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Bone Marrow, Abstract 262–270, Symposium
Bone Marrow · 247 was amplifiable only in tumour cells and not in the co-cultivated fibroblasts indicating no cell-cross-contamination during microdissection.
Symposium: Bone Marrow
262
Aberrant Methylation and impaired expression of the p15INK4b gene in chronic myelomonocytic leukemia (CMML)
M. TESSEMA, F. LÄNGER, J. DINGEMANN, A. GANSER1, H. KREIPE, U. LEHMANN Institut für Pathologie, Medizinische Hochschule Hannover Institut für Hämatologie/Onkologie, Medizinische Hochschule Hannover 1
Aims: The important cell cycle regulatory gene p15INK4b has been shown to be inactivated in acute myeloid leukaemia and myelodysplastic syndrome. Little is known about the expression and epigenetic modification of this gene in chronic myelomonocytic leukaemia (CMML) that belongs to the myelodysplastic-myeloproliferative disorders with a high proportion of blastic transformation. Methods: DNA and RNA were extracted from formalin-fixed paraffin-embedded (FFPE) bone marrow trephines from a series of CMML patients (n = 33). For methylation analysis methylationspecific PCR (MSP) and genomic bisulfit-sequencing as well as sequencing of cloned PCR products were employed. Expression was measured using quantitative real-time RT-PCR for mRNA levels and immunohistochemistry, which was also performed on the FFPE bone marrow biopsies. Results: Analysis of bone marrow trephines in a series of 33 CMML cases showed an aberrant p15INK4b gene methylation in up to 58% of cases. It turned out to be spread over a broad area of the 5′ region and exhibited substantial heterogeneity between cases and even in individual patients. The degree of aberrant methylation was correlated with a reduced mRNA as well as reduced protein expression and was associated with a higher expression of DNA methyltransferase DNMT 3A. It correlated also with the blast content of the trephines. Conclusions: We conclude that aberrant gene methylation is a frequent event in CMML that might contribute to the pathogenesis of this myelodysplastic-myeloproliferative disorder.
orders, the expression levels of H19 and IGF-2 were quantified by real-time PCR. Transcript levels of IGF-2 in CMPD and normal hematopoiesis were correlated with staining pattern and intensity by immunohistochemistry using micro-tissue arrays (System Multiblock). Results: The expression of tumor suppressor H19 in CMPD was significantly reduced up to 20fold compared to non-neoplastic states. In contrast, IGF-2 expression levels were demonstrated to be up to 3fold higher in CMPD compared to normal hematopoiesis. Conclusions: We conclude that downregulation of H19 is a constant feature of all CMPD subtypes and may contribute to an upregulation of IGF-2 expression with resulting growth-promoting effects in CMPD.
264
CD117 Immunohistochemistry of bone marrow biopsies in the diagnosis of myelodysplastic syndromes S.M. KRÖBER1, O. AICHELE2, H. MÜLLER1, E. KAISERLING1
1
Institut für Pathologie, Universität Tübingen Abteilung Hämatologie und Onkologie, Medizinische Klinik, Universität Tübingen 2
Tumor-suppressor H19 and Insulin-like growth factor-2 are inversely expressed in bone marrow cells of chronic myeloproliferative disorders
Background: CD117 immunostaining has proven to be a useful tool for distinguishing acute myeloid leukaemias from acute lymphoblastic leukaemias by flow cytometry and in bone marrow biopsies (BMB). Systematic analysis of the CD117 expression of blasts in myelodysplastic syndromes (MDS) cases have not been performed on parraffin embedded BMB. Methods: 99 formalin fixed, paraffin embedded BMB were investigated. The cases were classified as normal or reactive bone marrow (nBM, n = 16), lymphoproliferative disorders, i.e. non-Hodgkin’s lymphoma of low grade malignancy or Hodgkin’s lymphoma (lyBM, n = 6), myelodysplastic syndrome (MDS, n = 49, including 6 cases where refractary anaemia could neither be established nor ruled out), hypocellular acute myeloid leukaemia (hAML, n = 15), and residual acute myeloid leukaemia with oligoblastic bone marrow (rAML, n = 13). Immunostaining with T595 (Anti-CD117) and QBEND10 (Anti-CD34) using the ABC-Method was performed. The percentage of CD117+ and of CD34+ haemopoietic cells in 10 HPF and the occurrence of clusters of CD34+ and/or CD117+ cells were determined in each case. Results: The average number of CD117+ cells was very low in nBM (1.28%) and lyBM (1.16%) and showed only a little increase (4.07%) in the MDS subtypes with blast count below 10%, the percentage of CD117+ cells usually not exceeding the number of CD34+ cells. However the CD117+ cell count (11.93%–22.2%–27.9%) and cluster values (13/15, 12/13, and 15/15 vs. 0) were higher in MDS RAEBII, rAML, and hAML than in nBM and lyBM with reactive changes. Conclusions: The addition of CD117 immunostaining in BMB is useful in the identification of MDS RAEB II, hAML, and rAML.
