Chronic Myeloproliferative Disorders (CMPD)

Path. Res. Pract. 179, 131-186 (1984) ,

Diagnostic Seminar

Chronic Myeloproliferative Disorders (CMPD)

Rolf Burkhardt Abteilung fiir Knochenmarksdiagnostik, Medizin. Klinik Innenstadt der Universitat Miinchen Gesellschaft fOr Strahlen- und Umweltforschung mbH, Abteilung fOr Hamatomorphologie, Neuherberg bei Miinchen

Reiner Bartl Abteilung fOr Knochenmarksdiagnostik, Medizin. Klinik Innenstadt der Universitat Miinchen Gesellschaft fOr Strahlen- und Umweltforschung mbH, Abteilung fOr Hamatomorphologie, Neuherberg bei Miinchen

Klaus Jager Abteilung fOr Knochenmarksdiagnostik, Medizin. Klinik Innenstadt der Universitat Miinchen

Bertha Frisch Institute of Hematology, Tel-Aviv Municipal Governmental Medical Centre, and Sackler School of Medicine, Tel-Aviv University, Israel

Gerhard Kettner Medizinische Klinik Innenstadt der Universitat Miinchen

Georg Mahl Gesellschaft fOr Strahlen- und Umweltforschung mbH, Abteilung fOr Hamatomorphologie, Neuherberg bei Miinchen

Malte Sund Gesellschaft fOr Strahlen- und Umweltforschung mbH, MEDIS-Institut, Neuherberg bei Miinchen

I. General Aspects

A. Natural History

1. Definition and Nosology

Chronic myeloproliferative disorders (CMPD) are monoclonal neoplasias that arise from stem cells and expand primarily within the bone marrow, producing a surplus of one or more of the cell lines normally contributed to the peripheral blood by the bone marrow.

© 1984 by Gustav Fischer Verlag, Stuttgart

This overproduction sooner or later also involves parts of the skeleton inactive in the adult, as well as organs that were haematopoietic during the fetal life. The excess floods the blood with red blood corpuscles, granulocytes, or platelets as long as the neoplasia produces mobilizable elements, and the bone marrow remains capable of their emission. This increase causes disturbances of the larger vessels and capillaries due to obstruction or thrombosis, depending on the amount of erythrocytosis, leukocytosis,

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132 . R. Burkhardt, R. Bartl, K. Jiiger, B. Frisch, G. Kettner, G. Mahl and M. Sund

or thrombocythaemia. The functional deficiency of the cells may also perturb resistance against infection and haemostatic mechanisms, while the products of their degradation cause dysfunction of metabolism and renal excretion. Gout, amyloidosis, and renal diseases are among the more frequent consequences.

When the neoplasia develops into more immature or fibrotic stages, anaemia, granulocytopenia, or thrombocytopenia result in varying proportions, further modified by the rates of consumption and survival of these cells. These in turn may be affected by immune-haemolysis and -thrombocytolysis mainly when the CMPD is complicated by myelofibrosis. The expansion to extramedullary sites manifests itself most frequently as hepatosplenomegaly, though almost all organs may be affected. A very rare modification is represented by sarcomatous growth of myeloid tissue, mostly pleomorphic megakaryocytes, which may also involve the lymph nodes and serous membranes in the peritoneal and pleural regions76, 110, 14.

The diseases are found in all age groups, though mainly in the second half of life with a moderate to marked male predominance among all subgroups59 except for thrombocythaemia124. Because of similarity of the neoplastic myeloproliferation to normal haematopoiesis, and the slow propagation rate, the diagnosis is generally preceded by a prolonged oligo symptomatic period, unless detected by chance.

2. Course and Development

Two events closely connected with the myeloproliferative process modify the disease itself and its consequences to normal haematopoiesis:

The first is the change from one type into another differentiated one, or, more frequently, into a more immature type3, 9, 83, 94, 178. In both cases the haematologic symptoms change, in the latter into the fatal "blast crisis". The incidence of BC has been reported as 1-100% for PV16, 39, 71, 92, 132, 134, 217; as 18-95% for CML39, 50, 86, 89, 92,153,190; however much lesser for PT; data for this group are hardly comparable due to problems of defini-

In the current text the following abbreviations will be used: BC = blast crisis CMGM = chronic megakaryocytic granulocytic myelosis CML = chronic myeloid leukeamia IDP = interstitial deposits of platelets CMPD = chronic myeloproliferative disorders MixM = mixed cellular myelosis Meg = megakaryocytes MegM = megakaryocytic myelosis MF = myelofibrosis OMS = osteomyelosclerosis MPD-MF, respectively-OMS myelofibrosis/osteomyelo-sclerosis due to MPD non-MPD-MF/OMS = myelofibrosis osteomyelosclerosis due to cause other than MPD PT = primary thrombocythaemia PV = polycythaemia vera

tion39, 92, 100, 163, 175. The blast cells may be lymphoid as well as myeloblasts, erythroblasts, and megakaryoblasts; recent observations have shown that the lymphoid cells are B-cells, thus indicating an origin of the CMPD close to the beginning of haematopoietic differentiation6.

The second event consists of fibrotic bone marrow changes in close association with the neoplastic proliferation, sometimes including primitive bone formation with obstruction of the marrow sEaces. The frequency of MFI OMS is 5-52% for PV16, 3,39,70,92,134,183, 30-40% for CML39, 50, 58, 65, 92, 97,107, and 6-10% for PT39,92. Fibrosis may also involve extramedullary sites of the disease process21, 27, 102, 168,214,223.

Blastic crisis may occur together with fibrosis. In this case the syndrome of "acute myelosclerosis,,12, 15,20, 75, 137, 141, 142, 162,218 is presented, character-ized clinically by acute illness with anaemia or pancytopenia, occurrence of immature cells in the peripheral blood and minimal splenic enlargement. More regularly BC develops in MF and OMS after a prolonged course of these diseases72, 181, 182, with a frequency of 5_28%39,72, 92,181,182,183. In the uncomplicated cases of MFIOMS the fibrous tissue condenses to form a tight meshwork, leaving nests of immature and pleomorphic haematopoietic elements. These may also be found within some enlarged and sclerotic sinusoidal capillaries that have been left among the fibres. Fibrotic transformation shows many variations, among these a paucicellular sclerotic-edematous stage.

The fibrotic process is accompanied by centrifugal expansion of haematopoietic marrow and extramedullary haematopoiesis in the spleen, the liver, and, rarely, the lymph nodes71, 168, 204. The clinical signs of hepatosplenomegaly and anaemia with a "leukoerythroblastic blood picture" usually precede the final collapse of haematopiesis by months or even years. Rarely the fibrotic process itself takes an acute course, similar to the syndrome of "acute myelosclerosis,,35, 142.

Life expectancy in the CMPD is determined by the original type of CMPD and its proneness to BC, but modified by the fibrotic complications - slowed down or accelerated - as in the comparatively rapid course of CML or the chronic progress of polycythaemia (pV)9. Other reasons for the large variations in the median survivals between and within these groups are not known. They have been attributed to differences in regulatory events in cells that appear later during the maturation process80, 91, 95, 200. Spontaneous remissions are very uncommon. Prolongation of life is obtained by therapy in PV, but less so for the other groupS189. The introduction of bone marrow transplantation for CML in selected cases now offers hope of cure136.

3. Haematologic Symptoms

As this concise natural history of CMPD has indicated, the various types of CMPD can adopt a great variety of haematologic symptoms and various combinations of increased or decreased peripheral blood counts and organomegaly.

4. Morphology and Pathophysiology

This schematic review does not reflect the manifold clinical expressions of CMPD during the many years they generally take. No wonder that an abundance of synonyms was introduced to describe these appearances, the most prominent of which were first recognized as myelogenous leukaemia by Neumann 1878155, as osteosclerotic anaemia with leukaemic blood picture by Heuck 1879106, as PV by Vaquez 1892206, as idiopathic thrombocythaemia by Epstein and Goedel193474, and as megakaryocitic myelosis by Hewer 1937 in the spleen108, and by Hittmair 1944 in the bone marrow111 • Table 1 gives some of the synonyms used in the literature.

Table 1. Synonyms (English and German only)

Polycythaemia rubra vera Polyzythaemie Primary erythraemia

Chronic granulocytic leukaemia (myelosis) Chronic myelocytic leukaemia Chronic myelogeneous leukaemia Chronische myeloische Leukamie Chronische granulozytare Myelose Reifzellige myeloische Leukamie

Primary, essential, or idiopathic thrombocythaemia Primare, essentielle oder idiopathische Thrombocythamie Reifzellige megakaryozytiire Myelose

Leukoerythroblastische Anamie mit Myelofibrosis Myelofibrosis Myelosclerosis with leukaemoid blood picture Myelosklerosis

Megakaryocytic myelosis with osteosclerosis Myelosis with osteosclerosis Osteomyelofibrosis Osteomyelosclerosis Osteosclerotic leukaemia

Agnogenic myeloid metaplasia Anaemia with myeloid splenomegaly Chronic nonleukaemic myelosis Chronische megakaryocytar-granulozytare Myelose (CMGM) Leukoerythroblastosis Megakaryocytic anaemia Megakaryocytic splenomegaly Myelogene Pseudoleukaemie Myeloid megakaryocytic splenomegaly Myeloid megakaryocytic hepatosplenomegaly Myeloid metaplasia Splenic aleukaemic myelosis

This wealth of nomenclature is the result of the impossibility to understand especially the subleukaemic variety of CMPD from the study of the clinical, haematologic, and necropsy manifestations alone. Bone marrow aspiration has no access to the structural composition of

Chronic Myeloproliferative Disorders (CMPD) . 133

haematopoietic and stromal compartments. The necropsy findings cannot reconstruct the preceding structural development of the abnormal changes which are blurred by the autolytic alterations of the cells. The synopsis of all these syndromes under the heading of "myeloproliferative disorders" by Dameshek62 would not have been realized without the introduction and use of bone marrow biopsy61, 149.

Experimental and genetic studies have greatly contributed to the understanding and confirmation of this concept. These have shown that PV, CML and PT are the main representatives of a group of closely related haematologic neoplasias. Genetic proof of monoe/onalit'! is established for CML by the Philadelphia anomalyl5 : the PhI-chromosome which has been found in CML and its variants53, 55, 57, 87, 219, in BC due to CML, in the prephase of BC49, 145, and in single cases of PV127, 136, PT88,208,221, and MF/OMS 125 • The Ph-chromosome is found in haematopoietic, but not in somatic cells. Substantial evidence for the B-cell nature of lymphoid blast crisis in CML has been provided by immunologic marker studies and enzyme analyses6, 98, 120, 121, 144, 148.

Moreover, additional proof of monoclonality has been furnished by study of the enzyme Glucose-6 phosphatdehydrogenase (G 6 PD) that occurs in two variants, both linked to the x-chromosome. Females heterozygous for G 6 PD have both variants randomly distributed in all somatic cells. But progeny derived from one stem cell have only that one variant of G 6 PD. This has been demonstrated for blood cell lines including platelets, B-lymphocytes and monocytes among female heterozygote patients with PV, CML, PT, agnogenic myeloid metaplasia, and acute myelofibrosis, while their fibroblasts had a mixed enzyme distribution 1,78,79,81,118. These results also correlate with the occurrence of the PhI-chromosome.

They have confirmed the previous assumption that myelofibrosis and osteomyelosclerosis are not manifestations of the myeloproliferative process itself but are secondary consequences produced by stimulation of the nonneoplastic fibrogenic tissues35, mediated by factors of the proliferating cells, mainly the megakaryocytes. Megakaryocytes and platelets have been shown to produce a fibroblast-activating factor and an agent that inhibits collagenase (probably factor 4)51, 177. In CMPD clusters of megakaryocytes and masses of platelets have been found in the interstitial spaces of the bone marrow, suggesting ineffective megakaryocytopoiesis; these are often surrounded with a tight net of fibrils 32,34,38. These observations have been confirmed by recent studies. It is therefore highly probable that myelofibrosis is triggered by the local excretion of megakaryocyte- and platelet1'roducts during abnormal megakaryocytopoiesis25, 52,19. The term "agnogenic" therefore does not apply to the myelofibrotic or osteomyelosclerotic transformations of CMPD especially when the diagnosis of the original CMPD was made from the biopsy and/or the case history39.

Whether a genuine "primary" myelofibrosis may be distinguished from the CMPD-fibrosis is subject to further discussion207. Possibly this group will disappear with the improvement not only in methods of histologic prepara-

134 . R. Burkhardt, R. Bartl, K. Jager, B. Frisch, G. Kettner, G. Mahl and M. Sund

tion but also in immunologic marker techniques, so that most cases showing fibrotic bone marrow changes or osteosclerosis will be assigned to CMPD that is MPD-MF, or to lymphoproliferative disorders, to immune- and other reactions against metastatic carcinoma, and to other diseases with inflammatory and immunologic activities, e. g. tuberculosis or autoimmune-states that is non-MPD-MF. An immunologic or inflammatory pathogenesis may apply to CMPD-myelofibrosis also, since the strength of its expression from slight to maximal and the speed of evolution varies among single cases as well as between the different subgroups, with no evident correlation with the number or type of megakaryocytes.

The view that inflammatory and immunologic reactions, and factors produced by other bone marrow cells, may participate in the pathogenesis of MF/OMS is supported by experimental studies48, 96, 139 and by the presence of eosinophils, mast cells, plasma cells, and lymphocytes in the bone marrow, especially in cases with fibrotic transformation35, 39, 84, 119,201.

Studies with sequential biopsies have demonstrated the structural development in CMPD during different stages, includinE fibrotic transformation and blastic metamorphosis9, ,92. These constitute a crucial instrument to prove the transformation of one subgroup of CMPD into another in individual cases as well as to assess therapeutic risks, haematologic complications, remission, cure, and relapse.

They have further shown that a histologic subclassification of CMPD, based on the findings in the iliac crest biopsy, has prognostic significance, and that histologic criteria, such as the increases in megakaryocytic and granulocytic proliferation, are of predictive value for estimation of the later development of MF/OMS and blastic crisis9, 10, 83, as shown in a previous study9. There is general agreement that changes in the peripheral blood in CMPD should be interpreted together with the 'haematopoietic changes in the bone marrow, irrespective of reservations about the significance of histologic changes especially in early cases of PV and CMLI7,107,173.

5. Unsolved Problems

a) Pathogenesis of Myeloproliferation: Inspite of these achievements there are many open ques

tions concerning CMPD. We do not know what initiates neoplastic development at the stem cell level. The causal role of the PhI-chromosomal anomaly has been ~uestioned, since it is not a conditio sine qua non of CML 1,87, and may well be an acquired anormalyl0l, lending a selective advantage to the PhI cells after the induction of the clonal anomaly at the stem celllevel28, 81. In some cases the PhI-chromosome has appeared during the course of disease, and it has been produced by mitogen-stimulation during tissue culture of Ph1-CML-cells81 . The monoclonal nature, the close relationship of all CMPD and their possible transformation to lymphoid B-cells are strong arguments in favour of the concept of a "pluripotent stem cell leukemia"123.

Radiation107 and benzene exposure172 are among the proven causes in particular cases, induction by virus infection is not107. There is most probably a multifactorial etiology, including spontaneous somatic mutationsl45. Probably normal as well as abnormal cell determination in the human depends on many circumstances that cannot be reproduced experimentally, e.g. the complex influences of the environment including the inductive interrelation between tissues of different origins103. These influences are hard to reveal as long as we cannot identify stem cells. It has been shown by marker techniques that the neoplastic myeloproliferation does not necessarily include all the haematopoietic tissue. When the neoplastic clones are eradicated, normal haematopoiesis may recoverI69,186,187. This has been interpreted as proof of the coexistence of normal and abnormal cell clones.

Normal haematopoiesis is subject to feedback-control. Inhibitory activity against cell-proliferation has been proven for leukemia cells29, 161 as well as for cells of PV in cell culture experimentsl85. Presumably the excessive neoplastic proliferation in CMPD suppresses the normal stem cells54, whereas the neoplastic cells are resistant to normal regulatory mechanisms. Thus, the persistence of normal haematopoiesis in CMPD leaves hope for conservative therapy. The close relation of neoplastic and normal haematopoiesis however renders the selective eradication of the malignant clone extremely difficult.

b) Pathogenesis of Extramedullary Expansion: Though some of the most striking symptoms and

hazardous complications of CMPD are produced by their expansion into extramedullary sites, especially the spleen, the reason for this expansion is unknown, and there is no way of its prevention. Whereas formerly (probably due to inadequate diagnostic access to the bone marrow), the "giant splenic tumour" was held to be the original site of some types of myeloproliferative disorder73

, it is now generally assumed that the process starts in the bone marrow and spreads to other organs, and especially those with embryonic haematopoietic activities.

However, the possibility of an initially pluricentric propagation cannot be excluded. We assume that the extramedullary haematopoiesis is normally only suppressed since it can be reinstituted in case of high compensatory demand, e.g. in haemolysis. Thus insensitivity of neoplastic blood cells to normal regulation could be sufficient explanation for their extramedullary propagation. The extramedullary occurence of various myeloproliferative entities could then be explained by the different mechanisms of the three cell lines involved and the cellular composition in the individual case.

The assumption of the secondary nature of splenic involvement in CMPD is also indicated by the fact that early splenectomy can prevent the late consequences of splenomegaly (i.e. thrombosis of the splenic veins, infarction, or rupture of the capsule), but not the progression of the disease114

,220. On the other hand, splenectomy may exert an unfavourable influence on the immunologic reaction against the neoplastic process, and on its degradation products. Splenectomy is strictly contraindicated in cases

of PT. The risk of aplastic anaemia when large spleens of the subleukaemic forms of CMPD are irradiated is well known.

The role of splenic haematopoiesis in CMPD is far from clear. Active erythropoiesis in the spleen has been observed in myelofibrosis when the iron uptake of the bone marrow was depressed; but turnover rates showed ineffective erythropoiesis77

• Therefore the existence of a compensatory, normal, haematopoiesis in the enlarged spleen of CMPD is not likely to Rlay the genuine supportive role previously presumed4o,2 6. By labeling granulocytopoietic precursors it was shown that the majority of immature granulocytes in the peripheral blood in CML may originate from the spleen. One year after splenectomy however there was no difference in the pattern of labeling compared with the status quo ante160. BC also may arise in the spleen152.

The elucidation of these problems is complicated by the fact that medullary and extramedullary haematopoiesis are not independent of each other, and that it is difficult to assess the amount of cellular exchange between these compartments. We did not deal with hepatomegaly in this context since hepatic haematopoiesis generally lags behind the splenic, and because its role is even more unclear than that of the spleen.

c) Pathogenesis of Leukaemia: Leukaemia may be present from the onset of symptoms,

absent from beginning to end, or develop during the course of disease with leukocyte counts ranging from 15 to more than 300.000/mm3

• The level of socalled subleukaemia to leukaemia is arbitrarily set at about 30.0001 mm3. A major part of the leukaemic cells is represented by granulocytes, exceptions exist mainly in the case of metamorphosis into blastic crisis. As long as this final event does not take place, the counts at the terminal stages often decrease to subleukaemic or even subnormal peripheral blood levels. In such cases, bone marrow biopsy generally reveals either overproduction of very immature cells, a hypocellular sclerotic edematous marrow, or fibrotic respectively osteomyelosclerotic transformation of the marrow spaces, thus offering more than one explanation for the peripheral cytopenia.

Not much light has been shed on the mechanism that causes a leukaemic blood picture in CMPD. Blood cells mature in the extracapillary marrow spaces and enter the venous sinusoids that drain the bone marrow like a system of channels4o, 216. Observations with the electron microscope have shown that the marrow cells migrate through the endothelial cells13,191. In the larger areas of semithin lightmicroscopic sections the impression is gained that gaps between sinusoidal endothelial cells are also frequently present, whereby a passive extrusion of bone marrow elements into the circulation may also occur46. Normally the maturity of the cells is probably a decisive factor in their egress.

