Folate receptor alpha (FRA) expression remains ...

YGYNO-974968; No. of pages: 8; 4C: Gynecologic Oncology xxx (2013) xxx–xxx

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Folate receptor alpha (FRA) expression remains unchanged in epithelial ovarian and endometrial cancer after chemotherapy E. Despierre a,⁎, S. Lambrechts a, K. Leunen a, P. Berteloot a, P. Neven a, F. Amant a, D.J. O'Shannessy b, E.B. Somers b, I. Vergote a a b

Gynecologic Oncology and Leuven Cancer Institute, University Hospitals Leuven, Department of Oncology, KU Leuven, Belgium Diagnostics Development, Morphotek Inc., Exton, PA, USA

H I G H L I G H T S • Both epithelial ovarian and endometrial cancer demonstrate high expression of FRA compared to normal ovarian and endometrial tissue. • FRA expression is not altered by chemotherapy exposure at interval debulking surgery or at recurrence. • Immunohistochemical FRA staining at diagnosis can guide the decision whether to use FRA targeted therapy upon recurrence.

a r t i c l e

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Article history: Received 21 December 2012 Accepted 23 March 2013 Available online xxxx Keywords: Ovarian cancer endometrial cancer folate receptor FRA immunohistochemistry chemotherapy

a b s t r a c t Objective. Based on its expression profile, folate receptor alpha (FRA) is an attractive candidate for targeted diagnostics and therapeutics. However, applicability of these agents in residual or recurrent disease could be influenced by chemotherapy. We evaluated whether chemotherapy modified FRA expression in non-mucinous epithelial ovarian (EOC) and endometrial carcinoma (EC). Methods. FRA staining was evaluated by immunohistochemistry, using MAb 26B3, in 81 patients (41 EOCs and 40 ECs) and 17 control tissues (5 benign ovarian cysts, 5 normal ovarian, and 7 normal endometrial tissues). Chemotherapy effect was evaluated in 42 patients (30 paired samples at primary and interval debulking surgery and 12 from primary and recurrent disease). FRA expression was assessed using a semi-quantitative staining algorithm, the M-score (range 0–50). Results. Median difference in M-score between tumor and control samples was 27.5 for EOC (95% CI 10.0 to 45.0) and 6.7 for EC (95% CI −6.7 to 21.7). Paired samples from both primary and interval debulking surgery did not differ in FRA expression in EOC (median difference of M-score between paired samples of 0.0 [95% CI −2.6 to 2.6]). Recurrent EOC tumors reflected FRA status at diagnosis (median difference of M-score between paired samples of 3.3 [95% CI −7.0 to 13.6]). Conclusions. This study shows no significant difference in FRA expression after chemotherapy, strengthening the rationale for FRA targeted diagnostics and therapeutics in FRA expressing tumors, whether newly diagnosed or at recurrence. © 2013 Elsevier Inc. All rights reserved.

Introduction Folate receptor alpha (FRA) is a glycosylphosphatidyl-inositol-linked cell surface glycoprotein with high affinity for binding and transporting physiological levels of folate into cells [1]. Cellular uptake of folate, a basic component of cell metabolism and DNA synthesis and repair, occurs under physiological conditions by an ATP-dependent reduced folate carrier (RFC), which is ubiquitously expressed in normal cells, binds

⁎ Corresponding author at: Division of Gynecologic Oncology, University Hospitals Leuven, Herestraat 49, 3000 Leuven, Belgium. E-mail address: [email protected] (E. Despierre).

folate with low affinity and represents the sole folate uptake for most normal cells. In contrast, the expression of FRA in normal tissues is highly restricted to the apical surfaces of polarized epithelial cells [2], which are not exposed to circulating folate. FRA enhances folate uptake through receptor-mediated endocytosis, allowing internalization of folate but also folate-conjugates. FRA expression increases or decreases in response to folate repletion or depletion and is distinct in normal and malignant tissue. High expression patterns have been described in a range of epithelial cancers, including non-mucinous ovarian, endometrial, non-small cell lung carcinomas and to a lesser extent in clear cell renal, colorectal and breast cancers [3–10]. Moreover, in ovarian cancer the level of FRA expression has been correlated with tumor grade, stage, aggressiveness and response to chemotherapy [5,6,11–13].

