Breast cancer (BC) is the most commonly diagnosed malignancy in women worldwide, accounting for substantial morbidity and mortality rates in both developed and developing countries. Despite significant advances in early detection and treatment strategies, BC remains a heterogeneous disease with diverse molecular subtypes and variable clinical outcomes. The interplay between genetic predisposition, epigenetic regulation, and the tumor microenvironment is central to its pathogenesis. Increasing evidence supports the role of immune regulation and chronic inflammation in the development and progression of BC. Cytokines, which are small regulatory proteins secreted by stromal and immune cells, have been shown to shape tumor behavior through mechanisms involving immune evasion, angiogenesis, cell proliferation, and metastasis. However, the precise contribution of individual cytokines to breast carcinogenesis remains unclear.

development and progression of BC. Cytokines, which are small regulatory proteins secreted by stromal and immune cells, have been shown to shape tumor behavior through mechanisms involving immune evasion, angiogenesis, cell proliferation, and metastasis. However, the precise contribution of individual cytokines to breast carcinogenesis remains unclear.

Among Iraqi women, BC is the most common killer. In 2019, BC accounted for approximately onethird of all cancer diagnoses in Iraq, making it the leading cause of cancer- related death among Iraqi women. Among the top 10 malignancies in 2019, BC accounted for \(22.58\%\) of all cancer cases, with an age- standardized incidence rate of 35.95 per 100 000 women.

Clinicopathological features have a significant impact on patient prognosis and therapy choices. These features include tumor size, histological grade, lymph node involvement, hormone receptor (HR) status, and molecular subtype. Furthermore, hematological parameters—including hemoglobin concentration, white blood cell count, platelet levels, and inflammatory markers—have been increasingly recognized as possible indicators of tumor burden, systemic response, and disease course. To determine the therapy type and prognosis, we characterized and classified the HR status of BC, namely, progesterone receptor (PR), estrogen receptor (ER), and human epidermal growth factor receptor 2 (HER2).

Interleukin- 40 (IL- 40), a recently identified cytokine encoded by the C17orf99 gene on chromosome 17q25.3, has drawn attention for its potential immunomodulatory functions. First described by Catalan- Dibene et al., IL- 40, which is primarily expressed in B cells, regulates IgA production, mucosal immunity, and gut microbiota homeostasis. It has also been implicated in various autoimmune and inflammatory diseases. Despite emerging evidence on the immunoregulatory roles of IL- 40 in inflammatory and autoimmune disorders, its contribution to BC pathogenesis and biomarker potential remains underexplored, particularly in populations with unique epidemiological and environmental risk factors, such as Iraqi women. This study aimed to evaluate the serum levels and gene expression profiles of IL- 40 in Iraqi females diagnosed with BC compared with frequency- matched healthy controls (HCs) stratified by age. By integrating molecular and clinical data, this research seeks to elucidate the role of IL- 40 in BC biology and assess its utility as a diagnostic or prognostic biomarker in a high- risk, understudied population.

Table 1 displays the results of the analysis of age and BMI. The 2 groups did not vary significantly in terms of mean age, although the BC group was older than the HC group (50.80 [10.78] vs 48.50 [6.69] years, respectively). Alternatively, the BC group exhibited a significantly higher BMI compared with the HCs (31.60 [7.65] vs 26.63 [5.33] kg/m2, respectively). The \(P\) value was highly significant \((P = 0.0007)\) , suggesting a potential association between higher BMI and the onset of BC.

A range of clinicopathological parameters were assessed to evaluate disease severity, inform prognosis, and guide personalized treatment strategies for patients with BC, thereby enhancing both survival rates and quality of care. As shown in Figure 1A, the majority of patients \((n = 84;84.09\%)\) were diagnosed with grade II tumors, which are considered intermediate grade and are typically associated with a more favorable prognosis. Tumor size, a critical determinant of cancer staging, is shown in Figure 1B. Most patients \((n = 54;53.49\%)\) presented with T2 tumors, reflecting moderate tumor growth. Lymph node involvement, a key indicator of cancer dissemination and a determinant of therapeutic approaches, is shown in Figure 1C. Regional lymph node metastasis (N1) involving 1 to 3 axillary lymph nodes was found in \(45.45\%\) of patients.