In CMPD there is sufficient evidence for a correlation between cellular maturity (but not as much for the mass of the abnormal cells in the bone marrow)64, and polycythaemia, leukaemia, or thrombocythaemia. On the


other hand immature cells may also enter the circulation under special circumstances, as in blastic crisis, whereas seemingly more mature elements, e.g. promyelocytes, leave the bone marrow only in comparatively small numbers even when they are greatly increased. Therefore mobility should be considered independently of cellular maturity. A defect in mobility may be responsible for those cases of CMPD, in which the amount of mature granulocytes within the marrow and the morphologically normal condition of the walls of the sinusoids do not correspond with the peripheral blood counts.

The megakaryocytes are also mobile cells that enter the sinusoids in order to release their platelets32, 34, 221. Differences in mobility could be responsible for the fact that in CMPD sometimes large clusters of seemingly mature megakaryocytes are found in direct contact with the sinusoids when at the same time there is thrombocythaemia in some cases, and normal platelet counts in others. However, other factors, such as extramedullary thrombocytolysis, have to be taken into consideration as well.

Mature erythrocytes on the other hand enter the sinusoids with the aid of extracellular forces, and by their ability to change their shape. In PV and PT there is a close correlation between increased erythropoiesis and megakaryocytopoiesis, and increase in marrow sinusoids. The same holds true for the correlation of arterial capillaries and granulocytic proliferation. In CML, though lesser than in PV, the number of the marrow sinusoids is also increased. These correlations reflect the functional connection of haematopoiesis and microcirculation in the bone marrow38.

This function is probably lost when the smaller sinuses are obliterated and the remaining capillaries transformed into large channels with sclerotic walls, as in advanced myelofibrosis. These vessels may contain large amounts of immature and mitotically active elements of all cell lines, especially megakaryocytes, megakaryoblasts, and erythroblasts. It could also be that these abnormal vascular channels serve as receptacles for blood cells of extramedullary origin that have recirculated to the bone marrow.

A disproportion between bone marrow cells and their corresponding representatives in the peripheral blood cannot be interpreted without taking into consideration the kinetics of their proliferation, and their survival times. The accumulation of typical haematopoietic cells in the bone marrow fits well in the concept of the prolonged survival of the mature granulocytes and increased self-replication of the progenitor cell population4, 11, 66. Even minimal changes of the turnover rates of these cells may be sufficient to create hypercellularity in the marrow spaces.

The inadequacy of morphology in the estimation of cell kinetics becomes clear when we consider on one hand the rarity of the observation of the passage of a blood cell through the sinus wall in semithin sections of an iliac crest biopsy, and on the other hand the millions leaving the bone marrow every second. Our knowledge of cell kinetics in CMPD is impaired by the problem of recirculation209. The question whether disturbed cell transport or intravascular haematopoiesis in the bone marrow, or extramedul-


lary blood formation in the spleen, are responsible for the erythroleukaemic blood picture in some advanced stages of CMPD will only be answered by functional methods. Since such a blood picture is also present in splenectomised patients, and independent of splenic size, the spleen cannot be the only source.

B. Diagnosis

1. Cytology and Histology

In CMPD it is difficult to distinguish the normal cells from their neoplastic counterparts. Chromosomal anomalies, as numerous as they are in CMPD, are of little value for the clinical diagnosis, except for the Ph l -chromosome113. There are many observations on metabolic disturbances of cells in CMPD67, 188 as well as functional defects, e.g. diminished phagocytic or migratory performances, or reactions to leukopheresis7,30. These are of little help in the diagnosis and prognosis in individual cases, except for the elevated (most cases of PV) and the decreased levels (most cases of CML) of leukocyte alkaline phosphatase activity. The latter is associated with defective phagocytosis, which is restored by maturation of the cells in culture56, 165, 166. In contrast to other tumours the CMPD exhibit an infiltrating but not a destructive growth pattern, both within the marrow and the extramedullary sites (except for very rare cases of megakaryocytic sarcomatous growths which have an expansive spread).

The diagnosis of the CMPD is based mainly on the abnormal peripheral blood counts especially when immature or pleomorphic cells are present; and on an abnormal amount and distribution of their precursors in the marrow, in particular numerous abnormally shaped or immature megakaryocytes. These signs develop slowly and at present neither morphologic methods nor functional analysis can identify small populations of CMPD at the early stages of the disease. Retrospective analysis of typical cases offers some access to evaluation of very early changes, provided that the clinical investigation included a bone marrow biopsy. Observations of this kind are few, but they show that the vague early haematologic signs are best interpreted in combination with bone marrow histology, and with chromosomal analysis.

In CML the bone biopsy is diarnostic even in the clinically oligo symptomatic stage39,19 , whereas early PV may be more clearly defined by the blood values in about 10% of cases213. The bone marrow changes preceed the extramedullary manifestations. In the majority of untreated as well as treated cases of CMPD the histologic findings are also characteristics when other haematologic or clinical signs are present; the biopsy adds valuable supplementary information. In atypical, subleukaemic or oligo symptomatic cases the histologic evaluation is indispensable. This applies especially to non-thrombocythaemic megakaryocytic myelosis, to fibrotic transformation, and to blastic metamorphosis.

In order to recognize BC, recognition of the "accelerated phase,,97, 126, 176, 183 is required, since increase in blast cells in the peripheral blood is not a prominent feature in

many patients213• The observation of ml

cromegakaryocytes and of additional chromosomal anomalies have been emphasized as more reliable tools than bone marrow biopsy24,202. However, we have used bone marrow biopsies with good results in earlier studies83, though occasionally a focal distribution of bone marrow changes may be misleading. If there is suspicion of an error, comparison with aspirated sternal marrow may assist the histologic diagnosis, in addition to the criteria mentioned before. Skeletal analysis with x-ray techniques is helpful, when osteomyelosclerosis is suspected. Erroneous interpretation of biopsy findings can be diminished by careful consideration of the histologic differential diagnosis of CMPD (see below).

2. Issue of Bone Marrow Biopsy

Previous morphologic studies of the CMPD-complex have been usefuP9,92, especially sequential biopsies to follow the course of the disease. Recognition of the development of marrow changes in the individual case, together with the comparative evaluation of these changes in large numbers of patients plus the clinical data are required for classification and prognostic evaluation. As closely related, primary diseases of the bone marrow, the CMPD cannot be understood without considering their interrelations with the rest of the bone and marrow tissues, and their structural arrangement within the bone marrow. This is true for the general aspects of CMPD as well as the special histologic features of their diagnosis, morphogenesis, and classification, which should be related to the clinical and haematologic data and to the classical entities of CMPD defined originally with these data alone.

3. Technical problems

An earlier survey of a total of 1072 publications dealing with myelofibrosis and osteomyelosclerosis to 1974 demonstrated that large amounts of clinical and morphologic data cannot be compared without serious reservations due to differences in the methods and the criteria applied for their evaluation35. A similar situation still exists today. A considerable number of recent histologic investigations on the CMPD is available, based on excellent techniques of biopsy and preparation, but their comparability remains questionable due to technical differences and to different criteria for evaluation9, 34, 41, 63-65 89-91109,115,116,130,154,162,167,179,196,211,212,224 , .

An even greater problem is posed by the variability of the disease itself, complicated by the influence of modern aggressive therapy. The histologic findings become comparable only when the previous history of the disease, the stage at the time of the biopsy, and possibly, follow-up biopsies are considered. The results and evaluations presented here are based mainly on our own observations of 3415 iliac crest biopsies of 2846 patients suffering from CMPD. * Applying the criteria of our previous

* The referral of patients and biopsies by many clinics and hospitals is gratefully acknowledged.

studies9, 10,40 they were classified as follows: PV = 833; CML = 796; Meg M = 344; and MF/OMS = 897. The majorirr of the biopsies was taken with the myelotomy drill24,3 , the rest by surgery and manual trephines (mostly the Jamshidi-needle)122.

All biopsies were embedded in a mixture containing the methylie ester of methacrylic acid as main component, after fixation in a solution of buffered formalin and methanol. In selected cases, small pieces of the biopsies were also processed for electron microscopy or enzymatic studies. At least five un decalcified sections of 3 f..t thickness were used for evaluation with the light micro-

Fig. 1. Unequal manifestation of disease in the bone marrow biopsy; upper part: fatty dystrophy; lower part: myelofibrosis. Gomori's stain; X 10.


scope, after staining with Gallaminblue Giemsa for general analysis, with Gomori's silver impregnation for fibres, with Ladewig's stain for connective tissues, with the PAS reaction for glycoproteins, and the Berlin blue reaction for iron. The superiority of methacrylate embedding and preparation of undecalcified sections for evaluation of bone marrow biopsies is now established as it minimizes artifacts due to shrinkage. The technical details have been reported previousll' 34, 44, 192. For routine assessment, the Giemsa stain and a good silver impregnation, can be recommended as standard procedures.

The following sections demonstrate the value of adequate methods of biopsy and preparation, enabling recognition of the haematopoietic precursors, which is indispensable for the appraisal of their distribution among the CMPD. This improvement is shown to best advantage in a biopsy of adequate size, permitting analysis of the spatial distribution and topographic relations of marrow, bone and microcirculation. At the same time the pitfalls due to haphazard occurrence of abnormal changes are lessened. In our series about 20% of the manual biopsies and about 2 % of the drill biopsies were inadequate. Fig. 1 shows that only a biopsy core of sufficient length allows recognition of the focal nature of an intermediate area of fatty tissue; and fig. 2 illustrates the fact that an unequal tissue dis-

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.,t., . ) ... -, ,

Fig. 2. Unequal manifestation of disease in the bone marrow biopsy; right half: total aplasia of haematopoiesis; left half: megakaryocytic myelosis. Gomori's stain. x 10.


tribution in the transverse dimension also may cause an erroneous impression, when only half of the biopsy were considered: the right half is totally depleted of haematopoiesis (as in aplastic anaemia), whereas in the left half the correct diagnosis of megakaryocytic myelosis is established. At a magnification of 200 x it becomes evident that smaller pieces of a single biopsy may present different features, for example fatty degeneration, myelofibrosis, osteomyelosclerosis, CML or megakaryocytic myelosis, since these changes frequently occur in foci. Therefore the risk of misinterpretation increases greatly with only fragmentary biopsies.

Of course the principal problem of possible nonhomogeneity of the biopsy site cannot be overcome by technical means. This has to be kept in mind when the diagnosis of CMPD is uncertain, and especially when a comparison is made either between groups of patients, or several biopsies from the same patient. In the first case the large size of the groups, in the second the clinical information help to avoid erroneous conclusions.

4. Clinico-Histologic Differential Diagnosis

a) Overall View The architecture of the cancellous bone and the enclosed

marrow in CMPD differs markedly from the normal when the disease is fully established (Fig. 3-7). The normal aspect of the bone and marrow at the peak age of CMPD is illustrated with the biopsy of a normal woman of 58 in Fig. 3, which as well as Fig. 4 represents about one half of the biopsy. The well known physiological age changes of bone and marrow are clearly visible. The osseous trabecles are only slightly attenuated, the fatty tissue has increased, especially in the subcortical area. Nevertheless, the generally equal distribution of the marrow, and the uniform spatial arrangement of the trabecles are maintained.

In polycythaemia vera not only an attenuation but also severe rarefaction of the trabecles is common, as shown in Fig. 4. In addition the marrow is densely cellular. Both changes are even more pronounced in CML. Inspite of considerable normal variation in these structures, the reproducibility can be shown in the averages of representative groups.

There are two hypothetical explanations for the myelogenous osteodysplasia (MOD)3 which accompanies myeloproliferation in PV, CML and PT. Bone resorbing activity by the proliferating cells has been demonstrated in vitro for myeloid leukemia cells93

, though there is generally no evidence of increased activity of the bone remodelling cells in the biopsies. Secondly capillarization of the marrow tissues is greatly increased45

, confirmed by functional analyses205

• This lends support to the assumption that the circulatory demands of the neoplastic haematopoiesis are supplied on the expense of the bone, both being dependent on the same nutritional source. In MF the average number of arterial capillaries is even higher; they accompany the fibrous strands that replace the cellular proliferation to occupy the wide marrow spaces between the attenuated trabeculae.

Fig. 3. Normal proportion of haematopoiesis: fatty tissue: cancel-1'4" bone in a female of 58 years. Gomori's stain. x 10.

Fig. 4. Increase in haematopoiesis, decrease of fatty tissue, rarefaction of cancellous bone in PV. Female patient of 61 years. Gomori's stain. x 10.

Fig.s. CMPD-osteomyelosderosis grade 1: moderate lamellar reinforcement of trabecular bone together with small appositional excrescences of osteoid; dense marrow fibrosis. Female patient of 71 years. Gomori's stain. x 10.


Fig. 7. CMPD-osteomyelosclerosis grade 3: filigrane-like osteocondensation, primarily consisting of primitive bone. Obstruction of marrow spaces. Focal increase in fatty tissue; fibrosis of resting haematopoiesis. Female patient of 66 years. Gomori's stain. x 10.

The relation of marrow to bone volume is reversed in osteomyelosclerosis (Figs. 5-7). When the changes are few, as in Fig. 5, we may find some mineralized fibrous strands accompanying the marrow vessels near the bone, and single sprouts of osteoid extending from the mature bone (arrows). More severe changes are characterized by rarefaction of the old bone and the extension of calcifying fibrous strands, filling the marrow space and connecting with the trabecular surfaces and with the sprouts of osteoid. The frequent occurrence of active osteoblasts and -clasts characterizes this form of MOD as active (Fig. 6).

This process may result in a bizarre osseous architecture as shown in Fig. 7. The old bone is almost totally replaced by a tight meshwork of primitive or immature osseous material. The overall view shows the impressive gross structural changes together with regression of the bone marrow, leaving large empty spaces that at higher magnification consist of a mixture of edema, fibres and fat cells.

.... Fig. 6. CMPD-osteomyelosclerosis grade 2: rarefaction of typical lamellar bone together with appositional and perivascular sprouting of primitive bone; increase in fatty tissue; fibrosis of the resting haematopoietic areas. Female patient of 71 years. Gomori's stain. x 10.


The capillaries are no longer concentrated in the marrow centres but in a zone of loose connective tissue seaming the osseous trabecles: the activated mesenchyme that accompanies increased osseous remodelling, while the proliferative activity in the marrow centres is reduced. Of a total of 359 MF/OMS cases, re-evaluated for these studies, 182 (= 51 %) were reclassified as MF, and 175 (= 49%) as OMS.

In OMS various expressions of the three forms represented in Figures 5, 6, and 7 were observed and the transition from one to another was documented in follow-up biopsies. In accordance with earlier observations159 it is highly probable that we are dealing with a continuous development of which the three stages were arbitrarily chosen for the purpose of the subsequent evaluation. Fig. 8 shows (for comparison) osseous remodelling due to metastatic carcinoma. There are two significant points: first the difference between the systemic spread of the CMPD and the focal expansion of the metastatis in the bone marrow, and second the similarity of the osteoido-plastic reaction in both.

These examples show that CMPD may change the overall structures of the bone and marrow in a characteristic manner. The histologic changes do not always correspond with clinical and haematologic symptoms. Whereas clinically typical cases of PV may exhibit only weak histologic signs, the reverse is the rule for all cases with more imma-

Fig. 8. Metastatic mammary carcinoma: filigrane-like osteoid formation at the tumour margin (bottom). Female patient of 58 years. Gomori's stain. X 10.

ture, with megakaryocytic, or with fibrosclerotic changes. In CML the grade of the cellular bone marrow changes and the leukaemia are generally consistent.

b) Details The more subtle structural analysis of CMPD in the

bone marrow biopsy is rewarding in the diagnosis of uncertain clinical cases, but complicated by the problem of a large variety of histologic borderline cases. This aspect is generally ignored in studies set up to compare histologic and clinical data or to test different therapeutic regimens in well defined groups. The purpose of this review is different, namely to assist the histopathologist to recognize the whole spectrum of the histologic changes in CMPD with which he is confronted in daily practise.

Normal: To introduce the role of bone marrow histology in the most common problems of differential diagnosis a few typical examples will be demonstrated: Fig. 9 shows the normal appearance of the bone marrow. Since routine histologic diagnosis is based on visual impression quantitative data are omitted. They have been documented in other studies9

• The visible margin of the mature osseous trabeculae is covered with a unicellular layer of flat endosteal cells. The arrangement of the marrow cells resembling a cloudy sky, the darker clouds caused by small groups of erythroblasts, the brighter consisting of imma-

Fig. 9. Normal bone marrow. Description see text. Female patient 28 years. Giemsa stain. X 200.

ture granulocytes, and polynuclear granulocytes, rimmed by clusters of erythrocytes, and interspersed with irregularly shaped sinusoids and deposits of round fat cells. A few megakaryocytes are recognizable by their size. The borders of the numerous sinusoids are hardly visible at this magnification, except for a few endothelial cell nuclei. On the left side of the picture a longitudinally and a transversially cut arterial capillary are seen, surrounded by a few typical plasma cells. Some reticular cells and macrophages are present but difficult to distinguish. The net of reticular fibres is hardly visible. That is almost all we need for the review. The figures of this chapter are shown at low magnification (200 fold) for the general survey. In practise, we also use a magnification up to 500 fold or more to establish the cytologic features with greater certainty.

PV: In polycythaemia vera (Fig. 10) the "clouds" are more bulky, mostly due to the increase of erythroblasts, the sinusoids are increased and enlarged, containing more erythrocytes than usual, the ratio parenchyme: fatty tissue is increased, and giant megakaryocytes are loosely distributed in the marrow. At higher magnification we distinguish the trilinear haemopoietic proliferation, and, after the iron stain, the absence of stainable iron. The reticular fibres are not prominent, except in association with the sinusoidal walls.

Fig. 10. Polycythaemia vera, typical. Description see text. Male patient of 57 years; blood: erythrocytes 6.9 millions, leukocytes 9.000, platelets 240.000/mml. Giemsa stain. x 200.


Fig. 11. Secondary erythrocytosis. Description see text. Male patient 54 years; blood: erythrocytes 7.9 milion, leukocytes 4.000, platelets 159.000/mml. Giemsa stain. x 200.

The typical picture of a case of secondary erythrocytosis looks different (Fig. 11). Only the erythroblasts are increased, the megakaryocytes are of regular shape and number, the enlarged sinusoids contain masses of red blood corpuscles and the volume of fat is normal or even increased. Also increased, in contrast to PV, is the iron content of endothelial cells, reticular cells, and macrophages. Though this picture itself is not characteristic, it provides strong evidence against the diagnosis of PV.

CML: In subleukemic cases of chronic myeloid leukaemia the question of reactive granulocytosis may arise. The biopsy picture of CML (Fig. 12) is highly characteristic even in the majority of early cases. The cellular clouds are closed to an almost homogeneous cover to the exclusion of fat cells, and consist mainly of myelocytes, metamyelocytes, and polymorphonuclear cells, mixed with dwarfed megakaryocytes that are not always distinctly increased (as in Fig. 12). The trabecular surfaces are seamed with immature elements, mostly promyelocytes. The erythroblastic islands are small, and the numerous capillaries narrowed and partially concealed by the masses of cells.

Fig. 13 shows a leukaemoid reaction: the para trabecular proliferation zone is occupied by immature granulocytes; the marrow centres contain masses of mostly mature, and


Fig. 12. CML, typical. Description see text. Male patient of 40 years; blood: erythrocytes 3.3 million, leukocytes 150.000, platelets 194.000/mm3• Giemsa stain. x 200.

