Iraqi J Pharm Sci, Vol.29(2) 2020 Serum hepcidin and ferroportin in breast cancer DOI: https://doi.org/10.31351/vol29iss2pp99-106 99 Assessment of Some Hematological Parameters in Iraqi Women with Different Breast Cancer Stages Zahra M. Ali* and Shatha H. Ali* and Forat Y. Mohsen** *Department of Clinical Laboratory Science, College of Pharmacy, University of Baghdad, Baghdad, Iraq **Ministry of Health and Environment, Medical City, Oncology Teaching Hospital, Baghdad, Iraq Abstract Breast cancer (BC) is the most commonly diagnosed cancer in women. The metabolism of iron is closely regulated by hepcidin which exerts its action by interacting with a ferroportin. The aim of the present study was to assess the alterations in the levels of some serum biomarkers that have a role in iron homeostasis (hepcidin and ferroportin) in addition to hematological parameters (hemoglobin, leukocyte and platelets count) in different stages of BC. This study included 66 women with BC. The patients were categorized as follows : group 1 includes :22 patients with stage I disease ,group 2 includes: 22 patients with stage II disease ,and group 3 include: 22 patients with stage III disease .Group 4 includes :22 apparently healthy women as control. Data analysis revealed a significant elevation of serum hepcidin levels of patients groups 1, 2, and 3 (437.2±26.4, 501.4±31.8 and 558.5±21.3 pg/ml respectively) ,vs (179.4±19.8 pg/ml) of control, with steady elevations from stage I to III . Furthermore, serum ferroportin levels were significantly lowered in groups 1 and 3 compared to control (0.589±0.107 and 0.733±0.1 vs 1.37±0.28 ng/ml respectively).While blood hemoglobin level of group 3 were lower (11.96±0.18 vs 12.7±0.13g/dl ) compared with controls . Blood leukocyte count of patients (all groups) (7.39±0.28 ,8.93±0.48,9.86±0.52 (^103/µl) respectively) were markedly increased compared to controls (6.06±0.23), while mean platelet count for patients in group 2&3 were significantly increased compared to controls (313.9±19.3,309.2±25.3 vs 233.3±9.1 respectively). In conclusion, hepcidin, ferroportin and hematological markers including hemoglobin, WBC count and platelets count are altered in women with BC compared to healthy control. The changes occur mostly in accordance with disease stages . Key words: Breast cancer, Hepcidin , Ferroportin. العراقيات المصابات بمراحل مختلفة من سرطان الثدي تقييم بعض المعلمات الدموية لدى المريضات **فرات يحيى محسن و *شذى حسين علي، 1*,زهراء محمد علي . العراق،بغداد ، جامعة بغداد ، كلية الصيدلة ،فرع العلوم المختبرية السريرية * . العراق،بغداد ، مستشفى االورام التعليمي ، دائرة مدينة الطب ، وزارة الصحة والبيئة ** الخالصة يعمل بالتنسيق مع الذي بواسطة بروتين الهبسدين بدقة ان ايض الحديد منظم.سرطان الثدي هو اكثر سرطان مشخص لدى النساء الفروبورتين. ( باالضافة الى قياس الهبسدين والفروبورتين ) التي لها دور في تنظيم معدالت الحديد بروتينات في مصل الدم فائدة قياستهدف هذه الدراسة الى تقييم وسائل تساعد في دراسة تقدم سرطان الثدي .كالعوامل الدموية )الهيموغلوبين وكريات الدم البيضاء والصفائح الدموية( بالمرحلة )مريضة 22:المجموعة االولى شملت حسب مراحل المرضمصابة بسرطان الثدي ,تم تقسيم المريضات انثى 66شملت هذه الدراسة بالمرحلة الثالثة من )مريضة 22,المجموعة الثالثة شملت (بالمرحلة الثانية من المرض)مريضة 22,المجموعة الثانية شملت (االولى من المرض . كمجموعة مقارنة تبدو بصحة جيدة انثى 22.المجموعة الرابعة شملت (المرض 21.3±558.5 و 31.8±501.4و 26.4±437.2) 3و2و1ت الهبسدين لدى كل مجاميع المرضى في معدال تحليل النتائج اظهرت ارتفاع مهم (pg/ml لمجموعة المقارنة وكانت هناك زيادة تتناسب مع تقدم المرض من المرحلة االولى الى المرحلة الثالثه . 19.8±179.4بالتتابع مقابل ) روبورتين لدى مرضى المرحلة االولى والثالثة بالمقارنة مع معدالت مجموعة االصحاءكان هناك نقصان مهم في معدالت الف باالضافة الى ذلك 0.18±11.96بالتتابع( .بينما اظهرت معدالت الهيموغلوبين للمجموعة الثالثة نقصان مهم 0.28±1.37مقابل 0.1±0.733و 0.589±0.107 0.28±7.39هي 3و2و1كان متوسط معدالت كريات الدم البيضاء لمجاميع المرضى .بالمقارنة مع مجموعة االصحاء 0.13g/dl±12.7مقابل مرتفعة ارتفاعا مهما مقارنة لمجموعة 3و2بينما كانت متوسطات معدالت الصفيحات الدموية للمجاميع بالتتابع 0.52±9.86و 0.48±8.93و بالتتابع . 