Metastatic status (M stage) is essential for determining whether the malignancy has extended beyond the breast and regional lymph nodes. As shown in Figure 1D, \(81.82\%\) of the patients were free of distant metastases (M0), suggesting the potential for curative treatment. The histological subtypes, which can significantly influence treatment decisions and prognosis, are shown in Figure 1E. Invasive ductal carcinoma (IDC), the most common form of BC, was diagnosed in \(72.73\%\) of the patients. Finally, tumor laterality is illustrated in Figure 1F. The left breast was affected slightly more frequently \((n = 42\) \(43.18\%)\) than the right breast \((n = 39;38.64\%)\)

The molecular classification of BC subtypes was determined based on the expression of key biomarkers, including HER2, PR, and ER (Table 2). Among the identified subtypes, Luminal A (ER \(^+\) /PR \(^+\) HER2 \(^+\) ) was the most prevalent, accounting for \(60\%\) of the cases. This subtype is generally associated with a favorable prognosis and strong response to hormone therapy. The Luminal B subtype (ER \(^+\) /PR \(^+\) HER2 \(^+\) ), which is characterized by a higher proliferative rate and potentially poorer outcomes than Luminal A, was observed in \(12\%\) of the cases. HER2- enriched BC (ER \(^-\) /PR \(^-\) ,HER2 \(^+\) ) constituted \(15\%\) of the cohort. This subtype is typically more aggressive but can benefit from targeted HER2- directed therapies. Lastly, the triple- negative BC (TNBC) subtype (ER \(^-\) /PR \(^-\) ,HER2 \(^+\) ), which lacks hormonal and HER2 expression and is often associated with poor prognosis and limited targeted treatment options, was identified in \(13\%\) of the cases.

The serum levels of IL- 40 were quantitatively assessed in female patients with BC and compared with those in HCs (Table 3). The analysis revealed that the mean serum IL- 40 concentration in the BC group \(\mathrm{n} = 100\) was significantly elevated, with a mean (SD) of 20.26 (6.90) ng/mL, compared with a mean (SD) of 14.08 (6.12) ng/mL in the HC group.

ROC curve analysis was conducted to evaluate the diagnostic potential of serum IL- 40 levels in distinguishing patients with BC from HCs, as illustrated in Figure 2. The analysis identified an optimal diagnostic threshold for serum IL- 40 of 16.77 ng/mL (Table 4). At this cutoff value, IL- 40 demonstrated a sensitivity of \(62.79\%\) and a specificity of \(61.36\%\) for distinguishing patients with BC from HCs. The Youden index, a measure of overall diagnostic effectiveness combining sensitivity and specificity, was 0.2415 at this optimal threshold. The analysis yielded an AUC of 0.75, suggesting moderate diagnostic accuracy for IL- 40. The \(95\%\) CI was 0.65 to 0.85.

Table 5 presents the comparative fold- change analysis of C17orf99 gene expression between female patients with BC \(\mathrm{n} = 100\) and HCs \(\mathrm{n} = 100\) .The findings revealed a significant upregulation of IL40 in patients with BC, with a mean fold change of 3.71 compared with 1.03 in the control group, a variation that was highly statistically significant \((P = 0.0002)\)

The analysis revealed no statistically significant difference in age between patients with BC and HCs, aligning with global trends where age does not consistently emerge as a differentiating factor in such comparisons. Previous studies have reported similar findings, underscoring that age alone may not distinctly separate patients with BC from healthy individuals in certain populations. However, contrasting observations from other regions suggest that ethnic and geographical factors may influence the relationship between age and disease onset. For instance, research in Western populations has documented a higher mean age among patients with BC than among controls, highlighting the potential variability linked to demographic or environmental contexts. These discrepancies emphasize the need to consider population- specific characteristics when evaluating age- related risk patterns. The current findings, consistent with several international cohorts, reinforce the notion that age may play a more nuanced role in BC epidemiology, particularly in understudied populations such as Iraqi women.

Regarding BMI, a significantly higher BMI was observed in patients with BC than in HCs, suggesting a potential role of obesity in BC development. Atom and Alparrey similarly reported a higher BMI in patients with BC, although their control group had an anomalously high BMI, which likely reflects data entry or reporting error. Despite this discrepancy, both studies underscored obesity as a significant modifiable risk factor. Although the mechanistic link between BMI and BC remains unclear, the association between elevated BMI and BC may reflect increased aromatase activity in adipose tissue, particularly in postmenopausal women. However, the menopausal status of our cohort was not stratified, limiting mechanistic interpretations. Environmental or lifestyle factors unique to Iraqi women, such as dietary patterns or physical inactivity, may also contribute to the results.20 However, these mechanisms require further exploration by molecular and hormonal studies.

