A method to detect fulvestrant interference in estradiol in breast cancer patients

Background Fulvestrant resembles estradiol in its structure. Reports have been published concerning fulvestrant measured as estradiol by the immunoassays. This may induce falsely elevated estradiol results and wrongly impact medical decisions. Our aim was to confirm the interference of fulvestrant on estradiol concentration and test a method to identify the false results. Methods Four serum samples with low estradiol levels were spiked with fulvestrant at various concentrations. Estradiol was then measured directly on serum (Dir), after a 1:5 dilution (Dil), and a ratio Dil/Dir was estimated. On the second part of the study, estradiol results (Dir, Dil and ratio Dil/Dir) from 14 women treated with fulvestrant were analysed, as well as from 14 patients not under this treatment. Results The addition of exogenous fulvestrant to the serum samples induced a gradual rise on estradiol concentration with a mean ratio for the Dil/Dir samples of 2.1 ± 0.4 (range 1.7–2.9). Patients on fulvestrant treatment experienced a mean ratio for the Dil/Dir estradiol sample of 2.4 ± 0.4 (range 1.6–3.0). In the control group, a mean estradiol ratio Dil/Dir of 1.1 ± 0.1 was observed (range 0.8–1.3). No correlation between the number of days after fulvestrant injection and estradiol result (r = 0.531) was observed. Conclusion Our study confirmed the interference of fulvestrant in the estradiol measurement by immunoassay. When fulvestrant was present, the estradiol ratio for Dil/Dir sample was about 2. In the control group, the ratio was around 1. The estradiol Dil/Dir ratio is a simple tool which can be used to identify fulvestrant false immunoassay estradiol results.


Introduction
Fulvestrant was the first selective estrogen receptor degrader (SERD) (1) to enter clinical practice and represents, alone or in combination with targeted therapies, a key compound for endocrine advanced breast cancer (ABC).As a drug, it competes with estradiol with high affinity to the estrogen receptor (ER); the complex fulvestrant-ER causes an unstable conformational change with ER degradation and inhibition of estrogen signalling (2).
Fulvestrant was mainly studied in postmenopausal patients (3).Yet, the recent positive results from the trials PALOMA 3 (4) and MONARCH 2 (5) in the ABC premenopausal groups ER+ HER2− have come to support its generalized use in these patients in association with CDK4/6 inhibitors and also in monotherapy.
In premenopausal patients, fulvestrant requires the combination with a gonadotropin-releasing hormone (GnRH) agonist therapy for ovarian suppression (OS) (6).However, studies show that 17-24% (7,8) of premenopausal women do not experience complete OS with GnRH agonists therapy and, although still not clear, this may contribute to poorer outcomes (7,9,10).Some authors (7,8) suggest that estradiol measurement should routinely be carried out in these patients to ensure that they are indeed fully castrated.
Fulvestrant resembles estradiol in its molecular structure (Fig. 1) leading to a cross-reactivity in estradiol immunoassays (11).A few case reports (12,13,14) have been published concerning fulvestrant measured as estradiol by the commercial immunoassays and inducing falsely elevated estradiol results.This is a problem for clinicians and laboratory staff and may wrongly impact medical decisions.
Our aim was to confirm the interference of fulvestrant on estradiol concentration and test a simple laboratory method to identify the false results.

Study design
This was a single-center, observational study with retrospective data collection, separated into two parts.Initially, four samples of serum with low estradiol levels, obtained from our laboratory serum bank, were spiked with fulvestrant at various concentrations.A serum sample with low estradiol concentration (16.5 pg/mL, sample 1) was aliquoted and spiked with fulvestrant 200 ng/mL to obtain the final concentrations of 4, 8, 12, 16, 20, 24, 28 and 32 ng/mL.These concentrations were chosen because, after administration, fulvestrant is slowly absorbed and maximum plasma concentrations are reached after approximately 5 days.At steady state, fulvestrant plasma concentrations are between 16.3 and 25.1 ng/mL (15).The three remaining serum samples had respectively estradiol concentrations of 22.3 pg/mL (sample 2), 40.1 pg/mL (sample 3) and 56.1 pg/mL (sample 4) and were also aliquoted and spiked with fulvestrant 200 ng/mL to get the concentrations of 8 and 20 ng/mL.Estradiol was measured on each aliquot, directly (Dir) and, to confirm results, after a 1:5 onboard dilution (Dil) with estradiol diluent (the 1:5 dilution factor was already pre-defined by the Centaur CP system assay for estradiol).To evaluate the proportion between the two measurements, a ratio Dil/Dir was also estimated.
On the second part of the study, we reviewed the medical records and analysed the estradiol results (Dir, Dil and ratio Dil/Dir) from 14 women with stage IV hormonereceptor-positive HER 2 negative advanced breast cancer treated with fulvestrant in our center.The estradiol levels of 14 female patients not under this treatment, previously measured for routine in our laboratory, were used as a control group.
The study was approved by our Institutional Ethics Committee.Due to the retrospective nature of the study, and because adequate measures to protect data confidentiality were provisioned, permission to waive informed consent was given.

Fulvestrant
A pre-filled syringe of commercial fulvestrant (250 mg/5 mL, EVER Valinject/Kent Pharma, Heathrow, UK) was used to prepare a solution of 200 ng/mL in ethanol (Merck).

Estradiol measurement assay
Estradiol was measured by a competitive chemiluminescent immunoassay (Enhanced Estradiol, Centaur CP, Siemens Healthcare Diagnostics Inc, Tarrytown, NY, USA).The test range is 10.7-3000 pg/mL, the limit of blank is 7.9 pg/mL and the limit of detection is 10.7 pg/mL.The intra-and inter-assay precisions are 9.2% and 4.5%, respectively, at a concentration of 39.3 pg/mL.The reference interval for considering post-menopausal females cutoff is <32.2 pg/ mL (16).The definition for complete ovarian suppression while on a GnRH agonist is not clearly defined (8).We used the estradiol cut-off levels for naturally postmenopausal women.