9.1±233.3مقابل 25.3±309.2و 19.3±313.9واالصحاء ) الهبسيدين والفروبورتين والعوامل الدموية )الهيموغلوبين واعداد كريات الدم البيضاء واعداد الصفائح الدموية( اظهرت نتج ان يمكن ان نستوبذلك انحرافات في النساء المصابات بسرطان الثدي بالمقارنة مع مجموعة االصحاء .معظم هذه االنحرافات كانت متناسقة مع مرحلة المرض. . الفروبورتين،الهبسدين ،: سرطان الثدي الكلمات المفتاحية 1Corresponding author E-mail: obaydizahra@yahoo.com Received: 21/10 /2019 Accepted: 20/5 /2020 Iraqi Journal of Pharmaceutical Science https://doi.org/10.31351/vol29iss2pp99-106 Iraqi J Pharm Sci, Vol.29(2) 2020 Serum hepcidin and ferroportin in breast cance 100 Introduction Breast cancer (BC) is the most commonly diagnosed cancer in women, it is estimated to affect (24.2%) of women worldwide. In Iraq, the number of new cases in 2018 for both sexes, and for all age groups was 5141 which represent 20.3% of all new cases for all cancer types (25320), while the number of prevalent cases of all cancer types over 5 years for all ages was 54809 cases (1). Breast carcinomas is the most common type of BC although different types of sarcomas and lymphomas also can be encountered (2). Chronic inflammation is a key participant in cancer development and progression (3). The mechanism by which chronic inflammation is related to BC prognosis is not fully clear .However , complex processes through which chronic inflammation may promote carcinogenesis such as polarization of M2 tumor-associated macrophages via inflammatory cytokines with the subsequent production of tumor growth factors or promotion of angiogenesis (4,5).In addition, several prognostic factors are correlated with inflammatory status such as body fatness, physical activity, and cardiovascular comorbidity, which may affect tumor prognosis through alternate mechanisms. Meanwhile, the presence of undetected cancer cells may result in inflammation, thus inflammation may not only contributor to tumor promotion, but also result of these cancer cells (6). The American Society of Clinical Oncology (ASCO) recommends the use of tumor markers in prevention, screening, treatment and surveillance of BC. The most common clinically applied tumor markers for breast cancer are CA 15-3, CA 27.29, Carcmoembryonic antigen (CEA), Estrogen receptor (ER), Progesterone receptor (PR), Human epidermal growth factor receptor 2 (HER2), urokinase plasminogen activator (uPA), plasminogen activator inhibitor 1 (PAI-1) and multiparameter assays for gene expression (7) . The metabolism of iron is closely regulated by hepcidin which is liver-derived peptide. It exerts its action by interacting with a ferroportin (FPN), a transmembrane protein implicated in iron efflux from the body iron stores (8).Binding of hepcidin with FPN exerts a negative effect on erythropoiesis by inducing internalization and then subsequently destruction of FPN (8,9). Increased erythropoiesis suppresses hepcidin production, while iron loading and inflammation induce its production. Blood loss, anemia, increased erythropoietin or hypoxia stimulate bone marrow erythropoiesis and then reduce hepcidin (10,11) . Increase in serum hepcidin has been reported increasingly from diverse clinical states, (neoplastic diseases, inflammation, and sepsis) (10,12,13). Sideropenic anemia is frequently reported in cancer patients, (14) that is mediated by the inflammatory cytokines produced by cancer cells and by macrophages infiltrating the cancer tissue (15). These cytokines stimulate the hepatic production of hepcidin, which in turn inhibit iron absorption in the duodenum and promote iron sequestration by macrophages limiting its recycling (16). Moreover, the inflammatory cytokines decrease FPN expression in duodenal enterocytes blocking the transport of iron from enterocytes to transferrin (16). IL-10 also induces an increase in serum ferritin and soluble transferrin receptor (sTfR) levels causing anemia (17) . The role of peripheral WBC in predicting incident BC remains to be determined in spite of the established role of