Leukemia (1997) 11, 1234–1237  1997 Stockton Press All rights reserved 0887-6924/97 $12.00

Expression of FLT4 and its ligand VEGF-C in acute myeloid leukemia

W Fielder1, U Graeven1,2, S Ergu¨n3, S Verago, N Kilic3, M Stockschla¨der1 and DK Hossfeld1

Department of 1Hematology/Oncology and 3Institute of Anatomy, University Hospital Eppendorf, Hamburg, Germany

FLT4 represents a recently cloned member of class III in angioblasts of head mesenchym, the cardinal vein and tyrosine kinases which include receptors for the angiogenic extra-embryonally in the allantois.10 In late embryos and growth factor VEGF, namely FLT1 and KDR. The ligand of FLT4 has been identified as VEGF-C which shares sequence homo- adults FLT4 expression seems to be restricted to endothelial logy with VEGF and P1GF. In the adult FLT4 shows a restricted cells of lymph vessels and to some high endothelial venules 9 expression pattern that is limited to lymphatic endothelia and (HEV). Additionally FLT4 has been found in pericardium, endothelia of some high endothelial venules (HEV). FLT4 has pleura, lung, kidneys and placenta.7,11 FLT4 is also expressed also been detected in some tumor cell lines including the hema- in a variety of cell lines derived from Wilms’ tumor, retino- topoietic line HEL. We therefore investigated expression of blastoma and teratocarcinoma.12 Since the leukemic hemato- FLT4 and its ligand VEGF-C in fresh samples from patients with 12 AML. Using a sensitive PCR method we detected FLT4 m-RNA poietic cell line HEL is positive for FLT4, we investigated in 15 of 41 patients with de novo AML at diagnosis or relapse expression of FLT4 and its ligand VEGF-C in human bone and in three of 12 patients with secondary AML. FLT4 marrow or peripheral blood samples from normal volunteers expression was confirmed by immunocytochemistry in a sub- and patients with acute myeloid leukemia (AML) with the PCR group of the studied patient population. FLT4 was also found technique and immunocytochemistry. This may lead to in leukemic cell line U937, but not TF-1 and KG1a. VEGF-C possible new insights into autocrine or paracrine growth expression was found in leukemic samples of four of seven FLT4-positive and four of six FLT4-negative patients. U937 cells mechanisms. also produced VEGF-C m-RNA. Interestingly, FLT4 expression was not detected in bone marrow samples of 15 normal volun- teer donors or in CD34-positive cells from three additional Material and methods donors. Possible autocrine and paracrine growth stimulation of leukemic blasts by VEGF-C is currently being investigated in our laboratory. Isolation of peripheral blood or bone marrow Keywords: AML; VEGF-C; FLT4; angiogenesis mononuclear cells

