DNA Double Strand Breaks as Predictor of Efficacy of the Alpha-Particle Emitter Ac-225 and the Electron Emitter Lu-177 for Somatostatin Receptor Targeted Radiotherapy

TLDR

γH2AX‑foci formation, induced by beta and alpha irradiation, is an early key parameter for predicting response to internal radiotherapy. The study compares the biologic effects of Ac‑225‑DOTATOC and Lu‑177‑DOTATOC to assess the predictive value of γH2AX‑foci formation. The authors incubated AR42J cells with Ac‑225‑DOTATOC and Lu‑177‑DOTATOC up to 48 h, quantified DNA double‑strand breaks by γH2AX immunofluorescence, assessed cell‑cycle arrest by flow cytometry, and measured tumor uptake and growth in subcutaneous AR42J xenografts. Ac‑225‑DOTATOC achieved a 700‑fold lower ED50 (14 kBq/ml) than Lu‑177‑DOTATOC (10 MBq/ml), induced more DNA double‑strand breaks, caused a 60 % G2/M arrest at 24 h, produced an apoptotic sub‑G1 peak at 72 h, and led to higher γH2AX‑foci in tumors despite identical uptake, resulting in delayed tumor growth (25 days vs 21 days).

Abstract

Key biologic effects of the alpha-particle emitter Actinium-225 in comparison to the beta-particle emitter Lutetium-177 labeled somatostatin-analogue DOTATOC in vitro and in vivo were studied to evaluate the significance of γH2AX-foci formation.To determine the relative biological effectiveness (RBE) between the two isotopes (as - biological consequence of different ionisation-densities along a particle-track), somatostatin expressing AR42J cells were incubated with Ac-225-DOTATOC and Lu-177-DOTATOC up to 48 h and viability was analyzed using the MTT assay. DNA double strand breaks (DSB) were quantified by immunofluorescence staining of γH2AX-foci. Cell cycle was analyzed by flow cytometry. In vivo uptake of both radiolabeled somatostatin-analogues into subcutaneously growing AR42J tumors and the number of cells displaying γH2AX-foci were measured. Therapeutic efficacy was assayed by monitoring tumor growth after treatment with activities estimated from in vitro cytotoxicity.Ac-225-DOTATOC resulted in ED50 values of 14 kBq/ml after 48 h, whereas Lu-177-DOTATOC displayed ED50 values of 10 MBq/ml. The number of DSB grew with increasing concentration of Ac-225-DOTATOC and similarly with Lu-177-DOTATOC when applying a factor of 700-fold higher activity compared to Ac-225. Already 24 h after incubation with 2.5-10 kBq/ml, Ac-225-DOTATOC cell-cycle studies showed up to a 60% increase in the percentage of tumor cells in G2/M phase. After 72 h an apoptotic subG1 peak was also detectable. Tumor uptake for both radio peptides at 48 h was identical (7.5%ID/g), though the overall number of cells with γH2AX-foci was higher in tumors treated with 48 kBq Ac-225-DOTATOC compared to tumors treated with 30 MBq Lu-177-DOTATOC (35% vs. 21%). Tumors with a volume of 0.34 ml reached delayed exponential tumor growth after 25 days (44 kBq Ac-225-DOTATOC) and after 21 days (34 MBq Lu-177-DOTATOC).γH2AX-foci formation, triggered by beta- and alpha-irradiation, is an early key parameter in predicting response to internal radiotherapy.