344 Derivation of a New Hematopoietic Cell Line with Endothelial Features from a Patient with Transformed Myeloproliferative Syndrome A Case Report Walter Fiedler, M.D.1 R. Peter Henke, M.D.2 Süleyman Ergün, M.D.3 Udo Schumacher, M.D.3 Ursula M. Gehling, M.D.1 Gabi Vohwinkel, M.Sc.1 Nerbil Kilic, M.D.3 Dieter K. Hossfeld, M.D.1 1 Department of Oncology/Hematology, University Hospital Eppendorf, Hamburg, Germany. 2 Institute of Pathology, University Hospital Eppendorf, Hamburg, Germany. 3 Institute of Anatomy, University Hospital Eppendorf, Hamburg, Germany. BACKGROUND. During embryonal development primitive hematopoiesis can be observed first in the yolk sac, in which both hematopoietic and endothelial cells are derived from a common precursor, the hemangioblast. Whether cells with this dual differentiation potential persist during postnatal life is unknown. METHODS. A cell line was derived from a patient with secondary acute leukemia. Because of its ability to grow in soft agar and in SCID mice, this cell line was analyzed for expression of differentiation antigens by fluorescence-activated cell sorter analysis, immunocytochemistry, fluorescent in situ hybridization (FISH) analysis with simultaneous cell surface staining, and polymerase chain reaction (PCR). RESULTS. A new cell line was established from a patient with essential thrombocytosis that transformed into acute leukemia. The patient’s initial clinical presentation included skin and lymph node infiltrations that were taken for an angiosarcoma due to positivity for CD34, CD31, and von Willebrand factor on immunohistology. In addition to hematopoietic markers, leukemic cells expressed endothelial antigens such as CD62E, CD105, and bound Ulex europäeus lectin-1. Immunocytochemistry revealed positive staining for vascular endothelial growth factor receptor type 2 (KDR), Tie-2/Tek, the angiopoietin receptor, and vascular endothelial cadherin. These results were confirmed by PCR analysis. Simultaneous staining for CD62E and FISH analysis showed that cells with endothelial characteristics belonged to the leukemia. FISH analysis of histologic sections of the lymph node infiltration confirmed this manifestation as part of the leukemic process. The derived cell line, UKE-1, forms colonies in soft agar and is tumorigenic in SCID mice. CONCLUSIONS. This new cell line, UKE-1, appears to combine hematopoietic and endothelial features, indicating the close ontogenic relation of both lineages. Cancer 2000;88:344 –51. © 2000 American Cancer Society. KEYWORDS: essential thrombocytosis, leukemia, endothelial cell, cell line. Supported in part by grants from Deutsche Forschungsgemeinschaft Fi 389/4-1 and Roggenbuck Stiftung. Address for reprints: Walter Fiedler, M.D., Department of Oncology/Hematology, University Hospital Eppendorf, Martinistraße 52, 20246 Hamburg, Germany. Received April 26, 1999; revision received August 16, 1999. © 2000 American Cancer Society D uring embryonal development mesenchymal cells give rise to the formation of both endothelial and hematopoietic cells.1–3 This process first can be observed in the mouse embryo at Day 7 of gestation when blood islands are being formed in the yolk sac.4 The close developmental relation between endothelial and hematopoietic cells within this extraembryonic structure led to the hypothesis that both cell systems are derived from a common stem cell, the hemangioblast.5,6 This concept has been supported by several mouse knockout models in which both endothelial and hematopoietic cells were Hematopoietic Cell Line with Endothelial Features/Fiedler et al. found to be deficient.7,8 In addition, hemangioblasts have been derived from embryonic stem cells in vitro.9 Because of their common origin, endothelial and hematopoietic cells share several cell surface antigens, such as CD34, CD31, and CD10510 and produce or possess receptors for cytokines such as granulocytecolony stimulating factor (G-CSF), granulocyte-macrophage– colony stimulating factor, stem cell factor, interleukin-6, tumor necrosis factor-␣, or vascular endothelial growth factor (VEGF).11–13 Whether this common precursor cell of hematopoiesis and angiogenesis persists during postnatal life still is a