High-throughput viable circulating tumor cell isolation using tapered-slit membrane filter-based chipsets in the differential diagnosis of ovarian tumors

When an adnexal mass is found, surgical treatment direction may vary depending on the final pathological result; therefore, it is important to differentiate malignant from benign or borderline. Intraoperative rupture of ovarian cancer confined to the unilateral ovary (i.e., stage IA) is associated with poor prognosis in the early stages, upstaging to IC1, and requiring adjuvant chemotherapy [ 1 ]. If an adnexal tumor initially presumed to be benign is confirmed to be malignant after ovarian cystectomy or salpingo-oophorectomy, an additional staging surgery should be performed [ 2 ]. Approximately 70% of ovarian cancer is diagnosed at an advanced stage, and advanced ovarian cancer has poor prognosis, with a 5-year survival rate of <50% [ 3 , 4 ]. Therefore, a diagnostic tool capable of the early detection of ovarian malignancies before pathological confirmation through surgery is needed. Several diagnostic tools can aid in the initial diagnosis of adnexal masses before surgery. Cancer antigen (CA)-125 has been used as a leading protein biomarker for the screening, treatment, and follow-up of ovarian cancer for almost four decades. Elevated serum CA-125 occurs in >90% of advanced ovarian cancer cases and in 50–60% of stage I ovarian cancer cases [ 5 ]. CA-125 may also be elevated in benign gynecologic conditions, such as adenomyosis, endometriosis, myomas, and non-gynecologic conditions [ 6 ]. Consequently, the high false-positive rate and modest sensitivity of CA-125 contribute to unnecessary surgical procedures and psychological consequences in these women [ 7 ]. Imaging tests included ultrasound with Doppler, computerized tomography (CT), and magnetic resonance imaging (MRI) [ 8 ]. In addition, methods for predicting the likelihood of malignancy in an adnexal mass, including the Risk of Malignancy Index (RMI) and Risk of Malignancy Algorithm (ROMA), have been reported [ 9 , 10 ]. However, the routine use of these tools is limited by their inadequate diagnostic performance in certain situations and high costs. Circulating tumor cells (CTCs) are cancer cells shed from primary or metastatic tumors into the bloodstream. CTC detection has several advantages over tissue biopsy:1) convenience, 2) minimal invasiveness, 3) serial evaluation, and 4) evaluation of the entire tumor burden instead of a limited part [ 11 ]. Previous studies have shown that CTCs serve as prognostic factors for overall survival and progression-free survival (PFS) in breast, colorectal, prostate, and lung cancers [ 12 – 15 ]. Several technologies have been developed to isolate CTCs from whole blood using the differential expression of biological factors (tumor-associated antigens or simple markers of epithelial vs. mesenchymal/hematopoietic derivation) or physical properties of cancer cells (size, weight, or density) [ 11 , 16 ]. CellSearch ® system, using a magnetic ferrofluid containing antibodies against epithelial cell adhesion molecule, has been approved by the Food and Drug Administration as a CTC enumeration method in patients with metastatic breast, prostate, and colorectal cancer [ 17 ]. However, only epithelial cell types of CTCs can be selected because chemical pre-/post-treatment does not allow other cells to remain viable [ 18 ]. We developed a CTC isolation chipset using a tapered-slit membrane filter (TSF) based on multiple physical properties, such as size and elasticity that can detect cell types other than epithelial cells while maintaining CTC viability. Recently, studies have been conducted on CTCs as a predictive or prognostic factor for ovarian cancer. This study aimed to evaluate CTC isolation using TSF-based chipsets for the differential diagnosis of adnexal tumors.

