Immunohistochemical expression of Claudin18.2 in borderline and malignant gynecologic tumors: Results from a pilot study

Immunohistochemical expression of Claudin18.2 in borderline and malignant gynecologic tumors: Results from a pilot study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Immunohistochemical expression of Claudin18.2 in borderline and malignant gynecologic tumors: Results from a pilot study Bettina Blau-Schneider, Sonja Stallmann, Boris Gabriel, Esra Bilir, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8756545/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Claudin 18.2 (CLDN 18.2) is an emerging therapeutic target in gastrointestinal malignancies, but its expression in gynecologic mucinous tumors remains poorly characterized. Our study aimed to evaluate CLDN18.2 expression in borderline and malignant mucinous tumors of the female genital tract and to explore its potential clinical relevance. Methods Formalin-fixed paraffin-embedded tumor specimens from 20 patients with mucinous tumors of the ovary, corpus uteri, or cervix uteri treated between 2013 and 2024 were included. CLDN18.2 expression was assessed by immunohistochemistry (IHC) and scored using the H-score system based on standardized digital evaluation. Clinical data, including tumor stage, grade, and outcomes, were retrospectively obtained. Associations between CLDN18.2 expression and clinicopathologic parameters were analyzed descriptively using SPSS. Results CLDN18.2 expression was detectable in 50.0% of evaluable tumors. Among histologic subtypes, 60% mucinous borderline tumors and 46.2% mucinous adenocarcinomas were positive. Staining intensity was generally mild to moderate, with strong expression (H-score > 200) observed in only one case. No statistically significant associations were observed between CLDN18.2 positivity and tumor stage, grade, patient demographics, or borderline versus invasive status; however, the small sample size limits the interpretability of these findings. Conclusions CLDN18.2 is expressed in a subset of gynecologic mucinous tumors, but high-level expression is uncommon. Given the limited number of cases, these preliminary results should be interpreted with caution, and no firm conclusions regarding clinical correlations can be drawn. Only a minority of patients may meet thresholds for CLDN18.2-targeted therapies established in gastrointestinal malignancies. Larger, multicenter studies are warranted to better define the prevalence, biological significance, and potential therapeutic relevance of CLDN18.2 in these tumors. Claudin18.2 Gynecologic mucinous tumors Immunohistochemistry Tissue microarray Zolbetuximab Translational oncology Borderline tumor Figures Figure 1 Figure 2 Background Mucinous tumors of the female internal genital tract represent a distinct and heterogeneous group of neoplasms, located in the ovary, endometrium, cervix, and other Mullerian-derived sites. Although relatively rare compared to serous or endometrioid subtypes, mucinous carcinomas pose significant diagnostic and therapeutic challenges due to their unique biology and frequent molecular heterogenity. Ovarian mucinous carcinoma is a rare subtype of epithelial ovarian cancer characterized by a molecular profile that differs from that of high-grade serous carcinomas. It often presents at an early stage and in younger women, and shows frequent copy-number loss in CDKN2A, as well as TP 53- and KRAS mutations and, in some cases, HER2 amplifications, with limited responsiveness to standard platinum-based chemotherapy. ( 1 ) In the endometrium, a rare but increasingly recognized subtype is the gastric (gastrointestinal)-type mucinous adenocarcinoma (EmGA). In the largest series published to date, EmGA exhibits frequent pathogenic variants in TP53 , KRAS , PIK3CA , and STK11 , among other genes, placing many cases into the p53-abnormal molecular class according to The Cancer Genome Atlas framework. Immunophenotypically, these tumors often express markers such as MUC6, CK7, CK20, and CDX2, reflecting their gastric/gastrointestinal differentiation. Clinically, EmGA may exhibit aggressive behaviour; however, given the limited available data, this appears to be variable, and adverse outcomes have been reported even in some low-grade or early-stage cases. ( 2 ) In the cervix, the gastric-type (also called gastric-type mucinous) adenocarcinoma (GAC) constitutes a rare, HPV-independent variant. It is associated with more aggressive behavior and worse prognosis than the usual HPV-related endocervical adenocarcinoma, even when diagnosed at an early stage. ( 3 ) Molecularly, GAC shows considerable genetic heterogeneity, with frequent TP53 mutations, and recurrent alterations in STK11 , CDKN2A/B , ARID1A , and DNA damage repair genes. ( 4 ) Beyond single-site tumors, synchronous mucinous metaplasia and neoplasia of the female genital tract (SMMN-FGT) can occur, in which mucinous lesions may appear simultaneously at multiple Mullerian sites (e.g., ovary, endometrium, cervix). ( 5 ) A recent case report identified shared STK11 mutations in synchronous lesions, suggesting a possible common clonal origin, though additional mutational differences (e.g., in KRAS ) have been observed between sites. ( 6 ) This multifocal presentation underscores the complexity of pathogenesis and the need for careful molecular and pathological evaluation. The diagnostic challenge of mucinous tumors is further characterizised by their morphological overlap with metastatic gastrointestinal adenocarcinomas. Immunohistochemical markers such as CK7, CK20, and CDX2 can be informative: in a series of gynecologic mucinous adenocarcinomas, the majority expressed CK7, while expression of CK20 and CDX2 correlated with intestinal differentiation, but the overlap limits discriminatory power. ( 7 ) The more recent marker SATB2 may help resolve such cases. ( 8 ) Given their rarity, diverse molecular alterations, and distinct clinical behavior, mucinous tumors of the internal genital tract warrant detailed characterization. Improved understanding of their molecular pathology can guide tailored therapeutic approaches, refine prognosis and support better diagnostic discrimination from metastatic disease. Claudin 18.2 (CLDN18.2) is a tight-junction protein isoform normally expressed in gastric epithelium. In malignant transformation, CLDN18.2 becomes aberrantly exposed on the tumor cell surface, making it a promising target for therapeutic antibodies. ( 9 ) ( 10 ) ( 11 ) In primary ovarian mucinous carcinomas, CLDN18.2 expression has been reported in up to 84% of cases, whereas expression in metastatic gastrointestinal mucinous carcinomas varies by origin: ~70% in upper gastrointestinal tract metastases, but generally absent in lower gastrointestinal metastases. ( 10 ) Within tubo-ovarian mucinous tumors, CLDN18.2 expression is largely restricted to the mucinous subtype, while non-mucinous subtypes show little to no expression. Importantly, this expression pattern is often retained in metastases, highlighting its potential as both a diagnostic and therapeutic marker. ( 11 ) Therapeutically, CLDN18.2 has already emerged as a target in gastric and gastroesophageal cancers, where monoclonal antibodies such as zolbetuximab were recently approved in combination with chemotherapy for first-line therapy in locally advanced and metastatic disease. ( 12 ) Mechanistically, CLDN18.2 may contribute to tumor progression by promoting