Bone marrow histopathology in chronic myelogenous leukemia (CML)--evaluation of distinctive features with clinical impact.

Bone marrow features in stable-phase chronic myelogenous leukemia (CML) are characterized by a striking heterogeneity which is determinable by appropriate means including representative pre-treatment trephine biopsies, immunohistochemistry and morphometry. Cell lineages involved to a variable extent consist not only of neutrophil granulopoiesis, but include also megakaryocytes, erythroid precursors, resident macrophages and lymphocytes. Moreover, the stromal compartment, in particular reticulin and collagen fibers, plays a pivotal role in the disease process. Following morphometric analysis significant correlations may be calculated between histological parameters and clinical-laboratory findings. Relevant interactions are detectable between number of megakaryocytes and their precursors with fiber density. This findings is in line with the close functional relationships between megakaryopoiesis and fibroblasts regarding the complex pathomechanisms of myelofibrosis. Moreover, other correlations are observable between reduction of erythropoiesis or increase in fibers with clinical features like anemia, percentages of myelo- and erythroblasts in the peripheral blood, spleen size or LDH level. These variables are in keeping with more advanced stages of CML which indicate a transition to myeloid metaplasia and thus exert a significant impact on survival. Consequently, the different risk profiles of patients are determined by both clinical and morphological parameters of predictive value. Regarding the latter, extent of myelofibrosis, amount of erythroid precursors and numbers of myeloerythroblasts in the peripheral blood are significantly associated with prognosis. For this reason, it should be mandatory to enter morphological criteria into prospective clinical trials on CML, not only for diagnostic purpose, but also for a proper evaluation of different survival patterns.     

CD34+ stem cells in chronic myeloproliferative disorders

Contrasting the wealth of information that is available about various biological and therapeutic aspects of human CD34+ stem cells, little data exist concerning their quantity and dynamics as well as their mutual relationships with other hematopoietic constituents in the bone marrow of patients with chronic myeloproliferative disorders. In comparison with a control group frequency of progenitors is significantly increased in chronic myeloid leukemia (CML). Following different therapeutic modalities their quantity reflects therapeutic efficacy (responder and non-responder patients) and therefore exerts a predictive value regarding acceleration and blastic crisis. The significant correlations between fiber content and number of these precursors elucidates the complex interactions between stroma and progenitor cell differentiation and maturation. Following allogeneic bone marrow transplantation there is a rapid recovery of the CD34+ stem cell population in the first month. A higher number of these cells is related with graft size, an earlier independence for platelet transfusion and a more extended regeneration of erythro- and megakaryopoiesis. The slight increase in reticulin fibers in these patients may be associated with the complex and so far ill-defined pathomechanism of homing (adherence to the fibrous matrix). In idiopathic myelofibrosis (IMF) an increased number of CD34+ stem cells is found predominantly in the early (prefibrotic or mild fibrotic) hypercellular stages and probably indicates a higher proliferative activity of the precursor cell pool. According to sequential biopsies most patients with early IMF that later evolved into an overt fibrosclerotic stage usually display a reduction of progenitor cells during the development of myelofibrosis. The unequal distribution of CD34+ stem cells in the bone marrow versus spleen in IMF (advanced fibrosclerotic stage) is in support of the currently discussed hypothesis of splenic filtration and concentration of precursor cells as an essential feature of myeloid metaplasia. Regarding prognosis in CML a higher amount of CD34+ stem cells is significantly associated with an unfavorable survival and thus confirms the assumed implication of an accelerated phase of disease at onset. On the other hand, in polycythemia vera (PV) and IMF a low number of progenitors is probably due to a decreased proliferation rate (reduced hematopoietic turnover index) and therefore reflects a reduction in the regenerative capacity of hematopoiesis. For this reason, a presumptive defect in the recovery of normal and clonally transformed stem cells is speculated to add to the worsening of prognosis by causing the well-known bone marrow insufficiency in terminal stage PV and IMF.     

