الفهرس 
INTRODUCTION
MammaglobinOnly 14 pages are availabe for public view
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Abstract
Breast cancer is the major common malignancy in women, and its treatment is possible if diagnosed at an early stage. Traditional prognostic factors include the axillary LN status, the tumor size, and histological grade. The prognosis for BC generally depends on its stage, typically graded as I to IV with sub-stages. Although at diagnosis, up to 90% of the patients have disease limited to the breast and axillary lymph nodes, in nearly half the disease will relapse later on, due to progression of clinically occult disease.
Detection of CTCs can provide prognostic as well as therapeutic information and help in identifying patients at high risk for developing metastases. The detection of CTCs in BM is an obvious choice in BC, since a majority of patients will develop bone metastases once dissemination has been detected clinically. Although BM aspiration is an acknowledged clinical method, it remains cumbersome, especially if repetitive examinations are considered. Peripheral blood is another alternative to evaluate a patient for the presence of disseminated epithelial cancer cells.
The aim of this study was to detect human Mammaglobin (h-MAM) mRNA in PB of BC patients as a marker of CTCs. Mammaglobin is a breast specific member of uteroglobin gene family. The h-MAM gene is localized to chromosome 11q13, a region that is amplified in breast carcinoma. The expression of h-MAM is restricted to the adult mammary gland and to mammary tumor cell lines.
This study utilizes a sensitive method to quantify h-MAM mRNA expression in PB samples, using real-time RT–PCR and the fluorescent Taqman assay by using Quantifast predesigned probe assays. This mRNA is supposed to be derived from CTCs, performing critical steps of the metastatic cascade.
Peripheral blood samples were obtained from 32 patients with BC; 22 patients with primary disease (M0:68.7%) and 10 patients with metastatic disease (M+:31.3%). Samples were collected before surgery or implementation of therapy. All patients were treated in Alexandria Main University Hospital and Higher Institute of Research. Clinical and histopathological data from each patient was entered in a database.
Total RNA was extracted from PB leucocytes of the patients and controls and was then reverse transcribed to c-DNA. Then, Quantifast Probe Assay PCR kit (Qiagen, Germany) was used for Mammaglobin gene expression (Secretoglobin SCGB2A2) analysis using predesigned assays together with QuantiFast Kits (2 step quantitative RT-PCR).
Results per PCR reaction were expressed as relative gene expression (RGE), using the delta–delta Ct method. GADPH was used as a house keeping gene (HKG) also called reference gene.
Our results showed that 9 (28.1 %) out of the 32 BC cases were positive for h-MAM. No correlation was found between RGE of h-MAM and menopausal status, ER, PR status and HER2\ neu expression. However, direct significant correlation was found between h-MAM RGE and metastasis, LN status and tumor stage. The h-MAM was detected in the PB of 3 (13.6%) out of 22 patients with non-metastatic operable BC before adjuvant chemotherapy and 6 (60%) out of 10 patients with MBC (p=0.012). Also, it was found that h-MAM RGE increases with increasing tumor stage (p=0.005); stages III & IV have significantly higher h-MAM RGE than stage II (p=0.022). Regarding LN status, a direct correlation was found between RGE and LN status where significant difference was observed between those with <4 LNs involved showing lower RGE than those with ≥4 LNs (p=0.03).
This study has its strengths and limitations. Among its strong points is that blood samples were collected prior to adjuvant chemotherapy or surgery, thus eliminating the effect of drugs and surgery on CTCs levels.
The major limitation of the study was its relatively small sample size, which might be the cause of variation with the results of some other studies. Another limitation of this study was that there was no follow up for the non-metastatic patients who were positive for the CTCs to detect whether and when metastasis might appear clinically.
In conclusion, detection of CTCs by h-MAM gene expression is important in the early detection of metastasis in cases of operable BC. It can influence early intervention and selection of therapies in those patients, thus prolonging OS.