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Leukemia (1999) 13, 1790–1803 1999 Stockton Press All rights reserved 0887-6924/99 $15.00 http://www.stockton-press.co.uk/leu The Tel-PDGFR fusion gene produces a chronic myeloproliferative syndrome in transgenic mice KA Ritchie1,2 AAG Aprikyan1,3, DF Bowen-Pope2, CJ Norby-Slycord1,2, S Conyers1, E Sitnicka4,5 SH Bartelmez4,5 and DD Hickstein1,3 1 Medical Research Service, VA Puget Sound Health Care System, Seattle, WA; 5Seattle Biomedical Research Institute Seattle, WA; Department of 4Pathobiology School of Public Health, and Departments of 2Pathology, and 3Medicine, School of Medicine, University of Washingon, Seattle, WA, USA Chronic myelomonocytic leukemia (CMML) is a pre-leukemic syndrome that displays both myelodysplastic and myeloproliferative features. The t(5;12) chromosomal translocation, present in a subset of CMML patients with myeloproliferation fuses the amino terminal portion of the ets family member, Tel, with the transmembrane and tyrosine kinase domains of platelet-derived growth factor receptor  (PDGFR) gene. To investigate the role of this fusion protein in the pathogenesis of CMML, we expressed the Tel-PDGFR fusion cDNA in hematopoietic cells of transgenic mice under the control of the human CD11a promoter. Transgenic founders and their offspring express the transgene specifically in hematopoietic tissues and develop a myeloproliferative syndrome characterized by: overproduction of mature neutrophils and megakaryocytes in the bone marrow; splenomegaly with effacement of splenic architecture by extramedullary hematopoiesis; an abnormal population of leukocytes co-expressing lymphoid and myeloid markers; and increased numbers of colonies in in vitro bone marrow CFU assays. All mice expressing the transgene exhibited at least one of these features of dysregulated myelopoiesis, and 20% progressed to a myeloid or lymphoid malignancy. This murine model of CMML parallels a myeloproliferative syndrome in humans and implicates the Tel-PDGFR fusion protein in its pathogenesis. Keywords: Tel-PDGFR; myeloproliferative syndrome; transgenic mice Introduction Myelodysplastic syndromes (MDS) represent heterogeneous clinicopathologic conditions generally characterized by ineffective hematopoiesis.1,2 Peripheral blood counts are low despite a usually hypercellular marrow. Also, peripheral blood erythrocytes, granulocytes, and platelets, and their precursors in the marrow demonstrate abnormal morphologies (dysplasia) (see Bain for review and description of the French– American–British (FAB) classification scheme57). Those MDS with increased numbers of blasts in the marrow may progress to acute myeloid leukemia, and therefore represent pre-leukemic states. The chronic myeloproliferative syndromes (MPS) are hematologic diseases characterized by hematopoietic hyperproliferation.3 The bone marrow is hypercellular, but hematopoiesis is effective and maturation is progressive, producing increased numbers of mature elements in the peripheral blood. Classification of a particular MPS is based on the lineage that is predominant in the periphery, ie erythrocytes in polycythemia vera (PV), platelets in essential thrombocythemia (ET), megakaryocytes (in the spleen) with myelofibrosis in myelosclerosis Correspondence: KA Ritchie, Pathology and Lab Medicine Service 113, VA Puget Sound Health Care System, 1660 South Columbian Way, Seattle, WA 98108, USA; Fax: 206-764-2001 A Aprikyan and K Ritchie contributed equally to this work Received 16 March 1999; accepted 20 May 1999 with myeloid metaplasia (MMM), and granulocytes in chronic myeloid leukemia (CML). All the MPS are characterized by splenomegaly, and may display subacute clinical problems associated with the hyperplastic lineage (eg hyperviscosity in PV, thrombosis in ET). Over time, these diseases may evolve into marrow failure. All of the MPS are associated with an increased risk of developing leukemia, best exemplified by CML, which progresses from a chronic phase to an acute leukemia in almost all patients. The progression of myelodysplastic syndromes to increasingly severe dysplasias, cytopenias, and ultimately to acute myeloid leukemia (AML) signifies these conditions as pre-leukemic states, and supports the multi-hit hypothesis of malignant transformation. Similarly, the increased risk of leukemia associated with the chronic myeloproliferative syndromes suggests that they, too, are pre-leukemic states. In both syndromes, the dysregulation of hematopoiesis predisposes to leukemia. Investigation of possible mechanisms of this dysregulation might elucidate early events in leukemogenesis. Chronic myelomonocytic leukemia (CMML) is an unusual myelodysplastic syndrome with features of both myelodysplasia and myeloproliferation. It is characterized by increased numbers of monocytes and sometimes granulocytes in the peripheral blood, and monocyte precursors in the marrow. CMML may be difficult to distinguish both clinically and histopathologically from CML, however, the presence of the Philadelphia chromosome and the bcr/abl fusion cDNA are diagnostic of CML. CMML may be further subtyped into a myelodysplastic form (eg abnormal morphologies in several lineages), or a myeloproliferative form, similar to CML yet negative for the Philadelphia chromosome. A subset of CMML shows a distinctive t(5,12) chromosomal translocation, and shows features of both MDS and chronic myeloproliferative syndrome.4 Insight into CMML was obtained by the cloning and sequencing of the t(5;12) chromosomal breakpoint, associated with a subset of CMML.5,6 This translocation creates a fusion gene between Tel, a newly identified member of the ETS family of transcription factors, and the platelet-derived growth factor receptor beta gene (PDGFR).7 The fusion gene contains the first 154 amino acids of Tel, including a putative helixloop-helix domain thought to mediate protein–protein interactions. The breakpoint occurs such that the fusion mRNA encodes a protein which lacks the extracellular ligand binding domain of PDGFR, but includes the transmembrane and intracellular domains of PDGFR, including the split tyrosine kinase domains that mediate intracellular signalling. Both the oligomerization domain of Tel and the tyrosine kinase activity of PDGFR are operative in the transformation of Ba/F3 cells.8,9 Both Tel10–19 and PDGFR20,21 have been found in a number of different translocation fusion genes associated with hematopoietic malignancies. The participation of PDGFR in malignant transformation and normal signalling pathways is Tel-PDGFR transgenic mice develop a myeloproliferative syndrome KA Ritchie et al well characterized.7,22–24 However, the respective roles of Tel and PDGFR in the pathogenesis of CMML has yet to be elucidated. We