The combination of mammaglobin A and TWIST-1 increases detection of circulating tumor cells in breast cancer / A combinação de mamaglobina A e TWIST-1 aumenta a detecção de células tumorais circulantes no câncer de mama

ABSTRACT Objectives: Breast cancer cells that are released into the bloodstream are called circulating tumor cells (CTCs). CTCs can express different genes, like TWIST-1 and mammaglobin A (MGA). The aims of this study were to analyze the expression of TWIST-1 and MGA in the blood of breast cancer patients to detect CTCs and to assess the association between the presence of CTCs and prognostic parameters of breast cancer. Methods: Prospective study. Total ribonucleic acid (RNA) from blood mononucleated cells was obtained from breast cancer patients (n = 36; age: 51.5 ± 12.5 years) and healthy donors (n = 14; age: 49.4 ± 9.4 years). Real-time polymerase chain reaction (RT-PCR) was performed to analyze the expression of TWIST-1 and MGA. Results: Patient carcinomas - ductal (86.7%), other types (13.3%). MGA gene expression was not detected in the donors' samples, while it was detected in 14% of the patient samples. Overexpression of TWIST-1 gene was observed in 17% of the patient samples. The combined analysis of both markers allowed the detection of CTCs in 27.8% of the samples, resulting in a significant (p < 0.05) sensitivity increase of detection. No significant associations (p > 0.05) were found between expression of the analyzed genes and the breast cancer prognostic factors. Conclusion: Combined analysis of TWIST-1 and MGA increased the sensitivity of CTCs detection compared to the single analysis of each gene. The detection of CTCs was not associated with known prognostic factors, suggesting that it is able to provide clinical information in addition to routine breast cancer clinicopathological parameters.

RESUMEN Objetivos: Las células de cáncer de mama liberadas al torrente sanguíneo se llaman células tumorales circulantes (CTCs). Las CTCs pueden expresar diferentes genes, como TWIST-1 y mamaglobina A (MGA). Los objetivos de este estudio fueron analizar la expresión de TWIST-1 y MGA en la sangre de pacientes con cáncer de mama (CM) para detectar CTCs y evaluar la asociación entre la presencia de CTCs y los parámetros pronósticos del CM. Métodos: Estudio prospectivo. Se obtuvo el ácido ribonucleico (ARN) de las células mononucleadas en la sangre de pacientes con CM (n = 36, edad: 51,5 ± 12,5 años) y donantes sanas (n = 14; edad: 49,4 ± 9,4 años). Se realizó reacción en cadena de la polimerasa en tiempo real (RT-PCR) para analizar la expresión de TWIST-1 y MGA. Resultados: Carcinoma ductal (86,7%), otros tipos (13,3%). No se detectó la expresión del gen MGA en las muestras de las donantes, pero en el 14% de las muestras de las pacientes. Se observó elevada expresión de TWIST-1 en el 17% de las muestras de pacientes con CM. El análisis combinado de ambos marcadores permitió detección de CTCs en el 27,8% de las muestras, resultando en un aumento significativo (p < 0,05) en la sensibilidad de detección. No se encontraron asociaciones significativas (p > 0,05) entre la expresión de los genes y los factores pronósticos. Conclusión: El análisis combinado de TWIST-1 y MGA aumentó la sensibilidad de detección de CTCs en comparación con el análisis de cada gen. La detección de CTCs no se asoció a factores pronósticos conocidos, sugiriendo que podría ofrecer informaciones clínicas adicionales a los parámetros clínico-patológicos de rutina del CM.

RESUMO Objetivos: As células cancerígenas da mama liberadas na corrente sanguínea são chamadas de células tumorais circulantes (CTCs). As CTCs podem expressar diferentes genes, como TWIST-1 e mamaglobina A (MGA). Os objetivos deste estudo foram analisar a expressão de TWIST-1 e MGA no sangue de pacientes com câncer da mama (CM) para detectar CTCs e avaliar a associação entre a presença de CTCs e os parâmetros prognósticos do CM. Métodos: Estudo prospectivo. O ácido ribonucleico (RNA) das células mononucleadas no sangue foi obtido de pacientes com CM (n = 36, idade: 51,5 ± 12,5 anos) e doadoras saudáveis (n = 14; idade: 49,4 ± 9,4 anos). Reação da cadeia da polimerase em tempo real (RT-PCR) foi realizada para analisar a expressão de TWIST-1 e MGA. Resultados: Carcinoma ductal (86,7%), outros tipos (13,3%). A expressão do gene MGA não foi detectada nas amostras das doadoras, mas foi observada em 14% das amostras das pacientes. Superexpressão de TWIST-1 foi observada em 17% das amostras dos indivíduos com CM. A análise combinada de ambos os marcadores permitiu a detecção de CTCs em 27,8% das amostras, resultando em um aumento significativo (p < 0,05) na sensibilidade da detecção. Associações significativas (p > 0,05) entre a expressão dos genes e os fatores prognósticos não foram encontradas. Conclusão: A análise combinada de TWIST-1 e MGA aumentou a sensibilidade da detecção de CTCs em comparação com a análise de cada gene. A detecção de CTCs não foi associada a fatores prognósticos conhecidos, sugerindo que ela pode fornecer informações clínicas adicionais aos parâmetros clinicopatológicos de rotina do CM.