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Please cite this article as: Despierre E, et al, Folate receptor alpha (FRA) expression remains unchanged in epithelial ovarian and endometrial cancer after chemotherapy, Gynecol Oncol (2013), http://dx.doi.org/10.1016/j.ygyno.2013.03.024

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E. Despierre et al. / Gynecologic Oncology xxx (2013) xxx–xxx

Based on its highly tumor restricted expression profile, FRA represents an attractive candidate for targeted diagnostics and therapeutics. FRA expression has been observed in nearly 90% of the non-mucinous ovarian cancers [2,5,14] with enhancement of growth of tumorigenic cancer cells in vitro and in vivo [15], providing a rationale for targeting the folate receptor in non-mucinous EOC. In the widely accepted dualistic model of endometrial carcinogenesis, 80% of the tumors are early stage, estrogen-dependent endometrial cancers with a low grade endometrioid morphology (Type I) [16]. In contrast, high grade non-endometrioid endometrial carcinomas encompassing mainly serous and clear cell carcinomas, characterize the endometrial cancers associated with adverse outcome and are classified as “Type II”. The association of FRA expression with high risk endometrial cancer features, as well as worse survival [17], suggests that FRA may also be a novel target for endometrial cancer therapy. Preclinical studies have demonstrated that Farletuzumab (MORAb-003), a humanized, high-affinity monoclonal antibody against FRA, mediates robust antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity in vitro, and inhibits tumor growth in xenograft models [18]. In a phase I study (NCT00428766), Farletuzumab administered as an intravenous infusion did not demonstrate dose-limiting toxicities or severe adverse events in heavily pretreated patients with EOC [19]. A phase II study (NCT00318370) of Farletuzumab with carboplatin and taxane in patients with platinumsensitive EOC in first relapse, has shown an improved response rate and time to progression compared with historical data. Also the duration of second remission was increased compared to first remission [20]. Currently, a randomized, double-blind, placebo-controlled phase III study with Farletuzumab plus chemotherapy has been recently closed and presentation of these results is awaited. In vitro studies demonstrated that EC-145 (vintafolide), a conjugate of folic acid and the vinca alkaloid desacetylvinblastine hydrazide, also selectively binds to cells that express FRA, causing dose-dependent cytotoxicity [21]. Initial results from a phase II randomized study of EC145 in combination with pegylated liposomal doxorubicin (PRECEDENT study; NCT00722592) indicate a significant improvement in progression-free survival in patients with platinum-resistant ovarian cancer compared with pegylated liposomal doxorubicin alone [22]. Although promising, identification of patients who may benefit from FRA-targeted therapy depends on the presence of FRA expression. While chemotherapy remains one of the keystones in the treatment of EOC and EC, there is a paucity of data regarding the effect of chemotherapy on FRA expression. Chemotherapy could influence FRA expression, reflecting the increased need for folate to support rapid repair of DNA damage caused by platinum. The goal of the present study was to evaluate the expression of FRA in epithelial ovarian and endometrial cancer specimens and to explore whether FRA expression is altered by chemotherapy. Materials and methods Patient and tissue characteristics In this retrospective cohort study, 41 patients with ovarian carcinoma, 40 patients with endometrial cancer and 17 control patients were selected from the historical tumor bank of the Department of Obstetrics and Gynaecology of the University Hospitals Leuven for participation in this study. Tumor samples were taken at primary diagnosis, at interval debulking surgery after 3 courses of chemotherapy and/or at recurrent disease, snap frozen in liquid nitrogen after prelevation and stored at − 80 °C until further processing. All patients with at least 2 sequential representative frozen tumor tissues were selected for the analysis. These patients were diagnosed and underwent surgery between May, 1996 and November, 2010. Tissues were accepted independent of its origin, for example, surgical specimens from primary ovarian or

endometrial tumor, lymph nodes or distant metastases. Clinical and pathologic data were abstracted from the surgical, pathologic and medical records. Histological diagnosis followed the classification of the World Health Organization [23] and the grading as well as staging were based on FIGO 2009 guidelines [24]. The collection of tissue samples was approved by the Institutional Review Board of the University Hospitals Leuven and written informed consent was obtained from all patients enrolled (ML2524).

