| Tumor is the major disease plaguing human health. Due to the highcomplexity, diversity and variability of tumor, it is very difficult to understandthe development mechanism and find the therapeutic methods formalignancies. With the deep understanding for the relationship between tumorand host, especially for the antitumor immune response and tumor immuneescape mechanism, some interventions based on the immune cells, moleculesand genes have been become the hot spots for cancer prevention. Tumorimmunotherapy which has significant clinical therapeutic effect and theadvantages has been proven to be the fourth strategy following surgery,radiotherapy, chemotherapy for malignancies. Recent research shows thatmelanoma antigen genes (MAGEs) have been considered to be the idealmolecular target of tumor immunotherapy due to its unique expression pattern.In recent years, our group committed to the expression pattern ofMAGE-A family in breast cancer. In the present study, we prepared themonoclonal antibodies (mAbs) targeting MAGE-A family membersMAGE-A9and-A11. By using these antibodies, the protein expressions ofMAGE-A9and-A11in breast cancer tissue and the corresponding normalbreast tissue were detected, and their correlation with the clinical/biologicalfactors of breast cancer was also analyzed. In addition, the mRNA expressionsof MAGE-A genes in peripheral blood of breast cancer were detected bymultiple nested RT-PCR. These studies provided a theoretical basis for theprognosis and early detection of breast cancer. The main research contents andresults were shown as follows:Part I Expression and clinical significance of MAGE-A9and-A11inbreast cancer tissue detected with self-made monoclonal antibodiesObjective: To detect the protein expression of MAGE-A9and-A11inbreast cancer tissue using self-made monoclonal antibodies, and investigate their clinical significance in breast cancer patients.Methods:1Antigen epitope screening, expression vector construction, positivecloning identification, positive clone plasmid extraction and transformation,induction expressed production, Ni column affinity purification, animalimmunity, cell fusion fusion and ELISA were used to make MAGE-A9andMAGE-A11mAb, respectively.2Western blot analysis was used to detect mAb specificity.3Immunohistochemical staining was used to detect mAb specificity innormal testis tissue.4Self-made MAGE-A9,-A11mAb were used to detect the proteinexpression of MAGE-A9and MAGE-A11in malignancies of differenthistological origin.5Immunohistochemical staining was used to detect the proteinexpression of MAGE-A9,-A11in breast cancer tissue and the correspondingnormal breast tissue and the clinical/biological factors of breast cancer wereanalyzed.Results:1MAGE-A9and-A11mAbs were successfully obtained.2MAGE-A9protein was detected in normal testis tissues andmalignancies of different histological origin including esophageal cancer,stomach cancer, cervical cancer, endometrial cancer, renal cancer and breastcancer by MAGE-A9mAb. In normal testis tissues, MAGE-A9was mainlyexpressed in cell nuclears of spermatogonium and spermatocyte, particularly.In breast cancer tissues, MAGE-A9protein was mainly expressed incytoplasm, and part of them in cell nuclear. MAGE-A9protein was notexpressed in corresponding normal breast tissue,56.7%(34/60) breast cancertissues showed MAGE-A9protein expression.In breast cancer tissue, MAGE-A9protein expression was correlated withthe expression rates of HER-2(χ~2=6.058, P=0.014) and ER(χ~2=4.344,P=0.037), with increase expression rates of HER-2and ER the MAGE-A9 protein expression rate was also increased, suggesting MAGE-A9protein maybe an important marker for guiding the treatment and prognosis of breastcancer. There were no significant differences between MAGE-A9proteinexpression and patient’s age (χ~2=2.986, P=0.225), pathological types(χ~2=0.162,P=0.983), histological grades (χ~2=0.873, P=0.646), clinical stages(χ~2=0.471,P=0.790), tumor size(χ~2=0.727, P=0.695), metastasis of axillary lymphnodes(χ~2=0.090, P=0.764), PR (χ~2=0.463, P=0.463)and AIB-I (χ~2=0.558,P=0.455).3MAGE-A11were detected by MAGE-A11mAb, expressed in normaltestis tissues and malignancies of different histological origin includingesophageal cancer, stomach cancer, cervical cancer, endometrial cancer, renalcancer and breast cancer. In normal testis tissues, MAGE-A11protein wasexpressed in cell nuclears of spermatogonium and