O. BOCK, J. SCHLUÉ, R. VON WASIELEWSKI, M. MENGEL, H. KREIPE
265
263
Institut für Pathologie, Medizinische Hochschule Hannover Aims: In most adult tissues, H19 and its chromosomal neighbour IGF-2, are reciprocally imprinted. In normal hematopoiesis, however, IGF-2 imprinting was shown to be disrupted with consecutive biallelic expression. H19 is known to be a negative regulator of IGF-2 expression. Our aim was to elucidate the expression profile of the hematopoietic growth promoter insulin-like growth factor-2 (IGF-2) and its designated repressor, the untranslated tumor-suppressor RNA H19, in non-neoplastic hematopoiesis and chronic myeloproliferative disorders (CMPD). Methods: After RNA extraction from total bone marrow cells of non-neoplastic hematopoiesis and chronic myeloproliferative dis-
Early Indication of Therapy Failure and Prognosis by Evolving or Progressive Myelofibrosis in Chronic Myeloid Leukemia
G. BÜSCHE, R. HEHLMANN1, M. FREUND2, H. HECKER3, T. BUHR, A. GEORGII, H. KREIPE, German CML Study Group Institut für Pathologie, Medizinische Hochschule Hannover 1 III. Med. Klinik, Klinikum Mannheim, Universität Heidelberg 2 Klinik für Hämatologie und Onkologie, Universität Rostock 3 Institut für Biometrie, Medizinische Hochschule Hannover Aims: Consideration of marrow fibrosis (MF) is unusual in therapy trials on chronic myeloid leukemia (CML) explaining the lack of long-term observations on the basis of sequential bone marrow
248 · Symposium biopsies (BMB) taken prospectively during controlled trials on CML. Methods: 1198 BMB from 493 patients recruited in two German therapy trials on Ph+ CML were examined for MF before and during therapy comprising histopathologic follow-up periods up to 9 years. Results: MF was a typical complication of Ph+ CML affecting the majority of patients when therapy was not effective. Oncoming or progressive MF was a reliable predictor of therapy failure one to two years earlier as indicated by changes in the peripheral blood, spleen size, marrow blast count and cytogenetics, correlating with an increase of the relative risk of acceleration by a factor of 14 independent of the type of therapy applied. The survival time of patients suffering from evolving or progressive MF was significantly shortened independently of risk score, hematologic and cytogenetic remission and type of therapy applied including allografting (multivariate analysis; P < 0.00005). Conclusions: The analyzed long-term observations strongly indicate that MF is a complication related to CML, not to therapy. MF is prevented when therapy is effective whereas oncoming or progressive MF is an early and reliable indicator of therapy failure and acceleration independent of peripheral blood findings, spleen size, marrow blast count and cytogenetics.
266
Bone marrow fibrosis in CML treated with Imatinib mesylate (Glivec®)
A. LUGLI, S. B. COGLIATTI1, M. TINGUELY2, B. BORISCH2, S. VON JUERGENSONN3, S. DIRNHOFER Institut für Pathologie, Universität Basel, Schweiz 1 Institut für Pathologie, Kantonsspital St.Gallen, Schweiz 2 Division de Pathologie clinique, Hopitaux Universitaires de Genève, Suisse 3 Novartis Pharma Schweiz AG, Bern, Switzerland Aims: Imatinib mesylate (IM, Glivec®) is effective in the treatment of CML. We analyzed semiquantitatively the effect of IM on bone marrow fibrosis in CML patients. Methods: Bone marrow biopsies of 39 patients with CML in chronic phase (n = 21) and in accelerated phase/blast crisis (n = 18) were examined. Evaluation of reticulin fiber (RF) content was based on the scoring system proposed by Manoharan (Br J Haematol, 1979, 43, 185–190). Four diagnostic categories were defined as follows: i) RF scores 0 and 1+: Complete response; ii) RF scores 2+ and 3+: Partial response; iii) RF scores 4+: Stable or progressive disease. Results: After 13 weeks of treatment, patients with chronic phase CML had a RF score 1+ in 79%. The number of the patients with RF score 1+ was at all time-points over 65%. Two patients revealed a RF score 3+ after 13 and 49 weeks, respectively. Patients with advanced phase CML had considerably more often elevated RF scores 2+ and 3+ (range: 29%–73% compared to 18%–33% in patients with chronic phase CML). Overall, there was a significant decrease in fiber content between baseline and week 13 as assessed by Manoharan Grading (p = 0.012). Conclusions: IM induces a persistent reduction of bone marrow fibrosis in CML patients already after 13 weeks of treatment. It can be speculated that IM might also be effective in other diseases with bone marrow fibrosis.