Mammary analogue secretory carcinoma: An Indian experience of a novel entity

Aims: To explore clinical, histopathological and immunohistochemistry (IHC) features of mammary analogue secretory carcinoma (MASC) with systematic literature review. Settings and Design: Hospital based cross-sectional study. Subjects and Methods: The data of all cases of MASC diagnosed over a period of 1 year i.e., from July 2017 to July 2018 were retrieved. The haematoxylin and eosin (H and E) sections, and IHC sections were studied. A strict histological and recently updated criteria were applied and patients with a confirmed diagnosis of MASC were included in the study. A systematic literature review was conducted by searching the PubMed and National Centre for Biotechnology Information database. Statistical Analysis Used: Microsoft Excel 2010. Results: The present case series is 27th in the English literature and 1stcase series describing its histopathology in the Indian literature. The mean age of presentation is 43 years. Female preponderance was found i.e., M:F ratio of 0.5. Conclusion: Histopathology and if necessary, followed by IHC is required for the confirmation of diagnosis of MASC. We should be aware about this recently described entity which is usually mistaken for other low grade salivary gland carcinomas like Acinic cell carcinoma (AciCC) and Mucoepidermoid carcinoma (MEC). The knowledge about its typical morphology, high degree of suspicion and IHC confirmation with both S-100 and Mammaglobin help in precise diagnosis.

Cytotoxic activity of mammaglobin A-specific CD8+ cytotoxic T lymphocytes and cytokine-induced killers against breast cancer cells in vitro / 西安交通大学学报(医学版)

Objective: To compare the cytotoxic activity of mammaglobin A-specific CD8+ cytotoxic T lymphocytes (CTLs) and cytokine-induced killers (CIKs) against breast cancer cells in vitro. Methods: PBMCs were isolated in vitro from the peripheral blood of healthy volunteers. Then DCs, CTLs and CIKs were isolated, induced and cultured from PBMCs in vitro. CD8+ CTLs were purified with immunomagnetic beads from CTLs. DCs were infected with recombinant adenovirus encoding mammaglobin A(Ad-MGBA). CTLs and CIKs were co-cultured with DCs being infected with Ad-MGBA. The cytotoxic activity of mammaglobin A-specific CD8+ CTLs and CIKs against breast cancer cells was compared by flow cytometry. Results: The apoptosis rate of breast cancer cell MDA-MB-415, which expressed MGBA was 63.07% by CD8+ CTL killing and 48.35% by CIK killing (P<0.05). The apoptosis rate of breast cancer cell MDA-MB-231, which could express MGBA was 14.62% by CD8+CTL killing and 29.29% by CIK killing (P<0.05). Conclusion: The cytotoxic activity of antigen-specific CD8+ CTLs for the same antigen-expressing oncology cells is higher than CIKs. However, for different antigen-expressing oncology cells, the cytotoxic activity of CD8+CTLs is lower than that of CIKs.

Detection of serum hMAM and CD147 levels of patients with breast cancer and its clinical significance / 吉林大学学报(医学版)

