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Clinical Investigation |
1 Department of Radiology, University of Washington/Seattle Cancer Care Alliance, Seattle, Washington; 2 Department of Pathology, University of Washington/Seattle Cancer Care Alliance, Seattle, Washington; 3 Phenopath Laboratories, Seattle, Washington; and 4 Department of Nuclear Medicine, University of Iowa, Iowa City, Iowa
Correspondence: For correspondence or reprints contact: David A. Mankoff, MD, PhD, Department of Radiology, Seattle Cancer Care Alliance, 825 Eastlake Ave. East, Mail Stop 62-600, Seattle, WA 98109. E-mail: dam{at}u.washington.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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Key Words: estrogen receptor • breast cancer • 18F-FES PET • IHC • tracer uptake
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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Studies have shown that PET with 18F-fluoroestradiol (18F-FES) can be used to noninvasively assess regional ER expression (10,11) and has the potential to overcome the sampling errors that arise from disease heterogeneity and that are associated with in vitro assays for cancer. PET can simultaneously measure the in vivo delivery and binding of estrogens and thus ER expression at multiple tumor sites. Earlier studies showed that 18F-FES uptake at a tumor site correlates with ER expression assayed in vitro by radioligand binding (11) and that the level of uptake predicts the likelihood of a response to tamoxifen and aromatase inhibitor treatment (12–14). In this study, we extended this work to compare 18F-FES uptake with ER expression assayed in vitro by IHC, the method currently used for making clinical therapy decisions at most centers (9,15,16). We also report alternate measures of 18F-FES uptake that account for 18F-FES blood clearance to account for rapid 18F-FES metabolism and variable 18F-FES blood levels (17). Our hypotheses were that 18F-FES uptake correlates with ER expression measured in vitro as a semiquantitative index of ER immunostaining and that measures of 18F-FES uptake accounting for blood clearance or metabolism may improve the correlation with in vitro assays of ER expression.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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An additional 21 patients who underwent both tissue sampling and an 18F-FES scan were considered for inclusion. They were excluded from the analysis because the tissue samples were outside the field of view of the 18F-FES study (n = 4), the tumor was too small (<1.5 cm) to accurately quantify with PET (n = 2), no tumors were present in the tissue samples (n = 4), the patients had intervening breast cancer treatment of several weeks or more between the scan and the biopsy (n = 8), the specimens were from lung cancer patients with earlier breast cancer and possible lung metastases (n = 2), or the specimen was a vertebral lesion (n = 1) with too few tumor cells present for a histologic assay of ER expression.
Tissue was sampled from the breast (n = 12), lymph nodes (n = 1), axilla (n = 2), sternum (n = 1), and chest wall (n = 1). The mean tumor size was 3.3 cm (range = 1.5–12 cm).
18F-FES Synthesis and Quality Control
Reagents and solvents for synthesis and purification were obtained from Aldrich Chemical Co. or from U.S. Pharmacopeia–approved suppliers and were used without further purification unless otherwise noted. 18F was produced by use of either a Siemens Eclipse or a Scanditronix MC-50 cyclotron. 18F-FES was prepared according to modifications of published methods (20). A typical injection consisted of approximately 222 MBq of radiopharmaceutical in 20 mL of isotonic phosphate-buffered saline containing less than 15% ethanol by volume.
High-performance liquid chromatography–mass spectrometry analysis [Waters 2690 and MicroMass ZMD (ES–)] was performed immediately after each synthesis to evaluate radiochemical and chemical purities and to measure the mass of 18F-FES in the dose. In all instances, the radiochemical purity was found to be 
98%. The specific activity was typically greater than 37 x 1012 Bq/mmol at the time of injection. In no instance was more than 5 µg of 18F-FES injected.
PET
All patients were positioned supine and imaged with a GE Healthcare Advance PET scanner. Performance details for the tomograph were previously reported (21,22). Calibration of the PET scanner was routinely done by imaging vials containing a known quantity of 18F assayed in a dose calibrator (model CRC-12; Capintec). The vial images were reconstructed with the same reconstruction parameters as those used in the patient studies to allow cross-calibration of MBq/mL from region-of-interest (ROI) analysis of the images.