Immunohistochemistry FRA membrane staining was evaluated by IHC, using the highly specific mouse monoclonal anti-FRA antibody MAb 26B3 in formalinfixed, paraffin-embedded (FFPE) tissue sections, as previously described [25]. Signals were detected using a MACH4 Universal HRP-Polymer Detection Kit (Biocare Medical, Concord, CA).

Immunohistochemical analysis Scoring for staining was performed by an expert gynecologic pathologist (Dr. Y-S Fu, Laboratory Corporation of America, Los Angeles, CA), using conventional scoring for intensity and the percent of the tumor stained at each intensity. FRA IHC membrane and cytoplasmic staining intensities were scored using the following criteria: 0 = no staining, 1+ = weak staining, 2+ = moderate staining and 3+ = strong staining, as previously described [12]. Also the percentage of cells stained by MAb 26B3 was determined. Estimates to the nearest 5% were used. If the percentage of the tumor area positive for membranous staining was greater than or equal to 5% at any intensity, FRA expression was considered positive. A sample was rejected for further analysis if it was composed of necrotic tissue or was deemed to represent normal tissue.

Table 1 Patient and tumor characteristics.

Tumor grade, n (%) Grade 1 Grade 2 Grade 3 Tumor histology, n (%) Serous Endometrioid Clear cell Mucinous Mixed Carcinosarcoma FIGOa stage, n (%) I II III IV Debulking status, n (%) No residual disease Residual disease > 1 mm Inoperable Follow–up, median (95% CI) Time to recurrence (months) Time to death (months) Vital status, n (%) Alive Deceased Missing a

Epithelial ovarian cancer n = 41

Endometrial cancer n = 40

1 (2.4) 9 (22.0) 31 (75.6)

12 (30.0) 9 (22.5) 19 (47.5)

36 (87.8) 1 (2.4) 2 (4.9) – 2 (4.9) –

10 (25.0) 23 (57.5) 2 (5.0) 1 (2.5) 3 (7.5) 1 (2.5)

2 (4.9) – 30 (73.1) 9 (22.0)

18 (45.0) 2 (5.0) 6 (15.0) 14 (35.0)

34 (82.9) 5 (12.2) 2 (4.9)

35 (87.5) 3 (7.5) 2 (5.0)

9.0 (6.3–15.3) 39.8 (26.3–50.0)

16.8 (2.9–Inf) 34.0 (7.1–Inf)

4 (9.8) 36 (87.8) 1 (2.4)

29 (72.5) 11 (27.5) –

Fédération Internationale de Gynécologie et d’Obstétrique.



The M-score: a semi-quantitative staining algorithm A weighted score for FRA IHC tumor cell membrane staining (M-score), capturing both the proportion of FRA positive cells and staining intensity, was calculated using the formula [8,12]:

3

Mi ¼

∑j¼1 W j X ij 3 ∑j¼1 W j

3

¼

∑j¼1 W j X ij 6

In the equation, Xij is the percentage of tumor stained at intensity j for patient i and Wj is the absolute value of the intensity (ranging from 0 to 3+). The M-score can theoretically range from zero (no positive staining) to a maximum of fifty (100% of cells staining at 3+ intensity).

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Definition of response to treatment and recurrence Response Evaluation Criteria in Solid Tumors (RECIST), supplemented by CA125 response, as developed by the Gynecologic Cancer Intergroup [26], was used to classify response to first line chemotherapy and to define progressive disease. The platinum free interval was defined as the interval between the last date of platinum dose and recurrence of disease. Patients with a platinum free interval > 6 months were considered platinum sensitive, while patients with a platinum free interval b 6 months were deemed platinum resistant. Statistical analysis Patient demographic data were summarized using descriptive statistics. To compare pre- and post-chemotherapy samples, the

A

IQR: Interquartile range

B

IQR: Interquartile range Fig. 1. M-score in control samples and in epithelial ovarian cancer (A) and endometrial cancer (B) at primary diagnosis, at interval debulking and at recurrent disease.


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difference between these samples was computed for patients for whom a post-chemotherapy sample was available. The median of these differences was computed with a 95% confidence interval (CI) using Bonett and Price’s method [27]. To compare the pre-chemotherapy samples with control samples, the Hodges–Lehmann estimator of location shift was computed together with the exact 95% CI. The resulting value is the median difference in M-score between a cancer patient and a control patient. The Kaplan–Meier method was used to estimate progression free survival (PFS) and overall survival (OS). PFS was defined as the time from primary diagnosis until progression of the disease or death. OS was calculated as the period from diagnosis until death from any cause. FRA expression was tested as a prognostic factor adjusting for grade, histology (serous vs other), FIGO stage, and residual disease using a multivariable Cox proportional hazards model. All statistical analyses were performed using SAS v9.2 (SAS Institute, Cary, USA). Results Patient and tumor characteristics Ovarian cancer The demographic characteristics of the 41 epithelial ovarian cancer patients represented in this study are shown in Table 1. Of the 39 patients who received adjuvant chemotherapy, all received a platinumbased regimen. Of the 41 included epithelial ovarian cancer patients, 79 tumor samples were available, of which 41 were obtained during primary surgery, 27 during interval debulking surgery and 11 during surgery for relapse. Twenty-four cases had tumor tissue from both primary diagnostic laparoscopy and interval debulking surgery. Finally, 9 patients had a secondary debulking from which tissue samples from primary surgery were available, allowing analysis of matched pairs of primary carcinomas and recurrent disease following chemotherapy. Median time between primary and secondary surgery for recurrent disease was 74.5 months (range 38.5–103 months). Endometrial cancer The characteristics of the 40 endometrial cancer patients are summarized in Table 1. Half of the patients received adjuvant chemotherapy, all platinum-based. In the endometrial cancer group, a total of 52 tumor samples were available, of which 40 were taken at primary diagnosis, 8 at interval debulking surgery and 4 samples at the time of recurrent disease. Chemotherapy effect was evaluated in 6 endometrial cancer patients with matched tissues from both primary and interval debulking surgery, and in 3 patients with samples from both primary and recurrent disease. Median time between primary surgery and biopsy at recurrent disease was 103 months (range 53–182 months). Control samples Control samples consisted of benign ovarian cysts (n = 5), normal ovarian (n = 5) and normal endometrial tissues (n = 7) from patients undergoing surgery for benign gynecological problems (prolaps, prophylactic surgery, bleeding problems, etc.). FRA expression in primary epithelial ovarian and endometrial cancers The M-score distribution for FRA staining of epithelial ovarian and endometrial cancers is shown in Fig. 1 and Table 2. The median M-scores for epithelial ovarian cancers and endometrial carcinomas stained with MAb 26B3 were 41.7 (interquartile range [IQR] 26.7 to 45.0) and 18.3 (IQR 3.3 to 38.3), respectively. EOC tumor tissues were associated with a higher FRA expression than control tissues (Hodges–Lehmann estimator of location shift 27.5, 95% CI 10.0 to 45.0) and the same tendency was observed in EC (Hodges–Lehmann estimator 6.7, 95% CI −6.7 to 21.7) (Fig. 2, Table 3). All patients with a newly diagnosed epithelial ovarian cancer showed FRA expression in

Table 2 Descriptive results of M-score at primary diagnosis in epithelial ovarian and endometrial cancer.

Grade 1 2 3 Histology Serous Endometrioid Other FIGOc stage 1 2 3 4 Residual disease No Yes Platinum response Resistant Sensitive Response to first line chemotherapy Complete remission Partial remission Stable disease Progressive disease

Epithelial ovarian cancer

Endometrial cancer

n

Median

Range

n

Median

Range

1 9 31

50.0 40.0 41.7

– 15.0–50.0 2.2–50.0

12 9 19

5.4 16.7 26.7

0.0–43.3 0.0–45.0 0.0–50.0

36 1 4

42.5 15.0 40.9a

2.2–50.0 – 4.2–50.0

10 23 7

40.8 18.3 0.7b

7.5–50.0 0.0–45.0 0.0–38.3

2 – 30 9

9.6 – 43.3 40.0

4.2–15.0 – 2.2–50.0 10.0–50.0

18 2 6 14

7.1 15.8 15.0 31.7

0.0–45.0 13.3–18.3 0.0–43.3 2.5–50.0

34 7

40.8 43.3

2.2–50.0 36.7–48.3

35 5

20.0 7.5

0.0–50.0 0.0–45.0

10 28

40.0 44.2

4.2–45.0 2.2–50.0

6 14

21.7 31.7

2.5–48.3 0.0–50.0

29 7 2 1

43.3 43.3 41.7 36.7

4.2–50.0 2.2–48.3 40.0–43.3 36.7

13 3 1 1

35.0 16.7 45.0 2.5

13.3–50.0 7.5–26.7 45.0 2.5

a Distribution of M-score: ovarian cancer: clear cell (n = 2): M-score: 4.2 and 36.7; mixed serous–clear cell (n = 2): M-score: 45 and 50. b Distribution of M-score: endometrial cancer: mucinous (n = 1): M-score: 0; clear cell (n = 2): M-score: 0 and 0; carcinosarcoma (n = 1): M-score: 2.5; mixed serous– endometrioid (n = 2): M-score: 0 and 38.3; mixed mucinous–endometrioid (n = 1): M-score: 0.67. c Fédération Internationale de Gynécologie et d’Obstétrique.

our cohort, whereas 12.5% of the endometrial cancers were FRA negative (M-score = 0). Evaluation of M-scores by histology, grade and FIGO stage within the endometrial cancers suggested that high grade, serous histology and advanced FIGO stage were all associated with strong FRA expression. Because our epithelial ovarian cancer cohort was enriched with high grade advanced stage serous carcinomas, associations with grade, stage and histology were not possible. However, advanced stage tumors (median M-score 43.3 for stage III and 40.0 for stage IV) were more likely to express FRA than early stage disease (median M-score 9.6). The data did not suggest a strong correlation between FRA expression and response to first line chemotherapy.

FRA expression in primary versus chemotherapy exposed disease In considering FRA as a therapeutic target, we evaluated whether FRA expression is altered after exposure to chemotherapy. Twentyfour patients within the group of epithelial ovarian cancers and 6 patients with endometrial cancer had matched samples from both primary diagnosis and interval debulking surgery (Fig. 3.i). All patients had advanced stage disease (FIGO stage IIIc–IV) and all received platinum based chemotherapy. Comparison of M-scores between primary tumors and tissues from interval debulking surgery, using the Bonett and Price procedure [27] suggests small differences in FRA expression (Table 3). We also analyzed FRA expression in matched samples from primary and recurrent disease in 9 epithelial ovarian and 3 endometrial cancer patients (Fig. 3.ii, Table 3). Again, no strong differences in FRA expression were suggested, supporting the hypothesis that FRA expression is not altered by chemotherapy exposure in EOC. Due to the low sample size, the effect of chemotherapy could not be evaluated in the EC group.



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Fig. 2. FRA membrane staining using the monoclonal antibody MAb 26B3 at primary diagnosis and in non-malignant control samples of epithelial ovarian (A) and endometrial (B) cancer (image magnification 20×).

were very wide (PFI: HR per 10 points increase 0.79, 95% CI 0.37–1.67; OS: HR per 10 points increase 0.80, 95% CI 0.42–1.54).

Survival analysis For the EOC patients, data on M-score, covariates and follow-up were available for 38 patients, of which all relapsed and 35 died. The median progression free interval (PFI) was 9.0 months (95% CI 6.3–15.3). The median OS time was calculated as 39.8 months (95% CI 26.3–50). We did not find strong evidence for an effect of FRA expression on PFI (HR per 10 points increase 1.00, 95% CI 0.78–1.28) or OS (HR per 10 points increase 0.78, 95% CI 0.61–1.00). Follow-up data were only available in 18 EC patients, 10 with recurrence and 8 patients who died. The median PFI was calculated as 16.8 months (95% CI 2.9–Inf) and median OS was 34.0 months (95% CI 7.1–Inf). The effects were of similar magnitude as for EOC but CIs

Discussion We examined FRA expression with MAb 26B3 in a cohort of 41 women with epithelial ovarian cancer and 40 endometrial cancer patients. All epithelial ovarian cancers and 87.5% of endometrial cancers were positive for FRA expression. Mucinous EOCs, generally characterized by low or absent FRA expression, were not included in our cohort, probably explaining the higher frequency of FRA expression in our EOC cohort than previous reports [2,5–7,14] but in agreement with a recent study using MAb 26B3 [12]. In EC, tumors with poor prognostic features

Table 3 Comparison of FRA expression pre- versus post-chemotherapy and primary tumor versus benign disease. Epithelial ovarian cancer n