Fig. 14. Primary thrombocythaemia, typical. Description see text. Female patient 34 years; blood: erythrocytes 4.7 million, leukocytes 8.000, platelets 916.000/mm3• Giemsa stain. x 200.

Fig. 13. Leukaemoid reaction due to myositis. Description see text. Male patient of 20 years; blood: erythrocytes 4.1 millions, leukocytes 20.000, platelets 477.000/mm3• Giemsa stain. x 200.

frequently pyknotic cells. Erythropoiesis and fat cells are only moderately decreased.

PT: Many conditions may cause thrombocythaemia and the diagnosis of the "primary" form is generally made by exclusion9

, in combination with the bone marrow biopsy. The typical histologic change (Fig. 14) is the striking increase in diffusely distributed megakaryocytes the majority of which belong to the giant, highly polyploidic class. The marrow sinusoids are increased but generally not to the degree seen in PV. The rest of the marrow shows no characteristic features, but variations which are considered later. Fig. 15 shows for comparison a case of secondary thrombocythaemia with megakaryocytosis in the bone marrow due to metastic carcinoma; a few giant megakaryocytes are also seen. The other megakaryocytes are less pleomorphic, and more immature. There is a diffuse increase in plasma cells, and the sinuses are inconSpICUOUS.

It has now been accepted that PT belongs to CMPD, and is derived from megakaryocytic myelosis 19,36,82,92,

100, 128, 138, 174, 180, 184. Its distinction from secondary thrombocythaemic conditions is not a major problem even in the few cases with an equivocal histologic diagnosis. Its recognition as a separate group among CMPD is contestedlO4, 146, 199 and the clinical criteria for this distinction are arbitrary: the diagnostic level for thrombocythaemia was set at 600.000 or 700.000 or one million, when maintained for a prolonged period of time; the spleen should be only moderately enlarged immature cells in the peripheral

Fig. 15. Megakaryocytosis due to metastatic carcinoma. Description see text. Male patient of 56 years.


blood rare, and the leukocyte counts limited to the subleukaemic range9,184. These criteria (including the chromosomal abnormalities92

) represent a collection of distinctive features but not a clear separation of PT from PV, CML or even MF, which all may exhibit thrombocythaemia 17,85,104,133,146,151,183,213. Therefore retro-spective criteria have been considered, such as an especially benign course, paucity of fibrotic transformation, and the virtual absence of BC92, 18\ and attempts are made to correlate the histologic diagnosis with these prognostic features 10,83,92. The comparative rarity of such cases and the prolonged course however impairs the collection of sufficient data.

Meg M: Parallel to the former group that typically shows prominent proliferation of large, mature megakaryocytes there are cases with abundant proliferation of megakaryocytes but of a different sort: sheets of much smaller cells, with pleomorphic, round, or slightly lobulated nuclei and different grades of cytoplasmic maturation (Fig. 16). Clinical signs (of myeloproliferation) are not regularly present; thrombocythaemia has been observed, even leukaemia (pancytopenia is more frequent); but severe anaemia is the rule. The recognition of these cases as a separate clinical entity has been debated; by some it has been included with agnogenic myeloid metaplasia, BC or atypical leukaemia. As its distinction is primarily a problem of histology the differential diagnosis will be discussed in the next section. In previous publications this type was integrated in the group of immature

Fig. 16. Immature megakaryocytic myelosis (Meg M-sI). Description see text. Male patient of 35 years; blood: erythrocytes 3.1 million, leukocytes 27.000, platelets 620.000/mm3• Giemsa stain. x 200.


Fig. 17. CMPD-myelofibrosis following CML. Description see text. Male patient of 71 years; blood: erythrocytes 6.5 million, leukocytes 48.000, platelets 102.000/mm3• Gomori's stain. x 250.

megakaryocytic myelosis34, 38, 41 to mark its distinction from "mature" MegM = PT, and the megakaryoblastic myelosis that is classified with the acute leukaemias.

MF: Lastly we have to compare the qualities of fibrosis and osteomyelosclerosis due to CMPD with the nonMPD-MF. Fig. 17 shows typical MF complicating CML. The same pattern of fibrosis is seen in PV, and PT. There are thin collagenous bundles surrounding the marrow capillaries and demarcating strands of granulocytes. The foremost diagnostic criterion is the persistence of the myeloproliferative disease, recognized by the increased cellularity, the cell type, and especially the large numbers of megakaryocytes including many abnormal forms and pyknotic nuclei. Frequently in MPD-MF there is an accumulation of megakaryocytes in the form of clusters, separated from their surroundings by a tighter fibrotic meshwork. The more fibrotic the marrow, the greater the difficulty in recognizing the original disorder. Even in the most fibrotic cases however islands of degenerating megakaryocytes may remain as diagnostic signposts, and groups of immature haematopoietic cells located in the sinusoids.

For comparison we have chosen a less advanced case with close similarity to a fibrotic marrow caused by inflammatory disease, in a patient with immune-vasculitis

Fig. 18. Non-CMPD-myelofibrosis due to pulmonary tuberculosis. Description see text. Male patient of 63 years; blood: erythrocytes 2,8 million, leukocytes 1.800, platelets 84.000/mm3•

Gomori's stain. x 250.

(Fig. 18). In this case the fibres are more tightly bound to the capillaries, and between the strands there are impressive signs of inflammation, such as edema, plasma cells, lymphocytes, and tissue mast cells.

Very significant is the rich capillarization of MF in combination with increases in lymphocytes, plasma cells and tissue mast cells which in many cases resemble the fibrotic stage of a granulation tissue of inflammatory origin. Our follow-up studies indicate that MF in CMPD is a continuous process, the structural grades reflecting different stages of disease progression, with the tendency to obliterate the marrow spaces at sites of adult haematopoiesis. This assumption is supported by the causal relationship of haematopoiesis to MF. The end stages of MF may exhibit broad fibrotic bands or, more often, resemble the acellular stage of scar tissue. The myeloproliferative process is finally more and more suppressed by the fibrosis that it has created.

That means that MF, once initiated, is not an autonomous process, but depends on the existence of CMPD. Recent transplantation studies have questioned the previous view of the irreversibility of MF and OMS 150, 15 • Our observations on CMPD-MF support the view that this idea was erroneously created by the irreversibility of the basic myeloproliferative process in the past, and that MF

might be basically subject to the same reorganization that is active in every region of the body with a rich capillarization, as soon as the fibroplastic stimulus ceases.

It is probable that not only the mass but also special properties of the CMPD are responsible for the slow or rapid progression of MF. The two different histologic aspects of acute myelosclerosis have already been mentioned. Among our material there are 16 cases to which the criteria of acute myelosclerosis applied. About 6 of them exhibit a picture identical with a erythro-megakaryoblastic proliferation and 8 a myelodysplastic syndrome, accompanied by reticulin-sclerosis or minimal MF, suggesting that the myelosclerosis in most of these cases was not the essential reason for the acute illness l42

• Nevertheless there are also cases of rapidly progressive MF and OMS, in our experience especially those in which large clusters of megakaryocytes are found together with a marked inflammatory reaction. The biopsy contributes further evidence for the possible participation of not only megakaryocytes, platelets, and granulocytes in the pathogenesis of MF, but also of inflammatory cells.

OMS: Osteomyelosclerosis presents in general the bone marrow features of MF together with the osseous remodelling described before (Figs. 5-7). Our example shows a detail of newly formed immature bone with osteoblasticclastic activities and large clusters of pleomorphic, partially degenerating megakaryocytes (Fig. 19). As the comparative figure (Fig. 20) shows, an almost identical type of osseous remodelling may be found in metastatic carcinoma, the tumour cells taking the place of the megakaryocytes.

Fig. 19. CMPD-osteomyelosclerosis. Description see text. Male patient of 58 years; blood: erythrocytes 3.0 million, leukocytes 18.000, platelets 41D.000/mm3. Giemsa stain. X 200.


A striking similarity is offered also in the case of complete scirrhous transformation of the marrow. In CMPD the persisting abnormally wide and sclerotic sinusoids may contain groups of immature cells, mostly megakaryocytes; in the scirrhous carcinoma, the same place is occupied by tumour cell clusters. When the fibrosis is almost acellular, tumour cells (Fig. 21 ), or degenerating megakaryocytes (Fig. 22) must be sought for as the scirrhous tissue itself does not bear distinctive histologic features. This similarity between the marrow fibrosis due to CML and metastatic carcinoma has been recently confirmed by fluorescent monospecific antibodies against collagen types I and III (Fig. 23). In addition, immunhistochemic tests with antibodies against pro-III-collagen and fibronectin point to a rapid metabolism of collagens in MF99.

The correlation of OMS and MF is difficult to assess from the biopsy. Among our cases we have never met an example of isolated osteosclerosis, on the other hand the reverse, namely MF without signs of OMS, is frequent (51 % of a total of 1044 MF/OMS biopsies). We cannot exclude the possibility that the findings in the biopsy may not represent those in the rest of the skeleton. However, together with observations of MF in the first, and OMS in the follow-up biopsy, and the reverse finding only in single cases we can assume that OMS is very often preceeded by MF. We hesitate nevertheless to consider OMS as the usual consequence of MF, since we have seen single cases with a history of more than 10 years, and persistent MF in the biopsy, and a number of cases with a short history and the histologic evidence of grade 3 OMS together with only grade 1 MF.

Fig. 20. Non-CMPD-osteosclerosis due to metastatic carcinoma. Description see text. Female patient of 45 years; blood: erythrocytes 3,5 million, leukocytes 5.200, platelets 180.000/mm3. Giemsa stain. X 250.


Fig. 21. Scirrhotic marrow tissue due to metastatic carcinoma. Description see text. Female patient of 65 years; blood: erythrocytes 2,5 million, leukocytes 2.400. Giemsa stain. x 250.

Fig. 23 a. Network-like structure of collagen type III with chronic osteomyelofibrosis. Immunhistochemistry with monospecific antibodies against collagen type III (300 X).99

Fig. 22. Scirrhotic marrow tissue due to CMPD-OMS. Description see text. Male patient of 51 years; blood: erythrocytes 2.8 million, leukocytes 6.000, platelets 133.000/mm3

• Giemsa stain. x 250.

Fig. 23 b. Collagen type III as a diffuse irregular array of fibers in the bone metastasis of a breast carcinoma. Immunflorescence with monospecific antibodies against collagen type III (500 x ).99

A further argument against a usual evolution of OMS from MF is the fact that we have found nearly identical survival rates for both. Moreover the average age of 359 untreated patients with MF/OMS was 60 years for grade 3 OMS; 62 years for grade 1 and 2 cases and 63 for advanced MF. It is likely that MF and OMS have one or more causes in common, but OMS needs some additional stimulation for its development. The further elucidation of these problems may be of interest for understanding the mechanism of connective tissue and bone formation. For this purpose greater numbers of patients of MF and OMS should be studied with sequential biopsies during the prolonged period of their disease.

The problems of differential diagnosis of MF/OMS and blast crisis have already been mentioned. Since the increase of immature cells in the peripheral blood is not a reliable indicator, a number of clinical, genetic, and histologic criteria have been more successfully used to define the accelerated phase of CMPD. There is increasing evi-

II. Problems of Classification

These problems cannot be solved without consideration of some structural variations found in the biopsies of CMPD with as yet uncertain clinical expression. Several attempts have already been made to classify them relying on the most prominent features common to all CMPD namely the variations of the proliferating cell lines, their maturity, and certain megakaryocytic anomalies which have lead to a more precise understanding of megakaryocytic myelosis and to the establishment of chronic megakaryocytic §ranulocytic myelosis (CMGM) as a new entity respectively 9,92. These may be further specified by a tentative classification of the megakaryocytes.

A. Classification of Megakaryocytes

1. Distribution: Figs. 24, 43, 44 and 46 show three different forms of distribution: diffuse (d), clusters (c), and sheets (s);

d-form: this term was applied to all cases in which the majority of megakaryocytes were diffusely spread over the section, with direct contact between single cells only;

c-form: the occurrence of at least three clusters of more than five megakaryocytes in the section;

s-form: when at least one large or two small colonies of megakaryocytes, forming an almost homogeneous layer, are seen in the biopsy.


dence that not only progression of the maturation arrest19

in CMPD to more and more immature stages may be accompanied by a certain degree of fibrosis (provoking the syndrome of acute myelosclerosis, p. (132, 144) but also some relationship exists between the fibrotic transformation and the accumulation of blast cells in the bone marrow, especially abnormal megakaryoblasts5

, 23, 69 and erythroblasts. Mixtures of promyelocytes, myeloblasts, proerythroblasts and megakaryoblasts have been found in BC143, and myelofibrosis has been documented to preceed BC in a considerable proportion of cases (861). Recently it has been shown that platelet derived growth factor also stimulates erythroblastic progenitor cells in vitr060. This observation could be a clue to the understanding of the association of MFIOMS and BC that can no longer be considered as alternative events. The distinction between BC with concomitant reticulin fibrosis and MF with concomitant accumulation of blast cells is therefore an important issue of the bone marrow biopsy.

These definitions were arbitrarily based on observations made on about 3415 biopsies of CMPD. The d-form is the rule for the distribution of normal megakaryocytes; giant and dwarf types may also be diffusely spread in the~bone marrow. The c-form is mostly composed of giant cells (Fig. 44), more seldom of dwarfs and normals; the sheets consist exclusively of accumulations of either imftIature or pleomorphic megakaryocytes (Fig. 45, 46). Clusters or sheets of megakaryocytes have previously been described by many in PV, PT, chronic megakaryocytic granulocytic myelosis (CMGM), MegM and MF25, 32, 3 4, 3.f, 128.

2. Cell types: Different megakaryoctic cell types are shown in the Fig. 24-33 and defined according to the following features, as observed in the lightmicroscope:

N-type = "normal": cell with tri- to multilobulated nucleus, and between 25 and 50 micron diameter, with round to polymorphous margin, signs of cytoplasmatic demarcation, and a fine, equal distribution of the stainable nuclear protein. The designation "normal" means normality of the cellular aspect (Fig. 24, 25e, f).

D-type = "dwarf": cell with at least tri-Iobulated nucleus, between 15 and 25 micron in diameter with mostly roundish margin, and otherwise similar signs as described for the N-type. However, due to the small size, less clearly distinguishable (Fig. 24, 25a-d, 26). The category "dwarf" was chosen instead of the more familiar "micromegakaryocyte" or "microkaryocyte,,197 not only as the opposite to


CMPD - MEGAKARYOCYTES

Forms of distribution in the bone marrow

.~ ... • • • . ~

d=diffuse c = clusters

Types of development

N = normal

@

B= blast

0= dwarf

@

I = immature

py= pycnot ic

the giant form, but also to comprise all apparently mature megakaryocytes with a size markedly smaller than the normal. Recent investigations have identified a number of blast- and microforms as well in the bone marro~, in the peripheral blood and in cell culture23

,147. The crriteria established by enzyme markers and electron-microscopy have contributed greatly to the better recognition of these cells in a semithin lightmicroscopic section. With respect to the role of megakaryocytes in CMPD some more recently established facts are of special importance: the correlation between the cellular size and nuclear ploidy68, 158; the incongruence between cytoplasmic maturity and nuclear ploidy or cellular size22, 68,16 • These results have provided indications of possible inactivity of giant cells131 and possible platelet production of micro-

s= sheets

G= giant

P = pleomorphic

Fig. 24. Schematic representation of the forms of distribution and the types of development of megakaryocytes in the bone marrow in CMPD.

forms with a ploidy level of only 4-8 N 2, 68, which has

greatly assisted our understanding of CMPD. Micro- or dwarf megakaryocytes therefore may be productive cells at a more primitive level of evolution than normal or giant cells203

•

G-type = "giant": cell with multilobulated nucleus, between 50 and 120 micron in diameter; nuclear substance and signs of cytoplasmic demarcation as described above, tendency to extremely lobulated margin, and to elongation of the whole cell (Fig. 24,27). The giant megakaryocytes are easily distinguished. They are among the most prominent features of CMPD in aspiration smears as well as in biopsy sections, and they are mentioned in every publication dealing with the morphologic

Fig. 25. Megakaryocytes, types a) = dwarf; b) = dwarf in mitotic division; c) = dwarf; d)-f) = normal range of cells. Description see text. Giemsa stain. x 1000.

aspects of these diseases. The frequency and ultrastructural anomalies of this special type in a subgroup of CML - chronic megakaryocytic-~ranulocric myelosis - have been especially emphasized 2, 193-19 . There are frequent multipolar mitotic figures in giant megakaryocytes especially in PV, PT and the mixed forms with the G-MEG/ GRAN-type. Mitotic nuclear division in ploidy stages beyond N-8 is uncommon. Whether abnormal mitotic activity is a special feature of CMPD has yet to be ascertained.

Fig. 27 shows the phenomenon of emperipolesis43; its

significance is unknown. It is not correlated with the number of mature granulocytes in the bone marrow nor their size, but with their cytoplasmic maturity.

I-type = "immature": uni-to binuclear cell between 15 and 25 micron in diameter, very fine nuclear structure, round to ovoid cytoplasm, generally without signs of cytoplasmic demarcation (Fig. 24,28).

P-type = "pleomorphic": various forms, especially with normal to dwarf size, and comparatively large, round nuclei distinguished by a coarsely staining nuclear substance (Fig. 29), or cells of irregular size, containing numerous round, separate nuclei, situated in the form of a ring close to the cell membrane, or multinucleated cells with blastlike nuclei (Figs. 16,30,31,57).


Fig. 26. Megakaryocyte, dwarf type (MEG-D) showing mature cytoplasmic organelles and demarkation of platelets. Megakaryocytic myelosis (Meg M-sI). EM 10 Carl Zeiss. X 3200.

B-type: = "blastic": round cells of about 8-10 micron in diameter with deeply staining cytoplasm and large nuclei, showing very fine nuclear structure, and 1 to 3 medium sized nucleoli, frequently united to bandlike forms (Figs. 28,57).

Py-type = "pyknotic": either isolated clumps of nuclear substance structured like a dried plum, or bizarre nuclear configurations surrounded with cytoplasmic debris (Figs. 32,33). Pyknotic nuclei of megakaryocytes, isolated or surrounded with remnants of their cytoplasm, are considered to represent the spent-stages of these cells, after exhaustion of platelet production. Their morphology and fate has been described in detail17l, 225. .

These types and sizes comply roughly with those described in the literature18, 26, 124. Our results with the electron microscope are in line with those of the light microscopy, as is shown in Figs. 26, 31-33 and 35. Exact comparability is not accomplished due to the different preparation techniques and methods of evaluation. In addition the error due to the difference between the diameters of the sections (3 ± 0.5 micron) and the megakaryocytes (15-120 micron) has to be taken into account105

• In an earlier study of our group the possibility of a wrong distinction between three groups of a size between 15 and


.. Fig. 27. Megakaryocytes, giant type (MEG-G), with intussusception (emperipolesis) of granulocytes. Description see text (PV). Giemsa stain. x 400.

Fig. 29. Megakaryocytes, pleomorphic type (MEG-P) in a case of Meg M-P. Female patient of 78 years; blood: erythrocytes 2.1 million, leukocytes 2000, platelets 15000 mm3

• Gomori's stain. X 200.

Fig. 28. Megakaryocytes, different stages of maturation and mitoses around a sinus. Megakaryoblasts (bottom left side), immature megakaryocytes (bottom) and mature normal and dwarf cells (middle to top). Female patient of 64 years - development from PI (a) to Mix M; (b) to OMS; (c) = this picture; blood: a) erythro~tes 4.6 million, leukocytes 11000, platelets 2.5 million mm ; c) haemoglobin 11 g%, leukocytes 32000, platelets 422000 mm3• Giemsa stain. x 400.