A histomorphometric analysis of trephine biopsies of bone marrow from 65 patients with chronic myeloid leukemia. Classification of patients into subgroups with different survival patterns

A histomorphometric (planimetric) study was performed on trephine biopsies of the bone marrow taken at presentation from 65 patients (31 males and 34 females, with a median age of 48 years) with chronic myeloid leukemia (CML). Specimens from 20 patients (9 males and 11 females, with a median age of 53 years) without any hematologic disorders served as controls. Of the various histologic variables tested, only the counts of neutrophilic granulocytes per 1 sq mm, the ratio of granulocytopoiesis to megakaryopoiesis and the density of reticulin (argyrophilic) fibers revealed a significant correlation with the prognosis. The CML patients were separated into two groups with different survival patterns. Group I (34 patients with a median survival of 24 months) mostly contained cases with the so-called "megakaryocytic subtype" of CML, which is accompanied by variable degrees of fibrosis; group II (31 patients with a median survival of 36 months) mainly contained cases with the "granulocytic subtype," which is not accompanied by myelofibrosis. Among the morphometric parameters, a positive correlation existed between the megakaryocyte count and the reticulin fiber density, which underlines the important role of that cell lineage in fibrillogenesis. There were multiple interrelationships between the histomorphometric variables and the laboratory data. Consequently, multivariate regression methods (using Cox's proportional hazards model) were applied to assess the relative predictive value of the patient characteristics for survival. The derived prognostic model divided the patients into two risk groups, with median survivals of 14 and 41 months, respectively. In order of their entry into the regression model, these variables were percentage of neutrophils in the differential blood count, amount of granulopoiesis, liver size, percentage of peripheral myeloblasts and density of reticulin fibers in the bone marrow. In comparing the two patient groups, based on bone marrow histomorphometric parameters, this model revealed that two of those factors (amount of granulopoiesis and density of reticulin fibers) had a significant correlation with the prognosis.     

Strategic nonmyeloablative conditioning: CD154:CD40 costimulatory blockade at primary bone marrow transplantation promotes engraftment for secondary bone marrow transplantation after engraftment failure

There is an increased risk of failure of engraftment following nonmyeloablative conditioning. Sensitization resulting from failed bone marrow transplantation (BMT) remains a major challenge for secondary BMT. Approaches to allow successful retransplantation would have significant benefits for BMT candidates living with chronic diseases. We used a mouse model to investigate the effect of preparative regimens at primary BMT on outcome for secondary BMT. We found that conditioning with TBI or recipient T cell lymphodepletion at primary BMT did not promote successful secondary BMT. In striking contrast, successful secondary BMT could be achieved in mice conditioned with anti-CD154 costimulatory molecule blockade at first BMT. Blockade of CD154 alone or combined with T cell depletion inhibits generation of the humoral immune response after primary BMT, as evidenced by abrogation of production of anti-donor Abs. The humoral barrier is dominant in sensitization resulting from failed BMT, because almost all CFSE-labeled donor cells were killed at 0.5 and 3 h in sensitized recipients in in vivo cytotoxicity assay, reflecting Ab-mediated cytotoxicity. CD154:CD40 costimulatory blockade used at primary BMT promotes allogeneic engraftment in secondary BMT after engraftment failure at first BMT. The prevention of generation of anti-donor Abs at primary BMT is critical for successful secondary BMT.     

Cytogenetic studies in bone marrow transplant recipients

Chromosome studies were performed in 24 patients who underwent allogeneic bone marrow transplantation (BMT) for severe aplastic anaemia (8), chronic myeloid leukemia (5 in chronic, 2 in accelerated phase and 1 in lymphoid blast crisis), acute myeloid leukemia (6), acute lymphoblastic leukemia in relapse (1) and Hodgkin's disease (1). Donor-cell type engraftment was demonstrated in 21 patients: in all 17 sex-mismatched transplants and - as demonstrated by reconstitution with Ph-negative cell populations - in 4 CML patients with a sex-matched donor. Recipient-type mitoses were seen in the bone marrow of 5 cases (1 SAA, 3 CML, 1 AML) after transplantation. They were only observed on one occasion in patients with SAA (4 of 25 on day 33) and AML (44 of 50 on day 14). Despite the continued demonstration of some Ph-positive mitoses in 3 patients with CML up to day 28, 323 and 451 after BMT, respectively, all surviving CML patients are still in complete haematological and clinical remission. So far the significance of these cytogenetically abnormal persisting host cells remains unknown.     

