A comparative analysis of tumor markers reveals EDA fibronectin as a promising target in high-grade serous ovarian cancer

Abstract

Background

Ovarian cancer remains a major clinical challenge with more than 40.000 annual deaths in Europe and in the United States, highlighting the need for better diagnostic and therapeutic strategies. This study first presents an immunohistochemical evaluation of the extra-domains A and B containing fibronectin (EDA-FN, EDB-FN), fibroblast activation protein (FAP), and carcinoembryonic antigen (CEA) in ovarian cancer specimens. Based on the initial results, the analysis was subsequently expanded to provide a comprehensive assessment of EDA-FN expression in human epithelial ovarian cancer tissue samples.

Methods

An initial exploratory immunohistochemical analysis was performed on 60 formalin fixed paraffin embedded (FFPE) epithelial ovarian cancer (EOC) tissue sections from 47 patients, including 47 specimens collected at first diagnosis and 13 matched relapsed lesions. Tissue sections were stained using previously validated antibodies specific to EDA-FN, EDB-FN, FAP and CEA, to evaluate the stromal immunoreactive score (sIRS Part 1). Following the completion of Part 1, the study was expanded to specifically analyze the most abundant antigen found (EDA-FN) on 204 FFPE High Grade Serous ovarian cancer (HGSOC) tissue samples from 102 subjects, including primary and metastatic sites from the same patient (Part 2).

Results

In Part 1, stromal expression of EDA-FN, EDB-FN and FAP was observed in epithelial ovarian cancer with no significant differences between matched primary and relapse tumor tissues. CEA was exclusively found in mucinous ovarian cancer (MOC). EDA-FN was the most abundant antigen among the ones investigated, prompting a deeper investigation in Part 2. In the expanded EOC cohort, EDA-FN remained highly abundant across all molecular subgroups (HRp, HRd/BRCAwt, and BRCAmut) and clinical subgroups (naïve vs. pretreated patients), but was found at elevated level in metastases compared to the corresponding primary tumors.

Conclusions

These findings highlight that EDA-FN is an excellent target for HGSOC, while CEA could serve as a potential target for MOC. Clinical investigations are warranted to evaluate innovative treatments in ovarian cancer targeting these antigens.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13048-025-01772-6.

Introduction

Epithelial ovarian cancer (EOC) remains a highly lethal disease with projections for 2025 estimating approximately 40.000 deaths in Europe and the United States [1, 2]. The introduction of targeted therapies in front line treatment of advanced high-grade serous ovarian cancer (HGSOC), the most common EOC histotype, has significantly improved patient outcomes. In 2018, the addition of bevacizumab, a VEGF inhibitor, was shown to significantly prolong progression-free survival (PFS), leading to its incorporation into frontline therapy [3]. Since 2019, the incorporation of upfront maintenance therapy with PARP inhibitors (PARPi) has further transformed the therapeutic landscape, particularly benefiting patients with BRCA mutations or homologous recombination deficient (HRd) tumors [4]. However, despite these advances, approximately 70% of patients experience disease recurrence within 18 months, highlighting the urgent need for novel and more effective therapeutic strategies [5]. Although the integration of immunotherapy in the upfront therapeutic algorithm of newly diagnosed EOC patients is gaining momentum, current evidence suggests its clinical benefit may be limited to specific molecular subgroups, particularly those with HRd [6, 7]. Antibody–drug conjugates (ADCs) have emerged as a promising class of therapeutics in oncology, enabling the targeted delivery of cytotoxic payloads to tumor cells while minimizing off-target toxicity. Following encouraging results in platinum-resistant EOC, the folate receptor alfa (FRα)-targeting ADC mirvetuximab soravtansine-gynx is now being evaluated in combination with bevacizumab for patients with platinum-sensitive recurrent disease [8]. However, less than 50% of EOC patients express FRα at levels sufficient to get access to mirvetuximab soravtansine-gynx [9]. Neoangiogenesis and tissue remodeling may give rise to or enhance the expression of tumor-associated antigens, offering additional avenues for generating novel targeted therapeutics [10]. EDA-containing fibronectin (EDA-FN), EDB-containing fibronectin (EDB-FN), Fibroblast Activation Protein (FAP) and Carcinoembryonic antigen (CEA) are antigens which have been considered for the diagnosis and treatment of various solid tumors. However, to date no comparative analysis of such targets has been conducted in EOC. EDA-FN and EDB-FN are splice variants of fibronectin that are undetectable in most adult healthy tissues, but become highly abundant in the majority of aggressive malignancies. CEA expression in normal tissues is restricted to the apical surface of enterocytes, making it inaccessible to antibodies present in circulation. However, malignant enterocytes lose polarity and start displaying CEA on the entire cell surface [11–13]. CEA is thus a key marker of metastatic colorectal cancer and is also overexpressed in breast, liver, lung, stomach, pancreatic, and ovarian cancers [14]. FAP is a transmembrane serine protease that is highly expressed by cancer-associated fibroblasts (CAFs) and has been extensively validated as tumor-associated marker by Nuclear Medicine techniques [15].

