Evaluation of breast cancer regarding molecular and immunochemical markers

Breast cancer is ranking the fi rst cause of mortality after urinary bladder tumors and malignant lymphomas [1]. A previous study showed that 5-10% of the cases of breast cancer in women can be attributed to inheritance, whereas 90-95% are sporadic, i.e., cases that appear randomly and are not predetermined genetically [2]. Accomplishment of cancer is predictable as a multistep development of the infection where cellular modifi es may cause undesired reactivity related to the uncontrolled growth and division of cells. Many critical mutations are required to convert a normal cell into a malignant cell. Malignant tumor developing is a several stages out growth that involves genetic alterations such as activating of oncogene and inactivation of tumor suppressor genes [3]. The most commonly engaged receptors and growth factors in human breast cancer are members of the epidermal growth factor receptor subfamily of tyrosine kinase receptors. The type I subfamily comprises HER-2, HER-3, and HER-4 [4]. These receptors assign a common molecular design; they all seize a large glycosylated extracellular ligand-binding domain, a single hydrophobic transmembrane domain and a cytoplasmic tyrosine kinase domain. HER-2 (also known as neu) is a protooncogene which encodes a 185-kDa tyrosine kinase glycoprotein. Amplifi cation of the HER-2 gene acts an important task in breast cancer pathogenesis [5]. Furthermore, amplifi cation or over-expression of the HER-2 oncogene is associated with a poor disease free survival rate in infected individuals with axillary node-positive breast cancer [6]. Large exertion have been dedicated to unravel these genetic modifi cation and Abstract


Introduction
Breast cancer is ranking the fi rst cause of mortality after urinary bladder tumors and malignant lymphomas [1]. A previous study showed that 5-10% of the cases of breast cancer in women can be attributed to inheritance, whereas 90-95% are sporadic, i.e., cases that appear randomly and are not predetermined genetically [2]. Accomplishment of cancer is predictable as a multistep development of the infection where cellular modifi es may cause undesired reactivity related to the uncontrolled growth and division of cells. Many critical mutations are required to convert a normal cell into a malignant cell. Malignant tumor developing is a several stages out growth that involves genetic alterations such as activating of oncogene and inactivation of tumor suppressor genes [3].
The most commonly engaged receptors and growth factors in human breast cancer are members of the epidermal growth factor receptor subfamily of tyrosine kinase receptors. The type I subfamily comprises HER-2, HER-3, and HER-4 [4]. These receptors assign a common molecular design; they all seize a large glycosylated extracellular ligand-binding domain, a single hydrophobic transmembrane domain and a cytoplasmic tyrosine kinase domain. HER-2 (also known as neu) is a protooncogene which encodes a 185-kDa tyrosine kinase glycoprotein. Amplifi cation of the HER-2 gene acts an important task in breast cancer pathogenesis [5]. Furthermore, amplifi cation or over-expression of the HER-2 oncogene is associated with Citation: Sabra  to fi nd affi liations between specifi c genetic revision and the clinical or pathological characteristics of the tumor. There has been quick progression of the accessible method to categorize new genetic modifi cations; technical advancement will also place the detection of genetic alterations in breast carcinomas within the clutch of usual clinical testing. As tumor behavior is largely determined by this genetic modifi cation, it is expected that clinical decisive manner will be increasingly infl uenced by awareness of the genetic shuffl ing-up of a tumor. In breast cancer, the main genetic modifi cations are amplifi cation of approximately ten oncogenes and inactivation of an unknown number of tumor suppressor genes [7]. In this study, the random amplifi ed polymorphic DNA (RAPD) technique, a simple PCR-based assay system was used to detect genomic instability in breast cancer tissues. Even as there is growing interest in the verifi cation for immune initiation to cancer in human beings, recognized in part by the detection of autoantibody against a number of intracellular and surface antigens in affected role with different neoplasm types. The detection of tumor sector antigens that elicit an antibody response may have value in cancer screening, diagnosis or prediction. Such antigens may also have utility in immunotherapy against the disease.
Response to specifi city of an observed humoral or cellular immunity to malignant cells has always been the critical concern in human cancer immunopathology [8]. A lot of efforts have been focused so far on developing a strict association between distinct autoimmunity and cancer, on one script, and the mien of autoimmune response in affected role with solid tumors or malignancies. On the other, this relationship can be ascribed to different reasons: a genetic and/or immunological susceptibility; oncogene activation and/or tumor suppressor gene modifi cation with an abnormal expression of the relevant yields or atypical conditions common to both autoimmunity and cancer, such as an inherited or attained immunodefi ciency [9]. Antibodies, which bind other protein, may add another aspect to the asymmetrical immunity of individual patients with abnormal mass of tissue or cancer. Deviation in immune reactivity and a limited window for the tenacity of antibodies to various epitope may account for such critical failure. The present study aims to investigate the genetic alterations in breast cancer tissue compared to the genomic blood DNA of the same patient and to characterize distinct changes in patterns of immunoglobulin levels in breast cancer patients. Bovine serum albumin is one of the most broadly considered proteins; its structure is identifi ed and its antigenic characteristics have been identifi ed in several works [10,11]. In our study, we have therefore designed to address heterophile antibody interference in our enzyme-linked immunosorbent assay detection and to propose scheme for resolving this research question. We aimed to evaluate using enzyme-linked immunosorbent assay whether antibody that interact with Bovine serum albumin present any diagnostic signifi cance as a risk of exposure factor for breast cancer. Furthermore, the sensitivities of immunochemical assay for antibody to breast cancer affected role may be enhanced by counting antigens as Bovine serum albumin. To evaluate the potential of Bovine serum albumin contribution to breast cancer, we examined the specifi ty of antibodies in breast cancer patient plasma samples against Bovine serum albumin by preabsorption, clarifi cation and quantifi cation using enzyme-linked immunosorbent assay [10][11][12][13].