Peripheral blood or bone marrow samples from 53 consecu- Introduction tive patients with newly diagnosed or relapsed AML were obtained prior to chemotherapy. Mononuclear cells were sep- Several members of closely related receptor tyrosine kinases arated by density gradient centrifugation on Ficoll–Hypaque (RTK), involved in angiogenesis, have recently been cloned (Pharmacia, Uppsala, Sweden). These preparations contained by virtue of their homology to the FMS oncogene, as has been at least 90% myeloblasts as judged by morphological criteria recently reviewed.1 They include FLT1 and KDR/FLK1 which on Papenheim smears. A control group of 15 healthy volun- are the receptors for vascular endothelial growth factor teer bone marrow donors was included. (VEGF).2 A third RTK, FLT3, has been shown to be expressed by leukemia cells as has been reviewed in this journal.3 FLT4, another member of this class of receptors, forms the high Preparation of CD 34-positive cells affinity for VEGF-C.4 The latter represents a novel angiogenic growth factor with to VEGF Mononuclear cells from about 30 ml of bone marrow from and placenta growth factor (P1GF).4 The coding for sev- normal human volunteers were recovered from a Ficoll– eral members of this class of RTKs such as FLT4, KIT, FMS, Hypaque gradient, washed twice and counted. To select for FLT3, PDGFRA have presumably been derived from a com- CD34-positive cells about 108 mononuclear cells were mon ancestor by duplications of immunoglobulin-like applied to the MiniMacs column (Miltenyi Biotech, Bergisch domains.5 FLT4, like other RTKs, has been mapped to the long Gladbach, Germany) according to the directions of the sup- arm of 5.6 plier. Recovered cells were evaluated for purity by FACS Two transcripts of FLT4 with a size of 4.5 kb and 5.8 kb are analysis employing a CD34 monoclonal antibody recognizing expressed in adult and fetal tissues leading to a short and a a different epitope from the one used for the MiniMacs col- long form of the FLT4 .7,8 The long form possesses 65 umn (Anti HPCA-2; Becton Dickinson, San Jose, CA, USA). additional amino acids including three tyrosine residues. One Purity of obtained cells was at least 93% (not shown). of these, tyrosine 1337, seems to be necessary for ligand- dependent transformation and signal transduction to the cyto- 9 plasmatic factors GRB2 and SHC. Source of cell lines Expression of FLT4 has been studied in mouse embryos and in adult human tissues. In early embryos FLT4 can be detected All three cell lines, TF-1, U937 and KG-1a, were kindly pro- vided by W Ostertag, Heinrich-Pette Institute, Hamburg, Ger- many. U937 and KG-1a were cultured in RPMI 1640 sup- Correspondence: W Fiedler, Dept Oncology/Hematology, University plemented with 10% fetal calf serum. TF-1 cells were grown Hospital, Eppendorf, Martinistraße 52, 20246 Hamburg, Germany 2Present address: Medizinische Universita¨tsklinik Knappschaftskrank- in the same medium after addition of 100 ng/ml GM-CSF enhaus, In der Schornau 23-25, D-44892 Bochum/Germany (kindly provided by E Henning, Essex Pharma GmbH, Received 21 December 1996; accepted 3 April 1997 Mu¨nchen, Germany). FLT4 and VEGF-C in AML W Fiedler et al 1235 Preparation of c-DNA and RT-PCR activity was visualized by means of the nickel-glucose oxidase technique.15,16 Controls included: blocking of anti-FLT4 anti- Mononuclear cells were washed twice in PBS and collected body by specific peptide, replacement of primary and second- by centrifugation. Total cellular RNA was prepared using ary antibody with PBS, visualization of peroxidase only, incu- Qiagen minicolumns (Qiagen, Hilden, Germany) as described bation of cells with normal rabbit serum (Sigma, Deisenhofen, by the manufacturer. One microgram of RNA was used for c- Germany) in concentrations ranging from 