 26.2 ± 15.4 f.lm 2, diameter 5.7 ± 1.3 f.lm). Noteworthy was that these measurements concurred with the size (diameter 8-10 f.lm) of stem cell derived very early megakaryocyte precursors described after employment of the glycoprotein IlIa staining method 59 . Accordingly, in sections of normal marrow tissue (control specimens) pro-megakaryoblasts were defined as Y2/51-positive nucleated elements with a size less than 50 f.lm 2 (diameter < 8 f.lm), a round nucleus (size < 20 f.lm 2 and diameter < 5.5 f.lm) displaying a dispersed chromatin pattern and frequently one or two prominent nucleoli and a small rim of cytoplasm (see also Fig. 2c-e). To avoid an erroneous counting of peripheral sections of more mature megakaryocytes or hypoploid micromegakaryocytes, no elements were regarded showing irregular shapes of cells or nuclei (form perimeter> 0.84 respectively 0.87 - see below) and a clumping or margination of chromatin. Calculation of the number of pro-megakaryoblasts and the content of argyrophilic fibers: To determine the immature small as well as medium-sized to large megakaryocytes, i.e. stages I to IV5 within the bone marrow tissue, the total area of the trephine biopsy was divided into five segments of the same size. More than 20 randomly selected Y2/51-positive megakaryocytic elements were measured in each field at a magnification of 1250x with calculation of morphometric variables. The total number of 100 megakaryocytic elements per biopsy proved to be high enough for the characterization of pro-megakaryoblasts (see above). Quantification of pro-megakaryoblasts per unit of bone marrow tissue volume (mm 3 ) was performed by employment of a previously described algorithm 15. The form perimeter or circular deviation (CD) of the megakaryocyte precursors and their nuclei was defined as CD = 4n NO (C = circumference and A = area), giving the value 1.00 for a circular shape and a lower factor indicating an ellipsoid outline or increased irregularity. The reticulin and collagen fiber content was determined following silver impregnation (Gomori's stain) by counting the number of intersections (i) with the lines of a grid ocular at a magnification of 500x in 20 randomly selected trabeculae-free fields (equalling 1.14 mm 2). The area covered by fat cells was subtracted and the density of argyrophilic fibers expressed as number of intersections per square millimeter fat cell-free hematopoietic tissue (i/mm 2).
Statistical Evaluation The statistical analysis included the computation of correlation coefficients between the number of pro-megakaryoblasts and the
total count for megakaryocytes with the density of argyrophilic (reticulin plus collagen) fibers and the platelet values. For calculating the correlation coefficients we used the method of Pearson 20.
Results An overview on bone marrow tissue stained by antiglycoprotein lIla revealed a distinctive reaction of all megakaryocytic elements and thrombocytes (Figs. 1a-d; 2a-e). In comparison with control specimens (Fig. 1a) the number of positively reacting megakaryocytes was significantly increased in OMF, particularly in those specimens displaying an early hyperplastic stage (Fig. 1b). Advanced stages of OMF were characterized by conspicuous fibroosteosclerotic changes and a prominent clustering of megakaryocytes along sinusoidal structures (Fig. 1c) or adjacent to the newly formed endophytic bone (Fig. 1d). In addition to groupings of pleomorphic and often bizarre appearing large megakaryocytes (Fig. 2a, b), infrequently very small Y2/51-positive cells could be observed intermingled with other hematopoietic elements (Fig. 2c-e). These cells were identified as pro-megakaryoblasts and showed a size of 42.1 ± 2.6 ftm 2 (diameter 7.5 ± 0.3 ftm) with a relatively large nucleus (size 16.6 ± 3.2 ftm 2, diameter 4.9 ± 0.5 ftm) and a nuclear cytoplasmic ratio of 0.45 ± 0.04. The overall size distribution of all Y2/51-positive megakaryocytic elements in the bone marrow tissue of the control group and our cohort of 40 patients with OMF are given in Fig. 3. In OMF large to giant megakaryocytes with a size exceeding 500 ftm 2 occurred in more than 20% in contrast to less than 10% in the normal marrow. Due to this predominance of large atypical, probably overaged and hyperpolyploid megakaryocytes (Fig. 2a, b), a shift to the right could be encountered in specimens showing OMF. A detailed morphometric evaluation failed to disclose significantly increased numbers of precursor cells in OMF, when compared with samples from control patients (Table 2). Only 3 of 40 patients with OMF had a pro-megakaryoblast count in excess of the upper limit of the normal value. In congruence with the marked increase in the total megakaryocyte count, the relative frequency of pro-megakaryoblasts was also reduced. As could be expected, the density of argyrophilic (reticulin and collagen) fibers proved to be conspicuously greater in OMF cases (Table 2). Statistical analysis could not reveal any correlations between number of pro-megakaryoblasts and total megakaryocyte count with the density of fibers or the corresponding value for platelets. Discussion Our immunomorphometric study on marrow tissue from patients with OMF extends results which were derived from in-vitro cultures of megakaryocyte progenitor cells from peripheral blood 9 , 12, 14, 26,29, 30, 33 and more important, also from the bone marrow 12 ,29. In these
592 . Thiele, J. et al.
