Janet S. Rasey

Quantifying regional hypoxia in human tumors with positron emission tomography of [18F]fluoromisonidazole: A pretherapy study of 37 patients

PURPOSE To assess pretreatment hypoxia in a variety of tumors using positron emission tomography (PET) after injection of the hypoxia-binding radiopharmaceutical [18F]fluoromisonidazole ([18F]FMISO). METHODS AND MATERIALS Tumor fractional hypoxic volume (FHV) was determined in 21 nonsmall cell lung cancer patients, 7 head and neck cancer patients, 4 prostate cancer patients, and 5 patients with other malignancies by quantitative PET imaging after injection of [18F]FMISO (0.1 mCi/kg). The FHV was defined as the proportion of pixels in the imaged tumor volume with a tissue:blood [18F] activity ratio > or = 1.4 at 120-160 min postinjection. A FHV > 0 was taken as evidence for tumor hypoxia. RESULTS Hypoxia was observed in 36 of 37 tumors studied with FMISO PET imaging; FHVs ranged from 0 to 94.7%. In nonsmall cell lung cancers (n = 21), the median FHV was 47.6% and the range, 1.3 to 94.7%. There was no correlation between tumor size and FHV. In the seven head and neck carcinomas, the median FHV was 8.8%, with a range from 0.2 to 18.9%. In the group of four prostate cancers, the median and range were 18.2% and 0 to 93.9%, while in a group of five tumors of different types the median FHV was 55.2% (range: 21.4 to 85.8%). CONCLUSIONS Hypoxia was present in 97% of the tumors studied and the extent of hypoxia varied markedly between tumors in the same site or of the same histology. Hypoxia also was distributed heterogeneously between regions within a single tumor. These results are consistent with O2 electrode measures with other types of human tumors. The intra- and intertumor variability indicate the importance of making oxygenation measures in individual tumors and the necessity to sample as much of the tumor volume as possible.

l Brief Communication QUANTIFYING REGIONAL HYPOXIA IN HUMAN TUMQRS WITH POSITRON EMISSION TOMOGRAPHY OF ('*F)FLUUROMISONIDAZOLE: A PRETHERAPY STUDY OF 37 PATIENTS

PURPOSE To assess pretreatment hypoxia in a variety of tumors using positron emission tomography (PET) after injection of the hypoxia-binding radiopharmaceutical [18F]fluoromisonidazole ([18F]FMISO). METHODS AND MATERIALS Tumor fractional hypoxic volume (FHV) was determined in 21 nonsmall cell lung cancer patients, 7 head and neck cancer patients, 4 prostate cancer patients, and 5 patients with other malignancies by quantitative PET imaging after injection of [18F]FMISO (0.1 mCi/kg). The FHV was defined as the proportion of pixels in the imaged tumor volume with a tissue:blood [18F] activity ratio > or = 1.4 at 120-160 min postinjection. A FHV > 0 was taken as evidence for tumor hypoxia. RESULTS Hypoxia was observed in 36 of 37 tumors studied with FMISO PET imaging; FHVs ranged from 0 to 94.7%. In nonsmall cell lung cancers (n = 21), the median FHV was 47.6% and the range, 1.3 to 94.7%. There was no correlation between tumor size and FHV. In the seven head and neck carcinomas, the median FHV was 8.8%, with a range from 0.2 to 18.9%. In the group of four prostate cancers, the median and range were 18.2% and 0 to 93.9%, while in a group of five tumors of different types the median FHV was 55.2% (range: 21.4 to 85.8%). CONCLUSIONS Hypoxia was present in 97% of the tumors studied and the extent of hypoxia varied markedly between tumors in the same site or of the same histology. Hypoxia also was distributed heterogeneously between regions within a single tumor. These results are consistent with O2 electrode measures with other types of human tumors. The intra- and intertumor variability indicate the importance of making oxygenation measures in individual tumors and the necessity to sample as much of the tumor volume as possible.

Evaluation of oxygenation status during fractionated radiotherapy in human nonsmall cell lung cancers using [F-18]fluoromisonidazole positron emission tomography

PURPOSE Recent clinical investigations have shown a strong correlation between pretreatment tumor hypoxia and poor response to radiotherapy. These observations raise questions about standard assumptions of tumor reoxygenation during radiotherapy, which has been poorly studied in human cancers. Positron emission tomography (PET) imaging of [F-18]fluoromisonidazole (FMISO) uptake allows noninvasive assessment of tumor hypoxia, and is amenable for repeated studies during fractionated radiotherapy to systematically evaluate changes in tumor oxygenation. METHODS AND MATERIALS Seven patients with locally advanced nonsmall cell lung cancers underwent sequential [F-18]FMISO PET imaging while receiving primary radiotherapy. Computed tomograms were used to calculate tumor volumes, define tumor extent for PET image analysis, and assist in PET image registration between serial studies. Fractional hypoxic volume (FHV) was calculated for each study as the percentage of pixels within the analyzed imaged tumor volume with a tumor:blood [F-18]FMISO ratio > or = 1.4 by 120 min after injection. Serial FHVs were compared for each patient. RESULTS Pretreatment FHVs ranged from 20-84% (median 58%). Subsequent FHVs varied from 8-79% (median 29%) at midtreatment, and ranged from 3-65% (median 22%) by the end of radiotherapy. One patient had essentially no detectable residual tumor hypoxia by the end of radiation, while two others showed no apparent decrease in serial FHVs. There was no correlation between tumor size and pretreatment FHV. CONCLUSIONS Although there is a general tendency toward improved oxygenation in human tumors during fractionated radiotherapy, these changes are unpredictable and may be insufficient in extent and timing to overcome the negative effects of existing pretreatment hypoxia. Selection of patients for clinical trials addressing radioresistant hypoxic cancers can be appropriately achieved through single pretreatment evaluations of tumor hypoxia.

