Introduction

Cancer antigen 125 (CA125) is an antigenic tumor marker expressed by epithelial ovarian neoplasms and cells lining various organs such as the endometrium, fallopian tubes, pleura, peritoneum, and pericardium.[1][2] CA125 is used as one of the serological tests in cases when an ovarian neoplasm is suspected and for monitoring patients who have already been diagnosed with epithelial ovarian cancers.[2][3] However, due to its low sensitivity, the test has limited use in diagnosing early ovarian cancer. The specificity is particularly low in premenopausal women; thus, it is most useful in postmenopausal women.[4]

Etiology and Epidemiology

CA125 is an epitope found on mucin 16 (MUC16), a glycoprotein antigen present on the cell surface. This antigen is normally expressed in tissues derived from coelomic epithelia, such as the ovary, fallopian tube, peritoneum, pleura, pericardium, colon, kidney, and stomach.[2][5] Currently, 3 antibodies can help identify the CA125 antigen, and all 3 groups recognize nonoverlapping epitopes.[2] The first group involves OC125-like antibodies, the second involves M11-like antibodies, and the third involves OV197-like antibodies.[3][6]

In a study carried out by Bast et al in 1983, elevated levels of serum CA125 (>35 U/mL) were present in 82% of patients with epithelial ovarian cancer, 28.5% of patients with non-gynecological cancers such as pancreatic, lung, breast, and colorectal, and 6% of the patients with benign diseases such as an ovarian cyst.[7] Among gynecologic malignancies, elevated levels of CA125 correlate with adenocarcinoma of the endometrium and endocervix.[8]  CA125 also becomes elevated under certain physiological conditions, such as during menstruation, the first trimester of pregnancy, the postpartum period, fibroids, and pelvic endometriosis.[3][9][10][11]

Pathophysiology

The inherent function of CA125 membrane protein is still not completely clear. The latest studies suspect that the oligosaccharides associated with CA125 might play a role in cell-mediated immunity.[12] CA125 may have a role in inhibiting cytotoxic responses of the natural killer cells.[13][14] Under physiological conditions, CA125 is expressed on the cell membrane. However, due to the presence of the cells' junctional complexes, it cannot cross into the bloodstream. Pathological states associated with the disruption of this membrane barrier lead to the antigen being shed into the blood and a consequent serological rise in the levels of CA125.[1] CA125 has been considered to play a role in promoting tumorigenesis and metastasis. This mechanism is believed to occur due to the binding between CA125 and mesothelin, a glycoprotein expressed on the mesothelial cells of the peritoneum.[15] Elucidating this role of CA125 in oncogenesis has proposed a potential therapeutic avenue through the creation of monoclonal antibodies targeting CA125.[15][16][15]

In the case of ovaries, it appears that CA125 expresses when the ovarian epithelium undergoes metaplasia into a Müllerian-type endothelium or a neoplastic transformation.[1][2] Upon their malignant transformation, the tumor cells invade and disrupt the architecture to enter the bloodstream.[17] In benign ovarian cysts, although the antigen may be shed into the cystic fluid, it is not present in the bloodstream.[8]

Specimen Requirements and Procedure

CA125 is detectable through serological-based tests or tissue-based studies of malignant ovarian or endometrial tissue specimens.[18] In addition, it may be present in body fluids such as pleural fluid, peritoneal fluid in benign or malignant conditions, and ovarian cystic fluid.[8] Serological assays on serum or plasma samples are the most commonly used quantitative analysis method for analyzing CA125 levels in the blood. To maintain the stability of the specimen, the serum must be separated from the clot and stored at −30 °C (long term) or 4 °C (short term).[19] 

When the CA125 assay is used for cancer diagnosis, sampling should not be conducted immediately before or during menstruation because the physiological elevation of the CA125 levels may provide false-positive results.[9][20] The serum samples for CA125 should not be collected within 2 weeks of surgery, as the levels may become falsely elevated secondary to tissue damage. CA125 levels have a half-life of 6 days and may require a few weeks to return to normal levels after surgery. A pre-treatment sample should be used as a reference for evaluating CA125 levels postoperatively.[21]

