ORIGINAL ARTICLE 
Annals of Nuclear Medicine Vol 14. No 5, 323-328, 2000 

Evaluation of 62Cu labeled diacetyl-bis(N4-methylthiosemicarbazone) as a hypoxic tissue tracer in patients with lung cancer 

Norio TAKAHASHI,* Yasuhisa FUJIBAYASHl,**. Yoshiharu YONEKURA,**
 Michael J. WELCH,*** Atsuo WAKI,** Tatsuro TSUCHIDA,*
 Norihiro SADATO,** Katsuya SUGlMOTO* and Harumi ITOH* 

*Department of Radiology and **Biomedical Imaging Research Center, Fukui Medical University, Fukui, Japan
 ***Mallinckrodt Institute of Radiology Washington University School of Medicine, 
St. Louis, MO, USA 
62Cu labeled diacetyl-bis(N4-methylthiosemicarbazone) (62Cu-ATSM) has been proposed as a generator-produced, positron-emitting tracer for hypoxic tissue imaging. From basic studies, the retention mechanism of 62CU-ATSM is considered to be closely related to cytosolic/microsomal bioreduction, a possible system for hypoxic bioreductive drug activation, In order to evaluate the characteristics of 62Cu-ATSM, PET studies were performed in 4 normal subjects and 6 patients with lung cancer. 62Cu-ATSM cleared rapidly from the blood with little lung uptake (0.43 +- 0.09, uptake ratio; divided by the arterial input function) in normal subjects. Intense tumor uptake of 62Cu-ATSM was observed in all patients with lung cancer (3.00 +- 1.50). A negative correlation was observed between blood now and now-normalized 62Cu-ATSM uptake in three of four patients. In contrast, 62Cu-ATSM uptake was not related to that of 18F-fluorodeoxyglucose. The negative correlation between blood flow and flow normalized 62Cu-ATSM uptake suggests an enhancement of retention of 62Cu-ATSM by low now 62Cu-ATSM is a promising PET tracer for tumor imaging, which might bring new information for chemotherapeutic treatment as well as radiotherapy of hypoxic tumors.
 
Key words: 62Cu-ATSM, hypoxia, lung cancer 18F-FDG, PET 

INTRODUCTION 
HYPOXIA in tumors may be an important factor in resistance to radiotherapy as well as chemotherapy.1,2 Nitroimidazole compounds are of great interest because they are reduced enzymatically and trapped in regions of low oxygen tension.3 Based on these considerations, various groups have attempted to design nitroimidazole-based drugs labeled with 18F,4 123I,5 or 99mTc6 for imaging hypoxia, but these drugs had low target accumulation due to slow blood clearance and low membrane permeability.7
 62Cu labeled diacetyl-bis(N4-methylthiosemicarbazone) (62Cu-ATSM) has been proposed as a generator-pro

Received March 2, 2000, revision accepted May 26, 2000.
 For reprint contact: Tatsuro Tsuchida, M.D., Department of Radiology, Fukui Medical University. Shimoaizuki, Matsuoka-cho, Yoshida-gun, Fukui 910-1137. JAPAN.
 E-mail: tsucchy@fmsrsa.fukui-med.ac,jp
 
duced, positron-emitting tracer for imaging hypoxia.8 62Cu-PTSM, developed as a perfusion tracer, is easily reduced by the electron transport system of mitochondria, which explains its retention.9 On the other hand, 62Cu-ATSM, an analogue of 62Cu-PTSM, cannot be reduced by normal mitochondria due to its low redox potential. As a result, although it has high membrane permeability, it is not retained in normal brain and heart tissue, but accumulates in hypoxic tissue where it is more easily reduced.10 Therefore, 62Cu-ATSM is a better candidate for a hypoxia imaging agent than nitroimidazole-based drugs because of its higher membrane permeability. It has been reported that 64Cu-ATSM has been selectively trapped in vitro in EMT6 cells under hypoxic conditions and in vivo in solid EMT6 tumors.11 To evaluate the characteristics of 62Cu-ATSM in humans, PET studies were performed on 4 normal subjects and 6 patients with lung cancer. 

