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Idiopathic myelofibrosis (IMF) is generally characterized by bone marrow (BM) fibrosis, anemia, splenomegaly and a leuko-erythroblastic blood picture. Although, histopathology is in keeping with the assumption of a stepwise evolution of the disease, little hematological data are available for patients with prefibrotic and early stages of disease. Therefore a clinicopathological study was performed that included firstly an exploratory sample of 68 patients with minor supportive therapy in whom BM biopsies during follow-up (41 +/- 32 months) revealed an evolution of a prefibrotic or very early fibrotic lesion into overt IMF. The validation sample consisted of 556 patients with pretreatment marrow specimens on admission. Diagnostic features and BM lesions were identical compared with the patients of the exploratory sample at their first examination. BM biopsies were processed by routine stainings including silver impregnation (reticulin fibers) and frequently also by immunohistochemistry to identify megakaryocytes and erythroid precursor cells more properly. Apart from minor hemorrhage and peripheral thrombosis patients with early stage IMF presented with non-specific symptoms including varying degrees of leukocytosis (51%), anemia (38%), a platelet count exceeding 600 x 10(9)/l (86%), splenomegaly (15%) and increase in leukocyte alkaline phosphatase (LAP) (24%) and serum lactate dehydrogenase (LDH) (20%). BM histology confirmed a moderate increase in hematopoiesis with a mixed granulocytic and megakaryocytic myeloproliferation, a reduction of erythroid precursors and significant megakaryocytic abnormalities. In keeping with the first BM examination of the exploratory sample no or only a borderline to slight increase in reticulin fibers was detectable, however, in 68 of 134 patients follow-up biopsies revealed a transition into overt IMF (intervals about three years). Median survival of this cohort with early-stage IMF was 129 months thus contrasting manifest IMF with an usually more unfavorable prognosis. Recognition of early stage IMF certainly alters the generally applied diagnostic criteria of this disorder. Regarding patients with associated thrombocytosis, differentiation from essential thrombocythemia is recommended. Moreover, characterization of early stage IMF probably exerts an impact on survival and may influence the decision to perform a BM transplantation.

A multicentre clinicopathological study was performed on 495 patients with chronic-phase Ph1+ chronic myelogenous leukaemia (CML) to determine bone marrow characteristics that exert a significant impact on survival under standard treatment regimens. Immunohistochemical and morphometric techniques were applied to identify nucleated erythroid precursor cells in the bone marrow and to quantify argyrophilic fibre density. Application of the Sokal index and another recently proposed CML score failed to distinguish three clearly defined risk groups. A borderline increase in fibre content (i.e. doubling of the normal density) and a relevant reduction of medullary erythropoiesis proved to be important predictors for survival, even in low-risk classified patients, according to both clinical scores. With regard to optimal treatment strategies, patients with manifest myelofibrosis showed no significant difference in survival rates under interferon or hydroxyurea treatment. Multivariate analysis confirmed the prognostic value of histological features. A risk model based on three variables (fibre density, erythropoietic precursors and spleen size) was constructed that enabled a distinct discrimination of risk profiles. In conclusion, the presented data provide compelling evidence that bone marrow features at diagnosis exert a significant impact on prognosis in CML. In this context, the generally clinical-based multivariate risk classification can be improved by consideration of morphological variables that are acting independently of treatment modalities.