Diagnostic Tests

Two tests are available to measure the levels of CA125. The original test is a radioimmunoassay using OC125 monoclonal antibody, which recognizes the antigenic determinants on the CA125 glycoprotein. The second-generation CA125 test uses two antibodies, OC125 and M11.[22] This test has shown a higher level of precision and improved sensitivity with a lower number of false-positive results. However, subsequent research comparing the two tests did not indicate the superiority of one over the other.[23]

These tests are made available by different commercial manufacturers in different versions, such as immunoradiometric assay (IRMA) and enzyme immunoassay (EIA). When using these tests to monitor patients longitudinally, the tests should preferably be from the same manufacturer and the same version, as different kits and versions can result in different absolute values and test sensitivity.[8]

Efforts have been made to enhance the specificity of the CA125 test by testing for CA125 glycoforms more strongly associated with ovarian malignancy. For instance, the CA125-Tn ELISA-based test demonstrated almost a two-fold increase in specificity with no change in sensitivity.[24] Similarly, Other studies have shown an improved performance of certain CA125 glycoforms over the conventional CA125 test.[25]

Testing Procedures

The original CA125 test is a homologous double-determinant assay with the same capture and tracer antibodies. The OC125 antibody is adsorbed onto the solid phase (capture antibody), followed by the addition of the serum. The OC125 moieties on the CA125 antigen then bind to the antibody. Finally, a second radiolabeled (IRMA) or an enzyme-labeled (EIA) antibody (tracer antibody) is added, which binds to the antigen-antibody complexes.[8] The second-generation CA125 test is a heterologous double-determinant assay, where the capture antibody is a monoclonal M11 antibody, and the tracer antibody is the OC125 antibody. Since the two antibodies do not have to bind to the same epitope, there is no competition for the same binding site, thus allowing for potentially higher sensitivity.[26]

Interfering Factors

The original CA125 assay uses murine monoclonal OC125 antibody as both the capture and the tracer antibodies. Although it should bind to the corresponding epitope on the CA125 antigen, some individuals demonstrate the presence of the human anti-mouse antibody or human anti-murine antibody.[26] These antibodies may interfere with the test by binding to the capture and tracer antibodies, leading to falsely elevated CA125 levels. Human anti-murine antibodies are observed in patients exposed to murine monoclonal antibodies for therapeutic or diagnostic purposes. This interference is less observable in the newer assays.[27]

Pre-analytical errors while testing for tumor markers such as CA125 are related to simple specimen-related errors such as incorrect sample processing, an insufficient sample, and inappropriate handling. When conducting tests involving CA125, specific attention should be paid to the timing of specimen collection.[19] Blood collection should be avoided during menstruation, which can lead to falsely elevated levels (up to three-fold). Caution is needed when interpreting elevated CA125 results in women with suspected endometriosis, pregnancy, and ascites.[28][29][28]

Human anti-murine antibodies are known to interfere with the test, and adding non-immune murine serum can neutralize this interference.[26][23] For patients undergoing monitoring with serial CA125 levels, it is important to use the same manufacturer and laboratory, preferably. If a different manufacturer or testing method needs to be employed, the recommendation is to carry out parallel testing by both methods to establish a new baseline for the patient.[30]

Results, Reporting, and Critical Findings

In the original CA125 IRMA test, the cut-off point for the upper limit of normal was arbitrarily set at 35 U/mL. This threshold was chosen because only 1% of the healthy population had CA125 levels greater than 35 U/mL. However, this was reduced to 0.2% of the healthy population when the cut-off point for the upper limit was raised to 65 U/mL.[7] The second-generation CA125 has shown to be more clinically reliable with greater precision in values less than 35 U/mL, leading to higher values compared to the original CA125 test.[22] A meta-analysis comparing various studies using a CA125 threshold of greater than 35 U/mL for preoperative identification of an adnexal mass suspicious for ovarian cancer revealed an overall sensitivity and specificity of 78.7% and 77.9%, respectively.[31] Studies have shown that the test exhibits a higher sensitivity and specificity in postmenopausal women compared to premenopausal women.[32][33][32] The low sensitivity is due in part to the fact that CA125 is elevated only in half of the patients with early-stage epithelial ovarian cancer and rarely elevated in patients with mucinous carcinomas of the ovary. The low specificity, especially among premenopausal women, is due to the elevation caused by benign conditions such as fibroids, endometriosis, and pregnancy.[4]

