The Clinicopathological and Molecular Characterization in Ascites-derived Paraffin- embedded Cell Blocks of Ovarian Cancer

The Clinicopathological and Molecular Characterization in Ascites-derived Paraffin- embedded Cell Blocks of Ovarian Cancer | 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 The Clinicopathological and Molecular Characterization in Ascites-derived Paraffin- embedded Cell Blocks of Ovarian Cancer Chunfang Zhang, Wenting Jia, Shu Dong, Pengxin Zhang, Jingchun Gao This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8818719/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 This study evaluated immunohistochemical (IHC) markers and homologous recombination repair (HRR) gene mutations in 58 ovarian cancer patients using ascites-derived paraffin-embedded cell blocks. IHC analysis revealed high expression of Pax-8 (87.5–100%), WT-1 (87.8–90%), CA-125 (100%), and CK7 (100%) across subtypes, supporting their diagnostic utility. HRR gene mutations were detected in 77.6% of cases, with TP53 (55%) and PIK3CA (21.7%) as the most frequent alterations. Nine BRCA1/2 mutations were identified. PIK3CA variants included E545K (28.6%) and E542K (28.6%), suggesting potential therapeutic targets. These findings highlight the clinical relevance of ascites cell blocks for molecular profiling, with HRR status guiding PARP inhibitor therapy and PIK3CA mutations indicating PI3K/AKT pathway inhibition. This approach may optimize personalized treatment strategies for advanced ovarian cancer. Ovarian cancer Ascites Cell block HRR PIK3CA Figures Figure 1 Figure 2 Introduction Ovarian cancer (OC), a highly lethal gynecologic malignancy, presents significant clinical challenges due to recurrence and drug resistance [ 1 , 2 ]. Epithelial ovarian cancer (EOC) constitutes approximately 90% of ovarian cancer cases, with high-grade serous ovarian cancer (HGSC) comprising 70% of EOC instances. The absence of early symptoms and effective screening strategies often leads to diagnosis at advanced stages (III-IV) in the majority of patients [ 3 – 5 ]. While standard treatment, including debulking surgery and platinum-based chemotherapy, many patients experience therapeutic limitations due to drug resistance and disease recurrence, underscoring the urgent need for novel treatment approaches [ 6 ]. Homologous recombination repair (HRR) is integral to the diagnosis, therapeutic decision-making, and prognostic assessment of ovarian cancer. The HRR is a critical mechanism for repairing DNA double-strand breaks (DSBs). Deficiencies in HRR result in compromised DNA damage repair, making cancer cells particularly susceptible to agents targeting DNA repair, such as poly (adenosine diphosphate-ribose) polymerase (PARP) inhibitors. Therefore, accurate assessment of a patient’s HRR status is of substantial clinical importance for tailoring individualized ovarian cancer treatments and forecasting the effectiveness of PARP inhibitors [ 7 – 9 ]. Malignant ascites is a prevalent complication among patients with advanced ovarian cancer and is closely associated with a poor prognosis. Ascitic fluid harbors a substantial number of tumor cells and immune cells within the tumor microenvironment, rendering it a valuable resource for investigating ovarian cancer metastasis, mechanisms of drug resistance, and the immune microenvironment [ 10 , 11 ]. The cell block technique is a widely employed method for the diagnostic evaluation of cellular samples, particularly in the cytological diagnosis of ascites in ovarian cancer. The preparation of cells from ascitic fluid into paraffin-embedded cell blocks facilitates subsequent histopathological and molecular pathological analyses akin to those performed on solid tissues, which is crucial for long-term storage and retrospective studies [ 12 ]. It has been suggested that ascites-derived cell block specimens may be instrumental in evaluating predictive markers of platinum resistance or sensitivity in HGSC peritoneal lesions. In this study, paraffin-embedded cell blocks were obtained from the ascitic fluid of 58 patients diagnosed with ovarian cancer. The diagnostic markers for ovarian cancer, such as Pax-8 and WT-1, were examined utilizing immunohistochemical (IHC) methods. Furthermore, HRR-related gene mutations were identified in all cases through next-generation sequencing (NGS) technology, to inform targeted therapeutic strategies based on the HRR-related gene mutation profiles. Materials and Methods 1. Patient selection Patients with primary or recurrent ovarian cancer treated in the Department of Pathology, the First Affiliated Hospital of Dalian Medical University, from January 2016 to June 2021, were collected for inclusion in the study (age > 18 years). The cohort comprised 45 cases of HGSC, 10 cases of low-grade serous carcinoma (LGSC), and 3 cases of endometrioid carcinoma (EC). The disease staging was conducted in accordance with the 2024 Federation of International of Gynecologists and Obstetricians (FIGO) staging classification [ 13 ]. The following clinicopathological characteristics were collected from all patients: age, disease stage, family history, CA-125 level, ascites volume and follow-up time. All of 58 patients were diagnosed as ovarian cancer by histopathological hematoxylin-eosin (H&E) staining. This study was approved by the Ethics Committee of the First Affiliated Hospital of Dalian Medical University (approval number PJ-KS-KY-2025-916). Informed consent was waived because of the retrospective nature of this study and anonymized clinical data was used for analysis. 