In this article, we present the results of the first comparative immunohistochemical (IHC) analysis using four validated antibodies named F8 [16], L19 [17], anti-FAP (Abcam), and F4 [18] specific to EDA-FN, EDB-FN, FAP, and CEA, respectively, on 60 EOC specimens from patients treated at Fondazione Policlinico Universitario Agostino Gemelli in Rome, Italy (Part 1). EDA-FN was found to be the most abundant marker and was therefore further investigated in a larger cohort of 204 matched primary and metastasis tumor samples from 102 HGSOC patients (Part 2).

Materials and methods

Patients and study design

This is a non-profit, observational, retrospective study, approved by the Institutional Ethics Committee (ID 6144). The inclusion criteria required patients to have a histological diagnosis of epithelial EOC, available formalin-fixed, paraffin-embedded (FFPE) tissue samples, updated clinical information, and a signed informed consent form. Patients were excluded if they had a non-epithelial histology or if the FFPE specimen contained less than 30% tumor cells. For eligible patients, clinical data were retrieved from their medical records. An IHC analysis with a panel of antibodies for the immuno-oncology targets EDA-FN, EDB-FN, FAP and CEA was performed on one representative tissue sample from a tumor block of all cases, primary and/or metastases tissues and, when available, at relapse. Pseudo-anonymized data were collected and managed using REDCap electronic data capture tools hosted at https://redcap-irccs.policlinicogemelli.it/ [19]. All data were processed in compliance with European Regulation on the Privacy of data (UE 2016/679).

Data analysis

A total of 60 tissue samples were estimated for the Part 1. Additionally, 102 paired tumor tissue samples were analyzed using the F8 antibody for the Part 2. This sample size has the power of 80% to identify small effect size (i.e. ES = 0.3) using the Wilcoxon Signed-Rank test at a significance level of 5%. Continuous variables were summarized as medians and interquartile ranges and differences between variables were analyzed using non-parametric tests such as Mann-Whitney U test, Wilcoxon Signed-Rank Test or Kruskal-Wallis Test followed by post hoc pairwise Dunn’s tests with Holm correction for multiple comparisons. Statistical analysis was performed in R (version 4.2.2) [20] using the rstatix and ggplot2 packages.

Before applying for the specific test, Shapiro-Wilk test has been used to assess whether a dataset follows a normal distribution. Categorical variables were used to express the number of cases and percentages (%). Median follow up was calculated with the inverse Kaplan-Meier technique. Survival outcomes were presented in terms of both disease-free survival (DFS) and overall survival (OS). DFS was defined as the time that elapsed from the first pathological diagnosis to recurrence or last follow-up, whereas OS was defined as the time from the first pathological diagnosis to death or last follow-up. Median OS and DFS were estimated with the Kaplan–Meier product limit method and were presented with a 95% confidence interval. All statistical tests used a two-tailed α of 0.05. All analyses were performed using SPSS version 24.0.

Sample processing

The FFPE tissue blocks were collected and cut into 3-µm thick sections. Haematoxylin and eosin (H&E) stained sections from FFPE were double-blind reviewed by two expert pathologists. IHC staining of FFPE tissues was performed with the F8, L19, F4 antibodies (Philogen), and anti-FAP (Abcam) followed by appropriate secondary monoclonal rabbit anti-human antibodies using the BOND Polymer Refine Detection on the Bond III automated immunostainer (Leica Microsystems, Bannockburn, IL). The fully human monoclonal antibodies F8 and L19, isolated from encoded combinatorial libraries are directed against the alternatively spliced EDA and EDB domains of fibronectin, respectively [16, 17]. These antibodies have been shown to selectively localize around tumor neo-vasculature structures in animal cancer models and in patients, making them valuable tools for cancer research and potential therapeutic applications [21].

