Breast cancer (BC) is the most common type of cancer among women worldwide. The International Agency for Research on Cancer reported that in 2022, 2.31 million women were diagnosed with BC. According to World Health Organization (WHO) data, more than 1 billion adults were living with obesity in 2022. Obesity is a main risk factor for different types of cancer. Studies suggest that for every 5 - kg / m² increase in body mass index (BMI), the probability of developing BC increases by 2% . The association between metabolic syndrome or obesity and postmenopausal BC has been confirmed in several studies. However, views on the link between premenopausal BC and obesity are controversial. Several studies have suggested that overweight and obesity confer a preventive benefit against BC in premenopausal women, except for those with a familial history of the disease. 1,2 A few studies have found that obesity increases the risk of BC in premenopausal women. 3,4 A meta- analysis of cohort studies indicated that for Asian women, a 5 - kg / m² increase in BMI was associated with a 16% increased risk of premenopausal breast cancer. In contrast, the same BMI increase was associated with a 9% lower risk in North American women. 3 The development of BC in individuals with obesity is often explained by a high level of active estrogen in the blood. Adipocytes exhibit endocrine activity by synthesizing various substances, including estrogen. These substances can promote cancer cell growth by stimulating cell proliferation. 5

Obesity is often accompanied by hyperinsulinemia and insulin resistance (IR), which have a multifaceted effect on cancer development. 2,3 Several studies have also confirmed that IR and hyperinsulinemia are risk factors for BC. 6,7 Insulin may have a crucial mitogenic role in BC formation. In vitro studies suggest that insulin stimulates breast and colon cancer proliferation and migration. 7,8 Research has shown that insulin could promote cell proliferation in normal and cancerous breast tissue. For instance, the MDA- MB- 157 BC cell line was stimulated by exogenous insulin, and strong mitogenic responses were observed. 2 On the other hand, in women with obesity, hypercholesterolemia affects increased estrogen synthesis in the ovaries. 3 In addition to stimulating androgen production, insulin ensures the long- term biological viability of androgens and estrogens by decreasing the production of sex hormone- binding globulin. 9 However, the link between insulin and cancer development has been refuted in some clinical studies of both pre- and postmenopausal patients. 2 IR leads to an increase in blood glucose levels, which may result in an increased nutrient supply to tumor cells. 10 This is significant because studies have shown that tumor cells can use glucose as nourishment and for energy. A hyperglycemic environment can simultaneously foster both immune evasion and the development of drug resistance in cancer cells. 11 The formation of IR is also regulated by the production of proinflammatory cytokines—adipokine and interleukin- 6 (IL- 6), secreted by adipocytes. 12 IL- 6 affects glucose metabolism and insulin signaling pathways in β - cells and leads to a chronic inflammatory process, which plays a key role in cancer development. 9,12,13 Preclinical studies concluded that IL- 6 stimulates carcinogenesis, angiogenesis, and metastasis 14 , while clinical studies confirmed the correlation of serum IL- 6 levels with tumor invasion, metastasis to lymph nodes and distant organs, and advanced TNM stage. 15,16 Chronic inflammation and IR, in turn, also affect lipid metabolism regulation, leading to dyslipidemia. There is evidence that cholesterol and its metabolites accelerate the proliferation of estrogen receptor (ER)- positive BC cells. A correlation was also found between an increase in 27- hydroxycholesterol (27HC) and disease recurrence in luminal A cancer. It has also been reported that the concentration of 27HC is higher in BC tissue than in healthy breast cells. This indicates the necessary role of serum and tumor tissue cholesterol in tumor development. 17 All the above- mentioned metabolic disorders interact with each other. Each of these changes can lead to cancer development both independently and together. This is clearly illustrated in BC, where studies indicate that obesity mainly affects the development of the postmenopausal subtype. 1,18 Although often associated with postmenopausal breast cancer, obesity is also linked to premenopausal BC, which has a high mortality incidence in women aged 20 to 50 years. 18,19 The metabolic disorders in obesity drive this poor outcome by promoting tumor progression, metastasis, drug resistance, and an aggressive phenotype through effects on the tumor microenvironment. This hypothesis of a correlation between obesity and premenopausal BC led us to assess alterations in serum metabolic biomarkers and evaluate their association with disease progression across molecular subtypes, comparing patients with and without obesity. BC is divided into molecular subtypes depending on the presence or absence of ER, PR, and HER2 and the expression of the Ki- 67 protein. Table 1 presents the molecular subtypes of BC.20