Objective: To explore the serum levels and the positive expression rates of human mammaglobin (hMAM) and extracellular matrix metalloproteinase inducer (CD147) of the patients with breast cancer, and to clarify their clinical significnaces in the diagnosis of breast cancer. Methods: A total of 122 patients with breast cancer (breast cancer group), 21 patients with breast fibroadenoma (breast fibroadenoma group) and 16 healthy controls (healthy control group) were selected as the subjects. The serum samples of subjects in various groups were collected. The serum levels and the positive expression rates of hMAM and CD147 of the subjects in various groups were measured by ELISA method. The serum levels and the positive expression rates of hMAM and CD147 of the breast cancer patients with different clinicopathological features were analyzed. The cut-off values of serum levels of hMAM and CD147 of the patients with breast cancer and their sensitivities and specificities in the dignosis of breast cancer were comfirmed by receiver operating characteristic (ROC) curve. Results: The serum levels of hMAM and CD147 of the patients in breast cancer group were higher than those in healthy control group and breast fibroadenoma group (P<0. 05). The positive expression rates of hMAM and CD147 of the patients in breast cancer group were higher than those in healthy control group and breast fibroadenoma group (P<0. 01); while the positive expression rates of serum hMAM combined with CD147 of the patients in breast cancer group were higher than those in healthy control group and breast fibroadenoma group (P<0. 01). There were significant differences in the positive expression rates of serum hMAM and CD147 of breast cancer patients with lymph node metastasis or not (χ2=10. 375, P<0. 01; χ2=15. 556, P<0. 01). There were significant differences in the positive expression rates of serum CD147 of the breast cancer patients with different TNM stages, ER and Her-2 (χ2= 8157, P<0.05; χ2= 6. 035, P<0. 05;χ2 = 5. 385, P<0. 05). With the increasing of TNM stages, the positive expression rates of serum hMAM and CD147 were increased. The area under ROC curve (AUC) of serum level of hMAM was 0. 809, 95%CI; 0.703-0.916; the AUC of serum level of CD147 was 0. 721, 95% Cl: 0.582-0.861. When the cut-off value of hMAM was 6. 51 μg · L-1, its sensitivity and specificity were 61. 1% and 87. 5%, respectively. The sensitivity and specificity were 73.6% and 68.7% respectively when the cut-off value of CD147 was 144. 92 ng · L-1. The AUC of detection of hMAM combined with CD147 was 0. 880, 95% Cl: 0. 798-0. 962; the sensitivity and specificity were 80. 6% and 81. 2%, respectively. Conclusion; The serum level of hMAM has the high sensitivity and specificity in the diagnosis of breast cancer. Combined detection of hMAM and CD147 can improve the positive rate of diagnosis.

Synthesis and Characterization of Immunomagnetic Nanoparticles Modified by Monoclonal Antibodies Against Human Breast Mammaglobin / 中国药学杂志

OBJECTIVE: To synthesize immunomagnetic nanoparticles with uniform particle size, strong superamagnetism as well as strong immune activity which can be specifically and sensitively combined with circulating tumor cells in peripheral blood of patients with breast cancer.METHODS: Superparamagnetic oxide iron nanoparticles containing active carboxyl groups (SMNP-COOH) were synthesized by polyol methods, thermogravimetric analysis was used to determine the amount of carboxyl groups on the surface of SMNP-COOH, while the content of iron was determined by o-phenanthroline. Mediated by 1-ethyl-3,3-dimethylaminopropyl carbodiimide(EDC) and N-hydroxysuccinimide (NHS), immunomagnetic nanoparticles(IMNP) against human breast carcinoma cell line were constructed by binding the monoclonal antibodies against hMAM with SMNP-COOH. X-Ray diffraction was used to confirm their synthesis,meanwhile,transmission electron microscope (TEM), dynamic light scattering (DLS), and vibrating sample magnetometry (VSM) were applied to characterize their physicochemical properties. The conjugation amount of the antibodies and the activity of IMNPs were evaluated by enzyme linked immunosorbent assay (ELISA).RESULTS: X-Ray diffraction showed that the chracteristic peaks of the crystalline powder of SMNP-COOH and IMNP agreed with the Fe3O4 standard. The concentration of iron in SMMP-COOH and IMNP were 0.205 and 0.164 mol•L-1, respectively.TEM showed that both synthesized SMNP-COOH and IMNP were almost spherical or ellipsoidal. The sizes of SMNP-COOH and IMNP were (13.7±3.6) and (15.4±4.5) nm, respectively. Dynamic light scattering(DLS) demonstrated the intensity particle size and polydispersity index (PDI) of SMNP-COOH and IMNP were 23.4 nm and 0.303, and 71.2 nm and 0.175,respectively. VSM results showed that both SMNP-COOH and IMNP had strong superamagnetism, and the saturation magnetization of SMNP-COOH and IMNP were 71.37 and 67.68 emu•g-1Fe, respectively, which confirmed antibody binding may reduce the magnetism of SMNP-COOH. The ELISA results showed the conjugation amount of antibody was about 93 μg on 1 mg SMNP-COOH by covalent bond. The obtained immunomagnetic nanoparticles (IMNP) which were bound with the hMAM monoclonal antibodies could specifically and sensitively combine with breast cancer cell line MDA-MB-415.CONCLUSION: IMNP with strong superparamagnetic property,excellent stability and perfect antibody activity were successfully synthesized, which demonstrate the potential to magnetically separate circulating tumor cells in peripheral blood from patients with breast cancer, thus providing a favorable weapon to accurately detect CTCs in breast tumor patients.

