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In Egypt breast cancer constitutes 33% of all female cancer at NCI. The median age is 46 years, one decade younger than the corresponding age in western countries. In Egyptian patients 60.5% of patients are pre menopausal. The female to male ratio is 44:1 (El-Bolkainy, 2000).There has been growing interest in the use of biological markers in breast carcinomas to asses the prognosis (Schonborn et al., 1994).The present study was carried out on fifty female breast cancer patients and fifteen (age matched) apparently healthy females as a control. Patients were randomly selected from surgery department, Zagazig University from June 2003 to June 2004.All studied patients proved to have breast cancer by history, clinical examination, radiological investigation, biopsy and confirmed pathological analysis of resected specimen. All the tumors were diagnosed as invasive ductal carcinoma. These patients had received neither chemotherapy nor radiotherapy prior to the surgery. The patients were divided, according to absence, presence of axillary L.N or presence of distant metastasis into three groups; group I (N-) included 15 patients, group II (N+) included 20 patients and 15 patients in group III (M+), 58% of patients were less than 50 years old and 42% were more than 50 years old at time of diagnosis. They were subjected to thorough history, clinical examination. Routine laboratory investigations were carried out for all subjects.For every patient, fasting, morning, venous blood sample was withdrawn preoperatively and delivered into 2 separate tubes. Serum was separated from one tube and used for special investigation as, serum CEA, CA15-3, serum for nucleosomes assay was rapidly added to 10 mM EDTA (pH 8) (1:10 ratio) in a sterile microcentrifuge tube (1.5ml) and mixed thoroughly in order to stabilize the nucleosomes in the serum, and stored at -70C until the day of assay. The rest of venous sample (1ml) was delivered into EDTA containing polypropylene tube, CBC was performed. Fresh tumor and its corresponding safety margin normal tissue was obtained at the same time of surgical resection from primary breast cancer females who underwent radical mastectomy, tissue was rapidly frozen to - 80°C.In our study, HER-2 gene status was determined in breast tissue using differential PCR technique. Amplified HER-2 gene was detected in 14/50 (28%) of all patients, (3/15 (20%) of group I, 5/20 (25%) of group II and 6/15 (40%) of group III), this support its role in oncogenesis. HER-2 gene amplification was correlated with tumor grade; however no significant correlation was found between HER-2 gene amplification with each of studied tumor markers (CEA and CA15-3) as HER-2gene status is considered as independent prognostic marker. In addition there was no significant correlation with tumor size or axillary L.N.Serum nucleosomes levels were assayed in all subjects, using cell death detection ELIZAplus technique, as a marker of apoptosis, our work showed significant increase of nucleosomes levels in patients compared with healthy control, however no significant differences in-between patients groups. Our study showed a negative significant correlation between serum nucleosomes levels with HER-2 gene amplification and positive significant correlation with CA15-3.Whereas no correlation was found between nucleosomes values and other studied parameters.When nucleosomes levels have been compared with other studied tumor markers such as CEA and CA15-3 for specificity and sensitivity, we detected that, the serum nucleosomes was the most sensitive but the least specific.CEA and CA15-3 were preoperatively determined and showed less sensitivity in node negative patients (13.3% and 33.3% respectively), increased in node positive patients to levels ( 20% and 40% respectively) and the sensitivity was high in metastatic patients for CEA and CA15-3, (66.6%and 80% respectively).There was positive significant correlation between CEA and each of following parameters (CA15-3, tumor size, tumor grade and axillary L.N involvement).There was positive significant correlation between CA15-3 and axillary L.N involvement. However no significant correlation between CA15-3 and each of other studied parameters as tumor size and tumor grade, this is may be due to CA15-3 is considered independent prognostic and predictive marker especially in advanced breast cancer.CONCLUSIONHER-2 gene amplification was detected in 28% of breast cancer, and this genetic alteration mostly was among breast cancer patients who rapidly developed distant metastasis before first diagnosis, so amplification of this gene is associated with an increased risk of developing distant metastasis.Nucleosomes levels were increased in breast cancer patient’s sera and had negative correlation with HER-2 gene amplification. In comparison with CEA and CA15-3, nucleosome (as a tumor marker) was more sensitive but less specific, so nucleosomes levels could be used in monitoring of breast cancer patients instead of CEA and CA15-3.RECOMMENDATIONS• Breast cancer is a heterogeneous disease; the tumors grow metastasis and resist therapy through different mechanisms, so identification of genetic markers that sub-classify these tumors could identify patients who would benefit from adjuvant therapy.• HER-2 gene status determination should be routinely done for all patients diagnosed as breast cancer to develop specific therapeutic plane suitable for HER-2 gene status.• The combination of HER-2 gene and protein detection would facilitate recognition of gene/protein discordances.• Further studies to evaluate the correlation between HER-2 gene status in breast tissues and Her-2 shed antigen level in peripheral blood and its role as a prognostic and predictive factor.• HER-2 gene is localized to 17q12.1, so, we recommend further studies for evaluation of HER-2 /neu protein in peripheral blood in cases with malignancies associated with chromosome 17 abnormalities.• Because the determination of nucleosomes can be done in serum or plasma, that is easily obtainable, this method of quantification could also be used in serial measurements; for monitoring patients with malignancies, during therapeutic regimen to evaluate their responsiveness to therapy.
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