Breast Neoplasms ; chemistry ; genetics ; Chromogenic Compounds ; Female ; Genes, erbB-2 ; Humans ; In Situ Hybridization ; methods ; Receptor, ErbB-2 ; analysis

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

BACKGROUNDAccurate detection of human epidermal growth factor receptor 2 (HER2) expression and gene amplification is crucial for the application of HER2-specific therapy and for evaluating the response of patients with breast cancer. A uniform and standard procedure of immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) needs to be established for evaluating the HER2 status in breast cancer tissues for the treatment of patients with real HER2-positive tumors. The present multicenter study was aimed to examine the HER2 status in breast cancer specimens from Chinese patients using both IHC and FISH methods.

METHODSA multicenter study was performed on the HER2 status in 3 149 breast cancer specimens from different ethnic populations and areas in China by IHC and FISH assays. The potential association of HER2 status with demographic and clinical characteristics was analyzed.

RESULTSThe positive rates for HER2 over-expression and HER2 amplification were 23.3% and 27.5% in this study, respectively. The concordance between IHC and FISH was 71.2% (κ = 0.494, P < 0.001). Furthermore, 72.9% of specimens with IHC 2+ were negative to FISH. The discordance rates among laboratories were from 5% to 28% for IHC and 1% to 16% for FISH. HER2 amplification was associated significantly with advanced tumor stage (III or IV, P = 0.002), large tumor size (>5 cm, P = 0.002), moderate and poor histological grades (P < 0.0001), post-menopause (P < 0.0001), ER-PR- (P = 0.002), and having ≥ 4 lymph nodes affected (P < 0.0001) in this population. The positive rates of HER2 amplification in specimens from Man and Hui Chinese were significantly higher than that in other Chinese populations. There are slightly higher positive rates of HER2 expression and amplification in Chinese patients with breast cancer.

CONCLUSIONThese findings may provide new insights into understanding the epidemiological features of HER2 expression and amplification, and may be valuable for clinical practice.

Biomarkers, Tumor ; analysis ; genetics ; metabolism ; Breast Neoplasms ; metabolism ; Female ; Humans ; Immunohistochemistry ; methods ; In Situ Hybridization, Fluorescence ; methods ; Middle Aged ; Receptor, ErbB-2 ; analysis ; genetics ; metabolism

OBJECTIVETo study the difference in the expressions of C-erbB-2 gene between the coal miners with pneumoconiosis complicated by pulmonary cancer and the controls with single pulmonary cancer, and its relation to clinical pathology.

METHODMeasuring the expressions of C-erbB-2 in 32 cases of pneumoconiosis complicated by pulmonary cancer and those in 30 cases of pulmonary cancer by means of immunohistochemistry.

RESULTSThe positive expression rate in 32 cases of pneumoconiosis complicated by pulmonary cancer was 53.13% whereas that in 30 cases of single pulmonary cancer was 26.67% (P < 0.05); the positive expressions of C-erbB-2 in patients with lymph node metastasis (70.59% in pneumoconiosis group, 50.00% in controls) were significantly different from those without lymph node metastasis (33.33% in pneumoconiosis group, 11.11% in controls) (P < 0.05). The prognosis on patients with positive expressions of C-erbB-2 was poor, and was not related to pathologic category.

CONCLUSIONC-erbB-2 gene may be an important regulating gene in the coal miners with pneumoconiosis complicated by pulmonary cancer, and as a reference index to determine lymph node metastasis and prognosis.

Aged ; China ; Coal Mining ; Gene Expression ; Genes, erbB-2 ; Genotype ; Humans ; Lung Neoplasms ; complications ; genetics ; pathology ; Lymphatic Metastasis ; Male ; Middle Aged ; Pneumoconiosis ; complications ; genetics ; pathology ; Prognosis ; Receptor, ErbB-2 ; genetics ; metabolism ; Survival Analysis

BACKGROUNDTargeted tumor therapies have been making rapid progress in recent years, and the erbB-2 oncogene is a suitable target. There was much discussion about the level of erbB-2 in osteosarcoma. The aim of this study was to investigate the erbB-2 amplification or expression status in osteosarcoma.