267
Expression of fibrogenic cytokines in Microdissected megakaryocytes from chronic Myeloproliferative disorders O. BOCK, J. SCHLUÉ, R. VON WASIELEWSKI, M. MENGEL, H. KREIPE Institut für Pathologie, Medizinische Hochschule Hannover
Aims: Because myelofibrosis in CMPD might be due to aberrant expression of cytokines, quantitative assessment of cytokine expression in total bone marrow cells and megakaryocytes from chronic myeloproliferative disorders (CMPD) and reactive hematopoiesis was performed. Methods: After extraction of RNA from total bone marrow trephines and laser-microdissected megakaryocytes, quantitative expression analysis of basic fibroblast growth factor (bFGF) and transforming growth factor β-1 (TGFβ-1) were performed by quantitative real-time PCR. Molecular expression levels were correlated with staining pattern of corresponding proteins bFGF and TGFβ-1 by immunohistochemistry using tissue micro-arrays (System Multiblock). Results: In total bone marrow cells, RNA expression of bFGF and TGFβ-1 was up to 5fold higher in CMPD compared to reactive bone marrow. Using laser-microdissection for quantitative celltype specific analysis of megakaryocytes, CMPD megakaryocytes showed exaggerated high bFGF levels which were up to 10fold higher compared to megakaryocytes in reactive cases. Immunohistochemistry revealed a strong nuclear staining of bFGF in CMPD megakaryocytes except for CML and reactive cases. Using antiTGFβ-1, only weakly stained CMPD megakaryocytes could be detected in contrast to a strong staining of the granulopoiesis. Conclusions: Megakaryocytes in CMPD were shown to be a relevant source of bFGF, but not TGFβ-1. But the strong nuclear staining of bFGF in CMPD megakaryocytes from CMPD rather suggests exaggerated growth stimulation in an autocrine fashion than direct induction of fibroblasts by bFGF with consecutive bone marrow fibrosis.
268
Differential in vivo and in vitro expression of provisional extracellular matrix proteins in human megakaryocytes
A. BERNDT, W. VOGEL1, A. MÜLLER2, R. DAHSE3, K. M. HAAS4, L. BORSI5, D. KATENKAMP, H. KOSMEHL6 Institut für Pathologie, 2Klinik für Kinder- und Jugendmedizin, 3Institut für Humangenetik und Antropologie, 4Klinik für Mund,Kiefer- und Gesichtschirurgie, Friedrich-Schiller-Universität Jena; 1 Klinik für Innere Medizin II, Eberhard-Karls-Universität Tübingen; 5 Istituto Nazionale per la Ricerca sul Cancro, Genua; 6Institut für Pathologie, HELIOS-Klinikum Erfurt Aims: Megakaryocytes play a central role in wound healing and can be affected or involved in several leukaemic disorders, both associated with an abundant accumulation of a provisional matrix including oncofoetal fibronectin isoforms, tenascin-C and laminin. The aim was to define the expression of ED-B+ fibronectin (ED-B+ fn), tenascin-CL (Tn-CL) and laminin-5 γ2 chain (Ln-5) in human megakaryocytes of normal human bone marrow, human long-term bone marrow cultures (LTBMC), cytokine supported cultures of peripheral CD34+ cells and leukemic cell lines with megakaryocytic characteristics (K562, CMK). Methods: Immunocytochemistry, flow cytometry and reversetranscriptase polymerase chain reaction. Antibodies: BC-1 for EDB+ fibronectin, BC-2 and BC-4 for tenascin-C, D4B5 for Laminin γ2 chain. Results: Demonstration of ED-B+ fn, Tn-CL and Ln-5 exclusively in bone marrow megakaryocytes and in megakaryocytic progenitor cells generated ex vivo. Strong expression in LTBMC. TPO induced megakaryocytic differentiation of CMK cells is associated with an increase of large megakaryocytes followed by intracellular accumulation of ED-B+ fn, Tn-CL and Ln-5. Conclusions: In normal human haemostase megakaryocytes are suggested as carriers for provisional matrix proteins. Low-abundant synthesis, intracellular accumulation and failure of membrane exposition speak for a function during early events of wound heal-
Modern Methods in Cytology · 249 ing (formation of a provisional extracellular matrix). In case of megakaryocyte-dependent fibroblast proliferation in myelofibrosis, megakaryocyte derived provisional matrix proteins may be important for the initiation of the fibrotic process.
269
Stroma-dependent differentiation of myeloid progenitors during the perinatal period R. HUSS, R. RUPEC 1
lung, nervous system, stomac, testis, omentum majus, parotid gland, uterus and bone. The tumors were predominantly composed of myeloblasts, granulocytic cells with evidence of maturation, myelomonoblasts, monoblasts or megakaryoblasts. A co-expression of CD43, CD56 and CD 79a with myelomonocytic markers was seen in some cases. Conclusions: MS show a spectrum of features which may mimick malignant lymphoma. Different subgroups can be defined based on immunophenotypic and genetic features. An accurate workup and an early diagnosis reduce the risk of subsequent leukemic AML.