2. Immunohistochemistry Immunohistochemistry was performed on 4-µm-thick paraffin cell blocks sections using a Leica automatic immunohistochemistry detection instrument. WT-1, Pax-8, CK20, CK7 and CA-125 antibody reagents were purchased from Fuzhou Maixin Biotechnology Development Co., Ltd. Immunohistochemical staining was performed according to the instructions. 3. Genomic Mutation Detection HRR related genes mutation detection was performed using the HRR gene combination detection library preparation kit provided by AmoyDx (AmoyDx, Xiamen, China) according to the manufacturer’s protocol. DNA extraction was performed after confirming that the tumor cell content was > 30%. The extracted DNA (≥ 30 ng) was used for library preparation and then sequenced on the Illumina NextSeq 500 platform (Illumina, San Diego, CA, USA). The panel was tested for mutations in 32 genes including 13 genetic and therapy related genes (PIK3CA, TP53, AR, CDH1, ERBB2 (HER2), ESR1, HOXB13, KRSA, NRAS, PTEN, STK11, BRAF, HDAC2) and 19 HRR genes (ATM, ATR, BARD1, BRCA1/2, CHEK1/2, FANCA, FANCL, PALB2, RAD51B, RAD51C, RAD51D, RAD54L, CDK12, NBN, PPP2R2A, BRIP1, MRE11A). Results 1. Characteristics of study participants A total of 58 patients were enrolled in our study and the clinicopathological characteristics are summarized in Table 1. The cases included 45 (77.6%, 45/58) HGSC with a median age of 62 (range: 36-90), 10 (17.2%, 10/58) LGSC with a median age of 62.6 (range: 52-82) and 3 (5.2%, 3/58) EC with a median age of 64 (range: 43-65). The majority of patients (98.3%, 57/58) were FIGO stage III/IV, with 75.9% (n = 45/58) HGSC, 15.5% (n = 9/58) LGSC and 5.2% (n = 3/58) EC. There was no family history in any of these cases, except for one HGSC case. The median levels of the serologic marker CA-125 were 1194 U/mL, 1541.5 U/mL and 233 U/mL in HGSC, LGSC and EC respectively. The median values of ascites were 1000 mL, 925 mL and 240 mL in HGSC, LGSC and EC respectively. 58 patients were followed up for 2-52 months without loss to follow-up. No patient’s diagnosis changed after review. 2. Histopathological and immunohistochemistry The immunohistochemical results were displayed in Table 2 and Fig. 1. The results showed that the positive expression rate of Pax-8 in 45 HGSC was 87.5% (n = 21/24), that of WT-1 was 87.8% ( n = 36/41), that of CA-125 was 100% (n = 41/41), that of CK7 was 100% (n = 44/44) and that of CK20 was 6.67% (n = 3/45); in 10 cases of LGSC, the positive expression rate of Pax-8 was 100% (n = 7/7), the positive expression rate of WT-1 was 90% (n = 9/10), the positive expression rate of CA-125 was 100% (n = 9/9), the positive expression rate of CK7 was 100% (n = 10/10) and the negative expression rate of CK20 (n = 0/10); in 3 cases of EC, the positive expression rate of Pax-8 was 100% (n = 3/3), WT-1 was 100% (n = 2/2), CA-125 was 100% (n = 2/2), CK7 was 100% (n = 3/3) and CK20 was negative (n = 0/3). 3. HRR Genes Mutation Analyses The HRR-related gene mutations in 58 patients are shown in Fig. 2A. 77.6% (45/58) of the patients carried HRR-related gene mutations. Among them, TP53 gene was the commonly mutated gene with 55% mutation percentage, followed by PIK3CA gene with 21.7% mutation percentage. The mutation percentage of BRCA1/2 is 6.67%, BRCA1 (5%) and BRCA2 (1.67%). The main observation of PIK3CA mutations is shown in Fig. 2B. In this work, there were seven main PIK3CA mutation types, namely E545K (28.6%), E542K (28.6%), E762K (14.3%), E545D (7.1%), Q546K (7.1%), H1047L (7.1%), H1047R (7.1%). In this work, PTEN, BRCA1, CDK12, BRAF, BRCA2, CHEK2 and KRAS mutations were also found. Nine BRCA1/2 mutations identified in ascites cell blocks of 58 ovarian cancer patients in Table 3: 6 BRCA1 and 3 BRCA2. 7 BRCA1/2 mutations matched 7 germline BRCA1/2 mutations identified in blood samples. Two additional BRCA1/2 mutations, which were not identified in blood samples, were considered as somatic: one BRCA1 and one BRCA2. Both somatic mutations BRCA1 exon11 c.975_976delinsAT p.(E326*) and BRCA2 exon22 c.8839_8851del p.(E2947Pfs*25) are known pathogenic mutations. Both patients with somatic mutations had a negative family history and did not have ovarian cancer. Discussion WT-1 and PAX-8 are established biomarkers in ovarian carcinoma diagnosis due to their high sensitivity and specificity. PAX-8 has been shown to be a highly sensitive marker for identifying ovarian cancer cells, especially in the context of distinguishing them from non-gynecologic origins in ascitic fluid specimens. PAX-8, in combination with Calretinin, is valuable in identifying Müllerian-derived epithelia, assisting in diagnosing ovarian carcinomas pre-neoadjuvant chemotherapy [ 14 ]. PAX-8’s consistent expression in ovarian serous tumors makes it a reliable marker for distinguishing them from other tumor types like mucinous tumors [ 15 ]. Similarly, WT-1 has been recognized for its diagnostic value in ovarian cancer. It is particularly useful in distinguishing serous ovarian carcinomas from other histological subtypes. WT-1 immunoreactivity is prevalent in serous tumors, which are the most common subtype of epithelial ovarian cancer, and its expression is significantly higher in these tumors compared to non-serous subtypes [ 16 ]. The combined use of WT-1 and PAX-8 has been shown to improve the detection rates of metastatic ovarian carcinoma in serous effusion specimens, further supporting their utility as diagnostic markers [ 17 ]. Additionally, the expression of PAX-8 in high-grade serous ovarian carcinoma has been linked to its potential as a therapeutic target, given its overexpression in a significant