Methylated ARHGAP40 DNA as a potential biomarker for early diagnosis in high-grade ovarian serous cancer.

The expression of ARHGAP40 protein was detected in normal fallopian tube, ovarian benign tumor, borderline tumor, and serous cancer FFPE specimens using an immunohistochemical staining method. The detailed result of expression of ARHGAP40 was listed in Table  1 . The ARHGAP40 protein was positively stained in cytoplasm of cells with yellow or brown color (Fig.  1 ). ARHGAP40 staining was positively expressed in all fallopian tube specimens (15/15, 100%; Fig.  1 A shows a representative example of IHC staining, and Fig.  1 B shows the corresponding H&E staining) and benign ovarian tumor specimens (8/8, 100%; Fig.  1 C shows a representative example of IHC staining, and Fig.  1 D shows the corresponding H&E staining) as score 2 + or 3+. ARHGAP40 protein was positively expressed in most of borderline tumor (16 out of 18, 88.9%; Fig.  1 E shows a representative example of IHC staining, and Fig.  1 F shows the corresponding H&E staining) and LGSC specimens (6/7, 85.7%; Fig. G shows a representative example of IHC staining, and Fig.  1 H shows the corresponding H&E staining). ARHGAP40 protein was seldom expressed at a moderate level in HGSC (2/32, 6.3%; Fig.  1 I shows a representative example of IHC staining, and Fig.  1 J shows the corresponding H&E staining), and was negatively expressed in most of HGSC (30/32, 93.7%; Fig. K shows a representative example of IHC staining, and Fig.  1 L shows the corresponding H&E staining). There is a significant expression of ARHGAP40 between normal fallopian tube, ovarian benign tumor, borderline tumor, LGSC to HGSC ( P  < 0.001). The detailed expression of ARHGAP40 and clinicopathological parameters of HGSC were summary in Table  2 . Fig. 1 The expression of ARHGAP40 protein was immunohistochemically checked in normal fallopian tube, ovarian benign and borderline tumor, LGSC and HGSC. Representative examples of IHC staining and the corresponding H&E staining of normal fallopian tube, ovarian benign and borderline tumor, LGSC and HGSC were showed as follow: ARHGAP40 protein was positively expressed in normal fallopian tube ( A ), ovarian benign ( C ) and borderline tumor ( E ), and LGSC ( G ); The histology of normal fallopian tube ( B ), ovarian benign ( D ) and borderline tumor ( F ) and LGSC ( H ) was diagnosed with H&E staining. ARHGAP40 protein was negatively expressed in HGSC. Representative examples showed ARHGAP40 protein was weakly expressed ( I ) and negatively expressed in HGSC ( K ). The histology of HGSC was showed with H&E staining ( J and L ). All magnification 40× The expression of ARHGAP40 protein was immunohistochemically checked in normal fallopian tube, ovarian benign and borderline tumor, LGSC and HGSC. Representative examples of IHC staining and the corresponding H&E staining of normal fallopian tube, ovarian benign and borderline tumor, LGSC and HGSC were showed as follow: ARHGAP40 protein was positively expressed in normal fallopian tube ( A ), ovarian benign ( C ) and borderline tumor ( E ), and LGSC ( G ); The histology of normal fallopian tube ( B ), ovarian benign ( D ) and borderline tumor ( F ) and LGSC ( H ) was diagnosed with H&E staining. ARHGAP40 protein was negatively expressed in HGSC. Representative examples showed ARHGAP40 protein was weakly expressed ( I ) and negatively expressed in HGSC ( K ). The histology of HGSC was showed with H&E staining ( J and L ). All magnification 40× Table 2 The expression of ARHGAP40 in High-grade ovarian serious cancers Parameters ARHGAP40 expression 0/1+ 2+ 3+ 30 2 0 Age (years) ≤ 50 3 1 0 > 50 27 1 0 TNM stage I + II 3 0 0 III + IV 27 2 0 Lymph node No 10 0 0 Yes 7 0 0 NA 13 2 0 Ki-67 ≤ 20% 1 0 0 > 20% 29 2 0 FIGO stage I + II 5 0 0 III + IV 25 2 0 NA: not available The expression of ARHGAP40 in High-grade ovarian serious cancers NA: not available There is a CpG island around transcription start site (ATG) of ARHGAP40 gene. We designed bisulfite sequencing PCR primer set to analyze the methylation status of CpG island associated to ARHGAP40 promoter (Fig.  2 A). Genomic DNA was extracted from FFPE specimens of fallopian tube, ovarian serous cancer and carried out BSP amplification. The PCR products were subjected Sanger sequencing. As shown in Fig.  2 B and C, the unmethylated ARHGAP40 DNA was detected in fallopian tube specimen which positively expressed ARHGAP40, while methylated ARHGAP40 DNA was detected in HGSC specimen which negatively expressed ARHGAP40. The methylation status of CpG island in ARHGAP40 promoter region is significantly associated with the expression level of its protein. Fig. 2 The hypermethylation status of ARHGAP40 in CpG was significantly associated with protein expression. A: A BSP primer set was designed to detect methylated CpG sites in promoter region of ARHGAP40 gene. B: Unmethylation of CG sites were detected in fallopian tube tissue that positive expression of ARHGAP40 protein. C: Methylated CG sites were detected in HGSC tissue that negative expression of ARHGAP40. Arrows indict CG sites The hypermethylation status of ARHGAP40 in CpG was significantly associated with protein expression. A: A BSP primer set was designed to detect methylated CpG sites in promoter region of ARHGAP40 gene. B: Unmethylation of CG sites were detected in fallopian tube tissue that positive expression of ARHGAP40 protein. C: Methylated CG sites were detected in HGSC tissue that negative expression of ARHGAP40. Arrows indict CG sites To explore the clinical application of methylated ARHGAP40 in screening and early diagnosis of ovarian serous cancer, we preliminarily tested detection of methylated ARHGAP40 using MethyLight method in cfDNA in a total of 23 peripheral blood were collected from healthy control [ 15 ], patients with ovarian benign tumor [ 3 ] and HGSC patients [ 5 ]. Methylated ARHGAP40 DNA was not detected in all healthy control (0/15) and ovarian benign tumor (0/3), while was detected in 4 out of 5 (80%) HGSC patients. The methylated ARHGAP40 DNA detected by MethyLight method was confirmed by using Sanger sequencing to check MethyLight PCR products.

A total of 80 FFPE fallopian and ovarian tissue specimens were collected from Jinling Hospital, Nanjing Medical University (China) and Nanjing JunXie Hospital, Nanjing, China between September 2020 and June 2024, including normal fallopian tube [ 15 ], benign tumor [ 8 ], borderline tumor [ 18 ], and serous cancer (LGSC 7 and HGSC 32). Patients with ovarian serous cancer were not required to receive neoadjuvant chemotherapy prior to surgery. International Federation of Gynecology and Obstetrics (FIGO) stage was used to classify and stage clinicopathological features. Histological grade, presence of lymphnode invasion and pathological type were diagnosed according to World Health Organization (WHO) Classification of Female Genital Tumors (5th ed.). The detailed clinicopathological information of HGSC was listed in Table  1 . This study based on tissues was a retrospective one and informed consent was not available. In addition, a total of 23 peripheral blood were collected from healthy control [ 15 ], patients with ovarian benign tumor [ 3 ] and HGSC patients [ 5 ]. All healthy control and patients provided their informed consent. This study was approved by the ethics board of Nanjing JunXie Hospital, Nanjing, China (20241204-019) and fulfilled the Declaration of Helsinki. Table 1 The expression of ARHGAP40 protein in fallopian tube and ovarian tumors Type of tumor No. ARHGAP40 expression P value Negative (-) Positive (+) 0 1+ 2+ 3+ Fallopian tube 15 0 0 1 14 < 0.001 Benign tumor 8 0 0 2 6 Borderline tumor 18 0 2 3 13 LGSC 7 0 1 1 5 HGSC 32 16 14 2 0 The expression of ARHGAP40 protein in fallopian tube and ovarian tumors Paraffin-embedded Sect. (4 μm) were cut from tissue blocks, air-dried at 60 °C for 2 h, and processed through xylene