A negative and positive control staining versus reactivity with the mAbs was performed in each series. The antibody clones did not have the same affinity towards their cognate antigen, so they were used at different concentrations.

Sample evaluation

The F8, L19, and anti-FAP staining were evaluated based on the intensity signal (absent, 0; weak, 1+; moderate, 2+; strong, 3+) and the percentage of positive immunoreactive cells for the respective target (EDA-FN, EDB-FN, and FAP) on HGSOC, low-grade serous (LGSOC) and clear cell (CCOC) tissue samples. The results of CEA immunohistochemical staining were interpreted based on the localization pattern of the antigen in MOC tissues samples: score 0; negative/absent staining, score 1; staining restricted to the apical membrane: apical-type, score 2; staining on the apical/basolateral cellular membrane, score 3; staining on the apical and luminal. For statistical purposes results of F8, L19, and anti-FAP staining were defined by a semi-quantitative stromal immunoreactive score (sIRS) as follow: stromal immunoreactive score of primary (sIRS_P), stromal immunoreactive score of relpase (sIRS_R) and stromal immunoreactive score of metastasis (sIRS_M), wherein the staining percentage in a 5-tiered scale (0% = 0, 1–10% = 1, 11–33% = 2, 34–66% = 3 and 67–100% = 4) is multiplied with the staining intensity on a 4-tiered scale (no staining = 0, weak staining = 1, moderate staining = 2, strong staining = 3), resulting in an sIRS ranging between 0 (completely negative) and 12 (strongly positive) [22, 23, 24].

Results

The study design, which was divided into two parts, is presented in Fig. 1. The initial exploratory phase (Part 1) focused on a comparative analysis of EDA-FN, EDB-FN, FAP, and CEA expression in 47 EOC specimens, 13 of which had matched relapse lesions that were also included in the analysis. The investigation was subsequently expanded, focusing on EDA-FN with an additional 102 HGSOC subjects (the most common EOC) with matched primary and metastatic lesions (Part 2).

Fig. 1.

Study design, comprising an exploratory phase (Part 1) followed by an expanded analysis focused on EDA-FN expression (Part 2). The study cohort, the collected biological samples and analyzed biomarkers for each part are indicated in the figure

Part 1

A total of 47 EOC patients diagnosed between March 2016 and March 2018 were retrospectively enrolled for Part 1 of the study. Baseline tumor tissue samples were accessible for all patients, with 13 of them additionally having matched tumor biopsy samples taken at the time of relapse. The clinicopathological characteristics of the enrolled EOC patients are reported in Supplementary Table 1. Most of the analyzed patients had HGSOC (74.5%) and were diagnosed at an advanced stage (FIGO stage III-IV, 81%). 63.8% experienced a tumor relapse.

Fig. 2 A and B presents representative images of the immunohistochemical analysis of EDA-FN, EDB-FN, FAP and CEA stained with the F8, L19, anti-FAP (Abcam), and anti-CEA (F4) antibodies, respectively.

Fig. 2.

A) Three representative sections from different HGSOC surgical specimens showing strong immunohistochemical staining with anti-EDA-FN (F8), anti-EDB-FN (L19), anti-FAP, and anti-CEA (F4) antibodies. Corresponding hematoxylin-eosin (HE) and negative control (ctrl) images are included. (Magnification ×10). B) Three representatives, strong immunohistochemical staining obtained with the anti-CEA antibody in sections derived from different surgical specimens of MOC. Black arrows indicate the apical/basolateral localization of CEA on MOC tissue samples that were distributed around the cell surface. (magnification, ×10)

The F8 antibody showed a strong and diffuse staining pattern in the stroma and neovascular smooth muscle cells.Samples derived from HGSOC, LGSC and CCC exhibited a diffuse stromal and neovascular staining of EDA. A positive EDA staining was observed in forty-six (92%) of 50 neoplasm samples. In detail, F8 immunoreactivity appeared weak (1+) in 7 (14%), moderate (2+) in 22 (44%) and strong (3+) in 17 (34%) of cases. The L19 antibody presented a similar pattern but with lower intensity compared to F8. Tumor cells largely exhibited a negative staining with both antibodies. FAP was primarily expressed by CAFs within the tumor stroma. When considering all 47 specimens, the expression of EDA-FN was substantially higher compared to EDB-FN and FAP (sIRS median value 6 vs. 2 and 2 respectively; Supplementary Fig. 1A). The BRCA1/2 gene status did not have an impact on the expression levels of EDA-FN, EDB-FN, and FAP (Supplementary Fig. 1B. Except for FAP, whose expression was higher in patients who experienced relapse compared to those who did not, the expression levels of EDA-FN and EDB-FN were similar between the groups (Supplementary Fig. 1C).