Prognostic significance of the histomorphometric features of bone marrow trephine biopsies in patients with chronic myeloid leukemia

OBJECTIVE: To study the correlation of histomorphometric data of bone marrow trephine biopsies at the time of initial diagnosis in chronic myeloid leukemia (CML) patients with the patient prognosis. STUDY DESIGN: A total of 40 CML patients were divided equally in group I (developed accelerated phase or blast crisis within 18 months of initial diagnosis of chronic phase of CML) and group II (developed accelerated phase or blast crisis > 30 months after initial diagnosis of chronic phase of CML). The clinical, hematologic and histomorphometric data were compared in the 2 groups of CML patients. RESULTS: The percentage of bone marrow promyelocytes was significantly increased in group I. On morphometry, the number of blasts per square millimeter, the area of reticulin fibers per square millimeter and the percentage area occupied by reticulin fibers were statistically significant. CONCLUSION: There seems to be grounds for hope for predicting prognosis of CML patients at initial diagnosis based on histomorphometric findings. The percentage area of reticulin fibers and the number of blasts per square millimeter are important prognostic predictors in histomorphometry data.     

Dynamics of bone marrow changes in patients with chronic idiopathic myelofibrosis following allogeneic stem cell transplantation

Scant knowledge exists about the dynamics of fibro-osteosclerotic bone marrow (BM) lesions and regeneration of hematopoiesis following allogeneic peripheral stem cell transplantation (SCT) in chronic idiopathic myelofibrosis. Therefore, an immunohistochemical and morphometric study was performed on BM biopsies in 20 patients before and at standardized intervals (days 30 through 384) following SCT. In responding patients, a total regression of the pretransplant increased fibrosis was completed in the posttransplant period after about six months, while the extent of osteosclerosis did not change significantly during observation time. The quantity of CD61+ megakaryocytes including precursors was strikingly variable after SCT and, by using planimetric methods, atypical microforms exhibiting a dysplastic aspect could be demonstrated. These anomalies may be responsible for posttransplant thrombocytopenia. CD34+ progenitor cells were increased before transplantation, however, their number declined rapidly to normal values in responding patients. Nucleated erythroid precursors revealed a decreased amount before and after SCT accounting for anemia. Large clusters of this cell lineage indicated an initial hematopoietic reconstitution comparable with the expansion of the neutrophil granulopoiesis. Proliferative activity and apoptosis showed an increase until one year after SCT that implied a still regenerating hematopoiesis in keeping with an enhanced cell turnover.     

Effects of the tyrosine kinase inhibitor imatinib mesylate (STI571) on bone marrow features in patients with chronic myelogenous leukemia

Preliminary data are available about bone marrow (BM) changes in patients with chronic myeloid leukemia (CML) who received the molecularly targeted and highly effective tyrosine kinase inhibitor Imatinib mesylate (STI571). This review is focused on a systematic assessment of BM features detectable at different stages of CML (stable, accelerated, blastic) following long-term (more than 10 months) treatment. By applying enzyme- and immunohistochemistry including monoclonal antibodies visualizing proliferating cell nuclear antigen (PCNA) and apoptosis (anti-apostatin), a more elaborate insight into alterations affecting hematopoiesis and the stroma compartment was gained. In patients with stable-phase CML therapy resulted in a significant reduction in cellularity, neutrophil granulopoiesis and number of megakaryocytes, accompanied by a retrieval of erythroid precursors. In patients with Imatinib as the only treatment morphometric analysis of CD61+ megakaryopoiesis was in keeping with a significant decrease in maturation defects implying a lesser amount of atypical micromegakaryocytes almost consistent with normalization. Moreover, a reduction of the initially enhanced (CD34+) microvessel density was detectable associated with a decrease in luminal distension. Regression of marked to moderate myelofibrosis was recognizable in about 70% of patients especially in the accelerated and blastic phases. The amount of myeloblasts, CD34+ progenitor cells and lysozyme-expressing immature myelomonocytic cells declined with treatment, but recurred in about 19% of patients that developed a leukemic relapse after 21+/-6 months of therapy. Data on proliferative activity and apoptosis in general supported in vitro findings concerning the inhibitory effect of this agent on growth associated with a tendency for stimulated apoptosis, at least in responding patients.     

