MO‐D‐I‐609‐08: Validation of PET Hypoxia Tracers by Autoradiography and Fluorescent Microscopy

Purpose: To develop a method for PET hypoxia tracer validation based on statistical analysis of the spatial correspondence between the intratumoral distributions of tracer uptake (assessed by digital autoradiography) and pimonidazole, an independent marker of hypoxia (assessed by immunofluorescent microscopy). The utility of the method was demonstrated by applying it to three PET hypoxia tracers, IAZGP, FMISO and Cu‐ATSM. Method and Materials: Eight rats bearing R3327‐AT tumors were divided into four groups of two animals each. Group♯1 was injected with ¹⁸F‐FMISO and sacrificed 2hr later. Group♯2 was injected with ¹²⁴I‐IAZGP and sacrificed 3hr later. Groups♯3 and ♯4 were injected with ⁶⁴Cu‐ATSM and sacrificed, respectively, 24hr or 1hr later. Pimonidazole was administered to all animals 2hr before sacrifice. Tumors were excised, frozen and sectioned. Digital autoradiograms of the tracer distribution were obtained from selected sections and co‐registered with images of pimonidazole‐associated immunofluorescence derived from adjacent sections. The statistical analysis of association between PET tracer uptake and pimonidazole immunofluorescent staining intensity was then performed on a pixel‐by‐pixel basis. Results: For rats from group♯1 correlation coefficients between pimonidazole‐associated immunofluorescent staining intensity and ¹⁸F‐FMISO uptake were 0.84 and 0.75. For group♯2 rats correlations between pimonidazole and ¹²⁴I‐IAZGP were 0.85 and 0.77. For group♯3 (⁶⁴Cu‐ATSM administered 24hr prior to sacrifice), the correlations with pimonidazole were 0.61 and 0.64. However, for group♯4 (⁶⁴Cu‐ATSM administered 1hr prior to sacrifice), the correlation coefficients were −0.76 and −0.77, demonstrating that in this tumor model ⁶⁴Cu‐ATSM uptake was not indicative of hypoxia at early times post injection. Conclusion: The proposed method enables evaluation of the degree of spatial correspondence between distributions of a PET tracer and alternative markers (either endogenous or exogenous) or tracers of the biological process under study. This method can be used for the in‐vivo validation of any nuclear medicine tracer.