Correlation between Serum and Tissue Markers in Breast Cancer Iraqi Patients

: Breast cancer is the most prevalent malignancy among women worldwide, in Iraq it ranks the first among the population and the leading cause of cancer related female mortality. This study is designed to investigate the correlations between serum and tissue markers in order to clarify their role in progression or regression breast cancer. Tumor Markers are groups of substances, mainly proteins, produced from cancer cell or from other cells in the body in response to tumor. The study was carried out from April 2018 to April 2019 with total number of 60 breast cancer women. The blood samples were collected from breast cancer women in postoperative and pretherapeutic who attended teaching oncology hospital of the medical city in Baghdad and the serum markers evaluated by ELISA technique are Carbohydrate Antigen 15-3 (Ca 15-3), Carbohydrate Antigen 27.29 (Ca 27.29), Anti-Mullerian Hormone (AMH), Tumor Necrosis Factor-Alpha (TNF-α), Interleukin-6 (IL-6), Interleukin-10 (IL-10) and Human Epidiymis Protein-4 (HE4). Tissue samples were collected for the same breast cancer women who attended medical city, Baghdad with total number 30. The tissue markers evaluated by Immunohistochemical technique are Estrogen Receptor (ER), Progesterone Receptor (PR), Human Epidermal Growth Factor Receptor 2 (Her 2/neu) and Cyclin E. The results showed a positive significant correlation (p = 0.017) between Ca 27.29 and Her-2/neu, (p = 0.038) between IL-6 and cyclin E phenotype, (p = 0.051) between TNF-α and Cyclin E intensity, (p = 0.005) between HE4 and Her-2/neu, and negative significant correlation (p = 0.058) between IL-10 and ER score and (p = 0.045) between HE4 with Cyclin E intensity. We conclude from these correlations that positive correlations increasing disease progression, like correlation between Ca 27.29 and Her-2/neu, cyclin E with IL-6 and cyclin E with TNF-α. And the negative correlations may contribute to delay disease, like correlation between IL-10 and ER. From the correlations results in this study, it is clear that the Ca 27.29, Her-2 / neu, cyclin E markers play an important role in disease progression.