Dynamics of lineage-restricted mixed chimerism following sex-mismatched allogeneic bone marrow transplantation

Scant knowledge is available about the dynamics of lineage-specific mixed chimerism (Ch) following bone marrow transplantation (BMT). This review is focused on findings derived from bone marrow (BM) biopsies in patients with chronic myeloid leukemia (CML) including a sex-mismatched host/donor constellation. Appropriate techniques involved immunophenotyping by monoclonal antibodies to identify the various cell lineages, dual color fluorescence in situ hybridization (FISH) with x- and y-chromosome-specific DNA-probes and a proper detection system for a simultaneous labeling of the bcr/abl locus. A significant degree of Ch with more than 20% host CD34+ progenitors was found in the early and late (up to 200 days after BMT) posttransplant period. However, only 10% of these cells harbored the bcr/abl translocation gene. This result fits well with corresponding molecular biological findings of so-called minimal residual disease. Conversion of Ch evolved during leukemic relapse with 90% host progenitors of which 50% revealed the bcr/abl locus. A Ch of nucleated erythroid percursors (5%) and CD68+ macrophages (8%) was expressed to a significantly lower degree. The slightly increased frequency found in CD61+ megakaryocytes (16%) was probably due to the polyploid state of these cells. Similar to the CD34+ progenitor cells abrupt changes from donor to host type was associated with an insidious transformation into recurrent leukemia. The CD34+ endothelial cells showed a minor degree of Ch, because donor-derived elements ranged from 18% to 25%. Leukemic relapse was characterized by an almost complete conversion of the endothelial cells to a host type. These findings point towards a CD34+ progenitor cell origin of the (leukemic) endothelial cell layer and suggests that their dysfunction may contribute to an expansion of the neoplastic clone.     

Usefulness of Y-body study on bone marrow smears to demonstrate the origin of fibroblast stromal cells in allogeneic bone marrow transplantation

The value of Y-body study for assessment of stromal cell engraftment was analyzed in 25 patients submitted to allogeneic bone marrow transplantation (BMT) (sex-matched in 12 cases and sex-mismatched in 13). The study was performed weekly on bone marrow smears until day +35, and the results were compared with those obtained in a control group of 20 patients submitted to autologous BMT (12 males and 8 females). Engraftment of haemopoietic cells was documented in all cases. The results of Y-body study on the recipients' fibroblast cells showed a pattern identical to that observed prior to BMT, independent of donor's sex. On the other hand, there were no differences between allogeneic and autologous BMT recipients in regard to percentage of Y-body positive cells. These results indicate that in allogeneic BMT there is no engraftment of the fibroblastic component of bone marrow stroma.     

Dissociation of hemopoietic chimerism and allograft tolerance after allogeneic bone marrow transplantation.

Creation of stable hemopoietic chimerism has been considered to be a prerequisite for allograft tolerance after bone marrow transplantation (BMT). In this study, we demonstrated that allogeneic BMT with bone marrow cells (BMC) prepared from either knockout mice deficient in both CD4 and CD8 T cells or CD3E-transgenic mice lacking both T cells and NK cells maintained a high degree of chimerism, but failed to induce tolerance to donor-specific wild-type skin grafts. Lymphocytes from mice reconstituted with T cell-deficient BMC proliferated when they were injected into irradiated donor strain mice, whereas lymphocytes from mice reconstituted with wild-type BMC were unresponsive to donor alloantigens. Donor-specific allograft tolerance was restored when donor-type T cells were adoptively transferred to recipient mice given T cell-deficient BMC. These results show that donor T cell engraftment is required for induction of allograft tolerance, but not for creation of continuous hemopoietic chimerism after allogeneic BMT, and that a high degree of chimerism is not necessarily associated with specific allograft tolerance.     