Hsa-miRNA-143-3p Reverses Multidrug Resistance of Triple-Negative Breast Cancer by Inhibiting the Expression of Its Target Protein Cytokine-Induced Apoptosis Inhibitor 1 In Vivo / 한국유방암학회지

PURPOSE: Multidrug resistance (MDR) remains a major obstacle in the treatment of triple-negative breast cancer (TNBC) with conventional chemotherapeutic agents. A previous study demonstrated that hsa-miRNA-143-3p plays a vital role in drug resistance of TNBC. Downregulation of hsa-miRNA-143-3p upregulated the expression of its target protein cytokine-induced apoptosis inhibitor 1 (CIAPIN1) in order to activate MDR, while upregulation of hsa-miRNA-143-3p effectively enhances the sensitivity of drug-resistant TNBC cells to chemotherapeutics. The present study aimed to further verify these findings in vivo. METHODS: We established a hypodermic tumor nude mice model using paclitaxel-resistant TNBC cells. We expressed ectopic hsa-miRNA-143-3p under the control of a breast cancer-specific human mammaglobin promoter that guided the efficient expression of exogenous hsa-miRNA-143-3p only in breast cancer cells. Thereafter, we overexpressed hsa-miRNA-143-3p in xenografts using a recombinant virus system and quantified the expression of hsa-miRNA-143-3p, CIAPIN1 protein, and proteins encoded by related functional genes by western blot. RESULTS: We successfully completed the prospective exploration of the intravenous virus injection pattern from extensive expression to targeted expression. The overexpression of hsa-miRNA-143-3p significantly alleviated chemoresistance of TNBC by inhibiting viability. In addition, we observed that the expression of CIAPIN1 as a hsa-miRNA-143-3p target protein was remarkably decreased. CONCLUSION: We partly illustrated the mechanism underlying the hsa-miRNA-143-3p/CIAPIN1 drug resistance pathway. HsamiRNA-143-3p as a tumor suppressive microRNA may be a novel target to effectively reverse MDR of TNBC in vivo.

Application of detection of expressions of hMAM combined with MMP-9 and C-erbB2 mRNA in peripheral blood in diagnosis of micrometastases of breast cancer / 吉林大学学报(医学版)

Objective:To explore the positive expressions of biological markers human mammaglobin (hMAM) combined with matrix metallopeptidase 9 (MMP-9) and human epidermal growth factor receptor 2 (C-erbB2) mRNA in peripheral blood of the breast cancer patients with micrometastases,and to clarify its clinical application value in diagnosis of the micrometastases in peripheral blood of the breast cancer patients.Methods:A total of 74 patients with breast cancer,21 patients with breast fibroadenoma and 10 healthy controls were selected as the subjects.All the patients received surgical treatment and the peripheral blood was collected.The mRNA expression levels of hMAM,MMP-9 and C-erbB2 in peripheral blood were measured by the real-time fluorescent quantitative PCR.The positive expression rates of detection of hMAM,MMP-9 and C-erbB2 were compared,and the differences in detection of hMAM combined with MMP-9 and C-erbB2 between the patients with different clinicopathologic features were analyzed.Results:In the breast cancer patients with lymph node metastasis,the differences of positive expression rates of MMP-9 and C-erbB2 mRNA were significant (x2=6.450,P＜0.05;x2=5.636,P＜0.05),and the difference of positive expression rate of hMAM mRNA was sigificant between HER-2 positive and negative patients (x2=5.804,P＜0.05).The positive expression rates of individual hMAM and combined with MMP-9 and C-erbB2 were 37.84％ (28/74),59.46％ (44/74) and 48.65％ (36/74) in the breast cancer patients,the combined postive expression rate of these three kinds of markers was 64.86 ％ (48/74),which were higher than those in healthy controls group (x2=5.676,P＜0.05;x2=3.102,P＞0.05;x2=5.339,P＜0.05;x2 =2.310,P＞0.05),fibroadenoma of breast group (x2 =8.438,P＜0.01;x2 =4.491,P＜ 0.05;x2 =7.982,P＜0.01;x2 =4.844,P＜0.05) and non-breast cancer group (healthy controls group+ breast fibroadenoma group) (x2 =13.093,P＜0.01;xx2 =6.471,P＜0.05;x2 =11.837,P＜0.01;x2 =6.103,P＜ 0.05).The positive expression rates of individual hMAM and the joint detection in the breast cancer patients at stage Ⅲ + Ⅳ were higher than those in the patients at stage Ⅰ + Ⅱ;the positive expression rates of individual hMAM and combined with C-erbB2 were statistically significant (x2 =5.157,P＜0.05;x2 =4.912,P＜0.05).Conclusion:hMAM has a low positive rate in the diagnosis of micrometastases in the breast cancer patients,while hMAM combined with MMP-9 and C-erbB2 detection could improve the positive rates.which presents some clinical application value for the early diagnosis of breast cancer micrometastases.

