[CANCER RESEARCH 63, 2681–2687, May 15, 2003] 3-[ 18 F]Fluoro-3-Deoxythymidine ([ 18 F]-FLT) as Positron Emission Tomography Tracer for Imaging Proliferation in a Murine B-Cell Lymphoma Model and in the Human Disease Martin Wagner, 2 Ulrike Seitz, 1,2 Andreas Buck, Bernd Neumaier, Stefan Schultheiß, Markus Bangerter, Martin Bommer, Frank Leitha ¨user, Edgar Wawra, Gerd Munzert, and Sven N. Reske Departments of Nuclear Medicine [U. S., A. B., B. N., S. S., S. N. R], Internal Medicine I [M. W., M. Ba.], Internal Medicine III [M. Bo., G. M.], Pathology [F. L.], University of Ulm, 89081 Ulm, Germany and Vienna Biocenter, Institute of Molecular Biology, University of Vienna, A-1030 Vienna [E. W.] ABSTRACT Here we describe the evaluation of 3-[ 18 F]fluoro-3-deoxythymidine {[ 18 F]-FLT} as a tracer for positron emission tomography (PET) in a murine model of B-cell lymphoma and in human malignant lymphoma. The human B-cell line DoHH2 expressed high levels of active thymidine kinase 1 (TK-1) as the key enzyme of [ 18 F]-FLT metabolism. Immuno- staining confirmed high levels of TK-1 in DoHH2 derived xenograft tumors in SCID/SCID mice. In vitro studies demonstrated a time-depen- dent uptake of [ 18 F]-FLT, an efficient phosphorylation to the respective monophosphate and the incorporation of [ 18 F]-FLT into the perchloric acid insoluble fraction in DoHH2 cells, indicating the incorporation of this tracer into the DNA. After incubation with [ 18 F]FLT for 240 min, 12.5%  1.0% of radioactivity applied to the medium was intracellularly trapped in DoHH2 cells. Specific accumulation of [ 18 F]-FLT in the ma- lignant cell clone was confirmed in biodistribution studies in SCID/SCID mice bearing DoHH2-derived tumors. The percentage of injected dose of [ 18 F]-FLT per gram of tumor tissue correlated with the tumor-prolifera- tion index as evaluated in BrdUrd-labeling experiments. In a pilot study of 11 patients with both indolent and aggressive lymphoma, [ 18 F]-FLT was suitable and comparable to [ 18 F]-FDG in the ability to detect malignant lesions by PET scan. Furthermore, we found a close correlation (r  0.95, P < 0.005) of the [ 18 F]-FLT standardized uptake values with the Ki67- labeling index of tissue biopsies (n  10) in these patients. These results suggest that [ 18 F]-FLT represents a novel tracer for PET that enables imaging of proliferation in human lymphoma in vivo. INTRODUCTION PET 3 represents an analytical technology that uses compounds labeled with positron emitting radioisotopes as molecular probes to image and measure biochemical processes in vivo. Currently, FDG is the most widely used PET tracer in clinical oncology. Viable tumor cells display an increased intracellular influx of the glucose analogue FDG via overexpression of glucose transporters and a higher rate of intracellular phosphorylation through the hexokinase pathway which results in an intracellular trapping of FDG-6-phosphate in cancer cells (1–3). The strength of FDG-PET in diagnosis and staging of malignant lymphoma has been evaluated and compared with conventional stag- ing methods as CT, ultrasonography, or magnetic resonance tomog- raphy in several studies (4 – 6). FDG-PET has been shown to be more sensitive in the detection of nodal (6), extranodal (7), and bone marrow (5, 8) involvement as compared with CT scans. The lack of FDG uptake in residual masses after completion of therapy is asso- ciated with a high probability of long term disease free survival (9). However, the diagnostic accuracy of FDG-PET in the evaluation of indolent malignant lymphoma remains under discussion (10). In this regard, a quantitative estimate of the proliferative activity of a tumor obtained through noninvasive imaging would aid in selecting sides for biopsy, optimal