cell–cell adhesion and interaction with the tumor microenvironment, including cancer-associated fibroblasts. ( 13 ) Given the promising new targets in gastrointestinal mucinous tumor, we aimed to evaluate CLDN18.2 expression in borderline and malignant mucinous tumors of the female genital tract and to assess its potential clinical relevance. Methods The primary endpoint was the frequency and distribution of CLDN18.2 expression in mucinous tumors of the female genital tract, assessed using H-score based on standardized digital evaluation of immunohistochemical (IHC) staining. Secondary endpoints included the association between CLDN18.2 expression and clinicopathological variables such as tumor subtype (borderline vs. carcinoma), tumor stage, tumor grade, and patient demographics. Patient selection All patients treated for mucinous carcinoma or mucinous borderline tumors of the ovary, corpus uteri, or cervix uteri at the Gynecologic Cancer Center of St. Josefs-Hospital, Wiesbaden, Germany between 2013 and 2024 were screened for inclusion. Inclusion criteria were: age ≥ 18 years at the time of diagnosis; histopathological diagnosis of a primary mucinous carcinoma or mucinous borderline tumor of the ovary, corpus uteri, or cervix uteri; treatment and/or surgical management performed at our institution; and availability of formalin-fixed paraffin-embedded (FFPE) tumor tissue for immunohistochemical analysis. availability of clinical and pathological data. Exclusion criteria comprised: non-mucinous histological tumor subtypes; metastatic mucinous tumors of extra-gynecologic origin; recurrent disease without available primary tumor tissue; and insufficient or unavailable tumor material for further analysis. Among the 35 identified patients with primary mucinous carcinoma or mucinous borderline tumor of the ovary, corpus uteri, or cervix uteri, formalin-fixed paraffin-embedded (FFPE) tumor tissue was available for 20 cases which were included in the final study cohort. Diagnoses were established according to the World Health Organization classification of female genital tumors based on surgical resection specimens. ( 14 ) The study was conducted in accordance with the Declaration of Helsinki. All samples were provided by the Centre for Histology, Cytology, and Molecular Diagnostics in accordance with local regulations and with approval from the institutional review board of the University Hospital Heidelberg (#2463 and #S315-2020). To achieve standardized staining accross all samples, a tissue microarray (TMA) was constructed. Clinical data, including patient age, tumor stage, tumor grade, and clinical outcomes, were retrieved from medical records. Retrospectively collected variables included age, follow-up duration, recurrence, histology, tumor grade, surgical treatment status and International Federation of Obstetrics and Gynecology (FIGO) classification for each eligible case. ( 15 ) ( 16 ) ( 17 ) ( 18 ) Immunohistochemistry staining and evaluation Tissue microarrays allow simultaneous processing of a large number of tumor samples on a single slide. ( 19 ) Because all cores on a TMA are processed under the same experimental conditions (e.g., same antigen retrieval, antibody concentration, incubation times), they enable greater standardization and reduce inter-assay variability. ( 20 ) The TMA was stained with an Claudin18.2 Antibody (Clon: ZR451, RTU) from Zeta Corporation (Monrovia, USA) as follows: pretreatment with pH 9 buffer for 30 minutes and incubation of the antibody for 30 minutes. Slides were scanned with a slide scanner (MidiII, Epredia, Dreieich, Germany). Data was visualized and analyzed using QuPath (v.0.6.0)( 21 ) In QuPath a representative region of the tumor was marked and „cell detection“ was performed with the following settings: pixel size 0.5 µm, background radius 8 µm, use opening by reconstruction, median filter radius 0 µm, sigma 1.5 µm, minimum area 10 µm^2, maximum area 400 µm^2, intensity threshold 0.1, max background intensity 2, split by shape, cell expansion 5 µm, include cell nucleus, smooth boundaries, and make measurements. After cell detection, a classifier was trained for each individual tumor by using an object classifier with the classes tumor and stroma, after manual annotation of a representative area, in order to be able to retrieve results for tumor cells only. Finally, cell intensity classification was used with three thresholds for mild, moderate and strong staining intensity using Cell: diaminobenzidine (DAB) optical density (OD) mean with standard variables. The amount of tumor cells with no, mild, moderate and strong staining intensity was documented. H-Score was calculated as follows: 0–50 = negative, 51–100 mild positiv, 101–200 moderate positive, and 201–300 strong positive. Additionally, the number of samples with ≥ 75% of tumor cells showing moderate-to-strong positivity was determined. Cut-off values were adopted from previously published studies in gastrointestinal malignancies, as no standardized thresholds for gynecologic mucinous tumors are currently available Statistical Analysis All analyses were performed using Statistical Package for Social Sciences (SPSS) Version 30.0 for Windows (Chicago, IL, USA). Data were presented as mean (± standard deviation). The analyses of secondary endpoints were considered exploratory. Binary and categorical variables were compared using the chi-square test. P-values < 0.05 were considered statistically significant. No formal correction for multiple testing was applied, as the analyses were exploratory. Results A total of 30 patients were screened during the study period. Eighteen patients met the inclusion criteria and were included in the final analysis. Reasons for exclusion were X (n = X) and X (n = X). In two cases no tumor could be detected after TMA creation. Patient characteristics Mean patient age was 55.7 years (range 25–88; SD:16.2). Mean follow-up time was 30.9 months (range 2–88; SD:28.678). At the data cutoff, all patients were alive. One case of recurrence was observed in a patient with cervical carcinoma, occurring 25 months after initial diagnosis. Histologically, 25.0% (n = 5) of cases were borderline ovarian tumors, 60.0% (n = 12) ovarian cancers, 10.0% (n = 2) cervical cancers, and 5.0% (n = 1) endometrial cancer. FIGO staging was as follows: 55.0% (n = 11) FIGO IA, 20.0% (n = 4) FIGO IB, 10.0% (n = 2) FIGO IC1, 5.0% (n = 1) FIGO IC2, 5.0% (n = 1) FIGO IC3, and 5.0% (n = 1) FIGO IIA. Borderline tumors were detected in 25% (n = 5), and tumor grade 1,2, and 3 was found in 45.0% (n = 9), 20.0% (n = 4), and 10.0% (n = 2) of cases respectively. All patients were diagnosed and treated between 2015 and 2024, and all underwent tumor surgery. Immunohistochemistry Tumor cell count was representative in the 18 evaluable tumors (mean: 3542, min: 324, max: 7071). Regarding CLDN18.2 immunohistochemical staining, nine out of 18 (50%) tumors were completely negative, and nine samples were positive (H-Score mean: 125, range 57,202). Five tumors (27.8%) showed moderate staining intensity (H-Score: 101–200), and three tumors showed mild staining intensity (17%; H-Score 51–100). Only one case (5.6%) demonstrated strong staining intensity (H-Score: 201–300). None of the samples showed moderate and strong staining intensity in > = 75% of tumor cells. Three out of five mucinous borderline tumors (60.0%) showed a positive immunreaction. Six out of 13 (46.2%) mucinous