A total of 201 patients were analyzed in this study. The clinical characteristics of the study population are shown in Table 1 , and patient characteristics according to the final pathological reports after surgery are shown in Table 2 . Eighty-one (40.3%), 31 (15.4%), and 89 (44.3%) were pathologically confirmed with benign, borderline, and malignant tumors, respectively. The mean age of the study population was 48.0 years and the mean tumor size was 11.4 cm. The frequency of age > 48 years (60.7% vs. 34.6%, p = 0.001) and the frequency of tumor size > 11cm (46.1% vs. 24.7%, p = 0.004) were significantly higher in the malignant group compared to the benign group. The frequency of abnormal CA-125 was significantly higher in the malignant group compared to the benign and borderline groups (malignant vs. benign, 78.7% vs. 46.9%, p<0.001; malignant vs. borderline, 78.7% vs. 22.6%, p<0.001). Of 89 patients with malignant ovarian tumors, 46 (51.7%) were diagnosed at early stage (I and II) and 43 (48.3%) were diagnosed at advanced stage (III and IV). The most common histologic type was high-grade serous (HGS) carcinoma (43.8%), followed by clear cell carcinoma (19.1%) and endometrioid carcinoma (16.9%). In the comparison between the benign and stage I/II ovarian cancer groups, the significant difference in the frequency of abnormal CA -125 disappeared ( S1 Table ). There were significant differences in the frequencies of age > 48 years, tumor size > 11 cm, and malignant diagnoses on CT/MRI. Values are presented as mean ± standard deviation or number of patients (%). CA, cancer antigen; CT, computerized tomography; MRI, magnetic resonance imaging; CTC, circulating tumor cells Values are presented as mean ± standard deviation or number (%). * 7 missing P a : benign vs. malignant P b : borderline vs. malignant CA, cancer antigen; CT, computerized tomography; MRI, magnetic resonance imaging; FIGO, International Federation of Gynecology and Obstetrics In the study population, the median number of CTCs was 1.0 (range, 0–23) and CTCs were detected in 116 patients (57.7%). Table 3 shows a comparison of preoperative CTCs according to the final pathological results. The mean number of CTCs in the benign group had no significant differences from those in the overall, early-stage, and advanced-stage cancer groups (mean ± standard deviation, 1.09 ± 2.74 vs. 1.51 ± 1.48 vs. 1.30 ± 1.13 vs. 1.72 ± 1.76). The CTC detection rates in the benign, borderline, and cancer (early stage/advanced stage) groups were 42.0%, 48.4%, and 75.3% (69.6%/81.4%), respectively. These values were significantly different between the benign and cancer groups, regardless of stage, and between the borderline and cancer groups. Values are presented as mean ± standard deviation or number (%). P a benign vs. cancer P b borderline vs. cancer P c benign vs. cancer, early stage P d benign vs. cancer, advanced stage CTC, circulating tumor cells Table 4 and Fig 3 show the diagnostic performance of preoperative CTC, CA-125, and CT/MRI for the differential diagnosis of adnexal masses. Preoperative CTC had a sensitivity of 75.3%, specificity of 58.0%, and accuracy of 67.1% for differentiating ovarian cancer from benign ovarian tumors, excluding borderline ovarian tumors. The values were similar to CA-125 (sensitivity, specificity, accuracy: 78.7%, 53.1%, 66.5%, but lower than CT/MRI (94.2%, 77.9%, 86.5%). When CTC positive or CA-125 > 35 U/mL was defined as “CTC or CA-125” positive, the sensitivity increased to 91.0% and was comparable to that of CT/MRI. When both CTC positive and CA-125 > 35U/mL were defined as “CTC and CA-125” positive, specificity increased to 77.8%, similar to CT/MRI. The AUC (95% confidence interval [CI]; p-value) of CTCs to differentiate ovarian cancer from benign tumors was 0.651 (0.566–0.736; 0.001), which was similar to that of CA-125 (0.656 [0.571–0.741; 0.001]), “CTC and CA-125” (0.691 [0.609–0.773; <0.001]), and “CTC or CA-125” (0.616 [0.529–0.703; 0.011]) and lower than that of CT/MRI (0.861 [0.798–0.923; <0.001]) ( Fig 3A ). Even when borderline tumors were included (ovarian cancer vs. benign-to-borderline tumors), the sensitivity, specificity, accuracy, and AUC of all modalities were similar to those when borderline tumors were