dewaxing followed by graded ethanol dehydration (75%, 85%, 95%, and 100%). Antigen retrieval was performed using citrate buffer (pH 6.0) in a pressure cooker at 120 °C for 2 min. After microwave retrieval, sections were allowed to cool to room temperature, washed three times with PBS (5 min per wash), and blocked with 1% BSA for 30 min. Primary antibody against ARHGPA40 (NBP1-94112, Novus Biologicals, LLC 10771 E Easter Ave Centennial, CO 80112, USA; dilution 1:1000) was applied overnight at 4 °C in a humidified chamber. Subsequent immunodetection utilized the Dako REAL EnVision Detection System (Dako, UK) with horseradish peroxidase-conjugated secondary antibodies, followed by chromogenic development using 3,3’-diaminobenzidine (DAB) substrate. Nuclear counterstaining was performed with Mayer’s hematoxylin for 1 min. IHC staining results were evaluated according to intensity: 0 (negative), 1+ (weak), 2+ (moderate), and 3+ (strong). Positive staining of ARHGAP40 was defined with score 2 + and 3+, while negative staining was score 0 and 1+. Two independent pathologists blinded to clinical data performed histopathological evaluations, with discrepancies resolved through joint re-evaluation using multiheaded microscopy. BSP was employed to assess the methylation status of ARHGAP40. Genomic DNA was initially extracted from FFPE specimens using a commercial DNA extraction kit (Tiangen Biotech, Beijing, China) following the manufacturer’s protocol. Subsequently, bisulfite conversion of 500 ng genomic DNA was performed using the EZ DNA Methylation-Gold Kit (Zymo Research, Irvine, CA, USA) to ensure complete cytosine deamination. Target regions were amplified by PCR using gene-specific primers (Forward: 5’-GAGGTTGGTTTTAGGGTTTTTAGTT-3’; Reverse: 5’-AACTCTCCAAACCTCTATTTCCTC-3’) with ExTaq DNA polymerase (Takara Bio, Dalian, China). The thermal cycling protocol consisted of an initial denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s, with a final elongation step at 72 °C for 5 min. PCR products were electrophoresed on 2% agarose gels for quality verification prior to bidirectional Sanger sequencing performed by Sangon Biotech (Shanghai, China). MethyLight is a quantitative, fluorescence-based real-time PCR method to sensitively detect DNA methylation of candidate region of the genome. It is suited for detecting low-frequency methylated DNA regions against a high background of unmethylated DNA, because it combines methylation-specific priming with methylation-specific fluorescent probing. MethyLight uniquely well suited for detection of low-frequency DNA methylation biomarkers as evidence of disease. In this study, we designed a set of methylight methylation-specific PCR (MSP) forward and reverse primers and a methylation-specific probe to detect small amount of methylated ARHGAP40 in ctDNA. Briefly, 10 ml of peripheral blood were stored in cell-free storage tube (CWBIO, Nanjing China) to separate cell-free DNA using a QIAamp Circulating Nucleic Acid kit (QIAGEN55114, Qiagen, Hamburg, Germany) according to protocol provided by the manufacturer. cfDNA was modified with the EZ DNA Methylation-Gold Kit (Zymo Research, Irvine, CA, USA). Methylight was carried out using a BIOER FQD-96 A Fluorescent PCR Analyzer (BIOER, Hangzhou, China). The primer and probe for detection of ARGHP40 and internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were designed using website MethPrimer ( https://www.methprimer.com/ ). The detailed sequences of sets were as follow: forward primer set, 5’- ATTTTCGAGGTGTTAGTTTATGGTC- 3’; reverse primer set, 5’- CCTATACCTACAACCCAAATCGA-3’; and probe sequence, 5’- TAAACGCAACGCCTAACAATACGAACCCGC-3’. Statistical analyses were performed using IBM SPSS Statistics version 25.0 (Armonk, NY, USA). Intergroup comparisons of categorical variables were conducted using the chi-square (χ²) test, with the significance level (α) set at 0.05. Statistical significance was defined as a two-tailed P value < 0.05.

