Utility of 3'-[(18)F]fluoro-3'-deoxythymidine as a PET tracer to monitor response to gene therapy in a xenograft model of head and neck carcinoma

Abstract

Noninvasive imaging methodologies are needed to assess treatment responses to novel molecular targeting approaches for the treatment of squamous cell carcinoma of the head and neck (SCCHN). Computer tomography and magnetic resonance imaging do not effectively distinguish tumors from fibrotic tissue commonly associated with SCCHN tumors. Positron emission tomography (PET) offers functional non-invasive imaging of tumors. We determined the uptake of the PET tracers 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) and 3'-[(18)F]Fluoro-3'-deoxythymidine ([(18)F]FLT) in several SCCHN xenograft models. In addition, we evaluated the utility of [(18)F]FLT microPET imaging in monitoring treatment response to an EGFR antisense approach targeted therapy that has shown safety and efficacy in a phase I trial. Two of the 3 SCCHN xenograft models tested demonstrated no appreciable uptake or retention of [(18)F]FDG, but consistent accumulation of [(18)F]FLT. The third tumor xenograft SCCHN model (Cal33) demonstrated variable uptake of both tracers. SCCHN xenografts (1483) treated with EGFR antisense gene therapy decreased tumor volumes in 4/6 mice. Reduced uptake of [(18)F]FLT was observed in tumors that responded to epidermal growth factor antisense (EGFRAS) gene therapy compared to non-responding tumors or tumors treated with control sense plasmid DNA. These findings indicate that [(18)F]FLT PET imaging may be useful in monitoring SCCHN response to molecular targeted therapies, while [(18)F]FDG uptake in SCCHN xenografts may not be reflective of the level of metabolic activity characteristic of human SCCHN tumors.

Keywords: 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG); 3’-[18F]Fluoro-3’-deoxythymidine ([18F]FLT); Squamous cell carcinoma of the head and neck (SCCHN); epidermal growth factor receptor (EGFR); positron emission tomography (PET); region of interest (ROI); standardize uptake values (SUV); volume of interest (VOI).

Figure 1

microPET imaging of SCCHN tumor xenografts demonstrate uptake of [¹⁸F]FLT and [¹⁸F]FDG. Representative summed microPET images taken 40-90 min, post-injection of radiotracer in mice bearing 1483, OSC19 or Cal 33 tumors with arrows indicating the location of the tumor. The uptake was assessed 2-3 times with each tumor type.

Figure 2

SCCHN cell lines express hexokinase II and thymidine kinase. SCCHN cell lines 1483, OSC19 and Cal 33 were lysed and the proteins were fractionated on an 8% PAGE. Immunoblotting was carried out and the levels of hexokinase, thymidine kinase and EGFR were determined in the three cell lines. β-Actin levels demonstrate equal loading of protein in all lanes. The experiment was repeated twice with similar results.

Figure 3

Intratumoral administration of EGFR antisense plasmid DNA has antitumor efficacy. One million SCCHN cells (1483) were injected subcutaneously into six athymic nude mice on both flanks. EGFR sense plasmid DNA (■) or antisense plasmid DNA (▲) (25 μg/tumor) was administered daily into the left and right tumor respectively. Tumors were measured twice a week. The graph depicts growth of the tumors over the treatment period. B: EGFR antisense gene therapy reduces SCCHN xenograft cell proliferation. SCCHN tumors treated with either EGFR antisense or sense gene therapy were paraffin-embedded and examined for proliferation using Ki-67 staining. Positive cells demonstrated by the dark brown nuclear staining were counted under 400X magnification in 10 separate fields. There was a significant difference between tumors treated with EGFR antisense oligonucleotides compared to the control (p= 0.0001). Error bars indicate ± SEM.

Figure 4

PET imaging of SCCHN tumors using [¹⁸F]FLT correlates with SCCHN response to EGFR gene therapy. Animals bearing SCCHN tumors treated intratumorally with either EGFR antisense or sense plasmid DNA were imaged using [¹⁸F]FLT PET before and after treatment to monitor response to therapy. The graph demonstrates (A) change in tumor volumes for each individual mouse and (B) change in the total proliferative volume of tumors of individual mice pre- and post-treatment with either EGFR sense or EGFR antisense plasmid DNA. Animal ID’s of 6 mice are indicated in the graphs. (C) Representative [¹⁸F]FLT microPET images of 1483 xenografted mice treated at baseline and 2 weeks post-therapy with EGFR sense (control) or antisense (EGFR AS) plasmid DNA.