A clinicopathological study was performed to define initial-prefibrotic precursor stages of idiopathic (primary) myelofibrosis (IMF) by presenting laboratory and histological bone marrow features. Contrary to the usually accepted diagnostic requirements for IMF, including bone marrow fibrosis and a leukoerythroblastic blood picture, we found that 80 patients did not completely comply with these criteria. In particular, this cohort displayed no increase in the reticulin-collagen fiber content of the bone marrow at onset. Therefore, these cases were occasionally regarded as unclassifiable chronic myeloproliferative disorders (MPDs), or presumptively as essential thrombocythemia (ET). Patients were characterized by a certain set of clinical parameters comprising a borderline to slight leukocytosis and therapy-refractory anemia, minimal to modest splenomegaly, and often an elevated platelet count. Peripheral blood films revealed, only very sparsely, tear drop cells and a few erythroid and myeloid precursors, but no definite leukoerythroblastic reaction. Bone marrow histopathology was consistent with an increase in cellularity and a prominent left-shifted neutrophil granulopoiesis. Erythropoiesis disclosed a slight reduction with small to medium-sized islets. Megakaryopoiesis was the most prominent diagnostic hallmark to distinguish initial-prefibrotic IMF from the allied subtypes of MPDs. This cell lineage was not only characterized by a conspicuous growth and abnormal clustering, but also by a pronounced deviation from nuclear-cytoplasmic differentiation (dysplastic appearance). Cytological anomalies were compatible with a large variety of size and shape, ranging from giant- to atypical micromegakaryocytes with compact and bulky, cloud-like nuclei, due to a coarse lobulation and a frequent occurrence of naked (denuded) nuclei. Follow-up examinations, including sequential trephine biopsies in 22 patients, revealed a transition into myelofibrosis accompanied by laboratory findings in keeping with manifest IMF. In conclusion, morphological and clinical parameters have been validated by this study, which are consistent with a set of diagnostic criteria to recognize initial or prefibrotic precursor stages of IMF.

Background and Objectives: Diagnosis of essential thrombocythemia (ET) remains a challenging problem and has been predominantly established by exclusion of other thrombocythemic disorders. In this context the updated diagnostic criteria of the Polycythemia Vera Study Group (PVSG) are generally accepted, although histopathologic features of the bone marrow were only marginally considered., Design and Methods: A retrospective evaluation was performed of 168 patients presenting with ET in accordance with the criteria of the PVSG. Analysis was focused on the discriminating impact of bone marrow morphology., Results: Histopathology revealed that our cohort of patients could be divided into three distinct groups (true ET, questionable ET and false ET). These groups were characterized by certain diagnostic constellations of clinical data on admission. True ET was found in 53 patients presenting with no or a borderline splenomegaly and no relevant anemia or leuko-erythroblastic blood picture. The other patients showed clinical signs and symptoms which were more compatible with initial-prefibrotic (52 patients) or early (68 patients) idiopathic-primary myelofibrosis (IMF) with severe thrombocythemia. In true ET no significant hypercellularity of the bone marrow including myeloid precursors or an increase in reticulin fibers was detectable. Most prominent were changes of megakaryopoiesis which revealed large to giant-sized cells lacking a definite maturation defect. Their appearance in true ET contrasted with the clusters of abnormally differentiated, often bizarre elements of this lineage in patients with initial and early IMF (questionable or false ET). Calculation of survival disclosed a relevant disparity with a non-significant loss in life expectancy of 10.9% in true ET compared to 29.6% in questionable and 51.3% in false ET. Follow-up studies and repeated bone marrow biopsies revealed no transition into myelofibrosis in true ET, whereas this did occur in 22 of 27 patients with questionable and false ET. In the latter cohort bone marrow changes were accompanied by increasing anemia, splenomegaly, tear-drop poikilocytosis and reduction of the platelet count consistent with IMF., Interpretation and Conclusions: A detailed evaluation of bone marrow features, in particular megakaryopoiesis is recommended to establish positive criteria for the diagnosis of ET and thus to accomplish a significant improvement of the PVSG postulates. In this context ongoing clinical trials on ET must regard pretreatment bone marrow biopsies as a major clue to diagnosis.

A retrospective multicentre clinicopathological study was performed on sequential bone marrow trephine biopsies in 100 patients with Ph1+-chronic myelogenous leukaemia (CML) to elucidate the effect of interferon (IFN) alpha 2b and hydroxyurea (HU) treatment on myelofibrosis and megakaryopoiesis. According to strictly defined therapeutic regimens, 38 patients received IFN as monotherapy, 23 patients a combination of IFN and HU and 39 patients HU only. Using standardized intervals of biopsies and histochemical and morphometric methods, a significant increase in reticulin fibre density and in the number of CD61+ megakaryocytes was detectable in the majority of IFN-treated patients. To a lesser degree, these changes were also expressed in the cohort with a combined IFN and HU regimen. In contrast to these findings, in the group of patients with HU as single-agent treatment, a stable state or reversal of myelofibrosis was detectable together with corresponding changes in megakaryopoiesis. Further evaluations revealed that these effects had occurred within the first year, mostly after 6 months of treatment, and were prominently expressed in those patients with a slight to relevant grade of myelofibrosis at presentation. In conclusion, this study provides persuasive evidence that monotherapy by IFN exerts a fibrogenic effect, while HU treatment seems to prevent and even resolves bone marrow fibrosis in CML. Probably, in relation to the complex pathomechanisms responsible for the generation of myelofibrosis, the changing content of reticulin fibres was usually accompanied by corresponding alterations in the number of CD61+ megakaryocytes, including atypical microforms and precursor cells.