adenocarcinomas exhibited positivity. Despite the apparent difference in positivity between borderline tumors and adenocarcinomas, Chi-square analysis did not reveal any significant associaton between CLDN18.2 expression and invasive bahaviour of tumor (p-value = 0.44). In line with this, no significant correlations were observed between CLDN18.2 staining and other clinical parameters, including tumor stage (p = 0.23), grade (p = 0.59) or patient demographics (age p = 0.7, follow up p = 0.4). The evaluation process is shown in Fig. 1 . Representative images of Claudin18.2 staining are shown in Fig. 2 . Discussion In this pilot study of borderline and malignant mucinous tumors of the female genital tract, CLDN18.2 expression was generally scarce. Only 50% of evaluable tumors showed any immunoreactivity, and none exhibited moderate or strong staining in ≥ 75% of tumor cells. Mucinous borderline tumors exhibited a higher rate of positivity (75%) than mucinous adenocarcinomas (46.2%), although staining intensity in both subgroups was mostly mild to moderate, with only a single case showing strong expression (H-score > 200). These findings suggest that whereas CLDN18.2 is detectable in a subset of gynecologic mucinous tumors, high-level expression is rare. No statistically significant associations were observed between CLDN18.2 expression and tumor invasiveness, stage, grade, or patient demographics. However, given the exploratory nature of this pilot study, the limited sample size, and the low frequency of strong staining, the statistical power was restricted. Therefore, the absence of significant correlations should be interpreted with caution, and the results should be regarded as hypothesis-generating rather than confirmatory. The literature on CLDN18.2 expression in gynecologic malignancies remains limited, with small cohorts and heterogeneous scoring systems. Nevertheless, available studies consistently indicate that CLDN18.2 overexpression is largely restricted to mucinous subtypes. Wang et al. reported CLDN18.2 overexpression in 56% of mucinous tumors across various sites ( 10 ), whereas Wagner et al. found positivity in 45% of mucinous ovarian carcinomas, compared with only 4.1% in their overall ovarian cancer cohort. High-grade serous and endometrioid carcinomas were almost uniformly negative. ( 11 ) Similarly, Halimi et al. found nearly universal CLDN18.2 positivity (98%) in intestinal-type mucinous borderline ovarian tumors, whereas endocervical-type tumors were largely negative. ( 22 ) Comparisons between studies are limited by differing cut-offs: Wang et al. classified cases as positive if 50% of cells showed at least moderate staining, while Wagner et al. applied a stricter threshold of ≥ 60% of cells with ≥ 2 + intensity. ( 10 ) ( 11 ) This variability underscores the need for standardized scoring systems, particularly given the therapeutic implications. Wagner et al. further suggested that CLDN18.2 loss may correlate with dedifferentiation, as tumors with an expansile growth pattern showed higher expression than infiltrative carcinomas. ( 11 ) The prognostic significance of CLDN18.2 in gynecologic malignancies, however, remains unclear. Overall, CLDN18.2 represents a promising biomarker and potential therapeutic target in mucinous tumors of the female genital tract, particularly in ovarian mucinous carcinomas, warranting further investigation. Therapeutically, CLDN18.2 has gained prominence due to the development of targeted monoclonal antibodies such as Zolbetuximab. Phase II data from Türeci et al. suggest that treatment responses are greatest in tumors with ≥ 75% of cells exhibiting ≥ 2 + staining, although a definitive pan-tumor threshold has yet to be established. ( 23 ) Based on these criteria, only a minority of patients with gynecologic mucinous tumors in our cohort would be eligible for CLDN18.2-directed therapies. Taken together, the lower and heterogeneous expression observed in our study likely reflects biological differences between gynecologic and gastrointestinal mucinous tumors, including distinct molecular pathways, cellular origins, and tumor microenvironments. These differences may explain both the lower prevalence of strong CLDN18.2 expression and the lack of association with clinical parameters in our cohort. An additional limitation concerns the definition of positivity and clinically relevant cut-off values for CLDN18.2 expression. In the present study, H-score categories and a ≥ 75% moderate-to-strong staining threshold were applied based on criteria established in gastrointestinal malignancies. However, these thresholds have not yet been validated for gynecologic mucinous tumors. Their applicability in this specific tumor entity therefore remains uncertain. Alternative cut-off values were not systematically explored due to the limited cohort size, which may have influenced the classification of potentially eligible patients for targeted therapy. Strengths and limitations This study provides one of the first systematic assessments of CLDN18.2 expression in gynecologic mucinous tumors, including borderline and malignant subtypes, using well-characterized FFPE specimens with clinical data. Standardized H-score evaluation enabled semi-quantitative comparisons across tumor subtypes. Limitations include the small, single-center, retrospective design and the low frequency of strong CLDN18.2 expression, which restricted statistical power. Reliance on a single antibody clone, semi-quantitative scoring, and tissue microarrays may underestimate intratumoral heterogeneity. Only IHC was performed; complementary molecular analyses were lacking, and comprehensive tumor profiling was not conducted. No a priori sample size calculation was done. Despite these limitations, the findings provide preliminary insights and underscore the need for larger, multicenter studies with standardized immunohistochemical and molecular approaches to validate CLDN18.2 expression and its potential biological and therapeutic relevance. Conclusion CLDN18.2 is detectable in a subset of gynecologic mucinous tumors, with higher positivity in borderline tumors than in adenocarcinomas; however, strong expression is rare. CLDN18.2 expression did not correlate with tumor aggressiveness or other clinical parameters. Based on gastrointestinal cancer thresholds, only a minority of patients would qualify for CLDN18.2-targeted therapies. Given the exploratory design, limited sample size, and potential sampling bias from TMA analysis, these results should be interpreted cautiously. Larger, prospective, multicenter studies with standardized scoring and comprehensive tissue sampling are required to validate the clinical relevance and therapeutic potential of CLDN18.2 in gynecologic mucinous tumors. Abbreviations CLDN 18.2 Claudin 18.2 GAC gastric-type mucinous adenocarcinoma EmGA Endometrial gastric (gastrointestinal)-type mucinous adenocarcinoma SMMN-FGT,synchronous mucinous metaplasia and neoplasia of the female genital tract IHC immunohistochemistry TMA Tissue microarry Declarations The datasets used and analysed during the current study are available from the corresponding author on reasonable request. Competing interests BBS, SS, BG, EB, AW, MK, JS: none related to this work Ethics Approval This study was approved by the Institutional Review Board of the University Hospital Heidelberg, Germany (approval numbers: #2463 and #S315-2020). Consent to Participate Due to the