Ovarian cancer ranks as the eighth most common malignancy in women worldwide and is the leading cause of death among gynecologic cancers in the United States, where it is also the second most prevalent gynecologic malignancy [ 1 ]. Due to its asymptomatic nature in early stages and the absence of effective screening strategies, ovarian cancer is frequently diagnosed at advanced stages, which significantly complicates treatment and reduces overall survival rates. Epithelial ovarian cancer (EOC) constitutes the majority of cases and is subclassified into five histological subtypes: high-grade serous carcinoma (HGSC), low-grade serous carcinoma (LGSC), clear cell carcinoma, endometrioid carcinoma, and mucinous carcinoma. Among these, HGSC is the most common histological subtype. HGSC and LGSC exhibit distinct morphological, pathogenetic, molecular, and prognostic profiles. HGSC typically originates from the distal fimbrial end of the fallopian tube, whereas LGSC arises from benign or borderline serous tumors within the ovary. Genetically, 96% of HGSC cases harbor TP53 mutations, often accompanied by high-copy-number alterations and germline BRCA1/2 mutations [ 2 ]. In contrast, LGSC is characterized by frequent BRAF and KRAS mutations [ 2 ]. The Rho family, a subfamily of the Ras superfamily of guanosine triphosphatases (GTPases), regulates critical cellular processes including cell morphology, gene transcription, cell cycle progression, apoptosis, carcinogenesis, migration, and invasion [ 3 ]. The Rho GTPase-activating proteins (RHOGAPs), also known as the ARHGAP family, function as negative regulators of Rho GTPase signaling [ 4 ]. Dysregulation of ARHGAP family members has been implicated in tumorigenesis and cancer progression across diverse malignancies. For instance, ARHGAP7 suppresses hepatocellular carcinoma progression by inhibiting Rho protein-mediated cell proliferation, anchorage-independent growth, and xenograft tumor formation [ 5 ]. Conversely, ARHGAP6/ARHGAP26 fusion proteins attenuate apoptosis in gastric cancer [ 6 ]. Our previous work demonstrated that ARHGAP40 is downregulated in basal cell carcinoma due to CpG island hypermethylation within its promoter region [ 7 ]. Recent studies extend this finding, identifying ARHGAP40 hypermethylation in breast cancer [ 8 ]. Here, we investigate ARHGAP40 epigenetic regulation in ovarian cancer and report its promoter hypermethylation in high-grade serous ovarian carcinomas (HGSC), contrasting with unmethylated status in normal fallopian tube epithelia and benign ovarian tumors. These observations align with emerging evidence that DNA methylation-mediated epigenetic silencing of tumor-suppressive genes contributes to ovarian carcinogenesis [ 9 ]. However, the clinical utility of ARHGAP40 methylation as a diagnostic biomarker requires further validation. Ovarian cancer is characterized by a poor prognosis, with a 5-year survival rate below 50% and a 10-year survival rate of approximately 35% [ 10 ]. Prognosis is strongly correlated with disease stage at diagnosis: patients with stage I tumors exhibit 5-year survival rates of 70–92%, whereas those with stage IV disease have survival rates < 6% [ 11 ]. Early detection remains a critical unmet need in gynecologic oncology. Current diagnostic workflows for symptomatic patients (e.g., presenting with ascites or gastrointestinal dysfunction) rely on pelvic and rectovaginal examinations, imaging modalities (e.g., transvaginal ultrasound), and serum CA125 testing combined with other biomarkers [ 12 ]. However, screening strategies using serum CA125 and transvaginal ultrasound have shown limited efficacy in differentiating benign from malignant pelvic masses, even after algorithmic optimization [ 13 , 14 ]. Liquid biopsy approaches, including circulating cell-free DNA (cfDNA) and circulating tumor DNA (ctDNA) analysis, offer promising alternatives for ovarian cancer screening and management. cfDNA refers to extracellular nucleic acid fragments released into the bloodstream through cellular necrosis, apoptosis, or active secretion [ 15 – 18 ]. ctDNA, typically defined as fragmented DNA < 157 base pairs in length, has demonstrated utility in early cancer detection, prognosis prediction, and therapeutic monitoring [ 19 – 22 ]. These noninvasive biomarkers may address the limitations of conventional screening methods.

