Therapy-related myeloid leukemias (t-ML) have emerged as severe late complications of cancer treatment in as many as 5–10% of patients who are otherwise cured of a primary neoplasm with aggressive multi-modal regimens. A new alkylating agent, temozolomide (TMZ), has shown efficacy in the treatment of adult high-grade glioma and refractory leukemia. Due to this early success, TMZ is now being incorporated as upfront therapy into more than 40 studies for a range of cancers including glioblastoma and melanoma. TMZ’s method of action depends on the methylation of guanine bases at the O6 position, resulting in O6-methylguanine and G:C‡A:T transitions. While some early in vivo studies of TMZ demonstrated tumorigenicity after contact with normal tissues, until recently no cases of secondary malignancies from TMZ exposure had been reported in the literature. However, two groups have now reported secondary myeloid malignancies after TMZ treatment (De Vita, De Matteis et al. 2005; Su, Chang et al. 2005). As TMZ moves into the frontline of our chemotherapeutic armamentarium, further investigation of its in vivo mutagenic potential are warranted.
We have employed a transgenic genomic mutation indicator mouse strain (small blue mouse) to determine the in vivo mutagenic potential of TMZ on bone marrow (BM) cells in comparison to cyclophosphamide (CP). In this mouse model, the mutational target is the non-transcriptionally active lacZ portion of the plasmid pUR288. The plasmid is stably integrated in multiple copies into the genome, and the mutation frequency in the lacZ gene is determined with a plasmid rescue procedure applied to genomic DNA derived from BM (or any other tissue) and a subsequent selection for lac-Z negative clones. The type of mutation can then be further determined by PCR amplification and restriction digestion.
For this study, animals were treated with TMZ (175 mg/kg/day i.p for 5 days), CP, or PBS, and BM was harvested 10 days after the last treatment. CP treatments were 200 mg/kg i.p. either once or weekly for 6 weeks. Determination of the mutation frequency revealed that the 1-day CP treatment increased the mutational load in BM approximately 2–3 fold over control, whereas the TMZ regimen resulted in an almost 40-fold increase over control. BM cells in animals treated 6 times with CP did not show an increase in the mutation frequency over animals treated only one time with CP. As expected, over 80% of all mutations in response to TMZ treatment were point mutations. BM cells from animals treated with CP showed less than 30% point mutations, with the remaining mutations being either translocations or deletions. This data emphasizes the strong mutagenic potential of TMZ on BM cells in vivo in our mouse model system. Further investigation is needed to determine whether the increased mutation rate seen with TMZ exposure is indicative of an elevated risk of therapy-induced leukemia.