used the human CD11a promoter, which directs tissuespecific expression of a human CD4 reporter gene in the leukocytes of transgenic mice,25 to express the human TelPDGFR fusion gene in transgenic mice. Parallel to the endogenous mouse CD11a gene, this promoter directs expression of the reporter gene early in hematopoiesis, and also in both myeloid and lymphoid leukocyte lineages. By 4 months of age, mice expressing the Tel-PDGFR fusion protein exhibit dysregulated hematopoiesis with features similar to a human myeloproliferative syndrome. Older mice demonstrate a low frequency of hematopoietic malignancies. Materials and methods Gene construct and transgenic mice The construct used to generate the Tel-PDGFR transgenic mice consists of a 1.7-kb fragment from the human CD11a proximal promoter, a 463 bp cDNA fragment encoding the first 154 amino acids of Tel, a 3.3-kb cDNA fragment containing the entire transmembrane and intracellular domains of PDGFR (amino acids 511–1106 and 1787 bp of 3⬘ untranslated region),26 and a 1.9 kb fragment containing the human growth hormone (hGH) mini-gene (Figure 1). The human CD4 cDNA was released from the CD11a promoter-hCD4-hGH construct25 by BamHI digestion, and the portion containing the human CD11a promoter, the human growth hormone mini-gene and p0GH (Nichols Institute, San Juan Capistrano, CA, USA) was gel isolated and purified on a Qiagen column. A custom multicloning site (MCS) containing 5⬘-BamHI–SalI– HincII–NotI–BglII-3⬘ restriction sites was synthesized (Applied Biosystems Model 392, Foster City, CA, USA) and ligated into the purified plasmid between the CD11a promoter and the hGH mini-gene. Orientation was confirmed by sequencing. Primer (b) with a 5⬘ HincII site and primer (d) with a 3⬘ NotI site were used to amplify by RT-PCR the 463 bp Tel fragment from mRNA prepared from the HL-60 cell line. This fragment Figure 1 Construct used to generate the Tel-PDGFR transgenic mice. The PvuI–NsiI fragment containing 1.7 kb of the human CD11a promoter, the portions of human Tel and human PDGFR cDNAs corresponding to the Tel-PDGFR fusion gene, and a human growth hormone mini-gene was isolated and sent to DNX for microinjection. Arrows indicate oligos used during construction: (a) 5⬘GCCAGTGTCACCAGCCTGT-3⬘; (b) 5⬘-CCCCGAATATTGGCGTCGACATCTCTCTCGCTGTGAGACATG-3⬘; (c) 5⬘-CTCAAGTGGGCTGAAAATGAGTT-3⬘; (d) 5⬘-TGTCACGTGCTGTGCTGCGGCCGCCTTCTTCATGGTTCTGATGCAG-3⬘; (e) 5⬘-ACGTAGATGTACTCATGGCCGTCA-3⬘; (f) 5⬘-TTGGGAAGGCACTGCCCTGAA-3⬘. was then ligated into the HincII site of the MCS. Orientation and possible mutations were ruled out by DNA sequencing using the primer pairs (a–d) and (c–e), and an automated DNA sequencer (Applied Biosystems, Model 373). To preserve the reading frame and amino acid composition of the junction, the NotI site in the MCS was digested and filled in with Klenow and dNTPs. Finally, the FspI–SspI fragment containing the transmembrane and tyrosine kinase domains of PDGFR was isolated from full length PDGFR cDNA26 and ligated into this NotI site. Orientation and maintenance of the reading frame across the junction were confirmed by DNA sequencing using primer pair (c–e). The hGH mini-gene was included in the construct because it is reported to increase mRNA stability.27 The entire construct (7.4 kb) was excised by Pvul–NsiI digestion, isolated from an agarose gel, purified using a Qiagen column, quantitated by absorbance at 260 nm, and sent to DNX (Princeton, NJ, USA) for microinjection. Mice received from DNX were screened for the presence of the transgene as described,25 using a probe for the hGH portion of the construct. Detection of Tel-PDGFR expression by Western blotting Approximately 100 l of mouse peripheral blood were obtained by retro-orbital bleeding. Erythrocytes were lysed by an NH4 Cl-Tris hypotonic lysis method28 and the peripheral blood leukocytes were washed twice in cold PBS, resuspended in lysis buffer (50 mm HEPES, pH 7.5; 150 mm NaCl; 1% Triton X-100) containing protease inhibitors (1.0 mm PMSF, 1 g/ml leupeptin, 1 g/ml aprotinin), and centrifuged at 10 000 g for 10 min at 4°C. Resultant supernatants were used for analysis. Protein lysates from tissues were similarly prepared. Protein concentration was determined by absorbance at 280 nm. Fifty to 100 g of total protein were loaded on to a reducing, 10% SDS-polyacrylamide gel. Prestained molecular weight markers were run in one lane of each gel. After electrophoresis, the proteins were electroblotted on to either a polyvinylidene difluoride (PVDF) membrane (BioRad, Hercules, CA, USA) or nitrocellulose-nylon membrane (Genescreen; BioRad) in electroblot buffer (25 mm Tris, 192 mm glycine, 20% methanol) in an electroblotting apparatus (BioRad). The membrane was then incubated 30 min at room temperature with blocking buffer (50 mm Tris pH 7.4, 150 mm NaCl, 0.1% Tween-20, 0.2% BSA), and probed with a 1/2000 dilution of a rabbit polyclonal antibody (7649) that reacts with the C-terminal portion of PDGFR.29 The blots were washed four times with blocking buffer, incubated with a 1/5000 dilution of alkaline phosphatase conjugated goat antirabbit IgG secondary antibody (GIBCO-BRL, Gaithersburg, MD, USA) for 1 h, washed again, and developed with either a chromogenic substrate (p-nitrobluetetrazolium chloride (NBT) and 5-bromo-4chloro3-indoylphosphate-toluidine salt (BCIP) (BioRad) or chemiluminescent substrate (Chemisubstrate; Pierce, Rockford, IL, USA). Histology Mouse tissues were fixed in 10% buffered formalin, pH 7.0, sectioned and processed in a Miles Scientific (Naperville, IL, USA) Tissue Tek according to manufacturer’s instructions. Routinely sampled tissues were spleen, thymus, femur, lung, 1791 Tel-PDGFR transgenic mice develop a myeloproliferative syndrome KA Ritchie et al 1792 heart, kidney, liver, pancreas, brain, and skeletal muscle. Bones were fixed in formalin, decalcified in perfix (1.8% zinc chloride, 2% trichloroacetic acid, 4% paraformaldehyde, 17% isopropyl alcohol in water), then processed routinely. Hematoxylin and eosin-stained histologic sections were prepared according to routine procedures.30 Histologic sections were photographed using a Zeiss photomicroscope (Carl Zeiss, Oberkochen, Germany) and Nikon UFX exposure metering system (Tokyo, Japan). Flow cytometry The following directly conjugated antibodies were used to analyze spleen cells and peripheral blood leukocytes: fluoroscein isothiocyanate (FITC) rat anti-mouse CD45R/B220 (B lymphocyte marker, RA3-6B2, IgG2a, PharMingen, San Diego, CA, USA); FITC conjugated rat anti-mouse CD2 (T lymphocyte marker, RM2–5, IgG2b, PharMingen); FITC conjugated hamster anti-mouse CD3 (T cell marker, 145-2C11, hamster IgG, PharMingen); phycoerythrin (PE) conjugated rat anti-mouse granulocyte (GR-1, RB6-8C5, IgG2b PharMingen); phycoerythrin (PE) conjugated rat anti-mouse CD11b (myeloid marker, M1/70, IgG2b, PharMingen); FITC conjugated rat anti-mouse F 4/80 (monocyte/macrophage marker, C1:A3-1, IgG2b, Serotec, Washington, DC, USA); FITC conjugated rat anti-mouse CD14 (monocyte marker, rmC5-3, IgG1, PharMingen); FITC conjugated rat IgG1 (R3-34, isotype control, PharMingen); FITC conjugated rat IgG2a (R35–95, isotype control, PharMingen); FITC conjugated rat IgG2b (R53–38, isotype control, PharMingen); PE conjugated rat IgG2b (R53-38, isotype control, PharMingen). Rat IgG (Sigma Chemicals, St Louis, MO, USA) or Fc BLOCK (Pharmingen) were used to block non-specific binding. Spleen and peripheral blood leukocytes from transgenic founders and normal littermates were analyzed by flow cytometry. Peripheral blood was obtained from anesthetized mice by orbital bleeding. Mice were sacrificed by cervical dislocation, tissues were removed for histology and cell suspensions were made by gently rubbing one half spleen over a fine stainless steel screen. Erythrocytes were lysed as described,28 leukocytes were washed twice in PBS, and resuspended in PBS, 2% fetal bovine serum (FBS), and 0.1% sodium azide. Fifty l of cell suspension (5 × 105 cells) were dispensed into a 5 ml polystyrene test tube, non-specific binding was blocked with 5 l of rat IgG (10 mg/ml stock) or 2 l of Fc BLOCK (0.5 mg/ml stock), 1 l of appropriate antibody was added to each tube, and the cells were incubated on ice for 60 min. The cells were then washed twice with PBS, 2% fetal bovine serum, 0.1% sodium azide, fixed with 1% paraformaldehyde, and analyzed on a FACScan (Becton Dickenson, San Jose, CA, USA) flow cytometer using CONSORT 30 software. A gate was drawn around all live leukocytes on the forward scatter (FSC) vs side scatter (SSC) plot, and analyzed on an FL-2 (PE) vs FL-1 (FITC) two dimensional plot. In some experiments antibody capping was inhibited by 4°C instead of sodium azide, propidium iodide was used to label dead cells, and the cells were analyzed using FACSCAN Research software. Both procedures yielded the same results. Analysis quadrants were placed such that 98% of the cells stained with isotype controls fell into the lower left quadrant. Single antibody controls showed the location of each different, positive population on the contour graph. The relative fluoresences of the FITC-CD2 positive cells and the FITC-B220 positive cells were sufficiently different to clearly separate T cells from B cells on the x axis. Simultaneous addition of PE-GR-1, FITC-CD2 and FITC-B220 to a single tube allowed for distinct separation of granulocytes, T cells and B cells on the two-dimensional contour graph. Colony-forming cell (CFC) assays Bone marrow cells were harvested from one femur by flushing the marrow cavity with 3 ml of cold PBS, 1% FBS using a 1 cc syringe and a 25 gauge needle. Cells were counted and were grown in a double layer nutrient agar culture in the presence of stem cell factor (SCF) 50 ng/ml, interleukin-3 (IL-3), 50 ng/ml, interleukin-6 (IL-6), 20 ng/ml, and granulocyte– macrophage colony-stimulating factor (GM-CSF), 5 or 20 ng/ml, as described.31 In most experiments cells were plated at 50 000 cells per plate and these data were then normalized to CFC per 5000 cells. Plating densities of either 5000 or 50 000 gave similar results. All CFC assays were set up in triplicate. Plates were incubated in 5% CO2, 95% ambient air gas mixture at 37°C for 10 days, and colonies counted at low magnification by individuals blinded to the plates’ composition. Raw data were statistically analyzed and graphed using Excel (Microsoft, Redmond, WA, USA) or Kaleidagraph (Synergy Software, Reading, PA, USA) or Instat for DOS (Graphpad, San Diego, CA, USA) softwares. To determine the types of cells in colonies for each growth condition, 10 individual colonies from each growth condition were picked with Pasteur pipettes, separately resuspended in 0.5 ml of sterile PBS, 2% FBS, cytospun (Cytospin 2, Shandon, Pittsburgh, PA, USA), Wright–Giemsa-stained (Hemastainer; Geometric Data, Wayne, PA, USA), and a 200 cell differential count was performed on each colony. Copy number determination and Southern blots Genomic DNA was prepared from tissues and treated with RNase according to standard procedures.32 DNA concentration was determined by OD.260 To accomodate a range of copy numbers, 10, 5, 2.5, and 1 g of transgenic and normal littermate control DNAs were applied to nylon membranes (Genescreen Plus; New England Nuclear, Boston, MA, USA) using a dot-blot apparatus (BioRad) according to the manufacturer’s directions. Dot blots were hybridized with a radiolabelled (Ready-to-Go DNA Labelling Beads; Pharmacia, Piscataway, NJ, USA) fragment of the human growth hormone gene25 and autoradiographed. Densitometry was performed using a Beckman DU640 spectrophotometer (Palo Alto, CA, USA). To derive relative copy numbers, all values were normalized to the transgenic mouse with the lowest signal (Transgenic 935). Immunoglobulin heavy chain (IgH) and T cell receptor beta (TCR) gene rearrangements were detected by Southern blotting. Purified genomic DNA was separately digested with EcoRI, BamHI, of BglII (New England Biolabs) and probed with either pJ11 (mouse IgH J region, kindly provided by Dr Ursula Storb)33 or TCR (1.4 kb EcoRI–SacI fragment, kindly provided by Dr Dennis Willerford).34,35 Because the transgenic mice are derived from both the C57B1/6 and SJL inbred mouse strains, unrearranged bands were determined by including kidney DNA from both parent strains. These controls ruled out restriction fragment length polymorphisms that might otherwise be misinterpreted as rearranged genes. Tel-PDGFR transgenic mice develop a myeloproliferative syndrome KA Ritchie et al Results Generation of transgenic mice The human CD11a promoter was used in the transgene construct to direct expression of the Tel-PDGFR fusion cDNA (Figure 1). The 1.7 kb CD11a proximal promoter directs the expression of a human CD4 reporter gene in all leukocytes of transgenic mice.25 Fifteen out of 44 mice received from DNX were positive for the transgene, as determined by tail DNA slot blotting or by PCR amplification of tail DNA using primers that flank the Tel-PDGFR junction (c and e, Figure 1). One female and all of the male founders were bred to establish transgenic lines. Normal littermates were reserved for controls. Two founders died prior to the time of analysis and two founders died at over 2 years of age without evidence of malignancy. Eleven founders, 11 littermates and a number of offspring from different transgenic lines were analyzed in detail by Western blotting, histopathology, FACS analysis, and in vitro bone marrow colony-forming cell (CFC) assay. (Figure 2a, left two lanes). The additional bands