The ARHGAP40 protein was ​absent in the majority of high-grade serous carcinomas (HGSCs), variably expressed in a subset of low-grade serous carcinomas (LGSCs) and borderline tumors, but consistently detected in ​all normal fallopian tube epithelia and ovarian benign tumors. This expression pattern inversely correlated with promoter CpG island methylation status: ARHGAP40 methylation was significantly enriched in HGSCs compared to non-malignant tissues, leading to transcriptional silencing of the gene. These findings establish ​methylated ARHGAP40 ctDNA as a promising biomarker for early detection of HGSC. The methylation-driven loss of ARHGAP40 protein aligns with its tumor-suppressive role and highlights the epigenetic dysregulation specific to aggressive ovarian carcinomas. While validation in larger, independent cohorts is required, this approach could enable non-invasive diagnosis of HGSC at earlier stages, where therapeutic outcomes are more favorable.

Ovarian serous carcinomas often lack specific symptoms in early stages, with nonspecific presentations such as pelvic pain, urinary urgency/frequency, abdominal bloating, loss of appetite, or unintentional weight loss. These vague symptoms frequently lead to delayed diagnosis, with most cases identified at advanced stages (III–IV), associated with a poor prognosis [ 2 ]. Current statistics indicate that only 15–20% of ovarian cancers are diagnosed at an early stage (confined to the primary site), conferring a 5-year survival rate exceeding 90%. In contrast, the majority of patients present with metastatic disease (e.g., regional lymph node involvement or distant metastases), for which the 5-year survival rate drops to 20–41% [ 23 ]. Current diagnostic guidelines from the American College of Obstetricians and Gynecologists (ACOG) recommend imaging modalities (e.g., transvaginal ultrasound, computed tomography) to detect pelvic masses and biochemical tests such as serum CA125 glycoprotein analysis. However, CA125 exhibits limited utility in early-stage ovarian cancer due to poor sensitivity (e.g., false negatives in early lesions) and low specificity (e.g., elevated levels in benign conditions like endometriosis or pregnancy) [ 24 , 25 ]. This diagnostic gap underscores the urgent clinical need for biomarkers or multimodal approaches with improved sensitivity and specificity for early-stage detection and differentiation of malignant versus benign pelvic masses. Epigenetic alterations—heritable changes in gene expression without DNA sequence modifications—play a critical role in malignant transformation and cancer progression [ 26 ]. DNA methylation, the addition of a methyl group to the 5’-carbon of cytosine in CpG dinucleotides, is the most prevalent epigenetic modification. Aberrant hypermethylation of CpG islands, which leads to transcriptional silencing of tumor-suppressor genes, is a hallmark of cancer and one of the earliest events in carcinogenesis [ 27 ]. Such methylation changes hold promise for detecting early-stage or premalignant disease. In ovarian cancer, numerous genes are silenced by promoter hypermethylation, including BRCA1 , a key tumor suppressor implicated in DNA repair and genomic stability [ 26 , 28 – 32 ]. BRCA1 mutations are well-established drivers of hereditary breast and ovarian cancers, but epigenetic silencing via promoter hypermethylation is also implicated in sporadic cases [ 33 ]. Notably, BRCA1 hypermethylation occurs at significantly higher frequencies in high-grade serous carcinomas (HGSCs), the most lethal ovarian cancer subtype, compared to other histotypes [ 33 ]. Despite extensive research on epigenetically altered genes in ovarian cancer, many findings remain unvalidated across independent cohorts. To date, no single gene has been identified as universally hypermethylated