John R. Grierson

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.

Tumor Hypoxia Imaging with [F-18] Fluoromisonidazole Positron Emission Tomography in Head and Neck Cancer

Purpose: Advanced head and neck cancer shows hypoxia that results in biological changes to make the tumor cells more aggressive and less responsive to treatment resulting in poor survival. [F-18] fluoromisonidazole (FMISO) positron emission tomography (PET) has the ability to noninvasively quantify regional hypoxia. We investigated the prognostic effect of pretherapy FMISO-PET on survival in head and neck cancer. Experimental Design: Seventy-three patients with head and neck cancer had pretherapy FMISO-PET and 53 also had fluorodeoxyglucose (FDG) PET under a research protocol from April 1994 to April 2004. Results: Significant hypoxia was identified in 58 patients (79%). The mean FMISO tumor/bloodmax (T/Bmax) was 1.6 and the mean hypoxic volume (HV) was 40.2 mL. There were 28 deaths in the follow-up period. Mean FDG standard uptake value (SUV)max was 10.8. The median time for follow-up was 72 weeks. In a univariate analysis, T/Bmax (P = 0.002), HV (P = 0.04), and the presence of nodes (P = 0.01) were strong independent predictors. In a multivariate analysis, including FDG SUVmax, no variable was predictive at P < 0.05. When FDG SUVmax was removed from the model (resulting in n = 73 with 28 events), nodal status and T/Bmax (or HV) were both highly predictive (P = 0.02, 0.006 for node and T/Bmax, respectively; P = 0.02 and 0.001 for node and HV, respectively). Conclusions: Pretherapy FMISO uptake shows a strong trend to be an independent prognostic measure in head and neck cancer.

[18F]FMISO and [18F]FDG PET imaging in soft tissue sarcomas: correlation of hypoxia, metabolism and VEGF expression

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 [18F]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 (≥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 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.

Tumor 3′-Deoxy-3′-18F-Fluorothymidine (18F-FLT) Uptake by PET Correlates with Thymidine Kinase 1 Expression: Static and Kinetic Analysis of 18F-FLT PET Studies in Lung Tumors

We report the first, to our knowledge, findings describing the relationships between both static and dynamic analysis parameters of 3′-deoxy-3′-18F-fluorothymidine (18F-FLT) PET and the expression of the proliferation marker Ki-67, and the protein expression and enzymatic activity of thymidine kinase-1 (TK1) in surgically resected lung lesions. Methods: Static and dynamic analyses (4 rate constants and 2 compartments) of 18F-FLT PET images were performed in a cohort of 25 prospectively accrued, clinically suspected lung cancer patients before surgical resection (1 lesion was found to be benign after surgery). The maximal and overall averaged expression of Ki-67 and TK1 were determined by semiquantitative analysis of immunohistochemical staining. TK1 enzymatic activity was determined by in vitro assay of extracts prepared from flash-frozen samples of the same tumors. Results: Static 18F-FLT uptake (partial-volume–corrected maximum-pixel standardized uptake value from 60- to 90-min summed dynamic data) was significantly correlated with the overall (ρ = 0.57, P = 0.006) and maximal (ρ = 0.69, P < 0.001) immunohistochemical expressions of Ki-67 and TK1 (overall expression: ρ = 0.65, P = 0.001; maximal expression: ρ = 0.68, P < 0.001) but not with TK1 enzymatic activity (ρ = 0.34, P = 0.146). TK1 activity was significantly correlated with TK1 protein expression only when immunohistochemistry was scored for maximal expression (ρ = 0.52, P = 0.029). Dynamic analysis of 18F-FLT PET revealed correlations between the flux constant (KFLT) and both overall (ρ = 0.53, P = 0.014) and maximal (ρ = 0.50, P = 0.020) TK1 protein expression. KFLT was also associated with both overall (ρ = 0.59, P = 0.005) and maximal (ρ = 0.63, P = 0.002) Ki-67 expression. We observed no significant correlations between TK1 enzyme activity and KFLT. In addition, no significant relationships were found between TK1 expression, TK1 activity, or Ki-67 expression and any of the compartmental rate constants. Conclusion: The absence of observable correlations of the imaging parameters with TK1 activity suggests that 18F-FLT uptake and retention within cells may be complicated by a variety of still undetermined factors in addition to TK1 enzymatic activity.