matter of debate. Although endothelial cells have been detected in peripheral blood and endothelial-like cells have been derived from progenitor cells isolated from peripheral blood, to our knowledge definite proof of the existence of the putative hemangioblast still is lacking.14 –16 In the current study, we present the medical history of a patient with leukemic transformation of a myeloproliferative syndrome and the derivation of a cell line that shows both endothelial and hematopoietic features. This observation adds further support to the concept of a hemangioblast being the precursor of both endothelial and hematopoietic cells. 345 tion of peroxidase only, and incubation of cells with normal rabbit serum (Sigma, Deisenhofen, Germany) in concentrations ranging from 0.1– 0.01% rather than primary antiserum. Monoclonal or polyclonal antibodies were purchased as follows: KDR (Sigma), von Willebrand factor (Dakopatts), vascular endothelial (VE)-cadherin (Immunotech, Hamburg, Germany), CD31 (Pharmingen, Hamburg, Germany), and Tie2/ Tek (Santa Cruz Biotechnology, Santa Cruz, CA). Fluorescence-Activated Cell Sorter Analysis Fluorescein isothiocyanate (FITC) or phycoerythrin (PE) labeled monoclonal antibodies against CD antigens were purchased as follows: CD34, CD45, and HLA-Ia from Becton Dickinson (Heidelberg, Germany); CD13, CD33, and CD36 from Coulter/Immunotech (Hamburg, Germany); CD15, myeloperoxidase, and CD61 from Dakopatts; CD41 and CD42b from Serotech (Kidlington, UK); and CD62E, CD105, and CD106 from Cymbus Biotechnology (Hants, Canada). Ulex europäeus agglutinin-1 coupled with FITC was purchased from Sigma. Staining of the cells was performed as recommended by the supplier. Fluorescence-activated cell sorter (FACS) analyses were run on a FACScan using Cell Quest software, both from Beckton Dickinson. MATERIALS AND METHODS Histology and Immunohistology Polymerase Chain Reaction Analysis Immunohistology was performed using the alkaline phosphatase-antialkaline phosphatase technique. The immunostaining procedure was performed on a TechMate 500 immunostainer (Dakopatts, Copenhagen, Denmark). Antibodies against leukocyte common antigen (LCA), CD30, CD31, CD61, factor VIII, myeloperoxidase, desmin, S-100 (Dakopatts), CD34 (Novocastra, Newcastle-upon-Tyne, UK), actin (Enzo, Farmingdale, NY), vimentin (Linaris, Wertheim, Germany), and cytokeratins (KL1; Dianova, Hamburg, Germany) were used. Sections were counterstained with hematoxylin. Total cellular RNA from fresh leukemic and cultured cells was prepared using Qiagen minicolumns (Qiagen, Hilden, Germany) as described by the manufacturer. One microgram of RNA was used for c-DNA synthesis employing avian myeloblastosis virus reverse transcriptase and oligo dT as primer. Different aliquots of c-DNA were amplified with specific primers for KDR, Tie-2/Tek, von Willebrand factor, VEcadherin, and actin as controls for successful c-DNA synthesis. For KDR, Tie-2/Tek, von Willebrand factor, and cadherin 5, 2 rounds and for actin 1 round of 35 cycles of polymerase chain reaction (PCR) were performed in a programmable heat block at 94 °C for 1.5 minutes, at 60 °C for 3 minutes, and at 72 °C for 4 minutes. PCR products were separated on 1% agarose gels, stained with ethidium bromide, and visualized under ultraviolet light. Primer sequences are available on request. To avoid cross-contamination, PCR reactions and gel electrophoresis were performed in different rooms using different sets of pipettes. Appropriate control reactions always remained negative. Immunocytochemistry Cells were cultured in chamber slides, washed twice with phosphate-buffered saline (PBS) for 10 minutes, fixed for 15 minutes with freshly prepared paraformaldehyde (4%) at room temperature, and further processed for immunocytochemistry. A detailed description of the methods used was reported elsewhere.17,18 In brief, an amplification combination of the peroxidase antiperoxidase and the avidin-biotin-peroxidase complex techniques was used. The peroxidase activity was visualized by means of the nickel-glucose oxidase technique.19,20 Controls included replacement of primary and secondary antibodies with PBS, visualiza- Fluorescent In Situ Hybridization Analysis On histologic sections, enumeration of chromosomes 7 and 8 was performed using biotinylated alpha satellite probes (D7Z1 and D8Z1; Oncor Inc., Gaithersburg, 346 CANCER January 15, 2000 / Volume 88 / Number 2 MD) and a nonfluorescent technique as described earlier.21,22 Briefly, 6-m paraffin sections were adhered to silanized glass slides and air-dried. Sections were dewaxed, heated by exposure to microwaves in citric acid monohydrate, and treated with sodium thiocyanate, followed by a pepsin digestion step. Each section was covered with 0.1 ng/mL biotinylated probe in freshly prepared hybridization solution. Sections were covered with coverslips and denatured for 10 minutes in a 78 °C waterbath. Hybridization was performed overnight at 37 °C. Labeled DNA was detected with subsequent incubations with a mouse monoclonal antibiotin antibody, a biotinylated goat antimouse antibody, peroxidase-conjugated streptavidin, and diaminobenzidine. The slides were counterstained with hematoxylin, dehydrated, and then permanently mounted. For evaluation of each probe, the signal numbers in 200 nonoverlapping neoplastic cells were counted. Nonneoplastic cells were used as controls. Cell Culture Primary leukemic cells were obtained from peripheral blood after readmission. After separation on FicollHypaque they were cultured in Iscove modified Dulbecco medium (IMDM) supplemented with 10% fetal calf serum (FCS), 10% horse serum, and 1 M hydrocortisone. Fluorescent In Situ Hybridization Analysis with Simultaneous Cell Surface Staining After trypsinization, 2 ⫻ 104 cells were incubated in PBS with 2% FCS with anti CD62E (Cymbus Biotechnology). After a washing step, cells were stained with a Cy 3 labeled anti-mouse antibody and after further washing spun onto glass slides with a cytocentrifuge. Glass slides were dried overnight in the dark and then treated with Carnoy fixative supplemented with 1% paraformaldehyde. Cells were hybridized with a digoxigenin labeled alpha satellite probe (D7Z1; Oncor, Inc.) To visualize the chromosome 7 probe, cells were treated with an FITC labeled antidigoxigenin antibody (Oncor, Inc.). 4⬘6-diamidine-2⬘ phenylindole dihydrochloride (DAPI) was used as counterstain. Ultraviolet microscopic images were documented with the Metasystem software package (Metasystems, Heidelberg, Germany). SCID Mouse Experiments One million tumor cells dissolved in 200 L tissue culture medium were injected subcutaneously between the scapula of each of 5 SCID mice. After 12 weeks, tumors developed and the mice were sacrificed. The tumors, lungs, livers, spleens, and bone samples were excised and fixed in neutral-buffered formalin and processed for routine histologic examination. RESULTS Case Report In 1968 a 30-year-old woman developed carcinoma of the uterine cervix that was treated surgically without consecutive radiotherapy and had remained in remission since that time. In 1985, she was diagnosed with essential thrombocythemia. Thrombocytosis was controlled with hydroxyurea without bleeding or thrombotic episodes. In May 1997, she experienced pain and swelling of the right shoulder. Physical examination revealed a 5-cm right supraclavicular mass and enlarged cervical and axillary lymph nodes of approximately 1 cm in greatest dimension. Intracutane nodules were noted in the region of the affected shoulder and the ipsilateral lower thorax. The remainder of the physical examination was unremarkable except for a systolic murmur and no hepatomegaly or splenomegaly was found. Computed tomography scans of the chest and abdomen revealed osteolysis of the distal end of the right clavicle, a lymph node of 3 cm in greatest dimension at the bifurcation of the trachea, a 2-cm lymph node in the right lung segment 2/3, and a 2-cm hyperdense structure in the spleen that was considered to be a hemangioma. The right supraclavicular lymph node connected to the right external jugular vein was removed. Histologic examination was consistent with an epitheloid angiosarcoma of UICC Grade 2. Laboratory analysis revealed a leukocyte count of 7.2/nL, hemoglobin of 10.9 g/dL, and thrombocytes of 527/nL. Six cycles of ifosfamide and adriblastin were administered between May