number of cases [ 18 ]. The utility of PAX-8 and WT-1 as biomarkers is further supported by their ability to distinguish primary ovarian carcinomas from metastatic breast carcinomas, providing critical information for accurate diagnosis and treatment planning [ 19 ]. HRR defects are an important molecular feature of ovarian cancer, especially in HGSC, where mutations in HRR related genes (e.g., BRCA1/2, RAD51, and PALB2) are present in about 50% of cases [ 20 ]. In this study, 77.6% of patients carried mutations in HRR genes, of which TP53 mutations accounted for 55%, nine BRCA1/2 mutations were identified, further corroborating the critical role of HRR defects in ovarian cancer development. HRR defects lead to impaired repair of DNA double-strand breaks, which sensitizes the tumor cells to PARP inhibitors, and therefore the detection of HRR status has become the precise treatment of ovarian cancer as an PARP inhibitors (e.g., olaparib, niraparib) selectively kill HRR-deficient tumor cells through the mechanism of "synthetic lethality" and significantly prolong progression-free survival (PFS) [ 21 ]. In recent years, mutations in other HRR genes (e.g., RAD51C/D, ATM) have also been found to be predictive of PARP inhibitors efficacy, expanding the beneficiary population [ 22 ]. In addition, HRR status may affect the sensitivity of platinum-based chemotherapy because platinum drugs work by inducing DNA cross-linking, and HRR-deficient cells are more likely to accumulate DNA damage [ 23 , 24 ]. Therefore, HRR testing may not only guide the use of PARP inhibitors, but also optimize the choice of chemotherapy regimen. Despite the growing clinical use of HRR testing, challenges remain. For example, some HRR-unmutated patients may still benefit from PARP inhibitors (e.g., the “BRCAness” phenotype), suggesting the need to combine it with other biomarkers (e.g., genomic instability scores) to further improve predictive accuracy [ 22 ]. In addition, secondary mutations in the HRR gene may lead to PARP inhibitors resistance, thus dynamic monitoring of HRR status is essential for therapeutic adjustment [ 25 , 26 ]. PIK3CA is a key regulatory gene of the PI3K/AKT/mTOR pathway, and its mutations (e.g., E545K, H1047R) can lead to the sustained activation of the pathway and promote tumor cell proliferation, survival and metastasis [ 27 ]. In this study, 21.7% of ovarian cancer patients were found to have PIK3CA mutations, of which E545K and E542K accounted for 28.6%, suggesting that PIK3CA mutations are more common in ovarian cancer and may be a potential target for targeted therapies. In addition, the presence of PIK3CA mutations may be closely associated with the prognosis of ovarian cancer patients, and studies have shown that patients carrying PIK3CA mutations may have higher tumor aggressiveness and worse prognosis. The detection of PIK3CA mutation can provide more precise treatment strategies for ovarian cancer patients, and also lays important basis for further research on the relationship between PIK3CA mutation and biological behavior of ovarian cancer [ 28 , 29 ]. However, there are limitations to this work. First, the sample size of this study was small (n = 58) and it was a single-center retrospective analysis, which may be subject to selection bias. Second, there was no multi-omics validation of HRR functional defects (e.g., methylation), and there was a lack of correlation analysis of treatment response and survival data. Future sample expansion and prospective studies are needed to further validate the findings. Conclusion In this study, immunohistochemical results showed that WT-1, PAX-8, CA-125, and CK7 were stably expressed in serous ovarian cancer and could be used as diagnostic markers, while the low expression of CK20 could help to identify metastatic cancers. The molecular pathological analysis of paraffin-embedded cell blocks of ascites from 58 ovarian cancer patients revealed a high incidence of HRR-related gene mutations in ovarian cancer (77.6%), with TP53 (55%) and PIK3CA (21.7%) as the commonly mutated genes. BRCA1 (5%) and BRCA2 (1.67%) were detected in ascites cell blocks of 58 ovarian cancer patients, who can benefit from PARP inhibitors targeted therapy. HRR assay provides an important basis for targeted therapies, such as PARP inhibitors, and the PIK3CA mutation suggests that inhibitors of the PI3K/AKT pathway may be a potential treatment option. This study supports the value of ascites cell blocks in molecular typing of ovarian cancer and provides a clinical reference for the development of individualized treatment strategies. Additional benefits when cytological samples are tested, could be in faster processing of genotyping and earlier informative report for clinician as well as in potentially reduced number of genetic testing. Abbreviations IHC immunohistochemical HRR homologous recombination repair OC Ovarian cancer EOC Epithelial ovarian cancer HGSC high-grade serous ovarian cancer DSBs DNA double-strand breaks PARP poly (adenosine diphosphate-ribose) polymerase NGS next-generation sequencing LGSC low-grade serous carcinoma EC endometrioid carcinoma FIGO Federation of International of Gynecologists and Obstetricians H&E histopathological hematoxylin-eosin PFS prolong progression-free survival. Declarations Ethics approval and consent to participate The collection of patient’s clinical data was approved by the Ethics Committee of the First Affiliated Hospital of Dalian Medical University in accordance with the Declaration of Helsinki (approval number PJ-KS-KY-2025-916). Informed consent was waived because of