The use of 19F spectroscopy and diffusion-weighted MRI to evaluate differences in gene-dependent enzyme prodrug therapies.

To evaluate noninvasive measures of gene expression and tumor response in a gene-dependent enzyme prodrug therapy (GDEPT), a bifunctional fusion gene between Saccharomyces cerevisiae cytosine deaminase (CD) and Haemophilus influenzae uracil phosphoribosyltransferase (UPRT) was constructed. CD deaminates 5-fluorocytosine (5FC) to 5-fluorouracil (5FU), and UPRT subsequently converts 5FU to fluorouridine monophosphate, and both of these reactions can be monitored noninvasively in vitro and in vivo using 19F magnetic resonance spectroscopy (MRS). Following transient transfection the CD-UPRT fusion protein exhibited both UPRT and CD enzymatic activities as documented by 19F MRS. In addition, an increase in CD activity and thermal stability was witnessed for the fusion protein compared to native CD. Stable expression of CD-UPRT in 9L glioma cells increased both 5FC and 5FU sensitivity in vitro compared to CD-expressing and wild-type 9L cells. Noninvasive 19F MRS of both CD and UPRT gene function in vivo demonstrated that in animals bearing CD-expressing tumors there was limited conversion of 5FC to 5FU with no measurable accumulation of cytotoxic fluorinated nucleotides (F-nucs). In contrast, CD-UPRT-expressing tumors had increased CD gene activity with a threefold higher intratumoral accumulation of 5FU and significant generation of F-nucs. Finally, CD-UPRT yielded increased efficacy in an orthotopic animal model of high-grade glioma. More importantly, early changes in cellular water mobility, which are felt to reflect cellular death, as measured by diffusion-weighted MRI, were predictive of both durable response and increased animal survival. These results demonstrate the increased efficacy of the CD-UPRT GDEPT compared to CD alone both biochemically and in a preclinical model and validate both 19F MRS and diffusion-weighted MRI as tools to assess gene function and therapeutic efficacy.     

Onset, progression, and plateau of skeletal lesions in fibrous dysplasia and the relationship to functional outcome.

Most lesions in FD and their attendant functional disability occur within the first decade; 90% of lesions are present by 15 years, and the median age when assistive devices are needed is 7 years. These findings have implications for prognosis and determining the timing and type of therapy. INTRODUCTION: Fibrous dysplasia of bone (FD) is an uncommon skeletal disorder in which normal bone is replaced by abnormal fibro-osseous tissue. Variable amounts of skeletal involvement and disability occur. The age at which lesions are established, the pace at which the disease progresses, if (or when) the disease plateaus, and how these parameters relate to the onset of disability are unknown. To answer these questions, we performed a retrospective analysis of a group of subjects with FD. MATERIALS AND METHODS: One hundred nine subjects with a spectrum of FD were studied for up to 32 years. Disease progression was assessed in serial (99)Tc-MDP bone scans by determining the location and extent of FD lesions using a validated bone scan scoring tool. Physical function and the need for ambulatory aids were assessed. RESULTS: Ninety percent of the total body disease skeletal burden was established by age 15. Disease was established in a region-specific pattern; in the craniofacial region, 90% of the lesions were present by 3.4 yr, in the extremities, 90% were present by 13.7 yr, and in the axial skeleton, 90% were present by 15.5 yr. Twenty-five of 103 subjects eventually needed ambulatory aids. The median age at which assistance was needed was 7 yr (range, 1-43 yr). The median bone scan score for subjects needing assistance was 64.3 (range, 18.6-75) compared with 23.1 (range, 0.5-63.5) in the unassisted subjects (p < 0.0001). Among subjects needing assistance with ambulation, 92% showed this need by 17 yr. CONCLUSIONS: The majority of skeletal lesions and the associated functional disability occur within the first decade of life. The implication is that the window of time for preventative therapies is narrow. Likewise, therapeutic interventions must be tailored to where the patient is in the natural history of the disease (i.e., progressive disease [young] versus established disease [older subjects]). These findings have implications for prognosis, the timing and type of therapy, and the development of trials of new therapies and their interpretation.