Before PET, patients had intravenous catheters placed for radiopharmaceutical injection and for blood sampling. Using short transmission scans, patients were placed in the tomograph in a position to view a known tumor site for which biopsy results were available for comparison. Chest sites were preferred to enable image-based measurement of the arterial blood clearance curve by use of the left ventricular blood pool (23). A 20- to 25-min transmission scan was performed for attenuation correction. 18F-FES at 125.8–233.1 MBq (3.4–6.3 mCi) (average = 196.1 MBq [5.3 mCi]) was infused over 2 min in a volume of 20 mL. Dynamic emission imaging began with the start of infusion and continued for 60 min with the following time bins: 4 x 20 s, 4 x 40 s, 4 x 60 s, 4 x 180 s, and 8 x 300 s. Data obtained at 30–60 min were summed to measure the 18F-FES standardized uptake value (SUV) and to aid in drawing ROIs.
Plasma Processing for Metabolite Analysis
Samples of whole venous blood were collected from patients at 5, 20, and 60 min after injection. The blood was centrifuged at 3,000 rpm for 5 min, and the plasma was withdrawn. The plasma was used for high-performance liquid chromatography metabolite analysis to determine blood radioactivity present as 18F-FES versus labeled metabolites as previously described (17).
PET Image Analysis
ROIs were placed over the central portion of the tumor site by reference to an 18F-FDG PET scan, correlative anatomic imaging (mammogram, ultrasound, or CT), and the PET transmission scan. Square ROIs (1.5 cm in diameter) were placed over the center of the tumor site on 3 adjacent imaging planes by use of 18F-FES and 18F-FDG PET images as guides. We used a similar approach in earlier PET studies as a compromise between partial-volume effects seen with larger regions and the high statistical image noise experienced with smaller regions (18). For patients undergoing core needle biopsy, the site of the biopsy was considered in placing the ROIs. Three adjacent 1.5-cm ROIs were placed over the left ventricle to measure the blood clearance curve. We previously showed that this approach results in a blood clearance curve comparable to that obtained by arterial blood sampling (24).
The estimated uptake of 18F-FES at the tumor site was corrected for partial-volume sampling by use of tumor sizes obtained from the anatomic imaging study (mammogram, ultrasound, or CT) that best depicted the tumor boundaries. As in earlier studies (19), the partial-volume-corrected (PV) uptake was calculated with the following expression:
 | (Eq. 1) |
Tumor 18F-FES uptake was quantified by use of 3 different uptake measures: the average SUV of the hottest portion of the tumor (SUV), total flux (fluxtot), and flux corrected for labeled metabolites (fluxcorr). The SUV was calculated by a commonly used formula:
 | (Eq. 2) |
is the average tissue uptake at 30–60 min after injection, ID is the injected dose (in MBq), and wt is the patient weight (in kilograms).
The fluxtot measure of 18F-FES uptake, which accounts for variable 18F-FES blood clearance, was defined as follows:
 | (Eq. 3) |
 | (Eq. 4) |
IHC Analysis
Tissue blocks from sections of the 18F-FES-imaged tumors were prepared, sectioned in 4- to 5-µm-thick slices, and placed on glass slides. IHC of tumor samples was performed with monoclonal antibody 1D5 (M 7047/JA/03.03.99; Dako), directed against the ER. All reagents were purchased from Dako, and testing was done by use of a Dako autostainer. All samples were processed in a single batch with a positive control. For each sample, a pathologist, unaware of the 18F-FES results, identified 3 areas on each slide for digital photography and storage. All photomicrographs were recorded with the same magnification and lighting to minimize differences. The results of the immunoassay for ER were qualitatively assessed by a pathologist using a scale from 0 to 3+, with a sample being reported as positive if greater than 5% of the cells in the sample were positively immunostained. If a discrepancy in the score existed between the original pathology report at the time of the surgery and the score determined from the digital photographs (n = 2), then the photographs were reviewed by another pathologist. The samples were also scored by a pathologist using the Allred scoring system, which was recently adopted as the standard method of reporting ER staining at the University of Washington (7,27). Each photomicrograph was evaluated, and the score for the 3 photomicrographs was averaged to produce one value per patient.