METHODSFluorescence in situ hybridization (FISH) and DNA probes for erbB-2 and centromere 17 were used to examine the erbB-2 gene amplification status in 32 osteosarcoma samples, and expression of erbB-2 was analyzed by immunohistochemistry (IHC) and reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSNone of the 32 osteosarcomas was observed by FISH to have the erbB-2 gene amplified, and no distinguishable membrane staining was seen in any case yet, nevertheless, erbB-2 overexpression was present in 6 tumor samples by RT-PCR.

CONCLUSIONSThe status of erbB-2 gene amplification and membrane overexpression is rare in osteosarcomas, and might suggest that the erbB-2 target agent should not be applied to osteosarcomas as single treatment.

Adolescent ; Bone Neoplasms ; genetics ; therapy ; Child ; Dimerization ; Female ; Gene Amplification ; Genes, erbB-2 ; Humans ; In Situ Hybridization, Fluorescence ; Male ; Osteosarcoma ; genetics ; therapy ; Receptor, ErbB-2 ; analysis ; antagonists & inhibitors ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo summarize the morphological features of basal-like subtype of invasive breast carcinoma (BLSIBC), and to look for diagnostic clues for its recognition.

METHODSImmunohistochemistry was performed in 109 cases of invasive ductal carcinoma, with CK5/6, CK14, CK8/ 18, 34betaE12, calponin, p63, CD10, ER, PR and c-erbB-2 monoclonal antibodies. Five subtypes were classified according to immunophenotypes: luminal A subtype (ER+/HER2-), luminal B subtype (ER+/ HER2+), normal breast-like subtype (ER/HER2-), HER2-overexpressing subtype and BLSIBC which was identified with at least one kind of basal-like cytokeratins or markers of myoepithelium and ER/HER2. The microscopic features of basal-like subtype were also analyzed.

RESULTSThe number of luminal A case was 48 (44.0%), luminal B 15 (13.8%), HER2 over-expressing 15 (13.6%), normal breast-like 10 (9.1%), basal-like subtype 19 (17.4%). Besides, the other two cases expressed c-erbB-2 or/and ER plus markers for myoepithelium, thus were excluded from all the five mentioned subtypes. Of the 19 basal-like subtype, CK5/6 was expressed in 16 cases, CK8/18 in 17 cases, CK14 in 11 cases, 34betaE12 in 18 cases, p63 in 5 cases, CD10 in 6 cases, and calponin in 1 case. The diameter of the BLSIBC cases was 1.2-7 cm (averagely 3.9 cm) , and in 6 cases, the tumor diameter was >5 cm. Only one case displayed extensive in situ component, 9 cases were grade 2, and 9 cases were grade 3. Compared to non basal subtype, there were significantly more high grade cases (P <0.01). The morphological features of basal-like subtype were summarized as the followings: pushing margin (13 cases), lymphocytic tissue hyperplasia (18 cases), nest or sheet arrangement (18 cases), nucleus grade 3 and scattered giant or bizarre nuclei (17 cases), syncytial growth (7 cases), and comedo-like necrosis (17 cases). The frequency of these features were significantly more common than non basal subtype (P <0.01).

CONCLUSIONThe morphologic diagnostic features of BLSIBC are pushing margins, lymphocyte infiltration, comedo-like necrosis, gigantic cell and syncytial growth.