Myelofibrosis in chronic myeloproliferative disorders--dynamics and clinical impact

In chronic myeloproliferative disorders, presenting or evolving myelofibrosis (MF) is associated with significant morbidity and mortality. A systematically conducted evaluation of previous studies and data from our own material reveals a strikingly expressed heterogeneity of findings. Assessment of MF should be performed by a recently established semiquantitative scoring system regarding quantity and quality (reticulin versus collagen). It is important to differentiate between a fiber increase in bone marrow specimens and the clinical diagnosis that is explicitly based on extramedullary hematopoiesis (myeloid metaplasia). For this reason, prodromal stages of (reticulin) fibrosis are overlooked by the clinicians. Up to 30% of patients with chronic myelogenous leukemia show a minimal to advanced MF that is significantly associated not only with corresponding clinical parameters but more importantly with prognosis. In polycythemia vera about 20% of patients may display some degree of reticulin fibrosis at diagnosis, depending on stage of the disease. Transformation into (collagen) MF after more than 10 years is accompanied by clinical signs of myeloid metaplasia (spent phase). Essential thrombocythemia (ET) is characterised by the absence of increased reticulin at onset and an insignificant progression into MF, provided diagnosis is performed by the WHO criteria. Discrimination of prefibrotic and early stages of chronic idiopathic myelofibrosis (CIMF) from ET is relevant, especially concerning the rate and time usually required for the development of MF with myeloid metaplasia (full-blown CIMF). In conclusion, more elaborate evaluations including standardized grading of MF is warranted by regarding bone marrow biopsy specimens in association with clinical parameters including follow-up examinations.     

Alterations in Bone and Erythropoiesis in Hemolytic Anemia: Comparative Study in Bled,Phenylhydrazine-Treated and Plasmodium-Infected Mice

Sustained erythropoiesis and concurrent bone marrow hyperplasia are proposed to be responsible for low bone mass density (BMD) in chronic hemolytic pathologies. As impaired erythropoiesis is also frequent in these conditions, we hypothesized that free heme may alter marrow and bone physiology in these disorders. Bone status and bone marrow erythropoiesis were studied in mice with hemolytic anemia (HA) induced by phenylhydrazine (PHZ) or Plasmodium infection and in bled mice. All treatments resulted in lower hemoglobin concentrations, enhanced erythropoiesis in the spleen and reticulocytosis. The anemia was severe in mice with acute hemolysis, which also had elevated levels of free heme and ROS. No major changes in cellularity and erythroid cell numbers occurred in the bone marrow of bled mice, which generated higher numbers of erythroid blast forming units (BFU-E) in response to erythropoietin. In contrast, low numbers of bone marrow erythroid precursors and BFU-E and low concentrations of bone remodelling markers were measured in mice with HA, which also had blunted osteoclastogenesis, in opposition to its enhancement in bled mice. The alterations in bone metabolism were accompanied by reduced trabecular bone volume, enhanced trabecular spacing and lower trabecular numbers in mice with HA. Taken together our data suggests that hemolysis exerts distinct effects to bleeding in the marrow and bone and may contribute to osteoporosis through a mechanism independent of the erythropoietic stress.     