No statistically significant differences were observed when comparing staining results at baseline and at recurrence of matched tumor tissue samples (n = 13; Supplementary Fig. 2). The intensity of F8 was diminished in two cases, transitioning from sIRS_P of 8 to sIRS_R of 4 or negative, whereas in ten cases, it increased at the time of recurrence.

CEA expression was found to be negative in EOC tissues (Fig. 2A), while being highly abundant around the cell surface with apical/basolateral distribution in MOC samples (N = 10; Fig. 2B). Three samples exhibited both apical and basolateral staining (score 2), in five samples the staining was restricted to the apical membrane (score 1), and one sample was considered negative (Supplementary Table 2).

Part 2

One hundred and two advanced HGSOC patients diagnosed between December 2022 and December 2023, for whom matched primary and metastatic tumor tissue samples were available, were retrospectively enrolled for Part 2 of the study. Metastatic sites were the peritoneum (92%), omentum (7%) and intestine (1%).

Clinico-pathological characteristics of this cohort are presented in Table 1.

Table 1.

Clinical, pathological, surgical, treatment and survival characteristics of the part 2 cohort (all cases, subgroups according to genomic and disease status)

Characteristics	All cases	Genomic status			Disease status
		HRp	HR-d and BRCA wild type	HR-d and BRCA mutated	No recurrence	Recurrence
	n = 102	n = 38	n = 36	n = 28	n = 82	n = 20
Age at diagnosis, years	58 (40–83)	62.5 (43–83)	56.5 (40–76)	55.5 (44–72)	57.5 (40–83)	60.5 (43–75)
CA125 at diagnosis, U/mL *	366 (16.4-10000)	341 (16.4–8457)	507 (17.1–5744)	275.5 (36.1-10000)	324.7 (16.4-10000)	639 (32-8457)
FIGO stage
III	73 (71.5)	29 (73.3)	24 (66.6)	20 (71.4)	60 (73.2)	13 (65)
IV	29 (28.4)	9 (23.7)	12 (33.3)	8 (28.6)	22 (26.8)	7 (35)
Primary debulking surgery	102 (100.0)	38 (100.0)	36 (100.0)	28 (100.0)	82 (100.0)	20 (100.0)
Residual tumor
Absent	98 (96.1)	38 (100.0)	35 (97.2)	25 (89.3)	79 (96.3)	19 (95.0)
> 0 cm	4 (3.92)	0 (0.0)	1 (2.8)	3 (10.7)	3 (3.7)	1 (5)
Chemotherapy
No †	1 (1.0)	1 (2.6)	0 (0.0)	0 (0.0)	0 (0.0)	1 (5.0)
Yes	101 (99.0)	37 (97.4)	36 (100.0)	28 (100.0)	82 (100.0)	19 (95.0)
Chemotherapy regimen
Platinum based adjuvant setting	101 (100.0)	37 (100.0)	36 (100.0)	28 (100.0)	82 (100.0)	19 (100.0)
Maintenance therapy
No	14/102 (13.7)	9/38 (23.7)	2/36 (5.6)	3/103 (2.9)	6/82 (7.3)	8/20 (40.0)
Yes	88/102 (86.3)	29/38 (76.3)	34/36 (94.4)	100/103 (97.1)	76/82 (92.7)	12/20 (60.0)
Bevacizumab	21/88 (23.9)	18/29 (62.1)	3/34 (8.8)	0/25 (0.0)	14/76 (18.4)	7/12 (58.3)
Bevacizumab with Parp Inhibitors	28/88 (31.8)	1/29 (3.4)	26/34 (76.5)	1/25 (4.0)	26/76 (34.2)	2/12 (16.7)
Parp inhibitors	39/88 (44.3)	10/29 (34.5)	5/34 (14.7)	24/25 (96.0)	36/76 (47.4)	3/12 (25.0)
Patient status°
Alive	99 (97.1)	37 (97.4)	35 (97.2)	27 (96.4)	82 (100.0)	17 (85.0)
Dead of disease	3 (2.9)	1 (2.6)	1 (2.8)	1 (3.6)	0 (0.0)	3 (15.0)
Dead for other causes	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Presence of disease	20 (19.6)	13 (34.2)	5 (13.9)	2 (7.1)	0 (0.0)	20 (100.0)
Progression	2 (14.3)	0 (0.0)	2 (100.0)	0 (0.0)	-	2 (14.3)
Relapse	12 (85.7)	10 (100.0)	0 (0.0)	2 (100.0)	-	12 (85.7)
Median follow up (95% CI), months	17.8 (16.6–18.8)	19.8 (17.8–21.3)	17.2 (14.7–18.9)	15.6 (12.6–17.6)	17.2 (14.9–17.8)	20.6 (19.5–23.8)
Probability of disease-free survival (95% CI) at 12 months ǂ	90.3 (82.2–94.9)	83.2 (66.3–92.1)	97.2 (81.9–99.6)	91.6 (70.1–97.8)	100 (NA)	55.0 (31.3–73.5)
Probability of overall survival (95% CI) at 12 months ǂ	97.9 (91.9–99.5)	100 (NA)	97.2 (81.9–99.6)	95.5 (71.9–99.3)	100 (NA)	90.0 (65.6–97.4)

Results are presented as n (%) or median (min-max) as appropriate. BRCA: BReast CAncer gene. HR: Hormone Recombination. CA: Cancer Antigen. ECOG: Eastern Cooperative Oncology Group. FIGO: international federation of gynaecology and obstetrics. CI: Confidence Interval. NA: Not Applicable. * Information available for 101/102 cases. † One patient did not undergo chemotherapy for clinical condition. § Calculated with the inverse Kaplan-Meier technique. ǂ Calculated with the Kaplan-Meier technique. ° Last follow up: 24 January 2025

Among the 102 patients included, 37% were HRp, 35% were HRd/BRCA1/2 wild-type (HRd/BRCAwt), and 27% had BRCA mutations (BRCAmut). With a median follow-up of 17.8 months, 19.6% experienced recurrence. With respect to patients receiving maintenance treatment (n = 88), 23.9% received bevacizumab as single agent, while 31.8% received bevacizumab in combination with PARP inhibitors.

EDA-FN staining in primary tumor tissues revealed predominantly a moderate-to-strong and diffuse expression of the antigen (median sIRS 8). No statistically significant differences were observed in terms of sIRS among the three molecular subgroups (HRp, HRd/BRCAwt, and BRCAmut) or between patients with or without tumor relapse (Fig. 3A). Similar data were obtained in metastatic tumor tissues (Fig. 3B). The results are in line with the ones obtained in Part 1 of the study.

Fig. 3.

EDA-FN expression, represented as sIRS, in primary (A) and metastatic (B) tumor tissue samples of HGSOC patients (n = 102), collected at baseline, grouped by HRD status (upper panel) and relapse experience (lower panel)

When comparing EDA-FN expression between metastatic and matched primary samples, sIRS was significantly higher in metastases (P < 0.0001, Fig. 4A).

Fig. 4.

EDA-FN expression, represented as sIRS, in primary (P, red dots) and metastatic (M, light blue dots) tumor tissue samples of the entire cohort (A, n = 102), cases grouped by HRD status (B) and relapse experience (C) ****P < 0.0001 refer to the paired Wilcoxon signed-rank test comparison between primary vs. metastasis tumor tissue samples; *P < 0.05, **P < 0.01, ***P < 0.001 refer to the Wilcoxon signed-rank test comparison between primary vs. metastasis tumor tissue samples in each group

This difference remained significant when analyzing subgroups, including HRp patients (P < 0.01), HRd/BRCA wt patients (P < 0.05), BRCAmut patients (P < 0.01), those who experienced recurrence (P < 0.01) and those who did not (P < 0.001), as illustrated in Fig. 4B and C.

We subsequently grouped patients according to Bevacizumab treatment and recurrence experience. Results showed no statistically significant differences in EDA-FN expression in primary and metastatic tumor tissue samples among the analyzed groups (Fig. 5A and B). Patients who relapsed after Bevacizumab treatment (n = 9) and those who did not recur (n = 40) showed similar antigen expression.

Fig. 5.

EDA-FN expression, represented as sIRS, in primary (A) and metastatic (B) tumor tissue samples of HGSOC patients (n = 102) collected at baseline, grouped by relapse experience (yes/no below the graphs) and Bevacizumab treatment (yes/no above the graphs)

Discussion

This study provides a comprehensive comparison of four tumor-associated markers (EDA-FN, EDB-FN, FAP, and CEA) in EOC specimens using immunohistochemistry. Among these, EDA-FN emerged as the most abundantly expressed in EOC, with levels typically elevated in metastatic lesions compared to primary tumors. Interestingly, CEA was undetectable in EOC but highly expressed in MOC, a rare histological subtype. Regarding antigen expression, our findings are consistent with previously published data. In HGSOC, the anti-FAP antibody demonstrates stromal staining in approximately 86% of cases [25]. The anti-CEA antibody shows positivity in about 75% of MOC but is generally negative in serous subtypes [26]. The EDB splice variant of fibronectin is broadly expressed in the tumor stroma and neovasculature across a wide range of solid tumors, whereas the EDA variant is predominantly localized to the tumor stroma and, to a lesser extent, within tumor cells [27–29]. Splice variants of fibronectin, such as EDA-FN and EDB-FN, are attractive targets for tumor-targeted drug delivery, as they are strongly expressed in a variety of aggressive malignancies but largely absent in normal adult tissues (except for the placenta and the endometrium during the proliferative phase) [29]. The EDB-fibronectin splice variant is expressed in over 90% of solid tumors, while EDA-fibronectin is found in more than 70% of cases, predominantly in the tumor stroma and occasionally in tumor cells [10]. In line with previous reports in other cancer types [30], our findings indicate that EDA-FN is more abundant than EDB-FN in EOC. Comparative analyses of expression patterns for the extra domains EDB-FN and EDA-FN of fibronectin (L19 and F8 antibody, respectively) have been already reported in other cancer types [11, 29, 31–33]. In certain malignancies, EDA-FN was found to be more abundant than EDB-FN (e.g., kidney cancer, melanoma, cutaneous squamous cell carcinoma, leukemia), while in others the two antigens were expressed at comparable levels (e.g., glioblastoma). Our results showed that EDA-FN was found to be more abundant than EDB-FN in EOC, although the pattern, in terms of diffuse stromal staining, was similar. The F8 antibody, which selectively binds EDA-FN, has been employed successfully for the delivery of various therapeutic payloads [34].

ADCs such as mirvetuximab soravtansine and trastuzumab deruxtecan have demonstrated encouraging activity in EOC. Mirvetuximab targets FRα, which is overexpressed in fewer than 50% of EOC cases. Trastuzumab deruxtecan targets HER2, whose expression in EOC varies widely between studies (11–66%) [35]. In contrast, our study reveals that EDA-FN is consistently and strongly expressed in both primary and metastatic EOC lesions, supporting its potential as a broadly applicable target for ADC-based therapies. The delivery of cytotoxic payloads such as Monomethyl auristatin E (MMAE) or Exatecan (Dxd) via an EDA-FN-specific antibody could thus represent a promising therapeutic strategy that warrants further preclinical and clinical evaluation. In addition to cytotoxic agents, proinflammatory cytokines offer an alternative therapeutic modality. Intraperitoneal administration of interleukin-2 (IL-2) has shown durable responses in patients with platinum-resistant ovarian cancer [36]. Similarly, an interleukin-12 (IL-12) DNA plasmid vector (“IMNN-001”) has yielded promising results in combination with neoadjuvant and adjuvant chemotherapy in newly diagnosed EOC patients [37]. However, these approaches are limited to the treatment of local or locally advanced disease. The targeted delivery of pro-inflammatory cytokines using EDA-FN-directed antibodies such as F8 may enhance therapeutic efficacy and enable therapy of disseminated EOC.

To our knowledge, anti–EDA-FN antibodies have not yet been evaluated in imaging or therapy studies for EOC. Given EDA-FN’s distinct and consistent expression profile, further exploration of EDA-FN-targeted therapies (spanning both cytotoxic and immunomodulatory agents) may lead to more effective and selective treatment strategies for patients with EOC.

CEA is one of the most extensively validated cell surface markers for ligand-based pharmacodelivery, particularly in metastatic colorectal cancer (mCRC). In addition to mCRC, CEA is expressed in a variety of epithelial malignancies, including gastric, pancreatic, lung, and breast cancers, as well as in MOC [38]. In our study, immunohistochemical analysis using the F4 antibody [39] confirmed strong and localized expression of CEA on the cell surface of MOC specimens, supporting its potential as a therapeutic target in this rare ovarian cancer subtype.

MOC accounts for approximately 3–5% of all epithelial ovarian cancers and is considered a biologically and clinically distinct entity. Unlike HGSOC, MOC tends to present at earlier stages but often shows poor response to standard platinum-based chemotherapy in advanced stages. The prognosis of patients with metastatic or recurrent MOC remains dismal due to its resistance to chemotherapy and the lack of targeted treatment options. Moreover, clinical trials in ovarian cancer typically exclude or underrepresent patients with MOC. As such, MOC represents an area of significant unmet medical need, where molecularly guided treatment approaches are urgently required.

A broad range of CEA-targeting biopharmaceuticals has been developed and evaluated, particularly in mCRC. These include bispecific T-cell engagers (e.g., CEA-TCB or cibisatamab) [40], radiolabeled monoclonal antibodies (e.g., arcitumomab) [41], and CEA-directed antibody-drug conjugates (e.g., labetuzumab govitecan) [42]. Most recently, a novel biparatopic CEA-targeting ADC demonstrated promising preclinical and clinical efficacy across CEA-expressing tumors. However, CEA-targeting strategies have not yet been explored in the context of MOC, likely due to its rarity. Given the robust and selective expression pattern observed in our study, the application of CEA-directed therapeutics may offer a rationale and novel approach for MOC treatment. Additionally, the use of radiolabeled CEA-targeting agents for molecular imaging could aid in diagnosis, patient stratification, and response monitoring.

Future research should prioritize expanding the characterization of CEA expression in larger, multi-institutional MOC cohorts and evaluating the efficacy of CEA-targeted modalities in preclinical MOC models. These efforts may enable the repurposing or adaptation of existing CEA-based therapeutics for patients with this rare and poorly addressed malignancy.

The current study presents some limitations. The small number of matched relapse tumor samples in Part 1 and the lack of longitudinal samples in Part 2 limit the ability to comprehensively evaluate dynamic changes in EDA-FN expression over time. Moreover, the inclusion period predates the widespread adoption of PARP inhibitors for the therapy of EOC, which may influence the relevance of the findings to contemporary treatment landscapes.

Overall, our findings support the development of EDA-FN and CEA-targeted drugs for the therapy of EOC and MOC, respectively.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

EOC

Epithelial ovarian carcinoma

VEGF

Vascular Endothelial Growth Factor

PARP

Poly (ADP-ribose) polymerase inhibitors

HGSOC

High-Grade Serous OC

CCC

Clear-Cell Carcinoma

LGOC

Low-Grade Serous Ovarian Cancer

HRd

Homologous Recombination Deficiency

EDB

Spliced Extra-Domain B

EDA

Spliced Extra-Domain A

FAP

Fibroblast Activation Protein

CAFs

Cancer-Associated Fibroblasts

CEA

Carcinoembryonic antigen

mCRC

Metastatic Colorectal Cancer

MOC

Mucinous ovarian cancer

FFPE

Formalin-Fixed, Paraffin-Embedded

IHC

Immunohistochemistry

HE

Haematoxylin and eosin

wt

wild-type

CAR

Chimeric Antigen ReceptorT-cells

MIRV

Mirvetuximab soravtansine.

Author contributions

A.P., R.D.L., E.P., T.P., C.N., “Conceptualization, Writing - Original Draft”; A.P., R.D.L., C.N., “Methodology”; A.P., N.D.A, A.T., T.P., D.G., “Software”, “Formal Analysis”and “Data Curation”;M.B., F.S., “Project administration”A.P., R.D.L., E.P., C.N., “Investigations” F.P., L.P., G.F.Z., A.F., D.N., G.S., “supervision” and “writing–review and editing”All authors discussed and contributed to the final manuscript, approving the submitted version.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Patients’ written informed consent was obtained before the samples collection. This is a no-profit, observational, retrospective study, approved by the Institutional Ethics Committee of ‘Fondazione Policlinico Universitario Agostino Gemelli-IRCCS’ of Rome, Italy (ID 6144).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.