Pathologisches Institut der Universität München 1 Klinik und Poliklinik für Dermatologie der Universität München Aims: The regulation of myelopoiesis during perinatal life is poorly understand. Here we provide evidence that the interaction of the inhibitor of the transcription factors NF-κB, IκB-α and the Notch1ligand Jagged1 induces the differentiation of myeloid progenitors. Methods: We used conditional IκB-α knock-out mice and a stem cell transplantation model to investigate the role of the NF-κB/ IκB-α signalling pathway in myelopoiesis. Results: Although normal at birth, mice that are constitutively deficient of IκB-α in all organs, develop an accelerated myelopoiesis with extensive maturation of granulocytes immediately after birth with an consecutive overexpression of Notch1 and Hes1 in neutrophils and the Notch1-ligand Jagged1 in the liver stroma of five days old and severly growth retarded mice. NF-κB was found to be constitutively active in the liver and neutrophils of those IκB-α ∆/∆ mice, while granulopoiesis was normal in IgH –/– mice that received a stem cell transplantation from IκB-α ∆/∆ mice or in mice that selectively lacked IκB-α expression only in neutrophils using conditional mutagenesis (IκB-αflox/flox LysMCre). Although Jagged1 is strongly expressed in the prenatal liver stroma of embryonic day 12 (E12) mice, the expression declines in the liver stroma of wild-type immediately after birth, but it continously persisted in IκB-α deficient mice in their postnatal period. Conclusions: These data show that myelopoietic differentiation is not only controlled by an internal activation of hematopoiesis-associated transcription factors or conventional soluble growth factors and cytokines, but also by the interaction of progenitor cells (stem cells) with stroma-associated components, such as the Notch-ligand Jagged1, which seems to be a key regulator of perinatal hematopoiesis via the NF-κB/IκB-α pathway.
270
Primary myeloid sarcomas – initial or isolated extramedullary manifestations of acute myeloid leukemia
A. SCHMITT-GRÄFF 1, J. THIELE2, H. M. KVASNICKA2, W. FEIDEN3, J. ANAGNOSTOPOULOS4, H. STEIN4 Institutes of Pathology1,2,4 and Neuropathology3, Universities of 1 Freiburg, 2Köln, 3Homburg and of the 4University Medical Center Benjamin Franklin, The Free University of Berlin Aims: Myelosarcomas (MS) may accompany or signal relapse of AML or coincide with blastic transformation of a CMPD. However, primary or de novo MS may preceede AML by months or years, may occurr in MDS that never progress to full-blown leukemia or be associated with normal or reactive intramedullary hematopoiesis. In view of the high risk of a delayed or missed diagnosis we performed a co-operative study based on a series of de novo MS. Methods: We evaluated characteristic phenotypic, genetic and clinical features of 35 primary MS occurring in nonleukemic patients. Immunohistochemistry included antibodies to MPO, CD3, 4, 7, 8, 20, 34, 43, 56, 61, 68, 79a, 99a, 117 elastase, lysozyme. Results: Preferential sites of primary manifestations were oral mucosa, skin, lymph nodes, small intestine, retroperitoneum, pleura/
Symposium: Modern Methods in Cytology
271
Chromosome and gene analysis in cytology: a navigation tool for diagnosis and therapy L. BUBENDORF Institut für Pathologie, Universität Basel
Aims: We describe FISH as a powerful method to improve the performance of cytology in the diagnosis of malignant tumors and the prediction of prognosis or therapy response. Methods: Interphase FISH is used in cytology to enumerate individual chromosomes for aneusomy detection, or to visualize the copy numbers of specific genes for the detection of amplifications or deletions. FISH is applicable to almost any type of cytology specimen irrespective of cell type, staining or fixation modality. The sensitivity of FISH testing can be improved by the use of a mixture of differentially fluorescinated probes. Results: The multiprobe-test UroVysion (Vysis, Inc., Downer’s Grove, IL) for the simultaneous analysis of the chromosomes 3, 7, 17, and 9p21 has repeatedly been shown to be markedly more sensitive for the detection of urothelial tumors than cytology at a retained specificity. Moreover, FISH testing in urinary specimens during surveillance may stratify patients with different risk of recurrence or progression, thus allowing to reduce the number of follow-up cystoscopies in patients at low risk. Another assay (LA Vysion; chr. 6, 5p15, 7p12, and 8q24) has been designed to enhance the detection of lung cancer and to clarify equivocal findings in lung cytology. Testing for HER-2 amplification has become a standard tool for selecting patients with breast cancer for therapy with trastuzumab, and can readily be performed in cytological specimens of distant metastases. FISH is the preferred method to detect N-MYC amplification, which is an independent prognostic factor in neuroblastoma. In multiple myeloma, deletion at 13q14 (Rb gene) defines a group of highly aggressive tumors. Conclusions: FISH has become an established supplementary method in cytology with an increasing number of applications.
272
Multimodal and Monocellular Measurements of Markers and Morphology A. BÖCKING1, J. STOCKHAUSEN2, H. J. GROTE2, D. MEYER-EBRECHT2
1
Institute of Cytopathology, Heinrich-Heine-University, Düsseldorf Institute of Measuring Technology and Image Analysis, Technical University of Aachen, Germany 2
Aims: To develop a new type of cancer diagnosis based on sequential multimodal measurements of different cellular markers combined with subjective morphologic inspection on a few identical cells.
