Breast cancer (BC) is a significant global health concern. It is the most frequently diagnosed cancer worldwide and the leading cause of cancer-related mortality in women.¹ In 2022, BC accounted for 2.3 million new cases and approximately 670,000 deaths globally, with an age-standardized incidence rate (ASIR) of 26.88 per 100,000. Significant epidemiological disparities were observed across Socio-Demographic Index (SDI) regions, with high-SDI areas exhibiting the highest ASIR (66.89 per 100,000) compared with low-SDI regions (6.99 per 100,000).^2,3 Projections for 2023–2024 indicate a continued rise in global incidence, with pronounced inequities between the developed and developing regions. In countries with a very high Human Development Index (HDI), lifetime BC risk reached 1 in 12 women, whereas low-HDI regions reported lower incidence (1 in 27 women) but disproportionately higher mortality (1 in 48 deaths), reflecting systemic gaps in healthcare access, early detection, and treatment.⁴

Cancer Antigen 15-3 (CA15-3) was first identified in the early 1980s as part of a broader effort to identify markers that could aid in cancer diagnosis and monitoring. It is the product of the Mucin 1 gene, which encodes a mucin protein found in normal breast tissue. However, this protein structure differs in cancerous tissues, with enhanced levels of CA15-3 in the blood.⁵ CA15-3 is not employed for the initial diagnosis; however, it is frequently used to evaluate treatment response, recurrence, and disease progression. Araz et al. found that elevated levels of CA15-3 can be a sign of disease progression or recurrence in BC patients.⁶ Research has considered the combined use of CA15-3 with other tumor markers, including carcinoembryonic antigen (CEA), to increase diagnostic accuracy and prognostic value, suggesting that combined marker assays may show a higher predictive capability.⁷ CA15-3 levels can also be increased in other conditions such as lung, ovarian, and liver cancer. Furthermore, in benign conditions, including liver and breast disease, and even some autoimmune disorders, progress in liquid biopsy technologies has enabled more sensitive detection of tumor markers.⁸

Trefoil factors (TFF) are components of mucus barriers that can be found in the exocrine body fluids, including gastric juice, saliva, breast milk, urine, and tears.⁹ Trefoil factor 3 (TFF3) belongs to the trefoil factor family of small secreted proteins, which play important roles in mucosal protection, repair, and cell proliferation regulation. TFF3 shows the highest expression level in the gastrointestinal tract, especially in the epithelial cells of the intestine, where its activity is essential for the maintenance of the mucosal structure.¹⁰ TFF3 stabilizes the mucus layer, promotes epithelial cell migration, and aids in healing damaged tissues. It can stimulate the proliferation of intestinal epithelial cells, contributing to tissue regeneration after injury. TFF3 has been linked to gastrointestinal diseases such as inflammatory bowel disease, with altered expression observed in patients, suggesting its involvement in inflammatory response and tissue repair mechanisms.¹¹ Also, the protective role of TFF3 in mucosal healing may be relevant in ulcerative conditions, such as Peptic Ulcers. Its overexpression in gastric cancers correlates with tumor progression and poor prognosis.^12,13 Elevated TFF3 levels are associated with aggressive cancer and metastasis. TFF3 has been implicated in promoting tumor cell proliferation and survival in BC.¹⁴ TFF3 expression levels in tumor tissues and body fluids have been investigated as potential biomarkers for diagnosis and prognosis, offering insights into disease progression and therapeutic responses.¹⁵ The presence of TFF3 has been observed in invasive BC, with some cases exhibiting reduced expression and others showing elevated expression.¹⁶ Researchers have explored strategies to target TFF3 in cancer therapy, considering its role in tumor growth and metastasis. This involves monoclonal antibodies or small-molecule inhibitors that disrupt TFF3 signaling.¹⁷

Human Epididymal Protein 4 (HE4) is primarily expressed in the epithelial cells of the reproductive tract, particularly in the epididymis, as well as in various other tissues. It is encoded by the WAP Four-Disulfide Core Domain 2 (WFDC2) gene, located on chromosome 20. HE4 is a member of the whey acidic protein (WAP) family and is involved in various biological processes, including immune responses and epithelial differentiation.¹⁸ HE4 was first identified in the context of male fertility; however, it has gained prominence in cancer research, particularly in ovarian cancer. Its expression levels were found to be significantly elevated in patients with ovarian tumors. The discovery of HE4 as a potential biomarker for ovarian cancer was pivotal, leading to its inclusion in diagnostic protocols along with other markers, such as Cancer Antigen 125 (CA-125).¹⁹ HE4 has also been studied in the context of endometrial, lung, and BC. Elevated HE4 levels have been correlated with disease progression and poor prognosis in these malignancies.²⁰ Building on these findings, this study aimed to assess the effectiveness of CA15-3, TFF3, and HE4 in detecting and monitoring BC.