0.1% to 0.01% DNA synthesis employing avian myeloblastosis virus (AMV) instead of primary antiserum. reverse transcriptase and oligo dT as primer. RT reactions were diluted to 100 ␮l with distilled water and heated to 95°C for 10 min. Results Different aliquots of c-DNA were amplified with specific primers for FLT4, VEGF-C and actin as control for successful FLT4 expression was studied with a sensitive nested RT-PCR c-DNA synthesis. For FLT4 and VEGF-C two rounds and for reaction. The limit of detection is at least one FLT4-positive actin one round of 35 cycles of PCR were performed in a cell in 105 bone marrow cells (data not shown). With this programmable heat block at 94°C for 1.5 min, at 60°C for method c-DNA samples from bone marrow cells of 15 healthy 3 min and at 72°C for 4 min. For the first round 1 ␮l of the donors were investigated. None was positive for FLT4 RT reaction and for the second round of RCR 1 ␮l of a 1:100 expression. To confirm further the lack of FLT4 formation in dilution of the first round was used. Five microliters of PCR normal bone marrow cells, CD34-positive cells were prepared products were separated on 1% agarose gels, stained with by use of a mini MACS column from three additional normal ethidium bromide and visualized under UV light. Primer donors yielding a suspension of CD34-positive cells of at least sequences were as follows: FLT4: outer sense primer 5Ј- 93% purity (not shown). FLT4 specific amplification products CTGCTGGAGGAAAAGTCTGG-3Ј; outer antisense primer 5Ј- were not found in the three samples (Figure 1). CTTGCAGAACTCCACGATCA-3Ј; inner sense primer 5Ј- A series of 53 consecutive patients with AML and the leu- CGTATCTGTGCAGCGTGTG-3Ј; inner antisense primer 5Ј- kemic cell lines KG1a, TF-1 and U937 were investigated for GGTTGACCACGTTGAGGTG-3Ј; VEGF-C: outer sense primer expression of FLT4 by PCR analysis. U937, but not TF-1 and 5Ј-AGCTAAGGAAAGGAGGCTGG-3Ј; outer antisense primer KG1a cells were found to be positive for FLT4 m-RNA. Forty- 5Ј-TGTGTTTTCATCAAATTCTCGG-3Ј; inner sense primer 5Ј- one samples originated from patients with de novo AML at TGAACACCAGCACGAGCTAC-3Ј; inner antisense primer 5Ј- diagnosis or at relapse and 12 samples from patients with CATAAAATCTTCCTGAGCCAGG-3Ј. Actin: sense primer 5Ј- secondary AML. From the 53 AML patients overall 34% CGCTGCGCTGGTCGTCGACA-3Ј; antisense primer 5Ј- expressed FLT4. A representative example of a PCR analysis GTCACGCACGATTTCCCGCT-3Ј. The size of the PCR pro- is demonstrated in Figure 1. Table 1 shows the relative per- ducts corresponds to 625 bp for FLT4 outer primer pair, centages of FLT4 positivity of AML samples according to 500 bp inner primer pair, VEGF-C outer primer pair 764 bp, FAB type. inner primer pair 269 bp and for actin 619 bp. To avoid cross- The cell lines U937 and TF-1 and leukemic blasts from contamination the set-up of PCR reactions and gel electro- eight patients were investigated for FLT4 protein expression phoresis were carried out in different rooms using different sets of pipettes. Appropriate control reactions always remained negative. For each primer pair PCR products were subcloned using the TA cloning (Invitrogen, San Diego, CA, USA). Single bacterial colonies were picked and cultured overnight. Bacteria were diluted in distilled water and boiled for 10 min. PCR reactions were performed with primers recog- nizing sites flanking the PCR product (Dynal A and B; Dynal, Hamburg, Germany). Since one of the primers is biotinylated strand separation was carried out using streptavidin-coated Dynabeads (Dynal). DNA sequencing was performed with the Taq cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) and the automated DNA sequencer 373A (Applied Biosystems). The identified DNA sequences corresponded to the published ones for all primer pairs.