Symposium: Bone Marrow
262
Aberrant Methylation and impaired expression of the p15INK4b gene in chronic myelomonocytic leukemia (CMML)
M. TESSEMA, F. LÄNGER, J. DINGEMANN, A. GANSER1, H. KREIPE, U. LEHMANN Institut für Pathologie, Medizinische Hochschule Hannover Institut für Hämatologie/Onkologie, Medizinische Hochschule Hannover 1
Aims: The important cell cycle regulatory gene p15INK4b has been shown to be inactivated in acute myeloid leukaemia and myelodysplastic syndrome. Little is known about the expression and epigenetic modification of this gene in chronic myelomonocytic leukaemia (CMML) that belongs to the myelodysplastic-myeloproliferative disorders with a high proportion of blastic transformation. Methods: DNA and RNA were extracted from formalin-fixed paraffin-embedded (FFPE) bone marrow trephines from a series of CMML patients (n = 33). For methylation analysis methylationspecific PCR (MSP) and genomic bisulfit-sequencing as well as sequencing of cloned PCR products were employed. Expression was measured using quantitative real-time RT-PCR for mRNA levels and immunohistochemistry, which was also performed on the FFPE bone marrow biopsies. Results: Analysis of bone marrow trephines in a series of 33 CMML cases showed an aberrant p15INK4b gene methylation in up to 58% of cases. It turned out to be spread over a broad area of the 5′ region and exhibited substantial heterogeneity between cases and even in individual patients. The degree of aberrant methylation was correlated with a reduced mRNA as well as reduced protein expression and was associated with a higher expression of DNA methyltransferase DNMT 3A. It correlated also with the blast content of the trephines. Conclusions: We conclude that aberrant gene methylation is a frequent event in CMML that might contribute to the pathogenesis of this myelodysplastic-myeloproliferative disorder.
orders, the expression levels of H19 and IGF-2 were quantified by real-time PCR. Transcript levels of IGF-2 in CMPD and normal hematopoiesis were correlated with staining pattern and intensity by immunohistochemistry using micro-tissue arrays (System Multiblock). Results: The expression of tumor suppressor H19 in CMPD was significantly reduced up to 20fold compared to non-neoplastic states. In contrast, IGF-2 expression levels were demonstrated to be up to 3fold higher in CMPD compared to normal hematopoiesis. Conclusions: We conclude that downregulation of H19 is a constant feature of all CMPD subtypes and may contribute to an upregulation of IGF-2 expression with resulting growth-promoting effects in CMPD.
264
CD117 Immunohistochemistry of bone marrow biopsies in the diagnosis of myelodysplastic syndromes S.M. KRÖBER1, O. AICHELE2, H. MÜLLER1, E. KAISERLING1
1
Institut für Pathologie, Universität Tübingen Abteilung Hämatologie und Onkologie, Medizinische Klinik, Universität Tübingen 2
Tumor-suppressor H19 and Insulin-like growth factor-2 are inversely expressed in bone marrow cells of chronic myeloproliferative disorders
Background: CD117 immunostaining has proven to be a useful tool for distinguishing acute myeloid leukaemias from acute lymphoblastic leukaemias by flow cytometry and in bone marrow biopsies (BMB). Systematic analysis of the CD117 expression of blasts in myelodysplastic syndromes (MDS) cases have not been performed on parraffin embedded BMB. Methods: 99 formalin fixed, paraffin embedded BMB were investigated. The cases were classified as normal or reactive bone marrow (nBM, n = 16), lymphoproliferative disorders, i.e. non-Hodgkin’s lymphoma of low grade malignancy or Hodgkin’s lymphoma (lyBM, n = 6), myelodysplastic syndrome (MDS, n = 49, including 6 cases where refractary anaemia could neither be established nor ruled out), hypocellular acute myeloid leukaemia (hAML, n = 15), and residual acute myeloid leukaemia with oligoblastic bone marrow (rAML, n = 13). Immunostaining with T595 (Anti-CD117) and QBEND10 (Anti-CD34) using the ABC-Method was performed. The percentage of CD117+ and of CD34+ haemopoietic cells in 10 HPF and the occurrence of clusters of CD34+ and/or CD117+ cells were determined in each case. Results: The average number of CD117+ cells was very low in nBM (1.28%) and lyBM (1.16%) and showed only a little increase (4.07%) in the MDS subtypes with blast count below 10%, the percentage of CD117+ cells usually not exceeding the number of CD34+ cells. However the CD117+ cell count (11.93%–22.2%–27.9%) and cluster values (13/15, 12/13, and 15/15 vs. 0) were higher in MDS RAEBII, rAML, and hAML than in nBM and lyBM with reactive changes. Conclusions: The addition of CD117 immunostaining in BMB is useful in the identification of MDS RAEB II, hAML, and rAML.