peripheral WBCs in BC prognosis (18-19). Many studies showed the lack of association between total white blood cell count (TWBCC) and BC risk (20-21) ; yet, TWBCC serves as a significant predictor of certain types of BC and for other types of cancer like lung (20) ,gastric (22) ,and colorectal cancers ( 23). Furthermore, platelets have important roles in various physiological and pathophysiological processes, including inflammation, immunity, angiogenesis, wound healing, and cancer progression (24-25). Platelet dysfunction and thrombotic disorders are important manifestations of cancer progression (24-26). Thrombocytosis is associated with poor cancer prognosis, suggesting a potential role for platelets in the pathogenesis of the disease (27-28). The aim of the present study was to assess the alterations in the levels of some serum biomarkers that have a role in iron homeostasis (hepcidin and ferroportin) in addition to hematological parameters (hemoglobin, leukocyte and platelets count) in different stages of BC. Material and Methods This study was conducted at The Oncology Teaching Hospital / Baghdad Medical City from October/2018 to February/2019 . Subjects enrolled in the study were 66 women that were recently diagnosed as having BC at different stages of the disease; and 22 apparently healthy women of comparable age to serve as control group. The age range of the subjects was 30-70 years. Specialist oncologist performed diagnosis and staging of BC according to Breast Cancer Staging, NCCN guidelines Version 4.2017 (29); a specialized pathologist using the WHO grading system, (30) performed the histological confirmation and grading. BC patients at stage IV of the disease is characterized by metastasis to other organs, and those at stage 0 having either ductal carcinoma in situ (DCIS) or lobular carcinoma in situ (LCIS) of disease, were excluded from the study. Other excluded patients were those whom were already undergoing chemo-, radio-, or hormonal therapy; or those with autoimmune diseases or hemoglobinpatheis, except iron deficiency anemia. Iraqi J Pharm Sci, Vol.29(2) 2020 Serum hepcidin and ferroportin in breast cance 101 Subjects enrolled in the study were grouped as: Group 1 including 22 BC women at stage I of the disease, Group 2 including 22 BC women at stage II of the disease, Group 3 including 22 BC women at stage III of the disease and Group 4 including 22 apparently healthy women serving as control group. Blood specimens were obtained from all participants. Serum levels of hepcidin and FPN were estimated quantitatively by sandwich type of enzyme linked immune-sorbent assay (ELISA), (31) using kits purchased by R&D® (USA) for hepcidin and MyBioSource® (USA) for ferroportin. Measurements of total white blood cell count (32), platelet cell count (33) were performed utilizing whole blood samples based on DynaHelix Flow technology. Estimation of blood hemoglobin was performed utilizing colorimetric surfactant method (34). Statistical analysis Analysis of data was carried out using the available statistical package of SPSS-23 (Statistical Packages for Social Sciences- version 23). Data were presented in simple measures of mean, standard error, and range. Student t-test was used for testing the significant difference between two groups, whereas ANOVA among more than two groups by LSD post hoc multiple comparisons of data. Person’s correlation was calculated for studied quantitative variables. Statistical significance was considered whenever the P value was less than 0.05. Results Age range for group 1 was 32-67 years (51±2.3), for group 2 was 34-68 years, (47.63±1.87), and for group 3 was 32-65 year (45.5±1.75), While age range for control group was 31-60 years (44.3±1.5). Table-1 summarized the descriptive date of BC patients and healthy control regarding age, body weight, height, and body mass index (BMI). There was no significant difference between total patients group and healthy control group regarding age, body weight, and height (P value 0.064,0.502 ,0.064 respectively) and