retrospective nature of this study and the use of archived formalin-fixed paraffin-embedded tissue samples and anonymized clinical data, the requirement for written informed consent was waived by the Institutional Review Board. Human Ethics and Consent to Participate All procedures involving human participants were performed in accordance with the ethical standards of the responsible institutional and national research committees and with the Declaration of Helsinki. Ethical approval was obtained as stated above. Funding This study did not receive any specific funding from public, commercial, or not-for-profit organizations. Author Contribution Draft: MK, BBSReview of tissue samples: SS, MKContruction of the tissue microarray: SSImmunohistochemical staining evaluation: MKFigures: MKData Management: BBSWriting: BBS, MKReview for important intellectual content: SS, MK, AW, BG, EB, JS Acknowledgement We would like to thank the staff of the Gynecologic Cancer Center at St. Josefs-Hospital, Wiesbaden, for their support in collecting clinical data and tumor samples. We also acknowledge the pathology laboratory team for their assistance with immunohistochemical staining and digital image analysis. Data Availability The datasets used and analysed during the current study are available from the corresponding author on reasonable request. References Maiorano MFP, Maiorano BA, Cormio G, Loizzi V. Mucinous Ovarian Carcinoma: Integrating Molecular Stratification into Surgical and Therapeutic Management. Biomedicines. 2025;13(5):1198. 10.3390/biomedicines13051198 . PMID: 40427025; PMCID: PMC12. 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Türeci O, Sahin U, Schulze-Bergkamen H, Zvirbule Z, Lordick F, Koeberle D, Thuss-Patience P, Ettrich T, Arnold D, Bassermann F, Al-Batran SE, Wiechen K, Dhaene K, Maurus D, Gold M, Huber C, Krivoshik A, Arozullah A, Park JW, Schuler M. A multicentre, phase IIa study of zolbetuximab as a single agent in patients with recurrent or refractory advanced adenocarcinoma of the stomach or lower oesophagus: the MONO study. Ann Oncol. 2019;30(9):1487–95. 10.1093/annonc/mdz199 . PMID: 31240302; PMCID: PMC6771222. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8756545","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":588448583,"identity":"fc352950-1f08-47b3-bddf-014d8af52c67","order_by":0,"name":"Bettina Blau-Schneider","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+0lEQVRIiWNgGAWjYBACAwnmBggDiA/wMDDIMTYASQY2fFoYUbUYk6aFAag4sYGBgBZz6cbGzzwMdfLm0s0HD7ypuJfe3N578AFDmQ1OLZZzDjZL8zAcNtw551jCwTlninMbe84lGzCcS8PtsBuJDdK8/w4kGNzIMTjM25aQ2zgjx0yCse0wPi3Nv4EOA2rJ/3CY919COiNEy398WtqADmMG2cJwmLchIQGq5QA+v7RZzgH6ZcONNIODQP8YNvacMTZIOJeMUwswoA7feAMMMYMbyY8/vKlJkDds7zF88KHMDqcWTGDYACQSSNDAwCBPkupRMApGwSgYCQAAO6NZSpe+VAMAAAAASUVORK5CYII=","orcid":"","institution":"Department of Obstetrics and Gynecology, University Hospital of Wuerzburg","correspondingAuthor":true,"prefix":"","firstName":"Bettina","middleName":"","lastName":"Blau-Schneider","suffix":""},{"id":588448584,"identity":"4ac4c674-44be-4149-beab-8e772a2266ae","order_by":1,"name":"Sonja Stallmann","email":"","orcid":"","institution":"Center for Histology, Cytology, and Molecular Diagnostics Wiesbaden","correspondingAuthor":false,"prefix":"","firstName":"Sonja","middleName":"","lastName":"Stallmann","suffix":""},{"id":588448585,"identity":"194bfd10-227d-418e-9596-b6e504b6e402","order_by":2,"name":"Boris Gabriel","email":"","orcid":"","institution":"Department of Obstetrics and Gynecology, St. Josefs-Hospital Wiesbaden","correspondingAuthor":false,"prefix":"","firstName":"Boris","middleName":"","lastName":"Gabriel","suffix":""},{"id":588448589,"identity":"a73fd5fd-5b15-4817-bea1-90038229b5a5","order_by":3,"name":"Esra Bilir","email":"","orcid":"","institution":"Department of Gynecologic Oncology, Koç University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Esra","middleName":"","lastName":"Bilir","suffix":""},{"id":588448590,"identity":"071b02b5-b881-4e82-ab21-aab07ee1c8e5","order_by":4,"name":"Achim Wöckel","email":"","orcid":"","institution":"Department of Obstetrics and Gynecology, University Hospital of Wuerzburg","correspondingAuthor":false,"prefix":"","firstName":"Achim","middleName":"","lastName":"Wöckel","suffix":""},{"id":588448591,"identity":"a07f7e74-bd3e-4178-bab2-1a975244de5a","order_by":5,"name":"Jessica Salmen","email":"","orcid":"","institution":"Department of Obstetrics and Gynecology, University Hospital of Wuerzburg","correspondingAuthor":false,"prefix":"","firstName":"Jessica","middleName":"","lastName":"Salmen","suffix":""},{"id":588448592,"identity":"7d5ba063-3697-42e4-b8c6-9e68e2ff9805","order_by":6,"name":"Mark Kriegsmann","email":"","orcid":"","institution":"Center for Histology, Cytology, and Molecular Diagnostics Wiesbaden","correspondingAuthor":false,"prefix":"","firstName":"Mark","middleName":"","lastName":"Kriegsmann","suffix":""}],"badges":[],"createdAt":"2026-02-01 13:38:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8756545/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8756545/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102596219,"identity":"312d2b0e-5dae-402d-84fc-df18789fc276","added_by":"auto","created_at":"2026-02-13 12:15:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":232714,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDigital immunohistochemistry analysis workflow:\u003c/strong\u003e A: Mucinous tumor cells stain positive with antibody against Claudin18.2. B: Most of the cells are detected using cell detection in QuPath. C: Tumor and stroma can reliably be separated using an object classifier (red = tumor cells, green = stroma cells). D: The intensity of tumor cells can be objectified and visualized (blue = negative, yellow = mild positive, orange = moderate positive, red = strong positive). Mag.: 20X; Staining: Claudin18.2.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8756545/v1/6f7cfabb236e4ba0d15f993e.png"},{"id":102596220,"identity":"1a46b5a0-73cf-4243-ba7f-2e4b11e92209","added_by":"auto","created_at":"2026-02-13 12:15:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":158372,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExamples of Claudin18.2 staining:\u003c/strong\u003e A: Gastric foveolar cells show strong staining intensity (control). B: Mucinous adenocarcinoma negative for Claudin18.2. C: Mucinous boderline tumor with mild staining intensity. D: Mucinous borderline tumor with moderate staining intensity. Mag.: 20X; Staining: Claudin18.2.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8756545/v1/e899c98978bba8f76f1531bd.png"},{"id":104696237,"identity":"72352055-4662-4563-a432-ea896ea2062b","added_by":"auto","created_at":"2026-03-16 07:28:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1062413,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8756545/v1/dd6ef347-6120-42fc-b05a-855c2d383c12.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Immunohistochemical expression of Claudin18.2 in borderline and malignant gynecologic tumors: Results from a pilot study","fulltext":[{"header":"Background","content":"\u003cp\u003eMucinous tumors of the female internal genital tract represent a distinct and heterogeneous group of neoplasms, located in the ovary, endometrium, cervix, and other Mullerian-derived sites. Although relatively rare compared to serous or endometrioid subtypes, mucinous carcinomas pose significant diagnostic and therapeutic challenges due to their unique biology and frequent molecular heterogenity.