treatments for patients and could provide a more accurate assessment of the response to therapy. Pyrimidine nucleosides and several of their analogues are phospho- rylated to the respective monophosphate (MP) by thymidine kinase 1 (TK-1) and are incorporated into DNA, partly acting as chain termi- nators. More than 40 years after its invention, [ 3 H]thymidine remains the gold standard to measure cell proliferation in vivo. Positron- emitting radionuclides enable in vivo imaging of deregulated prolif- eration with PET as one of the key features of malignant disease. [ 11 C]-labeled thymidine has been proposed as a radio tracer for imaging tumor proliferation by several groups (11, 12). Furthermore, we have recently shown that uptake of [ 18 F]fluorodeoxyuridine in vivo correlates with the proliferation in a murine pancreatic cancer model (13). However, the metabolic instability of both tracers repre- sents the major drawback for routine clinical applications. As a more recent development, 3'-[ 18 F]fluoro-3'-deoxythymidine {[ 18 F]FLT} was used with highly promising results (14 –16). 3'-fluoro-3'- thymidine (FLT) has been identified as a nucleoside analogue with antiretrovirus activity (17). FLT permeates the cell membrane by a carrier-mediated mechanism, as well as by facilitated diffusion (18, 19), and is phosphorylated to 3'-fluorothymidine MP (FLT-MP) through the S-phase specific enzyme TK-1 (see Ref. 17; for review, see 20). This phosphorylation results in intracellular trapping of FLT-MP. High levels of TK1 activity have been reported for rapidly growing cells and tumor tissues (21–23). Here we report high levels of active TK-1 in DoHH2 cells (24, 25) and in DoHH2 cells derived xenograft tumors in SCID/SCID mice as a murine model of human B-cell lymphoma (26). In this model and in a pilot study of human malignant lymphoma, we have evaluated [ 18 F]FLT as a PET tracer for imaging proliferation in vivo. MATERIALS AND METHODS Cell Culture and SCID/SCID Lymphoma Model. Human non- Hodgkin’s B-cell lymphoma cell line DoHH2 (24, 25) and EBV-transformed human lymphocytes were maintained in RPMI 1640 medium supplemented with 10% FCS (Life Technologies, Inc., Berlin, Germany). The fibroblast cell line HT1080 (ATTC, Washington, D.C.) was grown to confluence to induce growth arrest through contact inhibition as described. According to Cotter et al. (26), tumor cells (10 6 cells in 100 l) were administered by s.c. route to CB-17 SCID/SCID mice weighing 30 –35 g. At 3– 4 weeks posttransplantation, the mice developed lymphomas weighing 2.0 g. Quantitative RNA Analysis. The ABI Prism 7700 Sequence Detection System (Perkin-Elmer Applied Biosystems, Foster City, CA) was used for real-time monitoring of PCR amplification of the c-DNA, and the target c-DNA was quantified using the delta-delta-CT method as described (27). The Received 8/14/02; accepted 3/13/03. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom requests for reprints should be addressed, at Department of Nuclear Medicine, University of Ulm, Robert-Koch-Str. 8, D-89081 Ulm, Germany. Phone: 49-731-500-33864; Fax: 49-731-500-24979; E-mail: ulrike.seitz@medizin.uni-ulm.de. 2 Both authors contributed equally to this work. 3 The abbreviations used are: PET, Positron emission tomography; FDG, 2-deoxy-2- [ 18 F]fluoro-D-glucose; CT, computed tomography; MRI, magnetic resonance tomography; TK, thymidine kinase; [ 18 F]FLT, 3'-[ 18 F]fluoro-3'-deoxythymidine; FLT, 3'-fluoro-3'- thymidine; MP, monophosphate; BrdUrd, bromodeoxyuridine; FP, forward primer; RP, reverse primer; high-performance liquid chromatography; ID, injected dose; SUV, standardized uptake value. 2681 Research. on January 5, 2016. © 2003 American Association for Cancer cancerres.aacrjournals.org Downloaded from