In terms of clinical parameters, the mean BC duration in patients was 2.81 (0.33) years, with a similar mean treatment duration of 2.80 (0.33) years. These values reflect trends reported by Gnant et al.,21 whose trial of extended- release anastrozole found that 3208 of 3484 women remained progression- free after 2 years of treatment. Nevertheless, the optimal therapy duration remains a topic of debate. These complications can lead to reduced treatment adherence and early discontinuation, thereby compromising outcomes.22 Interestingly, the current study found no significant association between family history and the incidence of BC. This is consistent with the findings of Ho et al.,23 who reported that \(84\%\) of the patients denied a positive family history. Swahn et al.24 found that responses were split, with \(33.2\%\) of patients with BC reporting no family history, \(38.2\%\) reporting a positive history, and \(30.0\%\) unsure. Although family history remains an important traditional risk factor, its predictive value may be limited in isolation.

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The current study found that the majority of patients were diagnosed with grade II tumors, which is consistent with previous reports by Salman et al.25 \((76.7\%)\) and Mohsin and Mohamad26 \((64.44\%)\) .A higher prevalence of grade I tumors was reported by Nayyef and Aziz,27 which is indicative of earlier detection. In contrast, more advanced tumor grades were documented by Andrikopoulou et al.28 and Nag et al.,29 in which grade III tumors predominated, suggesting later- stage diagnoses in these cohorts. Regarding tumor size (T stage), T2 tumors were the most prevalent in the current study, which is consistent with the findings of some previous studies \((61.7\%)\) 25,29 Similarly, Nayyef and Aziz27 reported that \(74\%\) of their cohort presented with T1 or T2 tumors. A slightly lower T2 rate was observed in the study by Ameen.30 These findings suggest that a significant proportion of BC cases are detected at intermediate stages, which may facilitate more favorable therapeutic outcomes. In terms of nodal involvement (N stage), \(45\%\) of the patients were classified as N1, reflecting limited regional lymph node spread. This is comparable to the findings of a study by Nag et al.,29 who reported the majority of patients with N1 cases, and the study by Nayyef and Aziz,27 who reported a higher N1 frequency. However, some studies have indicated that the majority of patients had no lymph node involvement (N0),25 suggesting a more favorable prognosis in that group. Regarding distant metastasis (M stage), the majority of patients had no evidence of metastasis (M0), corroborating the findings of some previous studies.25,27,29 These data support the notion that many patients are diagnosed before metastatic spread, which is an important factor in treatment planning and prognosis. IDC was the predominant histological type, accounting for \(72.73\%\) of the cases, which is consistent with global trends. Higher IDC frequencies have been reported by some studies,25,28,31 affirming that IDC is the most common BC subtype worldwide. Regarding tumor laterality, the left breast was more frequently affected, in line with the reports by a few studies.25,27,28 However, Mohsin and Mohamad26 noted a greater incidence in the right breast, possibly due to population- specific or sample size variations. Molecular classification revealed that Luminal A \(\mathrm{(ER^{+} / PR^{+}}\) \(\mathrm{HER2^{- }}\) ) was the most common molecular subtype. This aligns with various studies.27,32 Previous research29 reported an even greater prevalence of \(\mathrm{ER^{+} / PR^{+}}\) , suggesting a good response to hormonal therapy. Other studies have reported variability, including a study by Nitrenganya et al.33 The predominance of Luminal A tumors \((60\%)\) in our cohort contrasts with studies reporting Luminal B dominance, possibly due to genetic variations (e.g., CYP19 polymorphisms) or earlier detection practices in Iraq, whereas Andrikopoulou et al.28 identified Luminal B \(\mathrm{(ER^{+} / PR^{+}}\) \(\mathrm{HER2^{+}}\) ) as the dominant subtype in \(39\%\) of the cases.