Pro-Megakaryoblasts in Primary Myelofibrosis· 593
reports an expansion of the precursor pool of this lineage has been found in the majority of cases investigated. Similar findings have been described in P. vera and particularly in PTH14,26,29,32,33,36,37,39,4o,47. In these disorders spontaneous megakaryocyte colony formation was observed under suboptimal culture conditions or in the absence of any added stimulator9,29,3o,33,36,39,4o,47. However, patients with OMF having undergone splenectomy did not reveal an increased number of CFU-Mk in circulation30,32,3s. On the other hand, following removal of the spleen a spontaneous recovery in the amount of committed progenitor cells of the granulocyte - monocyte and erythroid lineages has been noticed in the peripheral blood after a short post-operative reduction s1 . For this
reason it was not entirely clear whether the enhancement of circulating megakaryocyte precursors was generated by the extramedullary hematopoiesis in the spleen as a site of colony forming cell production in OMF16. In this context our finding of a normal amount of pro-megakaryoblasts in the bone marrow implied that the increase in precursor cells in the peripheral blood may probably originate from an enhanced egress of these elements from the splenic pool. In several patients with OMF a considerable increase in the spontaneous growth of megakaryocyte colonies from bone marrow derived stem cells was reported 12,29, but there was no correlation detectable between the number of CFU-MK in the marrow and peripheral blood. One explanation for the increased colony formation of medullary progenitor
Fig. 2a-e. Mature megakaryocytes and pro-megakaryoblasts in bone marrow tissue of patients with primary osteo-myelofibrosis (agnogenic myeloid metaplasia arising "de novo") following immunostaining with an antibody against platelet glycoprotein lIla. Particularly in the early hyperplastic stages there are groupings of large and pleomorphic megakaryocytes containing a multi-lobulated nucleus surrounded by an extended veil-like portion of cytoplasm (a, b). For comparison with a large mature megakaryocyte (b), small (diameter 6.3 ~m - c), large (diameter 8.0 ~m - d) and medium sized (diameter 7.2 ~m - e) pro-megakaryoblasts (arrow heads) are shown. a - x 280; b - x 650. ... Fig. 1a-d. Survey of bone marrow tissue following immunostaining with an antibody against platelet glycoprotein lIla. Normal control specimen (a) in comparison with primary osteo-myelofibrosis (agnogenic myeloid metaplasia arising "de novo") in an initial hyperplastic (b), an early fibro-osteosclerotic (c) and a terminal, grossly osteosclerotic (d) stage. There are numerous large, often bizarre looking megakaryocytes in the early stage (b) frequently lying adjacent to dilated vascular structures (arrows in c) or clustered at the peritrabecular area of the the osteosclerotic bone tissue (d). a-d x 150.
594 . Thiele, J. et al.
Table 2. Immunomorphometric features of bone marrow tissue in controls and patients with primary osteo-myelofibrosis (agnogenic myeloid metaplasia) following anti-glycoprotein IlIa staining for the identification of megakaryocyte precursor cells Primary osteomyelofibrosis 40
Controls 15
n
± 29.9
Megakaryocytes total 24.4 ± 5.3 per mm 2
79.9
Pro-megakaryoblasts 1.6 ± 1.2 per mm 2 (range 0.3-5.0)
2.6 ± 3.3 (range 0.2-9.2) 3.1 ± 2.8
% of megakaryocytes 6.2 ± 4.5 per mm 3
140 (range 40-230)
170 (range 10-1200)
Density of fibers (i/mm 2 x 102)
19.6 ± 7.9
86.3 ± 23.5
Adipose tissue %
50.8 ± 8.7
21.0 ± 14.6
900
n
CONTROLS O/'o1F
... ...
