This study included 50 women diagnosed with benign breast mass (a mean age of 33.9 ± 1.9 years) and an equal number of women diagnosed with BC, mainly in the postmenopausal age group and not undergoing anticancer treatment (a mean age of 50.6 ± 1.5 years). This research involved selecting and examining patients at the cancer clinic of King Abdallah Medical City in Makkah, Saudi Arabia. The control group comprised 50 women volunteers (a mean age of 40.7 ± 0.93 years). All groups were matched in terms of age, weight, and menopausal status. Fasting blood samples were collected. The serum was separated by centrifuging the clotted samples at 3,500–4,000 rpm and stored at −20°C until analysis.

This study complied with the ethical standards specified in the 1975 Declaration of Helsinki, and clearance was obtained from the medical ethics committee of the College of Medicine at Umm Al-Qura University in Makkah, Kingdom of Saudi Arabia (Approval Number: HAPO-02-K012-2022-09-1183). Each participating patient and control provided written informed consent.

All participants underwent a clinical examination and completed a questionnaire that included aspects of their medical and family history. Individuals with positive neoadjuvant chemotherapy or a history of malignancy, radiotherapy, or/and chemotherapy were excluded. Blood samples were obtained before any surgical intervention.

The medical records of the participants involved in the study were examined to gather information from cytopathology reports, which contained details about the stage of the tumor, its features, and the status of ER and PR. Information regarding demographic features, including reproductive factors (such as age at menarche, age at menopause, age at first full-term pregnancy, number of full-term pregnancies, and prior use of external hormones such as hormone replacement therapy and oral contraceptives), medical history, and tobacco use, was collected via a thorough medical history and clinical examination. Data regarding cancer occurrence, including BC and other types, were collected from immediate (parents and siblings) and extended family members (grandparents, uncles, and aunts). BC was pathologically staged according to the TNM classification [12].

The serum levels of TAC were measured using a competitive inhibition enzyme immunoassay kit [MBS9304157, My BioSource, Sunny Southern California, San Diego (USA)] as per the given test protocol (https://www.mybiosource.com/).

The serum levels of Ox-LDL were quantified using a competitive inhibition enzyme immunoassay kit (SEA527Hu 96 Testes, Cloud-Clone Corp, Houston, USA) according to a predetermined assay protocol (http://www.Cloud.Clone.US).

The serum levels of CA15-3 were measured using an ELISA kit provided by My BioSource, Inc., located in San Diego, USA. The CA15-3 ELISA test is a modified version of the solid-phase sequential sandwich ELISA. Biotinylated monoclonal antibodies and samples are introduced into wells coated with streptavidin. The CA15-3 in the patient sample forms a complex with the capture antibody modified with biotin. Simultaneously, the biotinylated antibody binds to the streptavidin-coated plate. Following the wash stage, the anti-CA15-3-horseradish peroxidase (HRP) enzyme conjugate is introduced and binds to the trapped CA15-3, creating a sandwich structure. The unbound antibodies are rinsed away. Upon adding the TMB substrate, a blue color is produced. The level of CA15-3 is directly correlated with the intensity of the color. A standard curve is plotted to establish the relationship between the intensity of the color and the level of CA15-3.

The serum level of CEA was quantified using an ELISA kit manufactured by Cloud-clone Corp. and assembled by US Co Life Science Inc. USA. A sandwich enzyme immunoassay kit is presented here. A precoated antibody specifically targeting the CEA has been applied to the microtiter plate included in this kit. In the next step, standards or samples are added to the appropriate wells of a microtiter plate together with a biotin-conjugated antibody that selectively binds to the CEA. Furthermore, the avidin-HRP conjugate is added to each microplate well and incubated. Upon introducing the TMB substrate solution, only the wells containing CEA, biotin-conjugated antibody, and enzyme-conjugated avidin would exhibit a noticeable color change. The enzyme-substrate interaction is stopped by adding sulfuric acid, and the subsequent color change is measured quantitatively using a spectrophotometer at 450 ± 10 nm. The CEA levels in the sample are measured by comparing the optical density of the samples with the standard curve. The measured value varied between 19.6 ng/mL and 1,250 ng/mL.

The data were analyzed using the SPSS (version 20, Sydney, NSW, Australia) software. Quantitative data were reported as the mean values ± standard error (SE), whereas qualitative data were presented as frequencies and percentages. Statistical analysis was performed using an independent sample t-test for parametric variables to compare between two groups. One-way analysis of variance with post-hoc multiple comparisons test was used to evaluate the mean difference among the malignant, benign, and healthy control groups. Pearson’s correlation coefficient and linear regression analysis were performed to analyze the correlation between oxidative stress parameters (TAC and Ox-LDL) and tumor markers (CEA and CA15-3). The Kolmogorov-Smirnov test and normal plot were used to determine the normality of the data. To assess the diagnostic accuracy of the serum TAC, Ox-LDL, and CA15-3 levels, the receiver operating characteristic (ROC) curve analysis was performed on the dataset. The measurement accuracy was determined by calculating the area under the ROC curve, commonly referred to as AUC. A test with an area of 1 is considered ideal, whereas a test with an area of 0.5 is considered completely ineffective. The accuracy of a diagnostic test may be classified using a standard academic point system. An accuracy score of 0.9–1 is considered outstanding (A), 0.8–0.9 is acceptable (B), 0.7–0.8 is fair (C), 0.6–0.7 is bad (D), and 0.5–0.6 is failed (F). For each statistical analysis, P-values < 0.05 were deemed significant.