This was a case-control study. Samples were collected at the Al-Sadder Teaching Hospital in Al-Basrah Governorate, Oncology and Hematology Center, Tumor LAB Department, Basrah, Iraq, between December 2023 and May 2024. BC staging was contingent upon physician assessment using the Tumor-Node-Metastasis (TNM) classification system as per the American Joint Committee on Cancer (AJCC) guidelines. This framework stratifies disease progression through the systematic evaluation of three parameters: primary tumor dimensions (T), regional lymph node involvement (N), and distant metastatic spread (M). Staging was determined at the time of the initial diagnosis via multimodal diagnostic approaches, including clinical evaluation, imaging modalities (e.g., mammography and ultrasonography), and histopathological analysis of biopsy specimens. Overall, 72 women diagnosed with BC were subdivided into pre-treatment (n=15) and post-treatment (n=57, stages 1–3) groups. Also, 15 healthy women were included in the control group. The control group was sourced from healthy women selected from a general population similar in characteristics (age, gender, ethnicity, socioeconomic status, and lifestyle factors). CA15-3 levels were quantified as per the COBAS Integra 400 Plus protocol (Roche Diagnostics, Switzerland). The levels of the other two biomarkers in serum were evaluated using enzyme-linked immunosorbent assay (ELISA) and specific commercial kits (TFF3 and HE4) (Elabscience, USA), following the manufacturer’s instructions.

Inclusion criteria required women to have a confirmed BC diagnosis. The patients with chronic diseases other than BC, cardiovascular disease, infections, and endocrine disorders were excluded from the study.

The participants in this study (patients and controls) provided five millilitres of blood, which was then transferred to sterilized test tubes and allowed to coagulate at room temperature for 30 minutes. The blood samples were centrifuged at 3000 rpm for 15 minutes. Subsequently, the sera were separated and stored at −20 °C until further use.

The current case-control study recruited 72 women diagnosed with BC, who were categorized into 2 groups: the pre-therapy group (Group 2) with 15 participants and the post-chemotherapy group with 57 patients: 18 women at Stage 1 (Group 3), 32 women at Stage 2 (Group 4), and 7 women at Stage 3 (Group 5). Furthermore, a control group of 15 healthy women (Group 1) was recruited.

Table 1 shows the age range of the participants in different study groups, i.e., the control group (40.6±13.9), the pre-treatment group (49.27±6.2), and three chemotherapy groups (Stage 1: 49.28±7.48, Stage 2: 48.75±7.02, Stage 3: 51.14±5.01).

BC is the predominant type of cancer affecting women worldwide. It is a diverse collection of diseases that vary in their characteristics. Identifying reliable prognostic markers is crucial for determining the likelihood of recurrence and selecting appropriate treatment for patients with specific pathological and clinical characteristics.²¹

The present findings align with those of Li et al. and Hing et al., who reported that significant elevations in CA15-3 concentrations function as prognostic biomarkers for the early detection of metastatic progression in BC. Elevated CA15-3 levels have been positively correlated with adverse clinicopathological characteristics, including advanced tumor stage, higher histological grade, and metastatic involvement. The observed increase in CA15-3 concentrations in advanced-stage malignancies may reflect the underlying pathophysiological mechanisms associated with increased tumor burden and metastatic spread. This likely reflects increased shedding of tumor-associated antigens into the systemic circulation during neoplastic progression and metastatic evolution.^22,23 A study by Fu et al. showed that increased levels of CA15-3 in the blood at the time of diagnosis were linked to more advanced stages of BC, larger tumor size, and the presence of cancer cells in the axillary lymph nodes of the armpit.²⁴ Serum indicators were evaluated sequentially in multiple studies to determine their use in the early identification of the disease and tracking treatment response.²⁵ CA15-3 can be particularly useful for monitoring the response to treatment and detecting recurrence, as elevated levels often correlate with disease progression. Our study findings are consistent with the results reported in Taghizadeh et al.,²⁶ which found elevated CA15-3 levels exhibited a robust connection with the advanced stages of cancer. Lian et al. reported similar results, emphasizing the significance of tumor markers.²⁷ According to previous studies, serum CA15-3 can be used as an indicator of advanced disease and metastasis.^28–30 Both oncologists and surgeons are recommended to examine CA15-3 levels, as this might assist in determining the necessity of an intensive treatment plan.³¹

The diagnostic utility of CA15-3 as a BC biomarker has been contested owing to limitations in specificity and sensitivity, as evidenced by conflicting study findings such as those reported by Coppola et al. Discrepancies arise from nonspecific elevations in non-malignant conditions (e.g., benign breast lesions) and non-breast malignancies, increasing false-positive rates and reducing clinical validity. These cross-reactive elevations undermine its reliability as an independent diagnostic tool, necessitating complementary biomarkers or multimodal approaches to improve diagnostic accuracy.³² There is also some debate regarding CA15-3 consistency as a stage-specific marker. A study by Kabir et al. noted that while CA15-3 levels generally increase with cancer stage, this correlation is not uniform across all patients. Factors such as individual tumor biology, treatment history, and other patient-specific variables can influence CA15-3 levels, leading to variability in its utility for staging. Elevated CA15-3 levels can occur in benign conditions, and not all patients with BC show elevated CA15-3 levels. This complicates its use as a diagnostic tool.³³

