Hypoxia imparts resistance to radiotherapy and chemotherapy and also promotes a variety of changes in tumor biology through inducible promoters. The purpose of this study was to evaluate the use of positron emission tomography (PET) imaging with fluorine-18 fluoromisonidazole (FMISO) in soft tissue sarcomas (STS) as a measure of hypoxia and to compare the results with those obtained using [(18)F]fluorodeoxyglucose (FDG) and other known biologic correlates. FDG evaluates energy metabolism in tumors while FMISO uptake is proportional to tissue hypoxia. FMISO uptake was compared with FDG uptake. Vascular endothelial growth factor (VEGF) expression was also compared with FMISO uptake. Nineteen patients with STS underwent PET scanning with quantitative determination of FMISO and FDG uptake prior to therapy (neo-adjuvant chemotherapy or surgery alone). Ten patients receiving neo-adjuvant chemotherapy were also imaged after chemotherapy but prior to surgical resection. Standardized uptake value (SUV) was used to describe FDG uptake; regional tissue to blood ratio (>or=1.2 was considered significant) was used for FMISO uptake. Significant hypoxia was found in 76% of tumors imaged prior to therapy. No correlation was identified between pretherapy hypoxic volume (HV) and tumor grade ( r=0.15) or tumor volume ( r=0.03). The correlation of HV with VEGF expression was 0.39. Individual tumors showed marked heterogeneity in regional VEGF expression. The mean pixel-by-pixel correlation between FMISO and FDG uptake was 0.49 (range 0.09-0.79) pretreatment and 0.32 (range -0.46-0.72) after treatment. Most tumors showed evidence of reduced uptake of both FMISO and FDG following chemotherapy. FMISO PET demonstrates areas of significant and heterogeneous hypoxia in soft tissue sarcomas. The significant discrepancy between FDG and FMISO uptake seen in this study indicates that regional hypoxia and glucose metabolism do not always correlate. Similarly, we did not find any relationship between the hypoxic volume and the tumor volume or VEGF expression. Identification of hypoxia and development of a more complete biologic profile of STS will serve to guide more rational, individualized cancer treatment approaches.

Radiolabeled fluoromisonidazole (FMISO) is being investigated as an imaging agent for hypoxia in tumors and nonmalignant tissues in myocardial infarct or stroke. In this study in vitro cell cultures were used to characterize the oxygen dependency of FMISO uptake and to examine other modifying factors. The uptake of [3H]FMISO was measured in four cell lines in vitro: V-79, EMT-6(UW), RIF-1, and CaOs-1. The modifying effects of different O2 levels as well as cell growth state and concentration of glucose and nonprotein sulfhydryls were examined. In these cell types an O2 level between 720 and 2300 ppm inhibited FMISO binding by 50%, relative to binding under anoxic conditions. These values bracket the O2 level which confers full radiobiologic hypoxia, about 1000 ppm. Some bound label was released from cells in the first 1 to 3 h after a 3-h anoxic labeling with [3H]FMISO, but this does not represent tritium loss from the parent molecule. Cells from unfed plateau-phase cultures took up less [3H]FMISO than did exponentially growing cells incubated at comparable O2 levels. Reducing glucose to 1/10 or 1/100 of the usual concentration in medium had little effect on binding of micromolar levels of FMISO, except in V-79 cells, where reduced glucose levels were associated with increased FMISO accumulation. Adding cysteamine to the culture medium moderately increased FMISO uptake. We conclude that cell growth state, glucose, and nonprotein sulfhydryl concentrations affect FMISO binding, albeit less than varying O2 levels: anoxic/oxic binding ratios vary from 12.6 to 28 for the four cell types examined. Nonetheless these factors must be considered in evaluating the oxygen-dependent binding of this nitroimidazole in tumors or tissues.

The usefulness of radiolabeled nitroimidazoles for measuring hypoxia will be clarified by defining the relationship between tracer uptake and radiobiologically hypoxic fraction. We determined the radiobiologically hypoxic fraction from radiation response data in 36B10 rat gliomas using the paired cell survival curve technique and compared the values to the radiobiologically hypoxic fraction inferred from mathematical modeling of time-activity data acquired by PET imaging of [(18)F]FMISO uptake. Rats breathed either air or 10% oxygen during imaging, and timed blood samples were taken. The uptake of [(3)H]FMISO by 36B10 cells in vitro provided cellular binding characteristics of this radiopharmaceutical as a function of oxygen concentration. The radiobiologically hypoxic fraction determined for tumors in air-breathing rats using the paired survival curve technique was 6.1% (95% CL = 4.3- 8.6%), which agreed well with that determined by modeling FMISO time-activity data (7. 4%; 95% CL = 2.5-17.3%). These results are consistent with the agreement between the two techniques for measuring radiobiologically hypoxic fraction in Chinese hamster V79 cell spheroids. In contrast, the FMISO-derived radiobiologically hypoxic fraction in rats breathing 10% oxygen was 13.1% (95% CL 7.9-8.3%), much lower than the radiobiologically hypoxic fraction of 43% determined from the radiation response data. This discrepancy may be due to the failure of FMISO to identify hypoxic cells residing at or above an oxygen level of 2-3 mmHg that will still confer substantial protection against radiation. The presence of transiently hypoxic cells in rats breathing reduced oxygen may also be under-reported by nitroimidazole binding, which is strongly dependent on time and concentration.

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