Pretreatment blood samples were acquired from patients with BC diagnosed with the disease. The diagnosis was verified using histological and clinical data and medical records. Table 1 summarizes the clinical features and demographic data of patients with BC and those with benign conditions. Patients with BC and benign conditions were matched in terms of age with the control group. In a sample of 50 patients with BC, 6 (12%) patients were classified as grade I, 31 (62%) as grade II, 11 (22%) as grade III, and 2 (4%) as grade IV (Table 1). Immunohistochemical data showed that 70% of the samples were estrogen-receptor-positive (ER⁺), 56% were progesterone-receptor-positive (PR⁺), and 28% were human epidermal growth factor receptor 2 positive (Her2⁺) (Table 1). Of the 50 patients with BC, 48 (96%) presented with a mass, whereas 2 (4%) did not have a mass. In addition, 2 (4%) experienced discharge, which included blood, whereas 48 (96%) did not have any discharge. Among a group of 50 patients with benign breast masses, 39 were identified as having fibroadenoma. In contrast, the remaining 11 patients were diagnosed with various other forms of benign breast conditions, such as granulomatous mastitis, papilloma, fibroglandular tissue, and ductal ectasia (Table 1). Histopathological examinations of 50 patients with BC revealed that 47 (94%) had invasive ductal carcinoma, whereas 3 (6%) had lobular carcinoma. Of the patients with BC, 37 (74%) had lymph node involvement, whereas 13 (26%) did not have lymph node involvement. Moreover, 21 (42%) had cancer metastasis, whereas 29 (58%) did not exhibit metastasis, as indicated in Table 1.

The serum level of TAC was highly and significantly decreased in patients with BC and benign lesions, with mean values of 8.3 U/mL and 16.04 U/mL, respectively, as shown in Figure 1, in comparison with normal controls (43.4 U/mL) (P < 0.001).

Moreover, the serum level of Ox-LDL was highly and significantly increased in patients with BC compared with the benign disease group, with mean values of 3,831 pg/mL and 1,234 pg/mL (P < 0.001), respectively (Figure 1). Furthermore, patients with BC displayed significantly higher means of Ox-LDL in comparison with normal healthy controls (682 pg/mL) (P < 0.001).

Also, the BC group demonstrated a significant increase in the mean values of the other two parameters, i.e., CEA and CA15-3, compared with the control group. The mean CEA levels were found to be 472.56, 328.42, and 314.55 ng/dL in the BC, benign, and control groups, respectively (P < 0.001). In contrast, the means of CA15-3 were 57.28, 15.16, and 14.35 U/mL in the BC, benign, and control groups, respectively, with a significant difference (P < 0.001), as shown in Figure 1.

The studied marker TAC showed a weak negative correlation with CA15-3 (r = −0.255, P < 0.01). A significant positive correlation between Ox-LDL and CA15-3 was observed (r = 0.441, P < 0.001), as shown in Figure 2.

Figure 3 displays the variations in TAC, Ox-LDL, CA15-3, and CEA levels among patients with BC who had specific histopathological characteristics.

The ROC curve was examined to assess the ability of the serum levels of TAC to discriminate between samples with and without BC. Figure 4 depicts the AUC. The data analysis of the ROC curve revealed a significant AUC with a value of 0.975 and a statistically significant P-value of < 0.001. The sensitivity and specificity were determined to be 100% and 86.4%, respectively, using a cutoff value of TAC equal to 18.9 U/mL (Table 2).

In addition, the ROC curve analysis was performed to evaluate the discriminatory ability of the Ox-LDL test in distinguishing between samples with and without BC. Figure 4 displays the AUC.

The data analysis of the ROC curve revealed a significant AUC with a value of 0.986, indicating statistical significance (P < 0.001). In Table 2, the sensitivity and specificity were determined to be 97.9% and 100%, respectively, using a cutoff value of 998 pg/mL for Ox-LDL.

This study observed that patients with BC exhibited lower TAC levels and higher Ox-LDL levels in the serum, indicating elevated oxidative stress compared with the control group. This finding suggests a potential association between increased oxidative stress and BC. Based on our observations, TAC and Ox-LDL levels could be additional parameters for monitoring cancer prevention, diagnosis, and treatment. However, the exact mechanism behind the elevated oxidative stress, whether it stems from increased ROS production or reduced antioxidant defenses, is yet to be elucidated. Therefore, further studies on oxidative stress and antioxidant therapy in BC are recommended.