Fig. 30. Megakaryocytes, pleomorphic type (MEG-P) in a case of Meg M-P. Separate nuclei situated close to the cytoplasmic margin. Female patient of 65 years; blood: erythrocytes 3.3 million, leukocytes 8000, platelets 28000 mm3

• Giemsa stain. X 400.

Fig. 31. Pleomorphic megakaryocytes, separate nuclei in abnormal situation as shown in Fig. 34. Incomplete erythroplasmic maturation (Meg M-P). EM 10 Carl Zeiss. X 2520.



Fig. 33. Megakaryocyte, complete pyknosis. Roundish conglomeration of nucleus. Previous lobulation marked by narrow rims. Massive condensation of nuclear substance at nuclear margin. Cytoplasm in dissolution. EM 10 Carl Zeiss. x 4000.

Fig. 34. Thrombocytopoiesis within marrow sinus, in a case of ~ PV. Description see text. Female patient of 73 years; blood: erythrocytes 7.0 million, leukocytes 16000, platelets 510000 mm3• Giemsa stain. x 400.

Fig. 32. Megakaryocyte with incomplete pyknosis. Bizarre nuclear deformation and patchy condensation of chromatine substance; irregular margin of rests of mature cytoplasm (OMS). EM 10 Carl Zeiss. x 2520.

. ...

Fig. 35. Interstitial platelet deposition, initial. Cytoplasm of megakaryocyte (part of the nucleus seen bottom left) with almost complete demarkation of platelets in the interstitial marrow space (Meg M). EM 10 Carl Zeiss. x 5000.

Fig. 36. Interstitial platelet deposition (!DP), complete. Numerous megakaryocytes are seen in different stages of platelet shedding. Masses of platelets are deposited within the marrow space. Same case as Fig. 36. Giemsa stain. x 800.


Fig. 37. Interstitial platelet deposition (!DP), complete. Same situation as Fig. 41. With the silver impregnation, numerous fine fibrils are seen to surround the single platelets. Gomori's stain. x 400.


60 micron was calculated at 13% for distinction between small and medium, and 21% for distinction between medium and large. Under the same conditions a normal average of 7.6 (± 2.7) megakaryocytes per 10 mm2 of a section of 3 micron was counted129

• Earlier experience in our laboratory of quantitative analyses of the megakaryocytes showed that the distinction applied in this review was reproducible in sections of adequate quality. However it should be remarked that many variations of the haematopoietic cell lines, characteristics of CMPD, in particular the megakaryocytes, cannot be integrated into a classification without rendering it almost useless. In doubtful cases or cases with mixtures of different types t~e

type was chosen according to the predominant one. It was expected that the investigation of a large number of cases would minimize the possible errors including the misinterpretation of marginally cut cells.

3. Inefficient Platelet Production: Megakaryocytes, as all haematopoietic cells, are produced outside the vascular system. They migrate to the vessels, shedding their products into the lumina, after partially or totally entering them34,221. Fig. 34 shows a megakaryocyte inside a large sinusoid, surrounded with a mass of platelets, some still connected to its cytoplasm. This normal function is disturbed in CMPD as has been shown earlierl4, 25, 32, 35, 39,

Fig. 38. Polycythaemia vera, ERY-type. Description see text. Femal patient of 56 years; blood: erythrocytes 6.0 million, leukocytes 6000, platelets 132000 mm3• Giemsa stain x 200.

Table 2. CMPD - classification according to predominant cell lines

Predom. Clinical % of Median Cell-line(s) Diagnosis CMPD Survival

(months)

MEG GRAN ERY PV 22 79 (n = 275)

MEG -- ERY 21 84 GRAN ERY 3 99

ERY 2 115

GRAN --- CML 18 26 (n= 247)

MEG GRAN --- 27 35

MEG -- PT (n = 60) 7 82

Fig. 39. Polycythaemia vera, GRAN-type. Description see text. Male patient of 49 years; blood: erythrocytes 9.2 million, leukocytes 24 000, platelets 406 000 mm3


Most fre-quentMeta-morphosis

MF-OMS

MF-OMS a a

BC

MF-OMS

MF-OMS


136, 170. Fig. 35 shows the demarcation of parts of mature cytoplasm from a megakaryocyte in the interstitial space in the electron microscope with low magnification. The light microscopy (Fig. 36) shows this phenomenon spread over a wide marrow area: the so called interstitial deposition of platelets (IDP). In the same case, with Gomori's stain, it can be seen that a very fine, filigrine-like fibrous structure surrounds the platelet deposits together with the megakaryocytes (Fig. 37). This is interpreted as the effect of the fibroblast activating factor mentioned above. Since the c-form (cluster-form) of megakaryocytic proliferation is very frequent in CMPD, and many of these clusters show the IDP-phenomenon together with MF, it has been suggested that IDP is one of the causes of fibrotic transformation in CMPD25, 34, 35, 52. These results provide some additional criteria to classify the variety of CMPD-manifestations:


B. Histologic Subgroups/Variants

1. Ery M = Erythrocytic Myelosis, (din. polycythaemia vera)

Variants: ERY/GRAN/MEG GRAN MEG ERY

The significance of the abbreviations of ER Y, GRAN, MEG, and their combinations to indicate the predominance of one or more of the haematopoietic cell lines in

CMPD needs no explanation, any more than does the addition of "Blast" and of MF or OMS when immaturity or fibrotic transformations are referred to. Using the predominant cell-line, PV can be subdivided into the four variants demonstrated in the Table 2. Fig. 38 illustrates prominent erythroblastic proliferation, the ERY-type, Fig. 39 the GRAN-type, and Fig. 40 the MEG-type. The classical ERY/GRAN/MEG-(trilinear) type has already been demonstrated (Fig. 10). These types are variations of the basically trilinear cell proliferation that is typical for PV. They exhibit the following types of megakaryocytes: Normal, D = dwarf (rare cases), and G = giant (most

Fig. 40. Polycythaemia vera, MEG-type. Description see text. Male patient of 49 years; blood: erythrocytes 6.4 million, leukocytes 24000, platelets 358000. Giemsa stain. x 200.

cases, especially in ERY/GRAN/MEG and MEG-variants). In these cases the cluster-distribution predominates; in the majority of cases the megakaryocytes are diffusely arranged.

2. Gran M = Granulocytic myelosis, Dwarf MEGType, (clin. chronic myeloid leukemia)

Variants: GRAN (EOS)Gran/dDMEG (EOS)GRAN/cDMEG

Fig. 41. Chronic granulocytic myelosis, Gran M-cD. Description see text. Male patient of 49 years; blood: erythrocytes 3.6 million, leukocytes 74000, platelets 361000 mm3

•

Giemsa stain. X 200.


Eosinophilic variants occur in all subgroups, generally combined with proliferation of megakaryocytes. Another variant with few megakaryocytes can also be distinguished. Basophilic variants are extremely rare112

• The common neutrophilic/eosinophilic type shows considerable increase in dwarf-megakaryocytes (Fig. 12). Fig. 41 illustrates a variant of the common neutrophilic type distinguished by the arrangement of the D-megakaryocytes in clusters. Variants with a bandlike increase in promyelocytes have been distinguished as PRO-types (Fig. 42) which are especially prone to blastic metamorphosis. The significance of these variants has been demonstrated in Table 2.


3. Meg M-G = Megakaryocytic Myelosis, Giant Type, (din. primary thrombocythaemia)

Variants: dG cG

Typical PT is most frequently correlated in the bone marrow with the dG-MEG variant, without remarkable proliferation of the other cell lines (Fig. 14). The cG-variant probably represents a more advanced stage.

4. Mix M = Mixed Myelosis, Giant MEG Type, (din. subleukaemia, with or without thrombocythaemia)

Variants: dG-MEG/GRAN cG-MEG/GRAN

This preliminary distinction was made in order to separate some prominent variants that have already been recognized in previous classification studies39

, 89, 91, 92. They differ from typical CML and PT by an abnormal prolifera-

Fig. 42. Chronic granulocytic myelosis, Gran M-PRO. Description see text. Female patient 70 years; blood: erythrocytes 3.6 million; leukocytes 66 000, platelets 15 000 mm3

•

Giemsa stain. x 200.

tion of giant megakaryocytes either diffusely distributed (Fig. 43), or in clusters (Fig. 44) in combination with granulocytopoiesis. These variants have been generally classified either with PT or CML. In the latter case they have been understood as variants of CGM = chronic granulocytic myelosis: CML being the one, CMGM = chronic megakaryocytic granulocytic myelosis the other. As will be shown later (p.169, table 5) the blood cell counts of the dG-variant are closer to CML, of the cGvariant closer to PT. There are also other differences between these variants that are not easily reconciliated with their integration into one common group.

Fig. 43. Mixed myelosis, MegGran M-dG. Description see text; blood: erythrocytes 5.2 million, leukocytes 21000, platelets 1.0 million/mm3



5. Meg M-I1Meg M-P = Megakaryocytic Myelosis, Immature and Pleomorphic Types, (clin. nonsignificant)

Variants: cP-MEG sP-MEG sl-MEG

This group is distinguished by the unique predominance of immature and/or pleomorphous megakaryocytes. Because of its lesser frequency compared with PV and CML, and its generally nonsignificant clinical symptoms, it was for long not distinguished from agnogenic myeloid metaplasia. That the immature type of megakaryocytic


myelosis is a separate entity, is evident from its histology. Fig. 45 shows the comparatively uniform picture of this pleomorphic counterpart of PT, the sP-MEG variant. The latter shows incipient MF, a transformation that occurs especially frequently in this type. Follow-up biopsies have shown that this type often appears also as the transitional

stage between one of the classical groups of CMPD, especially CML, and myelofibrosis or osteomyelosclerosis. Fig. 46 gives an example of Meg M-sI developing from CML, separated by a girdle of blast cells, probably megakaryoblasts, from the typical changes of the original CML.

Fig. 44. Mixed myelosis, MegGran M-cG. Description see text; blood: erythrocytes 4.9 million, leukocytes 17 000, platelets 1.2 million/mm3• Giemsa stain. x 200.

Fig. 45. Megakaryocytic myelosis, Meg M-P. Description see text; blood: erythrocytes 3.6 million, leukocytes 11000, platele'ts 95000 mm3• Giemsa stain. X 400.

Fig. 46. Megakaryocytic myelosis, Meg M-I (top of picture), developing in CML (bottom of picture). Megakaryocytic proliferation departs from girdle of megakaryoblasts that separate Gran M from Meg M. Male patient of 33 years; blood: erythrocytes 2.8 million, leukocytes 144000, platelets 134000 mm3

.

Giemsa stain X 250.


C. Terminal Stages

1. MF = Myelofibrosis, Variants: strand-pattern; Grades 1-2

honeycomb-pattern; Grades 1-2

Fibrotic transformation occurs in each group of CMPD, though in different proportions. Since this development is of considerable practical interest, a sufficiently exact, and easily applicable distinction of the different grades, or stages, is necessary, as previously shown1o,47, 135. For comparison the reinforcement of the normal reticular structure (demonstrable only in silver impregnation) is shown in


Fig. 47. This socalled reticular-sclerosis has been designed as "early MF,,92.1t is also found in many other conditions, especially malignant lymphoma; it should not be confused with the collagenous type, since reticular-sclerosis may remain unchanged for periods of more than 10 years in CMPD46

, in contrast to the collagenous fibrosis that in most cases tends to progressive development (Table 3).

Considering the evident topical variations in the fibrotic process, only two grades for the semiquantitative characterization were used (Fig. 48). Grade 1 of MF was defined by the presence of at least three bundles of collagenous strands, using the Gomori stain, within a microscopic field

Table 3. CMPD - control of fibrotic bone marrow changes

First Biopsy

Reticular Sclerosis Collagen-Fibrosis

Control Biopsy

_MF-OMS _MF-OMS

25% 82%

at a magnification of 200 (Fig. 49). Grade 2 of MF was characterized by further increased fibre formation (Fig. 50). The pattern generally resembles a tight mat of

Fig. 47. Reticulin-sclerosis of bone marrow in a case of CML. Description see text. Female patient of 52 years; blood: erythrocytes 3.9 million, leukocytes 60000, platelets 162000 mm3• Gomori's stain. x 200.

Chronic Myeloproliferative Disorders (CMPO) . 163

CMPD - MYELOFIBROSIS

Reticular Seier. Grade 1 MF Grade 2 MF

CMPD - OSTEOMYELOSCLEROSIS *

Grade 1 OMS Grade 2 OMS Grade 3 OMS

* marrow fibrosis not indicated

Fig. 48. Schematic representation of different stages of CMPO - myelofibrosis and osteomyelosclerosis

crossing sisal fibres, mostly following the direction of capillary vessels, trabecular surfaces, or strands of cells. This arrangement is mostly found in the marrow regions where a partially immature eosinophilic granulocytopoiesis predominates. Focal areas of immature haematopoiesis or lymphocytic infiltrations are not affected by this type of fibrosis. A remarkable variant of both grade 1 and 2 shows honeycomb-like fibrous structures enclosing every cell instead of the straight bundles that are not as closely arranged (Fig. 51). The two variants are also found in different parts of the same biopsy, and it cannot yet be decided whether they represent consecutive stages of fibrous development, or signify a different pathogenesis. The honeycomb-structures are more often found between the interstitial deposits of platelets, accumulations of immature or pleomorphous megakaryocytes (and clusters of tumour cells of metastatic origin).

2. OMS = Osteomyelosclerosis, Grades 1-3

The distinction of three grades of osteomyelosclerosis has already been shown (Fig. 48). The fact that osseous remodelling is marked by the easily distinguishable osteoblasts and osteoclasts makes it easier to estimate the activity in osteosclerosis than in myelofibrosis, especially when grade 3 changes are present. Our example (Fig. 52) presents the general view of the characteristic filigree architecture caused by the packing of the marrow spaces with irregular sprouts of osteoid tissue, that replace the resorbed trabeculae. Obviously this process is dissimilar to the primary and secondary osteosclerotic disorders of the marble bone type, however it is similar to carcinomatous osteodysplasia4

• In a single biopsy section we observe areas with osteoblasts forming sprouts of primitive bone (Fig. 53), as well as osteoclasts resorbing mature


trabeculae and osteoid (Fig. 54), and large compartments of osteoid substance without a sign of remodelling (Fig. 55). The former areas generally coincide with residual myeloproliferation and inflammatory cells, the latter with the fatty and edematous regions more often found in OMS than in MF.

In the most "active" regions there is osteoblastic activity, inflammation, and endothelial sprouting in a narrow field of mesenchymal activation (as also seen in para-

trabecular areas in various conditions of rapid osseous remodelling); indicating that OMS is a secondary condition (Fig. 56). CMPD-OMS may reach some degree of stability together with reduction in myelopoiesis, possibly as a consequence of therapy. In this case the massive osseous sprouts exhibit only inactive surfaces, and enclose marrow spaces filled with a loose edematous and sclerotic tissue, containing many mast cells but only few remnants of myelopoiesis, mostly inside the sclerotic sinusoids.

Fig. 49. CMPD-mye!ofibrosis grade 1 in a case of previous PV. Description see text. Female patient; blood: 3.9 million erythrocytes, leukocytes 5000, platelets 100 000 mm3• Gomori's stain. x 200.

3. BC = Blast crisis

Significant is the increase in immature cells, generally concentrated to the para trabecular and perivascular bone marrow regions. There are cases with homogeneous cell population similar to acute leukemia. The majority however shows various aspects: the combination of immature megakaryocytes and megakaryoblasts either with pro myelocytes or with proerythroblasts are among the most frequent (Fig. 57).

Fig. 50. CMPD myelofibrosis grade 2 in a case of previous PV. Description see text. Male patient of 49 years, PV since age of 21 years, now leukaemic; blood: erythrocytes 5.7 million, leukocytes 120000, platelets 245000 mm3

• Gomori's stain. x 250.


D. Synopsis

1. Clinico-Histologic Correlations; General Survey

During the last years several attempts have been made to correlate the prognosis of PV, CML, PT, and the CMPD-MF/OMS, with the different histologic aspects of these groups39, 83, 92. They have shown that clinical and histologic criteria may be useful but that it is difficult to integrate them into a common classification that is accessi-


<III Fig. 51. CMPD-myelofibrosis grade 2, honeycomb-pattern, in a case of OMS. Description see text. Male patient of 65 years; blood: erythrocytes 4.0 million, leukocytes 11 000, platelets 80000 mm3• Gomori's stain. x 400.

Fig. 53. CMPD-osteomyelosclerosis grade 3. Region with osteoblastic osteoid formation. Only a few pyknotic megakaryocytes are seen (top of figure). Amidst fatty tissue (bottom left) one osteoclast and two preosteoclasts. Same case as Fig. 29. Giemsa stain. x 200.

<III Fig. 52. CMPD-osteomyelosclerosis grade 3. Description see text. Female patient of 66 years; blood: erythrocytes 3.7 million, leukocytes 15100, platelets 200000 mm3• Ladewig's stain. x 20.

Fig. 54. CMPD osteomyelosclerosis grade 3. Region with osteoclastic remodelling (middle of figure), osteoid sprouts covered with flat cells whowing no sign of activity. Same case as Fig. 29. Giemsa stain. X 200.

ble from the clinical as well as histopathologic sides. There are however numerous cases that need elucidation from both aspects. When the WHO-classification was applied to our cases in an earlier stage of our studies, we found the

Table 4. Chronic Myeloproliferation Review of own cases according to WHO-classification (1976)

Number Number Percentage of biopsies of patients

CML + subgroups 625 544 19 Chronic erythroblastosis 95 76 3 Polycythaemia vera 820 732 26 Idiopathic thrombocythaemia (platelets> 700 thousand) 218 188 7 Myelosclerosis with myeloid metaplasia 735 641 22 CMPD (unclassified) 922 668 23

Total 2849 100%

Number of patients with more than one biopsy control: 379


Fig. 55. CMPD osteomyelosclerosis grade 3. Region with sprouts of primitive bone and hardly any sign of remodelling. A few megakaryocytes and granulocytic haematopoiesis are seen. Same case as Fig. 29. Giemsa stain. X 200.

distribution as indicated in Table 4. About 23% of our series could not be fitted into the WHO-scheme. To study these more closely we have re-examined only the untreated cases in our series, selected according to a biopsy size of at least 4: 10 mm2 for drill, and 2: 20 mm2 for the manual trephine, and the availability of clinical information obtained at the time of the biopsy, in particular the blood counts. The study included 1009 biopsies of 850 patients, from a total of 3411 biopsies of CMPD patients collected between the years 1960 and 1982. The preselection of the whole group comprised all biopsy cases from a total of 2870 patients in whom clinically and/or histologically, the probability, or the certainty of CMPD could be documented retrospectively, and other diagnoses were excluded. Cases that were clinically only suspicious were also accepted, provided that CMPD was clearly identified in the biopsy. 16 clinical and 22 histologic criteria were registrated in each case, and statistically evaluated using BMPD computer programs (2D, 3D, 4F, lL).

The following clinical criteria: a) = obligatory, b) = facultative were used for preselection: PV: a) red cell counts> 6.5 millions respectively corre

sponding values of Hb and haematocrit, and exclusion of secondary erythrocytosis;


b) leukocyte and platelet counts, blood sedimentation rate, size of spleen and liver, the level of LAP, vitamin B12, and erythropoietin (if available).

CML: a) leukocyte count > 40 thousands/mm\ typical blood smear and exclusion of secondary leukocytosis; b) chromosomal analysis, LAP, size of spleen and liver, erythrocyte count, and level of vitamin B12 (if available).

PT: a) thrombocyte count> 700 thousands/mm3, and

exclusion of all other reasons of thrombocythaemia; b) splenic size, evidence of circulatory disturbances.

X: The non typical cases with various clinical and haematologic findings: a) clear proof of CMPD in the biopsy, or proof of untreated CMPD in the previous history; exclusion of severe infections or neoplastic disorders of other origin; b) clinical and haematologic status.