the retrospective nature of this study and anonymized clinical data was used for analysis. Consent for publication Not applicable. Competing Interest The authors declare no conflict of interest. Clinical trial number Not applicable. Funding Nil. Author Contribution Chunfang Zhang and Wenting Jia: Curate the data and draft the manuscript. Shu Dong: Acquisition and analysis of data. Pengxin Zhang and Jingchun Gao: Conception and design of the study. All authors read and approved the final manuscript. Acknowledgements The authors are grateful to all colleges (Department of Pathology, The First Affiliated Hospital of Dalian Medical University, China) for their valuable assistance during this study. Data Availability The study’s data can be obtained from the corresponding author upon reasonable request. References Sun Y, Yao L, Wang C, Xiong B, Guo J, Wang L et al. 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Genes (Basel). 2023;14. https://doi.org/10.3390/genes14081632 . Guo T, Dong X, Xie S, Zhang L, Zeng P, Zhang L. Cellular mechanism of gene mutations and potential therapeutic targets in ovarian cancer. Cancer Manag Res. 2021;13:3081–100. https://doi.org/10.2147/cmar.S292992 . Tables Tables 1 to 3 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.docx Table2.docx Table3.docx 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. We do this by developing innovative software and high quality services for the global research community. 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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-8818719","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":607247772,"identity":"0e0c0851-65df-4104-958f-d2445509e659","order_by":0,"name":"Chunfang Zhang","email":"","orcid":"","institution":"First Affiliated Hospital of Dalian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chunfang","middleName":"","lastName":"Zhang","suffix":""},{"id":607247774,"identity":"c4e6e00a-6958-42eb-a91f-54fcc2da6a4d","order_by":1,"name":"Wenting Jia","email":"","orcid":"","institution":"First Affiliated Hospital of Dalian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wenting","middleName":"","lastName":"Jia","suffix":""},{"id":607247776,"identity":"d024d82b-c3c6-4da7-b63c-209188be4e82","order_by":2,"name":"Shu Dong","email":"","orcid":"","institution":"The Fifth People’s Hospital of Dalian","correspondingAuthor":false,"prefix":"","firstName":"Shu","middleName":"","lastName":"Dong","suffix":""},{"id":607247777,"identity":"5d5dbc80-ff56-4bda-921a-a0353d800e0d","order_by":3,"name":"Pengxin Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFElEQVRIiWNgGAWjYDACCTiLsYEhocLGjp+Z+fAD4rU8OJOWLNnOlmZApBagpodthxk3nOdRkMCpHAjkZzc/e/h1h02efERy24PEtjRm48M8DAYMNTbRuLQwzjlmbix7Jq3Y8MzBdoOEczZ8Zod5DzxgOJaW24BDC7NEgpm0ZNvhxI3tjW0SCWVpzGaH+RIMGBsO49TCJpH+Dajlf+LGZkagFrbDjJubeQwk8Gnhkcgxk/zYdiBxPjvIFpD3mQlokZDIKZNmPJOcuIHnIFALMJAlDgMDOQGPX+RnpG+T/LnDLnH+jPRnkj9AUdl/+PCDDzU2OLWAg4AXKGtwAFkoAY9yEGD8CdQij8/QUTAKRsEoGNkAAK2VXsw01mV8AAAAAElFTkSuQmCC","orcid":"","institution":"First Affiliated Hospital of Dalian Medical University","correspondingAuthor":true,"prefix":"","firstName":"Pengxin","middleName":"","lastName":"Zhang","suffix":""},{"id":607247778,"identity":"326adff8-bfc4-4e5a-8e48-1f1935e73947","order_by":4,"name":"Jingchun Gao","email":"","orcid":"","institution":"First Affiliated Hospital of Dalian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jingchun","middleName":"","lastName":"Gao","suffix":""}],"badges":[],"createdAt":"2026-02-08 02:38:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8818719/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8818719/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104993786,"identity":"3959f016-cfdd-4e9e-b3b8-bcdeaf053813","added_by":"auto","created_at":"2026-03-19 15:57:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":20890241,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe hematoxylin-eosin and immunochemistry staining of cell block and tissue block in ovarian cancer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHGSC: high-grade serous ovarian cancer; LGSC: Low-grade serous ovarian cancer; EC: endometrioid carcinoma; HE: hematoxylin-eosin; CB: cell block; TB: tissue block; immunochemistry makers: CA125, Pax-8, WT-1.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8818719/v1/275d36806d0c44b247c27f0e.png"},{"id":104993785,"identity":"cf689b80-5284-42cf-a6f4-11acba055075","added_by":"auto","created_at":"2026-03-19 15:57:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":686636,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe molecular characterization in ascites -derived paraffin-embedded cell blocks of ovarian cancer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eThe HRR-related gene mutations in 58 patients\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B) \u003c/strong\u003eThe PIK3CA gene mutations in 58 patients\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8818719/v1/37c97305ec1fec83346b2aea.png"},{"id":107480311,"identity":"a8845a27-d7cb-4b39-af18-9b99e78fef1a","added_by":"auto","created_at":"2026-04-22 02:07:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":19157470,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8818719/v1/3d549adc-c4a1-4f44-a433-72449961cf15.pdf"},{"id":104993678,"identity":"f92ce494-f9e2-4ac1-b111-080deab68459","added_by":"auto","created_at":"2026-03-19 15:56:43","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":14589,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8818719/v1/f77a299b93ae65e988b6c773.docx"},{"id":104993686,"identity":"755beb84-b944-43fd-b4e0-cca15422f91a","added_by":"auto","created_at":"2026-03-19 15:56:47","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":17315,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.docx","url":"https://assets-eu.researchsquare.com/files/rs-8818719/v1/3e7e8f85bd206545611c2911.docx"},{"id":104993775,"identity":"e214037d-7cd3-48bb-8866-d86b70cd7ea2","added_by":"auto","created_at":"2026-03-19 15:57:01","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":18874,"visible":true,"origin":"","legend":"","description":"","filename":"Table3.docx","url":"https://assets-eu.researchsquare.com/files/rs-8818719/v1/2d0bc28e13810c6350e315a3.