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Clinical Investigation |
1 Methodist Hospital, Baylor College of Medicine, Houston, Texas; 2 Mallinckrodt Institute of Radiology and Siteman Cancer Center, Washington University School of Medicine, St. Louis, Missouri; 3 Delaware SPECT Imaging Center, Newark, Delaware; 4 Department of Neurosurgery, Emory University, Atlanta, Georgia; 5 Endocyte Inc., West Lafayette, Indiana; and 6 Genitourinary Oncology Program, Methodist Hospital Research Institute, Houston, Texas
Correspondence: For correspondence or reprints contact: Ronald E. Fisher, Methodist Hospital, 6565 Fannin, Houston, TX 77030. E-mail: rfisher{at}bcm.tmc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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1.5 cm underwent imaging with 99mTc-EC20. The majority of these patients (77%) had a diagnosis of renal cell carcinoma. The remaining patients had a variety of other solid tumors. Whole-body planar images were obtained 1–2 h after injection, followed by SPECT of the region containing index lesions. The uptake of 99mTc-EC20 in tumors was scored as no uptake, mild uptake, or marked uptake. The resultant 99mTc-EC20 data were analyzed for correlation with the expression of the 
-isoform of FR, as determined by IHC analysis, in tissue available from prior or subsequent surgery or biopsy. Results: The administration of 99mTc-EC20 was well tolerated. Tumors with increased 99mTc-EC20 uptake were identified in 68% of patients, and IHC results were positive for the expression of the -isoform of FR in 67% of patients. The agreement between methods was 61% overall (
= 0.096; 95% confidence interval = –0.085 to 0.277), with 72% agreement of positive results and 38% agreement of negative results. Conclusion: In vivo imaging with 99mTc-EC20 identified approximately two thirds of patients as having FR-positive tumors. Agreement between imaging and in vitro IHC was poor but was potentially confounded by a lack of correlation between the time of tissue sampling and the time of 99mTc-EC20 imaging, the heterogeneous expression of FR in metastatic lesions from the same patient, and the inability to detect the β-isoform of FR by IHC. This pilot study of 99mTc-EC20 scintigraphy indicates that the agent is safe and well tolerated and that this noninvasive procedure may have utility in selecting patients likely to benefit from FR-targeted therapy.
Key Words: folate receptor • solid tumors • diagnostic imaging agent • tumor imaging • 99mTc
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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Before facilitating any bioprocess, however, folate must gain entry into the cell, and in mammalian cells, entry proceeds via 2 distinct transport systems: the reduced folate carrier system, which is the primary method of folate entry into normal cells, and the folate receptor (FR) system. Although it has been noted that the FR glycoprotein is expressed in a minority of normal tissues, it is expressed at high levels on the surface of some epithelial tumors, such as ovarian cancer (2).
Studies evaluating over 8,000 tissue samples have shown that FR is overexpressed in cancers of the ovaries (>95% of samples tested exhibited FR expression at least 6 times greater than normal expression levels), mesothelium (
100%), lungs (>75%), kidneys (65%), endometrium (60%), breasts (>60%), and cerebrum (50%) and clinically nonfunctional pituitary adenomas (
80%). It has been hypothesized that the proliferative or metabolic state of malignant cell types causes the upregulation of FR to facilitate the entry of folate for accelerated synthetic and metabolic processes (3–15).
The overexpression of FR on tumor cells relative to that on normal cells provides a potential mechanism for targeted antitumor therapy (16). Various FR-targeted therapies intended to deliver potent cytotoxic antitumor agents to cells that selectively express FR are currently under development (17–19).
An important adjunct to the development of molecularly targeted therapies, including FR-targeted therapy, is the codevelopment of diagnostic tests to provide information on the presence or absence of the molecular target in question. Such diagnostic tests would allow for the "preselection" of individuals who might benefit from FR-targeted treatment and would likely play a critical role in the design and implementation of therapeutic trials. EC20 is a 99mTc-based folate-linked radiopharmaceutical that has been developed to identify patients whose tumors express FR. 99mTc-EC20 has the potential to provide the noninvasive detection of tissues expressing FR. 99mTc-EC20 targets FR with a mechanism that is identical to that used for targeting experimental therapeutic molecules (such as EC145 and EC0225) to the receptor.