The histologic criteria have been outlined in the previous sections. The re-evaluation was done by two independent investigators. It comprised 5 courses for the semiquantitative registration of 22 criteria, each course performed without considering the results of the previous ones.

In the first evaluation the overall view was considered in order to characterize haematopoiesis, fatty tissue, bone, vessels, and

Fig. 56. CMPD-osteomyelosc!erosis, zone of mesenchymal activation showing from top to bottom: osteoid substance, osteoblastic seam, loose connective tissue with sprouting fibroblasts and endothelial cells, plasma cells, and pyknotic megakaryocyte surrounded with !DP. Giemsa stain. x 400.


... Fig. 57. BC, pleomorphic type. Masses of immature and atypical haematopoietic precursor cells are accumulated in the paratrabecular bone marrow areas, among them megakaryoblasts, promyelocytes, and erythroblasts. Female patient of 59 years; blood: erythrocytes 2.5 million, leucocytes 3000, platelets 71 000 mm3


reactive overall changes; in the second the classification of MF, OMS, and osseous remodelling; in the third the quantity, distribution, and type of the megakaryocytes were estimated; the fourth was devoted to the interstitial deposits of platelets, and the fifth to the quantification, distribution, and localization of the mast cells. Each criterion was assessed using a semiquantitative scale set according to the standards derived from the study of 158 normal biopsies of all age groups. Generally 2-4 grades were used for registration of the range of one criterion, e.g. normal -increased - strongly increased; or absent - present - strongly present; or diminished - normal- increased - strongly increased. The reproducibility of this procedure was checked by re-testing of samples, which showed a high degree of conformity. This may be due to the fact that the attention was focussed on distinct groups of criteria at a time in order to keep the standard of comparison in mind.

To begin with, the evaluated clinical and histologic data were grouped according to the clinical diagnoses, the different values for the blood leukocyte and platelet counts, and the types of the megakaryocytes. By this way a table was designed for comparison of the prominent clinical and histologic criteria (Table 5).

Table 5. CMPD: 850 Biopsies of Untreated Patients. Correlation of Haematologic Data with Histologic Diagnoses

Vertical Columns: Blood Counts E = Erythrocytes (mill.); L = Leucocytes (thous.); Thr = Thrombocytes (thous.) fmm3

Al A2 E > 6.5 5.5-6.5 L < 30 < 30 Thr * *

Ery M n = 200 72 25

GranM n = 132

Meg M-G n = 58 3

Mix M-c n = 67 7

MixM-dG n = 61 5

Meg M-P n = 56 4

MF grade 2 n = 78 4 4

OMS grade 2 + 3 n = 160 1 2

BC n = 38 5

* Thrombocytes not considered

BI < 5.5 > 30 *

82

2

4

18

11

6

7

16

B2 < 5.5 15-30

0.5

13

14

24

48

18

12

11

CI C2 D < 5.5 < 5.5 < 5.5 <15 < 15 < 15 > 700 300-700 < 300

0.5

2

47 28

24

8 8 8

5 5

6

1 14

3 3

M Rest of Cases

2

3

6

10

5

5

1

%

%

%

%

%

%

%


The groups in the headings are defined exclusively by the erythrocyte, leukocyte, and platelet counts in the peripheral blood taken at time of biopsy. The purpose was to provide a scale with as much clinical significance as possible, applicable also to those cases that could not have been included in other groupings. The limits were set according to the overall survey of blood values among our patients. A practical identity of the group At with PV, Bt with CML and Ct with PT was attained. Thrombocythaemia of > 700 thousands/mm3 occurred in groups other than PT in the following percentages: At = 7%; Bt = 13%. Cases with less significant peripheral blood values were grouped accordingly into A2, B2, and C2. The percentages of thrombocythaemia in these groups were: A2 = 8%; B2 = 20%. The D-group comprises the cases with normal to low blood counts, many of them comparable clinically with "agnogenic myeloid metaplasia". The M-group is composed of a few cases exhibiting blood cell combinations of erythrocyte counts above 5.5 millions with leukocytes above 30 thousands/mm3

, and frequently also thrombocythaemia (12 from 29). These combinations could not be included in the other groups without rendering the chosen separation worthless.

The histologic groups (vertical left) were designed according to the definitions given in the previous section. Clinical criteria were not considered. Among PV, a subgroup of histologic borderline cases, consistent with the diagnosis, was provisionally separated, but not indicated in the table since its correlation with the blood values was identical with the typical cases. In each main group a ten-

tative subdivision was made to distinguish those cases that already showed myelofibrosis br osteomyelosclerosis of grade 1 together with the characteristic changes of CMPDgroups. Only small proportions of the classic groups PV, PT, CML were slightly fibrotic (5-9%), the mixed, pleomorphic and blastic groups however to much higher degree (31-68%), highest in Be. This subdivision was also omitted, because the correlations with the blood values were very similar to the non-fibrotic cases in each group. Ery M (PV), Gran M (CML), and MF/OMS are most numerous. Meg M-G (PT), the Mix-groups, and Meg M-P show almost equal numbers. BC is the weakest group. The classic groups are closely correlated with the corresponding blood values. Apart from Ery M, small proportions of all groups with the exception of Gran Mare erythraemic and apart from Meg M-G the same is true of thrombocythaemia, especially for Mix M-c. In the leukaemic and subleukaemic range we find again all groups except of Ery M, especially Mix M-d. The immature and fibrosisgroups are mainly cytopenic. BC range mainly among D, however smaller proportions among all, especially the leukaemic and sub leukaemic groups.

These results show that there is good correlation between the typical histologic Ery- and Gran-groups and their haematologic expression as PV and CML. Less impressive is the correlation between Meg M-G and pure thrombocythaemia. The rest of the histologic groups exhibit by no means clear haematologic correlates. Two circumstances should be mentioned: the haematologic uniformity of the immature and fibrosis-groups and the

Table 6. Correlation between Clinical and Histologic Subgroups, Clinical Data, and MF/OMS

Clinical Groups Histologic Groups Gran M: Mix Meg M-G: Mix Mix M-dG: Mix

BJ : Bz C1 : Cz Bz: Cz M-dG M-cG M-cG

Agej p = 0.0001 j P = 0.01 j

S?-Sex i p = 0.007 i p = 0.03 i Splenomegaly p = 0.0000 i p = 0.01 i p = 0.0001 i p = 0.0005 j

Hepatomegaly p= 0.0002 j

j 1

LAP P = 0.001 P = 0.01 j

Bone marrow j p = 0.0000 j p = 0.04 j P = 0.0000 j p = 0.01 j sinusoids

MFj p = 0.01 j p= 0.006 j p= 0.0000 j p = 0.0002 j

OMSj p=O.Ol j p =0.0000 j p = 0 .0006 i p = 0 .004 j

S?-sex also i for Mix M-cG: Gran M p = 0.04 Al :C1 p= 0.07 B1:C1 p=O.OO13

Gran M younger than other CMPD, p = 0 .007 (except of MF/OMS) Gran M younger than Mix M-dG, p= 0.0000 Definition of groups: see text p. 169

difference between the two Mix-groups. While 66% of the d-form are leukaemic or sub leukaemic, 40% of the c-form are thrombocythaemic.

2. Statistical Evaluation

The statistical differences between the strong and weak haematologic groups, and the histologic classic and mixgroups are shown in Table 6 the sub leukaemic group (B2) is distinguished from the leukaemic (B1) by older age, hepatomegaly, greater number of sinuses in the bone marrow, and complication with MF/OMS. The group with weak thrombocythaemia (C2) differs from typical thrombocythaemia (C1) in a similar way, except for splenomegaly instead of hepatomegaly and no evidence for age predilection. Among the weak haematologic cases, the thrombocythaemic (C2) comprise significantly more females and more OMS than sub leukaemic (B2) patients.

On the histologic side Mix M-d differs from Gran M (CML) in the sex proportion, in lower incidence of

Table 7. Correlation between MF/OMSIBC and Histologic Data

Histologic MF: OMS MF/OMS: other BC: other

Data CMPD CMPD

Fatty tissue p = 0.0000 i Mast cells p= 0.0006 i i

p = 0.0000

Inflam- i mation

p= 0.0000

BC i i p = 0.0005 P = 0.0000

MEG i -number p = 0.04

-!DP i p = 0.0000

-d i i p = 0.05 P = 0.0000

-c i p = 0.0000

-s i i p = 0.0000 p= 0.0000

-D i i i p = 0.0001 P = 0.0000 p= 0.03

-G i p = 0.0000

-PII i i i p = 0.0000 p = 0.0000 p= 0.0000

-Py i i p = 0,03 p = 0.0000

Definition of groups see p. 169


splenomegaly and greater proportions of bone marrow sinuses and OMS. The mix M-c differs from Meg M-G (PT) in greater proportions of splenomegaly, sinuses, and MF. Among the Mix-groups is Mix M-c distinguished by older age and greater proportions of splenomegaly and MF. Generally speaking, the weak haematologic and mixhistologic groups differ from their typical counterparts by hepatosplenomegaly (except Gran M), increased venous capillaries in the bone marrow, and much higher proportions of MF/OMS. The percentages of MFIOMS (grade 1) among the haematologic groups are: A1 (PV) = 3 %; A2 = 11 %; B1 (CML) = 11 %; B2 = 26%; C1 (PT) = 2%; C2 = 41 %; D = 67%; M = 21 %. The correspondent figures for histology (grades 1 MF/OMS only) are Ery M (PV) = 8%; Gran M (CML) = 7%; Meg M-G (PT) = 9%; Mix M-d = 10%; Mix M-c = 39%; Meg M-P/I = 52%; BC = 67%.

MF and OMS are in many ways correlated with histologic and clinical data. Table 7 shows that fatty bone marrow changes in association with increase in mast cells occur more frequently in OMS. Inflammatory changes are correlated with both MF/OMS. Equally impressive is the correlation of MF/OMS with anomalies of megakaryocytes: the cluster- and sheet-form is significantly more frequent in MFIOMS than the diffuse form, as well as interstitial deposits of platelets (IDP) and the giant, pleomorphic, immature, and pyknotic types, compared with the non-fibrotic variations of CMPD. MF on the other hand differs from OMS by higher proportions of megakaryo-

Table 8. Correlation between MF/OMSIBC and Clinical Data

Clinical MF: OMS Data

MF/OM : other S CMPD

C other B :

CMPD

Agej i p = 0.0000

Spleno- i megaly

p = 0.001

Hepato- i megaly p = 0.0000

LDH i i p = 0.0000

LAP i i p = 0.001

Leuko- p = 0.0000 i p = 0.0000 i cytosis

Thrombo- p = 0.0000 i p = 0.0000 i cytosis

Survival i p = 0.0000* i

Survival 33 30 4 (median! n=51jn=109 n=27 months)

* Breslow- and Mantel Cox tests


Table 9. CMPD: Correlations between variation of megakaryocytes (MEG) and other histologic changes in the bone marrow biopsy

MEG-Data Histologic Distribution Cell type Thrombo-Data d c s D G P/r Py IDP cytosis

Fatty i tissue p = 0.01

Siderin i p = 0.0000

Imflam- t mation p = 0.0000

BC i i t p = 0.0000 p= 0.0000 p = 0.0000

MFIOMS t i i t i i i i t p = 0.0000 p = 0.0000 p = 0.0000 p = 0.0000 p = 0.0002 P = 0.0000 P = 0.0000 P = 0.0000 P = 0.0000

Marrow i SInuses p = 0.0000

IDP i p = 0.0000

MEG-distribution: d = diffuse c = cluster s = sheets

i p = 0.0000

MEG-cell type: D = dwarf G = giant P/r = pleomorphic/immature Py = pyknotic cells IDP = interstitial deposits of platelets

i p = 0.0000

Table 10. CMPD: Correlations between variations of megakaryocytes (MEG) in the bone marrow biopsy and clinical data

MEG Data Clinical Increase Distribution Cell Type Data in number d c s D G P/I Py

Age t i p = 0.001 P = 0.001

~-Sex i i p = 0.0000 p = 0.0001

Spleno- i megaly p = 0.0000

APH i i p= 0.02 p= 0.02

LDH i i p= 0.02 P = 0.001

LAP t i i p = 0.01 P = 0.01 P = 0.0001

Erythro- i t i t cytosis p= 0.002 P = 0.05 P = 0.0000 P = 0.0000

Leuko- i i t t i t t t cytosis p = 0.0003 p = 0.0000 p = 0.0000 p = 0.003 P = 0.0000 P = 0.0001 P = 0.0000 P = 0.02

Thrombo- i t i t i t t cytosis p = 0.0000 p = 0.0000 p = 0.0005 P = 0.002 P = 0.0000 P = 0.0000 P = 0.01

Survival

MEG-distribution: d = diffuse c = cluster s = sheets

i i p = 0.005 P = 0.0001

MEG-cell type: D = dwarf G = giant P/r = pleomorphic/immature Py = pyknotic cells IDP = interstitial deposits of platelets

Thrombo-IDP cytosis

i P = 0.001

t p = 0.0000

cytes, expecially in d-form, and dwarf, immature and pyknotic types. The expansion in sheets belongs to the characteristics of BC; blastic proliferation exceeds also in production of dwarf, pleomorphic, and immature megakaryocytes, it is also correlated with MF. Not only in MF but also in MegM-P/I and in PV a remarkable increase in lymphoid nodules was found. This point is remarkable since the genetic relation between CMPD and B-celilymphoma is known and several studies have suggested that a more than random correlation especially of CLL and PV may exist39, 188 • Whether the lymphoid nodules are pre-stages of dedifferentiation of these rypes of CMPD, or merely products of an immunologic reaction has not yet been decided.

The correlations of MF/OMS with some clinical data are given in Table 8. This group is older, lives shorter, shows more pronounced hepatosplenomegaly, more cases with elevated levels of LDH in the blood and lower leukocyte and platelet counts than the rest of non-fibrotic CMPD. In BC the mean leukocyte and platelet counts are lower than in the rest of the groups. The survival rates of MF and OMS are almost equal, of BC lowest of all groups. Leucocyte alkaline phosphatase is more frequently elevated in MF than OMS.

A few additional details of a possible role of megakaryocytic anomalies in CMPD are demonstrated in Table 9. The correlation of the cG-MEG type (clusters of giant megakaryocytes) with MF/OMS is associated with increase in sinus capillaries and, in the case of clusterforms, with interstitial deposits of platelets. The formation of megakaryocytic clusters may impair the contact with the sinuses and exert some influence on platelet-production and/or release. Thrombocytosis is correlated with increase in marrow sinuses, in fatry tissue, and in siderin deposits, conditions that are common in Meg M-G. Inflammation, MF/OMS, and BC however are correlated with low platelet values. Significant correlations also exist between megakaryocyte anomalies and clinical data (Table 10). The outstanding frequency of splenomegaly when the megakaryocytes are diffusely distributed is


Table 11. CMPD: Histologic and haematologic variants occuring more frequently in female patients

~ Sex more frequent p-values m: compared with : (Fisher test)

PT - din. CMPD other ' 0.001 PT-C t CML- Bl 0.001 PI - C2 CML- B2 0.007

Mix M-dg Gran M 0.03 Mix M-cG Gran M 0.04

G-MEG (cell type) other MEG-types 0.0001 c-MEG (distribution) d-MEG 0.05

Thrombocytosis lower platelet 0.01 counts/blood

Normal leukocyte : leukaemic blood 0.001 counts/blood values

Median survival of females longer than that of males among CMPD in general:

Median survival of females longer than that of males among CMPD with normal numbers of megakaryocytes:

Median survival of females longer than that of males among CMPD with increase in megakaryocytes:

• except of MF/OMS and BC

p = 0.04 (Breslow test) p = 0.03 (M.-Cox test)

no significance

p = 0.06 (Breslow test) p = 0.04 (Mantel Cox test)

no female predominance for /DP, MF/OMS, stages of MF/ OMS

Definition of groups see p. 169

assumed to reflect mainly the situation of CML. Considering the cluster-form and the giant type of megakaryocytes together (cG) we find parallel correlations of both not only with thrombocytosis, leukopenia and longer survival, but also with elevated levels of APH and LAP, and female predominance. Giant megakaryocyte clusters are found

Table 12. Comparison of 295 primary and sequential bone marrow biopsies in 126 patients untreated at initial biopsy

Histologic Diagnosis Number of Biopsies Primary Follow-up Patients no Gran M MEG-G MEG-lIP MF OMS BC

change duster j immaturej

36 91 Ery M (PV) 10 12 3 2 3 6 16 38 Gran M (CML) 1 2 3 4 5 1 5 10 Meg M-G (PT) 1 2 2

17 41 Mix M-cG 4 2 2 4 3 2 6 14 Mix M-dG 1 1 1 1 1 1

16 38 Meg M-IIP 1 xMF 1

1 4 5 5

19% 13% 10% 12% 20% 22% 4%

11 23 MF 4 l+MF 3 j 1j 2 19 40 OMS 2 17 j

126 295 Mean interval between first and last biopsy = 36 months min. 1, max. 188 months


especially in CMPD with long survivals and older population (PV and PT) than dwarfs (CML), however only the giant type and not the cluster form is distinguished by its correlation with advanced age.

The female sex is strikingly more frequent not only among PT in our cases (in contrast to the male predominance among the rest of major groups, and to the literature) but also among the cG-MEG anomaly (Tables 6,10 and 11). This was not only proven for PT, but also for the giant megakaryocytes and cluster forms in other groups (e.g. Mix M and PV). No female predominance exists in MFIOMS (all stages), and in cases with ineffective thrombocytopoiesis. This seems contradictory to the frequency of the cG-anomaly especially in cases with marrow fibrosis and IDP. On the other hand, thrombocytosis is significantly more frequent in the females of our material and females with CMPD have a better prognosis than males, exspecially when the cases with large and normal numbers of megakaryocytes are compared (Table 11).

3. Conclusions of Statistical Evaluation (Tables 5-11)

These results suggest that females might be less prone to the consequences of neoplastic megakaryocytopoiesis, ineffective platelet production and MFIOMS, and better prepared to control the egress of platelets also under abnormal conditions. They add further evidence to the prominent role of abnormal megakaryocytopoiesis among all groups of CMPD. In accordance with the experimental studies on fibroblast stimulating and collagenase inhibiting factors of megakaryocytes and platelets, and with the electron microscopic observations (v.p. 133) it can be assumed that the significant statistical correlation of IDP and MFIOMS in our material indeed indicates a causal relationship. Clusters of giant megakaryocytes are among the sources of abnormally increased platelet production. They are correlated not only with IDP but also with thrombocythaemia. Thrombocythaemia is not a specific criterion of PT but of many other entities of CMPD. But thrombocythaemia is rare in MFIOMS and the permanent thrombocythaemia of PT shows the lowest rates of MFI OMS and BC among CMPD. From these facts a hypothetical natural history of MFIOMS and BC can be drawn: megakaryocytic proliferation in CMPD is prone to proceed from normal to giant polyploidic types, and from the diffuse increase to cluster formation. In this stage platelet formation gets ineffective, and the fibrotic transformation is induced. Exactly this development has been stated with numerous follow-up biopsies, as will be shown later. The correlation of BC with MFIOMS on the other hand is less obvious. The proof of erythroblast stimulating activity from platelet extracts (v.p. 146) points to the possibility of induction of the MPD to a more immature level by a similar factor of platelets, accumulated in abnormal amounts in the tissue when thrombocytes are not extruded into the circulation. Another possibility is the blastic metamorphosis of the neoplastic cell clones due to unknown reasons.