Application of detection of expressions of hMAM combined with MMP-9 and C-erbB2 mRNA in peripheral blood in diagnosis of micrometastases of breast cancer / 吉林大学学报(医学版)

Objective:To explore the positive expressions of biological markers human mammaglobin (hMAM) combined with matrix metallopeptidase 9 (MMP-9) and human epidermal growth factor receptor 2 (C-erbB2) mRNA in peripheral blood of the breast cancer patients with micrometastases,and to clarify its clinical application value in diagnosis of the micrometastases in peripheral blood of the breast cancer patients.Methods:A total of 74 patients with breast cancer,21 patients with breast fibroadenoma and 10 healthy controls were selected as the subjects.All the patients received surgical treatment and the peripheral blood was collected.The mRNA expression levels of hMAM,MMP-9 and C-erbB2 in peripheral blood were measured by the real-time fluorescent quantitative PCR.The positive expression rates of detection of hMAM,MMP-9 and C-erbB2 were compared,and the differences in detection of hMAM combined with MMP-9 and C-erbB2 between the patients with different clinicopathologic features were analyzed.Results:In the breast cancer patients with lymph node metastasis,the differences of positive expression rates of MMP-9 and C-erbB2 mRNA were significant (x2=6.450,P＜0.05;x2=5.636,P＜0.05),and the difference of positive expression rate of hMAM mRNA was sigificant between HER-2 positive and negative patients (x2=5.804,P＜0.05).The positive expression rates of individual hMAM and combined with MMP-9 and C-erbB2 were 37.84％ (28/74),59.46％ (44/74) and 48.65％ (36/74) in the breast cancer patients,the combined postive expression rate of these three kinds of markers was 64.86 ％ (48/74),which were higher than those in healthy controls group (x2=5.676,P＜0.05;x2=3.102,P＞0.05;x2=5.339,P＜0.05;x2 =2.310,P＞0.05),fibroadenoma of breast group (x2 =8.438,P＜0.01;x2 =4.491,P＜ 0.05;x2 =7.982,P＜0.01;x2 =4.844,P＜0.05) and non-breast cancer group (healthy controls group+ breast fibroadenoma group) (x2 =13.093,P＜0.01;xx2 =6.471,P＜0.05;x2 =11.837,P＜0.01;x2 =6.103,P＜ 0.05).The positive expression rates of individual hMAM and the joint detection in the breast cancer patients at stage Ⅲ + Ⅳ were higher than those in the patients at stage Ⅰ + Ⅱ;the positive expression rates of individual hMAM and combined with C-erbB2 were statistically significant (x2 =5.157,P＜0.05;x2 =4.912,P＜0.05).Conclusion:hMAM has a low positive rate in the diagnosis of micrometastases in the breast cancer patients,while hMAM combined with MMP-9 and C-erbB2 detection could improve the positive rates.which presents some clinical application value for the early diagnosis of breast cancer micrometastases.

Immunohistochemically stained sections against different anti-Mam-A epitopes for in situ breast cancer diagnosis / 军事医学

Objective To investigate the difference between mammary gland tissues and breast cancer tissues.Methods Monoclonal antibodies against Mam-A immunized epitopes were screened for immunohistochemical staining of normal breast tissues and breast cancer tissues.The average optical density was used as an index to identify the quantitative data by computer-aided technology to screen epitope-specific antibodies with significant difference in staining characteristics between two types of tissues.Furthermore the feasibility and effectiveness of breast cancer diagnosis were evaluated.Results Four anti-Mam-A epitope-specific monoclonal antibodies,mAb1152,mAb11617,mAb995 and mAb656,were obtained.Immunohistochemical staining showed that the average density of mAb1152,mAb11617 and mAb995 was significantly different between the two types of tissues.The difference was significant between normal breast tissues and breast cancer tissues under the same conditions.The results showed that mAb11617 was better than mAb1152 and mAb995.At the best working point,mAb11617 was the best,the specificity was 90％ and the sensitivity was 59.62％.Further analysis showed that the sensitivity of mAb11617 combined with mAb995 in the diagnosis of in situ breast cancer was 81.48％ and the specificity was 90％,which was of great diagnostic significance.Conclusion There is significant difference between breast tissues and breast cancer tissues in Mam-A protein immunological activity or expression.This difference,which can be recognized by the specific antibody staining and computer aided technology,is of important diagnostic value.

