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Arbeitsgemeinschaft Haematopathologie I. Thema: Knochenmark
Abstracts· 295
263 Molecular pathology of mitochondrial disorders J. Muller-Hocker, Institut fur Pathologie der Ludwig-Maximilians-Universitat, Thalkirchner StraBe 36, Munchen Defects of the respiratory chain most often affect the limb muscles and central nervous system, but also the heart, the liver, kidneys and the endocrine system. Specific mutations of mitochondrial DNA (mt DNA) have been identified for various mitochondrial diseases such as deletions, depletions and point mutations of transfer RNAs and of messenger RNAs. The mutations occur either spontaneously or are maternally inherited because mtDNA is exclusively derived from the mother. Besides mutations of mtDNA mutations of nuclear DNA play also an important role because most of the protein subunits of the respiratory chain enzymes are coded by nuclear DNA. Furthermore the replication, transcription and translation of mtDNA is controlled by various nuclear factors. The clinical picture of mtDNA mutations depends on various factors: the type of mutation, the organ distribution pattern, the proportion of mutated and wild-type mtDNA and the energy demand of the involved organs. Additional diseases and exogen influences probably may lower the threshold for clinical manifestation. During aging similar mutations of mtDNA accumulate especially in postmitotic organs and probably contribute to the reduced organ function in senescence.
Arbeitsgemeinschaft Haematopathologie I. Thema: Knochenmark Working Group Hematopathology I. Bone Marrow
1
Abstract not received
2 I\IYELODYSPLASTICS SYNDROMES - PROGNOSTIC VALUE OF BONE MARROW HISTOLOGY D Schneider (a.G.)*, lrith Baumann (a.G.)*, HK Muller-Hennelink*, AC Feller+ *lnstitute ofPatholot,'y, University Wlirzburg, Josef-Schneider-Str 2,97080 Wurzburg +lnstitut of Pathology, Medical University of Lubeck, Ratzeburger Allee 160,23538 Lubeck Aims. The prognostic value of histological marrow findings in myelodysplastic syndroms (MDS) is controversely discussed. FABclassification and Boumemouth score are believed to be reliable prognostic indices, whereas data about histological criteria are of inconsistant value. We examined in how far morphology of bone marrow trephines, bone marrow smears and peripheral blood smears can provide additinal infonnation of prognostic significance. Methods: Routinely processed and paraffin embedded trephines, bone marrow and blood smears of 86 patients with MDS were retrospectively analysed by three independent examiners, and classified according to the F AB-classification. In bone marrow lymphocytosis immunhistochemistry was perfonned. These results were correlated to clinical data Results By sequential analysis of the bone marrow blast cell count 2 months after diagnosis we generated distinct prognostic subgroups. 69% of patients with increase of blasts within 2 months after diagnosis developed acute leukaemia during a median follow-up of 58 weeks compared with 0% of the patients with stable blast cell count (p<0,001). Bone marrow lymphocytosis and myelofibrosis were both significantly more often found in patients with stable disease (p
3 The value of lymphoid antibodies for the evaluation of minimal lymphocytosis in bone marrow biopsies of patients with known low grade nonhodgkins lymphoma. Nina Hurwitz, A.Lohri (a.G.)** A.Tichelli (a.G.)***, Ch.McGandy (a.G.)*, E.Vrost (a.G.)****, A.Berrebi (a.G.)**** *Institute of Pathology, **Departments of Oncology and of ***Hematology University of Basle, Depanment of Hematology ****Kaplan hospital Rehovot, afill. to the Hebrew University, Jerusalem 40 decalcified bone marrow biopsies with mild focal and interstitial lymphocytosis from patients with known low grade nonhodgkins lymphoma were selected. The sections were incubated with the following antibodies:LCA(CD4S); MB2and KiB3,B-cells; CD3 and UCHLl(CD45RO),T-cells.Evaluated were: the quantity of interstitial B- and T-cells, the quantity and distribution of each subset within aggregates and cytologic details of the marked cells.The interpretation of the results was made according to own previous observations and recently published data. An increase of interstitial B-cells was regarded as highly
296 . Abstracts suggestive of minimal involvement. Compact homogenous aggregates of B-cells often surrounded by a margin of T -cells were regarded as neoplastic, even if they were small.Reactive infiltrates showed a more or less even admixture of both lymphocyte subsets. Lymphocyte marking was especially helpfull for the detection of minimal residual disease in hairy cell leukemia after chemotherapy, and for the recognition of minimal infiltrates by Tchronic lymphatic leukemia. Considering cytologic detail together with lymphocyte typing, discrimination between reactive and neoplastic infiltrates was possible in 28 cases (70%). 12 cases (30%) had to remain equivocal also after immunohistochemical examination. We conclude that application of lymphoid antibodies is a valuable help for the differentialdiagnosis between reactive and neoplastic lymphoid infiltrates in patients with nonhodgkins lymphoma, even though no clearcut diagnosis could be achieved in some of the cases.