significant difference regarding BMI (P value 0.041). Table1. Descriptive Data of Total Patients and Control Groups Parameter BC Patients (n=66) Healthy control (n=22) P value Age (years) 47.9±1.14 44.3±1.5 0.064 Body weight (Kg) 74.07±1.15 72.04±2.7 0.502 Height (cm) 159.8±0.81 163.04±2.05 0.064 BMI (kg/m2) 28.8±0.3 26.96±0.82 0.041* Data are expressed as mean± SE , BC: breast cancer, BMI: body mass index,* significant difference from control group (p˂0.05) Table 2 presents the data of studied groups regarding age, body weight, height and body mass index Table2.Descriptive data of studied groups. group parameter Group 1 Group 2 Group 3 Group 4 Age (years) 51.5±2.3 46.2±1.66 45.9±1.79 44.3±1.5 Body weight (Kg) 73.3±1.76 72.4±1.96 76.4±2.24 72.04±2.75 Height (cm) 160.1±1.42 157.9±1.46 161.6±1.26 163.04±2.05 BMI(kg/m2) 28.4±0.45 28.9±0.41 29.1±0.7 26.9±0.82 Data are expressed as mean± SE , BMI: body mass index Data of Table-3 describe the differences between patients and control groups regarding family history, a number of smoker subjects, presence of diabetes mellitus , presence of hypertension . Sixteen of BC patients were presented with one or more first degree relative with BC in contrast to two of female control, all healthy control females were selected as none of them having diabetes mellitus nor hypertension and all of them were nonsmoker, while five of BC patients were diabetic and eight of them were hypertensive. Table 3 . Medical history of studied groups Parameter BC patients n=66 Healthy control n=22 Family history 16 2 Smoker ** 2 None DM 5 None Hypertension*** 8 None BC: breast cancer, n=number, DM: diabetes mellitus, Iraqi J Pharm Sci, Vol.29(2) 2020 Serum hepcidin and ferroportin in breast cance 102 Table 4 describes the differences among studied groups of patients regarding hormonal receptors state [estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor 2 receptor (Her-2)], and histological classification of BC as invasive ductal carcinoma (IDC) was the predominant type followed by ductal carcinoma in situ (DCIS) with IDC , then invasive lobular carcinoma (ILC) with IDC and finally ILC . Table4. Basic criteria of studied patients in different stages of breast cancer Group Parameter Stage I (n=22) % Stage II (n=22) % Stage III (n=22) % Total (n=66) % ER positive 10 45.4 16 72.7 17 77.2 43 65.1 PR positive 12 54.5 16 72.7 16 72.7 44 66.6 Her-2 positive 9 40.9 10 45.4 9 40.9 28 42.4 IDC 16 72.7 15 68.1 14 63.6 45 68.1 DCIS&IDC 5 22.7 6 27.2 4 18.1 15 22.7 ILC 0 0 1 4.5 0 0 1 1.5 ILC&IDC 1 4.5 0 0 4 18.1 5 7.2 ER: estrogen receptor, PR : progesterone receptor, Her-2:human epidermal growth factor receptor, IDC :invasive ductal carcinoma, DCIS: ductal carcinoma in situ, ILC :invasive lobular carcinoma. As presented in Table-5, there were significant elevations in serum hepcidin level, blood WBC count and blood platelet count of total BC patients as compared to control group, while there was significant decrease in blood hemoglobin level of total patients as compared to control and no significant difference between these groups regarding a serum ferroportin level. Table5. Blood and serum levels of the studied parameters among total patients and control groups Group Parameter Total patients (n 66) Control group (n 22) P value S. hepcidin pg/ml 499.09±16.4 179.4±19.8 0.000* S.ferroportin ng/ml 0.794±0.11 1.37±0.28 0.07 B.hemoglobin gm/dl 12.1±0.16 12.7±0.13 0.009* B.WBC count (ˆ103/µl) 8.73±0.28 6.06±0.23 0.000* B. platelet count (ˆ103/µl) 301.8±12.4 233.3±9.16 0.000* Data are expressed as mean ±SE, WBC: white blood cell ,B: blood ,S: serum Table6. Blood and serum levels of the studied parameters among the different study groups Group Parameter Group 1 (n=22) Group 2 (n=22) Group 3 (n=22) Group 4 (control) (n=22) P value S. hepcidin pg/ml 437.2± 26.4 501.4± 31.8 558.5± 21.3 179.4± 19.8 0.000* S.ferroportin ng/ml 0.589± 0.107 1.06± 0.31 0.733 ± 0.102 1.37± 0.28 0.07 B.hemoglobin gm/dl 12.2± 0.28 12.4± 0.37 11.9± 0.18 12.7± 0.13 0.17 B . WBC count ^103/µL) 7.39± 