In addition, a semiquantitative index of ER staining (IHC index) was calculated by use of a previously described automated image analysis technique and was verified by comparison with a radioligand-binding assay (Photoshop; Adobe Systems) (28,29). In brief, the Photoshop Magic Wand tool was used to identify a representative cell and subsequently cells with a similar intensity. The histogram command was then applied to obtain the mean optical density, which was recorded as the mean value for the tumor. The inverse area was then selected, and the histogram mean density was recorded as the background. The final value recorded was the background minus the mean.
To test the variability of the IHC index, which has not yet been reported, we sent the digitized photographs to 2 independent reviewers for analysis. Each reviewer repeated the procedure 3 times on each of 3 photomicrographs, selecting a different initial cell. The 3 readings on each photomicrograph were averaged, and the averages for the 3 photomicrographs were then averaged to produce one value. The latter values from the reviewers were then averaged to obtain the final quantitative value.
Statistical Analyses
Although partial-volume correction adds complexity to the estimation of 18F-FES uptake, the intent of our study was to validate 18F-FES uptake as a measure of ER expression in comparison with ER expression determined by IHC independent of tumor size. Because of the expected influence of partial volumes for smaller tumors, partial-volume corrections were necessary to accurately measure 18F-FES uptake. Therefore, only PV 18F-FES uptake values were compared with the ER expression results. The measures of 18F-FES uptake (PV SUV [PVSUV], PV fluxtot [PVFES fluxtot], and PV fluxcorr [PVFES fluxcorr]) were compared with the measures of ER expression (qualitative ER scoring, Allred scoring, and IHC), and the results were reported by use of Spearman correlation coefficients (
). The correlation between 18F-FES uptake and the semiquantitative IHC index was also evaluated by use of a linear bivariate fit.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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Two patients had short-term exposure to antitumor agents before the 18F-FES scan and biopsy. One patient began aromatase inhibitor treatment less than 1 mo before the 18F-FES scan and biopsy. One patient received several doses of oral cyclophosphamide and 1 wk of fractionated radiation between the 18F-FES scan and surgery.
Blood was collected from all but one patient, for whom PVFES fluxcorr therefore could not be calculated. Thus, data from 16 patients were included in the PVFES fluxcorr analyses, and data from 17 patients were included in all of the other analyses.
IHC Index Observer Variability
Our analysis of the interobserver variability of the IHC index showed excellent agreement between the 2 observers who used the automated analysis to calculate ER uptake measured by IHC, as shown in Figure 1. The r value was 0.99 (P < 0.001), and the average difference between observers was one percentage point (coefficient of variation = 6%).
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18F-FES uptake measures were compared with qualitative ER uptake measured by IHC (Fig. 3). For 18F-FES uptake versus qualitative IHC results, = 0.62 (P = 0.009) for PVSUV, = 0.57 (P = 0.02) for PVFES fluxtot, and = 0.60 (P = 0.01) for PVFES fluxcorr. For non–PV SUVs versus qualitative IHC results, = 0.52 (P = 0.03). IHC indicated that 5 patients had ER-negative tumors and 12 patients had ER-positive tumors (3 with 2+ immunostaining and 9 with 3+ immunostaining); no patient in the series had 1+ immunostaining. With the exception of one patient, all patients with ER-negative tumors had PVSUVs of less than 1.0, whereas patients with ER-positive tumors had values above 1.1. When an SUV of 1.1 was used to discriminate ER-positive and ER-negative tumors, there was 94% agreement (16/17 patients) for 18F-FES uptake versus IHC results.
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Similar plots were seen for PVFES fluxtot, with ER-negative tumors having values of less than 0.01 mL/min/g (Fig. 3B), and for PVFES fluxcorr, with ER-negative tumors having values of less than 0.02 mL/min/g (Fig. 3C); again, the exception was the same patient with a higher SUV.