Immunocytochemistry

Cells were transferred from culture into the chamber slides where they were washed twice with PBS for 10 min. After- wards they were fixed for 15 min with fresh paraformaldehyde (4%) at room temperature. They were then washed in PBS and further processed for the visualisation of FLT4. Polyclonal antibodies against FLT4 were purchased from Santa Cruz (Santa Cruz, CA, USA) and used in a 1:1000 dilution. A Figure 1 PCR analysis upper panel: FLT4-specific primers with c- detailed description of the methods used was reported else- DNA from lane 1: normal CD34-positive cells; lane 2: normal bone 13,14 marrow; lanes 3–6: four different patients with AML with FAB types: where. In brief, we used an amplification combination of lane 3: M1; lane 4: M5; lane 5: M5; and lane 6: M3; lane 7: no c- the peroxidase anti-peroxidase (PAP) and the avidin-biotin- DNA; lane 8: 100 bp ladder size marker (large sizes at bottom). Lower peroxidase complex (ABC) techniques. The peroxidase panel: actin-specific primers, lanes same as above. FLT4 and VEGF-C in AML W Fiedler et al 1236 Table 1 Expression of FLT4 or VEGF-C in leukemic blasts from the four cases with positive staining for FLT4, a variable per- AML patients determined by PCR analysis centage ranging from about 20 to 80% positive cells was seen. VEGF-C RNA expression was studied with PCR in leukemic FAB type FLT4 expression VEGF-C expression blasts of 21 AML patients (Figure 3) and the leukemic cell lines positive/total positive/total (%) (%) KG-1a, TF-1 and U937. The only cell line positive for VEGF-C m-RNA was U937 which was also producing FLT4. From 11 M0 0/1 (0) 0/1 (0) patientsexpressingFLT4fourwerealsoproducingtheligand M1 2/7 (28) 0/4 (0) VEGF-C. Four receptor-negative patients expressed the ligand and M2 2/4 (50) ND six did not. These results suggest that ligand and receptor forma- M3 2/3 (67) 1/2 (50) tion is independent of each other in primary AML cells of the M4 4/12 (33) 1/3 (33) majority of patients. On the other hand, AML cells from three of M4 Eo 1/6 (17) 0/4 (0) four patients with FAB M5 subtype expressed VEGF-C indicating M5 5/7 (71) 3/4 (75) M7 0/1 (0) ND a high rate of coexpression of receptor/ligand in this AML subtype. Total 15/41 (37) 5/18 (28) Sec AML 3/12 (33) 3/3 (100) Discussion Total 18/53 (34) 8/21 (38) FLT4 represents a recently cloned receptor for the angiogenic growth factor VEGF-C which has sequence homology to VEGF and P1GF.4,11 In the adult organism FLT4 expression seems to be restricted to a few tissues such as lymph endothelia and with immunocytochemistry using a modified APAP method. HEV.10 On the other hand, FLT4 can be found in a variety of In concordance with the PCR analysis U937 cells were posi- tumor cell lines including the leukemic cell line HEL.12 We tive for FLT4 protein expression. In Figure 2 photomicrographs therefore studied FLT4 expression in normal bone marrow and of immunocytochemistry are shown from U937 cells and from leukemic blasts of 53 patients with AML. Using a sensitive a patient with FAB M5 AML, both positive for FLT4 protein nested PCR approach we were not able to detect FLT4 formation. Of the eight patients investigated four were positive transcripts in mononuclear cells from the bone marrow of 15 and four were negative for FLT4 immunostaining. In six healthy volunteers. Neither was FLT4m-RNA found in cells patients these results were concordant with PCR results. In one enriched in CD34-positive cells from three additional normal patient, PCR was positive and no protein expression was donors. In leukemic samples from 53 consecutive patients detected. In the other patient reverse results were found. In with AML, FLT4 expression was seen in 18 cases, correspond-