MATERIALS AND METHODS 
Preparation of 62Cu-ATSM 
62Cu was obtained with a 62Zn/62Cu generator system from [62Zn]ZnCl2 solution.12 Cu-ATSM was synthesized according to the method of Gingas et al., 13 and confirmed by elemental analysis and mass spectrometry. 62CU-ATSM was prepared as follows8: Briefly, four m/of 62Cu-glycine (non-carrier added 62Cu) solution obtained from the generator was mixed with 0.2 m/of ATSM solution (0.4 mM in dimethyl sulfoxide). The radiochemical purity of 62Cu-ATSM was confirmed with HPLC in combination with authentic Cu-ATSM. 
Subjects
The study involved 4 normal male volunteers (ages 34-64 yrs) and 6 patients with lung cancer (4 males and 2 females, ages 49-87 yrs). Altogether there were 2 adenocarcinomas, 3 squamous cell carcinomas and I metastasis from breast cancer (Table 1). All the patients were investigated with both 62Cu ATSM and 18F-fluorodeoxyglucose. Four of the 6 patients were also evaluated by 15O-water. The study was approved by the Ethical Committee of Fukui Medical University and written informed consent was obtained from all the subjects befbre the PET study. 
PET
PET was performed with a high-resolution, whole-body PET scanner with an 18-ring detector arrangement (Advance, GE Medical Systems, Milwaukee). The physical characteristics of this scanner have been described in detail by DeGrado et al.14 Briefly, the system permits the simultaneous acquisition of 35 transaxial images with an interslice spacing of 4.25 mm. Both axial and transaxial resolution are 4.2 mm, allowing multidirectional reconstruction of the images without loss of resolution. The FOV and the pixel size of the reconstructed images were 256 and 2 mm, respectively. A1O-min transmission scan was acquired with a 68Ge/68Ga source for attenuation correction, followed by intravenous injection of 370 to 740 MBq of 62Cu-ATSM over 30 sec. PET data acquisition was started at the time of 62Cu-ATSM injection and continued for 20 minutes in 10-sec frames for the first 120 sec, 60-sec frames for the next 8 min and a final l0-min frame. In order to evaluate the side effects of 62Cu-ATSM, physical examinations and hematological and biochemical data analysis were performed before and after administration of 62Cu-ATSM.
 To compare 62CU-ATSM images with blood flow and glucose metabolism of tumors 15O-water and FDG PET was performed within one week. After approximately 1110 MBq of 15O-water was injected intravenously, a serial dynamic PET scan was performed for 120 sec in 4 of 6 patients. In all 6 patients, approximately 370 MBq of FDG was injected intravenously after a 4-hr fast. A static scan was performed for 20 min (40-6O min postinjection). 
Data Analysis
In the normal volunteer study, circular regions of interest (ROIs) were placed on the dynamic 62Cu-ATSM PET images of the lung and left atrium (22.9 cm2 and 1 .8 cm2 respectively in area). The last frame, which was obtained 10-20 min after the injection, was used as a static image and the lung activity of 62Cu-ATSM was normalized by the arterial blood activity, which was derived from the ROI placed over the left atrium of the PET image (uptake ratio). The other circular ROls were placed on the static image of the left myocardium and liver (0.6 cm2 and 22.9 cm2 respectively in area) and the uptake ratio of those organs was obtained.
 In patients with lung cancer, multiple circular ROls (> 0.6 cm2 in area) were placed over the 62Cu-ATSM PET images of tumors, and the uptake ratio was calculated as described above. The blood flow images were calculated from 15O-water PET data in each subject by an autoradiographic method.15-17 The arterial input function was derived from the radioactivity in the left atrium by using the circular 1.8 cm2-ROI in area, instead of arterial blood sampling. The standardized uptake value (SUV) images of FDG were calculated with the following formula:
 
SUV = radioactivity concentration (Bq/ml) / { injected dose (Bq) / body weight (g) } 

The same ROls as used in the 62Cu-ATSM PET images of tumors were placed on both the blood now and SUV images, and the uptake ratio of 62CU-ATSM was compared with the blood now and SUV of FDG in each patient. In addition, the uptake ratio was normalized by its absolute blood flow value. The fiow-normalized uptake ratio was compared with the blood flow. 

RESULTS 
62Cu-ATSM rapidly cleared from the blood, reaching a stable activity level a few minutes after the Injection (Fig. 1). Little uptake was observed in the lung (uptake ratio: 0.43 +- 0.09). The left myocardial uptake was small (1.84 +- 0.35), but the liver uptake was considerable (2.45 +- 1.03). No side effects were observed in any of the four subjects.
 62Cu-ATSM rapidly accumulates in tumors, reaching plateau levels within a few minutes after the injection (Fig. 2). An abnormally intense uptake of 62Cu-ATSM was observed in all patients with lung cancer (uptake ratio: 3.00 +- 1.50), but the distribution pattern of 62Cu-ATSM is different from that of FDG or blood flow (Fig. 3). No correlation was observed between 62Cu-ATSM and the blood flow pattern except in one patient (Fig. 4). No correlation between 62Cu-ATSM and FDG was found (Fig. 5). In three of four patients, a negative correlation was observed between blood now and the flow-normalized 62Cu-ATSM uptake ratio (Pig. 6)
 