However, the test is relatively sensitive and specific among patients who have completed treatment for ovarian cancer. Patients who appear to be clinically free of the tumor with elevations in CA125 (>35 U/mL) almost always have a tumor in second-look surgery.[8]  

Clinical Significance

CA125 plays a significant role as a tumor marker, particularly in the preoperative assessment of patients with an adnexal mass and suspected to have an ovarian malignancy.[31] Around 80% of the patients diagnosed with ovarian epithelial carcinoma show elevated CA125 levels, and levels are monitored post-treatment to assess the progression of the disease.[34] The use of CA125 for preoperative assessment is more valuable among postmenopausal women compared to its use among premenopausal women.[4] Although an elevated CA125 level above 35 U/mL is utilized in assessing postmenopausal women, earlier guidelines from the American College of Obstetricians and Gynecologists had recommended using an arbitrary cutoff of 200 U/mL; this was, however, not supported by any research evidence.[35]

Current guidelines suggest using formal risk assessment tests such as the risk of malignancy index or the risk of ovarian malignancy algorithm (ROMA) while assessing the need for gynecological referral in premenopausal women.[4] CA125 is successfully used for disease monitoring and evaluating the progression of the disease. A significant correlation exists between the disease progression and serum CA125 levels, with doubling or halving serum values considered clinically significant.[8] 

CA125 levels also indicate the tumor status. Most patients with CA125 levels greater than 35 U/mL demonstrate disease recurrence on second-look surgery, and those with CA125 levels less than 35 U/mL have minimal residual disease among half of the patients.[8] CA125 levels also act as an early predictor of outcomes where a deviation from the ideal CA125 regression curve suggests a poor outcome. Among patients who had undergone complete remission, elevation in CA125 occurred before the tumor recurrence in 75% of the patients.[2] However, using CA125 as a screening test for patients with ovarian cancer is not feasible. In subsequent studies evaluating the benefit of using multimodal screening combined with transvaginal ultrasound for ovarian cancer screening, no benefit in improving mortality was found.[36]

CA125 may also have a role in monitoring response to chemotherapy. Serial measurements of CA125 are found to correlate with clinical disease outcomes in 89% of 531 patients.[21] There is a consensus among current guidelines to use CA125 to monitor the response to chemotherapy. The Gynecologic Cancer Intergroup (GCIC) defines response as a 50% reduction in CA125 concentration compared to a pretreatment sample.[37][38] The response should be confirmed and sustained for at least 20 days. Patients can only be diagnosed if they have a CA125 concentration in a pretreatment sample that is twice the upper reference limit and taken 2 weeks before initiation of therapy. Additional samples are recommended at 2 to 4 weeks during treatment and at intervals of 2 to 3 weeks during follow-up. The same method should be used to monitor the patient throughout, and patients who receive immunotherapy (mouse antibodies) cannot be diagnosed.[39] The Food and Drug Administration approved the serial measurement of CA125 to aid in monitoring therapeutic response.[40]

Preoperative and postoperative CA125 concentrations may be of prognostic significance.[41] After primary surgery and chemotherapy, persistent elevations of CA125 concentrations are associated with poor prognosis. Patients with preoperative CA125 concentrations greater than 65 U/mL are reported to have a lower 5-year survival rate and a 6.37-fold risk of death compared to patients with CA125 levels less than 65 U/mL.[21] The half-life of the CA125 antigen is reported to have an additional prognostic value. A half-life of less than 20 days is associated with improved survival compared to a half-life of more than 20 days.[26] Normalization of CA125 levels after 3 cycles of combination therapy also correlates with improved survival. Importantly, CA125 concentration is not elevated in 10% to 20% of patients with advanced ovarian cancer. For these patients, using radiological imaging techniques and monitoring other tumor markers are necessary.[42]