O. BOCK, J. SCHLUÉ, R. VON WASIELEWSKI, M. MENGEL, H. KREIPE
265
263
Institut für Pathologie, Medizinische Hochschule Hannover Aims: In most adult tissues, H19 and its chromosomal neighbour IGF-2, are reciprocally imprinted. In normal hematopoiesis, however, IGF-2 imprinting was shown to be disrupted with consecutive biallelic expression. H19 is known to be a negative regulator of IGF-2 expression. Our aim was to elucidate the expression profile of the hematopoietic growth promoter insulin-like growth factor-2 (IGF-2) and its designated repressor, the untranslated tumor-suppressor RNA H19, in non-neoplastic hematopoiesis and chronic myeloproliferative disorders (CMPD). Methods: After RNA extraction from total bone marrow cells of non-neoplastic hematopoiesis and chronic myeloproliferative dis-
Early Indication of Therapy Failure and Prognosis by Evolving or Progressive Myelofibrosis in Chronic Myeloid Leukemia
G. BÜSCHE, R. HEHLMANN1, M. FREUND2, H. HECKER3, T. BUHR, A. GEORGII, H. KREIPE, German CML Study Group Institut für Pathologie, Medizinische Hochschule Hannover 1 III. Med. Klinik, Klinikum Mannheim, Universität Heidelberg 2 Klinik für Hämatologie und Onkologie, Universität Rostock 3 Institut für Biometrie, Medizinische Hochschule Hannover Aims: Consideration of marrow fibrosis (MF) is unusual in therapy trials on chronic myeloid leukemia (CML) explaining the lack of long-term observations on the basis of sequential bone marrow
248 · Symposium biopsies (BMB) taken prospectively during controlled trials on CML. Methods: 1198 BMB from 493 patients recruited in two German therapy trials on Ph+ CML were examined for MF before and during therapy comprising histopathologic follow-up periods up to 9 years. Results: MF was a typical complication of Ph+ CML affecting the majority of patients when therapy was not effective. Oncoming or progressive MF was a reliable predictor of therapy failure one to two years earlier as indicated by changes in the peripheral blood, spleen size, marrow blast count and cytogenetics, correlating with an increase of the relative risk of acceleration by a factor of 14 independent of the type of therapy applied. The survival time of patients suffering from evolving or progressive MF was significantly shortened independently of risk score, hematologic and cytogenetic remission and type of therapy applied including allografting (multivariate analysis; P < 0.00005). Conclusions: The analyzed long-term observations strongly indicate that MF is a complication related to CML, not to therapy. MF is prevented when therapy is effective whereas oncoming or progressive MF is an early and reliable indicator of therapy failure and acceleration independent of peripheral blood findings, spleen size, marrow blast count and cytogenetics.
266
Bone marrow fibrosis in CML treated with Imatinib mesylate (Glivec®)
A. LUGLI, S. B. COGLIATTI1, M. TINGUELY2, B. BORISCH2, S. VON JUERGENSONN3, S. DIRNHOFER Institut für Pathologie, Universität Basel, Schweiz 1 Institut für Pathologie, Kantonsspital St.Gallen, Schweiz 2 Division de Pathologie clinique, Hopitaux Universitaires de Genève, Suisse 3 Novartis Pharma Schweiz AG, Bern, Switzerland Aims: Imatinib mesylate (IM, Glivec®) is effective in the treatment of CML. We analyzed semiquantitatively the effect of IM on bone marrow fibrosis in CML patients. Methods: Bone marrow biopsies of 39 patients with CML in chronic phase (n = 21) and in accelerated phase/blast crisis (n = 18) were examined. Evaluation of reticulin fiber (RF) content was based on the scoring system proposed by Manoharan (Br J Haematol, 1979, 43, 185–190). Four diagnostic categories were defined as follows: i) RF scores 0 and 1+: Complete response; ii) RF scores 2+ and 3+: Partial response; iii) RF scores 4+: Stable or progressive disease. Results: After 13 weeks of treatment, patients with chronic phase CML had a RF score 1+ in 79%. The number of the patients with RF score 1+ was at all time-points over 65%. Two patients revealed a RF score 3+ after 13 and 49 weeks, respectively. Patients with advanced phase CML had considerably more often elevated RF scores 2+ and 3+ (range: 29%–73% compared to 18%–33% in patients with chronic phase CML). Overall, there was a significant decrease in fiber content between baseline and week 13 as assessed by Manoharan Grading (p = 0.012). Conclusions: IM induces a persistent reduction of bone marrow fibrosis in CML patients already after 13 weeks of treatment. It can be speculated that IM might also be effective in other diseases with bone marrow fibrosis.