Patient participants
Informed consent was acquired personally by a health professional for all individuals prior to their addition in this report and the use of tumor tissue and plasma samples for scientifi c studies, which was approved by the Local Ethical  after surgery and kept at -80˚C until use for investigation. Three whole blood samples were collected on citrate, fi rst sample was taken just before surgery, second sample was taken postoperatively (before chemotherapy) and the third sample was taken after six cycles of chemotherapy. Plasma was collected from the three fresh blood samples and from controls, and then samples were kept at -80˚C until use.

DNA isolation and RAPD-PCR assay
Six random primers (decanucleotide GC-rich) were used to score the alterations in the isolated tumor DNA in comparison with corresponding blood DNA from the same patient. These primers have been reported previously by Singh, K.P. and Roy, D. 2001 [15]. The sequences of the used primers are shown in Table 2. The primers were synthesized by Biobasic. DNA of tumor tissue and corresponding blood DNA of the same patient before surgery and after chemotherapy were compared with each other to insure that the alterations found in tumor DNA are of cancerous origin. Genomic DNA extraction from tumor tissue was achieved using the genomic DNA purifi cation kit K0 512 (100 preps) that purchased from Fermentas; DNA from whole blood was isolated according to Sambrook et al 1989 [15]. Polymerase Chain Reaction (PCR) was followed according to Singh, K.P. and Roy, D.2001 [14]. Go Taq ® Green PCR Master Mix (Promega) was added to DNA samples and each primer (20ng/μl). Finally; the samples were loaded in thermocycler (Peqlab ® ) with heated lid. Primarily, samples were heated for 1 cycle (3.5min at 92°C for denaturation, 1min at 34°C, 2min at 72°C) and then cycled 44 times at 92°C for 1min, 34°C for 1min and 72°C for 2min followed by a fi nal extension cycle of 15min at 72°C. The yield of PCR amplifi cation was resolved by electrophoresis on 1.5% agarose gel and then visualized by ethidium bromide staining. A negative control reaction, without genomic DNA was run with every set of samples to validate that no contaminating nucleic acid was present in the reactions [16].
(0.02 M Tris-HCl, 1.5 M NaCl pH 9.0) and 100μl was added to each of the wells of ELISA plate [17,18]. After overnight incubation at 4 o C the plate was washed 4 times with PBST20  [19]. The absorption of individual plasma with Bovine serum albumin was able to remove preadsorbed proteins completely. Hence, the concentration of immunoglobulin in each of these plasma samples was determined by ELISA as described above.

Statistical analysis
The arithmetic mean for each group was calculated and the variation or variability in each group was represented by the Standard Deviation (SD). Data are presented as means with their standard deviation. Differences among the control, patients treated and non treated groups were assessed by Student's t-test to analyze specifi c differences between means.

Results
Six random ten-mer primers were used to analyze instability in the genome of breast cancer tissues using RAPD-DNA fi ngerprinting. Of these six primers, three of them produced similar amplifi cation patterns in all the DNA samples (data not shown). The remaining three primers detected genetic alterations in the tumor DNA. RAPD products were resolved on agarose (1.5%). Amplifi ed products resolved

Primer
Sequences Immunoglobulin measurement by enzyme-linked immunosorbent assay (ELISA)    noticeably reduced the levels of IgA and IgM in the plasma samples from cancer patients (group 4) while plasma samples from normal control individuals (group 2) were barely affected by this treatment. The quantitative analysis of plasma IgA level (mean±SD) showed that group 1 had a mean level of IgA of (2.56±0.25mg/ml) which was lower than group 3 (2.92±0.19 mg/ml). This demonstrated a signifi cant increase in immunoglobulin A concentration in the plasma of group 3 compared to group 1 (p<0.001). In contrast, the mean levels of immunoglobulin A in group 4 were (2.53±0.24mg/ml) and there was no signifi cant difference between group 4 and group 1 (p<0.142). The effect of Bovine serum albumin treatment as as a protective protein on the detection of immunoglobulin A in plasmas of the control group (group 2) was earlier