A patient found to be ER positive by IHC had a PVSUV of 1.1 and low PVFES fluxtot and PVFES fluxcorr values, 0.016 and 0.021 mL/min/g, respectively. Her tumor had a qualitative ER score of 3+ and an Allred score of 7. This patient had stopped hormone replacement medication approximately 1 wk before her 18F-FES scan. Her estradiol level was normal, at 28 pg/mL, at the time of the 18F-FES scan. Although her hormone replacement medication may have competed with 18F-FES uptake, this possibility is considered unlikely given the relatively low level of plasma estradiol at the time of scanning. The difference between 18F-FES PET results and in vitro assay results more likely reflects a difference between immune recognition (IHC) and functional binding, which has been reported for in vitro assays (7,30,31).
18F-FES uptake and semiquantitative Allred scores were compared as shown in Figure 4. The values for 18F-FES uptake versus Allred scores (PVSUV, PVFES fluxtot, and PVFES fluxcorr) were 0.72 (P = 0.001), 0.61 (P = 0.01), and 0.60 (P = 0.01), respectively. For non–PV SUVs versus Allred scores, = 0.65 (P = 0.005).
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= 0.73 (P < 0.001) for PVSUV, = 0.63 (P = 0.007) for PVFES fluxtot, and = 0.57 (P = 0.02) for PVFES fluxcorr. Correlation coefficients were not significantly different for the 3 measures when a linear bivariate fit was used: r = 0.64 for PVSUV, r = 0.63 for PVFES fluxtot, and r = 0.62 for PVFES fluxcorr. P values were 0.005 for PVSUV, 0.007 for PVFES fluxtot, and 0.01 for PVFES fluxcorr. For non–PV SUVs versus the IHC index, r = 0.35 (P = 0.17).
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include all patients.
To examine the possible influence of short-term exposure to noninterfering breast cancer therapy, we excluded the 2 patients with short-term exposure to either aromatase inhibitors or chemotherapy and radiation therapy at the time of the 18F-FES PET scan and biopsy. Correlations between 18F-FES uptake and IHC remained in this subset of patients (n = 15): for PVSUV versus qualitative IHC, = 0.60 (P = 0.02); for PVSUV versus Allred scores, = 0.66 (P = 0.007); and for PVSUV versus the IHC index, = 0.68 (P = 0.006).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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Three different methods of measuring ER expression by IHC were available on the same tissue slides for comparison with 18F-FES uptake. Initial ER scoring was a qualitative assessment of the slides, and they were given scores of 0–3+ on the basis of the intensity of the nuclear staining. In clinical practice, a score of 1+ or higher is taken to indicate a tumor that may be sensitive to endocrine therapy (8). We also used a commonly known method of semiquantitative evaluation, the Allred method, which includes an 8-point scoring system based on the intensity and percentage of positively stained cells on a slide (7,27). To take advantage of the quantitative nature of PET data with which the IHC results were compared, we applied a semiquantitative computerized analysis method (Photoshop) that we have used before (28). Although this method is not routinely used to assess ER expression in clinical practice, its reproducibility was excellent. The results of similar computerized analyses were previously tested and compared with the results of in vitro radioligand-binding assays for ER expression (32,33), like the method that we used (28). All methods of scoring ER expression by IHC correlated well with each other, as expected.
In 1988 (11), Mintun et al. reported good correlations between 18F-FES uptake and in vitro ER concentrations determined by radioligand binding. In 1995, Dehdashti et al. (34) compared the separation of ER-positive and ER-negative cancers with 18F-FES uptake in tumors and reported an overall agreement of 88%. There has been a shift in clinical practice from using radioligand binding to using IHC for routine ER content analysis of breast cancers (15). Several studies included comparisons of in vitro radioligand-binding assays for ER expression with IHC assays for ER expression (7,16,33,35). These studies showed that the agreement between the 2 methods ranged from 86% to 98%. Our rate of agreement between the 18F-FES uptake and IHC methods fell in this range.
In our earlier study comparing in vitro radioligand-binding assays and the IHC index, we found a linear correlation (r) of 0.70 (28). Other studies including quantitative comparisons of radioligand-binding assays and IHC found similar IHC indices (30,31,36–38). The correlation () between 18F-FES uptake and the IHC index that we found in the present study, 0.73 (linear correlation [r] of 0.64), fell in the range of values reported in earlier studies comparing in vitro assays.