Human mammaglobin in breast cancer: a brief review of its clinical utility.

Human mammaglobin is a member of the uteroglobin proteins family that has recently been tested as a specific marker for breast cancer. While low levels may be seen in normal breast tissue, expression is increased dramatically in breast cancer and is correlated with higher grade. Detection in blood and body fluids is also correlated with cancer metastasis, and its levels with prognosis. This promises to be a useful screen for early detection of breast cancer, especially in high risk individuals. Mammoglobin has also been used for immunotherapeutic targeting of breast cancer cells. However, there are some controversies regarding its diagnostic efficacy and prognostic value, which warrant further study.

Clinical value of hMAM combined with CK19 detection for circulating tumor cells in patients with breast cancer / 肿瘤

Objective: To explore the clinical value of cytokeratin 19 (CK19) and human mammaglobin (hMAM) in the detection of circulating tumor cells (CTCs) in peripheral blood of patients with breast cancer. Methods: Expressions of CK19 and hMAM in blood were identified by real-time fluorescent quantitative PCR, and the correlations between CK19, hMAM and the clinicopathologic features were analyzed. Results: In breast cancer patients, the rates of diagnostic positive detection of individual CK19 or hMAM and the combination of these two biomarkers were 45.5% (20/44), 38.6% (17/44) and 56.8% (25/44), respectively. The expression of combined biomarkers was correlated with lymph node metastasis (P = 0.031). The rates of diagnostic positive detection of CK19, hMAM and the combination biomarkers were significantly higher in breast cancer patients prior to treatment than those in the healthy control group (P = 0.000 3, P = 0.000 1 and P = 0.000 1, respectively). After two-cycle chemotherapy, CTCs which were positive at baseline presented as negative in 5 stage III patients and 4 stage IV patients (P = 0.025 3 and P = 0.045 5). However, the number of CTCs-positive patients at stagesI-II decreased no matter using CK19, hMAM or CK19 plus hMAM as biomarker, leaving more CTCs-positive patients in combined biomarker group than that in single biomarker group, but there was no statistical significance. Conclusion: The combined panel of both hMAM and CK19 may serve as representative biomarkers for CTCs, thus it presents potentially significant value for monitoring early metastasis, therapeutic efficacy and prognosis for the patients with breast cancer. Copyright © 2014 by TUMOR.

Expression of hMAM, VEGF-C and VEGFR-3 in breast cancer tissues and its relation with prognosis / 第二军医大学学报

Objective To study the expression of human-mammaglobin (hMAM) and vascular endothelial growth factor C (VEGF-C) and vascular endothelial growth factor receptor 3 (VEGFR-3) in breast cancer tissues, and to analyze its relationship with biological characteristics and prognosis of breast cancer. Methods The expression of hMAM, VEGF-C andVEGFR-3 was determined by immunohistochemical technique in 116 breast cancer tissues and 44 adjacent normal tissues (from cancer tissue≥ 5 cm) using tissue microarray technology (TMA). Results (1) The positive rates of hMAM, VEGF-C and VEGFR-3 in primary breast carcinoma tissues were significantly higher than those in the adjacent normal tissues (59. 48% [69/116] vs 0. 00%, 50. 86% [59/116] vs 9. 09% and 61. 20% [71/116] vs 18. 18%, respectively, all P<0. 01). (2)The positive rates of hMAM, VEGF-C and VEGFR-3 were significantly correlated with axillary lymph node metastasis, and the positive rates of hMAM, VEGFR-3 were significantly correlated with histological gradings (all P<0. 05). (3) Spearman rank correlation analysis showed that the positive rates of hMAM, VEGF-C and VEGFR-3 in breast cancer tissues were all significantly correlated with each other, with correlation coefficients being 0. 278, 0. 280, and 0. 244, respectively, all P<0. 05). (4)Kaplan-Meier survival analysis showed that the disease-free survival and 5-year overall survival of patients with negative expression of HMAM, VEGF-Cand VEGFR-3 were significantly prolonger compared to those with positive expression (Log-rank test, P< 0. 05). Conclusion hMAM, VEGF-C and VEGFR-3 are highly expressed in breast cancer patients, which might be associated with the invasion and metastasis of breast cancer, and hMAM expression might be related to the lymphangiogenesis of breast cancer.

Expression of human mammaglobin and clinicopathologic correlations in breast cancer: The findings in Malaysia.