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Clinicopathological and Molecular Characterization in Ascites-derived Paraffin- embedded Cell Blocks of Ovarian Cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOvarian cancer (OC), a highly lethal gynecologic malignancy, presents significant clinical challenges due to recurrence and drug resistance [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Epithelial ovarian cancer (EOC) constitutes approximately 90% of ovarian cancer cases, with high-grade serous ovarian cancer (HGSC) comprising 70% of EOC instances. The absence of early symptoms and effective screening strategies often leads to diagnosis at advanced stages (III-IV) in the majority of patients [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. While standard treatment, including debulking surgery and platinum-based chemotherapy, many patients experience therapeutic limitations due to drug resistance and disease recurrence, underscoring the urgent need for novel treatment approaches [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHomologous recombination repair (HRR) is integral to the diagnosis, therapeutic decision-making, and prognostic assessment of ovarian cancer. The HRR is a critical mechanism for repairing DNA double-strand breaks (DSBs). Deficiencies in HRR result in compromised DNA damage repair, making cancer cells particularly susceptible to agents targeting DNA repair, such as poly (adenosine diphosphate-ribose) polymerase (PARP) inhibitors. Therefore, accurate assessment of a patient\u0026rsquo;s HRR status is of substantial clinical importance for tailoring individualized ovarian cancer treatments and forecasting the effectiveness of PARP inhibitors [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMalignant ascites is a prevalent complication among patients with advanced ovarian cancer and is closely associated with a poor prognosis. Ascitic fluid harbors a substantial number of tumor cells and immune cells within the tumor microenvironment, rendering it a valuable resource for investigating ovarian cancer metastasis, mechanisms of drug resistance, and the immune microenvironment [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The cell block technique is a widely employed method for the diagnostic evaluation of cellular samples, particularly in the cytological diagnosis of ascites in ovarian cancer. The preparation of cells from ascitic fluid into paraffin-embedded cell blocks facilitates subsequent histopathological and molecular pathological analyses akin to those performed on solid tissues, which is crucial for long-term storage and retrospective studies [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. It has been suggested that ascites-derived cell block specimens may be instrumental in evaluating predictive markers of platinum resistance or sensitivity in HGSC peritoneal lesions.\u003c/p\u003e \u003cp\u003eIn this study, paraffin-embedded cell blocks were obtained from the ascitic fluid of 58 patients diagnosed with ovarian cancer. The diagnostic markers for ovarian cancer, such as Pax-8 and WT-1, were examined utilizing immunohistochemical (IHC) methods. Furthermore, HRR-related gene mutations were identified in all cases through next-generation sequencing (NGS) technology, to inform targeted therapeutic strategies based on the HRR-related gene mutation profiles.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\n\u003ch3\u003e1. Patient selection\u003c/h3\u003e\n\u003cp\u003ePatients with primary or recurrent ovarian cancer treated in the Department of Pathology, the First Affiliated Hospital of Dalian Medical University, from January 2016 to June 2021, were collected for inclusion in the study (age\u0026thinsp;\u0026gt;\u0026thinsp;18 years). The cohort comprised 45 cases of HGSC, 10 cases of low-grade serous carcinoma (LGSC), and 3 cases of endometrioid carcinoma (EC). The disease staging was conducted in accordance with the 2024 Federation of International of Gynecologists and Obstetricians (FIGO) staging classification [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The following clinicopathological characteristics were collected from all patients: age, disease stage, family history, CA-125 level, ascites volume and follow-up time. All of 58 patients were diagnosed as ovarian cancer by histopathological hematoxylin-eosin (H\u0026amp;E) staining. This study was approved by the Ethics Committee of the First Affiliated Hospital of Dalian Medical University (approval number PJ-KS-KY-2025-916). Informed consent was waived because of the retrospective nature of this study and anonymized clinical data was used for analysis.\u003c/p\u003e\n\u003ch3\u003e2. Immunohistochemistry\u003c/h3\u003e\n\u003cp\u003eImmunohistochemistry was performed on 4-\u0026micro;m-thick paraffin cell blocks sections using a Leica automatic immunohistochemistry detection instrument. WT-1, Pax-8, CK20, CK7 and CA-125 antibody reagents were purchased from Fuzhou Maixin Biotechnology Development Co., Ltd. Immunohistochemical staining was performed according to the instructions.