and postmenopausal BC has been confirmed in several studies. However, views on the link between premenopausal BC and obesity are controversial. Several studies have suggested that overweight and obesity confer a preventive benefit against BC in premenopausal women, except for those with a familial history of the disease. 1,2 A few studies have found that obesity increases the risk of BC in premenopausal women. 3,4 A meta- analysis of cohort studies indicated that for Asian women, a 5 - kg / m² increase in BMI was associated with a 16% increased risk of premenopausal breast cancer. In contrast, the same BMI increase was associated with a 9% lower risk in North American women. 3 The development of BC in individuals with obesity is often explained by a high level of active estrogen in the blood. Adipocytes exhibit endocrine activity by synthesizing various substances, including estrogen. These substances can promote cancer cell growth by stimulating cell proliferation. 5

Obesity is often accompanied by hyperinsulinemia and insulin resistance (IR), which have a multifaceted effect on cancer development. 2,3 Several studies have also confirmed that IR and hyperinsulinemia are risk factors for BC. 6,7 Insulin may have a crucial mitogenic role in BC formation. In vitro studies suggest that insulin stimulates breast and colon cancer proliferation and migration. 7,8 Research has shown that insulin could promote cell proliferation in normal and cancerous breast tissue. For instance, the MDA- MB- 157 BC cell line was stimulated by exogenous insulin, and strong mitogenic responses were observed. 2 On the other hand, in women with obesity, hypercholesterolemia affects increased estrogen synthesis in the ovaries. 3

In addition to stimulating androgen production, insulin ensures the long- term biological viability of androgens and estrogens by decreasing the production of sex hormone- binding globulin. 9 However, the link between insulin and cancer development has been refuted in some clinical studies of both pre- and postmenopausal patients. 2 IR leads to an increase in blood glucose levels, which may result in an increased nutrient supply to tumor cells. 10 This is significant because studies have shown that tumor cells can use glucose as nourishment and for energy. A hyperglycemic environment can simultaneously foster both immune evasion and the development of drug resistance in cancer cells. 11 The formation of IR is also regulated by the production of proinflammatory cytokines—adipokine and interleukin- 6 (IL- 6), secreted by adipocytes. 12 IL- 6 affects glucose metabolism and insulin signaling pathways in β - cells and leads to a chronic inflammatory process, which plays a key role in cancer development. 9,12,13 Preclinical studies concluded that IL- 6 stimulates carcinogenesis, angiogenesis, and metastasis 14 , while clinical studies confirmed the correlation of serum IL- 6 levels with tumor invasion, metastasis to lymph nodes and distant organs, and advanced TNM stage. 15,16 Chronic inflammation and IR, in turn, also affect lipid metabolism regulation, leading to dyslipidemia. There is evidence that cholesterol and its metabolites accelerate the proliferation of estrogen receptor (ER)- positive BC cells. A correlation was also found between an increase in 27- hydroxycholesterol (27HC) and disease recurrence in luminal A cancer. It has also been reported that the concentration of 27HC is higher in BC tissue than in healthy breast cells. This indicates the necessary role of serum and tumor tissue cholesterol in tumor development. 17 All the above- mentioned metabolic disorders interact with each other. Each of these changes can lead to cancer development both independently and together. This is clearly illustrated in BC, where studies indicate that obesity mainly affects the development of the postmenopausal subtype. 1,18 Although often associated with postmenopausal breast cancer, obesity is also linked to premenopausal BC, which has a high mortality incidence in women aged 20 to 50 years. 18,19 The metabolic disorders in obesity drive this poor outcome by promoting tumor progression, metastasis, drug resistance, and an aggressive phenotype through effects on the tumor microenvironment. This hypothesis of a correlation between obesity and premenopausal BC led us to assess alterations in serum metabolic biomarkers and evaluate their association with disease progression across molecular subtypes, comparing patients with and without obesity. BC is divided into molecular subtypes depending on the presence or absence of ER, PR, and HER2 and the expression of the Ki- 67 protein. Table 1 presents the molecular subtypes of BC.20