Radiolabeled fluoromisonidazole as an imaging agent for tumor hypoxia

Fluoromisonidazole labeled with H-3 or F-18 has been tested as a quantitative probe for hypoxic cells in vitro and in rodent and spontaneous dog tumors in vivo. In V-79, EMT-6(UW), RIF-1, and canine osteosarcoma cells in vitro, the binding of 50 microM [H-3]Fluoromisonidazole was 50% inhibited by 1000-2000 ppm O2, relative to binding under anoxic conditions. After a 3 hr incubation with labeled drug, the anoxic/oxic binding ratios ranged from 12 to 27 for the four cell types. Retention of [H-3]fluoromisonidazole 4 hr after injection was greater in large KHT tumors (400-600 mm3) with an estimated hypoxic fraction greater than 30%, than in smaller tumors (50-200 mm3) with an estimated hypoxic fraction of 7-12%. RIF-1 tumors, with an estimated hypoxic fraction of 1.5%, retained the least label, with tumor: blood ratios ranging from 1.7 to 1.9. Spontaneous dog osteosarcomas were imaged with a time of flight positron emission tomograph for up to 5 hr following injection of [F-18] fluoromisonidazole. Analysis of regions of interest in images allowed creation of dynamic tissue time activity curves and calculation of tissue uptake in cpm/gram. These values were compared to radioactivity in plasma. In all cases, retention in some tumor regions exceeded that in plasma and in normal tissue, such as muscle or brain, by 3 to 5 hr post injection. Uptake of fluoromisonidazole in tumors was heterogeneous, with ratios of maximum to minimum uptake as high as 4 in different regions of interest in the same tumor. Tumor:plasma values ranged from 0.28 to 2.02. The oxygen dependency of fluoromisonidazole retention was similar in a variety of cell types and was 50% inhibited by O2 levels in the transition between full radiobiological hypoxia and partial sensitization. The quantitative regional imaging of [F-18] fluoromisonidazole in spontaneous canine tumors at varying times post-injection lays the basis for imaging and modeling of oxygen-dependent drug retention in different regions of human neoplasms.

Characterization of Radiolabeled Fluoromisonidazole as a Probe for Hypoxic Cells

Radiolabeled fluoromisonidazole has been characterized as a probe for hypoxic cells in vitro and in vivo. The uptake and retention of [3H]fluoromisonidazole and [3H]misonidazole were compared in V-79 cell monolayers and spheroids by varying incubation time and O2 levels in contact with the medium. The two labeled drugs were retained similarly in cell populations isolated from different depths in spheroids, and the amount of each drug bound in cells at the spheroid periphery increased with decreasing O2 level. The labeling patterns in autoradiographs were similar for spheroids incubated with the two labeled drugs, with most silver grains located over a zone of viable and presumed hypoxic cells intermediate between the necrotic center and the periphery of the spheroid. Biodistribution of the two tritiated drugs was compared in C3H mice bearing KHT tumors with 15% radiobiologically hypoxic cells. Tumor:blood and tumor:muscle ratios greater than 5.0 were achieved in mice sacrificed 4 h after the last of three injections of 5 or 20 mumol/kg of [3H]fluoromisonidazole. These ratios are compatible with imaging and are higher than those obtained with 50 mumol/kg misonidazole in a similar administration protocol. TLC analysis of plasma from mice injected with [3H]fluoromisonidazole indicated that the drug was stable in vivo for up to 2 h and that the metabolites formed were too polar to be dehalogenation products. Fluoromisonidazole labeled with 18F at the end of the alkyl side chain would retain the label on metabolites that bind in hypoxic cells in vivo. Fluoromisonidazole binds stably in the same populations of hypoxic cells as does misonidazole, and we conclude that [18F]fluromisonidazole has potential use as a hypoxia imaging agent in vivo.

The effect of photon irradiation on blood-brain barrier permeability to methotrexate in mice.