Introduction:
Breast cancer is one of the most common cancers in women. It is the most diagnosed cancer that causes death in women with a higher incidence in developed countries and higher death rates in developing countries 1, 2 . In Iraq, breast cancer ranks the first among the women and the leading cause of cancer related female mortality. Numerous studies from Iraq indicated that up to the present time a considerable rate of female patients still present with breast cancer at younger age groups with more advanced stages and aggressive tumor compared to their western counterparts. Ultimately leading to undesirable prognosis 3 . In Iraq, about 34.27 % of new cases register for breast cancer in female in Iraqi women until 2016 4 .
Carbohydrate antigen Ca15 -3, is a glycoprotein antigen. It is found on surface of cancer cell and sheds into blood stream and can be measured in saliva that is considered as blood stream in oral cavity that can be used for the detection of cancer begin and followup 5 . CA 15-3 is useful for determining prognosis of breast cancer and also for monitoring the efficacy of the treatment since the elevated level of this tumor marker in serum tend to increase the severity of breast cancer 6 .
Tumor marker Ca 27.29 is a carbohydrate containing protein antigen that reported as a tumor marker for breast cancer and it is called " Breast Carcinoma-associated antigen" 7 . The Ca 27.29 considered is the tumor marker which breast cancer cell shed copies from Ca 27.29 protein to blood stream 8 .
Anti -Mullerian hormone (AMH, also called a Mullerian inhibiting substances (MIS) or Mullerian inhibiting factor) was described at first in 1947 by Alfred Jost 9 . It is produced by Sertoli cell in testis and it has a role during embryogenesis by inhibiting the development of Mullerian duct in male embryo so, it plays a role in male sex differentiation 10 . The AMH activity is exerted by two receptors: Type I receptor and Type II receptor present on the AMH target organs such as gonads and Mullerian ducts 11 . The AMH type II receptor expressed in normal and tumor tissue in breast and limited laboratory finding suggest a protective role of AMH in breast carcinogenesis 12 . In other study showed the lowered concentrations of AMH in breast cancer patients and chemotherapy have a high effect on AMH level 13 .
Tumor Necrosis factor alpha (TNF -α) is an inflammatory cytokine synthesized and secreted by many cell types especially macrophage, natural killer cell (NK) and Tcell. This cytokine has a central role in promoting inflammation, endothelial activation and has a significant role in cancer pathogenesis 14 . The TNF -α is expressed by normal breast epithelial cells, but high level expressed in tumor cells from most breast cancer patients and the high level of TNF -α correlated with recurrence and advanced disease 15 . The TNFα is a necrotic factor in tumor microenvironment that stimulates tumor growth and migration and the elevated level related with metastasis 16 . On the other hand, reports of the anti-proliferative and apoptotic effect of TNF-α on breast cancer have only been executed on the MCF-7 cell line 17 . Some studies have shown that TNF-α may have a doubleedged role in angiogenesis, depending on the used doses. High doses of TNF-α inhibit angiogenesis in mice while low doses promote vascularization of the area. The anti-angiogenic effect of TNF-α is related with the down-regulation of adhesion molecules, while pro-angiogenic responses have been associated with the increased expression of VEGF, VEGFR, IL-8, and FGF. Therefore, low levels of TNF-α increase tumor growth, induce angiogenesis of several tumors in mice, and stimulate tumor-associated myeloid cells and the co-expression of endothelial and myeloid markers with pro-angiogenic/pro-vascular properties 18 . Interleukin -6 (IL -6) is a pro-inflammatory cytokine, it is produced by macrophage and monocyte. IL -6 is expressed by multiple tumor tissue like breast, prostate, colorectal and ovarian cancer 19 . An elevated serum level of IL -6 is shown to be correlated with disease staging and undesirable outcomes in women with metastatic breast cancer and it has a role in tumor advancements 20 . The IL -6 has a central role in tumor behavior like tumor growth, cell proliferation, migration and invasion, angiogenesis and metastasis 19 .
Antiinflammatory cytokine, (IL -10) gene are located on chromosome 1 at q31-32. It is produced by immune cell like Tlymphocyte, macrophage and natural killer cell (NK) 21  Estrogen and progesterone receptors are hormone receptors found on breast cells that pick up hormone signals resulting in cell growth 27 . In breast cancer status the determination of these receptors is useful for therapeutic options and provide prognostic information 28 .
Human epidermal growth factor receptor (Her -2) was first discovered in 1984 by Weinberg and associated 29 . The gene that encoded to this receptor is situated on chromosome 17q 30 . It is called as a neu in rat, the gene expression result production 185kDa transmembrane glycoprotein known as her -2 proteins and have extracellular domain (ECD) 31 .
Cyclin E / CCNE1 gene is localized on chromosome 19q 32 . Cyclin E is nuclear protein identified through it is capacity of division abnormality in cyclindeficient yeast cells. Cyclin E has a high expression in many human cancers including breast, endometrium, lung, cervix, gastrointestinal tract and lymphoma. The high cyclin E expression was prognostic factor which correlated with worse patient outcome in women with breast cancer 33 . This research discusses a group of serum and tissue markers in a group of Iraqi women with breast cancer. The research clarifies the correlations between these markers because of their importance in the progression of the disease or its regression.

Materials and Methods:
Sixty patients (32-75 years old) who admitted the Oncology Teaching hospital, Medical City-Baghdad confirmed as breast cancer by physical examination, biopsy tacking, ultrasound and mammogram.
Postoperatively patients followedup for defining the histopathology classification of breast cancer stage and lymph node metastasis were recorded. The patients did not receive chemotherapy, radiotherapy or hormonal therapy yet.

Blood samples
The blood was collected from patients by 5 ml disposable syringe and then was put in gel clot activator tube and left room temperature to allow clotting after that entered to centrifugation 3000 rpm for 15 minutes then serum distributed in 4 eppendorf tubes in equal amount and stored in deep freeze until time to use.

Kits used
The all serum biomarkers (Ca 15-3, Ca 27.29, AMH, IL-6, IL-10, TNF-α and HE4) for breast cancer women were evaluated depending on sandwich ELISA principles using three antibodies, capture antibody, detection antibody and HRPlinked secondary antibody. The sample with unknown amount of antigens is immobilized on wells of the plate by bind with capture antibody. The antigen-antibody complex is linked to an enzyme-bound secondary antibody. In the final step a substrate is added that the enzyme can convert to some detectable signal like color 34 . The Ca15-3 kit purchased from Thermofisher (USA), Ca27.29 kit from Mybiosource (USA). The AMH kit is from Kamiya biomedical (USA). The TNF-α, IL-6 and IL-10 kits are from Thermofisher (Austria). The HE4 kit is from Cusabio (China). Tissue markers were evaluated by Immunohistochemical technique. The ER, PR and Her-2/neu kits from Zytomed system (Germany) and cyclin E kit from Bio SB (USA). The method of action of the serum markers was according to was mentioned in the kit used for each marker.

Clinico-pathological Features
The clinic-pathological features of patients female are illustrated in Tables 1 and 2.

Tissue samples
The immunohistochemistry is a technique for identifying cellular or tissue antigens by mean antigen-antibody interactions. The IHC staining is complete by using enzyme-labeled (immunoperoxidase) antibodies to identify proteins. The secondary antibody is reactive against the primary antibody conjugated or linked to an enzyme marker. Finally, the color of the reaction is determined by precipitating chromogen, usually Di Amino Benzidine (DAB) (brown color) with which the enzyme react 35 . Tissue samples (Formalin Fixed Paraffin Embedded Tissue "FFPE") were collected from 30 patients who admitted to tissue laboratory, Oncology Teaching hospital, Medical City-Baghdad. The desired information about the patients and histopathological information of the tumors were collected from the patients' files. The tissue section slides of ER, PR, Her -2 / neu and Cyclin E receptors were imaged by using light Microscope (leica) and camera (leica) by 10 x and 40 x objective lenses and magnification power at 100 X and 400 X.
The ER and PR section was read according Allred score (proportional percentage + intensity of staining) of tumor cells and according the intensity of staining cells for Her-2/neu and these reading according to Iqbal BM and Buch A, 2016 36 (  Tables 3 and 4).  (Tables 5 and 6). Phenotype III = + nuclear and + cytoplasm 4 Phenotype IV = no or weak nuclear and + cytoplasm

Statistical analysis
The statistical analysis was performed using the statistical package SPSS version 23. Results were considered at significant (P˂0.05) difference levels. The correlation test was used to find out significance of correlation between related variables.

Results:
The mean of serum markers of patients samples are clarified in Table 7     This research showed that 70 % from cases were ER and PR positive as shown in Table 8. The Her-2/neu results showed 33% from cases were negative as shown in Table 9. While cyclin E phenotype showed 46% from cases were phenotype III and 33% from cases were strong staining cells as shown in Tables 10,11.    The results revealed correlations between the means of serum markers and tissue markers in breast cancer patients. The results showed that was a positive significant correlation between Ca 27.29 with Her-2 P (0.017), HE 4 with Her-2 P (0.005), IL -6 and cyclin E phenotype P (0.038), TNF -α with cyclin E intensity P (0.051) and revealed a negative significant correlation between IL -10 and ER score P (0.058) and HE 4 with cyclin E intensity P (0.045) as in Table 12.

Tissue sections
In invasive breast carcinoma the negative status of estrogen receptor (ER) and progesterone receptor (PR) mean that no ER and PR expression (0%) staining cells and the nuclear staining ˂ 1% of total cancer cells as illustrated in Fig. 2, while the levels of (ER) and (PR) expression were positively 70% (90-100%) from cancer cells reaction with immunostaining and strong intensity as showed in Figs 3 and 4.
Her-2/neu is a cell membrane receptor and its staining depends on the intensity of cancer cells staining. Figure 5a shows no cancer cells staining because these cancer cells did not express of her-2/neu receptor, while in Figs 5b and c respectively shows reaction between her-2/neu receptor with immunostaining.
The Fig. 6a shows the phenotype II of cyclin E expression that occur nuclear staining exceeded cytoplasmic staining and Fig. 6b the phenotype III of cyclin E that was nuclear and cytoplasmic staining are equal. Figure 6c shows the moderate staining of cancer cells because the moderate levels of cyclin E expression. Figure 6d reveals a strong staining due to the high levels of cyclin E expression in breast cancer cells.