A key finding of this study was the markedly elevated serum IL- 40 levels observed in patients with BC compared with those in HCs, with a statistically significant difference between the groups. Comparative studies have reported elevated levels of other interleukins in patients with BC. For example, Sermaxhaj et al.34 found significantly greater IL- 7 levels in patients with BC than in HCs, suggesting robust immune activation in malignancy. By contrast, Wang et al.35 reported significantly lower IL- 1β levels in patients with BC than in controls, reflecting the heterogeneity of immune responses associated with different cytokines. Further supporting the immunological relevance of IL- 40, Jaber and Ad'haih36 found elevated IL- 40 levels in patients with ankylosing spondylitis compared with controls, while Al Ghuraibawi et al.37 reported similar results. In type 2 diabetes mellitus, Nussrat and Ad'haih38 observed significantly increased IL- 40 levels, reinforcing the role of IL- 40 in chronic inflammatory conditions.

The present study demonstrated statistically significant findings with a highly significant \(P\) value and a moderately narrow CI, indicating moderate diagnostic accuracy for IL- 40 in Iraqi females with BC. At its optimal threshold, IL- 40 showed moderate diagnostic accuracy (AUC), correctly identifying a moderate proportion of both cases and HCs. The AUC of 0.756 \(95\%\) CI, 0.6453- 0.8484) suggests a moderate diagnostic utility for IL- 40. Discrepancies with prior studies (e.g., Ahmed and Abed, 2024) may stem from differences in cohort size, ethnicity, or assay sensitivity, underscoring the need for standardized protocols for IL- 40 measurement. These comparative outcomes highlight the variability in diagnostic efficacy across studies, underscoring the need for further validation of IL- 40 clinical utility.39

The current study also demonstrated a significant upregulation of C17orf99 gene expression in female patients with BC compared with that in HCs, with a mean fold change of 3.707 vs 1.026, respectively \((P = 0.0002)\) . IL- 40, initially characterized as a Bcell- associated cytokine involved in humoral immune responses, has recently emerged as a potential immunomodulatory factor in various inflammatory and neoplastic conditions. Its overexpression in patients with BC, as revealed in this study, aligns with the growing evidence that cytokines contribute to tumor development by promoting chronic inflammation, angiogenesis, and immune evasion.40 The application of the 2- ΔΔCt method for relative quantification revealed a statistically robust fold- increase, further supporting the hypothesis that IL- 40 may serve as a molecular biomarker. Notably, previous transcriptomic studies have implicated upregulated cytokine expression, including interleukins such as IL- 6, IL- 8, and IL- 17, in breast tumor progression and poor prognosis.41 Although IL- 40 has not yet been fully characterized in the context of BC, its structural and functional similarities with other tumor- promoting cytokines warrant further investigation. The significantly elevated IL- 40 expression observed in the present BC cohort may reflect the role of cytokines in shaping the pro- tumorigenic microenvironment. While our findings demonstrated elevated IL- 40 levels and gene expression in patients with BC, the exact role of IL- 40 in tumorigenesis remains unclear. Future studies should investigate cellular sources (e.g., tumor- infiltrating B cells) and their interactions with immune checkpoints. Moreover, recent bioinformatics analyses have identified IL- 40 as a cytokine signature predictive of tumor immune subtypes and therapeutic response.42 The constitutive production of IL- 40 from lymphomatous B cells and its variable expression in distinct subtypes of lymphomas, as noted in previous reports in cell lines, imply that it promotes B- cell lymphomatous escape. The IL- 4/STATs pathway, which is expectedly maintained by IL- 40 production from lymphomatous naïve B cells, might potentially play a crucial role in the development of cancer, as shown in gene and networking studies.43

elevated IL- 40 expression observed in the present BC cohort may reflect the role of cytokines in shaping the pro- tumorigenic microenvironment. While our findings demonstrated elevated IL- 40 levels and gene expression in patients with BC, the exact role of IL- 40 in tumorigenesis remains unclear. Future studies should investigate cellular sources (e.g., tumor- infiltrating B cells) and their interactions with immune checkpoints. Moreover, recent bioinformatics analyses have identified IL- 40 as a cytokine signature predictive of tumor immune subtypes and therapeutic response.42 The constitutive production of IL- 40 from lymphomatous B cells and its variable expression in distinct subtypes of lymphomas, as noted in previous reports in cell lines, imply that it promotes B- cell lymphomatous escape. The IL- 4/STATs pathway, which is expectedly maintained by IL- 40 production from lymphomatous naïve B cells, might potentially play a crucial role in the development of cancer, as shown in gene and networking studies.43

Elevated serum IL- 40 levels and transcriptional upregulation were observed in Iraqi women with BC. While IL- 40 shows promise as a diagnostic biomarker, further validation in longitudinal cohorts is required to assess its prognostic value and potential integration into risk stratification frameworks.