Exploratory Study of ^99mTc-EC20 Imaging for Identifying Patients with Folate Receptor–Positive Solid Tumors

Patients

Eligible patients were at least 18 y of age and had known or suspected metastatic renal cell carcinoma (protocols EC20.3 and EC20.9), pituitary adenoma (protocol EC20.7), solid tumors (protocol EC20.8), or ovarian carcinoma or recurrent endometrial carcinoma (protocol EC20.4). Patients with at least one identifiable lesion of ≥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 (α-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 α-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 × 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 γ-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 α-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 × 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 κ-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.

Patients

A total of 155 patients were enrolled and underwent imaging with ^99mTc-EC20 at 6 centers between 15 April 2003 and 31 August 2005. One patient later withdrew his consent for his scan to be included in the study and therefore was included only in the safety data presented here, not the imaging data. One patient received a second administration of ^99mTC-EC20 approximately 77 d after his initial imaging to assess uptake after resection of his primary tumor. This patient's data for both assessments were included in assessments of efficacy and safety but were counted only once for the determination of baseline demographics. The study population was primarily white (92%) and male (64%) and ranged in age from 32 to 80 y (mean, 57.8 y). The tumor types were primarily renal cell carcinomas (119 patients), benign ovarian tumors (8 patients), ovarian carcinomas (7 patients), breast carcinomas (6 patients), and pituitary adenomas (6 patients). The remaining 8 patients had small cell lung carcinoma; lung carcinoma (type unspecified); colon, endometrial, and thyroid carcinomas; non-Hodgkin's lymphoma; sarcoma; and glioma (1 patient each). Approximately 88% of patients had metastatic disease at the time of study entry, with the lungs (74%), lymph nodes (45%), liver (29%), and bone (23%) representing the primary sites of metastasis.

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.

Efficacy Results

^99mTc-EC20 images were evaluated for 154 patients; conventional images for the identification of target lesions were unavailable for 1 patient for comparison with the ^99mTc-EC20 images. Of the 154 patients who were evaluated, 105 (68%) had at least one metastatic site that showed uptake of ^99mTc-EC20 on either planar imaging or SPECT and were therefore classified as having EC20+ tumors (Table 2). Of the 105 patients who had EC20+ tumors, 57 (54%) had tumors that showed marked uptake of ^99mTc-EC20 and 48 (46%) had tumors that showed mild uptake. A total of 374 lesions were evaluated by planar imaging, and 369 lesions were evaluated by SPECT. When assessed by planar imaging, 141 lesions (38%) showed ^99mTc-EC20 uptake, with 66% of these showing mild uptake and 34% showing marked uptake. When assessed by SPECT, 185 lesions (50%) showed ^99mTc-EC20 uptake, with 58% of these showing mild uptake and 42% showing marked uptake. Figure 1 shows representative whole-body planar scintigrams, SPECT images, and corresponding CT images that exhibit the localization of ^99mTc-EC20 in a radiographically demonstrable chest wall metastasis. Figure 2 shows marked uptake in a metastasis in the paraspinal muscles. Figures 1 and 2 also demonstrate subtle uptake in other, smaller metastases.

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FIGURE 1. 

Anterior and posterior whole-body planar (A) and transverse SPECT (B) ^99mTc-EC20 images in 59-y-old man with renal cell carcinoma revealed large, destructive right chest wall metastasis (arrow). Scintigraphic findings correlated well with those on CT image (C), including photopenia in area of central necrosis. These scintigraphic results suggested that lesion was FR+. Note faint uptake in left renal fossa, thought to represent postsurgical inflammation. Mild uptake in right lung and right paratracheal region corresponded to other sites of metastatic disease demonstrated on CT (not shown). Small foci of mild ^99mTc-EC20 uptake were noted in inguinal regions bilaterally; this finding was seen in many patients and may be secondary to FRs on activated macrophages.

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FIGURE 2. 

^99mTc-EC20 images of 57-y-old man with known pulmonary metastases from renal cell carcinoma. Anterior and posterior whole-body planar images (A) revealed faint uptake of tracer in right upper-lobe pulmonary nodule (1.8 × 2.1 cm) (thin arrow) that was demonstrated on CT (not shown). More intense tracer uptake was seen in mass (2.7 × 3.5 cm) (thick arrow) in left paraspinal muscles. Planar images also revealed faint bilateral inguinal nodal uptake of ^99mTc-EC20. Paraspinal mass also was demonstrated on transverse SPECT image (B) and corresponding CT image (C) (arrows).

Of the 155 patients who completed the study, 123 had samples available for IHC staining. As determined by IHC staining for the α-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 κ-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%.

Fewer than 5% of patients had metastatic disease detected by ^99mTc-EC20 imaging that was not detected by CT. None of these additional sites of metastatic disease would have changed the staging of the tumors (other metastatic sites had been detected by CT).

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.