0.28 8.93± 0.48 9.86± 0.52 6.06± 0.23 0.000* B.platelet count(^103/µL) 282.2± 20.09 313.9± 19.3 309.2 ±25.3 233.3 ±9.16 0.016* Data are expressed as mean ±SE, WBC: white blood cell , B: blood ,S: serum Figure-1 illustrates that serum hepcidin levels of patient groups are significantly higher than that of controls through different stages from I-III. Hepcidin levels tends to increase as the disease progresses; as presented by significantly higher levels in stage III compared to that in stage I. Serum levels of FPN of stage I and III were significantly lower than that of control group with no significant difference between patient groups (Figure-2). Blood hemoglobin levels of stage III was significantly lower than that of control group with no significant difference between patients groups (Figure-3). Iraqi J Pharm Sci, Vol.29(2) 2020 Serum hepcidin and ferroportin in breast cance 103 Figure1. Mean value of serum hepcidin among studied groups *= significantly different from control group (p˂0.05) Figure 3. Mean value of blood hemoglobin among studied groups *= significantly different from control group (p˂0.05) Correlation studies were done between all studied parameters among all groups including total patients, group 1, group 2, group 3, and group 4 and the significant results (that indicated positive or negative correlation) are tabulated in table-5. Serum hepcidin levels were significantly correlated with serum FPN levels in BC patients (total patient r=- 0.407, stage II r=-0.678, stage III r=-0.433). More advanced stages of BC (stage III) were presented with more significant correlation between FPN levels and blood platelet count (r=-0.438). Figure2. Mean value of serum ferroportin among studied groups *= significantly different from control group (p˂0.05). Table 5. Correlation study among studied groups x-axis y-axis r-value P value Group S.hepcidin S.ferroportin -0.678 0.001 Stage II S.hepcidin S.ferroportin -0.433 0.044 Stage III S.hepcidin S.ferroportin -0.407 0.001 Total patient S.ferroportin B.Platelet -0.438 0.041 Stage III S: serum, B: blood Discussion Globally, more than 95% of BCs diagnosed as adenocarcinomas (35) , while IDC is the most common form of invasive breast cancer (accounting for 55% of incidence upon diagnosis) (36) ,and ILC constitutes only 5%–15% of invasive breast carcinoma (37) .The results of this study agree with these results ,as 68.1% of the patients having IDC ,and only 1.5% of patients having ILC as mentioned in Table-2. The important role of iron in DNA synthesis could lead to cell cycle arrest after iron depletion, (38) whereas chronic sub toxic levels feeding of iron to cancer cells transform them into a the more aggressive phenotype which is prone to metastasis (39). Hepcidin induces FPN degradation, thus, interfering with iron efflux and cause iron sequestration in tumor cells. Moreover, hepcidin blocks iron export from cells such as macrophages and enterocytes (40). Serum levels of hepcidin are strictly controlled by different stimuli; iron status is the prime controller of hepcidin expression under basal conditions (41); yet, other factors can also control liver hepcidin expression, such as hormones, growth factors and heparins (42-44). In this study, serum levels of hepcidin in all patients groups are significantly higher than that of the control group and the elevation was coordinated with the stage of the disease (P value 0.000, 0.000, 0.000 respectively) with significant elevation of stage III as compared to stage I (P value 0.001), while there was no significant difference between levels of stage I 1&II, stage II&III (P value 0.076 Iraqi J Pharm Sci, Vol.29(2) 2020 Serum hepcidin and ferroportin in breast cance 104 ,0.115 respectively). While, serum FPN levels are significantly lower in patients with stage I and III than in the control group (P value 0.015 , 0.047 respectively). Serum FPN levels in patients with stage II was also lower than that of the control group but the difference was statistically non-significant (P value 0.325),and