600
... ,
,,
300
o
400
" ...
......
... ...
' ................
600
--800
Fig. 3. Size distribution of nucleated megakaryocytic elements in bone marrow tissue calculated from normal control specimens and patients with primary osteo-myelofibrosis (agnogenic myeloid metaplasia arising "de novo") following immunostaining with an antibody against platelet glycoprotein IlIa. In comparison with the controls there is a significant tendency for larger cell forms of this lineage (shift to the right) in primary osteomyelofibrosis.
cells may be an interaction of accessory cells from the bone marrow microenvironment, which may be present in the culture dish. Accessory cells have been assumed to playa role in hematopoietic proliferation, mediated by release of either stimulating or conversely inhibitory factors 29 . However, it should be mentioned that several patients involved in these cell culture studies on circulating megakaryocyte progenitors in CMPDs had previously received chemotherapy30,32,37 which is known to inhibit spontaneous colony formation 35 . In congruence with our finding of a normal amount of pro-megakaryoblasts in the bone marrow of patients presenting with various stages of OMF (Table 1, Fig. 1 b-d), the relative frequency was conspicuously smaller than in the control specimens (Table 2). This result
was certainly related to the absolute increase in megakaryocytes (counts per square or cubic millimeter) and the obvious predominance of larger, apparently more mature and atypical elements of this series (Fig. 2a, b; Fig. 3). The exact amount of medullary megakaryocyte precursors should be of a special interest, because of the role of this cell line in the development of fibrosis. According to a model proposed by several authors 10,28,44 abnormal megakaryocytes and platelets die within the marrow and release certain factors into the intracellular space. There are two principal factors involved in this process: a growth factor discharged by degraded megakaryocytes and platelet factor 4, which in a combined action conversely generate marrow fibrosis 7,1O,1l,31,38,53. This hypothesis on medullary fibrosis was supported by morphometric measurements in patients with OMF. As could be shown, a two-fold increase in fiber density was significantly associated with features consistent with an augmented pleomorphism of megakaryocytes and a higher amount of bare (pyknotic-naked) nuclei, i.e. degenerative elements of this celllineage57 . In consideration of this model, the failure to detect any relevant correlations between density of fibers and numbers of pro-megakaryoblasts was remarkable. In order to explain this finding one has to take into account that probably only overaged-degenerated and mature platelet shedding megakaryocytes release these mediators into the myeloid stroma, which is an essential requirement for the evolution of marrow fibrosis1o,28,44. It is tempting to speculate that precursors or immature cells of the megakaryopoiesis apparently were not able to synthesize these factors. Consequently a predominance of the largesized, possibly hypcrpolyploid subpopulation of this cell line was observahle, 111 addition to many degenerated forms as pyknotic (naked) nuclei57 . However, these denuded nuclei could not be assessed in the present study for their obvious lack of immunoreactivity. In-vitro studies have shown that interferon alpha has an antiproliferative capacity on myeloid precursor cells 18 ,22, 48. In patients with OMF, growth of circulating hematopoietic progenitors was suppressed in a dose-dependent manner by this reagent9,48. Noticeable was that no resolution of the bone marrow fibrosis could be achieved by this therapy2,49. On the other hand, chemotherapy with busulfan and 6thioguanin did not result in a significant reduction of megakaryocyte proliferation in OMF, but in a decrease of medullary fibrosis 45 . The assumed mechanism of action accomplished by cytotoxic therapy in the"se patients was therefore suggested to include a reduction in the synthesis and/or release of platelet - derived factors by the mature megakaryocytes, but particularly platelet factor 4 which inhibits collagenase activity28, 44. Following a quantitation of bone marrow fiber production in OMF no significant relationships between density of fibers and platelet count could be assessed 3,57. Because it is known that an enlarged spleen can pool a vast amount of the marrow platelets, this finding was not surprising. Consequently in P. vera and PTH there were no interactions calculable between counts for megakaryocytes and the corresponding platelet values 4,41. Determination of the thrombocyte production rates, degree of megakaryocyte
Pro-Megakaryoblasts in Primary Myelofibrosis . 595
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Received November 24, 1989 . Accepted December 5, 1989
Key words: Megakaryocyte precursors - Osteomyelofibrosis - Morphometry - Anti-glycoprotein IlIa - Bone marrow biopsies Juergen Thiele, M.D., Institute of Pathology, University of Cologne, Joseph-Stelzmann-Str. 9, D-5000 Cologne 41, FRG
