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

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

INTRODUCTIONOur study investigates whether an approximation of breast cancer molecular subtypes using the hormone receptors and HER-2 status prognosticates for disease control after breast conservation therapy (BCT) in node-negative Asian breast cancer patients.

METHODS AND MATERIALSWe retrospectively reviewed 541 women with node-negative breast cancers treated with BCT between 1989 and 2007. Hormone receptors and HER-2 status were obtained from patients' histological report. All patients received radiotherapy. Thirty-six percent and 68% of women received chemotherapy and hormonal treatment respectively.

RESULTSMedian follow-up of patients is 72 months. Five-year local recurrence free survival (LRFS) is 97.2% for the cohort but differs between subtypes: luminal A, 0.8%; luminal B, 1.4%; HER-2, 3.6% and basal-like, 12.7% (P = 0.047). The 5-year distant disease free survival (DDFS) is 96.4% for the cohort but differs between subtypes: luminal A, 98.2%; luminal B, 92.6%; HER-2, 89.5% and basal-like, 91.5% (P = 0.019). The 5-year disease free survival (DFS) is 94.4% for the cohort but differs between subtypes: luminal A, 97.4%; luminal B, 92.7%; HER-2, 86.3% and basal-like, 85.0% (P = 0.007). Univariate analysis with luminal A as baseline revealed an association of the other 3 subtypes with decreased DFS (P = 0.007), Hazard Ratio (HR) of 2.2, 4.4 and 3.3 to Luminal B, HER-2 and basal subtypes, respectively. On multivariate analysis, HER-2 subtype (AHR = 3.3, 95% CI, 1.1 to 9.8, P = 0.036) and basal-like subtype (HR = 3.5, 95% CI, 1.2 to 9.9, P = 0.019) prognosticate adversely for DFS.

CONCLUSIONThe combination of hormone receptors and HER-2 status can be used as surrogates for molecular subtypes in Asian breast cancer patients with node-negative disease to prognosticate LRFS, DFS and DDFS.

Adult ; Aged ; Aged, 80 and over ; Breast Neoplasms ; genetics ; pathology ; prevention & control ; Female ; Humans ; Mastectomy, Segmental ; Middle Aged ; Multivariate Analysis ; Neoplasm Recurrence, Local ; Prognosis ; Radiotherapy, Adjuvant ; Receptor, ErbB-2 ; genetics ; Receptors, Estrogen ; genetics ; Receptors, Progesterone ; genetics ; Retrospective Studies ; Risk Assessment ; Survival Analysis ; Treatment Outcome

Age Factors ; Bone Neoplasms ; secondary ; Breast Neoplasms ; chemistry ; genetics ; pathology ; Carcinoma, Ductal, Breast ; chemistry ; genetics ; pathology ; Carcinoma, Intraductal, Noninfiltrating ; chemistry ; genetics ; pathology ; Female ; Gene Expression Profiling ; Humans ; Ki-67 Antigen ; analysis ; Lymphatic Metastasis ; Prognosis ; Receptor, ErbB-2 ; analysis ; Receptors, Estrogen ; analysis ; Receptors, Progesterone ; analysis ; Tumor Burden

OBJECTIVETo study the expression of stomatin like protein-2 (SLP-2) at mRNA and protein levels in two kinds of malignant epithelial tumors, including laryngeal squamous cell carcinoma (LSCC) and invasive breast cancer, and to study the relations of SLP-2 expression and clinicopathologic parameters with the prognosis.

METHODSRT-PCR and Western blot were used to detect the expression of SLP-2 mRNA and protein in LSCC and their normal counterparts (46 and 10 pair, respectively). Immunohistochemistry was carried on tissue array constructed from LSCC (104 cases) and breast cancer (263 cases), respectively. The association between SLP-2 expression and clinicopathologic parameters was analyzed.

RESULTSLSCC showed a higher expression of SLP-2 than that of their normal counterparts (negative expression) at mRNA (83%, 38/46) and protein (7/10) level. Immunohistochemical analysis of LSCC showed that compared with negative expression in normal laryngeal epithelium (0/20), a higher SLP-2 expression was detected in LSCC (36/104, P=0.000) and associated with the advanced clinical stage (P<0.01) and lymph node metastasis (P=0.003). Immunohistochemical study of invasive breast cancer demonstrated that compared with negative expression in normal breast tissue (0/10), more than one half of the cases showed a high SLP-2 expression (52.5%, 138/263, P=0.000) in breast cancer, which correlated with the tumor size (P=0.020), lymph node metastasis (P<0.01), advanced clinical stage (P<0.01), distant metastasis (P=0.002) and HER2/neu protein expression (P=0.037). Survival analysis showed a shorter overall survival probability in patients with a high SLP-2 expression. It was considered that lymph node metastasis, positive HER2/neu expression, and high-level SLP-2 expression may act as the independent prognostic factors for those tumors.

CONCLUSIONSA high expression level of SLP-2 may be associating with the development of invasion and metastasis in LSCC and breast cancer, and SLP-2 is also considered working as an independent factor indicating a poor prognosis clinically in breast cancer.

Adult ; Blood Proteins ; genetics ; metabolism ; Breast Neoplasms ; metabolism ; pathology ; Carcinoma, Ductal, Breast ; metabolism ; pathology ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Female ; Humans ; Laryngeal Neoplasms ; metabolism ; pathology ; Lymphatic Metastasis ; Male ; Membrane Proteins ; genetics ; metabolism ; Middle Aged ; Neoplasm Metastasis ; Neoplasm Recurrence, Local ; Neoplasm Staging ; Prognosis ; Proportional Hazards Models ; RNA, Messenger ; metabolism ; Receptor, ErbB-2 ; metabolism ; Survival Analysis

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

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

We evaluated the efficacy and safety of weekly paclitaxel plus trastuzumab as firs-tline chemotherapy in women with HER2-overexpressing metastatic breast cancer (MBC), and we investigated the prognostic factors including magnitude of HER2/neu amplification in this population. We analyzed 54 patients with HER2-overexpressing MBC that were treated with weekly paclitaxel plus trastuzumab as first-line chemotherapy from February 2004 to December 2006. At a median follow-up of 28 months, median time to progression (TTP) was 16.6 months (95% CI, 9.4 to 23.7 months) and median overall survival was 25.6 months (95% CI, 21.8 to 27.3 months). Therapy was generally well tolerated, although three patients (5.5%) experienced reversible, symptomatic heart failure. Of the 27 patients evaluable for the HER2 FISH, patients with a HER2/CEP17 ratio of < or =4.0 had significantly shorter TTP than those with a HER2/CEP17 ratio of >4.0 (10.8 vs. 23.2 months, P=0.034). A HER2/CEP17 ratio of >4.0 was identified as significant predictive factor of TTP by multivariate analysis (P=0.032). The combination of weekly paclitaxel plus trastuzumab as first-line chemotherapy is an effective regimen in patients with HER2-FISH-positive MBC. Furthermore, the magnitude of HER2 amplification is an independent predictive factor of TTP.
Adult ; Aged ; Antibodies, Monoclonal/*administration & dosage/therapeutic use ; Antineoplastic Combined Chemotherapy Protocols/*administration & dosage/therapeutic use ; Breast Neoplasms/*drug therapy/mortality/pathology ; Disease Progression ; Drug Administration Schedule ; Female ; Gene Amplification ; Humans ; In Situ Hybridization, Fluorescence ; Middle Aged ; Paclitaxel/*administration & dosage/therapeutic use ; Predictive Value of Tests ; Receptor, erbB-2/*genetics/metabolism ; Survival Analysis

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

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