267
Expression of fibrogenic cytokines in Microdissected megakaryocytes from chronic Myeloproliferative disorders O. BOCK, J. SCHLUÉ, R. VON WASIELEWSKI, M. MENGEL, H. KREIPE Institut für Pathologie, Medizinische Hochschule Hannover
Aims: Because myelofibrosis in CMPD might be due to aberrant expression of cytokines, quantitative assessment of cytokine expression in total bone marrow cells and megakaryocytes from chronic myeloproliferative disorders (CMPD) and reactive hematopoiesis was performed. Methods: After extraction of RNA from total bone marrow trephines and laser-microdissected megakaryocytes, quantitative expression analysis of basic fibroblast growth factor (bFGF) and transforming growth factor β-1 (TGFβ-1) were performed by quantitative real-time PCR. Molecular expression levels were correlated with staining pattern of corresponding proteins bFGF and TGFβ-1 by immunohistochemistry using tissue micro-arrays (System Multiblock). Results: In total bone marrow cells, RNA expression of bFGF and TGFβ-1 was up to 5fold higher in CMPD compared to reactive bone marrow. Using laser-microdissection for quantitative celltype specific analysis of megakaryocytes, CMPD megakaryocytes showed exaggerated high bFGF levels which were up to 10fold higher compared to megakaryocytes in reactive cases. Immunohistochemistry revealed a strong nuclear staining of bFGF in CMPD megakaryocytes except for CML and reactive cases. Using antiTGFβ-1, only weakly stained CMPD megakaryocytes could be detected in contrast to a strong staining of the granulopoiesis. Conclusions: Megakaryocytes in CMPD were shown to be a relevant source of bFGF, but not TGFβ-1. But the strong nuclear staining of bFGF in CMPD megakaryocytes from CMPD rather suggests exaggerated growth stimulation in an autocrine fashion than direct induction of fibroblasts by bFGF with consecutive bone marrow fibrosis.
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Differential in vivo and in vitro expression of provisional extracellular matrix proteins in human megakaryocytes
A. BERNDT, W. VOGEL1, A. MÜLLER2, R. DAHSE3, K. M. HAAS4, L. BORSI5, D. KATENKAMP, H. KOSMEHL6 Institut für Pathologie, 2Klinik für Kinder- und Jugendmedizin, 3Institut für Humangenetik und Antropologie, 4Klinik für Mund,Kiefer- und Gesichtschirurgie, Friedrich-Schiller-Universität Jena; 1 Klinik für Innere Medizin II, Eberhard-Karls-Universität Tübingen; 5 Istituto Nazionale per la Ricerca sul Cancro, Genua; 6Institut für Pathologie, HELIOS-Klinikum Erfurt Aims: Megakaryocytes play a central role in wound healing and can be affected or involved in several leukaemic disorders, both associated with an abundant accumulation of a provisional matrix including oncofoetal fibronectin isoforms, tenascin-C and laminin. The aim was to define the expression of ED-B+ fibronectin (ED-B+ fn), tenascin-CL (Tn-CL) and laminin-5 γ2 chain (Ln-5) in human megakaryocytes of normal human bone marrow, human long-term bone marrow cultures (LTBMC), cytokine supported cultures of peripheral CD34+ cells and leukemic cell lines with megakaryocytic characteristics (K562, CMK). Methods: Immunocytochemistry, flow cytometry and reversetranscriptase polymerase chain reaction. Antibodies: BC-1 for EDB+ fibronectin, BC-2 and BC-4 for tenascin-C, D4B5 for Laminin γ2 chain. Results: Demonstration of ED-B+ fn, Tn-CL and Ln-5 exclusively in bone marrow megakaryocytes and in megakaryocytic progenitor cells generated ex vivo. Strong expression in LTBMC. TPO induced megakaryocytic differentiation of CMK cells is associated with an increase of large megakaryocytes followed by intracellular accumulation of ED-B+ fn, Tn-CL and Ln-5. Conclusions: In normal human haemostase megakaryocytes are suggested as carriers for provisional matrix proteins. Low-abundant synthesis, intracellular accumulation and failure of membrane exposition speak for a function during early events of wound heal-
Modern Methods in Cytology · 249 ing (formation of a provisional extracellular matrix). In case of megakaryocyte-dependent fibroblast proliferation in myelofibrosis, megakaryocyte derived provisional matrix proteins may be important for the initiation of the fibrotic process.
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Stroma-dependent differentiation of myeloid progenitors during the perinatal period R. HUSS, R. RUPEC 1
lung, nervous system, stomac, testis, omentum majus, parotid gland, uterus and bone. The tumors were predominantly composed of myeloblasts, granulocytic cells with evidence of maturation, myelomonoblasts, monoblasts or megakaryoblasts. A co-expression of CD43, CD56 and CD 79a with myelomonocytic markers was seen in some cases. Conclusions: MS show a spectrum of features which may mimick malignant lymphoma. Different subgroups can be defined based on immunophenotypic and genetic features. An accurate workup and an early diagnosis reduce the risk of subsequent leukemic AML.