DISCUSSION 
62Cu-ATSM was rapidly cleared from the blood with little lung uptake In normal subjects. As normal myocardial uptake was small. 62Cu-ATSM also can be used for the evaluation of myocardial hypoxia In patients with ischemlc heart disease. The Intense liver uptake was expected because It was reported that 62cu-ATSM was cleared through the liver and kidneys and the liver uptake was the highest among the all organs In mice after 5 min. 11
 Intense tumor uptake of 62Cu-ATSM was observed in all six patients with lung cancer. The 62Cu-ATSM uptake did not correlate with that of FDG. This finding suggests that 62Cu-ATSM uptake may represent characteristics of tumors independent of those represented by FDG uptake. A negative correlation between blood flow and flow-normalized 62Cu-ATSM uptake suggests increased retention of 62Cu-ATSM in low flow areas, but other factors may affect the tumor retention of 62Cu-ATSM since the slope of the correlation differed among subjects. From the results of in vitro studies of our group with cultured tumor cells, a reduction in 62Cu-ATSM was shown to be closely related to cytosolic/microsomal bioreduction and was enhanced by hypoxia.18 This is a possible system for hypoxic, bioreductive drug activation. Higher tumor uptake of 62Cu-ATSM may reflect a higher sensitivity to bioreductive drugs than that to irradiation. because the reduction In 62Cu-ATSM is closely related to bioreductive drug activation, which is enhanced by hypoxic conditions.18 Accordingly, it may be posslble to determine more effective therapies with 62Cu-ATSM PET before treatment.
 Although we have not compared 62Cu-ATSM and 18F-fluoromisonidazole (18F-FMISO) in this study. 62Cu-ATSM has three advantages. First, 62Cu can be obtained by a generator system from 62Zn, which has a 9 hr half-life and could be delivered long distance. The second advantage is that the faster tumor uptake of 62Cu-ATSM than of 18F-FMISO allows more rapid imaging of tumors. In 18F-FMISO PET, the difference between normal and hypoxic tissues does not become clear until 2 hours post injection due to slow blood clearance and the low tumor to soft tissue ratio.7 62Cu-ATSM PET imaging can be done within 20 min after injection due to its high membrane permeability,10 so that the more efficient uptake and washout kinetics of 62Cu-ATSM in lung tumors in comparison with 18F-FMISO offers the possibility of a faster and more efficient means of evaluating of tumor hypoxia by PET imaging. The third advantage is that the uptake of 62Cu-ATSM may be higher than that of 18F-FMISO. The mean tumor uptake ratio of 62Cu-ATSM was 3.00 and maximum uptake was 9.33. In contrast, the maximum tumor/plasma 18F-FMISO ratio was reported to be from 0.9 to 1.5 among 3 patients, although one patient's was 2.3.7 It was reported that with 18F-FMISO, binding to EMT6 cells starts at a higher oxygen concentrations than with 64Cu-ATSM and the percentage uptake of 18F-FMISO is also much lower than that of 64Cu-ATSM after a longer incubation time.11
 There are some limitations in this study. Hypoxia in the tumor tissue was not confirmed directly in this study, but electrode measurement of intratumoral pO2 in patients with lung cancer is highly invasive and technically demanding and it is impossible to analyze hypoxia in a number of segments of tumor, because of an increasing risk of pneumothorax. Increased myocardial retention of 62Cu-ATSM under hypoxic conditions also has been reported in perfused rat hearts.8 It was also proved that hypoxic retention of 62Cu-ATSM is a reversible phenomenon and is dependent only upon pO2 and not upon irreversible cellular damage such as membrane disruption. 8 In the in vitro study with the EMT6 carcinoma cell line, the uptake of 64Cu-ATSM was relatcd in a sigmoidal fashion to the pO2 of the media where retention was greatly increased under hypoxic and anoxic conditions I l Enhanced retention of 6lCu-ATSM by low now in this study may indicate that the intense uptake of 62Cu-ATSM reflects a hypoxic condition. Another limitation is that the number of patients was small and the results are preliminary, but intense tumor uptake of 6lCu-ATSM was observed in all six patients with lung cancer and imaging was completed only 20 minutes after injection. Higher tumor uptake of 62Cu-ATSM may reflect higher sensitivity to bioreductive drugs than to irradiation, because the retention mechanism of 62Cu-ATSM is closely related to the bioreductive drug activation, which is enhanced by hypoxic conditions 18 Accordingly, it may be possible to determine a more effective therapy with 62Cu-ATSM PET before treatment. Appropriate clinical trials are necessary to clarify this question.
 In conclusion, this preliminary study suggests that 62Cu-ATSM is a promising PET tracer for tumor imaging, which may provide new information on radiotherapy as well as chemotherapy of hypoxic tumors. 

ACKNOWLEDGMENT 
We thank Nihon Medi-Physics Co Ltd., Japan, for supplying 62Zn

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