Figure 2 Immunocytochemistry: left side staining with a polyclonal antibody against FLT4, right side control reactions after preabsorption of antibody with specific peptide. Upper panel: cell line U937; lower panel: AML patient with M5 AML. U937 and patient’s leukemic cells are positive for FLT4 expression (× 100). FLT4 and VEGF-C in AML W Fiedler et al 1237 Acknowledgements

This work was supported in part by grants from Deutsche For- schungsgemeinschaft Fi389/2-3, Gr 984/3-1 and Roggen- buck Stiftung.

References

1 Rosnet O, Birnbaum D. Hematopoietic receptors of class III recep- tor-type tyrosine kinases. Crit Rev Oncogen 1993; 4: 595–613. 2 Mustonen T, Alitalo K. Endothelial receptor tyrosine kinases involved in angiogenesis. J Cell Biol 1995; 129: 895–898. 3 Drexler HG. Expression of FLT3 receptor and response to FLT3 ligand by leukemia cells. Leukemia 1996; 10: 588–599. 4 Joukov V, Pajusola K, Kaipainen A, Chilov D, Lahtinen I, Kukk E, Saksela O, Kalkkinen N, Alitalo K. A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases. EMBO J 1996; 15: 290–298. 5 Rousset D, Agnes F, Lachaume P, Andre´ C, Galibert F. Molecular evolution of the genes encoding receptor with immunoglobulinlike domains. J Mol Evol 1995; 41: 421–429. 6 Armstrong E, Kastury K, Aprelikova O, Bullrich F, Nezelof C, Gogusev J, Wasmuth JJ, Alitalo K, Morris S, Huebner K. FLT4 mapping to chromosome band 5q35 Figure 3 PCR analysis with VEGF-C specific primers from c-DNA in relation to the t(2;5), t(5;6), and t(3;5) translocations. Genes from lanes 1–6: six representative AML patients with FAB types: lane Chromosom Cancer 1993; 7: 144–151. 1: M5; lane 2: M4; lane 3: M5; lane 4: M4 Eo; lane 5: M4 Eo; lane 7 Borg JP, de Lapeyriere O, Noguchi T, Rottapel R, Dubreuil P, 6: M1; lane 7: no c-DNA; lane 8: 100 bp ladder size marker (large Birnbaum B. Biochemical characterisation of two isoforms of FLT4, a sizes at bottom). Lower panel: actin-specific primers lanes, same as VEGF receptor-related tyrosine kinase. Oncogene 1995; 10: 973–984. above. 8 Pajusola K, Aprelikova O, Armstrong E, Morris S, Alitalo K. Two human FLT4 receptor tyrosine kinase isoforms with distinct car- boxy terminal tails are produced by alternative processing of pri- mary transcripts. Oncogene 1993; 8: 2931–2937. 9 Fournier E, Dubreuil P, Birnbaum D, Borg JP. Mutation at tyrosine residue 1337 abrogates ligand-dependent transforming capacity of FLT4 receptor. Oncogene 1995; 11: 921–931. ing to 34%. FLT4 was detected in all types of myeloid FAB 10 Kaipainen A, Korhonen J, Mustonen T, van Hinsbergh VW, Fang GH, classification with a slight preponderance of M5. Dumont D, Breitman M, Alitalo K. Expression of the fms-like tyrosine VEGF-C was coexpressed in 36% of FLT4-positive AML kinase 4 gene becomes restricted to lymphatic endothelium during patients. Interestingly, in AML FAB type M5 a high rate of coex- development. Proc Natl Acad Sci USA 1995; 92: 3566–3570. pression of FLT4 and VEGF-C was observed. Of the three cell 11 Aprelikova O, Pajusola K, Partanen J, Armstrong E, Alitalo R, Bai- lines studied, only U937 cells showed VEGF-C and FLT4 ley SK, McMahon J, Wasmuth J, Huebner K, Alitalo K. FLT4, a novel class III receptor tyrosine kinase in chromosome 5q33-qter. expression. Since U937 is the only cell line studied with mono- Cancer Res 1992; 52: 746–748. cytic differentiation, FLT4 and VEGF-C coexpression may fre- 12 Pajusola K, Aprelikova O, Korhonen J, Kaipainen A, Pertovaara L, quently occur in monocytic leukemia. Whether autocrine growth Alitalo R, Alitalo K. FLT4 receptor tyrosine kinase contains seven mechanisms for VEGF-C play a role in this subtype remains to be immunoglobulin-like loops and is expressed in multiple human determined. On the other hand, VEGF-C m-RNA was detected tissues and cell lines. Cancer Res 1992; 52: 5738–5743. in 40% of FLT4-negative AML samples, suggesting independent 13 Davidoff MS, Schulze W. Combination of the peroxidase anti- peroxidase (PAP)- and avidin-biotin-peroxidase complex. (ABC) receptor and ligand formation in most cases of AML. techniques: an amplification alternative in immunohistochemical VEGF-C may also exert indirect effects in human AML. In his- staining. Histochemistry 1990; 93: 531–536. tological examinations of bone marrow biopsies from patients 14 Davidoff MS, Middendorf R, Meyer B, Holstein AF. Nitric oxide with AML increased amounts of von Willebrand factor-positive synthase (NOS-I) in Leydig cells of the human testis. Arch Histol endothelial cells were scored as compared to normal bone mar- Cytol 1995; 58: 17–30. row.17 Increased mitotic activity of endothelial cells in bone mar- 15 Itoh Z, Akiva K, Namura S, Miguno N, NAkamura Y, Sugimoto T. Application of coupled oxidation reaction to electron microscopic row stroma may be induced by factors secreted by leukemic demonstration of horseradish peroxidase: cobalt-glucose oxidase cells. In multiple myeloma, increased microvessel density in method. Brain Res 1979; 175: 341–346. bone marrow biopsies was significantly correlated with labelling 16 Za´borszky L, Le´ ra´nth C. Simultaneous ultrastructural demonstration index and prognosis.18 Angiogenic factors such as VEGF-C may of retrogradlly transported horseradish peroxidase and choline acetyl- possibly influence proliferation of endothelial cells in bone mar- immunoreactivity. Histochemistry 1985; 82: 529–537. row. FLT4 m-RNA was detected in human umbilical vein endo- 17 Dilly SA, Jagger CJ. Bone marrow stromal cell changes in haema- tological malignancies. J Clin Pathol 1990; 43: 942–946. thelial cells (HUVEC) and stroma cells from long-term bone mar- 18 Vacca A, Ribatti D, Roncalli L, Ranieri G, Serio G, Silvestris F, row cultures treated with mycophenolic acid to eliminate Dammacco F. Bone marrow angiogenesis and progression in mul- hematopoietic cells (data not shown). Paracrine provision of tiple myeloma. Br J Haematol 1994; 87: 503–508. hematopoietic growth factors such as GM- and G-CSF by endo- 19 Oster W, Mertelsmann R, Herrmann F. Role of colony-stimulating thelial cells induced by secretion of IL-1, TNF-␣ and possibly factors in the biology of acute myelogenous leukemia. Int J Cell VEGF-C by leukemic blasts may contribute to the malignant pro- Clon 1989; 7: 13–29. 19,20 20 Griffin JD, Rambaldi A, Vellenga E, Young DC, Ostapovicz D, cess. Investigations about possible autocrine or paracrine Cannistra SA. Secretion of interleukin-1 by acute myeloblastic leu- growth promoting actions of the VEGF-C/FLT4 ligand receptor kemia cells in vitro induces endothelial cells to secrete colony system are being actively pursued in our laboratory. stimulating factors. Blood 1987; 70: 1218–1221. FLT4 and VEGF-C in AML W Fiedler et al 1238