A clinicopathological follow-up study including sequential bone marrow biopsies was performed on 79 patients with idiopathic (primary) myelofibrosis (IMF) to characterize initial (prefibrotic) stages and to elucidate whether development of fibrosis was accompanied by corresponding clinical findings. For this purpose our cohort of patients was divided into two groups of which the first presented with the generally accepted signs and symptoms of IMF (group I; n = 60). Most patients of the second group (group II; n = 19) showed mild to moderate therapy-refractory anemia, minimal to slight splenomegaly and frequently thrombocytosis, but no bone marrow fibrosis at onset. Hematopoiesis was consistent with a striking hypercellularity in comparison to the age-related involution by adipose tissue, a conspicuous clustering and histotopographic dislocation of megakaryocytes, a neutrophil granulocytic proliferation and a reduction of erythropoietic islets with arrest of maturation. Most remarkable was the dysplastic cytology of megakaryocytes with a definitive deviation of differentiation resulting in bizarre forms. Follow-up examinations revealed that at later stages group II patients were not distinguishable from the first group with more advanced IMF. For this reason, these patients were regarded as presenting initial, prefibrotic IMF characterized by distinctive bone marrow features at the beginning. The prominent abnormalities of megakaryopoiesis together with the granulocytic proliferation were extremely helpful to differentiate prefibrotic IMF with accompanying thrombocythemia from essential thrombocythemia (ET). Dynamics of fiber progression were calculated by regarding increase in density per time. Speed of progression during the first year of observation proved to be significantly higher in group II patients with prefibrotic IMF in comparison to full-blown cases (group I). In conclusion, with respect to prospective clinical trials, diagnostic criteria for IMF should be re-evaluated by also taking initial, prefibrotic stages into account.

A retrospective study of 120 patients with the clinically and histologically established diagnosis of idiopathic (primary) myelofibrosis (IMF) was performed to determine prognostic factors of predictive value, including parameters characterizing the dynamics of hematopoietic cell kinetics. In contrast to previous studies, our cohort comprised the full spectrum of the disease, from initial prefibrotic to advanced osteosclerotic stages. The in situ end-labeling (ISEL) technique was used to demonstrate apoptosis, in order to determine dynamic parameters of predictive value. Cell proliferation was evaluated by employing the monoclonal antibody PC10 directed against proliferating cell nuclear antigen (PCNA). Proliferative activity (PCNA index) and frequency of apoptosis showed significant differences between early and advanced fibrosclerotic stages of disease. Decrease in proliferation indicated a significantly shorter survival, whereas a higher frequency of apoptotic cells was associated with a better prognosis. It may be speculated that a normal or enhanced proliferation rate expressed by PCNA positivity (late G1- and S-phase of the cell cycle) that is accompanied by a higher incidence of apoptosis reflects the regenerative (turnover) capacity of hematopoiesis. This may apply especially to early hypercellular stages without relevant myelofibrosis. In consideration of a recently published multivariate risk model, a simplified synthesis score for stratification of a patient's prognosis was constructed. Age, degree of anemia, leukocytes, and platelet count were regarded as the most important parameters. A substantial improvement of prognostic efficiency was further achieved by including PCNA index and frequency of apoptosis. Our results are in keeping with the assumption that generalization, indicated by myeloid metaplasia, has a prodigious impact on prognosis in IMF. Furthermore, in this context dynamic features such as proliferative activity and frequency of apoptosis exert an additional predictive value.