Pathologisches Institut der Universität München 1 Klinik und Poliklinik für Dermatologie der Universität München Aims: The regulation of myelopoiesis during perinatal life is poorly understand. Here we provide evidence that the interaction of the inhibitor of the transcription factors NF-κB, IκB-α and the Notch1ligand Jagged1 induces the differentiation of myeloid progenitors. Methods: We used conditional IκB-α knock-out mice and a stem cell transplantation model to investigate the role of the NF-κB/ IκB-α signalling pathway in myelopoiesis. Results: Although normal at birth, mice that are constitutively deficient of IκB-α in all organs, develop an accelerated myelopoiesis with extensive maturation of granulocytes immediately after birth with an consecutive overexpression of Notch1 and Hes1 in neutrophils and the Notch1-ligand Jagged1 in the liver stroma of five days old and severly growth retarded mice. NF-κB was found to be constitutively active in the liver and neutrophils of those IκB-α ∆/∆ mice, while granulopoiesis was normal in IgH –/– mice that received a stem cell transplantation from IκB-α ∆/∆ mice or in mice that selectively lacked IκB-α expression only in neutrophils using conditional mutagenesis (IκB-αflox/flox LysMCre). Although Jagged1 is strongly expressed in the prenatal liver stroma of embryonic day 12 (E12) mice, the expression declines in the liver stroma of wild-type immediately after birth, but it continously persisted in IκB-α deficient mice in their postnatal period. Conclusions: These data show that myelopoietic differentiation is not only controlled by an internal activation of hematopoiesis-associated transcription factors or conventional soluble growth factors and cytokines, but also by the interaction of progenitor cells (stem cells) with stroma-associated components, such as the Notch-ligand Jagged1, which seems to be a key regulator of perinatal hematopoiesis via the NF-κB/IκB-α pathway.
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Primary myeloid sarcomas – initial or isolated extramedullary manifestations of acute myeloid leukemia
A. SCHMITT-GRÄFF 1, J. THIELE2, H. M. KVASNICKA2, W. FEIDEN3, J. ANAGNOSTOPOULOS4, H. STEIN4 Institutes of Pathology1,2,4 and Neuropathology3, Universities of 1 Freiburg, 2Köln, 3Homburg and of the 4University Medical Center Benjamin Franklin, The Free University of Berlin Aims: Myelosarcomas (MS) may accompany or signal relapse of AML or coincide with blastic transformation of a CMPD. However, primary or de novo MS may preceede AML by months or years, may occurr in MDS that never progress to full-blown leukemia or be associated with normal or reactive intramedullary hematopoiesis. In view of the high risk of a delayed or missed diagnosis we performed a co-operative study based on a series of de novo MS. Methods: We evaluated characteristic phenotypic, genetic and clinical features of 35 primary MS occurring in nonleukemic patients. Immunohistochemistry included antibodies to MPO, CD3, 4, 7, 8, 20, 34, 43, 56, 61, 68, 79a, 99a, 117 elastase, lysozyme. Results: Preferential sites of primary manifestations were oral mucosa, skin, lymph nodes, small intestine, retroperitoneum, pleura/
Symposium: Modern Methods in Cytology
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Chromosome and gene analysis in cytology: a navigation tool for diagnosis and therapy L. BUBENDORF Institut für Pathologie, Universität Basel
Aims: We describe FISH as a powerful method to improve the performance of cytology in the diagnosis of malignant tumors and the prediction of prognosis or therapy response. Methods: Interphase FISH is used in cytology to enumerate individual chromosomes for aneusomy detection, or to visualize the copy numbers of specific genes for the detection of amplifications or deletions. FISH is applicable to almost any type of cytology specimen irrespective of cell type, staining or fixation modality. The sensitivity of FISH testing can be improved by the use of a mixture of differentially fluorescinated probes. Results: The multiprobe-test UroVysion (Vysis, Inc., Downer’s Grove, IL) for the simultaneous analysis of the chromosomes 3, 7, 17, and 9p21 has repeatedly been shown to be markedly more sensitive for the detection of urothelial tumors than cytology at a retained specificity. Moreover, FISH testing in urinary specimens during surveillance may stratify patients with different risk of recurrence or progression, thus allowing to reduce the number of follow-up cystoscopies in patients at low risk. Another assay (LA Vysion; chr. 6, 5p15, 7p12, and 8q24) has been designed to enhance the detection of lung cancer and to clarify equivocal findings in lung cytology. Testing for HER-2 amplification has become a standard tool for selecting patients with breast cancer for therapy with trastuzumab, and can readily be performed in cytological specimens of distant metastases. FISH is the preferred method to detect N-MYC amplification, which is an independent prognostic factor in neuroblastoma. In multiple myeloma, deletion at 13q14 (Rb gene) defines a group of highly aggressive tumors. Conclusions: FISH has become an established supplementary method in cytology with an increasing number of applications.
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Multimodal and Monocellular Measurements of Markers and Morphology A. BÖCKING1, J. STOCKHAUSEN2, H. J. GROTE2, D. MEYER-EBRECHT2
1
Institute of Cytopathology, Heinrich-Heine-University, Düsseldorf Institute of Measuring Technology and Image Analysis, Technical University of Aachen, Germany 2
Aims: To develop a new type of cancer diagnosis based on sequential multimodal measurements of different cellular markers combined with subjective morphologic inspection on a few identical cells.