Several studies have reported that obesity (high BMI) is not a risk factor for premenopausal breast cancer and may instead be associated with a reduced risk.9,18,21,28 Korean researchers conducted a largescale study to assess the association between obesity and BC occurrence in approximately 6.5 million women18 and observed that high BMI affected the development of postmenopausal BC and decreased the occurrence of premenopausal BC. A key limitation is that this study only included women aged 40 years and older. Because the median age of menopause is 51 years,29 this age threshold fails to capture the entire premenopausal population, which is significant given the high rate of breast cancer mortality among women aged 20- 49 years.30

Recent studies confirm the positive association between obesity and BC in premenopausal women,31- 3331- 33 which is consistent with our findings. Our findings are also consistent with research reporting a significant association between obesity and the histological phenotype of tumors.34,35

Large tumor sizes and infiltrative, rare tumor forms were more common in patients with obesity. It is important to note that our results refute the evidence that ER- positive status is less common in premenopausal BC with obesity.28,36 No significant difference was found between the ER- positive status in patients with and without obesity.

Also, PR- negative status was more common in individuals with a high BMI (P = 0.01) . The proportion of patients with high Ki- 67 expression was significantly greater in patients with obesity than in those without obesity (P = 0.001) . This higher prevalence of invasive forms in patients with obesity may be related to chronic inflammatory processes. The observed higher proliferative activity could be mediated by the influence of factors such as insulin in the obese state. Our results are consistent with findings from other studies,22,24 including in vitro studies confirming that exogenous insulin can increase cancer cell division rates.2

Progesterone receptor- negative (PR- negative) status was significantly more common (P = 0.01)

2016, the incidence of ER- positive BC has been increasing, while ER- negative BC has been decreasing among young women in the premenopausal period.30

This aligns with research by Harris et al., which found that obesity decreases ER-positive and increases ERnegative breast cancer in premenopausal women.2828 However, US researchers have observed that since

Proliferation of tumor cells was more often observed in premenopausal BC with obesity (P = 0.001) . All this confirms the link between obesity and the formation of the aggressive tumor phenotype and tumor histopathology. However, the data on the prevalence of aggressive subtypes in premenopausal patients were not confirmed.31,3231,32 In our study, although aggressive subtypes (TNBC, HER2- enriched) were numerically predominant in patients with obesity, the intergroup difference was not statistically significant (P = 0.44) , which may be related to a small sample size. However, some recent studies have reported that BMI in premenopausal patients with BC is not always associated with the prevalence of TNBC and HER2- enriched subtypes.32

text[[79, 898, 474, 926], [522, 673, 918, 926]] Research on the positive link between dyslipidemia and the development of BC has found contradictory results. A recent meta- analysis revealed a positive correlation between low serum HDL and the development of BC.37 Large case- control studies have confirmed an association between dyslipidemia (low HDL and high LDL) and the risk of TNBC and luminal B breast cancers.38 This lipid profile may be particularly significant due to the fundamental biochemical differences between these cholesterol carriers. Unlike HDL, which has a balanced 50:50 mass ratio of lipid to protein, LDL has a lipid- rich 75:25 ratio.39 This structure allows LDL to carry at least twice the amount of free cholesterol and cholesterol esters, potentially contributing to the tumor- promoting microenvironment observed in these aggressive cancer subtypes.40 These results deserve deeper analysis of lipid composition and its effects in clinical studies. Our study confirmed the