Background: Prognostic variables for idiopathic (primary) osteomyelofibrosis (IMF) are ill-defined because of the lack of large control studies based on uniform diagnostic criteria., Methods: A retrospective clinicopathologic study was performed on 250 consecutively recruited patients (115 males and 135 females) with an established diagnosis of IMF. In contrast to previous studies, the current study cohort encompassed the full spectrum of initial to advanced stages of the disease process according to laboratory data and particularly histology. Because of the relatively high patient age on admission (median, 66.5 years), relative survival rates with corresponding life expectancies and disease specific life loss were calculated. Moreover, a classification and regression tree (CART) analysis was performed to segregate the study patients into subgroups with significantly different prognosis., Results: Analysis of the life expectancy and the proportion of deaths attributable to IMF showed a global reduction in life expectancy of 31%. Further calculation disclosed a consistently greater impact of disease in older patients. Age, hemoglobin level on admission, and leukocyte and thrombocyte counts remained as the most relevant parameters for prognosis in multivariate consideration (CART analysis) and facilitated a clear-cut separation into three risk groups. The life expectancy of low risk patients was approximately 10 times higher than that of high risk patients (22.07 years vs. 2.25 years)., Conclusions: These results are in keeping with the assumption that features signaling bone marrow insufficiency are associated with a worsening of survival. Generalization, indicated by myeloid metaplasia, can occur at every stage, even in so-called hypercellular phases of IMF. Conversely, myelofibrosis alone is not necessarily predictive of poor survival.

A retrospective clinico-pathological study was performed on 208 consecutively recruited patients (94 males, 114 females, median age 67 years) with idiopathic (primary) osteo-/ myelofibrosis (IMF). According to bone marrow histology (cellularity) as well as extent (semiquantitative grading) and quality (reticulin/collagen) of myelofibrosis, stages of the disease process were determined. At closure of this study (observation time 65 months) 133 patients were dead and 75 alive and median survival was 56 months. The wide spectrum of clinical signs and symptoms and laboratory data on admission was reflected by a corresponding variety of histological features. Significant differences of hematological values could be calculated between patients with or without early reticulin fibrosis (fiber scores 0 and 1) and advanced fibro-osteosclerotic stages (fiber scores 2 and 3). Evolution of disease features was elicited by longitudinal follow-up studies and sequential bone marrow biopsies. Morphometric assessment of fiber density in patients without preceding chemotherapy revealed an unpredictable and varying progression of myelofibrosis associated with alterations of certain laboratory parameters (hemoglobin level, spleensize, thrombocytosis). Differentiation from essential (primary) thrombocythemia (ET) was required in 25 patients who fulfilled the postulated diagnostic criteria. In fact, this group was consistent with hypercellular, early stages of IMF without relevant reticulin fibrosis and an excessively raised platelet count (> or = 1000 x 10(9)/1). Discrimination was only feasible by regarding histology carefully, particularly abnormalities of megakaryopoiesis and follow-up data. Parameters of predictive value indicating a significant loss in life expectancy in comparison with a sex- and age-adjusted normal population included: age (> or = 60 years), hemoglobin levels (< or = 10 g/dl), thrombocyte count (< or = 600 x 10(9)/1) and the presence of myeloblasts and promyelocytes. Statistical analysis disclosed that in the so-called early stages of IMF without relevant myelofibrosis, findings indicative for extramedullary hemopoiesis or generalization of the disease process exerted an unfavourable influence on survival.

An immunomorphometric study was performed on bone marrow biopsies from 40 patients with primary osteomyelofibrosis--OMF, (agnogenic myeloid metaplasia) by employment of a monoclonal antibody against glycoprotein IIIa (Y2/51) to determine the number of pro-megakaryoblasts. Specimens from 15 individuals without any hematological disorder served as controls. With reference to the pertinent literature on megakaryocyte precursors and following a pilot study on corresponding smears, in tissue sections pro-megakaryoblasts were characterized by a size of 42.1 +/- 2.6 microns 2 (diameter 7.5 +/- 0.3 microns). In comparison with controls, in OMF no relevant increase in the number of pro-megakaryoblasts per square and cubic millimeter bone marrow was evaluable. The relative frequency of these precursors was significantly reduced due to an increase in the total amount of conspicuously large and abnormal megakaryocytes. Statistical analysis failed to reveal any correlations between counts for pro-megakaryoblasts or the total number of Y2/51--positive megakaryocytic elements with the density of argyrophilic fibers (determined by morphometry) or the platelet values. Our findings imply that in OMF the marked increase in circulating progenitor cells of the megakaryocyte lineage may be generated by extramedullary, probably splenic hematopoiesis. Moreover, the evolution of medullary fibrosis is thought to be associated with the striking predominance of large atypical, possibly overaged and hyperpolyploid megakaryocytes and not with an increase in precursor cells.

