OBJECTIVETo evaluate the application of the immunohistochemistry (IHC) and the fluorescence in situ hybridization (FISH) in detecting the amplification and the expression of HER-2 gene in the breast cancer patients.

METHODSSixty-six cases of paraffin-embeded breast cancer samples with overexpression, low or no expression of HER-2 gene as detected by IHC were analyzed for HER-2 gene amplification using FISH.

RESULTSAmong the 42 samples with HER-2 gene overexpression (3+/2+) detected by IHC, 31 showed positive HER-2 gene amplification and 11 showed negative HER-2 gene amplification in FISH. In the 24 samples with low or no HER-2 gene expression (1+/-) detected by IHC, no HER-2 gene amplification was detected by FISH. The results of the two testing methods showed a good consistency with the kappa coefficient of 0.672 (P<0.001). We also found that the 17 chromosome polysomy in 42% of the samples and the incidence of 17 polysomy was significantly higher in the HER-2 gene overexpression (3+/2+) group than in low or no HER-2 gene expression (1+/-) group (chi(2)=4.688, P=0.03).

CONCLUSIONIHC can be used as a screening method for detecting HER-2 gene amplification, and FISH should be performed in cases of HER-2 gene overexpression (3+/2+) as detected by IHC.

Breast Neoplasms ; genetics ; metabolism ; Carcinoma, Ductal, Breast ; genetics ; metabolism ; Female ; Humans ; Immunohistochemistry ; In Situ Hybridization, Fluorescence ; Receptor, ErbB-2 ; analysis ; genetics

Breast Neoplasms ; genetics ; Carcinoma, Ductal, Breast ; genetics ; Female ; Genes, erbB-2 ; Humans ; In Situ Hybridization ; methods ; In Situ Hybridization, Fluorescence ; Silver

OBJECTIVETo evaluate the application of dual-probe chromogenic in situ hybridization (dual-probe CISH) in analysis of HER2 gene status of breast cancer patients by comparison with fluorescence in situ hybridization (FISH). The potential impact of chromosome 17 polysomy in the determination of HER2 status was also studied.

METHODSOne hundred and forty-six cases of paraffin-embedded breast cancer tissues were retrieved. Analysis of HER2 gene and chromosome 17 copy numbers using CE-approved commercial kits of dual-probe FISH (for 146 cases) and dual-probe CISH (for 73 cases) were carried out. The results were interpreted according to ASCO/CAP, 2007 either HER2 gene copy number or the ratio of HER2/centromere 17 (CEN17).

RESULTSOf the 73 cases analyzed by both FISH and dual-probe CISH, the concordance rates for negative and positive results was 91.7% (33/36) and 97.4% (37/38) respectively, while the overall concordance rate between the two methods was 95.9% (70/73). Of the 146 cases analyzed by FISH, 13 cases were interpreted as equivocal if only HER2 copies were counted, compared with 8 equivocal cases by calculating the ratio of HER2/CEN 17. Moreover, 3 cases (4.8%) of the 63 HER2-positive cases determined by HER2 copies turned out to be HER2-negative when determined by the ratio of HER2/CEN 17; while using dual-probe CISH, 1 (3.0%) of the 33 positive cases turned out to be negative. In addition, when using FISH, there were more chromosome 17 polysomy cases (63.5%, 40/63) in the HER2-positive subgroup than HER2-negative subgroup (37.3%, 28/75) (P = 0.002).

CONCLUSIONSDual-probe CISH can achieve similar results as compared to FISH, indicating that this technology is a reliable alternative to FISH in HER2 testing. The accuracy can further be improved when HER2 and chromosome 17 are simultaneously tested and counted.

Breast Neoplasms ; genetics ; Carcinoma, Ductal, Breast ; genetics ; Chromosomes, Human, Pair 17 ; genetics ; Gene Dosage ; Genes, erbB-2 ; genetics ; Humans ; In Situ Hybridization ; methods ; In Situ Hybridization, Fluorescence ; Paraffin Embedding ; Polyploidy

OBJECTIVETo establish the restriction fragment differential display-polymerase chain reaction (RFDD-PCR) as an efficient technique for constructing and studying the gene expression profile of human tissues.