Background: Human mammaglobin (hMAG) is a secreted protein which has been detected in breast epithelial cells of mammary glands and has been used as a specific marker for breast cancer. Objectives: This study aims at studying the hMAG expression and identifying the significant predictors of hMAG expression in breast cancer tissues. Materials and Methods: The tissue samples were obtained from two major teaching hospitals in the country. They were examined by immunohistochemistry (IHC) and the hMAG expression was evaluated using an established scoring system. Results: Out of 84 breast cancer tissue samples, hMAG was expressed in 50 samples (59.6%). The expression of hMAG was found to be increased with cancer grade. The output of logistic regression model showed that hMAG was overexpressed in breast cancer samples from the first hospital (P = 0.014), but not with those from the second hospital. Conclusions: It can be concluded that hMAG may serve in the diagnosis and the assessment of progression with the increased cancer grade. The dominance in hMAG expression in samples from HUSM may correlate with ethnic, environmental or genetic factors.

Immunohistochemical expression and correlation of mammaglobin with the grading system of breast carcinoma.

Objectives: In this study it was intended to study mammaglobin expression as a marker for the detection of breast cancer and correlate it with the Bloom-Richardson grading system of breast carcinoma. Materials and Methods: The study was conducted from May 2007 to May 2008. Tissue samples were collected from 50 patients of breast cancer in the various stages of their disease and correlated histologically with the Bloom-Richardson grading system for breast carcinoma. The clinical data of the patients were obtained from their respective files. Results: Positive immunostaining for mammaglobin was seen in 84% of breast carcinoma cases. This immunoreactivity did not correlate with histological and nuclear grades of the tumors, yet it varied according to the histological type of the tumor with ductal carcinoma showing stronger and diffuse staining than other varieties. Conclusion: These results elicit that mammaglobin is overexpressed in carcinoma breast as compared to the normal breast epithelium. This mammaglobin expression can act as a useful tool in the diagnosis of women with breast cancer.

The role of bone marrow micrometastasis in breast cancer in judging prognosis / 肿瘤研究与临床

Objective To investigate the relationship between bone marrow micrometastasis of patients with breast cancer and clinical pathological parameters, some molecular markers as well as prognosis.Methods The expression of hMAM mRNA in BM of patients with breast cancer was detected by RT-PCR.The expressions of ER, PR in cancer tissues were detected by immunohistochemical SP method. Results About 38.2 % positive expression rate of hMAM mRNA in 102 patients with stage Ⅰ -Ⅳ breast cancer was found.The expression of hMAM increased more in patients with T2-3 (＞2 cm) tumors than T1 (≤2 cm) (P =0.001)and with stage Ⅱ ,Ⅲ than stage Ⅰ (P =0.001). The expression of hMAM in the BM of breast cancer with grade I was lower than that of grade Ⅱ or Ⅲ (P =0.014). The expression of hMAM in the BM was related to the pathological type (P =0.032) and the axillary lymph node metastasis (P =0.001). The expressions of hMAM in BM were much higher with ER negative in breast cancer tissues (P ＜0.05). There was a correlation between patients with positive expression of hMAM in BM and distant metastasis (P =0.009). Conclusion The micrometastasis in BM is correlated with some clinical pathological parameters and some tumor markers. The patients with positive expression of hMAM in BM have more chances with distant metastasis and poor prognosis. The detection of micrometastasis may be as one of targets to predict the prognosis of breast cancer.

The Establishment and Application of the Method Detecting hMAM Gene Expression of Breast Cancer Cell Lines in Peripheral Blood / 医学研究杂志

Objective To develop a method detecting hMAM gene expression of breast cancer cell lines in peripheral blood.Methods The hMAM gene was cloned and confirmed by agarose gel electrophoresis and sequence analysise,the standard substance of real-time quantitative polymerase chain reaction (FQ-PCR )was prepared. The breast cancer cell lines MDA-MB453、MCF7 were cultured and the blood sample model was made.The hMAM expression of breast cancer cell lines blood model and 10 cases of breast cancer patient peripheral blood were detected by RT-PCR and FQ-PCR,GAPDH gene expression as contrast.Results The production of PCR was cloned and testified by sequence.The correlation coefficient(r) of standard curve line of FQ-PCR was -1.0. The x?s and CV were 718.9?120.5 and 16.7% respectively.The hMAM expression of one breast cancer cell lines MDA-MB453 in 104 blood cells could be detected, but not detected in the breast cancer cell lines MCF7. The hMAM expression was detected in 3 of 5 patients with sentinel nodes and in 1 of 5 patients with non-sentinel nodes.Conclusions It might be a good method for detecting breast cancer cell micrometastased in peripheral blood. The diffrernce of hMAM gene expression correlates with the type of breast cancer cell lines.