Glypican-3–Targeting F(ab′)2 for 89Zr PET of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is an increasingly lethal malignancy for which management is critically dependent on accurate imaging. Glypican-3 (GPC3) is a cell surface receptor overexpressed in most HCCs and provides a unique target for molecular diagnostics. The use of monoclonal antibodies (mAbs) that target GPC3 (αGPC3) in PET imaging has shown promise but comes with inherent limitations associated with mAbs such as long circulation times. This study used 89Zr-conjugated F(ab′)2 fragments directed against GPC3 (89Zr-αGPC3-F(ab′)2) to evaluate the feasibility of the fragments as a diagnostic immuno-PET imaging probe. Methods: Immobilized ficin was used to digest αGPC3, creating αGPC3-F(ab′)2 fragments subsequently conjugated to 89Zr. In vivo biodistribution and PET studies were performed on GPC3-expressing HepG2 and GPC3-nonexpressing RH7777 orthotopic xenografts. Results: Reliable αGPC3-F(ab′)2 production via immobilized ficin digestion was verified by high-performance liquid chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. 89Zr-αGPC3-F(ab′)2 demonstrated F(ab′)2-dependent, antigen-specific cell binding. HepG2 tumor uptake was higher than any other tissue, peaking at 100 ± 21 percentage injected dose per gram (%ID/g) 24 h after injection, a value 33- to 38-fold higher than GPC3-nonexpressing RH7777 tumors. The blood half-life of the 89Zr-αGPC3-F(ab′)2 conjugate was approximately 11 h, compared with approximately 115 h for historic mAb controls. This shorter half-life enabled clear tumor visualization on PET 4 h after administration, with a resultant peak tumor-to-liver contrast ratio of 23.3. Blocking antigen-expressing tumors with an excess of nonradiolabeled αGPC3 resulted in decreased tumor uptake similar to native liver. The kidneys exhibited high tissue uptake, peaking at 24 h with 83 ± 12 %ID/g. HepG2 tumors ranging from 1.5 to 7 mm were clearly visible on PET, whereas larger RH7777 tumors displayed signal lower than background liver tissue. Conclusion: This study demonstrates the feasibility of using 89Zr-αGPC3-F(ab′)2 for intrahepatic tumor localization with small-animal PET. Faster blood clearance and lower background liver uptake enable excellent signal-to-noise ratios at early time points. Increased renal uptake is similar to that as has been seen with clinical radioactive peptide imaging. 89Zr-αGPC3-F(ab′)2 addresses some of the shortcomings of whole-antibody immuno-PET probes. Further optimization is warranted to maximize probe sensitivity and specificity in the process of clinical translation.