METHODSThe tissues of mamma adenocarcinoma (T), cancerometastasis lymph node (L) and normal mammary (N) from one mammary infiltrating ductal carcinoma case were collected, and the gene expression profile of each kind of tissue was constructed using RFDD-PCR technique at equal pace according to the operating manual of Qbio-gene Company. Then all fragments of the three gene expression profiles were separated and displayed by electrophoresis. With the use of gene database at the website http://www.Qbio-gene.com/display, the authors identified the names of the probable fragments by bioinformatics analysis. Through comparison of the three profiles, the numbers and types of most differentially expressed gene fragments were displayed.

RESULTSThe expression profiles of the three kinds of tissue have been constructed covering 1716 fragments of mammary adenocarcinoma, 1769 of cancerometastasis lymph nodes and 1922 of normal mammary tissue. Among these 5407 fragments, 39.39% were exactly the same. While 33.9% sequences of T and L showed differences in abundance or presence, 40.9% of T and N and 39.6% fragments of L and N were observed differentially expressed. These differentially expressed gene fragments were found to relate with metastasis, differentiation, inflammation and so on.

CONCLUSIONRFDD-PCR is an efficient technique for research in human diseases genomics as a mass screening for complete gene expression profile with high-flux. Through comparison among three or more profiles, the screening for candidate genes of a certain disease can be accomplished, and there is probably a chance to identify novel gene or expressed sequence tag.

Adenocarcinoma ; genetics ; Breast Neoplasms ; genetics ; Carcinoma, Ductal, Breast ; genetics ; Computational Biology ; Electrophoresis ; methods ; Female ; Gene Expression Profiling ; methods ; Humans ; Polymerase Chain Reaction ; methods ; Sequence Analysis, DNA ; methods

OBJECTIVETo investigate the clinicopathological significance of chromosome 17 polysomy in breast cancer.

METHODSRetrospective study of 200 cases of breast cancer including 106 cases of invasive ductal carcinoma and 94 cases of in-situ carcinoma was performed by fluorescence in-situ hybridization (FISH) to explore the relationship between chromosome 17 polysomy and age, nuclear atypia, lymphatic metastasis, HER2 gene amplification and HER2 protein expression.

RESULTSTwenty-six percent (52/200) of chromosome 17 polysomy was detected in 200 cases of breast ductal carcinoma, all of which were invasive ductal carcinoma. Overall 52. 8% (52/180) of invasive ductal carcinoma cases showed chromosome 17 polysomy, which was correlated to HER2 gene amplification (P = 0.000) and HER-2 protein expression (P=0.000), and to HER2 expression combined with HER2 gene amplification (P=0.001). Chromosome 17 polysomy with or without HER2 gene amplification was also associated with high-grade nuclear atypia (P = 0.012 or P = 0.010) and lymphatic metastasis (P = 0.002 or P = 0.009 ). However, chromosome 17 polysomy with or without HER2 gene amplification was not correlative with the age of patients (P = 1. 000 or P = 0. 415).

CONCLUSIONChromosome 17 polysomy may be related to the nuclear atypia, metastasis, HER2 gene amplification of invasive ductal carcinoma and thus a worse prognosis of the patients.

Breast Neoplasms ; genetics ; pathology ; Carcinoma, Ductal ; genetics ; Chromosome Aberrations ; Chromosomes, Human, Pair 17 ; genetics ; Gene Amplification ; Gene Dosage ; Gene Expression Regulation, Neoplastic ; genetics ; Genes, erbB-2 ; genetics ; Humans

OBJECTIVETo investigate c-myc and CCNE2 gene amplifications and their relationship in breast cancer.

METHODSSixty-six infiltrating ductal breast carcinomas with foci of ductal carcinoma in situ components collected from January 2005 to December 2007 were selected for tissue microarray and quantitative multi-gene FISH for c-myc and CCNE2 gene amplification, and the relationship with the clinicopathologic features was analyzed.