4 LECTIN BINDING SITES AND PLATELET GLYCOPROTEINS - A COMPARATIVE STUDY ON HUMAN MEGAKARYOCYTES J. Thiel~ S.E. Baldus (a.G.), A. Charles (a.G.), F.-G. Hanisch (a.G.), R. Fischer Institut fiir Pathologie und Institut fiir Immunbiologie*, Universitiit zu K61n, Joseph-Stelzmann-Str. 9, 50924 K6ln Little information exists about the glycosylation of megakaryocyte glycoproteins (gps). We investigated routinely processed bone marrow trephines derived from 15 patients with reactive and neoplastic lesions by applying a large panel of lectins and carbohydrate-specific monoclonal antibodies (mabs). A biotin-streptavidin-alkaline-phosphatase assay was used to visualize the antigens. " Ulex-europaeus-agglutinin-I detecting H-type-2 blood group antigen Fuca(1-2)GalB(1-4)G1cNAc showed a strong cytoplasmic reactivity in all cases, whereas two mabs recognizing related a(1-2)-fucosylated Lewis type-2 chains (124LE, 19-0LE) exhibited a dot-like staining pattern. In most cases, expression of Lewis x antigen could be demonstrated by mab LeuMl. The binding sites of lectins from Erythrina cristagalli (ECA) , Arachis hypogaea (PNA) , Helix pomatia (HPA) and Glycine max (SBA) were accessible only after neuraminidase treatment. 18 other lectins and mabs showed no or an only questionable reactivity. Immunoblots of platelet 1ysates were stained with these reagents in order to identify those gps bearing their binding sites. We observed a strong reaction of UEA-I, 12-4LE and 19-0LE to gp lb. This antigen was also detected by ECA and PNA after desialylation. No binding of any of these 1ectins and mabs could be seen towards gp IlIa. We conclude that most lectins and mabs recognize gp Ib on megakaryocytes and platelets, but not IlIa, which is expressed earlier during megakaryocytic maturation. GP lb has been described to contain different 0- and N-glycans and therefore reveals corresponding lectin binding sites and epitopes.
5 IMMUNOHISTOCHEMICAL EVIDENCE OF EVEN A FEW CD34-EXPRESSING HEMOPOIETIC PROGENITOR CELLS IN HUMAN BONE MARROW IS STRONGLY SUGGESTIVE OF MYELODYSPLASIA. M. Wehrmann (a.G.), H.-P. Horny, U. Schlicker (a. G.), E. Kaiserling. Institute of Pathology, University of Tiibingen Immunohistochemical investigation of 279 routinely processed (formalin-fixed, paraffin-embedded) bone marrow biopsy trephine specimens taken from the iliac crest was performed with a monoclonal antibody against CD34 (DIANOV A-IMMUNOTECH GmbH,
Hamburg, FRG), a hemopoietic stem cell-related antigen. The diagnoses were grouped together for statistical evaluation (WILCOXON test): (I) normal state (n=89); (2) reactive states, e.g. myeloid hyperplasia, aplastic anemia (n=77); (3) myelodysplastic syndromes (n=IO); (4) acute leukemia (n=38: AML, n=24; ALL, n=14); and (5) lymphoproliferative disorders (n=65). Annular (i.e. membrane-associated) immunostaining was considered specific. Staining of vascular endothelium was used as an internal positive control. CD34-expressing progenitor cells were found to be virtually absent from the bone marrow in normal and reactive states and from marrow infiltrated by malignant lymphoma (except ALL) or plasmacytoma. By contrast, CD34-expressing progenitorlblast cells were detected in 8 of the 10 cases of myelodysplasia, and in 17 of the 36 cases of acute leukemia (AML, n=ll; ALL, n=6). While CD34+ blast cells were found in small numbers (about O.5-liHPF) in most cases of myelodysplasia, they were often seen in large numbers in acute leukemia (up to IOOIHPF). The differences in numbers of CD34-expressing cells between normal/reactive and myelodysplastic, and between myelodysplastic and leukemic bone marrow were each statistically significant (p
