Adipokine Omentin-1 : A Diagnostic Tool in Breast Cancer

Introduction: Breast cancer is the most frequent solid malignancy in women. Omentin-1 is synthesized by visceral adipose tissue, placenta, and ovary and its production is altered in some cancers. The aim of this study was to assess omentin-1 as a diagnostic marker in patients

accurate diagnosis.A far as is known and due to reported conflicting results in the omentin-1 level in cancers, the serum level of omentin-1 was measured in women with breast cancer. 6,7,12

Patient Selection
Forty-five patients with breast cancers (within the age range of 18-60 years) from Vali-Asr hospital (Arak, Iran) and 45 healthy unrelated subjects (with the age range of 22-56 years) from healthy blood donors referring to Arak Blood Transfusion Organization during August 2015 to May 2016 were included in this age-matched casecontrolled study.Selected patients were confirmed in terms of clinical and para-clinical signs of breast cancer.Women were included in case they were newly diagnosed and histologically proven to have stage I-III breast cancers.Patients underwent standard breast magnetic resonance imaging (MRI) or dedicated axillary MRI prior to the surgery.Since body mass index (BMI) may influence omentin-1 levels, the selected groups were managed to have BMI within the range of 18.5-25 kg/m 2 .Women who had undergone any type of neo-adjuvant chemo-, immune-or endocrine therapy were excluded from the study.Similarly, patients with a history of breast surgery or treatment, liver dysfunction, obesity, and diabetes mellitus were excluded from this study as well.
Anthropometric and Blood Parameters Analysis Anthropometric measures including height, weight, and BMI were recorded.BMI was calculated as body weight divided by height squared (kg/m 2 ). 6Blood parameters including blood sugar (BS), red blood cell (RBC), white blood cell (WBC), as well as platelet (PLT) and hemoglobin (Hb) concentration were measured in duplicates in venous blood samples taken after at least 10 hours fast.For this purpose, 10 mL of peripheral blood was taken from both groups (patient and healthy women) and were poured into clot and ethylenediaminetetraacetic acid (EDTA)-contained tubes separately and transferred to the laboratory, immediately.Clot tubes were centrifuged for 10 minutes at 2000 × g, serum samples were separated and the BS of all tubes were measured by the enzymatic colorimetric method (Pars Azmun, Tehran, Iran).EDTAcontained tubes were assessed for cell blood counting using Sysmex automated hematology analyzer KX-21 (Sysmex, Milton Keynes, UK).

ELISA Assay
The serum samples were stored at -80°C until omentin-1 measurement.Omentin-1 serum level was measured in duplicates using enzyme-linked immunosorbent assay (ELISA) kit (Eastbiopharm, Hangzhou, China) based on the manufacture's instruction.All specimens were measured with the microplate reader (Stat Fax 2100) at wavelengths of 450 nm.
Statistical Analysis Kolmogorov-Smirnov test was used for assessing normal distribution of the data.The t test was performed for comparison within and between the normally-distributed groups.Spearman rank correlation coefficient analyses were performed to show a correlation between omentin-1 concentration and anthropometric and blood parameters analysis.Data were analyzed using SPSS software version 16.0 (SPSS Inc., Chicago, USA) and were represented as mean ± standard deviation (SD).P values less than 0.05 were considered to be statistically significant.Receiver operator characteristic (ROC) curves and positive and negative predictive values (PPV and NPV) were calculated using MedCalc Statistical software version 14.8.1 (MedCalc Software bvba, Ostend, Belgium; http:// www.medcalc.org;2014) to define the diagnostic accuracy as determined by the area under the ROC curve (AUC) of omentin-1.

Comparison of Anthropometric and Blood Parameters
This study was conducted on 45 women with stage I-III breast cancers and 45 healthy women as the control group.In patients and healthy control groups, BMI anthropometric parameter as well as serum levels of blood parameters including BS, Hb, RBC, WBC, and PLT were measured (Table 1).Except for WBC count (P=0.03),there were no significant differences between blood parameters and BMI analysis of patients and healthy control groups.

Comparison of Omentin-1 Concentration
The results of omentin-1 concentration showed that its serum levels were significantly lower in patients (157 ± 66 ng/L) compared to healthy control group (217 ± 75 ng/L, P = 002) (Figure 1).Furthermore, there was no significant correlation between omentin-1 levels and BMI and blood parameters analysis including, BS, Hb, RBC, WBC, and PLT.

Diagnostic Value of the Omentin-1 Level
The diagnostic value of omentin-1 level was investigated through calculating ROC curve (Figure 2).The best cutoff point for the diagnosis of breast cancer was at 146 ng/L.The sensitivity and specificity for omentin-1 with 95% CI (0.611 to 0.837) were 60% and 85%, respectively.Calculated PPV and NPV as predictive values at 146 ng/L cut-off point were 88.8% (CI: 79.2 to 94.3) and 51.5% (CI: 42.2 to 60.8), respectively.