on the blot represent proteins which cross react with the polyclonal antiPDGFR antibody. These cross reacting proteins do not represent endogenous mouse PDGFR, which has a molecular weight of 180 kDa,7 and is not expressed on PBLs.23 The human CD11a promoter directs expression of transgenes in hematopoietic tissues.25 Western blots of different tissues from transgenic mice reveal that the Tel-PDGFR fusion protein is expressed only in spleen, thymus and bone marrow (Figure 2b). Seven different founders and offspring which were tested displayed expression only in the spleen, thymus and bone marrow. Although the amount of TelPDGFR protein expressed by each founder varied, expression in all transgenics was restricted to leukocytes, and was not related to transgene copy number (see below). These studies indicate that the human CD11a promoter directs tissue-specific expression of the Tel-PDGFR transgene in leukocytes of transgenic mice. Histology Analysis of Tel-PDGFR expression by Western blotting To establish that the Tel-PDGFR transgenic mice express the fusion protein, lysates from peripheral blood leukocytes (PBL) were analyzed by Western blotting. Western blots of PBL protein lysates from founders and normal littermates were probed with a rabbit polyclonal antibody that reacts with the C-terminal portion of PDGFR. All of the transgenic founders (these seven plus eight others not shown) express variable amounts of the 85 kDa Tel-PDGFR fusion protein in their PBLs (Figure 2a). The 85 kDa band representing the fusion protein is not present in leukocytes from normal littermates Figure 2 (a) Expression of the Tel-PDGFR fusion protein in peripheral blood leukocytes. PBL lysates from transgenic mice and normal littermates were blotted and probed with a rabbit polyclonal primary antibody that recognizes the C-terminal portion of human PDGFR and a secondary, alkaline phosphatase conjugated goat antirabbit antibody. (b) Expression of the Tel-PDGFR fusion protein in different tissues. Protein lysates from different tissues were blotted and probed with a rabbit polyclonal primary antibody that recognizes the C-terminal portion of human PDGFR and a secondary, alkaline phosphatase conjugated goat anti-rabbit antibody. Representative histology of a normal littermate and two transgenic founders is shown (Figure 3). Low power views of the spleens (Figure 3, top row) indicate that the normal littermate has intact follicular architecture with evenly distributed lymphoid follicles (darker areas) with paler, central germinal centers, and pale interfollicular areas. The spleens of rodents normally contain small amounts of extramedullary hematopoiesis (EMH), usually seen as a few megakaryocytes and erythroid precursors scattered in the subcapsular regions. EMH does not normally efface the splenic architecture, however the splenic architecture of the transgenics is effaced by EMH, with only irregular residual lymphoid regions (darker areas). The large, pale cells scattered throughout the effaced spleen and visible even at low power are megakaryocytes, best shown by transgenic 941. While the megakaryocyte nuclei in normal littermate 926 and transgenic 907 display normal morphology, those of transgenic 941 are dark and irregular, suggestive of myelodysplasia in 941. High power views of the spleens (second row) show intact follicular architecture in the normal littermate with a germinal center at the upper left corner, and surrounding small lymphocytes. High power views of both transgenic spleens show immature erythroid cells (smaller darker cells), granulocytes with irregular and doughnut-shaped nuclei, and large, pale, polyploid megakaryocytes. Eight out of the 11 transgenics analyzed show partial to total effacement of the splenic architecture by EMH. Although the EMH contained erythroid, myeloid and megakaryocytic precursors in all cases, six mice had a predominance of granulocytes at all stages of maturation (demonstrated by transgenic 907), and two had large numbers of megakaryocytes (exemplified by transgenic 941). Normal mouse bone marrow has a cellularity of greater than 90%, therefore, the increase in cellularity up to 100% that is seen in the transgenics is not readily demonstrable. However, high power views of the femoral marrows (bottom row) show that the marrow of normal littermates contains a mixture of trilineage hematopoiesis, while the marrows of the Tel-PDGFR transgenics show a predominance of either granulocytes or megakaryocytes. For example, transgenic 907 marrow is packed with mature neutrophils and scattered megakaryocytes but no erythroid precursors. Trilineage hematopoiesis with greatly increased numbers of dysplastic megakaryocytes is present in transgenic 941. The Tel-PDGFR 1793 Tel-PDGFR transgenic mice develop a myeloproliferative syndrome KA Ritchie et al 1794 Figure 3 Histology of the spleen and bone marrow from a normal littermate and two transgenic founders at 5 months of age. Normal littermate tissues are shown in the left hand column and transgenic tissues appear in the middle and right-hand columns. The first row contains low power views of spleens; the second row contains high power views of spleens, and the bottom row shows high power views of the bone marrows. transgene does not appear to cause any relative increase in erythroid precursors. Of the eight transgenics showing abnormal bone marrow histology, four show increased numbers of granulocytes, three increased numbers of megakaryocytes, and one shows a malignant histiocytosis (see below). The Tel-PDGFR founders and their normal littermates have been followed for evidence of hematologic abnormalities by peripheral blood smears, white blood cell counts (WBC), hematocrit and hemoglobin determinations and FACS analysis of peripheral blood leukocytes (data not shown). These studies did not reveal a significant difference in the peripheral blood between most transgenics and normal littermates, which is likely due to the wide variability of peripheral total WBC (2 to 20 × 109/liter) and percent granulocytes (5 to 25%) between individuals and within individuals over time.36 FACS analysis of transgenic mice Spleen cells and peripheral blood leukocytes from normal littermates and transgenics were analyzed by FACS. Profiles of representative founders and normal littermates are shown (Figure 4). Simultaneous staining of cells with the three antibodies PE-GR-1, FITC-CD2, and FITC-B220 allows separation and enumeration of granulocytes (upper left), T lymphocytes (lower right) and B lymphocytes (lower far right) on a single contour plot. A few transgenics displayed increased neutrophils in the peripheral blood and spleen. For example, normal littermate 926 had 5% neutrophils in its spleen, while transgenic 907 had 48% neutrophils (Figure 