6 CYTOKlNE RELEASE BY HUMAN MEGAKARYOCYTES: ANALYSIS AT THE SINGLE CELL LEVEL Claudia Wickenhauser (a.G.), A. Hillienhof (a.G.), K. Jungheim (a.G.), J. Lorenzen (a.G.), M.-L. Hansmann, J. Thiele, R. Fischer Institut fiir Pathologie, Universitiit zu KOln Joseph-Stelzmann-Str. 9; 50924 K61n, FRG It is generally accepted that in chronic myeloproliferative disorders
fibroblast proliferation and ensuing myelofibrosis has to be considered as a reactive process. In this context, the significant role of inappropriate growth factor release from megakaryo- and thrombocytes as mediators of fibrillogenesis has been recognized. However, little information exists, whether there is an active and/or spontaneous release of these cytokines by normal human megakaryocytes. To answer this question, we enriched megakaryocytes by Percoll density centrifugation and performed a Reverse Haemolytic Plaque Assay (RHPA). This method enables the assessement of secreted haemopoietic growth factors at the single cell level. Following incubation with pure serum-free medium less than 0.5 % of the Y2/5l-positive megakaryocytic cells secreted II-la, IL-3, IL-6, GM-CSF and TGFB, while 3 % of the megakaryocytic cells produced PDGF. After 8 h incubation with rhlL-la (10.0 ng/ml) or rhGMCSF (10.0 pg/ml) the number of cytokine-secreting cells increased significantly. On the other hand the release of PDGF decreased to 0.8 % (IL-la) resp. 0.6 % (GM-CSF). Thus, it can be concluded that megakaryopoietic cells show a low but distinctive spontaneous secretion particulary of PDGF in addition to other growth factors and change their releasing pattern after incubation with rhIL-la or rhGM-CSF.
263 Molecular pathology of mitochondrial disorders J. Muller-Hocker, Institut fur Pathologie der Ludwig-Maximilians-Universitat, Thalkirchner StraBe 36, Munchen Defects of the respiratory chain most often affect the limb muscles and central nervous system, but also the heart, the liver, kidneys and the endocrine system. Specific mutations of mitochondrial DNA (mt DNA) have been identified for various mitochondrial diseases such as deletions, depletions and point mutations of transfer RNAs and of messenger RNAs. The mutations occur either spontaneously or are maternally inherited because mtDNA is exclusively derived from the mother. Besides mutations of mtDNA mutations of nuclear DNA play also an important role because most of the protein subunits of the respiratory chain enzymes are coded by nuclear DNA. Furthermore the replication, transcription and translation of mtDNA is controlled by various nuclear factors. The clinical picture of mtDNA mutations depends on various factors: the type of mutation, the organ distribution pattern, the proportion of mutated and wild-type mtDNA and the energy demand of the involved organs. Additional diseases and exogen influences probably may lower the threshold for clinical manifestation. During aging similar mutations of mtDNA accumulate especially in postmitotic organs and probably contribute to the reduced organ function in senescence.
