[CANCER RESEARCH 62, 3331–3334, June 15, 2002] Advances in Brief 3-Deoxy-3-[ 18 F]Fluorothymidine-Positron Emission Tomography for Noninvasive Assessment of Proliferation in Pulmonary Nodules Andreas K. Buck, 1 Holger Schirrmeister, Martin Hetzel, Mareike von der Heide, Gisela Halter, Gerhard Glatting, Torsten Mattfeldt, Florian Liewald, Sven N. Reske, and Bernd Neumaier Departments of Nuclear Medicine [A. K. B., H. S., M. v. d. H., G. G., S. N. R., B. N.], Internal Medicine II [M. H.], Thoracic Surgery [G. H., F. L.], and Pathology [T. M.], Universit of Ulm, D-89081 Ulm, Germany Abstract We investigated whether uptake of the thymidine analogue 3-deoxy-3- [ 18 F]fluorothymidine ([ 18 F]FLT) reflects proliferation in solitary pulmo- nary nodules (SPNs). Thirty patients with SPNs were prospectively exam- ined with positron emission tomography. Standardized uptake values were calculated for quantification of FLT uptake. Histopathology revealed 22 malignant and 8 benign lesions. Proliferation was evaluated by Ki-67 immunostaining and showed a mean proliferation fraction of 30.9% (range, 1– 65%) in malignant SPNs and <5% in benign lesions. Linear regression analysis indicated a significant correlation between FLT- standardized uptake values and proliferative activity (P < 0.0001; r  0.87). FLT uptake was specific for malignant lesions and may be used for differential diagnosis of SPNs, assessment of proliferation, and esti- mation of prognosis. Introduction Evaluation of SPNs 2 remains one of the most common diagnostic problems in daily clinical practice (1). Only half of the resected lesions are related to malignant disease (1), and many of the nonma- lignant lesions represent inflammatory processes (2). PET using the glucose analogue FDG enables noninvasive differentiation between benign and malignant lesions because glucose consumption is ele- vated in malignant tumors (3). FDG uptake, however, is not specific for malignancies because false positive findings were also reported in inflammation, muscle activity, or sarcoidosis (2). A study in patients with elevated FDG uptake in pancreatic tumors indicated that deter- mination of the proliferation rate clearly differentiated cancer from inflammation (4). Recently, Shields et al. (5) developed the new PET tracer FLT for noninvasive measurement of tumor proliferation. This thymidine analogue was reported to accumulate in proliferating cells after intracellular trapping because of phosphorylation by thymidine kinase 1 (6). This first study in humans was conducted to evaluate the correlation between [ 18 F]FLT uptake and the proliferation rate in SPNs. Materials and Methods Patients. This prospective study consisted of 30 patients (20 men and 10 women) with a mean age of 61.9  6.8 years (range, 37– 88 years). Patients were included when pulmonary nodules on CT with and without contrast enhancement were suspicious for a malignant tumor. None of the patients had prior surgery, chemotherapy, or radiation therapy. Nineteen patients had re- sective surgery up to 10 days after FLT-PET. Core biopsy specimens were used to evaluate the proliferation in the other 11 patients. All patients gave written consent to participate in this study, which was approved by the local ethical committee. Histopathology. Histopathological examination of the resected specimens revealed 22 malignant tumors [16 NSCLC, 1 small cell lung cancer, 1 non- Hodgkin’s lymphoma, and pulmonary metastases in 4 patients (1 colorectal cancer, 2 renal cell carcinoma, and 1 osteosarcoma)] and 8 benign tumors (1 bronchopulmonary chondroma, 3 bronchiolitis, 1 tuberculoma, 1 focal fibrosis, and 2 undefined tumors; malignancy excluded by clinical course). Immunostaining. A standard peroxidase-conjugated streptavidin-biotin complex