The uptake of 99mTc-EC20 into solid tumors was assessed in a series of single-arm, open-label pilot studies to determine the percentages of patients whose tumors would show 99mTc-EC20 uptake; to determine the correlation of 99mTc-EC20 imaging results with the results of immunohistochemical (IHC) staining of tumor tissue for FR; and to provide information on the safety of a single injection of 99mTc-EC20 in patients with solid tumors.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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1.5 cm, as diagnosed by CT, MRI, or ultrasonography, and good renal function (serum creatinine level 
1.5 times the upper limit of the normal range) were enrolled in the study. Patients were asked to provide a formalin-fixed, paraffin-embedded tissue sample of the primary tumor or site(s) of metastatic or recurrent disease obtained by a previous surgical or biopsy procedure for the purpose of an IHC assay of FR (-isoform) or were asked to provide tissue obtained at surgery or biopsy scheduled after 99mTc-EC20 imaging. Patients were excluded from the study if they were participating simultaneously in the study of other investigational agents, had received an investigational agent or therapeutic chemotherapy within 7 d of enrollment in this trial, were unable to tolerate the conditions for radionuclide imaging, or had received another radiopharmaceutical that would have interfered with the assessment of 99mTc-EC20 distribution. Pregnant or breast-feeding women were also excluded from the study. The study protocol was approved by the institutional review board at each participating study center, and all patients gave written informed consent before participation.
Radiopharmaceutical Preparation
EC20 for injection was packaged as a sterile lyophilized powder in a single reagent vial containing the components EC20 (100 µg), sodium -D-glucoheptonate dihydrate (80 mg), and tin(II) chloride dihydrate (80 µg). 99mTc-EC20 was prepared by adding up to 1,850 MBq of 99mTc-sodium pertechnetate into the vial and heating the vial in a boiling water bath for 18 ± 2 (mean ± SD) min. The radiochemical purity of 99mTc-EC20 was determined by thin-layer chromatography (20) at the time of preparation and was required to be 90% for administration.
Imaging Procedure
Tracer dose and imaging time were chosen in part on the basis of previous studies of 99mTc-EC20 in humans (21). Pharmacokinetic studies showed that the disappearance of decay-corrected radioactivity from blood fits a 2-compartment model, with harmonic means of 25 min and 29 h. These data likely reflect rapid serum clearance and urinary excretion but slow movement of the tracer from a peripheral compartment back to the blood. On the basis of human biodistribution data, radiation dosimetry with 99mTc-EC20 is comparable to that with other diagnostic radiopharmaceuticals, with an average effective dose of 1.1 mrem/MBq and the largest absorbed dose of 9.2 mrem/MBq (to the kidneys).
Patients were instructed to discontinue taking folic acid supplements for at least 2 d before their scheduled imaging study. On the day of the imaging study, patients were administered 2 intravenous injections: 1 mg of folic acid (to reduce the uptake of 99mTc-EC20 in normal tissues) and, 1–3 min later, 0.1 mg of EC20 labeled with 555–925 MBq of 99mTc-pertechnetate over 30 s in a total injection volume of 1–2 mL. Each injection was given as a slow intravenous push via a free-flowing indwelling intravenous catheter in an upper-extremity vein (i.e., in the antecubital fossa). The mean administered activity was 851 MBq (range, 606.8–1,054.5 MBq). The mean activity injected per kilogram of body weight was 10.73 MBq (range, 4.81–19.98 MBq/kg).
At approximately 1–2 h after injection, anterior and posterior midthigh to head planar images were acquired in a 256 x 1,024 (minimum) matrix word-mode format with a total scan time of 20–35 min. The images were obtained with a dual-detector, large–field-of-view 
-camera that was equipped with low-energy, high-resolution parallel-hole collimators. A 20% symmetric energy window was centered over the 140-keV photopeak of 99mTc.
SPECT images of the region that was known to contain the target lesion(s), as identified by conventional imaging (CT, MRI, or ultrasonography), were obtained after planar imaging. The SPECT images included 120–128 total projections over 360° with 15–20 s per stop and were reconstructed in 3 orthogonal planes.
Image Interpretation
An on-site nuclear medicine physician, who was unaware of the outcome of the IHC assay, evaluated the planar and SPECT images for the uptake of 99mTc-EC20 in the identified lesions. The physician used the conventional imaging studies to identify up to 3 presumed malignant lesions of 1.5 cm (one dimension) for each patient and compared the uptake of 99mTc-EC20 in these lesions with the background. The uptake of 99mTc-EC20 in the lesions was scored as no uptake (less than or equal to background), mild uptake (slightly increased compared with background), or marked uptake (strikingly increased compared with background). A result of "no uptake" was scored as 99mTc-EC20 negative, whereas a result of "mild uptake" or "marked uptake" was scored as 99mTc-EC20 positive (EC20+). On the basis of the expected uptake observed in animal models and a phase I dosimetry study, the uptake of 99mTc-EC20 in the kidneys, liver, bladder, and spleen was assessed with the same 3-point scale as that used for malignant lesions. Additional areas of uptake were recorded when noted by the physician.