Statistical analysis
Data were analysed as mean ± s.d. and range.The Pearson correlation coefficient was used to evaluate the correlation between the number of days after fulvestrant injection and estradiol level.

Results
The addition of fulvestrant (0-32 ng/mL) to sample 1 induced a gradual rise in estradiol concentration, from 16.5 pg/mL (basal) to 930 pg/mL, corresponding to an increase of 5636%.A similar behavior was observed for samples 2, 3, and 4 (Table 1).In the spiked samples, the estradiol level of the 1:5 diluted sample, compared with the direct one, is about two times higher.The mean estradiol ratio obtained for the Dil/Dir samples is 2.1 ± 0.4 with a 1.7-2.9range.
In the second step of the study, we analysed 27 estradiol samples (Dir, Dil and ratio Dil/Dir) from   14 patients under fulvestrant therapy (patients 1 to 14).The days between fulvestrant injection and blood collection as well as the estradiol level prior fulvestrant therapy are shown in Table 2, when available.The patients under fulvestrant therapy had an estradiol level between 116 and 838 pg/mL.The estradiol ratio Dil/Dir ranged from 1.6 to 3.0 with a mean value of 2.4 ± 0.4.Prior to the beginning of fulvestrant treatment, these women exhibited a low estradiol concentration (≤46 pg/mL).We observed no correlation between the number of days after fulvestrant injection and estradiol result (r = 0.531).The data are summarized in Table 2.
In the group of patients not under fulvestrant (controls 1 to 14), the estradiol concentration was between 98 and 1444 pg/mL with a mean ratio Dil/Dir of 1.1 ± 0.1 (range 0.8-1.3)(Table 3).

Discussion
The chemical structure similarity between estradiol and fulvestrant makes it impossible for the antibodies used in the immunoassays to distinguish among them (15,16).Consequently, an increase in estradiol level due to crossreaction with fulvestrant is observed (12,14).In our study, we confirmed the interference of fulvestrant in the estradiol measurement by Centaur CP immunoassay and proposed a simple laboratory tool that allows to differentiate false cases from actual ovarian escape.The increase in serum estradiol level after the addition of exogenous fulvestrant showed that it was measured as estradiol in the used immunoassay.An alternative type of analysis to measure estradiol with no anticipated cross-reactivity to fulvestrant such as liquid chromatography-mass spectrometry could be used and has been recommended (15).However, it is not a practical method for a routine clinical laboratory.
The fulvestrant half-life is estimated to be 50 days (15,17) remaining in circulation for a longer period than the interval between administrations (28 days).This is an additional problem since the interference caused by this drug may persists for months (14,18).
The estimation of the estradiol ratio between the diluted and the direct serum sample (ratio Dil/Dir) permitted to identify the interference of fulvestrant on estradiol quantification, by immunoassay.In the serum where fulvestrant is present, we found that the estradiol ratio for Dil/Dir sample is about 2. In the serum of patients not under fulvestrant, this ratio is around 1. Fulvestrant forms spontaneous and reversible colloidal aggregates in an aqueous solution, which can be converted to the monomeric form by dilution (19,20,21).Moreover, 99% of fulvestrant binds, reversibly, to plasma proteins (mainly very low, low-and high-density lipoprotein fractions) (15,22).The fulvestrant feature of colloidal aggregates formation and its bind to plasma proteins can explain why, after sera dilution and consequent disaggregation, the measured estradiol increases in samples with the drug, leading to a ratio Dil/Dir of about 2.
The study results are also meaningful for clinical practice.Since efficacy data in premenopausal women became available (4, 5), fulvestrant (with GnRH for ovarian suppression) in combination with CDK4/6 or in monotherapy is a widely used option in ABC premenopausal women ER+.Failure to attain complete ovarian suppression with GnRH can however occur (7,8) and may conceivably be associated with higher recurrence risk (9, 10).So, although not a level I recommendation, routine monitorization of estradiol in these patients may be considered.Patients on fulvestrant with chemotherapyinduced amenorrhea and no GnRH support, especially when younger, may experience ovarian recovery initially without bleeding and can also be candidates to estradiol measurements.Assuming an ovarian escape in a patient, and not being aware of the fulvestrant falsely elevated estradiol levels and how to distinguish it, can create errors and mistakenly influence medical decisions.It may result in unneeded surgical castration, unnecessary interruption of fulvestrant, an increase in blood tests and costs, and inconvenience for the patient and the clinician.
In our center, we also observed unexpectedly high estradiol levels not consistent with the patient's clinical history.This triggered the discussion and the study resulted from a straight collaboration between the clinical and the laboratory team, emphasizing once again the critical role of multidisciplinary work and communication in the management of cancer patients.
In conclusion, we present the estimation of the estradiol Dil/Dir ratio as a simple tool to detect fulvestrant interference and identify the false Centaur CP immunoassay estradiol results.

Figure 1
Figure 1Chemical structure similarity between estradiol and fulvestrant.

Table 1
Estradiol concentration of the serum samples spiked with fulvestrant.

Table 2
Estradiol concentration of serum samples from patients under fulvestrant therapy.
For patients with more than one estradiol measurement, the results are presented chronologically.a Time since the beginning of fulvestrant until the blood collection.Dil, 1:5 dilution; Dir, direct; NA, not available.

Table 3
Estradiol concentration of serum samples from the control group (patients not under fulvestrant therapy).