4a). Also, the transgenic mouse (907) had markedly decreased numbers of CD2 positive T cells, as shown by the absence of the population of cells just to the right of the vertical line. FACS analysis of peripheral blood showed the same results (data not shown). In addition to histologic abnormalities, transgenic 907 also exhibited abnormal leukopoiesis as evidenced by greatly increased numbers of neutrophils and decreased numbers of T cells in the spleen and peripheral blood. Other transgenics displayed an abnormal population of leukocytes that simultaneously coexpress both lymphoid and myeloid markers. Transgenic founder (940) and normal littermate (913) were chosen at random and analyzed by histology and FACS analysis (Figure 4b). FACS analysis of their spleens showed a population of abnormal leukocytes in the transgenic abnormally co-expressing variable amounts of the granulocyte marker GR-1 and constant amounts of the T lymphocyte marker CD2 (Figure 4b). This abnormal population is not present in the normal littermate. The same result was obtained when CD3, another T cell marker, was used in place of CD2 (data not shown). FACS analysis of another normal littermate (910) and transgenic (929) again showed an abnormal population of cells in the transgenic which co-expressed GR-1 and CD2 (Figure 4c, upper panel). However in this Tel-PDGFR transgenic mice develop a myeloproliferative syndrome KA Ritchie et al 1795 Figure 4 FACS analysis of spleen cells from representative normal littermates (left panels) and representative transgenics (right panels). (a and b) Two-color fluorescence PE-GR-1 vs FITC CD2 + FITC B220. (c) Lower panels also show PE-CD11b vs FITC CD2+FITC B220. Single antibody controls demonstrate that the B220 positive cells are brighter than (to the right of) CD2 positive cells, and that CD2 positive cells are found to right of the vertical line. The scales of both axes are log10 fluoresence. The mice were analyzed at (a) 5; (b) 8; and (c) 13 months of age. transgenic, the expression of GR-1 was uniformly high, rather than variable, as seen in transgenic 940 (Figure 4b). Coexpression of both myeloid and lymphoid markers was confirmed by demonstrating that this abnormal population of leukocytes in trangenic 929 also co-expressed another myeloid marker, CD11b, along with CD2 (Figure 4c, lower panel). The nine transgenic founder mice that were analyzed by FACS demonstrated four immunophenotypes: one mouse displayed no abnormalities on FACS; three mice had a slightly increased percentage of granulocytes (data not shown); one mouse had a markedly elevated (48%) percentage of granulocytes, and four mice showed an abnormal population of leukocytes that simultaneously co-expressed both lymphoid and myeloid cell surface antigens. These findings, particularly the biphenotypic picture, suggest that the Tel-PDGFR transgene disrupts leukopoiesis and alters lineage fidelity. Progenitor cell levels To investigate whether the Tel-PDGFR transgenic phenotype reflects changes present in hematopoietic progenitors, colonyforming assays were performed on the marrow from normal littermates and transgenic founders (Figure 5). All of the transgenic founders (and their offsrping, see below) analyzed, regardless of age or sex, displayed more CFCs per 5000 cells plated than normal littermate controls. This increase in bone marrow CFCs was a consistent abnormality in Tel-PDGFR founders (Figure 5) and in their progeny (Figure 7). Tel-PDGFR offspring Six Tel-PDGFR founders were backcrossed into C57B1/6 normal mice to generate independent transgenic lines. Histopathologic, Western blot, and FACS analysis of these offspring Figure 5 Colony-forming cell (CFC) numbers of transgenic founders and age matched normal littermates at the folowing ages: 5 months (926, 907); 10 months (912, 943); 13 months (909, 937, 910, 929); and 15 months (921, 919). All founders tested showed a statistically significant increase in the number of CFC on soft agar colony forming assay. showed the same features of a myeloproliferative syndrome as described for the founders: leukocyte-specific expression, distortion of splenic architecture by EMH, biphenotypic leukocytes, and increased numbers of CFC. Consistent with a single integration site, approximately 50% of the progeny of transgenic founders inherit the transgene (data not shown). At 4 months of age, non-transgenic offspring Tel-PDGFR transgenic mice develop a myeloproliferative syndrome KA Ritchie et al 1796 a c b d Figure 6 Histopathology and FACS analysis of spleens from transgenic offspring at 4 months of age. (a, b, e and g) Offspring littermates that did not inherit the transgene. (c, d, f and h) Offspring that inherited the transgene. had a normal sized spleen with intact follicular architecture (Figure 6a), and interfollicular regions containing small lymphocytes (Figure 6b). Four-month-old transgenic offspring had enlarged spleens that maintained follicular architecture but showed enlarged interfollicular regions (Figure 6c). High power view demonstrates that the interfollicular areas are expanded by extramedullary hematopoiesis (large arrowheads point to megakaryocytes, small arrowheads to clusters of granulocytes, the smallest, dark cells are erythroid precursors, Figure 6d). Regardless of founder lineage, all offspring that inherited the transgene displayed the increased splenic extramedullary hematopoiesis seen in the parent. FACS analysis of offspring revealed that leukocyte biphenotypism follows the inheritance of the transgene. Non-transgenic offspring displayed normal granulocytes, T cells and B cells on FACS analysis of peripheral blood leukocytes (Figure 6e, g). (The 16% granulocytes demonstrated by this normal littermate is within the normal range of 5–25%.) Transgenic offspring showed biphenotypic leukocytes (Figure 6f, h) like those seen in their transgenic parent, and additionally, the pattern of biphenotypism was inheritable. Founders with CD2+, uniformly high GR-1+, CD11b+ leukocytes (Figure 4c) produced transgenic offspring that also displayed CD2+, uniformly high GR-1+, CD11b+, CD11b+ leukocytes (Figure 6f, h). Founders that displayed leukocytes that coexpressed CD2 with a range of GR1 positivity (Figure 4b) produced transgenic offspring that also displayed CD2+, variable GR-1+ biphenotypic leukocytes (data not shown). CFC analysis of offspring (Figure 7) showed that the transgenic lines had increased numbers of CFCs compared to normal littermate controls. One individual from each transgenic line was analyzed; two offspring that did not inherit the transgene served as controls; cells were plated out in triplicate. The offspring of founders 929, 941 and 919 showed the greatest increase in CFCs. These founders also demonstrated the earliest and most severe