A clinicopathological study was performed on 114 patients (46 male/68 female, median age 67 years) with the diagnosis of agnogenic myeloid metaplasia (AMM) respectively primary osteo-myelofibrosis which was not preceded by any other or allied subtype of chronic myeloproliferative disorders. On admission patients revealed a striking variability of laboratory data as well as different histopathological features of initial bone marrow biopsies. For this reason discrimination was done into two groups based on bone marrow findings: group I patients (n = 46, 19 male/27 female) showed a hypercellular marrow without or only borderline (n = 24) to slight (n = 22) reticulin fibrosis and group II cases (n = 68, 27 male/41 female) displayed coarse bundles of collagen fibres (n = 18) frequently accompanied by osteosclerosis (n = 50). Statistical analysis of the corresponding initial hematological findings resulted in significant differences. These differences concerned also the complications occurring during the lengthy course of disease, which included a higher incidence of pancytopenia and severe marrow failure with hemorrhage and blast crisis in group II patients. However, overall survival time was not different in both groups. This may be related to the similarity of age distribution (64 resp. 65 years) and its significant association with arteriosclerotic vascular lesions. Consequently acute myocardial infarction and congestive heart failure were frequent causes of death in addition to infections due to marrow failure and blast crisis. Repeated bone marrow biopsies in 24 patients revealed an insidious transition from hypercellular lesions (group I) into advanced fibro-osteosclerotic changes (group II) concurring with laboratory data. Therefore our discrimination into two groups of patients represents variable stages or static histological and corresponding hematologic features in the evolution of a dynamic disease process in AMM.

A clinicopathological follow-up study was performed on 17 patients with chronic myeloid leukaemia (CML) and 23 cases with so-called primary (idiopathic) osteo-/myelofibrosis (OMF) concentrating on a comparison between clinical data and multiple sequential biopsies of the bone marrow. Histological classification of bone marrow lesions was done according to the subtypes proposed by Georgii et al. At clinical diagnosis initial trephine biopsies in CML showed in only 6/17 cases a pronounced granulocytic proliferation or CGL. In 9/23 patients with OMF a so-called hyperplastic or early hypercellular stage was encountered with a mixed megakaryocytic-granulocytic pattern without or with minimal reticulin fibres (CMGM/EMS). The histopathology of this early stage OMF as well as the later evolving advanced fibrosclerotic lesions (AMS/OMS) were by morphological aspects alone not distinguishable from cases with CML showing prominent fibrosclerotic alterations. At presentation 5/17 patients with CML displayed already some degree of reticulin fibre formation (EMS). Following serial trephine biopsies in CML with an increased megakaryocyte proliferation (CMGM), a remarkable tendency for myelofibrosis was present. The dynamics of this fibrosclerotic transformation seem to be variable in CML and OMF likewise. However, they are related to abnormal megakaryopoiesis as well as to duration respectively progress of disease, paralleled by corresponding haematological parameters. This longitudinal case control study emphasizes that histopathology of the bone marrow taken at clinical diagnosis may reflect different stages of chronic myeloproliferative diseases and therefore should be always accompanied by relevant clinical and cytogenetic findings to enable a correct diagnosis.

A clinicopathological study was performed on 90 patients (39 males - 51 females, age 68 years) with primary (idiopathic) myelofibrosis - osteomyelosclerosis (OMF) in order to correlate laboratory and histomorphological parameters with each other and to calculate factors of prognostic impact on survival. In addition to multiple interactions between various laboratory features, there was a significant correlation between degree of medullary fibrosis and osteosclerotic changes with sizes of spleen and liver, level of LDH and duration of relevant prediagnostic symptoms. In trephine biopsies of the bone marrow, reduction of hematopoietic tissue was assessed by evaluating the amount of fat cells plus the degree of osteosclerotic lesions. This histological parameter did not reveal significant relationships with hepatosplenomegaly, duration of relevant symptoms or length of disease, but was correlated with the clinical findings of bone marrow failure. On univariate analysis, several clinical (age greater than 45 years, presence of relevant prediagnostic symptoms, hemoglobin level less than 9 g/dl, counts of myelo- and normoblasts, thrombocyte count less than 100 and greater than 700 x 10(9)/l, spleen size and LDH level) and histological features (reduction of hematopoiesis, counts for megakaryocytes and lymphoid nodules) were found to exert a predictive value on prognosis. However, on multivariate regression analysis only age remained significant. This result apparently reflects the numerous interactions between the various clinical as well as histological variables tested.