Glypican-3–Targeted 89Zr PET Imaging of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a devastating malignancy in which imperfect imaging plays a primary role in diagnosis. Glypican-3 (GPC3) is an HCC-specific cell surface proteoglycan overexpressed in most HCCs. This paper presents the use of 89Zr-conjugated monoclonal antibody against GPC3 (89Zr-αGPC3) for intrahepatic tumor localization using PET. Methods: Polymerase chain reaction confirmed relative GPC3 expression in cell lines. In vitro binding, in vivo biodistribution, and small-animal PET studies were performed on GPC3-expressing HepG2 and non–GPC3-expressing HLF and RH7777 cells and orthotopic xenografts. Results: 89Zr-αGPC3 demonstrated antibody-dependent, antigen-specific tumor binding. HepG2 liver tumors exhibited high peak uptake (836.6 ± 86.6 percentage injected dose [%ID]/g) compared with background liver (27.5 ± 1.6 %ID/g). Tumor-to-liver contrast ratio was high and peaked at 32.5. The smallest HepG2 tumor (<1 mm) showed lower peak uptake (42.5 ± 6.4 %ID/g) and tumor-to-liver contrast (1.57) but was still clearly visible on PET. Day 7 tissue activity was still substantial in HepG2 tumors (466.4 ± 87.6 %ID/g) compared with control RH7777 tumors (3.9 ± 1.3 %ID/g, P < 0.01), indicating antigen specificity by 89Zr-αGPC3. HepG2 tumor treated with unlabeled αGPC3 or heat-denatured 89Zr-αGPC3 demonstrated tumor activity (2.1 %ID/g) comparable to that of control xenografts, confirming antibody dependency. Conclusion: This study demonstrated the feasibility of using 89Zr-αGPC3 to image HCC in the liver, as well as the qualitative determination of GPC3 expression via small-animal PET. The ability to clarify the identity of small liver lesions with an HCC-specific PET probe would provide clinicians with vital information that could significantly alter patient management, warranting further investigation for clinical translation.

Radiosynthesis of 6-[C-11]-d-glucose

Availability of 6-[C-11]-D-glucose will permit positron emission tomography (PET) investigations of glucose utilization derived from the pentose shunt which supports biosynthesis in tissues. The first radiosynthesis of 6-[C-11]-D-glucose is described. As much as 1 mCi of 6-[C-11]-D-glucose, sufficient for animal studies, is obtained from [C-11]CO2 after 100 min with a 16% radiochemical yield (EOB). The radiosynthesis has many attractive features. The method uses [C-11]CH3I and combines a Wittig reaction and a stereoselective OsO4 catalyzed alkene hydroxylation. The OsO4 hydroxylation of the [C-11]-labeled alkene (9) is accomplished in less than 10 min with high stereoselectivity (94:6) in favor of the 6-[C-11]-D-gluco-isomer. HPLC purification (C-18) of the protected labeled sugar removes the undesired 6-[C-11]-L-ido-sugar at an early stage and avoids the use of an expensive low-capacity ion-exchange HPLC column. OsO4, a highly toxic reagent, is removed in the process by adsorption and inactivation on polymer-bound triphenylphosphine.

An improved HPLC system for the analysis and purification of organic amine radiopharmaceuticals

This paper describes the separation of lipophilic amine radiopharmaceuticals on normal phase silica using a reversed phase type eluant. This class of compound usually gives very poor peak symmetry on reversed phase columns. However, excellent separation of a number of amines of interest to Positron Emission Tomography, such as spiperone and its derivatives, has been achieved on this system.

The Role of Imaging in the Development of Oncologic Agents

P to the development of metabolic imaging, researchers had to use in vitro measurements of tumor proliferation made with biopsy specimens obtained from patients. Many studies found such information predictive of the response to therapy and disease aggressiveness. Biopsies, however, may reflect the metabolism of only a small area of a tumor; they may not be representative and cannot easily be done repeatedly. In contrast, the power of positron emission tomography (PET) imaging is that it can quantitatively image tissues repeatedly in a noninvasive way. One can probe different pathways in tumor masses as determined by the appropriate choice of a metabolic tracer. The tracers are delivered in small doses in vivo under normal physiological conditions. This type of tailored noninvasive experimental design is not available with any other technique. Therefore, PET has emerged as a research tool to improve the evaluation of tumor treatment. [F-18]labeled fluorodeoxyglucose (FDG) is the most commonly used agent to diagnose tumors. Preliminary investigations of the use of FDG to measure response to therapy have been promising. While measuring tumor energetics may be of interest, we think that more specific measures of tumor metabolic pathways and proliferation will provide better information to predict and monitor response to therapy. To that end, our studies have concentrated on imaging tumor proliferation with thymidine and its analogs. We have worked on developing the tools needed to measure treatment response with PET and begun to incorporate these tools in the early trials of new oncologic agents. DEVELOPMENT OF NEW ONCOLOGIC AGENTS