\u003c/p\u003e \u003cp\u003eOvarian mucinous carcinoma is a rare subtype of epithelial ovarian cancer characterized by a molecular profile that differs from that of high-grade serous carcinomas. It often presents at an early stage and in younger women, and shows frequent copy-number loss in CDKN2A, as well as \u003cem\u003eTP\u003c/em\u003e53- and \u003cem\u003eKRAS\u003c/em\u003e mutations and, in some cases, \u003cem\u003eHER2\u003c/em\u003e amplifications, with limited responsiveness to standard platinum-based chemotherapy. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eIn the endometrium, a rare but increasingly recognized subtype is the gastric (gastrointestinal)-type mucinous adenocarcinoma (EmGA). In the largest series published to date, EmGA exhibits frequent pathogenic variants in \u003cem\u003eTP53\u003c/em\u003e, \u003cem\u003eKRAS\u003c/em\u003e, \u003cem\u003ePIK3CA\u003c/em\u003e, and \u003cem\u003eSTK11\u003c/em\u003e, among other genes, placing many cases into the p53-abnormal molecular class according to The Cancer Genome Atlas framework. Immunophenotypically, these tumors often express markers such as MUC6, CK7, CK20, and CDX2, reflecting their gastric/gastrointestinal differentiation. Clinically, EmGA may exhibit aggressive behaviour; however, given the limited available data, this appears to be variable, and adverse outcomes have been reported even in some low-grade or early-stage cases. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eIn the cervix, the gastric-type (also called gastric-type mucinous) adenocarcinoma (GAC) constitutes a rare, HPV-independent variant. It is associated with more aggressive behavior and worse prognosis than the usual HPV-related endocervical adenocarcinoma, even when diagnosed at an early stage. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) Molecularly, GAC shows considerable genetic heterogeneity, with frequent \u003cem\u003eTP53\u003c/em\u003e mutations, and recurrent alterations in \u003cem\u003eSTK11\u003c/em\u003e, \u003cem\u003eCDKN2A/B\u003c/em\u003e, \u003cem\u003eARID1A\u003c/em\u003e, and DNA damage repair genes. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eBeyond single-site tumors, synchronous mucinous metaplasia and neoplasia of the female genital tract (SMMN-FGT) can occur, in which mucinous lesions may appear simultaneously at multiple Mullerian sites (e.g., ovary, endometrium, cervix). (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eA recent case report identified shared \u003cem\u003eSTK11\u003c/em\u003e mutations in synchronous lesions, suggesting a possible common clonal origin, though additional mutational differences (e.g., in \u003cem\u003eKRAS\u003c/em\u003e) have been observed between sites. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThis multifocal presentation underscores the complexity of pathogenesis and the need for careful molecular and pathological evaluation. The diagnostic challenge of mucinous tumors is further characterizised by their morphological overlap with metastatic gastrointestinal adenocarcinomas. Immunohistochemical markers such as CK7, CK20, and CDX2 can be informative: in a series of gynecologic mucinous adenocarcinomas, the majority expressed CK7, while expression of CK20 and CDX2 correlated with intestinal differentiation, but the overlap limits discriminatory power. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) The more recent marker SATB2 may help resolve such cases. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eGiven their rarity, diverse molecular alterations, and distinct clinical behavior, mucinous tumors of the internal genital tract warrant detailed characterization. Improved understanding of their molecular pathology can guide tailored therapeutic approaches, refine prognosis and support better diagnostic discrimination from metastatic disease.\u003c/p\u003e \u003cp\u003eClaudin 18.2 (CLDN18.2) is a tight-junction protein isoform normally expressed in gastric epithelium. In malignant transformation, CLDN18.2 becomes aberrantly exposed on the tumor cell surface, making it a promising target for therapeutic antibodies. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e) (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e) (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e) In primary ovarian mucinous carcinomas, CLDN18.2 expression has been reported in up to 84% of cases, whereas expression in metastatic gastrointestinal mucinous carcinomas varies by origin: ~70% in upper gastrointestinal tract metastases, but generally absent in lower gastrointestinal metastases. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eWithin tubo-ovarian mucinous tumors, CLDN18.2 expression is largely restricted to the mucinous subtype, while non-mucinous subtypes show little to no expression. Importantly, this expression pattern is often retained in metastases, highlighting its potential as both a diagnostic and therapeutic marker. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e Therapeutically, CLDN18.2 has already emerged as a target in gastric and gastroesophageal cancers, where monoclonal antibodies such as zolbetuximab were recently approved in combination with chemotherapy for first-line therapy in locally advanced and metastatic disease. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eMechanistically, CLDN18.2 may contribute to tumor progression by promoting cell\u0026ndash;cell adhesion and interaction with the tumor microenvironment, including cancer-associated fibroblasts. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eGiven the promising new targets in gastrointestinal mucinous tumor, we aimed to evaluate CLDN18.2 expression in borderline and malignant mucinous tumors of the female genital tract and to assess its potential clinical relevance.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eThe primary endpoint was the frequency and distribution of CLDN18.2 expression in mucinous tumors of the female genital tract, assessed using H-score based on standardized digital evaluation of immunohistochemical (IHC) staining.\u003c/p\u003e \u003cp\u003eSecondary endpoints included the association between CLDN18.2 expression and clinicopathological variables such as tumor subtype (borderline vs. carcinoma), tumor stage, tumor grade, and patient demographics.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatient selection\u003c/h2\u003e \u003cp\u003eAll patients treated for mucinous carcinoma or mucinous borderline tumors of the ovary, corpus uteri, or cervix uteri at the Gynecologic Cancer Center of St. Josefs-Hospital, Wiesbaden, Germany between 2013 and 2024 were screened for inclusion.\u003c/p\u003e \u003cp\u003eInclusion criteria were:\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eage\u0026thinsp;\u0026ge;\u0026thinsp;18 years at the time of diagnosis;\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003ehistopathological diagnosis of a primary mucinous carcinoma or mucinous borderline tumor of the ovary, corpus uteri, or cervix uteri;\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003etreatment and/or surgical management performed at our institution; and\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eavailability of formalin-fixed paraffin-embedded (FFPE) tumor tissue for immunohistochemical analysis.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eavailability of clinical and pathological data.