Biomarkers, Tumor ; analysis ; Breast Neoplasms ; diagnosis ; pathology ; Carcinoma, Basal Cell ; pathology ; Female ; Genes, erbB-2 ; physiology ; Humans ; Immunohistochemistry ; Keratin-5 ; analysis ; Magnetic Resonance Imaging ; Male ; Mammography ; instrumentation ; methods ; Neoplasm Invasiveness ; physiopathology ; Prognosis ; Receptor, Epidermal Growth Factor ; genetics ; Receptor, ErbB-2 ; analysis ; genetics ; Receptors, Estrogen ; analysis ; Receptors, Progesterone ; analysis ; Ultrasonography ; methods

To verify the reliability of targeted detecting HER2 positive cancer cells and clinical pathological tissue specimens with a recombinant anti HER2 single chain antibody in single chain Fv fragment (scFv) format, we have constructed the fusion variable regions of the ScFv specific for HER2/neu. labeled a green-fluorescent protein(GFP). The humanized recombinant Anti HER2 ScFv-GFP gene was inserted into pFast Bac HT A, and expressed in insect cells sf9. Then the recombinant fusion protein Anti HER2 ScFv-GFP was properly purified with Ni2+-NTA affinity chromatography from the infected sf9 cells used to test the specificity of the fusion antibody for HER2 positive cancer cells. Firstly, the purified antibody incubated with HER2 positive breast cancer cells SKBR3, BT474 and HER2 negative breast cancer cells MCF7 for 12 h/24 h/48 h at 37 degrees C, in order to confirm targeted detecting HER2 positive breast cancer cells by Laser Confocal Microscopy. Furthermore, the same clinical pathological tissue samples were assessed by immunohistochemistry (IHC) and the fusion antibody Anti HER2 ScFv-GFP in the meanwhile. The data obtained indicated that the recombinant eukaryotic expression plasmid pFast Bac HT A/Anti HER2 ScFv-GFP was constructed successfully In addition, obvious green fluorescent was observed in insect cells sf9. When the purified fusion antibody was incubated with different cancer cells, much more green fluorescent was observed on the surface of the HER2 positive cancer cells SKBR3 and BT474. In contrast, no green fluorescent on the surface of the HER2 negative cancer cells MCF7 was detected. The concentration of the purified fusion antibody was 115.5 microg/mL, of which protein relative molecular weight was 60 kDa. The analysis showed the purity was about 97% and the titer was about 1:64. The detection results of IHC and fusion antibody testing indicated the conformity. In summary, the study showed that the new fusion antibody Anti HER2 ScFv-GFP can test HER2 positive cancer cells, indicating a potential candidate method for clinical HER2 positive specimens detection.
Animals ; Breast Neoplasms ; diagnosis ; pathology ; Female ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; Humans ; MCF-7 Cells ; Receptor, ErbB-2 ; analysis ; Recombinant Fusion Proteins ; genetics ; Sf9 Cells ; Single-Chain Antibodies ; genetics

OBJECTIVETo study the BRCA1 mutations in patients with early-onset breast cancer and their affected relatives in Guangdong province and explore the relationship between BRCA1 mutation and the expressions of estrogen receptor(ER), progesterone receptor(PR), HER2 and ALN.

METHODSFrom 58 patients with early-onset breast cancer and their affected relatives, the genomic DNA was extracted from the peripheral blood mononuclear cells and the coding regions of the BRCA1 gene was amplified using polymerase chain reaction. BRCA1 gene mutations were screened by denaturing high performance liquid chromatography (DHPLC) and subsequent direct DNA sequencing. The expression of ER, PR, HER2 and ALN were detected with immunohistochemistry and their relations with the gene mutation were analyzed.

RESULTSDisease-related BRCA1 mutations were detected in 2 of the 58 patients, who were younger than 35 years old, including 1 with a novel splice-site mutation (IVS5-1 G-->A). No association was found between this novel mutation and the expressions of ER, PR, HER2 and ALN.

CONCLUSIONThe incidence of BRCA1 mutation is significantly lower in patients with early-onset breast cancer and their affected relatives in Guangdong province than in the Western populations. The novel mutation identified in BRCA1 gene may represent a mutation characteristic of the patients in Guangdong province. BRCA1 gene mutations may not have any relation with the expression of ER, PR, HER2 and ALN.