Methotrexate was administered by intraperitoneal injection (100 mg/kg) to unirradiated mice, and to mice receiving varying doses of cranial irradiation. The animals were sacrificed 24 hours after injection, and methotrexate assays were performed on brain tissue. No methotrexate was detected in the brains of the unirradiated animals. Detectable levels of methotrexate were present after 2000 rad cranial irradiation, but not after 500 rad, 1000 rad, or 1500 rad. The implications of these findings are discussed.

Radiation inhibition of intimal hyperplasia after arterial injury.

To demonstrate the effect of gamma radiation on proliferating smooth muscle cells in vivo, a standardized bilateral carotid balloon catheter arterial injury was produced in 45 rats and doses from 0-20 Gy were delivered to the right carotid artery at 24 h after injury. At 20 days after injury, cross-sectional area of intima was determined from axial histological sections. Compared to contralateral, nonirradiated balloon-injured arteries, radiation produced a significant dose-dependent reduction in intimal cross-sectional area, with a 50% decrease at 5-7.5 Gy. To determine the effect of timing of irradiation on intimal hyperplasia, 30 rats with bilateral carotid injury received unilateral cervical irradiation at doses of 1, 5 or 10 Gy administered at either 1, 3 or 5 days after injury. The radiation dose (P = 0.0002), timing of irradiation (P = 0.003) and an interaction between timing and dose (P = 0.0278) were significantly associated with reduction in neointimal cross-sectional area. To determine the effects of radiation on intimal hyperplasia at later intervals, rats irradiated with 15 (n = 5) or 20 Gy (n = 5) were euthanized at 3 months after injury. A significant persistent reduction in intimal cross-sectional area for irradiated arteries at 3 months was associated with minimal apparent radiation effects upon adjacent tissue. These data suggest that external gamma irradiation at the single doses used effectively inhibits smooth muscle proliferation and intimal hyperplasia in the rat balloon catheter injury model in a time- and dose-dependent manner.

Radioprotection of normal tissues against gamma rays and cyclotron neutrons with WR-2721: LD50 studies and 35S-WR-2721 biodistribution.

The ability of WR-2721 to protect mice against two modes of death following whole-body radiation with 137Cs gamma rays or d(22)+Be neutrons was examined. For single fractions, 400 mg/kg WR-2721 was administered prior to irradiation. In two-fraction exposures, the dose was 275 mg/kg given prior to each fraction. Dose modification factors (DMFs) were calculated as ratios of LD50 values. For single fractions of gamma rays, the DMF was 1.74 for the LD50/7 end point and for LD50/30, the DMF for single fractions was 2.25. For two fractions 3 hr apart, it was 1.88. For single fractions of cyclotron neutrons, the DMF was 1.32 for LD50/7. Measured with the LD50/30 end point, the DMF for single neutron doses was 1.41 and for two fractions, 1.19. Substantial radioprotection of bone marrow and intestinal epithelium against cyclotron neutrons was seen in these investigations. Biodistribution studies were done following ip injection of 35S-labeled WR-2721 into C3H mice bearing RIF-1 tumors. Blood levels peaked at 10 min after injection and declined thereafter. Most normal tissues achieved maximum levels of 35S at 30 to 60 min postinjection and high concentrations were retained in most tissues for up to 2 hr. Assuming that all 35S is in parent compound or dephosphorylated radioprotective metabolites, the concentration of protector (milligram per gram tissue) in various organs at 30 min postinjection ranked as follows: kidney greater than submandibular gland much greater than liver = lung greater than gut greater than heart much greater than blood greater than skin greater than tumor greater than brain. High levels of 35S were achieved and retention times were long in certain normal tissues which respond at early or late times postradiation and may be dose limiting in radiotherapy: kidney, liver, salivary gland, and lung. These combined observations suggest that there is potential for protecting dose-limiting, late-responding normal tissue in the radiotherapy of human cancer with both neutrons and conventional radiotherapy.

USE OF STEROIDS TO SUPPRESS VASCULAR RESPONSE TO RADIATION

A quantitative measure of the vascular permeability surface area product (PS) for albumin has been made using a double isotope technique. PS was significantly elevated in irradiated rat lung, heart, skin, and muscle, between 19 and 26 days following 18 or 25 Gray thorax irradiation. Administration of dexamethasone from 2 days before irradiation through the day of measurement suppressed the expected increase in PS in lung, heart, and muscle, but not in skin. Shorter periods of steroid administration were not as effective in suppressing this response to radiation exposure. Increased vascular permeability following radiation may be an essential element in the development of radiation fibrosis. We hypothesize that the ability to suppress this response could result in a long term reduction in the incidence of fibrosis.

Determining Hypoxic Fraction in a Rat Glioma by Uptake of Radiolabeled Fluoromisonidazole

Abstract Rasey, J.S., Casciari, J.J., Hofstrand, P.D., Muzi, M., Graham, M.M. and Chin, L.K. Determining the Hypoxic Fraction in a Rat Glioma by Uptake of Radiolabeled Fluoromisonidazole. 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 [18F]FMISO uptake. Rats breathed either air or 10% oxygen during imaging, and timed blood samples were taken. The uptake of [3H]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.