and September 1997. Due to febrile neutropenia developing after the first cycle, G-CSF was given after subsequent cycles. After two therapy cycles and at the end of chemotherapy, enlarged lymph nodes and dermal nodules had disappeared and the mediastinal lymph node had regressed to 1 cm; however, the intrapulmonary lymph node remained constant. Only slight pain over the osteolysis at the tip of the clavicle persisted. In November 1997, the patient presented with multiple skin infiltrations, enlarged cervical and axillary lymph nodes, and bilateral pleural effusions. Laboratory investigation showed a leukocyte count of 5.6/ nL, a hemoglobin of 6.4 g/dL, and thrombocytes of 14/nL. Bone marrow biopsy showed infiltration with 54% blast cells. Pleural effusions contained numerous blast cells. A few days after readmission, bilateral pneumonia developed causing respiratory insufficiency. The patient was transferred to an intensive care unit and became dependent on mechanical ventilation. Chemotherapy with cytosine arabinoside and Hematopoietic Cell Line with Endothelial Features/Fiedler et al. 347 FIGURE 1. (a) Blood film of the patient showing a circulating leukemic cell (Pappenheim, stain, ⫻ 1000). (b) Cell line obtained from this patient showing large, adherent vacuolized blast cells and some smaller cells, one resembling a granulocyte (Pappenheim, stain, ⫻ 1000). (c) Histologic section of the patient’s skin infiltration showing a highly vascularized sarcomatoid tumor with marked nuclear pleomorphism. A capillary can be seen at the center (H & E, ⫻ 630). (d) Histologic section of bone marrow demonstrating infiltration with atypical, polymorphic cells with similar nuclear features as seen in panel c. (H & E, ⫻ 630). mitoxanthrone did not result in regression of the skin infiltration or normalization of the blood findings. The patient refused further therapy and died in December 1997. Laboratory Investigations Biopsy of a right supraclavicular lymph node revealed a highly vascularized and pleomorphic tumor displaying areas with large atypical cells with hyperchromatic, often vesicular nuclei (Fig. 1). Immunohistology revealed that tumor cells were positive for vimentin, CD31, and factor VIII. Cytokeratins, S-100, CD30, actin, and desmin were not expressed. Based on these histologic findings, an epitheloid angiosarcoma was diagnosed. At the patient’s second presentation 6 months later, the peripheral blood contained 54% blast cells. These cells had a wide cytoplasm that was filled with multiple vacuoles. The nuclei contained one to three nucleoli (Fig. 1A). Bone marrow biopsy revealed infiltration by atypical immature cells with a massive concomitant fibrosis (Fig. 1D). Slides of the supraclavicular lymph node were reviewed after determining that the patient had from a myeloproliferative syndrome with leukemic transformation. Additional immunohistochemical staining revealed that tumor cells expressed CD34 and myeloperoxidase. Rare large cells were positive for CD61. Taking all data into consideration, the diagnosis of a myelosarcoma was found to be more appropriate. In the interim, the patient had developed a pleural effusion and blasts cells were obtained from this effusion. FACS analysis of leukemic cells obtained by pleural puncture revealed coexpression of myeloid and endothelial antigens. In addition to the presence of CD34 (81%) and CD31 (72%), as already mentioned earlier, the cells were positive for the myeloid markers CD33 (91%), CD13 (74%), CD15 (84%), CD36 (53%), CD45 (97%), HLA-DR (76%), and myeloperoxidase (9%). Endothelial antigens included CD62E (56%), CD105 (16%), and Ulex europäeus agglutinin-1 (33%) (Fig. 2). Megakaryocytic markers such as CD41, CD42b, and CD61 were expressed by ⬍3% of the cells, excluding the possibility that essential thrombocytosis had transformed into acute myeloid leukemia (AML) of French–American–British type M7. To further prove the endothelial nature of these leukemic blasts, cultured cells were investigated for expression of additional antigens by PCR and immunocytochemistry. By PCR analysis, m-RNA was detected for von Willebrand factor, VE-cadherin, VEGF receptor 2 (KDR), and the angiopoietin-1 receptor (Tie-2/Tek) (Fig. 