Adult ; Age of Onset ; Base Sequence ; Breast Neoplasms ; genetics ; China ; DNA Mutational Analysis ; Female ; Genes, BRCA1 ; Genotype ; Humans ; Molecular Sequence Data ; Mutation ; Receptor, ErbB-2 ; genetics ; Receptors, Estrogen ; genetics ; Receptors, Progesterone ; genetics

OBJECTIVETo prepare the superparamagnetic iron oxide (SPIO)-labeled antisense oligodeoxynucleotide (ASODN) probe and evaluate the application of this probe in cellular magnetic resonance imaging (MRI).

METHODSWe prepared the SPIO-labeled ASODN probe using chemical cross linking method to conjugate SPIO to ASODN, detected its configuration by atomic force microscopy, determined the conjugating rate and biology activation by high performance liquid chromatography, and detected the stability by polyacrylamide gel electrophoresis. After that, we transfected the SK-Br3 oncocytes which had over-expression of the c-erbB2 oncogene by this probes, observed the intracellular iron distribution by optical microscope, measured iron content by atomic absorption spectroscopy, and observed the signal change by MRI.

RESULTSAtomic force microscope showed that the SPIO-labeled ASODN probe was mostly spherical and well-distributed, with a diameter of 25-40 nm and a conjugating rate of 100%. This probe had inhered biological activity and stability. In addition, light microscopy revealed an intracellular uptake of iron oxides in the transfected SK-Br3 oncocyte, and the iron content of the group of transfected SK-Br3 oncocytes was significantly higher than those of other contrast groups (all P < 0.01). MRI showed that transfected SK-Br3 oncocyte had the lowest signal among all other cells (all P < 0.05).

CONCLUSIONSWe prepared the SPIO-labeled ASODN probe successfully. It can effectively transfect SK-Br3 oncocyte and enter SK-Br3 oncocyte, and thus reduce the signal intension in MRI.

Cell Line, Tumor ; DNA, Antisense ; chemistry ; genetics ; Ferric Compounds ; chemistry ; Humans ; Magnetic Resonance Imaging ; Magnetics ; Molecular Probe Techniques ; Oligodeoxyribonucleotides ; chemistry ; genetics ; Oxyphil Cells ; chemistry ; Receptor, ErbB-2 ; analysis ; genetics

BACKGROUNDHuman epidermal growth factor 2 (HER2) is one of the most important prediction factors, but only 25% - 30% of breast cancer patients HER2 are positive. It is unknown whether there are other molecular markers that could be used to predict prognosis and recurrence in HER2 negative patients. This study investigated correlations of cyclin A2 and HER2 levels with clinical outcomes in 281 patients with invasive breast cancer in order to identify whether cyclin A2 can serve as a prognostic factor in HER2 negative patients.

METHODSImmunohistochemical staining was used to detect cyclin A2 and HER2 expression in 281 patients. Cyclin A2 and HER2 gene amplifications were analyzed using gene analysis and RT-PCR in 12 patients. Risk and survival estimates were analyzed using Log-rank, Kaplan-Meier, and Cox regression analysis; cyclin A2 and HER2 consistency with survival were analyzed using Kappa analysis.

RESULTSPatients with higher cyclin A2 and HER2 expressions had significantly shorter disease-free survival periods (P = 0.047 and P = 0.05, respectively). Kappa analysis performed that cyclin A2 and HER2 showed a low Kappa index (kappa = 0.37), allowing us to conclude that cyclin A2 and HER2 detect different pathologies. Gene analysis and RT-PCR showed that cyclin A2 was upregulated in patients with early relapse; the average increase was 3.69 - 2.74 fold.

CONCLUSIONSCyclin A2 and HER2 are associated with proliferation and high recurrence, particularly when combined. Cyclin A2 is easily detected by nuclear staining and might be a useful biomarker for recurrence risk in HER2 negative patients.

Adult ; Aged ; Aged, 80 and over ; Breast Neoplasms ; genetics ; metabolism ; Cyclin A2 ; genetics ; metabolism ; Female ; Humans ; Immunohistochemistry ; Middle Aged ; Multivariate Analysis ; Receptor, ErbB-2 ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction