Abstract
Folate receptor alpha (FRα) is a promising therapeutic target due to its high expression in several tumor types and its rare expression in healthy tissue. Recently, the antibody‐drug conjugate mirvetuximab soravtansine has been approved for treatment of advanced platinum‐resistant high‐grade serous carcinoma (HGSC). Immunohistochemical expression of FRα has been extensively studied in HGSC, but most studies conducted before the clinical studies targeting FRα used variable antibodies and scoring criteria, which makes comparison of older literature data with recent studies difficult. Moreover, the data regarding its expression in other types of ovarian and other female genital tract tumors are limited or absent. In our study, we focused on immunohistochemical expression in 923 tubo‐ovarian and endometrial tumors (assessed on tissue microarrays), using standardized scoring criteria and the VENTANA FOLR1 CDx assay. The results of our study showed the highest FRα expression in serous carcinomas, specifically HGSC (45% positive cases), followed by low‐grade serous carcinoma (25%), endometrial serous carcinoma (11%), and serous borderline tumor (10%). Endometrioid and clear cell ovarian carcinomas showed rare positivity (2% and 1%, respectively). All other tumors examined were negative, including mucinous ovarian tumors, sex cord‐stromal tumors, endometrial endometrioid carcinomas, undifferentiated and dedifferentiated carcinomas, and endometrial clear cell carcinomas. In conclusion, these results confirm that FRα expression in HGSC and LGSC reaches similar values compared to published data, and is present in a minority of endometrial serous carcinomas. In other ovarian and endometrial tumors examined, FRα expression is absent or rare.
Keywords: folate receptor alpha, FOLR1, ovarian tumors, endometrial tumors
Introduction
Tubo‐ovarian carcinoma is one of the leading causes of cancer‐related death in women worldwide. The five main histological types of tubo‐ovarian carcinoma include high‐grade serous carcinoma (HGSC), low‐grade serous carcinoma (LGSC), ovarian clear cell carcinoma (OCCC), endometrioid ovarian carcinoma (EOC), and ovarian mucinous carcinoma (MOC) [1]. Most patients with HGSC and LGSC are diagnosed at an advanced stage, mainly due to the absence of early clinical symptoms. Although platinum‐based chemotherapy remains the standard treatment for tubo‐ovarian carcinoma patients, most patients' tumors eventually recur and ultimately develop platinum resistance [2]. The therapeutic options for these patients are limited and new treatment options are needed. One of the new treatments is mirvetuximab soravtansine (MIRV), which targets folate receptor alpha (FRα), encoded by the FOLR1 gene. FRα is a membrane glycoprotein that is often overexpressed in various cancers. At the same time, FRα is rarely expressed in healthy tissues, making it a good therapeutic target [3]. Clinical studies focusing on MIRV have shown better progression‐free survival (PFS) and overall survival (OS) in patients treated with MIRV compared with other therapeutic approaches and antibody‐drug conjugates [4, 5]. Based on this, MIRV was approved in 2022 as a treatment option for patients with platinum‐resistant HGSC [2].
Immunohistochemical expression of FRα has been extensively studied in HGSC, but data regarding its expression in other types of ovarian tumors are limited [6, 7, 8, 9]. However, most studies conducted before the clinical studies targeting FRα used variable antibodies and scoring criteria, which makes comparison of older literature data with recent studies difficult [1, 10]. Knowledge concerning FRα expression in tumors other than tubo‐ovarian female genital tract tumors is limited to a few previous studies suggesting that a subset of endometrial carcinomas may show FRα overexpression [11, 12, 13].
In our study, we focused on the expression of FRα in various histological subtypes of tubo‐ovarian and endometrial tumors using standardized scoring criteria and the VENTANA FOLR1 CDx assay.
Materials and methods
Ethics approval
The study was approved by the Ethics Committee of General University Hospital in Prague (Prague, Czech Republic) in compliance with the Declaration of Helsinki (number 2140/19 S‐IV). The Ethics Committee waived the requirement of informed consent according to Czech law (Act 373/11, and its amendment 202/17); it is not necessary to obtain informed consent in fully anonymized studies.
Samples
For the most part, the tubo‐ovarian tumor sample set represents cases used in our previous studies [14, 15, 16, 17]. Additionally, some cases of HGSC and all cases of endometrial carcinomas were selected from the archives of participating institutions for the purpose of this study. In total, 796 tubo‐ovarian tumors were selected for immunohistochemical analysis, including 104 HGSCs, 87 LGSC, 30 serous borderline tumors (SBTs), 47 EOC, 86 OCCCs, and 104 ovarian mucinous tumors, including 31 carcinomas (MOC) and 73 mucinous borderline tumors (MBT), as well as 338 sex cord‐stromal tumors, including 249 adult granulosa cell tumors (AGCTs), 4 juvenile granulosa cell tumors (JGCTs), 35 Sertoli‐Leydig cell tumors (SLTs), and 50 fibromas/thecomas (F/T). Additionally, 127 endometrial tumors were analyzed, including 51 endometrial endometrioid carcinomas (EEC; 16 grade 1, 30 grade 2, 5 grade 3), 27 endometrial serous carcinomas (ESC), 23 undifferentiated carcinomas (UC), 9 dedifferentiated carcinomas (DC), and 17 endometrial clear cell carcinomas (ECCC).
Most HGSC were primary tubo‐ovarian tumors, but three cases represented metastases (two peritoneal, one omental) as the primary tumor was not available. Most LGSC and SBT were primary tumors, although 7 LGSC samples represented metastases (three peritoneal, two omental, one from the vaginal stump, and one from a lymph node). In SBT, one case was a primary peritoneal tumor, and one was an omental implant in recurrent disease. Additionally, there were 48 metastases of AGCT and one of SLT, all in recurrent disease. It is important to note that, in cases of HGSC, the SEE‐FIM (Sectioning and Extensively Examining the FIMbriated end) protocol was not uniformly applied, and thus a tubal primary could not be excluded. This may have resulted in misclassification between FIGO I and II, justifying their combination in the survival analysis detailed below.
All cases were previously reviewed and classified by strict diagnostic criteria, as described in detail in our previous studies [14, 15, 16, 17]. Cases not included in previous studies were reviewed by two experienced gynecologic pathologists (PD and KN). The clinico‐pathological characteristics of tumor types with available data are summarized in Table 1.
Table 1.
Clinico‐pathological characteristics of tumor types with available data
| HGSC (n = 104) | LGSC (n = 87) | SBT (n = 30) | OCCC (n = 86) | EOC (n = 47) | MOC (n = 31) | MBT (n = 73) | |
|---|---|---|---|---|---|---|---|
| Age at diagnosis | |||||||
| Mean | 59.2 | 52.1 | 48.2 | 61.8 | 53.0 | 51.2 | 52.4 |
| Median | 59.0 | 56.0 | 48.0 | 63.0 | 52.0 | 57.5 | 52.5 |
| Range | 36–80 | 19–82 | 28–73 | 33–86 | 28–83 | 28–78 | 18–83 |
| T stage | |||||||
| T1 | 12 (11.5%) | 12 (15.2%) | 6 (25%) | 53 (69.7%) | 37 (86%) | 25 (80.6%) | 71 (97.3%) |
| T2 | 13 (12.5%) | 5 (6.3%) | 3 (12.5%) | 9 (11.8%) | 3 (7%) | 1 (3.2%) | 0 (0%) |
| T3 | 75 (72.1%) | 46 (58.2%) | 11 (45.8%) | 10 (13.2%) | 3 (7%) | 5 (16.1%) | 1 (1.4%) |
| Tx/NA | 4 (3.8%) | 16 (20.3%) | 4 (16.7%) | 4 (5.3%) | 0 (0%) | 0 (0%) | 1 (1.4%) |
| N stage | |||||||
| N0 | 28 (26.9%) | 15 (19%) | 9 (37.5%) | 34 (44.7%) | 33 (76.7%) | 12 (38.7%) | 8 (11%) |
| N1 | 35 (33.7%) | 19 (24.1%) | 2 (8.3%) | 5 (6.6%) | 1 (2.3%) | 3 (9.7%) | 0 (0%) |
| Nx/NA | 41 (39.4%) | 45 (57%) | 13 (54.2%) | 37 (48.7%) | 9 (20.9%) | 16 (51.6%) | 65 (89%) |
| M stage | |||||||
| M0 | 69 (66.3%) | 61 (77.2%) | 18 (75%) | 5 (6.6%) | 39 (90.7%) | 31 (100%) | 68 (93.2%) |
| M1 | 27 (26%) | 2 (2.5%) | 0 (0%) | 1 (1.3%) | 1 (2.3%) | 0 (0%) | 1 (1.4%) |
| Mx/NA | 8 (7.7%) | 16 (20.3%) | 6 (25%) | 70 (92.1%) | 3 (7%) | 0 (0%) | 4 (5.5%) |
| FIGO | |||||||
| I | 8 (7.7%) | 12 (15.2%) | 6 (25%) | 51 (67.1%) | 36 (83.7%) | 25 (80.6%) | 71 (97.3%) |
| II | 6 (5.8%) | 5 (6.3%) | 3 (12.5%) | 7 (9.2%) | 3 (7%) | 1 (3.2%) | 0 (0%) |
| III | 60 (57.7%) | 45 (57%) | 11 (45.8%) | 13 (17.1%) | 3 (7%) | 5 (16.1%) | 0 (0%) |
| NA | 30 (28.8%) | 17 (21.5%) | 4 (16.7%) | 5 (6.6%) | 1 (2.3%) | 0 (0%) | 2 (2.7%) |
| Grade | |||||||
| G1 | – | – | – | – | 16 (34%) | – | – |
| G2 | – | – | – | – | 24 (51.1%) | – | – |
| G3 | – | – | – | – | 3 (6.4%) | – | – |
| NA | – | – | – | – | 4 (8.5%) | – | – |
EOC, ovarian endometrioid carcinoma; HGSC, high‐grade serous carcinoma; LGSC, low‐grade serous carcinoma; MBT, mucinous borderline tumor; MOC, ovarian mucinous carcinoma; OCCC, ovarian clear cell carcinoma; SBT, serous borderline tumor.
Immunohistochemical analysis
Immunohistochemical staining and evaluation of all samples were performed in a single laboratory (Department of Pathology, First Faculty of Medicine, Charles University and General University Hospital in Prague). Immunohistochemistry (IHC) was performed on tissue microarrays (TMAs) using 4‐μm‐thick sections of formalin‐fixed paraffin‐embedded (FFPE) tissue. For the construction of the TMAs, eligible areas of each tumor were identified, and two tissue cores (each 2.0 mm in diameter) were taken from the donor block using the TMA instrument TMA Master (3DHISTECH Ltd., Budapest, Hungary).
The TMA sections were stained using the BenchMark ULTRA instrument. The primary antibody was a mouse monoclonal anti‐FOLR1 antibody (clone FOLR1‐2.1, ready‐to‐use; Ventana, Tucson, AZ, USA). Staining was performed according to the manufacturer's instructions without any modifications of the protocol. In brief, antigen retrieval was performed using Ventana ULTRA Cell Conditioning Solution 1 (CC1) at 100 °C for 64 min, and the primary antibody was incubated for 32 min at 36 °C. The results were interpreted using a light microscope and a VENTANA FOLR1 Stain Intensity Reference Slide. The immunohistochemical expression was double‐blindly evaluated by either two (PD, IT) pathologists or, in equivocal cases, also by a third pathologist (KN).
Immunohistochemical staining was scored according to the interpretation manual of the VENTANA FOLR1 CDx assay. In brief, a combination of intensity (0 = no staining, 1+ = weak, 2+ = moderate, 3 + = strong) and percentage of positive tumor cells (0–100%) was used. FOLR1‐positive cases were defined as moderate or strong (2+ or 3+) staining in ≥75% of viable tumor cells. Moreover, a semi‐quantitative H‐score was used, calculated as: 1x (% of cells 1+) + 2x (% of cells 2+) + 3x (% of cells 3+), yielding a final value of 0–300.
Statistical analysis
All statistical analyses were performed in R (version 4.5.1, 2025‐06‐13). FOLR1 expression was quantified as the percentage of positive tumor cells and as the H‐score. Descriptive statistics (mean ± SD, median, range) were calculated for each tumor type. Differences in H‐score across tumor categories were evaluated using the Kruskal–Wallis test, with post hoc pairwise comparisons performed using Dunn's test with Bonferroni correction. Tumor groups with uniformly absent FRα expression (e.g., AGCT, JGCT, SLT, MBT, F/T, UC, DC) were excluded. Analyses were conducted separately for tubo‐ovarian and endometrial tumors. Associations between FRα expression (positive versus negative) and stage, as well as survival, were evaluated in HGSC and LGSC, the only tumor types with sufficient clinical data and expression variability. For contingency tables with low expected counts, simulated p values were calculated based on 105 Monte Carlo replicates.
Survival analyses were performed using Kaplan–Meier estimates and log‐rank tests to compare FRα‐positive and FRα‐negative cases. The primary endpoints analyzed were 10‐year OS and 10‐year PFS. OS was defined as the time from diagnosis to death or last follow‐up. PFS was defined as the time from diagnosis to documented disease recurrence or progression, or death; cases coded as AWD (alive with disease) were counted as PFS events, whereas patients alive without evidence of disease at last follow‐up were censored. Numbers of events and patients at risk are shown in the survival plots.
Multivariable Cox proportional hazards regression models were used to estimate the hazard ratios of potential prognostic factors and evaluate their associations with OS and PFS. Models included FRα expression (positive versus negative), age (continuous), FIGO stage (I–II, III), and radicality of surgery (R0, R1, R2). FIGO stages I and II were combined and analyzed as a single reference category (early‐stage disease) and compared against FIGO stage III. Models were simplified by stepwise backward elimination based on the Akaike information criterion (AIC), ensuring that only variables with at least nominal statistical significance (p < 0.05) were retained. The proportional hazards (PH) assumption was verified using Schoenfeld residuals for all variables. Results are reported as regression coefficients (β), hazard ratios (HR), 95% confidence intervals (CI), and p values. Model performance was assessed using Harrell's concordance (C‐index). All survival analyses were performed on HGSC and LGSC with available clinical data.
All tests were two‐sided, and p < 0.05 was considered statistically significant. R packages used: survival, survminer, ggplot2, dplyr.
Results
Expression of FRα
FRα expression, both quantified as H‐score and analyzed on a binary level (positive versus negative), displayed marked variation across tumor types (Figures 1 and 2).
Figure 1.
FRα expression (H‐score) across tubo‐ovarian tumor types. Due to the presence of groups with exclusively negative cases (zero variability), no global p value is provided for this comparison.
Figure 2.
Proportion of FRα negative and positive cases across tubo‐ovarian tumor types. Due to the presence of groups with exclusively negative cases (zero variability), no global p value is provided for this comparison.
The highest FRα expression was observed in serous carcinomas, specifically HGSC (47 out of 104, 45% positivity) (Figure 3A–D), followed by LGSC (22 out of 87, 25% positivity), and a smaller subset of SBT (3 out of 30, 10% positivity) (Figure 3E) and ESC (3 out of 27, 11% positivity) (Figure 4A,B). EOC and OCCC had only one FRα‐positive tumor each (2% and 1% positivity, respectively) (Figures 3F and 4C). FRα expression was absent in all cases of MOC, MBT, sex cord‐stromal tumors, EEC, UC, DC, and ECCC. The detailed IHC results of individual tumor types are provided in Table 2.
Figure 3.
Folate receptor alpha (FRα) expression in tubo‐ovarian tumors. (A) High‐grade serous carcinoma (HGSC) with strong circumferential expression in most tumor cells (×200). (B) HGSC showing strong apical and moderate circumferential expression in most tumor cells (×200). (C) HGSC with strong luminal (apical) expression (×100). (D) Low‐grade serous carcinoma with mostly apical expression (×200). (E) Serous borderline tumor showing mostly apical expression in most tumor cells (×100). (F) Clear cell carcinoma with apically accentuated strong expression (×200).
Figure 4.
Folate receptor alpha (FRα) expression in endometrial and tubo‐ovarian tumors. (A) Endometrial serous carcinoma (ESC) showing diffuse strong expression (×200). (B) Another case of ESC with variable positivity in some tumor cells (×200). (C) Endometrial clear cell carcinoma showing expression of variable intensity in some tumor cells (×100). (D) Ovarian endometrioid carcinoma with focal apically accentuated expression (×200).
Table 2.
Summary of FRα expression and H‐score across tubo‐ovarian and endometrial tumor types
| Tumor type | n | Positive, n (%) | Negative, n (%) | FRα overall range | FRα overall mean ± SD | FRα overall median (Q1; Q3) | H‐score range | H‐score mean ± SD | H‐score median (Q1; Q3) |
|---|---|---|---|---|---|---|---|---|---|
| HGSC | 104 | 47 (45%) | 57 (55%) | 0–100 | 67 ± 35.2 | 82.5 (39; 100) | 0–300 | 157.5 ± 97.1 | 182.5 (66; 231) |
| LGSC | 87 | 22 (25%) | 65 (75%) | 0–100 | 48.1 ± 38.4 | 50 (10; 88) | 0–300 | 101.7 ± 94.9 | 90 (10; 200) |
| SBT | 30 | 3 (10%) | 27 (90%) | 0–100 | 51.8 ± 32.3 | 55 (20; 80) | 0–280 | 97.2 ± 76.5 | 90 (20; 138) |
| OCCC | 86 | 1 (1%) | 85 (99%) | 0–70 | 6.6 ± 14.6 | 0 (0; 3) | 0–130 | 10.1 ± 24.6 | 0 (0; 3) |
| EOC | 47 | 1 (2%) | 46 (98%) | 0–100 | 7.4 ± 20.1 | 0 (0; 4) | 0–270 | 13.9 ± 45.4 | 0 (0; 4) |
| MOC | 31 | 0 (0%) | 31 (100%) | 0–10 | 0.3 ± 1.8 | 0 (0; 0) | 0–10 | 0.3 ± 1.8 | 0 (0; 0) |
| MBT | 73 | 0 (0%) | 73 (100%) | 0–0 | 0 ± 0 | 0 (0; 0) | 0–0 | 0 ± 0 | 0 (0; 0) |
| AGCT | 249 | 0 (0%) | 249 (100%) | 0–0 | 0 ± 0 | 0 (0; 0) | 0–0 | 0 ± 0 | 0 (0; 0) |
| JGCT | 4 | 0 (0%) | 4 (100%) | 0–0 | 0 ± 0 | 0 (0; 0) | 0–0 | 0 ± 0 | 0 (0; 0) |
| SLT | 35 | 0 (0%) | 35 (100%) | 0–0 | 0 ± 0 | 0 (0; 0) | 0–0 | 0 ± 0 | 0 (0; 0) |
| F/T | 50 | 0 (0%) | 50 (100%) | 0–0 | 0 ± 0 | 0 (0; 0) | 0–0 | 0 ± 0 | 0 (0; 0) |
| EEC | 51 | 0 (0%) | 51 (100%) | 0–70 | 4.4 ± 11 | 0 (0; 5) | 0–130 | 6.7 ± 19.7 | 0 (0; 5) |
| ESC | 27 | 3 (11%) | 24 (89%) | 0–100 | 32.2 ± 33.7 | 25 (0; 55) | 0–290 | 66.1 ± 81 | 45 (0; 105) |
| UC | 23 | 0 (0%) | 23 (100%) | 0–0 | 0 ± 0 | 0 (0; 0) | 0–0 | 0 ± 0 | 0 (0; 0) |
| DC | 9 | 0 (0%) | 9 (100%) | 0–0 | 0 ± 0 | 0 (0; 0) | 0–0 | 0 ± 0 | 0 (0; 0) |
| ECCC | 17 | 0 (0%) | 17 (100%) | 0–35 | 2.6 ± 8.4 | 0 (0; 1) | 0–55 | 3.9 ± 13.2 | 0 (0; 3) |
AGCT, adult granulosa cell tumor; DC, dedifferentiated carcinoma; ECCC, endometrial clear cell carcinoma; EEC, endometrial endometrioid carcinoma; EOC, ovarian endometrioid carcinoma; ESC, endometrial serous carcinoma; F/T, fibroma/thecoma; HGSC, high‐grade serous carcinoma; JGCT, juvenile granulosa cell tumor; LGSC, low‐grade serous carcinoma; MBT, mucinous borderline tumor; MOC, ovarian mucinous carcinoma; OCCC, ovarian clear cell carcinoma; Q1; Q3, interquartile range; SBT, serous borderline tumor; SD, standard deviation; SLT, Sertoli‐Leydig cell tumor; UC, undifferentiated carcinoma; the horizontal line separates ovarian (above) and endometrial (below) tumors.
Overall, differences in FRα expression across tumor types were highly significant (Kruskal–Wallis test, p < 0.001). Among tubo‐ovarian tumors, HGSC had the highest expression, followed by LGSC. Both showed higher expression than OCCC, EOC, and MOC, which displayed uniformly low levels of expression. SBT showed intermediate expression, higher than OCCC, EOC, and MOC, but not significantly different from HGSC or LGSC. In endometrial tumors, ESC showed higher expression than EEC and ECCC, whereas EEC and ECCC did not differ (post hoc multiple comparisons, see supplementary material, Table S1).
Correlation of FRα expression with stage and grade
To determine the prognostic significance of FRα expression, cases were categorized as FRα‐positive or FRα‐negative, as described above. No significant association between FRα positivity and FIGO stage was observed in either HGSC (χ 2 = 1.418, simulated p = 0.742) or LGSC (χ 2 = 1.159, simulated p = 0.763).
In the EOC group, only a single case was FRα‐positive, which precluded a meaningful analysis of expression in relation to grade. Therefore, the association of the continuous H‐score with grade was assessed, but no statistical significance was found (Kruskal–Wallis χ 2 = 1.289, df = 2, p = 0.525).
Survival analyses
In HGSC, the median follow‐up was 52 months [interquartile range (IQR), 26–72]. At the last follow‐up date available, 52 patients were alive with disease (51%), 25 had no evidence of disease (25%), and 24 had died (24%). Univariate log‐rank analyses revealed no significant associations between FRα expression and either OS (χ 2 = 0.8, p = 0.362) or PFS (χ 2 = 0.1, p = 0.705) (Figure 5A,B). Subsequent multivariable Cox proportional hazards models, including potential prognostic factors, confirmed that FRα expression was not significantly associated with survival outcomes for either OS or PFS (supplementary material, Table S2A). In contrast, surgical radicality was a strong prognostic factor for PFS. Both R1 and R2 resection margins were associated with worse PFS compared with R0, with the strongest adverse effect observed for R2 resection [HR (95% CI) = 3.99 (1.70–9.39), p = 0.002]. In addition, increasing age at diagnosis was independently associated with shorter PFS in HGSC [HR (95% CI) = 1.03 (1.00–1.07), p = 0.027].
Figure 5.
Prognostic value of folate receptor alpha (FRα) expression in serous tubo‐ovarian carcinoma. Kaplan–Meier curves with numbers at risk show that, in high‐grade serous carcinoma (HGSC), FRα expression is not associated with (A) 10‐year overall survival or (B) progression‐free survival. In low‐grade serous carcinoma (LGSC), FRα expression (C) shows no link to overall survival, but (D) is significantly correlated with progression‐free survival. The p values are based on log‐rank tests; significant p values are marked in bold.
In LGSC, the median follow‐up was 49.5 months (IQR, 21–70). At the last follow‐up date, 21 patients were alive with disease (31%), 30 had no evidence of disease (44%), and 17 had died (25%). FRα expression showed no significant association with OS in univariate analysis (χ 2 = 1.7, p = 0.198) (Figure 5C), nor after adjustment for clinicopathological variables in multivariable Cox regression (supplementary material, Table S2B). In contrast, FRα‐positive tumors were associated with significantly shorter PFS (χ 2 = 8.9, p = 0.003), with a median of 47 months compared with 104 months in FRα‐negative cases (Figure 5D). This association remained significant in multivariable analysis [HR (95% CI) = 3.74 (1.52–9.16), p = 0.004]. Detailed results of the full Cox proportional hazards models are provided in supplementary material, Table S2.
Discussion
FRα, encoded by the FOLR1 gene, is a membrane protein that plays an important role in folate uptake [18]. This protein is overexpressed in several solid tumors, including gastrointestinal, breast, and lung cancer, but most studies have focused on its expression in tubo‐ovarian epithelial tumors [18, 19, 20, 21].
However, the older literature data concerning its expression are equivocal and not comparable, mainly because different antibodies and cut‐offs for positivity were used. For example, in the first immunohistochemical study from 2008, the authors analyzed a cohort of 186 samples, of which 104 were classified as primary tumors of serous histology, further unspecified [10]. In their study, 72% of all tumors and 81.7% of tumors with serous histology were FRα‐positive. Their criteria for positivity were any percentage of stained tumor cells of any intensity. Another study conducted in 2014 included 2,801 samples of tubo‐ovarian carcinoma, 1,507 of which were HGSC [1]. Any tumor with at least moderate membranous and cytoplasmic staining was regarded as positive. Overall, 1,523 tumors out of 2,801 were FRα‐positive (54.4%), including 76.2% of HGSC, 49.5% of LGSC, 29.4% of EOC, 31.6% of OCCC, and 11.4% of MOC.
The approach to FRα scoring has been standardized in all main clinical studies focusing on MIRV treatment and the same criteria and antibody were used. FRα positivity was defined in these studies as ≥75% of viable tumor cells stained at moderate and/or strong intensity. The VENTANA FOLR1 CDx assay was used and subsequently was FDA approved as a companion diagnostic test to identify FRα‐positive patients [2]. A recent study compared six commercially available antibodies with the VENTANA FOLR1 CDx assay [22]. Of the six antibodies selected for this study, four showed a lack of specific membrane staining and/or high background and were excluded from further comparison. Two antibodies, including the Leica Link48 assay (clone FOLR1 3.2) and the Biocare intelliPATH assay, showed a similar distribution of positivity across the continuum of FRα expression as detected by the VENTANA FOLR1 CDx assay. However, both showed stronger staining intensity, which resulted in an increased number of samples classified as positive compared to the VENTANA FOLR1 CDx assay. The overall percentage agreement of the Leica Link48 and the Biocare intelliPATH assays compared to the VENTANA FOLR1 CDx assay was 71% and 77%, respectively. Based on these results, additional calibration of these antibodies would be needed prior to their use in routine diagnostic practice, as both may over‐select patients for FRα‐targeted therapies.
Standardized scoring criteria were also used in five studies focusing on the expression of FRα in tubo‐ovarian tumors [6, 7, 8, 9, 23]. All used a TMA approach, and only one study used a different antibody than the VENTANA FOLR1 CDx Assay.
Manning‐Geist et al had a sample size of 131 and focused on LGSC (89 samples) and SBT (42 samples), of which 40% and 21% were FRα‐positive, respectively [6]. Rushton et al had a large sample size of 5,367 tumors, 5,086 of which were HGSC and 281 LGSC [7]. FRα was positive in 43.5% of HGSC and 24.6% of LGSC. Previs et al analyzed 425 tubo‐ovarian tumors, of which 199 were HGSC (51.3% FRα positivity), 113 were classified as tumors not otherwise specified (19.5% FRα positivity), 73 were serous tumors not otherwise specified (34.3% FRα positivity), and the rest were cases of other histotypes [9]. Lawson et al tested 216 samples of tubo‐ovarian cancer (162 HGSC, 17 LGSC, and 37 cases of other histotypes). FRα was positive in 54.3% of HGSC, 29.4% of LGSC, 33.3% of mixed epithelial ovarian carcinomas, and 60% of carcinosarcomas. None of the tested EOC, OCCC, or granulosa cell tumors were FRα‐positive [8]. Leung et al used a laboratory‐derived IHC assay for FRα on a large cohort of 1,547 tubo‐ovarian tumors (662 HGSC, 160 LGSC, 332 EOC, 264 OCCC, 117 MOC, and 12 mesonephric‐type adenocarcinomas). FRα positivity was 43.5% for HGSC, 30% for LGSC, 6.3% for EOC, and 8.3% for OCCC. None of the MOC or mesonephric‐type adenocarcinomas showed high FRα expression [23]. Although the results of this study are similar to others, they may be influenced by the antibody used, which overestimated the number of positive cases in a recent comparative study [22]. In summary, across these five studies, the average FRα positivity is 44% for HGSC, 28.6% for LGSC, 20.9% for SBT, 7.8% for OCCC, 6% for EOC, and only one case of MOC was positive [9]. Another recent study used a different antibody to evaluate 326 HGSC (30.4% FRα‐positivity), 95 EOC (7.4% FRα‐positivity), and 75 OCCC (9.3% FRα‐positivity) [24].
Our results for HGSC (45% positive cases) and LGSC (25% positive cases) are similar to previous studies [6, 7, 8, 9, 23]. Data for other ovarian tumor histotypes are limited due to small sample sizes. SBT were tested in only one prior study (21% positive) compared with 10% positive cases in our study [6]. All ovarian mucinous tumors in our study were FRα‐negative, consistent with prior work [23]. The reported range for FRα positivity in EOC was 0–6%, in line with our 2% positivity [8, 9, 23]. Apart from one negative AGCT case in prior literature, we analyzed sex cord‐stromal tumors for the first time, and all were negative [6]. For OCCC, two studies included 7 and 11 tumors, all FRα‐negative [8, 9], whereas one larger study reported 8% positivity in 264 OCCC tumors [23]. In our series, 1/86 OCCC was FRα‐positive.
The published data on FRα expression in endometrial cancer are scarce. Two studies from 2007 tested FRα expression in endometrial cancer by IHC using a different antibody and scoring system (moderate to strong staining in >10% of tumor cells) [11, 12]. One included 292 tumors, with FRα positivity of 56% in EEC, 10% in mixed epithelial carcinoma, 32% in ESC, and 2% in ECCC [11]. The second analyzed 48 uterine tumors, with FRα positivity of 69% for ESC, 38% for EEC, and 37% for malignant mixed Müllerian tumors [12]. Another study, also using a different antibody and scoring system (moderate to strong staining of more than 25% of tumor cells), found 48/95 EEC (50.5%) FRα‐positive [13]. Three recent studies used the VENTANA FOLR1 CDx Assay and standardized scoring criteria. One included only three EEC, all FRα‐negative [8]. Another one had a larger sample size of 111 endometrial carcinomas, reporting 6/67 EEC (9.0%), 4/9 ESC (44.4%), 3/9 ECCC (33.3%), 4/10 carcinosarcomas (40.4%), 4/12 mucinous carcinomas (33.3%), and no UC or DC as FRα‐positive [25]. The most recent study examined 53 ESC, out of which 5 (9.4%) were FRα‐positive, and 10 high‐grade EEC, which were all FRα‐negative [26]. Our study showed that 11% of ESC were FRα‐positive, while all other endometrial carcinoma types were FRα‐negative.
Regarding survival, several previous studies reported no significant association between PFS and FRα‐positivity in LGSC [1, 6]. In our study, however, FRα‐positive LGSC were associated with significantly shorter PFS, although the relatively small number of cases and events in this subgroup, as well as the inclusion of 7 metastasis samples, warrant cautious interpretation.
From a practical point of view, one should be aware of possible discordance in samples from different timepoints, as was shown in a recent study in which 44% of samples were discordant (33% converting from FRα‐negative to FRα‐positive) [6]. Nevertheless, the sample size in this study was small, and more data are needed to assess spatial and temporal heterogeneity of FRα expression.
The limitations of our study are mainly related to the usage of TMAs. This approach is advantageous in studies with a larger number of cases because it allows evaluation of tumors from multiple patients on one slide; however, it also theoretically raises the risk of under‐ or overestimating IHC staining due to tumor heterogeneity. To minimize this risk, two large cores (2 mm) were taken from each tumor sample and evaluated side‐by‐side.
Conclusion
The results of our study confirmed that FRα expression assessed by the VENTANA FOLR1 CDx assay using standardized scoring criteria is similar among studies focusing on HGSC and LGSC. Moreover, our results showed that FRα expression is present in a minority of ESC but is absent or rare in other ovarian and endometrial tumors examined. From a practical point of view, FRα expression is, with exceptions, limited to tumors with serous differentiation (both tubo‐ovarian and endometrial), which seems to be the category of tumors in which the testing is potentially most beneficial concerning therapeutic consequences. Nevertheless, the potential role of MIRV treatment in non‐HGSC patients is currently unknown, as only HGSC patients were included in clinical studies to date.
Author contributions statement
PD prepared the study concept and design. All authors participated in material preparation, data collection and/or data analyses. All authors have read and approved the final paper.
Supporting information
Table S1. Results of Dunn's multiple comparison test for folate receptor alpha (FRα) expression (H‐score) across tubo‐ovarian and endometrial tumor types
Table S2. Multivariable Cox proportional hazards models for overall survival and progression‐free survival in HGSC and LGSC
Acknowledgements
This work was supported by the Ministry of Health, Czech Republic (MH CZ DRO‐VFN 64165), by Charles University (Project UNCE 24/MED/018), and by the European Regional Development Fund (BBMRI_CZ LM2023033). The authors wish to thank Mgr. Zachary Harold Kane Kendall, B.A. (Institute for History of Medicine and Foreign Languages, First Faculty of Medicine, Charles University) for the English proofreading.
No conflicts of interest were declared.
Data availability statement
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Table S1. Results of Dunn's multiple comparison test for folate receptor alpha (FRα) expression (H‐score) across tubo‐ovarian and endometrial tumor types
Table S2. Multivariable Cox proportional hazards models for overall survival and progression‐free survival in HGSC and LGSC
Data Availability Statement
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.