\u003c/p\u003e\n\u003ch3\u003e3. Genomic Mutation Detection\u003c/h3\u003e\n\u003cp\u003e HRR related genes mutation detection was performed using the HRR gene combination detection library preparation kit provided by AmoyDx (AmoyDx, Xiamen, China) according to the manufacturer\u0026rsquo;s protocol. DNA extraction was performed after confirming that the tumor cell content was \u0026gt;\u0026thinsp;30%. The extracted DNA (\u0026ge;\u0026thinsp;30 ng) was used for library preparation and then sequenced on the Illumina NextSeq 500 platform (Illumina, San Diego, CA, USA). The panel was tested for mutations in 32 genes including 13 genetic and therapy related genes (PIK3CA, TP53, AR, CDH1, ERBB2 (HER2), ESR1, HOXB13, KRSA, NRAS, PTEN, STK11, BRAF, HDAC2) and 19 HRR genes (ATM, ATR, BARD1, BRCA1/2, CHEK1/2, FANCA, FANCL, PALB2, RAD51B, RAD51C, RAD51D, RAD54L, CDK12, NBN, PPP2R2A, BRIP1, MRE11A).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e1.\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Characteristics of study participants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 58 patients were enrolled in our study and the clinicopathological characteristics are summarized in Table 1. The cases included 45 (77.6%, 45/58) HGSC with a median age of 62 (range: 36-90), 10 (17.2%, 10/58) LGSC with a median age of 62.6 (range: 52-82) and 3 (5.2%, 3/58) EC with a median age of 64 (range: 43-65). The majority of patients (98.3%, 57/58) were FIGO stage III/IV, with 75.9% (n = 45/58) HGSC, 15.5% (n = 9/58) LGSC and 5.2% (n = 3/58) EC. There was no family history in any of these cases, except for one HGSC case. The median levels of the serologic marker CA-125 were 1194 U/mL, 1541.5 U/mL and 233 U/mL in HGSC, LGSC and EC respectively. The median values of ascites were 1000 mL, 925 mL and 240 mL in HGSC, LGSC and EC respectively. 58 patients were followed up for 2-52 months without loss to follow-up. No patient’s diagnosis changed after review.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Histopathological and immunohistochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe immunohistochemical results were displayed in Table 2 and Fig. 1. The results showed that the positive expression rate of Pax-8 in 45 HGSC was 87.5% (n = 21/24), that of WT-1 was 87.8% ( n = 36/41), that of CA-125 was 100% (n = 41/41), that of CK7 was 100% (n = 44/44) and that of CK20 was 6.67% (n = 3/45); in 10 cases of LGSC, the positive expression rate of Pax-8 was 100% (n = 7/7), the positive expression rate of WT-1 was 90% (n = 9/10), the positive expression rate of CA-125 was 100% (n = 9/9), the positive expression rate of CK7 was 100% (n = 10/10) and the negative expression rate of CK20 (n = 0/10); in 3 cases of EC, the positive expression rate of Pax-8 was 100% (n = 3/3), WT-1 was 100% (n = 2/2), CA-125 was 100% (n = 2/2), CK7 was 100% (n = 3/3) and CK20 was negative (n = 0/3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;HRR Genes Mutation Analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe HRR-related gene mutations in 58 patients are shown in Fig. 2A. 77.6% (45/58) of the patients carried HRR-related gene mutations. Among them, TP53 gene was the commonly mutated gene with 55% mutation percentage, followed by PIK3CA gene with 21.7% mutation percentage. The mutation percentage of BRCA1/2 is 6.67%, BRCA1 (5%) and BRCA2 (1.67%). The main observation of PIK3CA mutations is shown in Fig. 2B. In this work, there were seven main PIK3CA mutation types, namely E545K (28.6%), E542K (28.6%), E762K (14.3%), E545D (7.1%), Q546K (7.1%), H1047L (7.1%), H1047R (7.1%). In this work, PTEN, BRCA1, CDK12, BRAF, BRCA2, CHEK2 and KRAS mutations were also found.\u003c/p\u003e\n\u003cp\u003eNine BRCA1/2 mutations identified in ascites cell blocks of 58 ovarian cancer patients in Table 3: 6 BRCA1 and 3 BRCA2. 7 BRCA1/2 mutations matched 7 germline BRCA1/2 mutations identified in blood samples. Two additional BRCA1/2 mutations, which were not identified in blood samples, were considered as somatic: one BRCA1 and one BRCA2. Both somatic mutations BRCA1 exon11 c.975_976delinsAT p.(E326*) and BRCA2 exon22 c.8839_8851del p.(E2947Pfs*25) are known pathogenic mutations. Both patients with somatic mutations had a negative family history and did not have ovarian cancer.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWT-1 and PAX-8 are established biomarkers in ovarian carcinoma diagnosis due to their high sensitivity and specificity. PAX-8 has been shown to be a highly sensitive marker for identifying ovarian cancer cells, especially in the context of distinguishing them from non-gynecologic origins in ascitic fluid specimens. PAX-8, in combination with Calretinin, is valuable in identifying M\u0026uuml;llerian-derived epithelia, assisting in diagnosing ovarian carcinomas pre-neoadjuvant chemotherapy [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. PAX-8\u0026rsquo;s consistent expression in ovarian serous tumors makes it a reliable marker for distinguishing them from other tumor types like mucinous tumors [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Similarly, WT-1 has been recognized for its diagnostic value in ovarian cancer. It is particularly useful in distinguishing serous ovarian carcinomas from other histological subtypes. WT-1 immunoreactivity is prevalent in serous tumors, which are the most common subtype of epithelial ovarian cancer, and its expression is significantly higher in these tumors compared to