Tissue FR Assay
The formalin-fixed, paraffin-embedded tissue samples available from surgery or biopsy were analyzed for FR by an IHC assay specific for the -isoform of FR. Evaluation of the stained samples was accomplished in a semiquantitative manner with a light microscope to assess the degree of circumferential membranous staining in the tumor cell population. The findings were classified into 1 of 4 categories: 0 = no staining in tumor cells above background; 1+ = weak staining in 5% of tumor cells; 2+ = moderate staining in 5% of tumor cells; and 3+ = strong staining in 5% of tumor cells. A staining result of 0 was scored as IHC negative, whereas a staining result of 1+, 2+, or 3+ was scored as IHC positive.
Safety Assessment
Vital signs were obtained within 5 min of injection (baseline) and at 1 h after injection. Blood and urine specimens for clinical laboratories were obtained within 2 h of injection and at 22–28 h after injection. Treatment-emergent adverse events were monitored through the time of the patient's follow-up laboratory measurements at 22–28 h after injection. Additionally, the patients were instructed to report any adverse events that occurred up to 7 d after injection.
Statistical Methods
The number of patients characterized as having tumors with no uptake, mild uptake, or marked uptake of 99mTc-EC20 and the number of patients who had IHC staining results of 0, 1+, 2+, or 3+ were tabulated, and the percentage of patients who had EC20+ tumors, as determined by 99mTc-EC20 imaging (i.e., those whose tumors showed mild or marked uptake), and FR-positive (FR+) tumors, as determined by IHC staining (i.e., those who had a score of 1+, 2+, or 3+) were calculated. A 2 x 2 frequency table was constructed to compare the 99mTc-EC20 imaging and IHC staining results.
The overall percentage of agreement of the 2 methods, the agreement of positive 99mTc-EC20 imaging results and positive IHC results, and the agreement of negative 99mTc-EC20 imaging results and negative IHC results were calculated. A -coefficient with a 95% confidence interval (CI) was calculated to examine the agreement between the 2 methods (22,23).
Adverse events, clinical laboratory evaluations, and vital signs were summarized descriptively. Quantitative laboratory measurements were categorized according to the normal reference range (low, normal, and high), and shift tables of the frequencies for preinjection versus postinjection categories were prepared. Changes in laboratory values and vital signs that met predefined criteria for clinical significance were summarized.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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The mean time from the initial cancer diagnosis to the time of the 99mTc-EC20 scan was 45.2 mo (range, –0.8 to 221.3 mo), and the mean time from the most recent recurrence or progression to the time of the 99mTc-EC20 scan was 2.4 mo (range, –0.6 to 36.6 mo). A total of 155 patients completed the study (i.e., they received 99mTc-EC20) and had at least one scan (either planar imaging or SPECT).
Biodistribution
The percentage of patients with uptake in the liver, kidneys, bladder, spleen, and other organs are shown in Table 1. Physiologic (usually marked) uptake of 99mTc-EC20 was noted in the kidneys and bladder in almost all patients, consistent with the known expression of FR on the proximal tubules of the kidneys (24) and the urinary excretion of the tracer. Mild to marked uptake was also seen in the liver in all patients, causing hepatic lesions (when present) to appear as photopenic defects, even in patients with marked uptake in lesions elsewhere in the body. Hepatic uptake of 99mTc-EC20 has been seen in animal models (20); however, it can be partially blocked by inhibitors of organic anion transport, suggesting that it is mediated, at least in part, by mechanisms other than FR binding (C.P. Leamon, oral communication, July 2007). Uptake (usually mild) was frequently seen in the spleen and rarely in other organs not associated with the tumors, such as the thyroid, lymph nodes, bowel, stomach, and joints. Uptake in the thyroid, bowel, stomach, and choroid plexus was uncommon and likely was related to a small amount of free 99mTc-pertechnetate which was easily recognizable and had no appreciable effect on image interpretation. Spleen uptake is related to the known presence of the β-isoform of FR in normal splenic tissue, as well as the presence of FRs on activated macrophages (25,26). Uptake (usually mild) in bone marrow was observed in nearly all patients and was assumed to reflect the normal physiologic distribution of the tracer.
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-isoform of FR, 67% of these patients had FR+ tumors (Table 3). Overall, the 99mTc-EC20 image evaluations corresponded to the IHC staining results for 61% of the patients (Tables 4 and 5). The -coefficient was 0.096 (95% CI = –0.085 to 0.277), indicating poor chance-corrected agreement. The agreement of EC20+ results with FR+ results was 72%, whereas the agreement of 99mTc-EC20–negative results with FR-negative results was 38%.
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Approximately 30% of the patients were found to have mild to moderate uptake within numerous small foci in the inguinal and axillary regions. When present, these were always multifocal and bilateral. Lymph nodes in these regions were consistently considered normal on the basis of CT criteria.