Our correlations, however, were lower than the correlation reported by Mintun et al. (11), r = 0.96. Several factors could account for the differences in the results. There was a more uniform distribution of ER expression in the in vitro assays in our study than in the study of Mintun et al. (11); this difference could have altered the apparent correlation between the imaging and in vitro results. In addition, in the earlier study, in vivo radioligand binding (18F-FES) was compared with in vitro radioligand binding; however, in the present study, in vivo radioligand binding was compared with the presence of ER epitopes determined by in vitro IHC. We would expect a lower correlation for our present study (18F-FES uptake versus in vitro IHC) than for the study of Mintun et al. (11) (18F-FES uptake versus in vitro radioligand binding), in accordance with studies comparing in vitro radioligand-binding assays and in vitro IHC assays (27,30,31). Given the differences in sampling and quantification for in vivo imaging versus in vitro assays, we would not necessarily expect a perfect correlation in any instance. Importantly, in vivo measurement of ER expression by 18F-FES PET appeared to add predictive capability for the response to endocrine therapy, even in patients whose tumors were shown to express ER in in vitro assays (12–14).
We compared different measures of 18F-FES uptake and ER concentrations, including PVFES fluxtot and PVFES fluxcorr. In the present analysis, in distinction to our earlier, preliminary analysis (18,39), the flux measures did not have a clear advantage over the simple SUV measure of 18F-FES uptake. However, in studies of patients treated with agents that could alter estrogen levels or 18F-FES clearance rates, the flux measures may be important to consider.
We identified a PVSUV threshold of 1.1 for distinguishing tumors determined to be ER positive and ER negative by IHC. Our earlier studies (14) and others (13) suggested that an 18F-FES SUV of 1.5–2.0 (or greater) is predictive of a response to endocrine therapy. The IHC threshold for ER positivity is staining in 5% of nuclei or more. Other researchers, for example, Ogawa et al. (40), indicated that 10% staining may be an acceptable cutoff for predicting a response to endocrine therapy. Taken together, these results suggest that the quantitative threshold for the determination of endocrine responsiveness by 18F-FES PET may be slightly higher than the threshold for correlation with standard IHC criteria for ER positivity.
There are inherent difficulties in comparing in vitro and in vivo parameters, including the size of the tissue sample. ER distribution may be heterogeneous throughout a lesion, and a tissue sample may not be representative of the entire lesion. This was likely the case with one of the outliers in the present study; for this patient, the initial ER status, determined from her surgery, was scored as 3+, but the slides subsequently made for analysis were scored as ER negative.
We are unable to conclusively explain an outlier with low 18F-FES uptake in the group of patients found to have ER-positive tumors by IHC; this finding may simply reflect inherent differences between radioligand-binding assays for ER (18F-FES PET) and immunorecognition (IHC).
The present study has several limitations. Although the sample size (n = 17) is the largest used to date to compare 18F-FES uptake with in vitro IHC analysis of ER expression, the patient population is still small. Additional samples would likely increase the significance of the correlation of quantitative measures of ER expression with 18F-FES uptake.
The patient population was heterogeneous, with both male and female patients, newly diagnosed and recurrent breast cancers, a variety of types of previous therapy, tissue samples from different tumor sites, and 18F-FES studies done at various intervals from the time of tissue sampling. Two patients had short-term exposure to agents with activity against breast cancer but not known to block estradiol binding to ER. This exposure did not appear to affect the correlation of 18F-FES uptake with IHC of the biopsy material; however, this feature was not rigorously tested.
The need for partial-volume correction of 18F-FES uptake in smaller tumors is another limitation of the present study. Although recovery coefficients are necessary to account for the loss of signal attributable to finite spatial resolution, this correction is an approximate one based on an imperfect estimate of tumor size and likely adds variability to the 18F-FES uptake measures.
Despite these potentially confounding factors, the correlation between 18F-FES uptake and ER expression remained significant and at the expected level.
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CONCLUSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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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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-18F-fluoro-17β-estradiol in breast cancer: correlation with estrogen receptor status and response to systemic therapy. Clin Cancer Res. 1996;2:933–939.[Abstract]-[18F]-fluoroestradiol (FES) with sex steroid binding protein (SBP). Nucl Med Biol. 1999;26:905–913.[CrossRef][Medline]Related articles in JNM:
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