Genetic and immunological assessment of a bone marrow transplantation in a patient with a primary immune defect: leukocyte adhesion deficiency

Leukocyte adhesion deficiency (LAD) was suspected in a three weeks old girl from a family with an established history of LAD with a lack (less then 1%) of the beta 2 integrins CD 11a, b/CD 18 expression at the leukocytes surface, was engrafted with her mother HLA identical bone marrow at the age of 14 months. Repeated post transplantation (up to 22 months). Immunological assessments showed a good engraftment with 97% of the lymphocytes expressing CD11a/CD18. Cells proliferated normally in response to PHA and to Tetanus toxo?d after revaccination. The level of serum immunoglobulins was normal. Investigation of the CD18 intragenic polymorphic marker Avall before and after bone marrow transplantation (BMT) showed a transition from the Avall +/+ genotype to the mother's Avall +/- genotype. Similarly DNA fingerprints obtained with the patient genomic DNA, prepared from PBMC, prior and after transplantation, showed that the patient's DNA fingerprints pattern matched the mother's one. These findings are consistent with the good engraftment observed clinically. This study emphasizes the usefulness of the molecular techniques to evaluate the degree of chimerism in monitoring the outcome of bon marrow transplantation.     

Acellular bone marrow extracts significantly enhance engraftment levels of human hematopoietic stem cells in mouse xeno-transplantation models

Hematopoietic stem cells (HSC) derived from cord blood (CB), bone marrow (BM), or mobilized peripheral blood (PBSC) can differentiate into multiple lineages such as lymphoid, myeloid, erythroid cells and platelets. The local microenvironment is critical to the differentiation of HSCs and to the preservation of their phenotype in vivo. This microenvironment comprises a physical support supplied by the organ matrix as well as tissue specific cytokines, chemokines and growth factors. We investigated the effects of acellular bovine bone marrow extracts (BME) on HSC in vitro and in vivo. We observed a significant increase in the number of myeloid and erythroid colonies in CB mononuclear cells (MNC) or CB CD34+ cells cultured in methylcellulose media supplemented with BME. Similarly, in xeno-transplantation experiments, pretreatment with BME during ex-vivo culture of HSCs induced a significant increase in HSC engraftment in vivo. Indeed, we observed both an increase in the number of differentiated myeloid, lymphoid and erythroid cells and an acceleration of engraftment. These results were obtained using CB MNCs, BM MNCs or CD34(+) cells, transplanted in immuno-compromised mice (NOD/SCID or NSG). These findings establish the basis for exploring the use of BME in the expansion of CB HSC prior to HSC Transplantation. This study stresses the importance of the mechanical structure and soluble mediators present in the surrounding niche for the proper activity and differentiation of stem cells.     

Purification of Bone Marrow Clonal Cells from Patients with Myelodysplastic Syndrome via IGF-IR

Malignant clonal cells purification can greatly benefit basic and clinical studies in myelodysplastic syndrome (MDS). In this study, we investigated the potential of using type 1 insulin-like growth factor receptor (IGF-IR) as a marker for purification of malignant bone marrow clonal cells from patients with MDS. The average percentage of IGF-IR expression in CD34⁺ bone marrow cells among 15 normal controls was 4.5%, 70% of which also express the erythroid lineage marker CD235a. This indicates that IGF-IR mainly express in erythropoiesis. The expression of IGF-IR in CD34⁺ cells of 55 MDS patients was significantly higher than that of cells from the normal controls (54.0 vs. 4.5%). Based on the pattern of IGF-IR expression in MDS patients and normal controls, sorting of IGF-IR-positive and removal of CD235a-positive erythroid lineage cells with combination of FISH detection were performed on MDS samples with chromosomal abnormalities. The percentage of malignant clonal cells significantly increased after sorting. The enrichment effect was more significant in clonal cells with a previous percentage lower than 50%. This enrichment effect was present in samples from patients with +8, 5q-/-5, 20q-/-20 or 7q-/-7 chromosomal abnormalities. These data suggest that IGF-IR can be used as a marker for MDS bone marrow clonal cells and using flow cytometry for positive IGF-IR sorting may effectively purify MDS clonal cells.     