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eExclusion criteria comprised:\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003enon-mucinous histological tumor subtypes;\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003emetastatic mucinous tumors of extra-gynecologic origin;\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003erecurrent disease without available primary tumor tissue; and\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003einsufficient or unavailable tumor material for further analysis.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eAmong the 35 identified patients with primary mucinous carcinoma or mucinous borderline tumor of the ovary, corpus uteri, or cervix uteri, formalin-fixed paraffin-embedded (FFPE) tumor tissue was available for 20 cases which were included in the final study cohort.\u003c/p\u003e \u003cp\u003eDiagnoses were established according to the World Health Organization classification of female genital tumors based on surgical resection specimens. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e The study was conducted in accordance with the Declaration of Helsinki. All samples were provided by the Centre for Histology, Cytology, and Molecular Diagnostics in accordance with local regulations and with approval from the institutional review board of the University Hospital Heidelberg (#2463 and #S315-2020).\u003c/p\u003e \u003cp\u003eTo achieve standardized staining accross all samples, a tissue microarray (TMA) was constructed. Clinical data, including patient age, tumor stage, tumor grade, and clinical outcomes, were retrieved from medical records. Retrospectively collected variables included age, follow-up duration, recurrence, histology, tumor grade, surgical treatment status and International Federation of Obstetrics and Gynecology (FIGO) classification for each eligible case. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e) (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e) (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e)\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunohistochemistry staining and evaluation\u003c/h3\u003e\n\u003cp\u003eTissue microarrays allow simultaneous processing of a large number of tumor samples on a single slide. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e) Because all cores on a TMA are processed under the same experimental conditions (e.g., same antigen retrieval, antibody concentration, incubation times), they enable greater standardization and reduce inter-assay variability. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThe TMA was stained with an Claudin18.2 Antibody (Clon: ZR451, RTU) from Zeta Corporation (Monrovia, USA) as follows: pretreatment with pH 9 buffer for 30 minutes and incubation of the antibody for 30 minutes. Slides were scanned with a slide scanner (MidiII, Epredia, Dreieich, Germany). Data was visualized and analyzed using QuPath (v.0.6.0)(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e) In QuPath a representative region of the tumor was marked and \u0026bdquo;cell detection\u0026ldquo; was performed with the following settings: pixel size 0.5 \u0026micro;m, background radius 8 \u0026micro;m, use opening by reconstruction, median filter radius 0 \u0026micro;m, sigma 1.5 \u0026micro;m, minimum area 10 \u0026micro;m^2, maximum area 400 \u0026micro;m^2, intensity threshold 0.1, max background intensity 2, split by shape, cell expansion 5 \u0026micro;m, include cell nucleus, smooth boundaries, and make measurements. After cell detection, a classifier was trained for each individual tumor by using an object classifier with the classes tumor and stroma, after manual annotation of a representative area, in order to be able to retrieve results for tumor cells only. Finally, cell intensity classification was used with three thresholds for mild, moderate and strong staining intensity using Cell: diaminobenzidine (DAB) optical density (OD) mean with standard variables. The amount of tumor cells with no, mild, moderate and strong staining intensity was documented.\u003c/p\u003e \u003cp\u003eH-Score was calculated as follows: 0\u0026ndash;50\u0026thinsp;=\u0026thinsp;negative, 51\u0026ndash;100 mild positiv, 101\u0026ndash;200 moderate positive, and 201\u0026ndash;300 strong positive. Additionally, the number of samples with \u0026ge;\u0026thinsp;75% of tumor cells showing moderate-to-strong positivity was determined.\u003c/p\u003e \u003cp\u003eCut-off values were adopted from previously published studies in gastrointestinal malignancies, as no standardized thresholds for gynecologic mucinous tumors are currently available\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll analyses were performed using Statistical Package for Social Sciences (SPSS) Version 30.0 for Windows (Chicago, IL, USA). Data were presented as mean (\u0026plusmn;\u0026thinsp;standard deviation). The analyses of secondary endpoints were considered exploratory. Binary and categorical variables were compared using the chi-square test. P-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant. No formal correction for multiple testing was applied, as the analyses were exploratory.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eA total of 30 patients were screened during the study period. Eighteen patients met the inclusion criteria and were included in the final analysis. Reasons for exclusion were X (n\u0026thinsp;=\u0026thinsp;X) and X (n\u0026thinsp;=\u0026thinsp;X). In two cases no tumor could be detected after TMA creation.\u003c/p\u003e\n\u003ch3\u003ePatient characteristics\u003c/h3\u003e\n\u003cp\u003eMean patient age was 55.7 years (range 25\u0026ndash;88; SD:16.2). Mean follow-up time was 30.9 months (range 2\u0026ndash;88; SD:28.678). At the data cutoff, all patients were alive. One case of recurrence was observed in a patient with cervical carcinoma, occurring 25 months after initial diagnosis.\u003c/p\u003e \u003cp\u003eHistologically, 25.0% (n\u0026thinsp;=\u0026thinsp;5) of cases were borderline ovarian tumors, 60.0% (n\u0026thinsp;=\u0026thinsp;12) ovarian cancers, 10.0% (n\u0026thinsp;=\u0026thinsp;2) cervical cancers, and 5.0% (n\u0026thinsp;=\u0026thinsp;1) endometrial cancer. FIGO staging was as follows: 55.0% (n\u0026thinsp;=\u0026thinsp;11) FIGO IA, 20.0% (n\u0026thinsp;=\u0026thinsp;4) FIGO IB, 10.0% (n\u0026thinsp;=\u0026thinsp;2) FIGO IC1, 5.0% (n\u0026thinsp;=\u0026thinsp;1) FIGO IC2, 5.0% (n\u0026thinsp;=\u0026thinsp;1) FIGO IC3, and 5.0% (n\u0026thinsp;=\u0026thinsp;1) FIGO IIA. Borderline tumors were detected in 25% (n\u0026thinsp;=\u0026thinsp;5), and tumor grade 1,2, and 3 was found in 45.0% (n\u0026thinsp;=\u0026thinsp;9), 20.0% (n\u0026thinsp;=\u0026thinsp;4), and 10.0% (n\u0026thinsp;=\u0026thinsp;2) of cases respectively.\u003c/p\u003e \u003cp\u003eAll patients were diagnosed and treated between 2015 and 2024, and all underwent tumor surgery.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eTumor cell count was representative in the 18 evaluable tumors (mean: 3542, min: 324, max: 7071).\u003c/p\u003e \u003cp\u003eRegarding CLDN18.2 immunohistochemical staining, nine out of 18 (50%) tumors were completely negative, and nine samples were positive (H-Score mean: 125, range 57,202). Five tumors (27.8%) showed moderate staining intensity (H-Score: 101\u0026ndash;200), and three tumors showed mild staining intensity (17%; H-Score 51\u0026ndash;100). Only one case (5.6%) demonstrated strong staining intensity (H-Score: 201\u0026ndash;300). None of the samples showed moderate and strong staining intensity in \u0026gt;\u0026thinsp;=\u0026thinsp;75% of tumor cells.