influence of dyslipidemia on the formation of aggressive molecular subtypes (TNBC, HER2- enriched, and luminal B). However, we found that the high levels of lipid markers in these subtypes are dependent on the presence of obesity (Figure 2). This aligns with large- scale studies concluding that dyslipidemia is linked to poorer disease outcomes. Specifically, elevated TG levels have been identified as a marker of poor prognosis in early- stage breast cancer.41 A large retrospective cohort study conducted by researchers in China showed that dyslipidemia was less frequent in patients with cancer than in healthy individuals.42 Significantly, they assessed changes in lipid profile indicators under the influence of chemotherapy. The results showed that chemotherapy leads to an increase in TC and a decrease in HDL. One study reports that changes in the lipid profile under the influence of adjuvant chemotherapy were accompanied by an increase in BMI in premenopausal patients with BC. In contrast, in postmenopausal patients with BC, such changes were not recorded.43 These results suggest that chemotherapy may have influenced changes in the lipid profile in our study as well. However, our patients received neoadjuvant chemotherapy, and the blood sample was collected before receiving the treatment.

text[[80, 490, 475, 921], [523, 84, 918, 233]] Consistent with the work of Japanese researchers, our study observed no significant differences in insulin levels or obesity among molecular subtypes. This supports their conclusion that, regardless of menopausal status, insulin has no significant etiological role and is not a predictive biomarker for breast cancer. Furthermore, their findings confirm the absence of a significant association between insulin and the status of key hormone receptors (ER, PR, HER2).22 A meta- analysis conducted in China also showed that serum levels of insulin have no effect on the development of BC in both premenopausal and postmenopausal women.19 The role of hyperinsulinemia in the development of postmenopausal BC was also assessed in a case- control study of women in Mexico. The study evaluated the association between serum insulin level and BC depending on the C- peptide level. C- peptide is an element of insulin metabolism and a marker of hyperinsulinemia. Although the registration of new cases of diabetes mellitus in patients was monitored, information about exogenous insulin use was not provided.44 Another study involving 20 years of observation suggested the probability of developing BC in women with high IR in postmenopausal periods.45 Another study reported that fasting insulin is a marker predicting a poor disease outcome for early BC.46 This prospective cohort study in patients without diabetes showed that high levels of insulin are associated with metastasis to distant organs and increased mortality. It should also be noted that in several in vitro experiments, BC cell lines increased their ability to divide under the influence of exogenous insulin.47 According to the mitogenic effect of insulin, its serum levels can predict the outcome of the disease. However, research on insulin's influence on cancer development has yielded conflicting results, highlighting a critical need for further investigation.

Preclinical and epidemiological studies have found an association between glucose and cancer development. Glucose affects cancer development and progression independently of obesity.448 There is evidence that the development of several cancers, including BC, is also linked to sugar consumption.49,50 Some studies found that diabetes mellitus and high sugar levels contribute to BC development depending on BMI without the influence of insulin.51 On the other hand, recent analyses show that high levels of glucose can lead to cancer development depending on the Warburg effect, chronic inflammation, and IR.111 Among women aged 40 years or older, a moderate correlation between BC and sugar consumption has been suggested, which is associated with an insulin- related mechanism.50 Recent studies have also confirmed the positive influence of increased sugar consumption on the risk of developing BC in women in the premenopausal period.49 This study substantiates that increased fructose consumption is associated with a higher frequency of the TNBC subtype.52 Furthermore, our findings on glucose revealed a significant difference between patients with and without obesity across all groups except luminal A. This indicates that obesity- associated hyperglycemia may drive breast cancer development and influence the formation of its more aggressive molecular subtypes.