Discussion
Adipokines are white adipose tissue-derived peptides.As previously mentioned, omentin-1, which was defined by Yang et al, is a new adipokine and is known to have antiinflammatory roles in vascular smooth muscle cells and synovial fluid of patients with rheumatoid arthritis. 13,14 far as is known, due to reported conflicting results in the omentin-1 concentration in the previously studied, cancers' serum level of omentin-1 were measured in patients with breast cancer. 6,7,12espite introducing new screening tests including Her2/ neu or CA 15-3 antigens, mammography is the gold standard screening method for breast cancer diagnosis.
As a result, emerging screening laboratory tests together with early-approved methods can provide an accurate diagnosis. 11The present study demonstrated that serum levels of omentin-1 in patients with stage I-III breast cancers, as early breast cancer stages, were significantly lower than the serum levels in healthy control group.A cutoff value was assessed for omentin-1 to test its diagnostic performance as 146 ng/L.At this cut off value, omentin-1 had a sensitivity of 60% and specificity of 85% with PPV and NPV of 88.8% and 51.5%, respectively.In this study, in order to match the groups, BMI anthropometric parameter and serum levels of blood parameters including, BS, Hb, RBC, WBC, and PLT were measured and the results revealed that except for WBC count, there were no significant differences between blood parameters and BMI analysis of patients and healthy control groups.Fazeli et al indicated that concentrations of omentin-1, visfatin, and vaspin in the serum of patients with colorectal cancer (CRC) were significantly higher than those of healthy control subjects.Based on this study, these adipokines may play potential roles in the development of colorectal cancer by mechanisms unrelated to obesity through Akt signaling pathway as Akt signaling can play important roles in cell proliferation and apoptosis prevention. 12In another study, omentin-1 in the serum of 81 women, 41 leans, and 40 obese individuals with PCOS and 61 healthy subjects were measured.The results showed that the amounts of omentin-1 in all patients with PCOS were significantly higher compared to the control group.Similarly, the average concentration of omentin-1 in obese patients with PCOS was significantly higher compared to the obese subjects in the control group. 15esults of a study on 45 patients with third stage colon cancer treated with surgery and 35 healthy subjects showed that omentin-1 values in patients were higher compared to the control group. 7Zhang et al demonstrated that the omentin-1 considerably inhibits 2 types of liver cancer cell lines (HepG2 and Huh7) proliferation.Omentin-1 positively regulates p53 protein levels by reducing deacetylation of p53 and increases the stability of P53.The data from this study indicate that omentin-1 may contribute to the treatment of hepatocellular carcinoma. 16n addition, Uyeturk et al found that level of omentin-1 in    patients with prostate cancer was higher than its level in people with benign prostatic hyperplasia. 6onversely, however, the results of this study reporting lower concentration of omentin-1 in patients with breast cancer, the above mentioned studies showed higher levels of omentin-1 in CRC, PCOS, prostate cancer, and liver cancer cell lines.Therefore, the only difference between our study and the other findings might be the result of the different study population, type of cancer tissues and treatment.
Omentin-1 serum levels in some cancers were studied; however, so far there is only one study on the measurement of serum levels of omentin-1 in patients with breast cancer.In this regard, Alaee and colleagues have recently measured serum omentin-1 level in patients with breast cancer.Similar to the results of the current study, they reported reduced amounts of omentin-1 in patients; however, unlike our study, there was no evaluation of omentin-1 potential as a diagnostic tool. 17n the other study, Shen and colleagues confirmed the lower concentration of omentin-1 in patients with renal cell cancer. 8In consistent with the results of the present study, Mogal and colleagues showed reduced omentin-1 in patients with RCC. 18mentin-1 was involved in colorectal-tumor development through Akt phosphorylation/activation. 19There are some documents showing that omentin-1 plays a role in apoptosis induction in hepatocellular carcinoma cells via p53 and p21 upregulation or activation of caspase-3 signaling pathway through increasing bax/bcl2 ratio. 16educing levels of omentin-1 in patients with breast cancer could be for defense strategies of the patients or escape routes of cancers that remains to be investigated in the future studies.One of the limitation of the study was the small sample size (only 45 patients residing in Arak were included in the study).Furthermore, omentin-1 gene expression could be useful for better evaluating the results.Therefore, studies with higher samples and gene expression are recommended.

Conclusion
In summary, finding a potential diagnostic tool is critical in early cancer detection.Since the serum level of omentin-1 was significantly lower than its level in healthy group, thus, it can be used as a screening test along with the early-approved methods in diagnosis of patients with stage I-III of breast cancers, as early breast cancer stages.

Ethical Approval
Informed consent was obtained from all individual participants prior to their inclusion in the study.

Figure 1 .
Figure 1.The bar graph shows omentin-1 serum levels in patients with breast cancer compared to those of control group.* P value < 0.05.

Table 1 .
Comparison of Anthropometric and Blood Parameters in Patients With Breast Cancer and Healthy Control Groups The experimental protocol for blood collection was approved by Human Ethics Committee of Arak University of Medical Sciences, Arak, Iran (code number: ARAK.RAC.1394.147) in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and all subsequent revisions.