Detection of Apoptotic Tumor Response In Vivo After a Single Dose of Chemotherapy with 99mTc-Annexin V

Annexin V, a human protein with a high affinity for phosphatidylserine, has been labeled with 99m Tc to detect apoptosis in vivo. To determine the effectiveness of imaging with this agent as a reflection of the degree of apoptosis after the first dose of chemotherapy, we studied rats with an engrafted hepatoma. Methods: Annexin V was labeled with 99m Tc (specific activity, 3.0 MBq/μg protein). Eleven days after being inoculated with allogenic hepatoma cells (KDH-8) in the left calf muscle, the rats were randomized to receive a single dose of cyclophosphamide (150 mg/kg intraperitoneally) or to serve as controls. 99m Tc-annexin V was injected 20 h later. Radioactivity in tissues was determined 6 h after injection of 99m Tc-annexin V. Tumor uptake of 14 C-iodoanitpyrine was determined as a marker of tumor blood flow. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) of tissue harvested at necropsy was performed to detect apoptosis in the tumor. Results: Cyclophosphamide treatment significantly increased the tumor uptake (percentage activity of injected dose per gram of tissue after normalization to the animal's weight [%ID/g/kg]) of 99m Tc-annexin V (0.070 ± 0.007 %ID/g/kg for treated rats and 0.046 ± 0.009 %ID/g/kg for controls, P < 0.001). 14 C-iodoantipyrine uptake was similar in the treated and untreated groups. The number of TUNEL-positive cells in the tumor was significantly larger in the treated rats (297.70 ± 50.34 cells/mm 2 ) than in the control rats (168.45 ± 23.60 cells/mm 2 , P < 0.001). Tumor uptake of 99m Tc-annexin V correlated with the number of TUNEL-positive cells in the tumor (r = 0.712; P < 0.001). Conclusion: Tumor uptake of 99m Tc-annexin V was significantly increased by a single dose of cyclophosphamide treatment, and the increase was concordant with the number of TUNEL-positive cells in the tumor. The current results are suggestive of the utility of 99m Tc-annexin V as a noninvasive means to assess tumor response, although further testing, including clinical evaluation, is required.