3). The PCR results were confirmed by immunocytochemistry (Fig. 4). Cytogenetic analysis of cells from the pleural effusion showed biclonality with the following karyotype: 1) 48, XX, ⫹8, ⫹19  and 2) 45, XX, ⫺7, del(11)(p14).nish 7 cen (D7Z1x1)(data not shown). To demonstrate that cells expressing endothelial antigens belonged to the same clone, simultaneous fluo- 348 CANCER January 15, 2000 / Volume 88 / Number 2 FIGURE 2. Fluorescence-activated cell sorter analysis of cultured cells. Left upper panel shows staining with phycoerythrin (PE) and fluorescein isothiocyanote (FITC) labeled control antibodies. Detection of subgroups of positive cells for (upper right panel) CD13 and CD62E, (lower left panel) CD31 and CD15, and (lower right panel) CD34 and Ulex europäeus lectin-1 are shown. Ig: immunoglobulin. FIGURE 3. Polymerase chain reaction analysis of cultured cells indicating expression for all tested genes (actin, von Willebrand factor [VWF], vascular endothelial-cadherin [VE-CAD], Tie-2/Tek, and vascular endothelial growth factor receptor type 2 (KDR)] by patient cells. Lane indications: M: size marker 100 basepair; C: negative control lanes without c-DNA; H: positive control lanes in which c-DNA from human umbilical vein endothelial cells was used; P: lanes in which c-DNA from patient cells was used. rescent in situ hybridization (FISH) analysis with cell surface staining of cultured cells was performed. As shown in Figure 5A, cells positive for CD 62E displayed monosomy 7. To determine whether the initial skin and lymph node infiltration represented the same disease as the later developing leukemia, interphase cytogenetic analysis on histologic sections was performed using probes for chromosomes 7 and 8. Three signals for chromosome 8 were found in 35.8% of cells in the tumor, whereas only 3.7% of cells showed ⱖ4 signals for this chromosome. The probe for chromosome 7 displayed 1 signal in 70.7% of cells and 2 signals in 9.3% of cells in the tumor tissue. Thus, the signal distributions were consistent with a monosomy 7 and a trisomy 8 (Fig. 5B). Leukemic cells from peripheral blood were cultured in vitro in IMDM supplemented with 10% FCS, 10% horse serum, and 1 M hydrocortisone. After approximately 6 weeks cells started to proliferate at a higher rate so that they could be split once a week. At last follow-up the cells had been in culture for approximately 9 months and were continuing to grow. Morphologically, these cells can be subdi- Hematopoietic Cell Line with Endothelial Features/Fiedler et al. 349 FIGURE 4. Immunocytochemistry of cultured cells using the immunoperoxidase technique showing positive staining for (a) von Willebrand factor, (b) the vascular endothelial growth factor receptor type 2 (KDR), (c) the angiopoietin receptor Tie-2/Tek, and (d) negative control staining without specific primary antibody (⫻ 1000). FIGURE 5. (a) Analysis of cultured cells with simultaneous cells surface staining with a monoclonal antibody against CD62E and interphase fluorescent in situ hybridization analysis with a chromosome 7 specific probe. Both cells show monosomy 7, but only one cell was positive for CD62E (⫻ 1000). (b) Interphase cytogenetic analysis of the lymph node with a chromosome 8 specific probe shows a trisomic signal distribution. Several cells with three signals are indicated by arrows representing trisomy 8 (⫻ 1000). vided into large, adherent cells with prominent vacuoles and into smaller nonadherent cells. Some cells of the nonadherent fraction appear to be capable of differentiating spontaneously along the myelomonocytic lineage because a few cells resemble mature granulocytes (Fig. 1B). Adherent and nonadherent cells continuously show monosomy 7 by interphase FISH analysis. This cell line has been called UKE-1. Cultured cells form colonies in soft agar. Subcutaneous transplantation of these cells into SCID mice led to tumor formation after approximately 2–3 months. On histologic examination, tumors in SCID mice closely resembled the original skin infiltration (data not shown). No spread to the bone marrow, liver, or spleen was observed in SCID mice. However, in one of five SCID mice a solitary lung metastasis was detected. DISCUSSION