Discussion:
This research explained that the correlations between serum and tissue markers have an important role in the progression or regression of breast cancer through the effect of each marker on the other marker. The negative correlation (p ˂ 0.05) between ER and IL-10 in our research Corresponds with Zhuangwei et al, who reported a high serum IL-10 correlated with ER negative expression 38 . Bhattacharjee et al, also revealed a significant correlation between IL-10 with ER and PR -ve tumors and IL-10 expression correlated with ER,PR -ve and Her-2/neu +ve significantly (p = 0.01) and this subtype of breast cancer (ER,PR -ve and Her-2 +ve) considered as the worst prognosis among the other subtypes 39 . Sheikhpour et al, reported that IL-10 over-expressed in ER -ve and interpreted that expression of activator protein (AP -1) is high in ER -ve in compared with ER +ve due to increased AP-1 expression in related with IL-10 21 .
The AP-1 is transcription factor regulates gene transcription and it is implicated in regulating many physiological and pathological cellular process like proliferation, differentiation, growth, programmed cell death, cell migration and transformation. AP-1 mediated gene expression in response to inflammatory cytokine and it important for the pathogenesis of disease 40 . Carruba et al, concluded that estrogen hormone (E2) is essential regulators of cytokine in cultured macrophage, the results reported that E2 induced decrease IL-10 secretion and that estrogen play a role in reduce IL-10 production 41 .
The positive correlation (p ˂ 0.05) between Ca 27.29 and Her-2/neu markers in our study agrees with Yerushalmi et al, who explained a significant correlation between Ca 15-3 and Her-2/neu 42 . We staining intensity by immunohistochemical technique and demonstrated that in breast tumors, the increasing of Her-2/neu amplification was in parallel with HE 4 staining intensity, Her-2/neu amplification is known as poor prognostic factor for breast cancer and proved that HE 4 expression also unfavorable prognostic factor in breast cancer 46 . The HE 4 protein enhance the proliferation, invasion and metastatic ovarian cancer cells and have the same biological effects on endometrial cancer, HE 4 can improve cell viability and found that HE 4 may play an essential role in EGFR (epidermal growth factor receptor) activation 47 . The EGFR also known (Her-2), EGFR (Her-2) is over-expressed in breast carcinoma and this overexpression due to EGFR (Her-2) gene amplification that can observed in many cancer types like breast, lung, colorectal and esophageal cancers 48 . So the increase of HE 4 in patients can induce to increase effectiveness EGFR (Her-2) that also increased in breast tumor.
The positive correlation (p ˂ 0.05) between IL-6 and cyclin E agree with Wei et al, who demonstrated that IL-6 able to suppress cyclindependent kinase 2 (CDK 2) activity by accumulation P27 kip1 protein 49 . Pan, Y. and Claret, F. X. concluded that IL-6 interact with Jab1/Csn5 to regulate un-phosphorylated STAT3 and the decrease of Jab1/Csn5 expression caused decrease un-phosphorylated STAT3 and that STAT3 bind to Jab1/Csn5 to increased it promoter activity with increase Jab1/Csn5 transcription in breast cancer while, decrease STAT3 caused lowering Jab1/Csn5 promoter activity and Jab1/Csn5 protein expression. The IL-6 was important activators of STAT3 and contributed IL-6 to activation Jab1/Csn5 transcription and expression by STAT3 50 . The Jab1/Csn5 gene is located on chromosome 8 and the expression of Jab1/Csn5 was found aberrantly expressed about 50 % in primary breast tumors and 90 % in metastatic lesions 51 . The Jab1/Csn5 overexpression is negatively correlated with P27 expression and that P27 can inhibit CDK and suppress cell-cycle 50 . Also found that breast tumors with high levels of P27 expression were rarely positive for Jab1/Csn5 expression and Jab1/Csn5 protein expressed levels were higher in oncogenically transformed breast cells and tumors than normal mammary epithelium. The function of Jab1/Csn5 is re-localizing P27 from nucleus to cytoplasm that inducing degradation P27 in cytoplasm by ubiquitin / proteasome pathway and finally, allowing breast tumor cells to progress into S phase 51 .
The results reveled a positive correlation (p ˂ 0.05) between cyclin E and TNF-α in breast cancer patients. This correlation was interpreted by indirect effects of cyclin E on TNF-α and as shown from results of Dhillon, N.K. and Mudryj, M. that revealed that cyclin E over-expression cause decreasing of BcL-2 protein levels in MCF-7 and T47D breast cancer cell lines 52 . The MCF-7 is the first cancer cell line isolated from Catherine Frances in Michigan, and called (MCF-7) Michigan Cancer Foundation and after 7 attempts to generate a monolayer from cancer cells 53 . The BcL-2 is overexpression in MCF-7 cell has suppress TNF-α and the response to TNF-α is increased in cyclin E overexpression cells and reported that an enhanced sensitivity of cyclin E over-expression cells to cytokine like TNF-α 52 .