Arbeitsgemeinschaft Haematopathologie I. Thema: Knochenmark Working Group Hematopathology I. Bone Marrow
1
Abstract not received
2 I\IYELODYSPLASTICS SYNDROMES - PROGNOSTIC VALUE OF BONE MARROW HISTOLOGY D Schneider (a.G.)*, lrith Baumann (a.G.)*, HK Muller-Hennelink*, AC Feller+ *lnstitute ofPatholot,'y, University Wlirzburg, Josef-Schneider-Str 2,97080 Wurzburg +lnstitut of Pathology, Medical University of Lubeck, Ratzeburger Allee 160,23538 Lubeck Aims. The prognostic value of histological marrow findings in myelodysplastic syndroms (MDS) is controversely discussed. FABclassification and Boumemouth score are believed to be reliable prognostic indices, whereas data about histological criteria are of inconsistant value. We examined in how far morphology of bone marrow trephines, bone marrow smears and peripheral blood smears can provide additinal infonnation of prognostic significance. Methods: Routinely processed and paraffin embedded trephines, bone marrow and blood smears of 86 patients with MDS were retrospectively analysed by three independent examiners, and classified according to the F AB-classification. In bone marrow lymphocytosis immunhistochemistry was perfonned. These results were correlated to clinical data Results By sequential analysis of the bone marrow blast cell count 2 months after diagnosis we generated distinct prognostic subgroups. 69% of patients with increase of blasts within 2 months after diagnosis developed acute leukaemia during a median follow-up of 58 weeks compared with 0% of the patients with stable blast cell count (p<0,001). Bone marrow lymphocytosis and myelofibrosis were both significantly more often found in patients with stable disease (p
3 The value of lymphoid antibodies for the evaluation of minimal lymphocytosis in bone marrow biopsies of patients with known low grade nonhodgkins lymphoma. Nina Hurwitz, A.Lohri (a.G.)** A.Tichelli (a.G.)***, Ch.McGandy (a.G.)*, E.Vrost (a.G.)****, A.Berrebi (a.G.)**** *Institute of Pathology, **Departments of Oncology and of ***Hematology University of Basle, Depanment of Hematology ****Kaplan hospital Rehovot, afill. to the Hebrew University, Jerusalem 40 decalcified bone marrow biopsies with mild focal and interstitial lymphocytosis from patients with known low grade nonhodgkins lymphoma were selected. The sections were incubated with the following antibodies:LCA(CD4S); MB2and KiB3,B-cells; CD3 and UCHLl(CD45RO),T-cells.Evaluated were: the quantity of interstitial B- and T-cells, the quantity and distribution of each subset within aggregates and cytologic details of the marked cells.The interpretation of the results was made according to own previous observations and recently published data. An increase of interstitial B-cells was regarded as highly
296 . Abstracts suggestive of minimal involvement. Compact homogenous aggregates of B-cells often surrounded by a margin of T -cells were regarded as neoplastic, even if they were small.Reactive infiltrates showed a more or less even admixture of both lymphocyte subsets. Lymphocyte marking was especially helpfull for the detection of minimal residual disease in hairy cell leukemia after chemotherapy, and for the recognition of minimal infiltrates by Tchronic lymphatic leukemia. Considering cytologic detail together with lymphocyte typing, discrimination between reactive and neoplastic infiltrates was possible in 28 cases (70%). 12 cases (30%) had to remain equivocal also after immunohistochemical examination. We conclude that application of lymphoid antibodies is a valuable help for the differentialdiagnosis between reactive and neoplastic lymphoid infiltrates in patients with nonhodgkins lymphoma, even though no clearcut diagnosis could be achieved in some of the cases.
4 LECTIN BINDING SITES AND PLATELET GLYCOPROTEINS - A COMPARATIVE STUDY ON HUMAN MEGAKARYOCYTES J. Thiel~ S.E. Baldus (a.G.), A. Charles (a.G.), F.-G. Hanisch (a.G.), R. Fischer Institut fiir Pathologie und Institut fiir Immunbiologie*, Universitiit zu K61n, Joseph-Stelzmann-Str. 9, 50924 K6ln Little information exists about the glycosylation of megakaryocyte glycoproteins (gps). We investigated routinely processed bone marrow trephines derived from 15 patients with reactive and neoplastic lesions by applying a large panel of lectins and carbohydrate-specific monoclonal antibodies (mabs). A biotin-streptavidin-alkaline-phosphatase assay was used to visualize the antigens. " Ulex-europaeus-agglutinin-I detecting H-type-2 blood group antigen Fuca(1-2)GalB(1-4)G1cNAc showed a strong cytoplasmic reactivity in all cases, whereas two mabs recognizing related a(1-2)-fucosylated Lewis type-2 chains (124LE, 19-0LE) exhibited a dot-like staining pattern. In most cases, expression of Lewis x antigen could be demonstrated by mab LeuMl. The binding sites of lectins from Erythrina cristagalli (ECA) , Arachis hypogaea (PNA) , Helix pomatia (HPA) and Glycine max (SBA) were accessible only after neuraminidase treatment. 18 other lectins and mabs showed no or an only questionable reactivity. Immunoblots of platelet 1ysates were stained with these reagents in order to identify those gps bearing their binding sites. We observed a strong reaction of UEA-I, 12-4LE and 19-0LE to gp lb. This antigen was also detected by ECA and PNA after desialylation. No binding of any of these 1ectins and mabs could be seen towards gp IlIa. We conclude that most lectins and mabs recognize gp Ib on megakaryocytes and platelets, but not IlIa, which is expressed earlier during megakaryocytic maturation. GP lb has been described to contain different 0- and N-glycans and therefore reveals corresponding lectin binding sites and epitopes.