Median difference in M-score (95% CI)

Interval debulking versus primary Recurrence versus primary Primary versus control

24 9 51

0.0 3.3 27.5b

Endometrial cancer Interval debulking versus primary Recurrence versus primary Primary versus control

6 3 47

−4.2 0.0 6.7 b

a b

IQR

Range

(−2.6 to +2.6)a (−7.0 to +13.6)a (+10.0 to +45.0)

−3.3 to +6.7 −1.3 to +11.7 –

−10.0 to + 26.3 −3.3 to +25.8 –

(−17.1 to +8.6)a – (−6.7 to +21.7)

−67.0 to +3.3 – –

−10.0 to + 18.3 −17.5 to 0.0 –

95% CI based on Bonett and Price's method. Hodges-Lehmann estimator of location shift (exact 95% CI).


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Fig. 3. i. FRA membrane staining using the monoclonal antibody MAb 26B3 of matched samples at primary diagnosis and interval debulking of epithelial ovarian (A) and endometrial (B) cancer. ii. FRA membrane staining using the monoclonal antibody MAb 26B3 of matched samples at primary diagnosis and recurrence of epithelial ovarian (A) and endometrial (B) cancer.

(high grade, serous histology and advanced FIGO stage) showed a modest trend towards higher FRA expression. Furthermore, we analyzed whether FRA expression was associated with outcome. In accordance with previous studies [5,11], we did not detect clear associations between FRA expression in EOC and PFI and/or OS. A limitation of our study, however, is the absence of FRA-negative ovarian cancer tissues, which makes it difficult to assess the true prognostic role of FRA expression. In contrast to previous reports [17,28], FRA expression could not be validated as a negative prognostic factor in EC, possibly due to the sample size. The high expression of FRA in some tumors compared with normal tissues has been exploited over the last decade by two approaches. One involves folate-mediated targeting of macromolecules, anticancer drugs, imaging agents and nucleic acids to cancer cells. By using this approach, the folate receptor has been utilized as a vehicle for the transfer of these agents selectively into cancer cells by receptormediated endocytosis [29]. The other approach consists of humanized anti-FRA monoclonal antibodies, leading to direct targeting and tumor cell death. Especially in the case of ovarian cancer, Farletuzumab (MORAb-003), a humanized monoclonal antibody against FRA, based on the murine LK26 clone, seems promising and is currently undergoing phase III testing [18–20]. Although FRA is highly expressed in primary non-mucinous EOCs and (especially high risk) ECs, chemotherapy exposure could compromise the expression of FRA and the subsequent applicability of targeted therapies directed towards FRA. In our series, comparison of 30 matched samples from both primary and interval debulking surgery and 12 samples from both primary and recurrent disease in EOC and EC, showed that FRA expression was maintained after chemotherapy exposure. Review of the literature

reveals only two reports on the effect of chemotherapy on FRA expression in epithelial ovarian cancer. Both Kalli et al. [5] and Crane et al. [30] reported no significant difference in FRA expression in epithelial ovarian cancers after chemotherapy, whether at interval debulking surgery (n = 28 in both studies together) or at the time of recurrent disease (n = 36). To the best of our knowledge no previous study reported on the effect of chemotherapy on FRA expression in endometrial cancer. Our findings indicate that FRA targeted agents could be interesting in the treatment of EOC and high risk EC, at diagnosis as well as after chemotherapy exposure. In summary, the present study clearly demonstrated high expression of FRA in both epithelial ovarian and endometrial cancer, as previously reported. Our data suggest that FRA expression is not altered by chemotherapy, strengthening the rationale for FRA targeted diagnostics and therapeutics in the treatment of EOC and high risk EC, whether newly diagnosed or at recurrence. Furthermore, the preservation of FRA expression after chemotherapy exposure suggests that immunohistochemical FRA staining at diagnosis can guide the decision whether to use FRA targeted therapy upon recurrence. Conflict of interest No conflict of interest to declare.

Acknowledgments This analysis was supported by Morphotek Inc., a subsidiary of Eisai Corporation of North America. We gratefully acknowledge Mrs. Katrien Drijkoningen and Mrs. Godelieve Verbist for expert technical support. We thank Ben Van Calster for the statistical analyses.



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Fig. 3 (continued).

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