The negative correlation (p ˂ 0.05) between HE 4 and cyclin E. Wang et al, interpreted the results on cell lines that HE 4 and ANXA 2 both localized in cell membrane and cytoplasm were over expressed and broadly interact in many malignant tumor cells, the cell lines that highly  54 . The ANXA 2 is Annexin 2 (ANXA 2) 36 -KDa calcium-dependent phospholipid binding protein in breast cancer, ANXA 2 participates in many cellular process including endocytosis, exocytosis of intracellular proteins, motility of cell, fibrino-lysis and ion channel constitution 55 . The ANXA 2 is related to the occurrence and development of many malignant tumor and play a central role in angiogenesis, migration, apoptosis, adhesion, invasion and proliferation of tumor cells 54 .
The ANXA 2 over-expressed in many solid tumors like lung, colorectal, pancreatic, breast and hepatocellular cancer. Wu et al, explained that ANXA 2 significantly increase expression both P21 and P27, but decreased expression of CDK1, CDK2 and cyclin B 56 . The cyclin E / CDK2 are responsible for G1 to S phase transition in cell cycle 57 . So, from these finding can conclude that HE 4 by interaction with ANXA 2 can inhibit or decrease levels of cyclin E.
The hormonal receptors results clarified a positive (ER+/PR+) were 70 % with negative (ER-/PR-) 26 %. Several studies have indicated that patients with positive hormonal receptors have a significantly higher survival rate, since positive status have benefits from hormonal treatment, where this not demonstrated in negative side 58 . Several studies have shown survival advantages of ER/PR receptors in breast cancer, it has been found that this survival advantages is substantially enhanced by adjuvant hormonal therapy which was given to patient with ER/PR positive tumor by reducing the recurrence rate during treatment period and reducing breast cancer mortality, therefor ER/PR expression status are regarded a good prognostic factor and predictor factor for response to endocrine therapy. The patients with ER+/PR+ tumor had superior survival compared with patients with ER-/PR-tumor and the estimated risk of death was significantly higher in patients with ER-/PRtumor compared with patients with ER+/PR+ tumor 59 .
The results of Her-2/neu demonstrated that Her-2/neu negative were 53 % more than positive 46 %. It is well known that overexpression of Her2 gene is a significant predictor of both short overall survival and time to relapse in breast cancer patient, Trastuzumab (Herceptin), which is monoclonal antibodies against extracellular domain of Her-2/neu, became the standard adjuvant treatment in breast cancer patients who appeared overexpression of Her-2/neu. It has been proven that trastuzumab is an effective drug in improving survival for patients with early Her-2 positive breast cancer as well as metastatic Her-2 positive breast cancer 60 .
In our study the cyclin E expression was 46 % phenotype III ( + nuclear and + cytoplasm) and 36 % phenotype II ( + nuclear and weak cytoplasm), patients with breast cancer whose tumors had no cytoplasmic cyclin E staining had an overall favorable prognosis, and those with any cytoplasmic cyclin E staining had a poor prognosis. We reported that LMW-E not full-length cyclin E, is most active in phosphorylating substrates and that LMW-E has a higher affinity than full-length cyclin E for binding CDK 61 .
LMW-E is more tumorigenic and patients whose tumors expressed LMW-E were shown to be at the highest risk for recurrence and death due to breast cancer. Because LMW-E lacks the nuclear localization of full-length cyclin E, the LMW-E accumulates in the cytoplasm, where it binds to CDK2 and retains kinase activity 62 . The expression of cytoplasmic cyclin E is related to the aggressiveness of the disease. The function of nuclear cyclin E has been attributed to cell-cycle progression, and overexpression of nuclear cyclin E leads to deregulation of cell proliferation, while cytoplasmic cyclin E has alternate functions that can affect signal transduction and metabolism 61 .

Conclusion:
The breast cancer is the common cancer that affected Iraqi women. The patients with higher serum of Ca 15-3, Ca 27.29 and elevated expression of ER, Her-2 and cyclin E levels are more likely to have breast cancer metastasis. So, both markers and by these correlations it has been demonstrated that they are as an indicator for helping clinicians to evaluate disease progression of breast cancer, predicting patient outcome and determining adjuvant treatment for better outcome.