RESULTSOf the 66 cases, 18 (27.3%) showed c-myc amplification and 23 (34.8%) showed CCNE2 amplification. A strong correlation was found between c-myc and CCNE2 amplification (P < 0.01). The breast cancers showing c-myc and CCNE2 amplifications were all aneuploidy, and were HER2 positive (P < 0.05). Tumors with c-myc amplification also showed higher Ki-67 index (P < 0.05).

CONCLUSIONSC-myc and CCNE2 amplifications are common events in breast cancer, and they often coexist. C-myc and CCNE2 genes may play critical roles in the pathogenesis and development of breast cancer through unique and overlapping signaling pathways.

Aneuploidy ; Breast Neoplasms ; genetics ; Carcinoma in Situ ; genetics ; Carcinoma, Ductal, Breast ; genetics ; Carcinoma, Intraductal, Noninfiltrating ; genetics ; Cyclins ; genetics ; Female ; Gene Amplification ; Genes, myc ; Humans ; In Situ Hybridization, Fluorescence ; methods ; Tissue Array Analysis

OBJECTIVETo evaluate the sensitivity and specificity of chromogenic in-situ hybridization (CISH) in detecting HER2 gene amplification in breast carcinomas.

METHODSHER2 oncogene amplification and its protein expression in 165 cases of breast carcinoma were investigated by immunohistochemistry (IHC) and CISH.

RESULTS(1) CISH did not detect HER2 gene amplification in 107 cases of IHC negative tumors and 24 cases of IHC 1+ tumors. (2) CISH identified high copy numbers of HER2 gene amplification in 21/22 (95.5%) cases with IHC 3+. (3) In 12 HIC 2+ cases, CISH identified 3 cases of high copy number amplification, 6 cases of low copy number amplification and 3 cases without amplification.

CONCLUSIONSHER2 gene amplification detection by CISH is highly sensitive and has a high concordance with IHC detection of the protein expression. It is concluded that CISH is a tool to evaluate HER2 gene status in breast cancer and can be an implement in conventional pathology laboratories.

Breast Neoplasms ; genetics ; metabolism ; pathology ; Carcinoma, Ductal, Breast ; genetics ; metabolism ; pathology ; Carcinoma, Intraductal, Noninfiltrating ; genetics ; metabolism ; pathology ; Carcinoma, Lobular ; genetics ; metabolism ; pathology ; Chromogenic Compounds ; Female ; Gene Amplification ; Humans ; Immunohistochemistry ; methods ; In Situ Hybridization ; methods ; Receptor, ErbB-2 ; genetics ; metabolism

OBJECTIVETo compare dual-color in-situ hybridization (DISH) with fluorescence in situ hybridization(FISH) in evaluating the human HER2 gene status in invasive breast cancer.

METHODSHER2 gene status in 110 cases of breast invasive ductal carcinomas with a 2+ score on immunohistochemistry (IHC) was investigated by FISH and DISH. The 2007 and 2013 ASCO (American Society of Clinical Oncology)/CAP (College of American Pathologists) HER2 guideline were used respectively to evaluate the agreement between these two techniques.

RESULTS(1) Using the 2007 ASCO/CAP guideline, the overall concordance between FISH and DISH was 97.3% (107/110). Fifty of 51 samples with amplification by FISH were also detected by DISH and the remaining one sample was equivocal.Eight of 10 equivocal samples by FISH were equivocal by DISH and the remaining two samples were negative. Forty-nine samples with no amplification by FISH were all negative by DISH. The concordance was 98.0%, 8/10 and 100.0% respectively for the FISH positive, equivocal and negative groups. (2) Using the 2013 ASCO/CAP guideline, the overall concordance between FISH and DISH was 90.0% (99/110). Fifty-three of 55 samples with amplification by FISH were also detected by DISH and the remaining two were equivocal and negative respectively. Two of 12 equivocal samples by FISH were equivocal by DISH and the remaining ten samples were negative in 7 cases and equivocal in 3 cases. Forty-three samples with no amplification by FISH were all negative by DISH. The concordance was 96.4%, 3/12 and 100.0% respectively for the FISH positive, equivocal and negative groups. Pearson correlation analysis indicated that the HER2 status detected by FISH and DISH were significantly correlated with each other (R=0.584, P<0.01).