\u003c/p\u003e \u003cp\u003eThree out of five mucinous borderline tumors (60.0%) showed a positive immunreaction. Six out of 13 (46.2%) mucinous adenocarcinomas exhibited positivity.\u003c/p\u003e \u003cp\u003eDespite the apparent difference in positivity between borderline tumors and adenocarcinomas, Chi-square analysis did not reveal any significant associaton between CLDN18.2 expression and invasive bahaviour of tumor (p-value\u0026thinsp;=\u0026thinsp;0.44). In line with this, no significant correlations were observed between CLDN18.2 staining and other clinical parameters, including tumor stage (p\u0026thinsp;=\u0026thinsp;0.23), grade (p\u0026thinsp;=\u0026thinsp;0.59) or patient demographics (age p\u0026thinsp;=\u0026thinsp;0.7, follow up p\u0026thinsp;=\u0026thinsp;0.4).\u003c/p\u003e \u003cp\u003eThe evaluation process is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRepresentative images of Claudin18.2 staining are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this pilot study of borderline and malignant mucinous tumors of the female genital tract, CLDN18.2 expression was generally scarce. Only 50% of evaluable tumors showed any immunoreactivity, and none exhibited moderate or strong staining in \u0026ge;\u0026thinsp;75% of tumor cells. Mucinous borderline tumors exhibited a higher rate of positivity (75%) than mucinous adenocarcinomas (46.2%), although staining intensity in both subgroups was mostly mild to moderate, with only a single case showing strong expression (H-score\u0026thinsp;\u0026gt;\u0026thinsp;200). These findings suggest that whereas CLDN18.2 is detectable in a subset of gynecologic mucinous tumors, high-level expression is rare.\u003c/p\u003e \u003cp\u003eNo statistically significant associations were observed between CLDN18.2 expression and tumor invasiveness, stage, grade, or patient demographics. However, given the exploratory nature of this pilot study, the limited sample size, and the low frequency of strong staining, the statistical power was restricted. Therefore, the absence of significant correlations should be interpreted with caution, and the results should be regarded as hypothesis-generating rather than confirmatory.\u003c/p\u003e \u003cp\u003eThe literature on CLDN18.2 expression in gynecologic malignancies remains limited, with small cohorts and heterogeneous scoring systems. Nevertheless, available studies consistently indicate that CLDN18.2 overexpression is largely restricted to mucinous subtypes. Wang et al. reported CLDN18.2 overexpression in 56% of mucinous tumors across various sites (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), whereas Wagner et al. found positivity in 45% of mucinous ovarian carcinomas, compared with only 4.1% in their overall ovarian cancer cohort. High-grade serous and endometrioid carcinomas were almost uniformly negative. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e) Similarly, Halimi et al. found nearly universal CLDN18.2 positivity (98%) in intestinal-type mucinous borderline ovarian tumors, whereas endocervical-type tumors were largely negative. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eComparisons between studies are limited by differing cut-offs: Wang et al. classified cases as positive if 50% of cells showed at least moderate staining, while Wagner et al. applied a stricter threshold of \u0026ge;\u0026thinsp;60% of cells with \u0026ge;\u0026thinsp;2\u0026thinsp;+\u0026thinsp;intensity. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e) (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e) This variability underscores the need for standardized scoring systems, particularly given the therapeutic implications.\u003c/p\u003e \u003cp\u003eWagner et al. further suggested that CLDN18.2 loss may correlate with dedifferentiation, as tumors with an expansile growth pattern showed higher expression than infiltrative carcinomas. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e) The prognostic significance of CLDN18.2 in gynecologic malignancies, however, remains unclear.\u003c/p\u003e \u003cp\u003eOverall, CLDN18.2 represents a promising biomarker and potential therapeutic target in mucinous tumors of the female genital tract, particularly in ovarian mucinous carcinomas, warranting further investigation.\u003c/p\u003e \u003cp\u003eTherapeutically, CLDN18.2 has gained prominence due to the development of targeted monoclonal antibodies such as Zolbetuximab. Phase II data from T\u0026uuml;reci et al. suggest that treatment responses are greatest in tumors with \u0026ge;\u0026thinsp;75% of cells exhibiting\u0026thinsp;\u0026ge;\u0026thinsp;2\u0026thinsp;+\u0026thinsp;staining, although a definitive pan-tumor threshold has yet to be established. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e) Based on these criteria, only a minority of patients with gynecologic mucinous tumors in our cohort would be eligible for CLDN18.2-directed therapies.\u003c/p\u003e \u003cp\u003eTaken together, the lower and heterogeneous expression observed in our study likely reflects biological differences between gynecologic and gastrointestinal mucinous tumors, including distinct molecular pathways, cellular origins, and tumor microenvironments. These differences may explain both the lower prevalence of strong CLDN18.2 expression and the lack of association with clinical parameters in our cohort.\u003c/p\u003e \u003cp\u003eAn additional limitation concerns the definition of positivity and clinically relevant cut-off values for CLDN18.2 expression. In the present study, H-score categories and a\u0026thinsp;\u0026ge;\u0026thinsp;75% moderate-to-strong staining threshold were applied based on criteria established in gastrointestinal malignancies. However, these thresholds have not yet been validated for gynecologic mucinous tumors. Their applicability in this specific tumor entity therefore remains uncertain. Alternative cut-off values were not systematically explored due to the limited cohort size, which may have influenced the classification of potentially eligible patients for targeted therapy.\u003c/p\u003e\n\u003ch3\u003eStrengths and limitations\u003c/h3\u003e\n\u003cp\u003eThis study provides one of the first systematic assessments of CLDN18.2 expression in gynecologic mucinous tumors, including borderline and malignant subtypes, using well-characterized FFPE specimens with clinical data. Standardized H-score evaluation enabled semi-quantitative comparisons across tumor subtypes.\u003c/p\u003e \u003cp\u003eLimitations include the small, single-center, retrospective design and the low frequency of strong CLDN18.2 expression, which restricted statistical power. Reliance on a single antibody clone, semi-quantitative scoring, and tissue microarrays may underestimate intratumoral heterogeneity. Only IHC was performed; complementary molecular analyses were lacking, and comprehensive tumor profiling was not conducted. No a priori sample size calculation was done.\u003c/p\u003e \u003cp\u003eDespite these limitations, the findings provide preliminary insights and underscore the need for larger, multicenter studies with standardized immunohistochemical and molecular approaches to validate CLDN18.2 expression and its potential biological and therapeutic relevance.