5 IMMUNOHISTOCHEMICAL EVIDENCE OF EVEN A FEW CD34-EXPRESSING HEMOPOIETIC PROGENITOR CELLS IN HUMAN BONE MARROW IS STRONGLY SUGGESTIVE OF MYELODYSPLASIA. M. Wehrmann (a.G.), H.-P. Horny, U. Schlicker (a. G.), E. Kaiserling. Institute of Pathology, University of Tiibingen Immunohistochemical investigation of 279 routinely processed (formalin-fixed, paraffin-embedded) bone marrow biopsy trephine specimens taken from the iliac crest was performed with a monoclonal antibody against CD34 (DIANOV A-IMMUNOTECH GmbH,
Hamburg, FRG), a hemopoietic stem cell-related antigen. The diagnoses were grouped together for statistical evaluation (WILCOXON test): (I) normal state (n=89); (2) reactive states, e.g. myeloid hyperplasia, aplastic anemia (n=77); (3) myelodysplastic syndromes (n=IO); (4) acute leukemia (n=38: AML, n=24; ALL, n=14); and (5) lymphoproliferative disorders (n=65). Annular (i.e. membrane-associated) immunostaining was considered specific. Staining of vascular endothelium was used as an internal positive control. CD34-expressing progenitor cells were found to be virtually absent from the bone marrow in normal and reactive states and from marrow infiltrated by malignant lymphoma (except ALL) or plasmacytoma. By contrast, CD34-expressing progenitorlblast cells were detected in 8 of the 10 cases of myelodysplasia, and in 17 of the 36 cases of acute leukemia (AML, n=ll; ALL, n=6). While CD34+ blast cells were found in small numbers (about O.5-liHPF) in most cases of myelodysplasia, they were often seen in large numbers in acute leukemia (up to IOOIHPF). The differences in numbers of CD34-expressing cells between normal/reactive and myelodysplastic, and between myelodysplastic and leukemic bone marrow were each statistically significant (p
6 CYTOKlNE RELEASE BY HUMAN MEGAKARYOCYTES: ANALYSIS AT THE SINGLE CELL LEVEL Claudia Wickenhauser (a.G.), A. Hillienhof (a.G.), K. Jungheim (a.G.), J. Lorenzen (a.G.), M.-L. Hansmann, J. Thiele, R. Fischer Institut fiir Pathologie, Universitiit zu KOln Joseph-Stelzmann-Str. 9; 50924 K61n, FRG It is generally accepted that in chronic myeloproliferative disorders
fibroblast proliferation and ensuing myelofibrosis has to be considered as a reactive process. In this context, the significant role of inappropriate growth factor release from megakaryo- and thrombocytes as mediators of fibrillogenesis has been recognized. However, little information exists, whether there is an active and/or spontaneous release of these cytokines by normal human megakaryocytes. To answer this question, we enriched megakaryocytes by Percoll density centrifugation and performed a Reverse Haemolytic Plaque Assay (RHPA). This method enables the assessement of secreted haemopoietic growth factors at the single cell level. Following incubation with pure serum-free medium less than 0.5 % of the Y2/5l-positive megakaryocytic cells secreted II-la, IL-3, IL-6, GM-CSF and TGFB, while 3 % of the megakaryocytic cells produced PDGF. After 8 h incubation with rhlL-la (10.0 ng/ml) or rhGMCSF (10.0 pg/ml) the number of cytokine-secreting cells increased significantly. On the other hand the release of PDGF decreased to 0.8 % (IL-la) resp. 0.6 % (GM-CSF). Thus, it can be concluded that megakaryopoietic cells show a low but distinctive spontaneous secretion particulary of PDGF in addition to other growth factors and change their releasing pattern after incubation with rhIL-la or rhGM-CSF.