manifestations of the myeloproliferative Tel-PDGFR transgenic mice develop a myeloproliferative syndrome KA Ritchie et al Figure 7 CFC numbers for 4-month-old offspring of different transgenic founders. The founder number is indicated at the bottom. phenotype (see spleen of 941, Figure 3), and relatively larger increases in CFCs in the founders (see founders 919 and 929 CFCs, Figure 5). The founders whose offspring had milder increases in CFCs (907) also tended to have intermediate increases in CFCs themselves, milder splenomegaly, less extramedullary hematopoiesis and less striking abnormalities on FACS analysis. The onset and severity of the myeloproliferative features most closely correlate with the degree of increased bone marrow CFCs of each founder and its offspring. The particular features of the myeloproliferative syndrome breed true within each lineage and the myeloproliferative phenotype tracks with the inheritance of the TelPDGFR transgene. Malignancies in founders Three out of 13 (23%) of the Tel-PDGFR founders developed a malignancy. No malignancies were observed in agematched, normal littermate controls. At 14 months of age, transgenic 911 was found to have a large tumor in the mesentery of the small bowel (Figure 8a, large arrowhead), hepatomegaly with white nodules of tumor infiltration (medium arrowhead), and a spleen 10 times normal size (small arrowheads). The histology of the tumor showed a mixture of early myeloid cells, granulocytes and lymphocytes (Figure 8b). The malignant nature of the tumor was demonstrated by tumor infiltration of spleen and lung parenchyma (data not shown) and by tumor infiltration among hepatocytes (Figure 8c, L). Founder 902 was found to have an enlarged spleen and a retrosplenic tumor. The histology was identical to that of the tumor in transgenic 911 and consisted of a mixture of granulocytes and lymphocytes with numerous mitotic figures (Figure 8d). FACS analysis of the 911 and 902 tumors were identical and confirmed the histopathology (Figures 8e and f showing 902 tumor). The cells were uniformly positive for CD11a, which confirms the hematopoietic origin of the tumor and rules out a poorly differentiated carcinoma. Also, the tumors consisted of a mixture of CD3-positive T cells, B220-positive B cells, and a few GR-1-positive granulocytes (not shown). DNA dot blots confirmed the presence and Western blots confirmed the protein expression of the transgene in the tumors (not shown). Morphologically and immunophenotypically these tumors represent dysregulated hematopoietic neoplasms that defy classification into specific types or lineages. Because of the indefinite histology and FACS results of 902 and 911 tumors, antigen receptor gene rearrangement studies were performed to help determine the lineages of the malignancies. Genomic Southern blots of tumor DNAs were probed with either a mouse IgH J-region probe (Figure 9a) or a mouse TCR probe (Figure 9b). Kidney DNA of the parental mouse strains (C57B1/6 and SJL) indicate the unrearranged, germline bands (G). Mice 902 and 230 have rearranged IgH alleles, and germline TCR alleles. This established the existence of a monoclonal B cell lymphoma in the mixture of cells in the 902 tumor, and confirms the B cell immunophenotype (CD19) of the 230 tumor (see below). Mouse 911 has a definitive TCR gene rearrangement, and a probable IgH rearranged band slightly above the germline band. This indicates that the 911 tumor most likely contains a malignant lymphoma that has rearranged both IgH and TCR loci. This observation is occasionally seen in human lymphomas, and is a manifestation of ‘molecular biphenotypism’. Alternatively but less likely, the 911 tumor contains a mixture of two separate malignant lymphomas, one of B cell and the other of T cell lineage. Founder 937 suddenly developed hind limb paralysis and was sacrificed. Post-mortem analysis showed a population of biphenotypic leukocytes, increased bone marrow CFCs but no gross evidence of tumor. Therefore, only histologic assessment was possible. Histology of the spine showed that a histiocytic infiltrate replaced the marrow of some vertebral bodies (arrowhead, Figure 10a), while the marrow of adjacent vertebral bodies had normal hematopoiesis (asterisk, Figure 10a). In clinical terms this mouse had suffered a pathologic fracture of its spine. The malignant nature of this process is demonstrated by tumor infiltrating paraspinal skeletal muscle (Figure 10b, arrowhead). While femoral marrow had regions of normal hematopoiesis (Figure 10c, asterisk), the myeloproliferation replaces adjacent marrow of (Figure 10c, arrowhead), indicating the systemic nature of this process. High power view of the infiltrate (Figure 10d) shows a histiocytic appearance. A negative reticulin stain (data not shown), ruled out marrow fibrosis. Therefore, similar to human myeloproliferative syndromes, the Tel-PDGFR mice display a predisposition towards malignant transformation. Malignancies in offspring Although most offspring were sacrificed at a young age for analysis, several lineages were maintained for breeding and over time several of these offspring developed malignancies. At 4 months of age, transgenic mouse 424 (offspring of founder 937) died suddenly. Necropsy discovered a large, mediastinal tumor compressing the trachea. Histology showed a 1797 Tel-PDGFR transgenic mice develop a myeloproliferative syndrome KA Ritchie et al 1798 a c b d e Figure 8 f Histopathology and FACS analysis of 911 (a, b, c) and 902 (d, e, f) tumors. The mice were analyzed at 14 months of age. a b Figure 9 Gene rearrangements in tumors. Unrearranged bands (G) are indicated by kidney DNA from the parental mouse strains C57B1/6 and SJL. Tumors are denoted by their mouse number (902, 911, 230). (a) Genomic DNA was digested with BamHI and hybridized with a mouse immunoglobulin heavy chain J region probe; (b) Genomic DNA was digested with BglII and hybridized with a mouse T cell receptor beta chain probe. Tel-PDGFR transgenic mice develop a myeloproliferative syndrome KA Ritchie et al 1799 a c b d Figure 10 Founder 937 suffered a pathologic spine fracture and paralysis at 13 months of age, due to a systemic and aggressive histiocytic infiltration of bone marrow and paraspinal muscle. malignant lymphoid tumor that infiltrated around pulmonary vessels (Figure 11a). This same finding was recently described for a bcr-abl transgenic mouse (Figure 3).37 At age 14 months, transgenic mouse 230 (offspring of founder 929) developed an enlarged abdomen and was sacrificed. The mouse had large mesenteric and mediastinal tumors. Peripheral smear showed increased numbers of small, dark lymphocytes (Figure 11b). The tumors consisted of small, dark, lymphoblastoid cells with a high mitotic rate. These malignant cells also