Establishment of FQ-PCR for determining mammaglobin mRNA and implication for monitoring micrometastasis of patients with breast cancer / 临床检验杂志

0.05).There were obvious differences between breast cancer group and benign breast diseases group,other cancers group or healthy persons group in the expression of hMaM mRNA(?~2=8.96,13.49 and 10.32 respectively,P

Prognostic Value of Bone Marrow Micrometastasis Detected by Nested RT-PCR for Cytokeratin 19 and Mammaglobin in Breast Cancer

PURPOSE: Breast cancers frequently undergo distant metastasis during the early phase, on which the survival of patients is greatly dependent. It has been suggested that the occurrence of micrometastasis relates with other prognostic features of breast cancer, such as lymph node metastasis and the presence of vascular invasion. The aim of this study was to examine the presence of keratin-19 and mammaglobin mRNA in bone marrow aspirates obtained from breast cancer patients, and their possible correlation with tumor staging and disease free survival. METHODS: Bone marrow samples were obtained from 254 breast cancer patients at the time of surgery. We separated the mononuclear fraction from the samples and carried out nested reverse transcriptase polymerase chain reaction for the detection of keratin-19 and mammaglobin mRNA using two different pairs of primers. We also studied the possible correlations between the tumor size, nodal involvement, stage, and distant metastasis. RESULTS: Seventy-five of the 254 samples were studied for cytokeratin 19 and the others for cytokeratin and mammaglobin. The median follow-up time was 21.1 months. Sixty-five (26%) of the 254 samples were cytokeratin 19 positive and 25 (14.3%) of the 175 were mammaglobin positive. Eight cases (12.3%) in the cytokeratin positive group showed a recurrent disease in distant organs. Whereas, six (3.2%) out of 185 cytokeratin negative patients had distant recurrences. Mammaglobin positivity was not correlated with distant metastasis. The stage, nodal status, and estrogen receptor were independent of bone marrow micrometastasis. CONCLUSION: Bone marrow micrometastasis, detected by nested RT-PCR for cytokeratin 19, could be a useful predictive marker for the distant metastasis of breast cancer.

Molecular detection of the mammaglobin expression in the peripheral blood of Thai breast cancer patients.

Mammaglobin (hMAM) is expressed exclusively in the mammary glands of adult woman and mammary tumour cell lines. Thus, we examined hMAM expression as a market for the detection of carcinoma cells in the peripheral blood of patients with breast cancer in Thailand. In addition, we studied the correlation between hMAM expression in circulation mammary carcinoma cells and clinicopathological prognostic factors of breast cancer. Blood sample obtained from two hundred breast cancer patients at various stages of their disease and from sixty females without breast cancer (thirty healty individuals and thirty patients with various malignancies other than breast cancer) were screened for hMAM mRNA by a nested reverse transcriptase polymerase chain reaction (RT-PCR) assay. We found significant differences between patients with breast cancer and those with other malignancies or healthy controls. None of the samples from the peripheral blood of sixty females without breast cancer was positive, whereas sixty four (32%) of the two hundred sample from breast cancer patients tested positive for hMAM mRNA. While our hMAM nested RT-PCR approach has 100% specificity, its sensitivity is only 32%. The presence or absence of hMAM expression in these breast cancer patients was not associated with clinicopathological prognostic factors including stage, oestrogen and progesterone status, lymph node metastases, histological type, tumour size, differentiation, lymphatic invasion, vascular invasion, menopausal status or age. We summarized that the hMAM nested RT-PCR assay may be an effective tool for the detection of circulating mammary carcinoma cells of breast cancer patients. Nevertheless, the clinical relevance hMAM RT-PCR based tumour cell detections should be further evaluated in prospective studies.

Prokaryotic expression and purification of two human mammaglobin gene subtypes / 医学研究生学报

Objective:To clone the cDNA in full-length of human mammaglobin,do prokaryotic expression and purify hMAM protein,as a basis for early diagnosis of breast cancer.Methods :hMAM cDNA was amplified through RT-PCR from breast cancer tissue and breast cancer cell line MD-MB453,and the recombination pQE40-hMAM vector was constructed and expressed in E.Coli.M15 after induction by IPTG.The fusion protein was purified with Ni-NTA-His affinity chromatography. Results: Two subtypes of hMAM cDNA and the hMAM(Isoform) cDNA were found,in which consisted of 270 bp,different from the wildtype hMAM cDNA of 279 bp on nine continuous base pair missing.The fusion protein formed inclusion body in prokaryotic expression system and the renatured protein was purified which purity was about 97%.Conclusion: The recombinant hMAM was successfully purified.