Dwarf megakaryocytes also occur in clusters producing thrombocythaemia and IDP. This has been observed in several cases of CML. Sequential biopsies demonstrated the transformation of the normal Gran M type into Gran Meg cD, and to sheets of pleomorphic, small megakaryocytes with increasing immaturity, deposition of platelets, and fibrosis. Even at this stage there may exist thrombocythaemia. Follow-up studies indicate that fibrosis and blastic metamorphosis may develop at the same time. In this case, immature or pleomorphic megakaryocytes are arranged in sheets, surrounded with masses of blastic elements among which promegakaryocytes, proerythroblasts and promyelocytes may be distinguished. In a previous study we have shown that unilinear proliferation of neoplastic haematopoiesis in CMPD shows minimal rates of fibrotic transformation39

; MFIOMS were most frequently observed when granulocytes and megakaryocytes expanded together, as in PV of the MEGIGRAN subtype and in the Mix M types. An exception is formed by eosinophilic CML (36 cases). 11% had platelet counts of > 1 million mm3 in the blood and large amounts of dDMEG in the bone marrow. 0% were complicated with MFI OMS. The same figures for neutrophilic CML were 2% and 7%. Whereas increase in mast cells is correlated with MFiOMS (Table 7), eosinophilia of the bone marrow may exert some inhibitory influence on the development of fibroSIS.

4. Case Control Studies

We would have a better idea of the natural history of CMPD if we were able to follow a greater number of individual cases during the whole period of illness with sequential biofssies. Until now only few such studies have been reported 9,92. Table 12 gives the present stage of our study, in which only those cases were included in which a biopsy at time of initial diagnosis and before specific treatment was obtained. 126 patients were monitored with 295 biopsies for periods of 1 to 188 months. No change was seen in the controls of non-fibrotic CMPD in 19% of the cases; 13 % developed into Gran M, mostly from Ery M, and 22 % into the more abnormal stages of MEG-ca and -lIP, 42% into MFIOMS, and4% into Be. About the same percentages of unchanged biopsies were found when MFI OMS were present from the beginning; about 70% proceeded to more advanced stages of fibrosclerosis, and two cases ended with Be. No BC was observed in 36 cases of initial PV, 5 cases of PT, 16 cases of Meg M-I1P, and 19 cases of OMS. These numbers are too small and unequal to establish a different development for the single groups. In general, progression of the disease is indicated by change from the typical histologic pictures to clustered or abnormal megakaryocytes and, finally, to MFIOMS in all groups. There are several cases in which a passage through each of these stages was documented with 3 to 5 sequential biopsies. The rates of blastic metamorphosis were comparatively low. When all cases were included in whom more than one biopsy was seen and previous treatment was disregarded, (as in our earlier study) about twice as

many biopsies could be evaluated. In this case blastic development was observed especially in Gran M, and the predominant transformation to MF/OMS in the mixed Meg-Gran types (Table 2). The same study has shown that PT (IT), when defined merely as thrombocythaemia in CMPD, comprises different combinations of megakaryocytic, erythrocytic, and granulocytic proliferation, that develop into MF/OMS and BC according to the predominant cell lines as in the rest of CMPD. With these results the prognostic role of the different cell lines in CMPD was demonstrated. For the purpose of the present study - complementary analysis of the correlations between defined haematologic groups and the most marked histologic changes in CMPD - a more restrictive definition of the typical groups was chosen. The comparison of both studies emphasizes the problems of a clinically and histologically compatible classification of CMPD. Most significant changes of the course of CMPD are demonstrated with three examples:

1. Male patient of 65 years, seen because of gastrointestinal bleeding. Blood counts at time of biopsy: erythrocytes 4.7 million, leukocytes 10000, platelets 684000 mm3

• No splenic enlargement. First biopsy (Fig. 5 8a:: slight increase in haematopoiesis, reaction after haemorrhage? Control at the age of 67: slight splenic enlargement; erythrocytes 4.8 million, leukocytes 10000, platelets 1.0 million. Second biopsy (Fig. 58b): increase in

Fig. 58 a. Increased haematopoiesis attributed to haemorrhages. Description see text. Gomori's stain. x 100.


Fig. 58 b. Primary thrombocythaemia (Meg M-Dg). Description see text. Gomori's stain. x 100.

Fig. 58 c. Myelofibrosis, incipient osteomyelosclerosis. Description see text. Gomori's stain. x 100.


Fig. 59 a. Primary thrombocythaemia (Meg M-dG). Description see text. Giemsa stain. x 200.

Fig. 59 c. Myelofibrosis grade 1; massive increase in pyknotic megakaryocytes and interstitial deposits of platelets. Description see text. Giemsa stain. x 200.

megakaryocytes, especially giant type, diffuse distribution. Rest of haematopoiesis inconspicuous: PT. Control at 69: large splenic tumour; erythrocrtes 5.8 million, leukocytes 20000, platelets 269000 mm . Third biopsy (Fig. 35c): CMPD-myelofibrosis and incipient OMS. In this case the development of PT from non-significant stage into MF was observed in the course of 4 years.

2. Male patient of 58, seen because of peripheral circulatory obstruction. Spleen moderately enlarged. Erythrocytes 4.3 million, leukocytes 5000, platelets 870000 mm3

• First biopsy (Fig. 59a): typical PT (Meg MDg). Controls one and three years later: little changes, increase in platelets to more than one million. Third biopsy (Fig. 59b): increasing pleomorphism and pyknoses of megakaryocytes. 7 years from first biopsy increased splenic size; erythrocytes 4.0 million, leukocytes 6000, platelets 1.1 million mm3; forth biopsy (Fig. 59c): inefficient thrombopoiesis with masses of pyknotic megakaryocytes, interstitial platelet deposits and MF grade 1-2. After a benign course of 7 years, PT ended in MF.

.... Fig. S9 b. Primary thrombocythaemia; increase in pleomorphic and pyknotic megakaryocytes. Description see text. Giemsa stain. x 200.

Fig. 60 a. Chronic granulocytic myelosis (Gran M-dD). Description see text. Giemsa stain. x 100.

3. Female patient of 40 years was seen with typical CML: erythrocytes 3.4 million, leukocytes 422000, platelets 508 000 mm3

• First biopsy (Fig. 60a): Gran M, MEG-dD, rarefaction of osseous trabecles. Two years later, after therapy with busulfane, increasing splenic tumour, erythrocytes 2.5 million, leukocytes 28 000, platelets 770000 mm3

• Second biopsy (Fig. 60b): massive OMS, haematopoiesis replaced by tight fibrous tissue of which large sheets of immature megakaryocytes are spared out. In the course of 25 months, CML was changed into OMS.

5. Analysis of Survival

Analyses of survival stress the difference between the haematologic and histologic aspects of CMPD. Table 13 shows that purely thrombocythaemic (C l ) cases range first when grouped according to the length of median survival. Erythraemia is next with hardly any difference regarding strong or weak expression (AI and A2)' The significance of the small group (M) with erythraemia plus leukaemia or thrombocythaemia is doubtful. Evident is the uniformity of survival among the cases with leukaemia, subleukaemia, and weak thrombocythaemia (Btl B2, Cl ). The cases with normal to subnormal blood cell levels (D) range last, with significant difference even from Bl . The com-


Fig. 60 b. Osteomyelosclerosis, sheets of immature megakaryocytes. Description see text. Giemsa stain. x 100.

Table 13. CMPD: Medium Survival of 455 patients according to different cell counts in the peripheral blood (definition of groups C-D see p. 169)

CMPD - Median Survival Rates of Haematologic Groups

Group Erythro - Le uk 0- Thrombo -cytes cytes cytes (mi III on sl (thousands) (thousands)

AI > 6 .5 < 30 X

A2 5.5-6.5 <30 X

8, < 5.5 > 30 X

62 < 5.5 15-30 X

C, <5.5 < 15 > 700

C 2 < 5.5 <15 300-700

0 <5.5 <15 < 300

M Rest of cases (n = 18)

Significant differences of survival:

Breslow-I.

0.0000 0.0156 0.0000 0.0035 0.0148

Survival (median) (months)

112

101

31

33

>124

31

22

39

Mantel-Cox-T.

0.0000 0.0572 0.0000 0.0106 0.0022


Table 14. CMPD: Median survival of 455 patients according to different changes in the bone marrow biopsy (definition of groupsEry M-BC see p. 169)

100"10

75

50

25

o 50 100

parison with the mean survival rates of the histologic groups, arranged in the same order (Table 14), shows that histology comprises a broader prognostic spectrum than that characterized by haematologic data, mainly due to distinction of the numerous cases with non-significant blood values into immature megakaryocytic myelosis, myelofibrosis, osteomyelosclerosis, and blastic metamorphosis. Practical identity of mean survival exists between the main haematologic and histologic groups: erythrocytosis (AI + 2) and Ery M, thrombocythaemia (Cl ) and Meg M-G, leukaemia (B l ) and Gran M. That confirms our assumption that the haematologic and histologic criteria chosen for this study are appropriate to characterize the main clinical entities of CMPD.

Predominantly erythrocytic and megakaryocytic proliferations show remarkable analogy according to their erythraemic and thrombocythaemic effects and benign prognosis. They are connected by the common precursor cell and by their correlation with the bone marrow sinuses. No different prognosis is shown for MF, OMS, and Mix M with megakaryocytic clusters. The longer survival of the Mix M-d has to be regarded with caution because this group is much smaller than the rest. A clear distinction

Significant differences Breslow-T. Mantel Cox-To of survival:

Ery M: Gran M 0.0000 0.0000 Ery M : Mix M-c 0.0006 0.0008 Ery M : Mix M-d 0.0107 0.0315 Gran M : Meg M-G 0.0018 0.0002

CMPD - MEDIAN SURVIVAL RATES

ERY M 130

ERY Matyp. 114

MEG M-G 114

GRAN M 34

MIX M 31

MF/OMS 30

MEG M liP 14

BC 6

150 months MF/BC 4

between this "intermediate" compartment and the immature Meg M-P/I cases, and between these and BC, is not marked at the statistical level probably due to the small number of cases.

6. Diagnostic Conclusions

The evaluation of megakaryocytic anomalies together with the results of experimental studies has greatly assisted our understanding of the relationship of inefficient megakaryocyto-/thrombocytopoiesis and MFIOMS in all groups of CMPD. Also some new insights were gained in the relationship of MF/OMS and BC

On the other hand the comparison of the cell counts of the peripheral blood and the histologic changes of the bone marrow including the survival statistics has proved that a good correlation exists between the haematologic and histologic definition of the well known groups of PV, CML and PT, to which about 35% of our CMPD cases can be assigned. A further proportion of 39% is haematologically non-significant. It can be classified by

histology into Meg M-P/I, MF/OMS, MF/BC and Be. The rest (26%) comprises mixed forms of megakaryocyticgranulocytic proliferation defined from histology by increase in giant megakaryocytes either in diffuse distribution or in form of clusters, and by sub leukaemia or weak thrombocythaemia.

Pure megakaryocytic proliferation with isolated thrombocythaemia is as rare as pure erythrocytosis, and equally compatible with otherwise undisturbed bone marrow function and good prognosis. PV and PT in restrictive definiton appear therefore as entities with a similar and prolonged stable clinical course. The isolated proliferation of granulopoiesis in the form of typical CML, or Gran M, implies greater impairment of the bone marrow function

Table 15. CMPD - WORKING

Histologic

I. Typical EryM

13%

n= 297 MegM-G

7%

GranM

15%

II. Inter- Ery M atyp. mediate

hransit- 11 % ional)

MixM-G

15%

n=282 MegM

7%

III. Terminal

28%

MF-QMS IBC

3%

n= 271 BC

* clinical aspect typical or borderline

1%


and a poor prognosis. It may be accompanied by megakaryocytic proliferation, usually of the dwarf type.

There are two forms of further megakaryocytic dedifferentiation: the development to highly ploidic giant forms, prone to form clusters and to produce masses of platelets with the effects of thrombocythaemia and IDP; or to sheets of immature and pleomorphic cells, also producing thrombocythaemia and IDP. Both may therefore be transformed to MF. The latter seems to proceed more frequently also to BC, and to originate more often from the dwarf type megakaryocytes. Sequential studies show however that both processes may as well evolve from PV, primarily from the GRAN and MEG-forms, as from PT and CML. The rarity of corresponding observations in PT

CLASSIFICATION

Haem. Groups

months)

130

PT C,

114

CML B,

34

PV* A21M

114

CML* B21C2

31

"AMM" 0

14

ill "AMM~ 0

30 OMS

"AMyS" 0/B2

4

BC* 0/B2-1

6


may be explained rather by the rare occurrence of this condition, than by specific properties of PT. Indirect proof of this assumption is obtained from the retrospective analysis of 314 cases with a history of either PV, PT, or CML. Almost equal amounts of these, 66, 61 and 63 % respectively, had MF or OMS in the biopsy.lO

In addition the biopsy studies add indirect evidence to the assumption that development to MF/OMS is promoted further by the combination of megakaryocytic and granulocytic proliferation. The Mix M-cG, the megakaryocytic-granulocytic proliferation with giant megakaryocytes in the form of clusters, is most frequently transformed into MF/OMS. As has been shown before, this variant is similar to, but not identical with, Mix MdG, the sub leukaemic form with diffuse spread of megakaryocytes. These groups are distinguished by their different propensity to transformation into MF/OMS, depending on the degree of inefficient platelet production, that is influenced by the abnormal accumulation of megakaryocytes in the form of clusters. It cannot be decided yet whether they represent distinct entities among CMPD from the beginning, or stages of megakaryocyticgranulocytic dedifferentiation that possibly can occur in the course of each of the classic types of CMPD. Our observations including sequential biopsies are much in favour of this assumption. Georgii92 has grouped similar cases as special form of CML: CMGM = chronic megakaryocytic granulocytic myelosis, frequently showing the Philadelphia chromosome (63%), but also developing from PV. Apart from these major variants there are small numbers of haematologically and histologically atypical cases that resist subclassification.

7. Working Classification

Thus the whole spectrum of CMPD appears to be composed of the three typical and comparatively stable types -PV, PT, CML, and a variety of less stable intermediate forms, of which the most frequent (Mix M-dG and -cG) probably are transitional stages on the way to fibrotic and blastic dedifferentiation. In order to provide a basis for the comparative evaluation of studies devoted to this problem in the future the following working classification is proposed: Table 15.

In this table, primarily under the aspect of bone marrow histology, the most prominent groups of CMPD are shown. These are arranged according to the different shares of proliferating cell lines, their maturity, the participation of fibrosis and osteosclerosis, and especially the variations of distribution and cell type of the megakaryocytes. Typical, intermediate and terminal forms are distinguished under the viewpoint of structural development as has been observed by numerous authors and confirmed with our study. Almost equal numbers of cases can be attributed to the three compartments, the first of which comprises the histologic equivalent of the three classical entities of CMPD, subdivided according to megakaryocytic characteristics. The intermediate compartment comprises the CMPD with a more atypical and mixed cellular-

ity, subdivided according to the predominant cell lines and megakaryocytic abnormalities, with separate consideration of a group showing prevalence of immature and pleomorphic megakaryocytes. The terminal compartment consists of the fibrotic and blastic end stages of the former groups, to which the intermediate are nearer than the typical ones. The first column to the right symbolizes the clinical entities most frequently correlated with the histologic groups; the second column to the right indicates the equivalent constellations of the peripheral blood, abbreviated according to the scheme given in the Table 5. This concept is proposed as a screen for the orientation of the various structural forms found in CMPD, parallel with their clinical and haematologic aspects. It corresponds to our present knowledge of the development of these more or less related diseases, and largely also to their clinical and haematologic characteristics. Its use as a diagnostic tool will depend on its capability to catalyze, absorb and coordinate from all other sources as much information as possible.

Summary

The wide clinical range of CMPD can be understood as leukaemia of pluripotent stem cells according to the pathogenic concepts reviewed above. Blastic metamorphoses of CMPD are regressions to a more primitive level of cellular differentiation. The predominant proliferative cell line characterizes the classical entities of PV, PT and CML, and their different prognoses. Pure erythrocytic and megakaryocytic proliferations are more compatible with sustained physiologic bone marrow functions than granulocytic proliferations. The combinations of granulocytic and megakaryocytic growth are especially prone to develop MF/OMS, in which participation of immune reactions, of granulocytic and of platelet factors is probable. An etiologic role for ineffective thrombocytopoiesis is supported by experimental as well as by histologic evidence. Myelofibrosis and osteomyelosclerosis may have similar causes, but develop independently. The prevalence of the female sex among thrombocythaemic patients was proven statistically also for the increase of giant type megakaryocytes in the form of clusters in the bone marrow, and for longer median survival of females in CMPD, especially when there is megakaryocytosis in the bone marrow. It is assumed that females may be better protected against the detrimentous effects of abnormal platelet production.

An arbitrary classification according to haematologic and histologic criteria was applied to PV, PT and CML, and groups with typical and atypical haematologic and histologic signs were distinguished. The latter cannot be separated from each other by their various haematologic manifestations, but by histology and their different propensity to progress into more immature and/or fibrotic stages. Three major groups are characterized by histology: mixed granulocytic-megakaryocytic myelosis with giant megakaryocytic clusters, a similar variant with diffuse dis-

tribution of giant megakaryocytes, and immature and/or pleomorphic megakaryocytic myelosis. Transitions from each of these groups have been observed as well as transitions from each of the typical CMPD-entities into these less typical forms. CML, frequently accompanied by dwarf-megakaryocytes, often develops into pleomorphic megakaryocytic or blastic myelosis. Blastic dedifferentiation and myelofibrosis manifest themselves as closely related end stages, to which principally all groups proceed after a longer or shorter period of time, modified by the proliferating cell lines in each group.

Clinical, experimental and histologic evidence of this natural history has been reviewed, with special emphasis on the re-evaluation of technically optimal bone marrow biopsies of untreated patients. The result is a working classification that allows recognition of typical and atypical cases, distinguished by haematologic and histologic criteria: erythrocyte, leukocyte and platelet counts in the blood, type and maturity of the predominantly proliferating cell line(s), and distinct anomalies of the megakaryocytes in the bone marrow. Between these criteria and the history of CMPD correlations are found and groups are established accordingly that are understood as typical, intermediate and terminal stages of the basic pluripotent stem cell leukaemia.