packed the spleen and bone marrow. DNA dot blot confirmed the presence of Tel-PDGFR DNA, and Western blot showed that the tumor expressed the Tel-PDGFR protein (not shown). FACS analysis showed that the tumor cells expressed CD2, CD5, CD19 and co-expressed variable amounts of the myeloid marker CD11b. The histology, peripheral smear, co-expression of CD5 and CD19, and the IgH gene rearrangement (Figure 9) are analogous to human chronic lymphocytic leukemia. The co-expression of the myeloid marker CD11b, T cell markers CD2 and CD5, and the B cell marker CD19 exemplify the biphenotypism exhibited by dysregulated leukemic cells. Because the presentation of several different founders and offspring can be difficult to follow, Table 1 is included to indicate the founder and offspring designations. Offspring of each founder were identified with numbers within a particular hundred series. Table 1 also summarizes relative transgene copy numbers. Similar to that found for CD11a-promoter-hCD4 transgenic mice, there is no correlation between copy number and levels of expression, CFC, phenotypic features, and the development of malignancy. Lack of correlation with copy number may be due to effects of different integration sites, timing of expression during hematopoiesis, and/or genetic variability in these non-inbred, F2 mice. Table 2 summarizes the number of founders showing the various features of the Tel-PDGFR phenotype. Discussion Transgenic mice expressing the Tel-PDGFR fusion protein display a phenotype characterized by splenomegaly, efface- ment of the splenic architecture by extramedullary hematopoiesis, increased granulocytes and/or megakaryocytes in the spleen and bone marrow, abnormal leukocytes that coexpress both myeloid and lymphoid markers, and increased numbers of colonies on bone marrow soft agar CFC assays. While normal littermates show normal lymphoid splenic architecture and normal, trilineage hematopoiesis in the marrow, Tel-PDGFR transgenics show markedly abnormal hematopoiesis consistent with a chronic myeloproliferative syndrome. Transgenics like 907 have histology very similar to that seen in the chronic phase of human chronic myeloid leukemia,3 and transgenics like 941 have histology very similar to that seen in human essential thrombocythemia or early stages of myelosclerosis with myeloid metaplasia.3 With long-term follow-up, 23% of the transgenic founders developed malignancy. Thus, the features of leukocyte-specific, yet both myeloid and lymphoid expression of Tel-PDGFR in transgenic mice are those of a chronic myeloproliferative syndrome, and are similar to those seen in transgenic mice harboring the CML-associated, bcr-abl fusion gene, and in human chronic myeloproliferative syndromes. Cytogenetic and other studies have shown that the underlying abnormality in the myeloproliferative syndromes exists in a pluripotent progenitor cell capable of differentiation into several lineages.38,39 However, the pre-leukemic syndromes and most of the associated leukemias are expressed as overgrowth in the myeloid lineages (granulocytes, monocytes). The lymphoid lineage is rarely affected in MDS, but often affected in MPS. Immunophenotyping of leukemias and MPS has shown that the leukemic clone can simultaneously or serially manifest itself as an overproliferation of both lymphoid and myeloid cells (termed a bilineage leukemia). Alternatively, the leukemic clone can simultaneously express both myeloid and lymphoid surface markers on the same cell (termed a biphenotypic leukemia). The ability of a leukemic process to exhibit lineage heterogeneity is well demonstrated by CML, in which the myeloid chronic phase of the leukemia transforms to an acute lymphoblastic leukemia with an immature B cell phenotype. Similarly, myeloid plus B cell and myeloid plus T cell biphenotypism has been described in human CML.40,41 Therefore, lineage heterogeneity and lineage Tel-PDGFR transgenic mice develop a myeloproliferative syndrome KA Ritchie et al 1800 a b Figure 11 (a) Histopathology of transgenic offspring 424 tumor (5 months of age). (b) FACS analysis and histopathology of transgenic offspring 230 tumor (14 months of age). promiscuity are features of the dysregulated leukopoiesis seen in human acute leukemia and MPS, as well as in bcr-abl42–44 and Tel-PDGFR transgenic mice. The CD11a promoter-Tel-PDGFR phenotype resembles the myeloproliferative phenotype seen in transgenic mice that express the bcr-abl fusion gene from the t(9,22) translocation of the Philadelphia chromosome positive CML.42–44 Both the Tel-PDGFR and bcr-abl transgenic mice develop myeloproliferative syndromes characterized by splenomegaly, effacement of the splenic architecture, extramedullary hematopoiesis, granulocytic and megakaryocytic hyperplasia, and biphenotypic leukocytes (GR-1 and B220 coexpression).44 This myeloproliferative pre-leukemia is not seen in transgenic mice in which Tel-PDGFR transgene expression is directed Tel-PDGFR transgenic mice develop a myeloproliferative syndrome KA Ritchie et al Table 1 Relative copy numbers and offspring designations for TELPDGFR mice Founder 902 905 907 911 919 928 929 935 937 941 Table 2 Offspring designation Relative copy number not bred 500 700 not bred 0–100 600 200 300 400 100 0.4 1.3 10 5.7 34 3.3 3.0 1.0 21.7 5.3 Features of the tel-PDGFR myeloproliferative syndrome Feature Splenomegaly (n = 11) Spleen histology (n = 11) Extramedullary hematopoiesis Intact architecture Bone marrow histology (n = 11) Increased granulocytes, megakaryocytes, or histiocytes No abnormality FACS analysis (n = 9) Biphenotypic leukocytes Increased granulocytes No abnormality Increased colonies on colony-forming cell assay (n = 6) Malignancy (n = 13) Number of founders showing feature 9 8 3 8 3 4 4 1 6 3 to the lymphoid compartment by the immunoglobulin heavy chain enhancer/promoter.45 It is also noteworthy that both bcr-abl and Tel-PDGFR transgenic mice exhibit the same histopathology of malignant lymphoid infiltrates surrounding pulmonary vessels (Figure 11a and Figure 337). Both bcr-abl and Tel-PDGFR transgenic mice have a myeloproliferative chronic phase that often evolves into lymphoid malignancy. These similarities suggest that bcr-abl and Tel-PDGFR share similar mechanisms and/or similar targets for transformation, and that like bcr-abl, myeloproliferation that evolves into lymphoid malignancy is an intrinsic property of the TelPDGFR fusion gene. The Tel-PDGFR and bcr-abl mice demonstrate some differences in their phenotype. The bcr-abl mice displayed peripheral blood granulocytosis and/or leukemia, while the TelPDGFR mice did not show significant abnormalities in their peripheral blood. The myeloproliferative syndrome of the TelPDGFR mice manifests itself primarily as histopathologic changes in spleen