excluded. Receiver operating characteristic curves of preoperative diagnostic methods in the differential diagnosis of adnexal mass (A) In differentiating ovarian cancer from benign ovarian tumors, excluding borderline ovarian tumors. (B) In differentiating stage I/II ovarian cancer from benign tumor. (C) In differentiating stage III/IV ovarian cancer from benign tumor. (D) In differentiating HGSC ovarian cancer from benign tumor. (E) In differentiating non-HGSC ovarian cancer from benign tumor. CTC, circulating tumor cells; CA, cancer antigen; CT, computerized tomography; MRI, magnetic resonance imaging; AUC, area under the curve; CI, confidence interval. Values are presented as % (95% confidence interval) CTC, circulating tumor cells; CA, cancer antigen; CT, computerized tomography; MRI, magnetic resonance imaging; HGS, high-grade serous For detecting stage I/II ovarian cancer from benign tumors, the sensitivity of CTCs, CA-125, CT/MRI, “CTC and CA-125”, and “CTC or CA-125” was 69.6%, 58.7%, 88.6%, 45.7%, and 82.6%, respectively. The specificity of CTCs, CA-125, CT/MRI, “CTC and CA-125”, and “CTC or CA-125” was 58.0%, 53.1%, 77.9%, 77.8%, and 33.3%. The AUCs (95% CI; p-value) of CTCs, CA-125, CT/MRI, “CTC and CA-125”, and “CTC or CA-125” were 0.620 (0.517–0.723; 0.028), 0.550 (0.444–0.657; 0.358), 0.833 (0.775–0.910; <0.001), 0.599 (0.492–0.706; 0.071), and 0.571 (0.467–0.676; 0.192), respectively ( Fig 3B ). The sensitivity of all modalities for differentiating stage III/IV ovarian cancer from benign tumors was higher than that for differentiating stage I/II ovarian cancer. The sensitivities of CTC, CA-125, and CT/MRI for differentiating stage III/IV ovarian cancer from benign tumors were 81.4%, 100.0%, and 100.0%, respectively. The AUCs (95% CI; p-value) of CTC, CA-125, CT/MRI, “CTC and CA-125”, and “CTC or CA-125” were 0.684 (0.586–0.782; 0.001), 0.766 (0.684–0.849; <0.001), and 0.890 (0.831–0.949; <0.001), 0.788 (0.700–0.876; <0.001), 0.662 (0.567–0.758; 0.004), respectively ( Fig 3C ). Regardless of the stage, CT/MRI showed the highest sensitivity and specificity among the single tests. The sensitivity increased with “CTC or CA-125” and the specificity increased with “CTC and CA-125,” showing similar values to CT/MRI. For differentiating HGS ovarian cancer from benign tumor, the sensitivity of CTCs, CA-125, CT/MRI, “CTC and CA-125”, and “CTC or CA-125” was 84.6%, 97.4%, 100.0%, 82.1%, and 100.0%, respectively. The AUCs (95% CI; p-value) of CTCs, CA-125, CT/MRI, “CTC and CA-125”, and “CTC or CA-125” were 0.702 (0.605–0.800; <0.001), 0.753 (0.667–0.840; <0.001), 0.890 (0.830–0.949; <0.001), 0.793 (0.704–0.882; <0.001), and 0.662 (0.565–0.759; 0.004), respectively. The sensitivity, accuracy, and AUC of all the modalities for distinguishing non-HGS ovarian cancer from benign tumors were lower than those for distinguishing HGS ovarian cancer ( Fig 3D and 3E ). A comparison of the clinical factors according to the presence of preoperative CTCs is presented in S2 Table . CA-125 levels > 35 U/mL (p = 0.013), CT/MRI findings of suspected malignancy (p = 0.004), and moderate-to-severe ascites (p = 0.034) were significantly associated with the presence of CTCs. In the univariate logistic analysis, CA-125 > 35 U/mL (hazard ratio [HR], 2.058; 95% CI, 1.162–3.643; p = 0.013), CT/MRI findings of suspected malignancy (HR, 2.347; 95% CI, 1.306–4.217; p = 0.004), and ascites (HR, 2.743; 95% CI, 1.051–7.162; p = 0.039) were significantly associated with CTC positivity. Multivariate logistic regression analysis showed that CT/MRI finding suspicious for malignancy (HR, 2.347; 95% CI, 1.306–4.217; p = 0.004) was the only independent risk factor associated with the presence of CTCs ( S3 Table ).

While the diagnostic performance of CTC alone using TSF platform in differentiating ovarian cancer from benign tumors was not substantial, the combination of CTC and CA-125 could potentially play a useful role in the diagnostic accuracy of ovarian cancer, similar to that of CT/MRI. Given the substantial costs associated with employing CT/MRI, it is imperative to conduct further comprehensive cost-effectiveness analyses that compare radiologic tests with the combined implementation of CTC and CA-125 for ovarian cancer diagnosis.