References

1 Adamson JW, Fialkow PJ, Murphy S et al (1976) Polycythemia vera: stem-cell and probable clonal origin of the disease. N Eng! J Med 292: 913-916

2 Albrecht M, Hille HH, Klenk U (1974) Morphologie der Megakaryozyten bei Blutkrankheiten. Blut XXVIII: 109-121

3 A1imena G, Dallapiccola B, Gastaldi P, Mandelli F, Brandt L, Mitelman F, Nilsson PG (1982) Chromosomal morphological and clinical correlations in blastic crisis of chronic myeloid leukaemia. A study of 69 cases. Scand J Haematol28: 103-117

4 Athens JW, Raab SO, Haab OP, Boggs DR, Ashenbrucker H, Cartwright GE, Wintrobe MM (1965) Leukokinetic studies. X. Blood granulocyte kinetics in chronic myelocytic leukemia. J Clin Invest 44: 765-777

5 Bain B, Catovsky D, O'Brien et al (1977) Megakaryoblastic transformation of chronic granulocytic leukemia. An electron microscopic and cytochemical study. J Clin Pathol 30: 235-242

6 Bakhshi A, Minowada J, Arnold A, Cossman J, Jensen JP, Whang-Peng J, Waldmann ThA, Korsmeyer StJ (1983) Lymphoid blast crisis of chronic myelogenous leukemia represent stages in the development of B-cell precursors. N Engl J Med 309: 826-831

7 Banerjee TK, Senn H, Holland JF (1972) Comparative studies on localized leucocyte mobilization in patients with chronic myelocytic leukemia. Cancer 29: 637-644

8 Bard R, Burkhardt R, Vondracek H, Sommerfeld W, Hagemeister E (1978) Rationelle Beckenkamm-Biopsie. Liingsteilung der Proben zur Anwendung von mehreren Praparationsverfahren ohne Materialverlust. Klin Wschr 56: 545-550

9 Bartl R, Frisch B, Burkhardt R (1982) Bone Marrow Biopsies Revisited - a New Dimension for Haematologic Malignancies. Karger, Basel

10 Bartl R, Burkhardt R, Jager K, Frisch B, Mahl G, Pappenberger R, Kettner G (1983) Histologische K1assifizierung der


chronischen myeloproliferativen Erkrankungen (MPD). Verh Dtsch Ges Path 67: 235-238

11 Baserga R (1965) The relationship of the cell cycle to tumour growth and control of cell division: a review. Cancer Res 25: 581-595

12 Bearman RM, Pangalis GA, Rappaport H (1979) Acute (malignant) myelosclerosis. Cancer 43: 279-293

13 Becker RP, De Bruyn PPH (1975) The transmural passage of blood cells into the lymphoid sinusoids and the entry of platelets into the sinusoidal circulation: a scanning electron microscopic investigation. Am J Anat 145: 183-205

14 Behnke 0, Pedersen NT (1974) Ultrastructural aspects of megakaryocyte maturation and platelet release. In: Baldini MG, Ebbe Sh (Eds) Platelets: Production, Function, Transfusion and Storage, 21. Grune and Stratton, New York

15 Bergsman KL, vanSlyck EJ (1971) Acute myelofibrosis. An accelerated variant of agnogenic myeloid metaplasia. Ann Int Med 74: 232-235

16 Berk PD, Goldberg JD et al (1981) Increased incidence of acute leukemia in polycythaemia vera associated with chlorambucil therapy. J Med 304: 441-447

17 Berlin NJ (1976) Diagnosis and classification of the polycythemias. Sem Hematol12: 339-351

18 Bessis M (1973) Living Blood Cells and their Ultrastructure. 367. Springer Verlag, Berlin-Heidelberg-New York

19 Bessis M (1973) Living Blood Cells and their Ultrastructure. 556. Springer Verlag, Berlin-Heidelberg-New York

20 Bird T, Proctor SJ (1977) Malignant myelosclerosis: myeloproliferative disorder or leukemia? Am J Clin Pathol67: 512-520

21 Bouroncle BA, Evan GA (1962) Myelofibrosis: clinical, hematological and pathologic study of 110 patients. Amer J Med Sci 243: 697-715

22 Brecher G (1974) Atypical megakaryocytes - a reflection of a stem cell disorder. In: Baldini MG, Ebbe Sh (Eds) Platelets: Production, Function, Transfusion and Storage: 93. Grune and Stratton, New York

23 Breton-Gorius J, Reyes F, Duhamel G, Najman A, Gorin NG (1978) Megakaryoblastic acute leukemia: identification by the ultrastructural demonstration of platelet peroxidase. Blood 51: 45-60

24 Breton-Gorius J, Reyes F, Vern ant JP et al (1978) The blast crisis of chronic granulocytic leukaemia: megakaryoblastic nature of cells as revealed by the presence of platelet peroxidase. A cytochemical ultrastructural study. Br J Haematol39: 295-303

25 Breton-Gorius J, Bizet M, Reyes F, Dupuy E, Mear C, Vannier JP, Tron P (1982) Myelofibrosis and acute megakaryoblastic leukemia in a child: topographic relationship between fibroblasts and megakaryocytes with an alpha-granula defect. Leukemia Res 6: 97-110

26 Breton-Gorius J (1983) Ultrastructure of leukemic cells. In: Gunz FW, Henderson ES (Eds) Leukemia, 161. Grune and Stratton New York

27 Briellmann A (1955) Beitrag zur Kenntnis der Osteomyeloretikulosen. Acta haemat (Basel) 13: 77-90

28 Broxmeyer HE, Grossbard E, Jacobsen et al (1978) Evidence for a proliferative advantage of human leukemia colonyforming cells in vitro. J Natl Cancer Inst 60: 513-521

29 Broxmeyer HE, Jacobsen N, Kurland Jet al (1978) In vitro suppression of normal granulocytic stem cells by inhibitory activity derived from human leukemia cells. J Natl Cancer Inst 60: 497-511

30 Buckner D, Graw jr RG, Eisch RJ et al (1969) Leukapheresis by continous flow centrifugation (CFC) in patients with chronic myelocytic leukemia (CML) Blood 33: 353-369


31 Burkhardt R (1966) Technische Verbesserungen und Anwendungsbereich der Histo-Biopsie von Knochenmark und Knochen. Klin Wschr 44: 326-334

32 Burkhardt R (1969) Die Morphologie der normalen und pathologischen Thrombopoiese im Knochenmark. In: Marx R (Ed) Der Thrombozyt, 34--43. ]F Lehmanns Miinchen

33 Burkhardt R, Pabst W, Kleber A (1969) KnochenmarkHistologie und Klinik der Polycythaemia vera. Arch klin Med 216: 64-104

34 Burkhardt R (1970) Farbatlas der klinischen Histopathologie von Knochenmark und Knochen. Springer Verlag, Berlin-Heidelberg-New York

35 Burkhardt R, Bartl R, Beil E, Demmler K, Hoffmann E, Irrgang U, Kronseder A, Langegger H, Saar U, Ulrich M, Wiemann H (1975) Myelofibrosis-osteomyelosderosis syndrome - review of literature and histomorphology. In: Advances in the Biosciences, vol 16: 9-56

36 Burkhardt R, Kronseder A (1977) Megakaryozytare Myelose - Ursache der "idiopathischen" Thrombozythamie. Fortschr Med 95: 1261-1266

37 Burkhardt R (1980) Myelogene Osteopathien. In: Handbuch der Inneren Medizin. Band 6: Kuhlencordt F, Bartelheimer H (Eds) Erkrankungen der Knochen, Gelenke und Muskeln (5. Aufl) Teil I: Klinische Osteologie. 1057-1188. Springer Verla~, Berlin-Heidelberg-New York

8 Burkhardt R (1981) Bone Marrow Histology. In: Catovsky D (Ed) The Leukemic Cell (Methods in Hematology, vol2) 49-86, Churchill Livingstone, London-Melbourne-New York

39 Burkhardt R, Bartl R (1982) Histobioptische Differentialdiagnose and Prognose der myeloproliferativen Storungen. In: Beitr Onkol, vol 13: 201-224, S. Karger, Basel

40 Burkhardt R, Bartl R, Frisch B, Jager K, Mahl G, Hill W, Kettner G (1982) The structural relationship of bone forming and endothelial cells of the bone marrow. Abstr Clin rheumatol1: 305

41 Burkhardt R, Frisch B, Bartl R (1982) Bone biopsy in haematologic disorders. ] Clin Pathol 8: 257-284

42 Burkhardt R, Frisch B, Schlag R, Sommerfeld W (1982) Carcinomatous osteodysplasia. Skel Radiol 8: 169-178

43 Burkhardt R, Kleinknecht R, Jager K, Frisch B, Mahl G, Bartl R (1983) Megakaryocytic emperipolesis - accidental or diagnostic sign? Verh Dtsch Ges Path 67: 266-271

44 Burkhardt R (1983) Histology of plastic embedded biopsies. Verh Dtsch Ges Pathol 67: 8-12

45 Burkhardt R, Schlotterbeck KP, Jager K, Kettner G (in preparation) Quantitative analysis of bone marrow capillaries in chronic myeloproliferative diseases - Comparison with haematopoiesis and volume of cancellous bone

46 Burkhardt R (unpublished) Own Observations 47 Buyssens N, Bourgeois NH (1977) Chronic myelocytic

leukemia versus idiopathic myelofibrosis. Cancer 40: 1548-1561 48 Cappio FC, Vigliani R, Novarino A, Camussi G, Campana

D, Gavosto F (1981) Idiopathic myelofibrosis: a possible role for immune complexes in the pathogenesis of bone marrow fibrosis. Br J Haematol49: 17-21

4 Canellos GP, Whang-Peng] (1972) Philadelphia-chromosome positive preleukemia state. Lancet 2: 1227-1228

50 Canellos GP, Whang-Peng ], DeVita VT (1976) Chronic granulocytic leukemia without the Philadelphia-chromosome. Am] Clin Pathol65: 467-470

51 Castro-Malaspina H, Rabellino EM, Yen A, Nachman RL, Moore MAS (1981) Human megakaryocyte stimulation of proliferation of bone marrow fibroblasts. Blood 57: 781-787

52 Castro-Malaspina H, Moore MAS (1982) Pathophysiological mechanisms operating in the development of myelofibrosis: Role of megakaryocytes. Nouv Rev Fr Hematol24: 221-226

53 Castro-Malaspina H, Schaison G, Briere], Passe Sh, Briere ], Pasquier A, Tanzer ], ]acquillat C, Bernard] (1983) Philadelphia-chromosome-positive chronic myelocytic leukemia in children. Cancer 52: 721-727

54 Chervenick PA, Boggs DR (1968) Granulocytic kinetics in chronic myelocytic leukemia. Ser Haemat 1: 24-37

55 Chervenick PA, Ellis LD, Pan SF, Lawson AL (1971) Human leukemic cells: in vitro growth of colonies containing the Philadelphia (Ph1) chromosome. Science 174: 1134-1136

56 Chikkappa G, Boecker WR, Borner G et al (1973) Return of alkaline phosphatase in chronic myelocytic leukemia cells in diffusion chamber cultures. Proc Soc Exp Bioi Med 143: 212-217

57 Cline M], Billing R (1977) Antigens expressed by human Blymphocytes and myeloid stem cells. ] Exper Med 146: 1143-1145

58 Clough V, Geary CG, Hashmi K et al (1979) Myelofibrosis in chronic granulocytic leukemia. Br] Haematol42: 516-526

59 Cutler S], Axtell L, Heise H (1967) Ten thousand cases of leukemia, 1940. ] Natl Cancer Inst 39: 993-1026

60 Dainiak N, Davies G, Kalmanti M, Lawler ], Kulkarni V (1983) Platelet-derived growth factor promotes proliferation of erythropoietic progenitor cells in vitro. ] Clin Invest 71: 1206-1214

61 Dameshek W (1935) Biopsy of the sternal bone marrow. Amer] Med Sci 190: 617-640

62 Dameshek W (1951) Some speculations on the myeloproliferative syndromes. Blood 6: 372-375

63 Dancey T, Deubelbeiss KA, Harker LA, Greenaway ] (1976) Section preparation of human marrow for light microscopy. ] din Pathol29: 704-710

64 Dancey]G, Vadnais-Metz LH (1978) A quantitation assessment of neutrophil marrow in seven patients with chronic granulocytic leukemia. Br] Haematol39: 325-338

65 Devred C, Diebold A, Bernadou A, Bielski-Pasquier (1976) L'histologie de la moelle osseuse dans la leucemie myeloide chronique. Nouv Press Med 5: 123-124

66 Dormer P (1982) Kinetik des Zellumsatzes bei chronischer myeloischer Leukamie. In: Beitr Onkol, vol 13: 225-237. S. Karger, Basel

67 Drings P (1978) Die chronische myeloische Leukamie. In: Queisser W (Ed) Das Knochenmark, 303. G. Thieme, Stuttgart

68 Ebbe Sh (1976) Biology of megakaryocytes. In: Spaet T (Ed) Progress in Hemostasis and Thrombosis, vol 3, 211. Grune and Stratton, New York

69 Efrati P, Nir E, Yaari A et al (1979) Myeloproliferative disorders terminating in acute micromegakaryoblastic leukemia. Br] Haematol43: 79-86

70 Ellis ]T, Silver RT, Coleman M, Geller SA (1975) The bone marrow in polycythemia vera. Sem Hematol12: 433-444

71 Ellis ]T, Petersen P (1979) The bone marrow in polycythemia vera. Pathol Ann 14: 383-403

72 Emberger ]H, Wagner A, Izaru P (1969) La terminaison en leucemie aigue de l'osteomyeIofibrose avec metaplasie myeloide hypersplenique. Nouv Rev Fr Hematol9: 375-390

73 Emile-Weil P, Chevalier P, See G (1933) Splenomegalie myeloide megacaryocytaire amyelocythemique. Sang 7: 773-789

74 Epstein E, Goedel A (1934) Hamorrhagische Thrombozythamie bei vaskularer Schrumpfmilz. Vir chows Arch Pathol Anat 292: 233-248

75 Fabich DR, Raich PC (1977) Acute myelofibrosis. A report of three cases. Am] Clin Pathol 67: 334-338

76 Fayemi AO, Gerber MA, Cohen ], Davis S, Rubin AD (1973) Myeloid sarcoma. Review of the literature and report of a case. Cancer (Philad) 32: 253-258

77 Ferrant A, Rodhain J, Cauwe F, Cogneau M, Beckers C, Michaux JL, Verwilghen R, Sokal G (1982) Assessment of bone marrow and splenic erythropoiesis in myelofibrosis. Sand J Haematol29: 373-380

78 Fialkow PJ, Jacobsen RJ, Papayannopoulou T (1977) Chronic myelocytic leukemia: clonal origin in a stem cell common to the granulocyte, erythrocyte, platelet and monocyte/macro~hage. Am J Med 63: 125-130

9 Fialkow PJ, Faguet GB, Jacobsen RJ, Vardya K, Murphy S (1981) Evidence that essential thrombocythemia is a clonal disorder with origin in a multipotent stem cell. Blood 58: 916-919

80 Fialkow PJ (1983) Clonal development and stem cell origin of leukemias and related disorders. In: Gunz FW, Henderson ES (Eds) Leukemia, 71. Grune and Stratton, New York

81 Fialkow PJ (1983) Clonal development and stem cell origin of leukemias and related disorders. In: Gunz FW, Henderson ES (Eds) Leukemia, 68. Grune and Stratton, New York

82 Frick PG (1971) Primary thrombocythemia. Clinical hematological and chromosomal studies in patients. Helv Med Acta 35: 20-29

83 Frisch B, Bartl R, Jager K, Pappenberger R (in press) Bone marrow histology in the chronic myeloproliferative disorders: criteria for recognition, classification and prognostic evaluation. A study of 3500 biopsies. S. Karger, Basel

84 Fritsch H (1978) Die Osteomyelosklerose. In: Queisser W (Ed) Das Knochenmark. Morphologie-Funktion-Diagnostik, 358. G. Thieme, Stuttgart

85 Galton DAG (1969) Chronic granulocytic leukemia. Sem Hematol 6: 323-343

86 Gardikas CD, Thomopoulos J, Hatzioannous J, Kanaghinis HT, Jordanoglou J, Lyberatos R (1971) Some data concerning the onset of the acute myeloblastic crisis in chronic myeloid leukemia. Acta haem at (Basel) 46: 201-206 .

87 Garson OM (1983) Cytogenetics ofleukemic cells. In: Gunz FW, Henderson ES (Eds) Leukemia, 168. Grune and Stratton, New York

88 Geller SA, Shapiro E (1982) Acute leukemia as a natural sequel to primary thrombocythemia. Amer J clin Path 77: 353-356

89 Georgii A, Vykoupil KF (1976) Histologisch-bioptische Klassifizierung myeloproliferativer Erkrankungen. In: Stacher A, Hacher P (Eds) Erkrankungen der Myelopoese, 47. Urban und Schwarzenberg, Miinchen

90 Georgii A, Thiele J, Vykoupil KF (1980) Osteomyelofibrosis/sclerosis: a histological and cytogenetic study on core biopsies of the bone marrow. Virch Arch AbtA Path Anat 389: 269-286

91 Georgii A, Vykoupil KF, Thiele J (1980) Chronic megakaryocytic granulocytic myelosis - CMGM. A subtype of chronic myeloid leukemia. Virch Arch AbtA Pathol Anat 389: 253-268

92 Georgii A (1983) Histopathology and clinical course of chronic myeloproliferative diseases. Verh Dtsch Ges Path 67: 214-234

93 Gerwitz G, Viguez A, Stewart A, Hoffmann B (1981) Production of a bone resorbing factor by myeloid leukemia cells. Blood 58 (suppl I): 139

94 Glasser RM, Walker RJ (1969) Transition among the myeloproliferative disorders. Am Int Med 71: 285-307

95 Gomez GA, Sokal JE, Walsh D (1981) Prognostic features at diagnosis of chronic myelocytic leukemia. Cancer Res 47: 2470-2477

96 Gordon BR, Coleman M, Cohen P, Day NK (1981) Immunologic abnormalities in myelofibrosis with activation of the complement system. Blood 58: 904-910

97 Gralnick HR, Harbor J, Vogel Ch (1971) Myelofibrosis in chronic granulocytic leukemia. Blood 37: 152-162


98 Greaves MF, Verbi W, Reaves BR et al (1979) Pre-B phenotypes in blast crisis of Phh positive CML: evidence for a pluripotential stem cell "target". Leuk Res 3: 181-191

99 Grundmann E, von Bassewitz DB, Roessner A, Voss B, Rauterberg J (1983) Structural proteins of bone marrow fibrosis. Verh Dtsch Ges Path 67: 72-79

100 Gunz FW (1960) Hemorrhagic thrombocythemia: a critical review. Blood 15: 706-723

101 Gunz FW (1983) Genetic factors in human leukemia. In: Gunz FW, Henderson ES (Eds) Leukemia, 318. Grune and Stratton, New York

102 Gunz FW, Henderson ES (1983) Leukemia. Grune and Stratton, New York

103 Hall BK (1981) Intracellular and extracellular control of the differentiation of cartilage and bone. Histochem J 13: 599-614

104 Hardisty RM, Wolf HH (1955) Haemorrhagic thrombocythaemia: a clinical and laboratory study. Br J Haematoll: 390-405

105 Harker LA (1960) Megakaryocytic alterations in thrombORoiesis. Blood 28: 1014-1015

06 Heuck G (1879) Zwei faile von Leukiimie mit eigenthiimlichem Blut- resp. Knochenmarksbefund. Arch path Anat, Berlin 78: 475-496

107 Henderson ES (1983) Clinical diagnosis. In: Gunz FW, Henderson ES (Eds) Leukemia, 397 ff., 431. Grune and Stratton, New York

108 Hewer TF (1937) Megakaryocytic myelosis with osteosclerosis. J Path Bact 45: 383-390

109 Hill K, Schafer R (1970) Die histologische Klassifizierung der Myelose im Myelotomiepraparat. In: Stacher A, Hocker P (Eds) Erkrankungen der Myelopoese, 59. Urban und Schwarzenberft, Miinchen

1 0 Hirschfeld H (1905) Uber atypische Myeloidwucherung. Folia Haematol 2: 665-670

111 Hittmair A (1944) Megakaryocytenleukamie und Osteomyelosklerose. Klin Wschr 23: 71-73

112 Horny HP, Parwaresch MR, Lennert K (1983) Basophilic leukemia and generalized mastocytosis. Verh Dtsch Ges Path 67: 192-197

113 Hossfeld DK, Wek HJ (1982) Chromosomenbefunde bei chronischen myeloproliferativen Erkrankungen. In: Beitr Oncol, vol 13: 249-256. S. Karger, Basel

114 Ihde D~, Canellos GP, Schwartz HJ et al (1976) Splenectomy in the chronic phase of chronic granulocytic leukemia. Ann Int Med 84: 17-21

115 Islam A, Catovsky D, Galton DAG (1980) Histological study of bone marrow regeneration following chemotherapy for acute myeloid leukaemia and chronic granulocytic leukaemia in blast transformation. Brit J Haematol45: 535-540

116 Islam A, Catovsky D, Goldman JM, Galton DAG (1983) Studies on cellular interactions between stromal and haemopoietic stem cells in normal and leukaemic bone marrow. 7th Meeting Intern Soc Haematol, Barcelona. Abstr. p. 24