and silenced in the majority of ovarian cancers. In this study, we report ​ARHGAP40 as a novel candidate biomarker. ARHGAP40 protein expression was lost in most HGSC cases, a finding strongly correlated with CpG island hypermethylation. In contrast, ARHGAP40 protein was robustly expressed in normal fallopian tube epithelium, benign ovarian tumors, borderline tumors, and the majority of low-grade serous carcinomas (LGSCs). While our cohort size is limited, these data suggest that ARHGAP40 methylation and protein loss may serve as a ​discriminative biomarker for HGSC versus LGSC, borderline tumors, or benign lesions. Further validation in larger, independent cohorts is warranted to confirm its clinical utility. Despite advances in ovarian cancer screening and prevention, early diagnosis remains a critical unmet challenge in gynecological oncology. Current approaches, such as serum cancer antigen 125 (CA125) testing and transvaginal ultrasound, have shown limited efficacy [ 34 – 36 ]. CA125 alone exhibits low sensitivity (23–50%) for early-stage detection [ 36 , 37 ], while transvaginal ultrasound is impractical for widespread screening due to its invasiveness, time requirements, and operator dependency. Human epididymis protein 4 (HE4) is expressed at low levels in healthy ovarian tissues but is elevated in ovarian cancer, and is better at differentiating epithelial ovarian cancer from benign ovarian tumor. HE4 used as an early detection is limited for its non-specificity [ 37 , 38 ]. Liquid biopsy, a minimally invasive technique that analyzes cfDNA, offers a promising alternative. cfDNA fragments released into the bloodstream by apoptotic or necrotic tumor cells include ctDNA, which carries tumor-specific epigenetic and genetic alterations. DNA promoter hypermethylation—a key epigenetic modification and early carcinogenic event—has shown potential as a diagnostic biomarker in ovarian cancer [ 31 , 39 – 41 ]. However, existing studies of cfDNA methylation for early detection are constrained by small sample sizes and a predominance of late-stage cases, leaving its utility in early-stage diagnosis underexplored. In this study, we developed a sensitive MethyLight assay to quantify methylated ARHGAP40 in cfDNA from healthy controls, patients with benign ovarian tumors, and those with high-grade serous carcinomas (HGSCs). Our results demonstrate that methylated ARHGAP40 ctDNA is detectable in HGSC patients and absent in non-malignant cases, suggesting its potential as a ​novel biomarker for early ovarian cancer diagnosis. A critical limitation of this study is the small sample size ( n  = 5 HGSC patients for plasma analysis), which may limit the statistical robustness of our findings. Given the preliminary nature of this work, our conclusions should be interpreted cautiously, and validation in larger, multi-center cohorts is urgently needed. While our study demonstrates promising sensitivity and specificity for ARHGAP40 methylation in early-stage HGSC detection, direct comparison with established biomarkers (CA125 and HE4) is limited by differences in study design and patient cohorts. Notably, CA125 exhibits suboptimal sensitivity (≤ 50%) in early-stage ovarian cancer, whereas HE4 shows improved performance but remains less widely adopted. Our pilot data suggest that ARHGAP40 methylation may complement these markers, particularly in early-stage disease where CA125 often fails. However, formal comparative studies are required to establish relative diagnostic utility. While our cohort size is limited by the preliminary nature of this report, these findings lay the groundwork for larger validation studies to confirm its clinical utility. Future work will expand patient enrollment and integrate ARHGAP40 methylation with other biomarkers to improve diagnostic accuracy.