Safety Results
A total of 17 adverse events were reported in 13 (8%) of the 155 patients receiving 99mTc-EC20. Only 3 events (lower abdominal pain in 1 patient and nausea and vomiting in 1 patient) were considered by the investigator to be "possibly related" to the study treatment. Adverse events in 7 patients resulted in hospitalization and, thus, were categorized as serious adverse events. None of the serious adverse events was considered by the investigator to be "related" to the administration of 99mTc-EC20.
No "clinically important effects" were observed with regard to hematology, serum chemistry, or urinalysis, as judged on the basis of the mean change in values from before injection to after injection of 99mTc-EC20. No systematic shifts from normal values before injection to abnormal values after injection were observed for any laboratory test, and none of the patients had postinjection laboratory values that the investigator considered to be clinically significant. No "clinically important effects" were observed for heart rate or for systolic or diastolic blood pressure at 1 h after injection of 99mTc-EC20.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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A single injection of 99mTc-EC20 was well tolerated. Only 8% of patients experienced an adverse event during administration and the follow-up period, and only 3 adverse events were considered to be "possibly related" to the injection. No drug-related serious adverse events occurred, and no clinically important effects of 99mTc-EC20 on laboratory assays or on the monitoring of vital signs were observed.
The results of the present study of 154 patients with a variety of solid tumors demonstrated that 68% of the patients showed uptake of 99mTc-EC20 in their tumors (i.e., EC20+ tumors). In comparison, IHC staining for the -isoform of FR in 123 patients demonstrated that 67% of these patients had FR+ tumors. In all patients, the correlation of the 99mTc-EC20 imaging results with the IHC staining results was 61%, although there was better agreement for positive imaging and staining results than for negative imaging and staining results (72% vs. 38%). Chance-corrected agreement between 99mTc-EC20 imaging results and IHC staining results was poor ( = 0.096; 95% CI = –0.085 to 0.277).
When considering the relatively poor agreement between the results of 99mTc-EC20 imaging and IHC, one must recognize that the present study was not designed as a lesion-to-lesion comparison of 99mTc-EC20 imaging and IHC. In most cases, the tissue samples available were limited to the primary neoplasm (often obtained months or years before study entry), and scanning was performed in patients with metastatic disease after resection of the primary neoplasm. Surgical removal or biopsy and IHC analysis of all of the metastatic lesions imaged in the present study would have been scientifically preferable but was not performed for practical and ethical reasons. Furthermore, the clinical relevance of such data is limited because of differences in the types of FRs detected by IHC and imaging, as discussed in the next paragraph.
Discrepant results for the 2 methods may reflect a difference in the FR status of the primary neoplasm versus the metastatic disease after excision of the primary tumor or heterogeneity in FR expression between metastatic lesions within a patient. In fact, evidence supporting both of these hypotheses exists. Studies comparing FR expression with tumor stage have suggested that FR expression is increased in more advanced (i.e., metastatic) disease (11). In the present study, 42% of patients with renal cell carcinomas had a heterogeneous mixture of lesions, including some with no uptake and others with mild or marked uptake. An additional consideration is that only 1 of the 2 FR isoforms (-isoform) is detected by IHC, and IHC may detect nonfunctional as well as functional receptors. In contrast, 99mTc-EC20 is capable of recognizing and binding to both the -isoform and the β-isoform of FR and only binds functional receptors that are accessible to the imaging agent. All of these issues suggest that 99mTc-EC20 imaging may be a more accurate means of predicting the response to FR-targeted therapy than is IHC, particularly IHC performed on the primary tumor. The ultimate test of this hypothesis will require a therapeutic clinical trial in which the response to FR-targeted therapy is correlated with 99mTc-EC20 scan results. Such a trial is already under way.
Other explanations for the lack of correlation include the possibility that uptake, particularly in larger and more destructive lesions, may have been secondary to associated inflammation, an observation consistent with the presence of FRs on the surface of activated macrophages (26) and the localization of 99mTc-EC20 uptake in the joints of patients with active rheumatoid arthritis (27). Finally, the high background activity found in the liver precluded an assessment of the FR status of hepatic metastases, as all metastases were either undetectable or photopenic.
Few previously unknown sites of metastatic disease were discovered on the scans. However, the present study was not designed to test the usefulness of 99mTc-EC20 in improving the staging of renal cell carcinomas or other cancers. The majority of patients enrolled in the present study already had known distant metastases; therefore, staging was not likely to be altered. A study addressing this topic would best be performed with patients who have lower stages of disease.
An interesting incidental finding was that of mild to moderate uptake within numerous foci in the inguinal and axillary regions bilaterally in some patients. Although the basis and clinical relevance of this uptake are uncertain, the uptake may identify lymph nodes harboring activated macrophages, known to express FRs.
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CONCLUSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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99mTc-EC20 imaging is a safe, noninvasive procedure that may identify FRs in recurrent or metastatic disease without the need for biopsy and that may have utility in identifying patients who may benefit from treatment with FR-targeted therapy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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References |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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