A total of 230 patients scheduled to undergo elective surgery for an indeterminate ovarian mass were prospectively recruited at Seoul National University Bundang Hospital from May 27, 2015 to April 4, 2016 and from November 05, 2020 to May 31, 2023. The exclusion criteria were as follows:1) patients with a history of other malignancies within the past five years from enrollment, 2) patients who received neoadjuvant chemotherapy before surgery, 3) patients with recurrent ovarian cancer, and 4) patients of non-ovarian origin in the final pathology report. Finally, 201 patients were analyzed in this study. Written informed consent was obtained from all participants. This study was approved by the institutional review boards of our institution (B-1408-263-003 and E-2008-630-001) and conducted in accordance with the Declaration of Helsinki. After the patient was placed under general anesthesia, 5 mL of peripheral blood was drawn from the antecubital vein before the start of planned surgery. All blood samples were collected in BD Vacutainer tubes and transferred to the Korea Advanced Institute of Science and Technology (KAIST) to identify and count the CTCs. The collected tubes were packed in ice and delivered within 6 hours of sampling to prevent cell lysis and destruction during delivery. Information on age, final pathological results (benign, borderline, or malignant), ovarian tumor size, International Federation of Gynecology and Obstetrics (FIGO) stage, cancer histology, preoperative CA-125 levels, and presumptive diagnosis on CT or MRI were collected. The 2014 FIGO staging system was used to stage ovarian cancer. The cutoff value for normal CA-125 levels was 35 U/mL. The presence of ascites was defined as moderate-to-severe ascites on preoperative CT or MRI. CTC positivity was defined as the presence of one or more detected CTCs, whereas CTC negativity was defined as the absence of detected CTCs. CTC isolation and counting were performed using a tapered-slit filter (TSF) platform [ 19 , 20 ] ( Fig 1 ). The TSF platform isolates CTCs based on their physical properties, such as size and deformability, irrespective of their surface protein expression. Moreover, owing to its unique slit design ( Fig 1 ) featuring a wide cell entrance and gradually narrowing cell exits, a high flow rate can be achieved while minimizing cell stress. Five mL of blood from an ovarian cancer patient was diluted in 10.0 mL of phosphate-buffered saline (PBS). The diluted sample was then directly processed into the TSF platform using a syringe pump for withdrawal without undergoing any pretreatment, such as Ficoll separation or cell fixation. After filtration, the captured cells were carefully released by applying a reverse flow of PBS using a syringe pump. The released cells were mounted onto glass slides using a cytocentrifuge (Shandon Cytospin III; Thermo Scientific, Wilmington, DE, USA). Cell-mounted glass slides were fixed, permeabilized, blocked, and subjected to immunofluorescent staining [ 20 ]. Subsequently, the fluorescent images ( Fig 2 ) were acquired using a fluorescence microscope system (Eclipse Ti, Nikon) and quantified using MetaMorph software (Molecular Devices, Sunnyvale, CA, USA). All immunofluorescent cells ( Fig 2 ) were examined and enumerated meticulously as CTCs based on the criteria of immunostaining (4′,6-diamidino-2-phenylindole, DAPI, positive; cluster of differentiation 45, CD45, negative; cytokeratin, CK, positive), morphology (such as higher degree of irregularity compared to background blood cells), cell size, and nucleus-to-cytoplasm ratio. CTCs were identified and counted from blood samples without patient information. Immunofluorescent cell images: A. circulating tumor cell (DAPI+/CK+/CD45-) and B. white blood cell (B, DAPI+/CK±/CD45+) from ovarian cancer patient’s blood, where the bar represents 20 μm, DAPI denotes 4′,6-diamidino-2-phenylindole, CD45 indicates the cluster of differentiation 45, and CK means cytokeratin. The sensitivity, specificity, and accuracy of the CTC detection chipsets were analyzed according to postoperative pathologic results and compared with those of other screening tools, such as CA-125 and CT/MRI. The area under the curve (AUC) was calculated to evaluate the ability of CTCs as a screening tool by creating a receiver operating characteristic (ROC) curve. Pearson’s χ 2 test or Fisher’s exact test was used to compare categorical variables, while Student’s t-test or Mann–Whitney test was used to compare continuous variables. Statistical analyses were performed using IBM SPSS Statistics for Windows (version 27.0; IBM Corp., Armonk, NY, USA) and MedCalc (version 22). Statistical significance was set at a P-value < 0.05.

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