method was used (DAKO Diagnostika, Hamburg, Germany), and 3,3'-diaminobenzidine (Sigma-Aldrich, Deisenhofen, Germany) served as chromogen (4). Briefly, formalin-fixed, paraffin-embedded sections (5 m) of resected specimens were dewaxed, rehydrated, and then microwaved in 0.01 M citrate buffer for 30 min. For immunostaining, MIB-1 antibody (Dianova, Hamburg, Germany), a monoclonal murine antibody specific for human nu- clear antigen Ki-67, was used as the primary antibody in a 1:500 dilution. Sections were lightly counterstained with hematoxylin. The primary antibody was omitted on sections used as negative controls. Sections obtained from highly proliferating lymph node tissue of a reference patient who was not included in our series served as a positive control for proliferating cells. An evaluation of MIB-1 immunostaining was carried out in an area with high cellularity. All epithelial cells with nuclear staining of any intensity were defined as positive. Proliferative activity was described as the percentage of MIB-1-stained nuclei. Morphometry. Histopathological slides were scored by a pathologist ex- perienced in this field who was blinded to the patients’ clinical data. The fraction of stained nucleus profile per total number of nucleus profiles was estimated by counting 600 nuclei/slide and three slides/case. For this purpose, the computer assisted imaging system Optimas 6.2 (Media Cybernetics, Inc., Silver Spring, MD) was used. Slides were analyzed by light microscopy, and three representative images of each slide were transferred to the computer frame by a video camera. [ 18 F]FLT Synthesis. [ 18 F]FLT was produced via benzoyl-protected anhy- drothymidine according to the method reported by Machulla (7). Radiosyn- thesis was carried out remotely and automatically in a PET tracer synthesizer from Nuclear Interface (Mu ¨nster, Germany). [ 18 F]Fluoride was produced via the 18 O(p,n) 18 F nuclear reaction by bombardment of isotopically enriched [ 18 O]water with an 18 MeV proton beam at the Cyclone 18/9 cyclotron (IBA, Louvaine-la-Neuve, Belgium). After recovery of [ 18 O]water using a QMA cartridge (Waters, Milford, MA), [ 18 F]fluoride was eluted with 360 l of K 2 CO 3 solution (3.3 mg K 2 CO 3 ). Twenty mg of Kryptofix 2.2.2 (Merck, Darmstadt, Germany) in 0.7 ml of acetonitrile was added, and the cryptate of [ 18 F]fluoride and potassium carbonate was evaporated to dryness. The evap- oration step was repeated with 1 ml of acetonitrile to remove any traces of moisture. Then a solution of 10 mg of 5'-benzoyl-3',2-anhydrothymidine in 1 ml of DMSO was added to the dry cryptate, and the resulting solution kept at 160°C for 10 min. Removal of the benzoyl-protecting group was achieved by hydrolysis with 1% 350-l1 M NaOH and heating it to 55°C for 10 min. The hydrolysate was transferred through an Alumina N (Waters) to retard unre- acted [ 18 F]fluoride. Subsequently, [ 18 F]FLT was purified using preparative high-performance liquid chromatography. For the separation of [ 18 F]FLT, a C-18 column (Phenomenex; Luna 5 , 250  10 mm) was used and eluted with isotonic sodium chloride solution and ethanol (90/10, v/v) at a flow rate Received 2/15/02; accepted 5/1/02. 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 Universit of Ulm, Robert- Koch-Strasse 8, D-89081 Ulm, Germany. Phone: 0731-500-24504; Fax: 0731-500-24503; E-mail: andreas.buck@medizin.uni-ulm.de. 2 The abbreviations used are: SPN, solitary pulmonary nodule; PET, positron emission tomography; FDG, 2-[ 18 F]fluoro-2-deoxy-D-glucose; FLT, 3-deoxy-3'-fluorothymidine; NSCLC, non-small cell lung cancer; SUV, standardized uptake value; CT, computed tomography. 3331 Research. on October 5, 2021. © 2002 American Association for Cancer cancerres.aacrjournals.org Downloaded from