Cytogenetic studies using Q-band polymorphisms in patients with AML receiving marrow from like-sex donors

Summary After bone marrow transplantation (BMT), it is important to monitor the bone marrow and lymphoid cell populations of the recipient to document engraftment. When donor and recipient are of unlike sex, the sex chromosomes serve as a useful marker to determine cellular origin. When donor and recipient are of like sex, autosomal heteromorphisms can be used to identify the origin of cells in metaphase. Using Q-banding, we found that 17 of 20 patient/donor pairs (85%) examined showed at least one chromosome heteromorphism that distinguished between recipient and donor cells with certainty. Five of the patients were followed up after BMT in order to document engraftment. Donor metaphases could be detected in the marrow within two weeks of BMT when the graft was successful. Chimaerism was detected in the lymphocyte population even when the graft persisted. In a case of graft failure, donor cells did not persist in the marrow, and the lymphocyte population did not convert to donor type. These studies demonstrate that autosomal heteromorphisms are useful in the study of myeloid and lymphoid chimaeric states after BMT.     

Activated fibronectin-secretory phenotype of mesenchymal stromal cells in pre-fibrotic myeloproliferative neoplasms

We characterized bone marrow stromal cells (BMSC) from patients with pre-fibrotic myeloproliferative neoplasms (MPN). MPN-BMSC showed decreased capacity to stimulate the proliferation of colony-forming units of normal hematopoietic stem and progenitor cells and displayed increased matrix remodelling (in particular fibronectin deposition) compared to control BMSC. This finding was confirmed in pre-fibrotic MPN bone marrow biopsies in a tissue microarray (n?=?34), which stained positive for fibronectin in the absence of reticulin as a standard myelofibrosis marker. Fibronectin expression correlated significantly with reduced haemoglobin levels in MPN-patients (p?=?0.007; R2?=?0.42). Our data show significant cell-intrinsic alterations in MPN-MSC and suggest that Fibronectin expression might be applicable as a biomarker for the identification of early myelofibrotic transformation in reticulin-negative MPN.     

Bone marrow angiogenesis: methods of quantification and changes evolving in chronic myeloproliferative disorders

Until now little information is available about bone marrow (BM) angiogenesis in chronic myeloproliferative disorders (CMPDs). Amongst the various immunohistochemical markers for endothelial cells CD34 and CD105 have proven to be most reliable since they exhibit no relevant co-staining. Determination of vascularity has to include pathophysiological aspects of perfusion. Therefore, quantification of the microvascular density (MVD) by the so-called hot spot method has to be improved by parameters that characterize blood flow more properly like microvessel area (luminal distension), shape (form factor), tortuosity, and branching (maximal vessel length). In comparison to the normal BM chronic myeloid leukemia (CML) revealed a significant increase in MVD which was functionally associated with elevated levels of angiogenic cytokines. Structure of vessels was significantly altered by showing an enhanced irregularity of shape and tortuosity and increase in fibers was conspicuously accompanied by a higher degree of MVD. Contrasting the group of patients with Imatinib (STI571) therapy interferon failed to reduce the number of vessels. Following bone marrow transplantation a significant enhancement of the MVD was found in the early post-transplant period, but after about 6 months normalization occurred. Anomalies of microvascular architecture were easily demonstrable by three-dimensional reconstruction and consisted of a complex branching network of irregular shaped sinuses. Chronic idiopathic myelofibrosis displayed a significant increase in the MVD only in the advanced fibrosclerotic stages. This feature was accompanied by an enhanced luminal distension and tortuosity, thus contrasting the prefibrotic and early fibrotic phases of this disorder. Similar to CML a relationship between evolving myelofibrosis and change in vascular architecture was encountered. This feature may present a possible target for future anti-angiogenic therapy. In essential thrombocythemia there is only a mild increase in MVD detectable while in polycythemia vera besides an enlarged number, a luminal dilation due to the densely packed erythrocytes is recognizable. In conclusion, contrasting the usually applied quantification technique more elaborate morphometrical methods are warranted to obtain a better insight into the vascular architecture of the BM. In CMPDs angiogenesis is significantly associated with the evolution of myelofibrosis and may be altered by therapeutic regimens probably due to changes in cytokine release.