Histomorphometry was performed on representative trephine biopsies of the bone marrow on admission of 50 patients (21 male, 29 female - age 67 years) with so-called primary osteomyelofibrosis/-sclerosis (OMF) not preceded by any other subtype of chronic myeloproliferative disorders. This study was firstly aimed at testing correlations between histological features (amount of haematopoiesis, cytological aspects of megakaryocytes, density of reticulin and collagen fibres and degree of osteosclerosis) and laboratory data, as well as spleen size and duration of relevant prediagnostic symptoms. Secondly, we concentrated on a discrimination of OMF patients into two subgroups according to bone marrow morphology and clinical variables. Statistical evaluation of histomorphometric variables and haematological findings disclosed that there was a progressive fibro-osteosclerotic process in the evolution of disease features. Increase in medullary fibrosis was significantly paralleled by an abnormal or pleomorphic megakaryopoiesis in the bone marrow: there was an increase in irregularity of perimeters for megakaryocytes and naked nuclei combined with smaller sizes of these elements including the nuclei. Additionally, there was a greater number of pycnotic bare nuclei. A number of morphometric features (density of fibres, degree of osteosclerosis, amount of haematopoiesis) were associated with corresponding clinical data (spleen size, length of preclinical history). By consideration of a set of basic histomorphometric variables our cohort of 50 patients could be divided into an early hyperplastic subtype with no or minimal medullary reticulin and another group with conspicuous fibrotic and osteosclerotic alterations of the bone marrow. It was noticeable that we found no significant correlation between amount of haematopoiesis or marrow cellularity with splenomegaly. This result suggests that splenic haematopoiesis (myeloid metaplasia) may represent an autonomous or neoplastic process and not only compensation for a failing fibro-osteosclerotic bone marrow.

In 40 patients (17 male, 23 female, median age 57 years) with the presumptive diagnosis of primary (essential) thrombocythemia (PTH) according to the diagnostic requirements of the Polycythemia-Vera-Study-Group (PVSG) a follow-up study and a histological evaluation of initial trephine biopsies of the bone marrow were performed. Thorough review of the hematological data during the lengthy course of disease (observation time ranging from 1.5-10.5 years) and the histomorphology of the bone marrow implied a discrimination into two groups of patients. Group I patients (n = 26; 10 male, 16 female) were compatible with PTH according to our follow-up studies. Group II patients consisted of 14 cases (7 male, 7 female) which suggested retrospectively early hyperplastic stages of agnogenic myeloid metaplasia (AMM) with concomitant thrombocytosis. In PTH (group I patients) there was a sustained elevation of the platelet count lasting for several years accompanied by stable other blood values. Early AMM (group II patients) was characterized by an insidious decline of the initially elevated thrombocyte count, starting in a few patients already 4-6 months after admission. In AMM there was further an increase in hepatosplenomegaly observable together with the level of LDH and the score of the leukocyte alkaline phosphatase, and finally an evolution of a leukoerythroblastic blood picture could be noticed. Initial histopathology of the bone marrow revealed a profound proliferation of a not severely dysplastic megakaryopoiesis in group I patients (PTH) and a normal content of reticulin fibers. In early thrombocythemic AMM (group II patients) conspicuous abnormalities of megakaryocytes were accompanied by a slight to moderate increase in argyrophilic fibers and a left-shifted neutrophilic granulocyto- as well as erythropoiesis. These differences of certain histomorphological features could be substantiated by morphometric analysis. Our findings suggest that even the rigid requirements for the diagnosis of PTH as proposed by the PVSG may not be sufficiently restrictive to exclude patients with early hyperplastic stages of thrombocythemic AMM.