Ganciclovir-mediated in vivo elimination of myeloid leukemic cells expressing the HSVtk gene induces HSVtk loss variants

The in vivo elimination of suicide gene-expressing tumor cells with prodrug treatment can induce protective immunity against wild-type tumors. In this study, we determined the efficacy and safety of the in vivo elimination of HSVtk expressing cells with ganciclovir treatment of a bystander cell killing-insensitive leukemic cell line. The retroviral construct pLTk+NeoΔMo, containing the HSVtk gene and the Neo(R) gene in a bicistronic unit, was introduced into rat leukemic LT12 cells. LT12/Tk+N cells showed a 1000- to 10,000-fold increased sensitivity to ganciclovir in vitro. In vitro mixing experiments demonstrated that LT12 cells were not susceptible to bystander cell lysis by LT12/Tk+N-2 cells exposed to ganciclovir. Rats injected s.c. with cloned LT12/Tk+N-2 cells developed tumors reaching a diameter of 3-4 cm after 40 days. Rats treated with ganciclovir twice daily for 5 consecutive days starting at day 7 did not develop s.c. tumors. Large established s.c. LT12/Tk+N-2 tumors completely regressed after ganciclovir treatment. However, recurrences of s.c. tumors were observed that were no longer sensitive to ganciclovir treatment. In vitro analysis of aspirates from the recurrent tumors demonstrated loss of HSVtk expression. In vitro culture of LT12/Tk+N-2 cells in soft agar in the presence of ganciclovir indicated that the frequency with which HSVtk-loss variants occurred is approximately one per 10 ⁴ cells. The in vivo occurrence of HSVtk-loss variants escaping ganciclovir-induced elimination may have important implications for vaccination protocols using HSVtk gene expressing tumor cells that are not susceptible to bystander cell killing.