spermatocyte, particularly.In breast cancer tissues, MAGE-A11was mainly expressed in cytoplasm, andpart of them in cell nuclears. MAGE-A11protein was not expressed incorresponding normal breast tissue,53.3%(32/60) breast cancer tissuesshowed MAGE-A11protein expression.In breast cancer tissue, MAGE-A11protein expression was correlatedwith the expression rates of HER-2(χ~2=11.082, P=0.01) and ER (χ~2=4.286,P=0.038), with increase expression rates of HER-2and ER the MAGE-A11protein expression rate was also increased, suggesting MAGE-A11proteinmay be an important marker for guiding the treatment and prognosis of breastcancer. There were no significant differences between MAGE-A11proteinexpression and the age of patients (χ~2=0.375, P=0.837), pathological types(χ~2=0.332, P=0.847), histological grades (χ~2=0.460, P=0.795), clinical stages(χ~2=1.283，P=0.527), tumor size (χ~2=0.662, P=0.718), metastasis of lymphnodes (χ~2=0.357, P=0.550), PR (χ~2=1.558, P=0.212) and AIB-I (χ~2=0.577,P=0.448).4In60breast cancer tissues, MAGE-A9and-A11showed uniformdistribution.The expression rates of MAGE-A9and MAGE-A11protein were56.7%and53.3%, respectively.28of60(46.7%) breast cancer tissues showed MAGE-A9and MAGE-A11expression. The expression rate of these twoproteins was not significantly different in60breast cancer tissues (χ~2=0.323,P=0.570>0.05), suggesting that MAGE expression showed an aggregativecharacteristic.Conclusion:As the tumor specific antigens, MAGE-A9and-A11may be importantmarkers for poor prognosis of breast cancer.Part II Expression of MAGE-A gene in peripheral blood circulatingtumor cells (CTCs) of breast cancer patientsObjective: To detect the expression of MAGE-A gene in peripheralblood circulating tumor cells of breast cancer patients, and investigate theirrelationship with the prognosis.Methods: Multiplex nested RT-PCR was used to detect the level ofMAGE-A mRNA in peripheral blood circulating tumor cells of106breastcancer patients and30health donors. Restriction endonuclease treatment wasused to detect the expression of each member of MAGE-A family, includingMAGE-A1,-A2,-A3,-A4and-A6genes.Results:1The expression rate of MAGE-A gene was40%(43/106) in breastcancer peripheral blood, which was detected by multi RT-nested PCR.Restriction endonuclease treatment was proceeded by Bcl I, EcoRI, Eco47III,Sph I and Afl III matching with MAGE-A1,-A2,-A3,-A4and-A6genes,respectively. The frequency of MAGE-A expression in breast cancerperipheral blood was the following order: A2>A3>A4>A1>A6.The expressionrate of MAGE-A1was10%(11/106), MAGE-A221%(22/106), MAGE-A315%(16/106) MAGE-A413%(14/106)，MAGE-A66%(6/106).28of106breast cancer peripheral blood were positive for only one MAGE-A gene,10of106breast cancer peripheral blood were positive for two genes,2of106breast cancer peripheral blood were positive for three genes,0of106breastcancer peripheral blood were positive for four genes. In three patient (GsY,CfJ, QxR), all five MAGE-A genes tested were expressed. 2MAGE-A gene expression was correlated with multipleclinical/biological factors. With increase of age, the MAGE-A gene expressionrate was also increased (χ~2=26.047, P=0.000). With increase of histologicalgrade, the MAGE-A gene expression rate was also increased (χ~2=5.709,P=0.017). The MAGE-A gene expression rate of breast cancer patients withdistal metastasis was significantly higher than patients without distalmetastasis (χ~2=15.402, P=0.000). The MAGE-A1gene expression rate ofbreast cancer patients with distal metastasis was significantly higher thanpatients without distal metastasis (χ~2=12.981, P=0.000). With increase ofclinical stage, the MAGE-A2gene expression rate was increased (χ~2=12.218,P=0.001). With increase of tumor size, the MAGE-A2gene expression ratewas also increased (χ~2=11.753, P=0.003). With increase of age, the MAGE-A4gene expression rate was also increased (χ~2=6.456, P=0.04). The above resultssuggest the mRNA levels of MAGE-A gene in peripheral blood of breastcancer can be used as an important maker for monitoring the prognosis ofbreast cancer.Conclusions:1MAGE-A gene expression in peripheral blood of breast cancer may beas a important maker for detection of breast cancer CTCs.2The expression of MAGE-A1,-A2and-A4may be correlated withprognosis of breast cancer, so as to do an important marker for monitoring thetreatment and prognosis of breast cancer. |
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