Serum IL- 6 has been confirmed in many studies as a predictor for the survival53,54 of patients with BC.53,54,55 In particular, a high level of IL- 6 predicts a poor prognosis for the overall survival of patients with metastatic BC.56 While several studies investigate the correlation between serum IL- 6 and tumor histology, some suggest it serves as a favorable prognostic marker in both early and advanced breast cancer.57 Milovanovic et al. claimed that in premenopausal patients with positive hormone receptors, IL- 6 is associated with a good prognosis, irrespective of HER2 status.14,58

While literature links elevated serum IL- 6 to advanced tumor stage, lymph node metastasis, and distant spread, our data reveal no significant difference in IL- 6 levels across molecular subtypes. Instead, high IL- 6 was significantly associated with obesity specifically in luminal B and HER2- enriched

patients. This suggests that in premenopausal women, the role of IL- 6 in these aggressive subtypes is potentially mediated by obesity.

Conversely, CA15- 3 is a highly sensitive biomarker for monitoring chemotherapy response and predicting survival in metastatic breast cancer,59,60 although it is ineffective for detecting early- stage disease. Recent scientific studies have also noted a prognostic value for early- stage BC,25 showing a correlation between disease recurrence and the level of CA15- 3 in all molecular subtypes. In follow- up monitoring of patients with early BC with CA15- 3, its serum levels have been found to significantly increase in 37% of patients with metastasis.61 While previous research has established that high serum CA15- 3 levels correlate with larger tumor size, lymph node metastasis, advanced disease stage, HER2 overexpression, and hormone receptor- negative status,62 our results did not show a significant difference in CA15- 3 levels across molecular subtypes. Instead, we identified a significant negative correlation between CA15- 3 levels and obesity, suggesting that CA15- 3 may not be an independent prognostic marker in patients with a high BMI. This finding aligns with the recognized low specificity of CA15- 3 and is supported by the work of Santillan- Benitez et al., who also found no direct link between obesity and CA15- 3 in their cohort of 88 patients (40 with breast cancer) investigating combined biomarkers.6363 Another study found that CA15- 3 levels were associated with tumor characteristics and serum resistin levels, although they did not directly assess the CA15- 3 level in relation to obesity or BMI. This suggests that metabolic alterations and adipokine profiles, rather than BMI itself, may influence CA15- 3 expression.6464 Also, although there are no specific studies related to CA15- 3, scientific studies have shown an inverse association between prostate- specific antigen (PSA) and BMI. Research has linked this association with hemodilution in individuals with obesity.6565 The observed inverse correlation between obesity and CA15- 3 may be unique to our cohort of premenopausal women with early- stage disease and distinct metabolic profiles, a hypothesis requiring validation in future studies.

The obtained results show that metabolic monitoring, especially glucose and lipid metabolism control, can be useful for patients with obesity and premenopausal BC in terms of reducing the risks specific to molecular types of tumors.

text[[80, 849, 475, 922], [523, 83, 917, 112]] A key limitation is the small luminal A subgroup, potentially due to random selection or the higher prevalence of subtypes associated with obesity in our cohort.66- 68 Furthermore, the absence of a healthy control group prevents comparison of biomarker levels to a baseline population, limiting a full assessment of their clinical significance.

The results of this study confirmed the presence of an association between obesity and BC in premenopausal women. There were also positive associations between luminal A, HER2- enriched, and luminal B subtypes and total cholesterol. In premenopausal patients with BC and obesity, the formation of the luminal B and HER2- enriched subtypes was influenced by chronic inflammation, high glucose levels, and IR. High glucose levels played a significant role in the development of TNBC. Our findings indicate that BMI is a significant confounding variable in the interpretation of the CA15- 3 tumor marker. To ensure accurate clinical assessment, CA15- 3 values in patients with a high BMI should be interpreted with caution due to the potential for reduced diagnostic sensitivity.