Since the advent of bone marrow transplantation it has been clear that human hematopoietic stem cells can differentiate into four lineages: lymphoid, erythroid, myelomonocytic, and megakaryocytic. Recently it has been suggested that during embryonal life both hematopoietic and endothelial cells are derived from a common precursor cell, the hemagioblast.5– 8 Experimental studies provide evidence that such cells 350 CANCER January 15, 2000 / Volume 88 / Number 2 persist during postnatal life. It has been shown that endothelial cells can be derived from immature cells that have been purified from peripheral blood.15 In a canine model, bone marrow transplantation was performed simultaneously with the implantation of a Dacron graft into the aorta. After 12 weeks the endothelial cells lining the Dacron graft were of donor origin.16 These experimental observations now have been extended by the description in the current study of a case of immature leukemia. Blast cells from this patient showed features of both hematopoietic and endothelial cells. This dual differentiation potential was observed in the clinical presentation, in which formation of a solid tumor resembling angiosarcoma and bone marrow infiltration with circulating malignant cells occurred. Accordingly simultaneous expression of hematopoietic and endothelial markers was detected by cell surface marker analysis. Although few cell surface antigens are completely specific for endothelial cells, the detection of multiple markers such as CD31, CD34, CD62E, CD105, von Willebrand factor, VE-cadherin, and Ulex europäeus agglutinin-1 on a subgroup of the cells clearly establishes the endothelial nature of the circulating cells. The receptors for VEGF and the angiopoietins (KDR and Tie-2/Tek, respectively) also were identified on cultured leukemic cells from our patient by means of PCR analysis and immunocytochemistry. They normally are expressed on endothelial cells such as those obtained from the human umbilical cord. Rarely, these receptors can be found on leukemic blasts from patients with acute myeloid leukemia.13,23 Furthermore, one of the endothelial markers, CD62E, could be located unambiguously on the leukemic cells by simultaneous FISH and immunofluorescent cell surface staining, indicating that cells with endothelial features belonged to the leukemic clone. Although it is tempting to speculate that this patient’s tumor represented transformation of the preexisting myeloproliferative syndrome, definitive proof is lacking. The leukemic stem cell of myeloproliferative syndromes including chronic myelogenous leukemia has been shown to be the malignant counterpart of very immature hematopoietic cells. Because the patient’s tumor showed features of hematopoietic and endothelial lineages, its clonogenic cell presumably represented an early common precursor. Conversely, the chromosomal status of the patient’s essential thrombocythemia was not known. The majority of patients with essential thrombocythemia have a normal karyotype although patients with trisomy 8 have been described.24 The complex chromosomal status of the disease in the patient in the current study also is compatible with a secondary neoplasm possibly in- duced by long term cytostatic therapy. The frequency of transformation of essential thrombocythemia caused by hydroxyurea monotherapy has been estimated to be between the spontaneous transformation rate and 3.5%.25,26 To our knowledge, the most recurrent chromosomal abnormality detected in hydroxyurea-induced transformation of essential thrombocythemia is rearrangement of chromosome 17 with possible alteration of the p53 gene.25 Because we did not find a chromosome 17 abnormality in the leukemic cells of our patient, the influence of hydroxyurea on the transformation process appears unlikely. To our knowledge, alterations of chromosome 8 in transformed essential thrombocythemia, as in the case presented in the current study, have been described in two patients,27,28 but the relation of these changes to previous therapy could not be established. To our knowledge, few chromosomal analyses have been reported in classic angiosarcoma. 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