Disussion
Antibodies to tumor-associated proteins may increase the number of obtainable tumor markers for breast cancer and may be used together in a serum profi le to improve sensitivity and specifi city. Serum autoantibody profi ling shows a potential advance for early detection and diagnosis of breast cancer. Our results reveal that RAPD is a reliable and reproducible assay that has the potential to detect a wide range of DNA damage (e.g. DNA adducts, DNA breakage) as well as mutations (point mutations and large rearrangements) and therefore can be applied to genotoxicity and carcinogenesis studies. Understanding of the genetic trial progress towards transformation from normal to malignant cell has now been approved. This hypothesis achieved by comparing the RAPD profi les obtained from healthy and malignant cells within the same person. Hence, the potential effects of study in human genetic variability have been addressed these argument questions among species [20,21]. In longitudinal studies of chemical or physical agents interaction with the genomic DNA may also be responsible for the DNA alterations. Therefore, changes observed in the DNA profi les such as changes in band intensity could be examined by the random amplifi ed polymorphic DNA analysis. Consequently fragments generated by the random amplifi ed polymorphic DNA analysis were visualized by agarose gel electrophoresis as the profi le may differ due to band shifts, missing bands or visibility of new bands [22]. Therefore the changes occurring in RAPD profi les show alterations in oligo-nucleotide priming sites and activity changes of the taq DNA polymerase [16]. It is note that changes in a particular band patterns reproduce DNA structure modifi cation in the genome from a single base changes point mutations to complex DNA alterations. In response to DNA destruction comprises hydrolysis, oxidation, alkylation, and mismatch of DNA bases which may notably interfere with the PCR steps either during denaturation, primer annealing or the polymerization step. Structural disorder may could be an important factor and so contribute to the effect on the dynamic nature of the polymerase chain reaction events. An age related disease caused by the accumulation of genomic instability in the genome and with increased risk of cancer occurrence. Exposure to ionizing radiation leads to oxidizing actions and causing disorders that created from exogenous sources and endogenous causes, particularly in the mitochondria [23]. They greatly reduce the primers annealing sensitivity to the reaction and they can reduce the DNA polymerization so obstruct the taq DNA polymerase [24]. The subsequent steps, however, involve variation in band intensity and disappearance, leading to broad DNA destruction. In the current study, a loss of an amplifi ed product (Amplicon) may attribute to the loss of the oligonucleotide priming site due to the oxidative stress activator including superoxide and hydrogen peroxide that affect cell membrane leading to mutagenicity and carcinogenicity due to extensive DNA damage. These results may demonstrate the enhancement in deoxyribonucleic acid molecule break and repair in breast cancer patients [25]. Our fi nding data showed that RAPD is a sensitive technique that has the potential to detect a broad range of deoxyribonucleic acid destruction as well as mutations and it is useful to study carcinogenesis. Furthermore, the related bands produced in RAPD profi le make it possible to recognize some of the molecular trials concerned in genomic disorder where DNA amplifi cation plays a role in initiation and malignancy in cancer cells [21]. It is fairly simple to understand and to use and produces results rapidly by using early with immune disorders [32]. Several approaches to address this issue have been developed a strict association between distinct autoimmunity and tumors [33,34]. This association can be attributed to different causes as genetic and/ or immune susceptibility, oncogene activation and/or tumor suppressor gene alteration, with an irregular expression of the appropriate products [9]. The results of our study therefore provide evidence that plasma IgA and IgM levels are related to the etiology or clinical course of breast cancer, suggesting a disturbance in the immune system and immune dysfunction even when the cancer is localized to the breast. One of the fundamental arguments of these antibodies that interact with  [10,35]. There are also a number of constituents that address the development to autoimmunity as environmental,  Citation: Sabra  genetic or immune disorders. Hormonal factors also have been included among events triggering autoimmunity or immune dysfunction. This combined fact highlights the involvement between autoimmunity and cancer [36,37]. This fi eld of study reveals that there are autoantibodies in the plasma of patient with breast cancer that interrelate with Bovine serum albumin. In this paper we try to use Bovine serum albumin probabilistic interaction model as a tool risk of infection factor for breast cancer and in indicating this disease at-risk populations. By focusing on this paper we develop and tested a predictable marker that can be used in clinical laboratories as a supernumerary competent tool for diagnosis of breast cancer.