This study demonstrates that TFF3 exhibits a diagnostic performance for BC comparable to the established biomarker CA15-3, supported by statistically significant findings, consistent with prior observations by Abdelrazek et al. Elevated serum TFF3 levels positively correlated with advanced clinical stage and poor prognostic indicators, suggesting the utility of TFF3 as a biomarker for disease progression. Mechanistically, TFF3 dysregulation may promote oncogenic signaling pathways, enhancing tumor proliferation and metastatic potential, which aligns with its association with aggressive disease phenotypes. These findings underscore TFF3’s dual diagnostic and prognostic relevance, highlighting its potential clinical value in BC management.³⁴ Another study reported statistically significant TFF3 expression differences (P < 0.05) between metastatic and non-metastatic breast cancer cases, demonstrating its potential as a discriminative biomarker with clinically relevant sensitivity and specificity.³⁵ Furthermore, Wu et al. identified TFF3 as a highly responsive biomarker for predicting the effectiveness of endocrine therapy in BC cells.³⁶ Also, Wang et al. found that TFF3 might facilitate tumor progression through its effects on cell adhesion and migration.³⁷ Elevated TFF3 levels can alter cellular interactions and contribute to the metastasis of cancer cells. Higher TFF3 levels in the pre-treatment and staged groups could mean that the disease is more advanced or aggressive. This dual role in both physiological maintenance and pathological progression underscores TFF3’s clinical relevance in BC diagnostics and prognostication.³⁷ TFF3’s role in cellular protection and repair may contribute to its elevated expression in cancerous tissues. Its overexpression could reflect the tumor's attempt to repair damaged tissues or to support cancer cell survival. Elevated TFF3 expression correlates with advanced breast cancer stage (p<0.01), suggesting a potential role in tumor progression. This observation is supported by multiple cohort studies demonstrating significant associations between high TFF3 levels and poor prognostic outcomes. Studies have suggested that TFF3 acts as a separate risk factor, contributing to the occurrence of lymph vascular invasion and spread of cancer cells to the lymph nodes.³⁸

Some researchers have reported results that are inconsistent with our findings; for example, a study by Shen et al. found no significant difference in TFF3 levels in BC patients and healthy controls, suggesting that TFF3 may not always be a reliable biomarker for BC. Some researchers attribute this variability to variations in study designs, patient populations, or methodological approaches.³⁹ Yan et al. founds that factors unrelated to BC, such as inflammation or other benign conditions, could influence TFF3 levels. This implies that elevated TFF3 levels might not be specific to BC, which challenges the use of TFF3 as a standalone biomarker.⁴⁰ Technical issues in measuring TFF3 levels, such as assay sensitivity and specificity, may also lead to inconsistent findings. According to Zhang et al., discrepancies in TFF3 measurements across different laboratories and techniques can affect the interpretation of its role in cancer.⁴¹

The study found no significant differences in serum HE4 levels across BC stages or chemotherapy statuses. These findings are consistent with the multivariate analysis conducted by Baba et al., who reported no positive association between HE4 levels and histological grade or clinical stage in BC patients.⁴² This finding supports the assumption that HE4 lacks utility as a stage-specific biomarker in BC. Corroborating this observation, Zhu et al. found no substantial variation in HE4 levels between healthy controls and BC patients, including those at different disease stages.⁴³ Collectively, these data suggest that HE4 exhibits limited sensitivity for early-stage detection and inadequate discriminatory capacity to stratify BC progression, underscoring its limited diagnostic applicability in staging or early diagnosis. The relatively stable levels of HE4 across different stages and pre-treatments may reflect its low sensitivity and specificity for BC. In other words, it may not be useful in detecting subtle changes that occur with disease progression, or it may not be as effective in distinguishing between different stages. Our findings contrast with prior reports of significant associations between circulating HE4 levels and prognostic factors in breast cancer (e.g., tumor size, nodal status). While these discrepancies may reflect methodological differences, the observed HE4 patterns could suggest context-dependent roles in tumor biology, potentially including pro-tumorigenic functions during disease progression.⁴⁴

The results revealed that serum levels of CA15-3 and TFF3 were markedly elevated in BC pre- and post-chemotherapy compared to healthy control subjects, highlighting their promising application as diagnostic biomarkers. Most importantly, CA15-3 levels were associated with disease progression, consistent with its well-established signaling for tumor mass and metastasis. In line with this, TFF3 upregulation correlated with advanced clinical stages, showing its role in tumor aggressiveness or in shaping metastatic pathways. Conversely, the limited diagnostic significance in BC stage or monitoring was confirmed by the insignificant changes in HE4 between the study groups.

Ethical approval was obtained from the ethical and research committee of the Department of Medical Laboratory Technology, College of Health and Medical Technology, Southern Technical University, Basrah, Iraq (No. 803 on 19/11/2023).