CONCLUSIONSThere is a high concordance between DISH and FISH for assessing the HER2 gene status in invasive breast cancer. DISH is a new option for assessing HER2 gene status of breast cancer in clinical practice. The clinical significance of the discordance between DISH and FISH in equivocal cases warrants further study.

Breast Neoplasms ; genetics ; Carcinoma, Ductal, Breast ; genetics ; Female ; Gene Amplification ; Genes, erbB-2 ; Humans ; In Situ Hybridization ; methods ; In Situ Hybridization, Fluorescence ; methods

OBJECTIVETo study the expression level and significance of glucose transporter 1 (Glut-1) in normal breast tissue, adenosis, adenoma and breast carcinoma.

METHODSA total of 147 cases of female breast tissue samples, including 92 cases of invasive ductal carcinoma, 26 cases of breast fibroadenoma, 24 cases of breast adenosis and 5 cases of normal breast tissues, were collected for quantitative detection of the expression of Glut-1 protein by immunohistochemistry (EnVision method) and Western blot, and its mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR).

RESULTSIn normal breast tissue and benign lesions of the breast, Glut-1 was undetectable or only weakly detectable in cytoplasm of ductal and acinar epithelia. In contrast, the intensity of Glut-1 staining was significantly higher in invasive ductal carcinomas (P = 0.0002) with protein expression predominantly in cellular membrane and lesser in cytoplasm. Western blot and RT-PCR analyses showed that the expression of Glut-1 protein and mRNA were significantly increased in invasive ductal carcinoma than fibroadenoma (P =0.001 for protein; P <0.05 for mRNA) and adenosis (P =0.001 for protein; P < 0.05 for mRNA). There was a significant difference among groups (P = 0.0002 for protein; P = 0.0001 for mRNA).

CONCLUSIONSGlucose transport activity, as indicated by Glut-1 protein and its mRNA expression, significantly increases in breast carcinoma than non-cancerous lesions. The over-expression of Glut-1 in breast carcinoma is tightly coupled with tumor cell proliferation, invasion and metastasis, implying that Glut-1 may serve as a new marker in the early diagnosis and prognostication of breast malignancy as well as a new therapeutic target.

Breast Neoplasms ; metabolism ; Carcinoma, Ductal, Breast ; metabolism ; Female ; Gene Expression Regulation, Neoplastic ; Glucose ; physiology ; Glucose Transport Proteins, Facilitative ; genetics ; metabolism ; Glucose Transporter Type 1 ; genetics ; metabolism ; Humans ; Prognosis

OBJECTIVE: To examine the expressions of JMJD3, matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF) in invasive ductal breast carcinoma, their association with the clinicopathological features of the patients and the effect of JMJD3 overexpression on proliferation and MMP-2 and VEGF expressions in breast cancer cells. METHODS: The protein and mRNA expressions of JMJD3, MMP-2, and VEGF in invasive ductal breast carcinoma and paired adjacent tissues were detected by immunohistochemistry and RT-PCR, respectively, and their correlation with the clinicopathological characteristics of the patients was analyzed. Kaplan-Meier survival analysis was used to evaluate the correlation of JMJD3, MMP-2 and VEGF expression levels with the survival of the patients. In breast cancer MDA-MB-231 cells transfected with a JMJD3-expression plasmid, the expression of Ki67 was examined immunohistochemically, the cell proliferation was assessed with CCK8 assay, and the mRNA expressions of MMP-2 and VEGF were detected with RT-PCR. RESULTS: Breast cancer tissues had significantly lower JMJD3 expression and higher MMP-2 and VEGF expressions at both the mRNA and protein levels than the adjacent tissue ( CONCLUSIONS The expressions of JMJD3, MMP-2 and VEGF in invasive ductal breast carcinoma are closely correlated to tumor proliferation, invasion, metastasis and prognosis and can be used for prognostic evaluation of breast cancer.
Breast Neoplasms/genetics* ; Carcinoma, Ductal, Breast/genetics* ; Humans ; Jumonji Domain-Containing Histone Demethylases ; Lymphatic Metastasis ; Matrix Metalloproteinase 2 ; Prognosis ; Vascular Endothelial Growth Factor A