there were no significant differences between stages I&II and stages I&III and stages II&III (P value 0.142 ,0.65 ,0.307 respectively ). Similar findings were reported in previous studies. Orlandi et al (45) reported that patients with BC and benign breast lesions had significantly higher hepcidin levels than healthy control group. Similarly, Ciniselli et al (46) found that when compared with patients with benign breast diseases, patients with BC had significantly higher hepcidin levels, Also BC patients were reported to have higher serum hepcidin that is associated with lower levels of FPN when compared to healthy controls (45,47) . Patients with stage III showed significantly lower blood hemoglobin levels compared to that of the control group (P value 0.032) and non- significant decrease of stage I and stage II as compared to control group (P value 0.138 ,0.325 respectively ) ,and there were non-significant differences among patients groups (stages I&II ,stages I&III ,and stages II&III ) (P value 0.613 ,0.498 ,0.238 respectively ) . These changes occur in accordance with the high hepcidin and low FPN levels. Twelve out of twenty two of BC patients at stage III have iron deficiency anemia at time of BC diagnosis. The number of anemic BC patients at stage I and stage II was much lower than that of patients at advance stage (stage III) (4/22 and 5/22) respectively; these findings point the important role of hepcidin in iron metabolism. Hepcidin overexpression is not only associated with various hematological malignancies and some solid organs tumors such as breast, prostate cancers, but it also participate in the development of anemia in those patients (10,45,48) . In this study, there was a steady increase in TWBCC throughout disease stages ,and there were significant elevations in all patients groups (stage I ,II ,and III ) as compared to control group (P value 0.022 ,0.000 ,0,000 respectively ) , even that the increase is still within normal range of blood WBC count [4- 10(ˆ103/µl)] (49).There were significant elevations of stage II and III than that of stage I (P value 0.008 ,0.000 respectively ) ,and no-significant elevation of stage III than that of stage II (P value 0.103). Elevated WBC count has been shown to be associated with increased risks of the lung (19), gastric (22), and colorectal (23) cancers. leukocytosis is associated with thrombocytosis which is negative prognostic indicator in patients with cancer (50-51).Thrombocytosis is associated with a poor cancer prognosis, suggesting a potential role for platelets in the pathogenesis of the disease (28) . In this study , platelet count of all patients group still within normal values. There were significant negative correlations between serum hepcidin levels and serum ferroportin levels of total patients, stage II patients group and stage III patient group, and these results may be related to negative regulatory role of hepcidin on FPN by induction of the later degradation (8,9) as these groups showed significant elevation of serum hepcidin level with decrease in FPN levels as compared to control. Also previous studies reported elevation of serum hepcidin in association with decreased FPN levels as compared to healthy control (45 ,52). Conclusion Hepcidin, ferroportin and hematological markers including hemoglobin, WBC count and platelets count are altered in women with BC compared to healthy control. The changes occur mostly in accordance with disease stage .Larger scale study testing validity of these markers in diagnosis and staging of BC is recommended. Acknowledgements I would like to thank all employees in The Oncology Teaching Hospital, doctors, nurses, lab staff and finally all the breast cancer patients and peoples for helping me to achieve this work. References 1. Bray F , Ferlay J, Soerjomataram I , Siegel RL, Torre LA, Jemal A .Global cancer statistics 2018: Globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries .Cancer J Clin.2018 ;68:394-424. 2. 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