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eCLDN18.2 is detectable in a subset of gynecologic mucinous tumors, with higher positivity in borderline tumors than in adenocarcinomas; however, strong expression is rare. CLDN18.2 expression did not correlate with tumor aggressiveness or other clinical parameters. Based on gastrointestinal cancer thresholds, only a minority of patients would qualify for CLDN18.2-targeted therapies.\u003c/p\u003e \u003cp\u003eGiven the exploratory design, limited sample size, and potential sampling bias from TMA analysis, these results should be interpreted cautiously. Larger, prospective, multicenter studies with standardized scoring and comprehensive tissue sampling are required to validate the clinical relevance and therapeutic potential of CLDN18.2 in gynecologic mucinous tumors.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCLDN 18.2 Claudin 18.2\u003c/p\u003e \u003cp\u003eGAC gastric-type mucinous adenocarcinoma\u003c/p\u003e \u003cp\u003eEmGA Endometrial gastric (gastrointestinal)-type mucinous adenocarcinoma\u003c/p\u003e \u003cp\u003eSMMN-FGT,synchronous mucinous metaplasia and neoplasia of the female genital tract\u003c/p\u003e \u003cp\u003eIHC immunohistochemistry\u003c/p\u003e \u003cp\u003eTMA Tissue microarry\u003c/p\u003e \u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003eThe datasets used and analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eBBS, SS, BG, EB, AW, MK, JS: none related to this work\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eEthics Approval\u003c/h2\u003e \u003cp\u003e This study was approved by the Institutional Review Board of the University Hospital Heidelberg, Germany (approval numbers: #2463 and #S315-2020).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent to Participate\u003c/strong\u003e \u003cp\u003e Due to the retrospective nature of this study and the use of archived formalin-fixed paraffin-embedded tissue samples and anonymized clinical data, the requirement for written informed consent was waived by the Institutional Review Board.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eHuman Ethics and Consent to Participate\u003c/h2\u003e \u003cp\u003e All procedures involving human participants were performed in accordance with the ethical standards of the responsible institutional and national research committees and with the Declaration of Helsinki. Ethical approval was obtained as stated above.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study did not receive any specific funding from public, commercial, or not-for-profit organizations.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eDraft: MK, BBSReview of tissue samples: SS, MKContruction of the tissue microarray: SSImmunohistochemical staining evaluation: MKFigures: MKData Management: BBSWriting: BBS, MKReview for important intellectual content: SS, MK, AW, BG, EB, JS\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe would like to thank the staff of the Gynecologic Cancer Center at St. Josefs-Hospital, Wiesbaden, for their support in collecting clinical data and tumor samples. 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Claudin-18 overexpression in intestinal-type mucinous borderline tumour of the ovary. \u003cem\u003eHistopathology.\u003c/em\u003e 2013;63(4):534\u0026thinsp;\u0026ndash;\u0026thinsp;44. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/his.12182\u003c/span\u003e\u003cspan address=\"10.1111/his.12182\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Epub 2013 Jul 26. PMID: 23905715.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eT\u0026uuml;reci O, Sahin U, Schulze-Bergkamen H, Zvirbule Z, Lordick F, Koeberle D, Thuss-Patience P, Ettrich T, Arnold D, Bassermann F, Al-Batran SE, Wiechen K, Dhaene K, Maurus D, Gold M, Huber C, Krivoshik A, Arozullah A, Park JW, Schuler M. A multicentre, phase IIa study of zolbetuximab as a single agent in patients with recurrent or refractory advanced adenocarcinoma of the stomach or lower oesophagus: the MONO study. Ann Oncol. 2019;30(9):1487\u0026ndash;95. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/annonc/mdz199\u003c/span\u003e\u003cspan address=\"10.1093/annonc/mdz199\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. PMID: 31240302; PMCID: PMC6771222.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Claudin18.2, Gynecologic mucinous tumors, Immunohistochemistry, Tissue microarray, Zolbetuximab, Translational oncology, Borderline tumor","lastPublishedDoi":"10.21203/rs.3.rs-8756545/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8756545/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eClaudin 18.2 (CLDN 18.2) is an emerging therapeutic target in gastrointestinal malignancies, but its expression in gynecologic mucinous tumors remains poorly characterized. Our study aimed to evaluate CLDN18.2 expression in borderline and malignant mucinous tumors of the female genital tract and to explore its potential clinical relevance.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eFormalin-fixed paraffin-embedded tumor specimens from 20 patients with mucinous tumors of the ovary, corpus uteri, or cervix uteri treated between 2013 and 2024 were included. CLDN18.2 expression was assessed by immunohistochemistry (IHC) and scored using the H-score system based on standardized digital evaluation. Clinical data, including tumor stage, grade, and outcomes, were retrospectively obtained. Associations between CLDN18.2 expression and clinicopathologic parameters were analyzed descriptively using SPSS.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eCLDN18.2 expression was detectable in 50.0% of evaluable tumors. Among histologic subtypes, 60% mucinous borderline tumors and 46.2% mucinous adenocarcinomas were positive. Staining intensity was generally mild to moderate, with strong expression (H-score\u0026thinsp;\u0026gt;\u0026thinsp;200) observed in only one case. No statistically significant associations were observed between CLDN18.2 positivity and tumor stage, grade, patient demographics, or borderline versus invasive status; however, the small sample size limits the interpretability of these findings.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eCLDN18.2 is expressed in a subset of gynecologic mucinous tumors, but high-level expression is uncommon. Given the limited number of cases, these preliminary results should be interpreted with caution, and no firm conclusions regarding clinical correlations can be drawn. Only a minority of patients may meet thresholds for CLDN18.2-targeted therapies established in gastrointestinal malignancies. Larger, multicenter studies are warranted to better define the prevalence, biological significance, and potential therapeutic relevance of CLDN18.2 in these tumors.\u003c/p\u003e","manuscriptTitle":"Immunohistochemical expression of Claudin18.2 in borderline and malignant gynecologic tumors: Results from a pilot study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-13 12:15:14","doi":"10.21203/rs.3.rs-8756545/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3d7baea6-bdf9-46de-891b-6941f2d379d2","owner":[],"postedDate":"February 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-16T07:28:26+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-13 12:15:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8756545","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8756545","identity":"rs-8756545","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}