117 Jacobsen N, Theilade K, Videbaek A (1982) Two additional cases of coexisting polycythaemia vera and chronic lymphocytic leukaemia. Scand J Haematol29: 405-410

118 Jacobson RS, Salo A, Fialkow PS (1978) Agnogenic myeloid metaplasia: a clonal proliferation of hematopoietic stem cells with secondary myelofibrosis. Blood 51: 189-194

119 Jager K, Burkhardt R, Bartl R, Frisch B, Mahl G (1983) Lymphoid infiltrates in chronic myeloproliferative disorders. Verh Dtsch Ges Pathol67: 239-242

120 Janossy G, Roberts M, Greaves MF (1976) Target cell in chronic myeloid leukemia and its relationship to acute lymphoid leukemia. Lancet 2: 1058-1611


121 Janossy G, Greaves MF, Sutherland R et al (1977) Comparative analysis of membrane phenotypes in acute lymphoid leukemia and in lymphoid blast crisis of chronic myeloid leukaemia. Leuk Res 1: 289-300

122 Jamshidi K, Swain WR (1971) Bone marrow biopsy with unaltered architecture. A new biopsy device. J Lab Clin Med 77: 335-342

123 Kersey JH (1983) Chronic myelocytic (multitpotent-stemcell) leukemia. (Editorial) N Engl J Med 309: 851-852

124 Kessler CM, Klein HG, Havlik RJ (1982) Uncontrolled thrombocytosis in chronic myeloproliferative disorders. Br J Haematol50: 157-167

125 Khan MH, Martin H (1968) Multiple chromosomal aberrations in a case of malignant myelosclerosis. Acta haematol (Basel) 39: 299-308

126 Koeffler HP, Golde DW (1981) Chronic myelogenous leukemia - new concepts. N Engl J Med 304: 1201-1209

127 Koulischer L, Friihling J, Henry J (1967) Observations cytogenetiques dans la Maladie de Vaquez. Europ J Cancer 3: 193-201

128 Krause JR (1983) An appraisal of the value of the bone marrow biopsy in the assessment of proliferative lesions of the bone marrow. Histopath 7: 627-644

129 Kronseder A (1973) Das Verhalten von Megakaryozyten im menschlichen Knochenmark unter normalen und pathologischen Bedingungen. Inauguraldissertation Miinchen

130 Kurnik J, Ward HP, Block MH (1972) Bone marrow sections in the differential diagnosis of polycythemia. Arch Path 94: 489-499

131 Lagerlof B (1972) Cytophotometric study of megakaryocytic ploidy in polycythaemia vera and chronic granulocytic leukaemia. Acta cytol (Baltimore) 16: 240-244

132 Landaw SA (1976) Acute leukemia in polycythemia vera. Sem Hematol13: 33-48

133 Laszlo J (1975) Myeloproliferative disorders (MPD): Myelofibrosis, myelosclerosis, extramedullary hematopoiesis, undifferentiated MPD, and hemorrhagic thrombocythemia. Sem Hematol12: 409-432

134 Lawrence JH, Winchell HS, Donald WG (1969) Leukemia in polycythemia vera. Relationship to splenic myeloid metaplasia and therapeutic radiation dose. Ann Intern Med 70: 763-771

135 Lennert K, Nagai K, Schwarze EW (1975) Patho-anatomical features of the bone marrow. Clin Haemat 4: 331-351

136 Levin WG, Houston EW, Ritzmann SE (1967) Polycythemia vera with Ph l chromosome in two brothers. Blood 30: 503-512

137 Lewis SM, Szur M (1963) Malignant myelosclerosis. Br Med J 1: 472-477

138 Lewis SM, Szur L, Hoffbrand AV (1972) ThrombOf];thaemia. Clin Hemat 1: 339-357

1 9 Lewis CM, Pegrum GD (1977) Immune complexes in myeloproliferative disorder. Lancet 2: 1151-1153

140 Liebermann PH, Rosvoll RV, Ley AB (1965) Extramedullary myeloid tumors in primary myelofibrosis. Cancer (Philad.) 18: 727-736

141 Lubin J, Rozen S, Rywlin M (1976) Malignant myelosclerosis. Arch Intern Med 136: 141-145

142 Mahl G, Frisch B, Bartl R, Burkhardt R, Jager K, Pappenberger R (1983) Acute myelofibrosis: only one extreme of the spectrum of idiopathic myelofibrosis? Verh Dtsch Ges Path 67: 272-275

143 Marie JP, Vernant JP, Dreyfus B et al (1979) Ultrastructurallocalization of peroxidase in "undifferentiated" blasts during the blast crisis of chronic granulocytic leukaemia. Br J Haematol43: 549-558

144 Martin PJ, Najfeld V, Hansen JA et al (1980) Involvement of the B-lymphoid system in chronic myelogenous leukaemia. Nature 287: 49-50

145 Martin PJ, Najfeld V, Fialkow PJ (1982)·B-lymphoid cell involvement in chronic myelogenous leukemia: implications for the pathogenesis of the disease. Cancer Genet Cytogenet 6: 359-368

146 Mayer RJ, Canellos GP (1983) Preleukemic syndromes and other myeloproliferative disorders. In: Gunz FW, Henderson ES (Eds) Leukemia, 752. Grune and Stratton, New York

147 Mazur EM, Hoffman R, Chasis J, Marchesi, Bruno E (1981) Immunofluorescent identification of human megakaryocyte colonies using an antiplatelet glycoprotein antiserum. Blood 57: 277-286

148 McCaffrey R, Harrison T A, Parkman R, Baltimore D (1975) Terminal desoxynucleotidyl transferase activity in human leukemic cells and normal human thymocytes. N Engl J Med 292: 775-780

149 McFarland W, Dameshek W (1958) Biopsy of bone marrow with the Vim Silverman needle. JAMA 166: 1464-1466

150 McGlave PB, Brunning RD, Hurd DD, Kim TH (1982) Reversal of severe bone marrow fibrosis and osteosclerosis following allogeneic bone marrow transplantation for chronic granulocytic leukemia. Br J Haemat 52: 189-194

151 Minot G, Buckman TE (1925) The blood platelets in the leukemias. Amer J med sci 169: 477-485

152 Mitelman F, Brandt L, Nilsson PG (1974) Cytogenetic evidence for splenic origin of blastic transformation in chronic myeloid leukaemia. Scan J Haematol13: 87-92

153 Moloney WC (1978) Chronic myelogenous leukemia. Cancer 42: 865-873

154 Nagai K, Kamata Y (1983) Morphologic studies on thrombocytes and megakaryocytes in primary thrombocythaemia and pol~cythaemia vera. Verh Dtsch Ges Path 67: 255-259

I 5 Neumann E (1878) Uber myelogene Leukamie. Berl Klin Wschr 15: 69-87; 115-131

156 Nowell PC (1960) A minute chromosome in human chronic granulocytic leukaemia. Science 132: 1497

157 Obion DJ, Elfenbein GJ, Braylan RC, Jones J, Weiner RS (1983) The reversal of myelofibrosis associated with chronic myelogenous leukemia after allogenic bone marrwo transplantation. Exp Haematol11: 681-685

158 Odell IT jr, Jackson CW, Friday TJ (1970) Megakaryocytopoiesis in the rat with special reference to polyploidy. Blood 35: 775-782

159 Oechslin RJ (1956) Osteomyelosklerose und Skelett. Acta Haemat (Basel) 16: 214-234

160 Ogawa M, Fried J, Sakai Y et al (1970) Studies of cellular proliferation in human leukemia. VI. The proliferative activity, generation time, and emergence time of neutrophilic granulocytes in chronic granulocytic leukemia. Cancer 25: 1031-1049

161 Olofsson T, Olsson J (1980) Suppression of normal granulopoiesis in vitro by a leukemia-associated inhibitor (LAI) of acute and chronic leukemia. Blood 55: 975-982

162 Ottolander GJ, teVeide J, Bredow P, Geraerdts JPM, Sle PHT, Willemze R, Zwaan FE, Haak HL, Muller HP, Bieger R (1979) Megakaryoblastic leukaemia (acute myelofibrosis) Brit J Haematol42: 9-20

163 Ozer FL, Truax WE, Miesch DC et al (1960) Primary hemorrhagic thrombocythemia. Am J Med 28: 807-823

164 Paulus JM (1970) DNA metabolism and development of organelles in Guinea-pig megakaryocytes: a combined ultrastructural, autoradiographic and cytophotometric study. Blood 35: 298-311

165 Pedersen B, Hayhoe FGJ (1971) Cellular changes in chronic myeloid leukemia. Br J Hematol21: 251-256

166 Pedersen B, Hayhoe FGJ (1971) Relation between phagocytic activity and alkaline phosphatase content of neutrophils in chronic myeloid leukaemia. Br J Haematol21: 257-260

167 Pfannkuch F, Koeppen KM, Scholman HJ (1983) The myeloproliferative syndrome in clinical diagnosis and biopsy. Verh Dtsch Ges Path 67: 243

168 Pitcock JA, Reinhard EH, Justus W, Mendelson RS (1962) A clinical and pathological study of seventy cases of myelofibrosis. Ann Intern Med 57: 73-84

169 Prchal JF, Adamson JW, Murphy S et al (1978) Polycythaemia vera. The in vitro response of normal and abnormal stem cell lines to erythropoietin. J Clin Invest 61: 1044-1047

170 Radley JM, Scurfield G (1979) Effects of 5-fluorouracil on mouse bone marrow. Br J Haematol43: 341-351

171 Radley JM, Haller CJ (1983) Fate of senescent megakaryocytes in the bone marrow. Br J Haemat 53: 277-278

172 Rawson R, Parker F, Jackson jr H (1941) Industrial solvents as possible etiologic agents in myeloid metaplasia. Science 93: 541-542

173 Reed RE (1980) Polycythemia vera and agnogenic myeloid metaplasia. Med Clin N Am 64: 667-681

174 Rhyner K, Blattler W, Bauer W, Bollinger A (1979) Die primare Thrombozythamie: Klinik, Pathophysiologie und Therapiemoglichkeiten. Schweiz Med Wschr 109: 467-471

175 Rosenthal DS, Moloney WC (1977) Occurrence of acute leukaemia in myeloproliferative disorders. Br J Haemat 36: 373-382

176 Rosenthal S, Canellos GP, deVita VT jr, Gralnick HR (1977) Characteristics of blast crisis in chronic granulocytic leukemia. Blood 49: 705-714

In Ross R, Vogel R (1978) The platelet-derived growth factor. Cell 14: 203-210

178 Rywlin AM (1976) Histopathology of the Bone. 71. Little, Brown and Company, Boston

179 Schulz A, Knor H (1983) Comparative histological and clinical study on the staging of polycythemia vera and osteomyelosclerosis. Verh Dtsch Ges Path 67: 247-254

180 Silverstein MN (1968) Primary or hemorrhagic thrombocythemia. Arch Intern Med 122: 18-22

181 Silverstein MN, Linman JW (1969) Causes of death in agnogenic myeloid metaplasia. Mayo Clin Proc 44: 36-39

182 Silverstein MN, Brown jr AL, Linman JW (1973) Idiopathic myeloid metaplasia: its evolution into acute leukemia. Arch Intern Med 132: 709-712

183 Silverstein MN (1974) Postpolycythemia myeloid metaplasia. Arch Intern Med 134: 113-117

184 Silverstein MN (1983) Primary thrombocythaemia. In: Williams WJ, Beutler E, Erslev AJ, Lichtman MA (Eds) Hematology 218-226. McGraw-Hill Book Company, New York

185 Singer JW, Fialkow PJ, Adamson JW et al (1979) Polycythemia vera: increased expression of normal committed granulocytic stem cells in vitro after exposure of marrow to tritiated thymidine. J Clin Invest 64: 1320-1324

186 Singer JW, Adamson JW, Ernst C et al (1980) Polycythemia vera: physical separation of normal and neoplastic committed granulocyte-macrophage progenitors. ] Clin Invest 66: 730-735

187 Singer]W, Arlin Z, Najfeld V et al (1980) Restoration of nonclonal hematopoiesis in chronic myelogenous leukemia (CML) following a chemotherapy-induced loss of phi chromosome. Blood 56: 356-360

188 Smyth JF (1983) Biochemistry of leukemic cells. In: Gunz FW, Henderson ES (Eds) Leukemia, 141. Grune and Stratton, New York


189 Sokal JE (1976) Evaluation of survival data for chronic myelocytic leukemia. Am J Hematol5: 493-500

190 Spiers AS (1979) Metamorphosis of chronic granulocytic leukemia: diagnosis, classification and management. Br J Haemat 41: 1-7

191 Tavassoli M, Aoki M (1981) Migration of entire megakaryocytes through the marrow-blood barrier. Br J Haemat 48: 25-29

192 teVelde], Burkhardt R, Kleiverda K, Leenhers-Binnendijk L, Sommerfeld W (1977) Methyl-methacrylate as an embedding medium in histopathology. Histopath 1: 319-330

193 Thiele J, Georgii A, Vykoupil KF (1976) Ultrastructure of chronic megakaryocytic-granulocytic myelosis. Blut 32: 433-438

194 Thiele J, Ballard AC, Georgii A et al (1977) Chronic megakaryocytic-granulocytic myelosis, an electron microscopic study. I. Megakaryocytes and thrombocytes. Virch Arch A Pathol Anat 373: 191-211

195 Thiele J, Ballard AC, Georgii A (1977) Freeze fracture of the normal and pathological megakaryocyte lineage in chronic megakaryocytic-granulocytic myelosis. Virch Arch B Cell Pathol23: 33-51

196 Thiele J, Vykoupil KF, Georgii A (1980) Myeloid dysplasia (MD) a hematological disorder preceeding acute and chronic myeloid leukemia. A morphological study on sequential core biopsies of the bone marrow in 27 patients. Virch Arch Abt A Path Anat 389: 343-368

197 Trautmann F (1961) Besondere Formen von Megakariozyten (Mikrokariozyten) im Sternalpunktat der chronischen myeloischen Leukose. Berl Medizin 21: 484-486

198 Trubowitz S, Davis St (1982) The Human Bone Marrow: Anatomy, Physiology, and Pathophysiology, vol II: 69. CRC Press Inc, Boca Raton

199 Trubowitz S, Davis St (1982) The Human Bone Marrow: Anatomy, Physiology, and Pathophysiology, vol II: 139, 140, 141. CRC Press Inc, Bocy Raton

200 Tura S, Baccarani M, Corbelli G (1981) Staging of chronic myeloid leukaemia. Br J Haemat 47: 105-119

201 Udoji WC, Razavi SA (1975) Mast cells and myelofibrosis. Amer J Clin Pathol63: 203-209

202 Udomratin T, Steinberg MH, Dreiling BJ, Loellhard L (1976) Circulating micromegakaryocytes signaling blast transformation of chronic myeloid leukemia. Scand J Haemat 16: 3 94-400

203 Undritz E, Nusselt-Bohaumilitzky K (1970) Uber das Auftreten von diploiden Megakaryoblasten, Promegakaryozyten und Megakaryozyten bei schweren Hamopathien, ein Riickgriff in die Phylogenese. Blut XX: 242-249

204 vanDyke D, Lawrence ]H, Anger HO (1970) Whole-body marrow-distribution studies in polycythemia vera. US Atomic Energy Commission Symposion Series no 19. Washington D. C.

205 vanDyke D, Anger HO, Parker H, McRae JM, Dobson EL, Yano Y, Naets JP, Linfoot J (1971) Markedly increased bone blood flow in myelofibrosis. J nuc! Med 12: 506-512

206 Vaquez HM (1892) Sur une forme speciale de cyanose s'accompagnant d'hyperglobulie excessive et persistante. Bull med Pas IV: 849

207 Varki A, Lottenberg R, Griffith R, Reinhard E (1983) The syndrome of idiopathic myelofibrosis. A clinicopathologic review with emphasis on the prognostic variables predicting survival. Medicine 62: 353-371

208 Verhest A, Monsieur R (1983) Philadelphia chromosomepositive thrombocythemia with leukemic transformation (Letter). N Engl] Med 308: 1603

209 Vincent PG (1983) Kinetics of leukemia and control of cell division and replication. In: Gunz FW, Henderson ES (Eds) Leukemia, 77. Grune and Stratton, New York

186 . R. Burkhardt, R. Bartl, K. jager, B. Frisch, G. Kettner, G. Mahl and M. Sund

210 Vykoupil KF, Thiele j, Stangel W, Krmpotic, Georgii A (1980) Polycythemia vera. I. Histopathology, ultrastructure and cytogenetics of the bone marrow in comparison with secondary pol~cythemia. Virch Arch Abt A Path Anat 389: 307-324

2 1 Vykoupil KF, Thiele j, Stangel W, Krmpotic, Georgii A (1980) Polycythemia vera. II. Transgression towards leukemia with special emphasis on histological differential diagnosis, cytogenetics and survival. Virch Arch Abt A Path Anat 389: 325-342

212 Vykoupil KF, Krech R, Thiele j, Windus G, Basse G, Georgii A (1983) Thrombocythaemia (thrombocytosis) as clinical diagnosis - histology of bone marrow biopsies. Verh Dtsch Ges Path 67: 260-265

213 Waitz R, Mayer S, Mayer G (1962) Aspects histologiques des myelosch~roses. Sem Hop Paris 38: 2758-2762

214 Wallerstein RO (1982) Myeloproliferative disorders. In: Newcom SR, Kadin ME (Eds) Hematologic Malignancies in the Adult, 89. Addison-Wesley Publ Comp Inc, Menlo Park/Calif

215 Ward HP, Block MH (1971) The natural history of agnogenic myeloid metaplasia (AMM) and a critical evaluation of its relationship with the myeloproliferative syndromes. Medicine (Baltimore) 50: 357-420

216 Weinfeld A, Westin j, Ridell B et al (1977) Polycythaemia vera terminating in acute leukaemia. A clinical cytogenetic and

morphologic study in 8 patients treated with alkylating agents. Scand j Haemat 19: 255-272

217 Weisenburger DD (1980) Acute myelofibrosis terminating as acute leukemia. Am j Clin Path 73: 128-132

218 Whangj, Frei E III, Tjio jH, Carbone PP, Brecher G (1963) The distribution of the Philadelphia chromosome in patients with chronic myelogenous leukaemia. Blood 22: 664-673

219 Wolf Dj, Silver RT, Coleman M (1978) Splenectomy in chronic myeloid leukemia. Ann Int Med 89: 684-689

220 Woodliff Hj, Onesti P, Dougan L (1967) Karyotypes in thrombocythaemia. Lancet 1: 114-115

221 Wright jH (1906) The origin and nature of the blood platelets. Boston Med Surg j 154: 643-645

222 Wyatt jP, Sommers SC (1950) Chronic marrow failure, myelosclerosis and extramedullary hematopoiesis. Blood 5: 329-347

223 Zambernard j, Block MH, Vatter A, Trenner L (1969) An adaptation of methacrylate embedding for routine histopatholo~ use. Blood 33: 444-450

4 Zucker-Franklin D (1981) Megakaryocytes and platelets. In: Zucker-Franklin D, Greaves MF, Grossi CE, Marmont AM (Eds) Atlas of Blood Cells. Function and Pathology, 559. E.E. edi. ermes Milano and Lea and Febiger Philadelphia

Received March 19, 1984 . Accepted in revised form june 25, 1984

Key words: Bone marrow - Biopsy - Classification - Chronic myeloproliferative disorders - Leukaemias - Myelosis -Osteomyelosclerosis

Prof. Dr. R. Burkhardt, Abt. f. Knochenmarksdiagnostik, Ziemssenstr. la, D-8000 Miinchen 2

Chronic Myeloproliferative Disorders (CMPD)

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