non-serous subtypes [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The combined use of WT-1 and PAX-8 has been shown to improve the detection rates of metastatic ovarian carcinoma in serous effusion specimens, further supporting their utility as diagnostic markers [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Additionally, the expression of PAX-8 in high-grade serous ovarian carcinoma has been linked to its potential as a therapeutic target, given its overexpression in a significant number of cases [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The utility of PAX-8 and WT-1 as biomarkers is further supported by their ability to distinguish primary ovarian carcinomas from metastatic breast carcinomas, providing critical information for accurate diagnosis and treatment planning [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHRR defects are an important molecular feature of ovarian cancer, especially in HGSC, where mutations in HRR related genes (e.g., BRCA1/2, RAD51, and PALB2) are present in about 50% of cases [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In this study, 77.6% of patients carried mutations in HRR genes, of which TP53 mutations accounted for 55%, nine BRCA1/2 mutations were identified, further corroborating the critical role of HRR defects in ovarian cancer development. HRR defects lead to impaired repair of DNA double-strand breaks, which sensitizes the tumor cells to PARP inhibitors, and therefore the detection of HRR status has become the precise treatment of ovarian cancer as an PARP inhibitors (e.g., olaparib, niraparib) selectively kill HRR-deficient tumor cells through the mechanism of \"synthetic lethality\" and significantly prolong progression-free survival (PFS) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In recent years, mutations in other HRR genes (e.g., RAD51C/D, ATM) have also been found to be predictive of PARP inhibitors efficacy, expanding the beneficiary population [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In addition, HRR status may affect the sensitivity of platinum-based chemotherapy because platinum drugs work by inducing DNA cross-linking, and HRR-deficient cells are more likely to accumulate DNA damage [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Therefore, HRR testing may not only guide the use of PARP inhibitors, but also optimize the choice of chemotherapy regimen. Despite the growing clinical use of HRR testing, challenges remain. For example, some HRR-unmutated patients may still benefit from PARP inhibitors (e.g., the \u0026ldquo;BRCAness\u0026rdquo; phenotype), suggesting the need to combine it with other biomarkers (e.g., genomic instability scores) to further improve predictive accuracy [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In addition, secondary mutations in the HRR gene may lead to PARP inhibitors resistance, thus dynamic monitoring of HRR status is essential for therapeutic adjustment [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePIK3CA is a key regulatory gene of the PI3K/AKT/mTOR pathway, and its mutations (e.g., E545K, H1047R) can lead to the sustained activation of the pathway and promote tumor cell proliferation, survival and metastasis [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In this study, 21.7% of ovarian cancer patients were found to have PIK3CA mutations, of which E545K and E542K accounted for 28.6%, suggesting that PIK3CA mutations are more common in ovarian cancer and may be a potential target for targeted therapies. In addition, the presence of PIK3CA mutations may be closely associated with the prognosis of ovarian cancer patients, and studies have shown that patients carrying PIK3CA mutations may have higher tumor aggressiveness and worse prognosis. The detection of PIK3CA mutation can provide more precise treatment strategies for ovarian cancer patients, and also lays important basis for further research on the relationship between PIK3CA mutation and biological behavior of ovarian cancer [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, there are limitations to this work. First, the sample size of this study was small (n\u0026thinsp;=\u0026thinsp;58) and it was a single-center retrospective analysis, which may be subject to selection bias. Second, there was no multi-omics validation of HRR functional defects (e.g., methylation), and there was a lack of correlation analysis of treatment response and survival data. Future sample expansion and prospective studies are needed to further validate the findings.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, immunohistochemical results showed that WT-1, PAX-8, CA-125, and CK7 were stably expressed in serous ovarian cancer and could be used as diagnostic markers, while the low expression of CK20 could help to identify metastatic cancers. The molecular pathological analysis of paraffin-embedded cell blocks of ascites from 58 ovarian cancer patients revealed a high incidence of HRR-related gene mutations in ovarian cancer (77.6%), with TP53 (55%) and PIK3CA (21.7%) as the commonly mutated genes. BRCA1 (5%) and BRCA2 (1.67%) were detected in ascites cell blocks of 58 ovarian cancer patients, who can benefit from PARP inhibitors targeted therapy. HRR assay provides an important basis for targeted therapies, such as PARP inhibitors, and the PIK3CA mutation suggests that inhibitors of the PI3K/AKT pathway may be a potential treatment option. This study supports the value of ascites cell blocks in molecular typing of ovarian cancer and provides a clinical reference for the development of individualized treatment strategies. Additional benefits when cytological samples are tested, could be in faster processing of genotyping and earlier informative report for clinician as well as in potentially reduced number of genetic testing.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIHC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eimmunohistochemical\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHRR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehomologous recombination repair\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOvarian cancer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEOC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEpithelial ovarian cancer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHGSC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehigh-grade serous ovarian cancer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDSBs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDNA double-strand breaks\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePARP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epoly (adenosine diphosphate-ribose) polymerase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNGS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enext-generation sequencing\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLGSC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elow-grade serous carcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eendometrioid carcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFIGO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFederation of International of Gynecologists and Obstetricians\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eH\u0026amp;E\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehistopathological hematoxylin-eosin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePFS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eprolong progression-free survival.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e \u003cp\u003e The collection of patient\u0026rsquo;s clinical data was approved by the Ethics Committee of the First Affiliated Hospital of Dalian Medical University in accordance with the Declaration of Helsinki (approval number PJ-KS-KY-2025-916). Informed consent was waived because of the retrospective nature of this study and anonymized clinical data was used for analysis.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting Interest\u003c/strong\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eClinical trial number\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eNil.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eChunfang Zhang and Wenting Jia: Curate the data and draft the manuscript. Shu Dong: Acquisition and analysis of data. Pengxin Zhang and Jingchun Gao: Conception and design of the study. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe authors are grateful to all colleges (Department of Pathology, The First Affiliated Hospital of Dalian Medical University, China) for their valuable assistance during this study.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe study\u0026rsquo;s data can be obtained from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSun Y, Yao L, Wang C, Xiong B, Guo J, Wang L et al. Involvement of cancer stem cells in chemoresistant relapse of epithelial ovarian cancer identified by transcriptome analysis. 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Cancer Manag Res. 2021;13:3081\u0026ndash;100. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2147/cmar.S292992\u003c/span\u003e\u003cspan address=\"10.2147/cmar.S292992\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 3 are available in the Supplementary Files section.\u003c/p\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":"Ovarian cancer, Ascites, Cell block, HRR, PIK3CA","lastPublishedDoi":"10.21203/rs.3.rs-8818719/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8818719/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study evaluated immunohistochemical (IHC) markers and homologous recombination repair (HRR) gene mutations in 58 ovarian cancer patients using ascites-derived paraffin-embedded cell blocks. IHC analysis revealed high expression of Pax-8 (87.5\u0026ndash;100%), WT-1 (87.8\u0026ndash;90%), CA-125 (100%), and CK7 (100%) across subtypes, supporting their diagnostic utility. HRR gene mutations were detected in 77.6% of cases, with TP53 (55%) and PIK3CA (21.7%) as the most frequent alterations. Nine BRCA1/2 mutations were identified. PIK3CA variants included E545K (28.6%) and E542K (28.6%), suggesting potential therapeutic targets. These findings highlight the clinical relevance of ascites cell blocks for molecular profiling, with HRR status guiding PARP inhibitor therapy and PIK3CA mutations indicating PI3K/AKT pathway inhibition. This approach may optimize personalized treatment strategies for advanced ovarian cancer.\u003c/p\u003e","manuscriptTitle":"The Clinicopathological and Molecular Characterization in Ascites-derived Paraffin- embedded Cell Blocks of Ovarian Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-19 15:55:50","doi":"10.21203/rs.3.rs-8818719/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":"99cbe02d-523d-496c-a2b0-4d85f1056fec","owner":[],"postedDate":"March 19th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-31T22:39:14+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-19 15:55:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8818719","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8818719","identity":"rs-8818719","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}