Quality Control and Lab Safety

For non-waived tests, laboratory regulations require, at the minimum, analysis of at least 2 levels of control materials once every 24 hours. If necessary, laboratories can more frequently assay quality control (QC) samples to ensure accurate results. QC samples should be assayed after calibration or maintenance of an analyzer to verify the correct method performance.[43] To minimize QC when performing tests for which manufacturers’ recommendations are less than those required by the regulatory agency, such as once per month, the labs can develop an individualized quality control plan (IQCP). This plan involves performing a risk assessment of potential sources of error in all testing phases and putting in place a QC plan to reduce the likelihood of mistakes.[44] Westgard multi-rules are used to evaluate the QC runs. In case of a rule violation, proper corrective and preventive action should be taken before patient testing.[45]

The laboratory must participate in the external QC or proficiency testing (PT) program because it is a regulatory requirement published by the Centers for Medicare and Medicaid Services (CMS) in the Clinical Laboratory Improvement Amendments (CLIA) regulations. This practice is helpful to ensure the accuracy and reliability of the laboratory compared to other laboratories performing the same or comparable assays.[46] The PT plan should be included in the quality assessment (QA) plan and the laboratory's overall quality program.[47]

The intricate tests conducted in clinical laboratories are the cornerstone of patient diagnosis and treatment. However, this crucial work does not come without risks. These facilities handle various potentially hazardous materials, from infectious agents to toxic chemicals. Strict adherence to safety protocols is essential to prevent accidental exposure or contamination, which includes proper handling, storing, and disposing of these materials to safeguard laboratory personnel, minimizing patient transmission risks, and ensuring the integrity of diagnostic tests.[48]

Beyond material safety, meticulous equipment maintenance plays a critical role. Keeping laboratory instruments and safety devices, such as eyewash stations and fume hoods, in top condition is crucial for optimal functionality and mitigating potential hazards. Equally important are established and practiced emergency response plans. These plans enable staff to react swiftly and effectively in the event of accidents or incidents.[49]

Continuous education and training are vital for the laboratory staff to stay abreast of evolving safety procedures and best practices. In addition, this fosters a culture of safety consciousness and accountability within the institution. Prioritizing lab safety does not just protect the workforce; it also strengthens the overall quality and trustworthiness of healthcare delivery. This comprehensive approach to safety includes employing appropriate personal protective equipment (PPE) such as gloves, lab coats, and respirators, adhering to biosafety levels (BSL) for specific infectious agents, following a dedicated chemical hygiene plan for safe chemical handling, and ensuring the proper segregation and disposal of biohazardous and chemical waste.[50]

Enhancing Healthcare Team Outcomes

CA125 serves a crucial role as a tumor marker in diagnosing patients suspected of having ovarian cancer and monitoring disease progression. According to guidelines, it is an essential criterion for referring patients with an adnexal mass suspicious of malignancy to a gynecological oncologist. Although it has a higher specificity when used in postmenopausal women, very high levels in premenopausal women require referral. In addition, patient outcomes have been observed to improve when treatment is provided by specialized gynecological oncologists and conducted in hospitals with necessary consult services and multidisciplinary collaboration.[51] 

Interprofessional team care involves multiple disciplines, such as primary care physicians, gynecological oncologists, nurses, and pathologists. Each of them has an essential role in providing care for individuals with suspected ovarian cancer or monitoring the disease status, thus influencing disease management. Clinicopathologic meetings involving discussions between pathologists and clinicians are a form of interprofessional care. Interprofessional team care has been shown to improve the accuracy of the diagnosis, achieve accurate staging and grading of the disease, and thus improve the management of the patient's disease. In addition to improving patient care, it provides a learning opportunity for the team members.[52]

Nurses involved in the care of a patient with ovarian cancer are in a unique position to provide information and education to the patient and their family about the disease. In addition, they may be able to identify patients needing psychological support and counseling, thereby improving the quality of life for their patients by coordinating referrals with nutritionists, physiotherapists, and psychologists.[53] Evidence shows that patients treated under interprofessional team care involving clinicians and physicians from different specialties had a significant survival advantage.[54]