and bone marrow. The majority of the bcrabl transgenic mice developed a malignancy within a short time, while a minority of the Tel-PDGFR transgenic mice developed a malignancy only after a long latent period. These differences between the Tel-PDGFR and bcr-abl mice probably reflect differences in the relative oncogenicity of these two fusion genes, or in the promoters used to express them in transgenic mice. Introduction of additional oncogenic mutations (‘second hits’) by either physical or chemical exposure or breeding with transgenic mice harboring other oncogenes might accelerate the development of malignancy in Tel-PDGFR mice. The similarities between the Tel-PDGFR and bcr-ab1 mice suggest that the activity of their tyrosine kinases are important in producing the myeloproliferative phenotype. The TelPDGFR and the bcr-abl fusion genes share some important features: the genes at N-terminal portions (bcr and Tel) were discovered solely by their participation in their respective translocations, and the C-terminal portions (abl and PDGFR) are tyrosine kinases that participate in multiple signalling pathways and also signal to ras.2,23,24,46,47 In addition, both of these fusion genes are associated with human myeloproliferative syndromes. v-fms, the viral homologue of the normal cellular proto-oncogene c-fms (the macrophage colony-stimulating factor-1 (MCSF-1) receptor), belongs to the same structural family as PDGFR.23 v-fms also produces chronic myeloproliferative syndromes and hematopoietic malignancies of different lineages in transgenic mice.48 These observations, plus the similar phenotypes in human myeloproliferative syndromes and in transgenic mice, suggest that v-fms, TelPDGFR and bcr-ab1 share similar mechanisms. Abnormal tyrosine kinase activity may account for the histologic and immunophenotypic abnormalities seen in the spleen, peripheral blood leukocytes and bone marrow of these mice. Aberrant tyrosine kinase activity acting through ras signalling pathways may deregulate growth control by either increasing intracellular signals to proliferate or hindering normal feedback signals that restrict proliferation. The CFC results demonstrate that, like human myeloproliferative syndromes, Tel-PDGFR transgenic mice have an intrinsic difference in hematopoietic progenitors, evidenced by increased numbers on soft agar cloning assays. Increased numbers of CFCs suggest that increased numbers of hematopoietic progenitors and their progeny ultimately pack the marrow and produce extramedullary hematopoiesis. Similarly, since myeloid and lymphoid lineages have distinct kinase signalling pathways, one hypothesis to explain the biphenotypism seen in bcr-ab1 and Tel-PDGFR transgenic mice as well as human leukemias, is that ectopic tyrosine kinase activity allows excessive ‘cross-talk’ between signalling pathways.49,50 The TEL-PDGFR fusion gene is associated with a pre-leukemic syndrome that progresses to myeloid leukemia in humans, but lymphoid malignancies in transgenic mice. This difference may reflect subtle differences between human and mouse hematopoiesis, an interspecies difference in the transformation of lymphoid or myeloid lineages, the nature of ‘second hits’ between mouse and man, the activity of endogenous retroviruses present in the murine genome, differences in signalling pathways, or the timing of transgene expression. Although the human CD11a promoter expresses transgenes relatively early in hematopoiesis (manuscript in preparation), it is not known when during hematopoiesis the tel-promoter-driven fusion gene is expressed. In general, the same genetic abnormality often produces similar, but not identical, phenotypes in humans and transgenic mice. Differences in the timing of expression were demonstrated when the human fetal globin gene behaved as an embryonic globin gene in transgenic mice.51,52 A bcl-2-Ig construct isolated from the t(14,18) translocation in human, monoclonal, malignant follicular lymphoma produces a polyclonal lymphoproliferation that does not progress to monoclonal malignancy in transgenic mice.53 Whereas mutations in dystrophin produce Duchenne muscular dystrophy in humans, mdx mice compensate for the lack of dystrophin, suffer only a transient myopathy, and live a normal lifespan without disease.54,55 1801 Tel-PDGFR transgenic mice develop a myeloproliferative syndrome KA Ritchie et al 1802 Such discrepancies are a feature of the transgenic approach to understanding human disease, and further investigation of these differences often elucidates the underlying mechanisms of disease. Further investigation of the TEL-PDGFR transgenic mice with respect to the possible mechanisms mentioned above may account for the TEL-PDGFR fusion gene association with myeloid leukemia in humans, but lymphoid malignancies in transgenic mice. Patients with the t(5;12) translocation exhibit a syndrome characterized by splenomegaly, eosinophilia, monocytosis and absence of the Philadelphia chromosome, and have been diagnosed as CMML. However, it is not clear whether this syndrome represents CMML-myelodysplastic or CMML-myeloproliferative. It has been suggested that the syndrome represents a clinical entity with features of both CMML and CML.4,56 Since the diagnosis of human myelodysplasia depends on recognition of specific dysmorphologies in hematopoietic elements (for example, binucleated erythroid presursors, Pseudo-Pelger–Huet neutrophils, micromegakaryocytes), and since the morphologies of mouse granulocytes differ from human granulocytes, and because a mouse MDS has never been described, the degree to which the changes seen in TelPDGFR mice represent a myelodysplastic syndrome is difficult to assess. The Tel-PDGFR mice exhibit splenomegaly, chronic myeloid or megakaryocytic proliferation in the spleen and bone marrow, effacement of splenic architecture, increased bone marrow CFCs, decreased T cells, and biphenotypic cells that co-express myeloid and lymphoid markers, and development of myeloid, lymphoid and biphenotypic malignancies. Therefore, the Tel-PDGFR mice demonstrate features of hematopoietic dysregulation more consistent with a myeloproliferative syndrome, and suggest that the human t(5;12) syndrome might be best classified as a myeloproliferative rather than a myelodysplastic disorder. Acknowledgements This work was supported by National Institutes of Health grants K08HL02959 (NHLBI; KAR) and DK48708-02 (NIDDK; SHB). DDH was supported by the Veterans Affairs Career Development and Merit Review programs, and the National Institutes of Health. The authors wish to acknowledge the expert technical assistance of Lisa Embree, the photographic expertise of Dale Tilly and Eden Palmer, and the statistical help of Dan Bankson. 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