Recently we experienced three cases of human epidermal growth factor receptor 2 (HER2)-amplified invasive breast carcinomas associated with co-amplification or gain of chromosome 17 centromere (CEP17) in silver-enhanced in situ hybridization (SISH) analysis. These cases revealed 2+ or 3+ staining for HER2 immunohistochemistry and >6 HER2 copies per cell on SISH analyses. However, the calculated HER2/CEP17 ratios were low (<2.2) and did not fit within the HER2-positive category. We interpreted those cases as HER2-positive tumors based on the number of HER2 copies per cell. There is a potential for misinterpretation of SISH analysis in cases showing increased CEP17 copy number, based on the criterion used for HER2 positivity (HER2 copies >6 per cell vs HER2/CEP17 ratio>2.2). We recommend reporting raw SISH or fluorescence in situ hybridization data, including number of cells counted, average numbers of HER2 and CEP17 signals, and the calculated HER2/CEP17 ratio to prevent underreporting of HER2 amplification.
Breast ; Breast Neoplasms ; Centromere ; Chromosomes, Human, Pair 17 ; Coat Protein Complex I ; Fluorescence ; Humans ; Immunohistochemistry ; In Situ Hybridization ; Receptor, Epidermal Growth Factor ; Receptor, erbB-2

This is a case report of 46,XY female phenotype (46,XY karyotype, no pubic hair, blind vagina and absence of uterus)in an 18-year-old patient. To confirm whether a Y chromosome has a structural abnormality, fluorescent in situ hybridization (FISH) with the chromosome X/Y cocktail probe was simultaneously performed, and the six loci [PABY, RPS4Y(sy16, sy17), ZFY, DYS14] on the short arm, one locus (DYZ3) on the centromere and one locus (DYZ1) on the long arm were amplified by polymerase chain reaction (PCR). The probes used FISH hybridized to centromere of the X chromosome and heterochromatin region (Yq12) of the Y chromosome, and all PCR related Y chromosome showed positive band like normal male. From the results obtained, it seemed that the Y chromosome from the 46,XY female was structurely normal. Especially, the SRY gene has been equated with the mammalian testis-determining factor, and absence or point mutation in the SRY gene causes XY female. To detect the point mutations of SRY sequencesn, single-strand conformation polymorphism (SSCP) assay was used. Our results confirm that this patient has no mutation in the SRY gene on the Y chromosome.
Adolescent ; Arm ; Centromere ; Female* ; Genes, sry ; Hair ; Heterochromatin ; Humans ; In Situ Hybridization, Fluorescence ; Karyotype ; Male ; Phenotype* ; Point Mutation ; Polymerase Chain Reaction ; Polymorphism, Single-Stranded Conformational ; Sex-Determining Region Y Protein ; Vagina ; X Chromosome ; Y Chromosome*

PURPOSE: This study aimed to investigate the clinical significance of chromosome 17 centromere (CEP17) multiplication (increased copy number of CEP17) related to human epidermal growth factor receptor 2 (HER2) and topoisomerase II alpha (TOP2A) status in patients with invasive breast cancer. METHODS: We constructed tissue microarrays using 594 invasive breast cancer samples and performed single-color silver-enhanced in situ hybridization (SISH) assay for HER2, TOP2A, and CEP17 to assess for copy number aberrations. The association of CEP17 multiplication with patient survival was analyzed according to HER2 and TOP2A status. RESULTS: Among 567 informative cases, HER2 amplification was noted in 22.8%, TOP2A amplification in 8.3% and TOP2A deletion in 11.1%. CEP17 multiplication was identified in 33.2% and was significantly associated with worse overall survival (OS) (p=0.02) and disease-free survival (DFS) (p=0.02). CEP17 multiplication correlated with patient survival in patients with normal TOP2A or non-amplified HER2 status, but the prognostic significance was lost in those with altered TOP2A or amplified HER2. On multivariate analyses, CEP17 multiplication was an independent prognostic factor for poorer OS (p=0.02) and DFS (p=0.01) in patients with normal TOP2A and non-amplified HER2. CONCLUSION: CEP17 multiplication was identified as a promising prognostic marker in patients with invasive breast cancer exhibiting either non-amplified HER2 or normal TOP2A status.
Antigens, Neoplasm ; Breast ; Breast Neoplasms ; Centromere ; Chromosomes, Human, Pair 17 ; Coat Protein Complex I ; Disease-Free Survival ; DNA Topoisomerases, Type II ; DNA-Binding Proteins ; Genes, erbB-2 ; Humans ; In Situ Hybridization ; Multivariate Analysis ; Prognosis ; Receptor, Epidermal Growth Factor ; Receptor, erbB-2

PURPOSE: This study aimed to investigate the clinical significance of chromosome 17 centromere (CEP17) multiplication (increased copy number of CEP17) related to human epidermal growth factor receptor 2 (HER2) and topoisomerase II alpha (TOP2A) status in patients with invasive breast cancer. METHODS: We constructed tissue microarrays using 594 invasive breast cancer samples and performed single-color silver-enhanced in situ hybridization (SISH) assay for HER2, TOP2A, and CEP17 to assess for copy number aberrations. The association of CEP17 multiplication with patient survival was analyzed according to HER2 and TOP2A status. RESULTS: Among 567 informative cases, HER2 amplification was noted in 22.8%, TOP2A amplification in 8.3% and TOP2A deletion in 11.1%. CEP17 multiplication was identified in 33.2% and was significantly associated with worse overall survival (OS) (p=0.02) and disease-free survival (DFS) (p=0.02). CEP17 multiplication correlated with patient survival in patients with normal TOP2A or non-amplified HER2 status, but the prognostic significance was lost in those with altered TOP2A or amplified HER2. On multivariate analyses, CEP17 multiplication was an independent prognostic factor for poorer OS (p=0.02) and DFS (p=0.01) in patients with normal TOP2A and non-amplified HER2. CONCLUSION: CEP17 multiplication was identified as a promising prognostic marker in patients with invasive breast cancer exhibiting either non-amplified HER2 or normal TOP2A status.
Antigens, Neoplasm ; Breast ; Breast Neoplasms ; Centromere ; Chromosomes, Human, Pair 17 ; Coat Protein Complex I ; Disease-Free Survival ; DNA Topoisomerases, Type II ; DNA-Binding Proteins ; Genes, erbB-2 ; Humans ; In Situ Hybridization ; Multivariate Analysis ; Prognosis ; Receptor, Epidermal Growth Factor ; Receptor, erbB-2

Animals ; Autoantigens ; genetics ; metabolism ; physiology ; Centromere Protein A ; Chromosomal Instability ; Chromosomal Proteins, Non-Histone ; genetics ; metabolism ; physiology ; Gene Expression Regulation, Neoplastic ; Humans ; Kinetochores ; metabolism ; Mitosis ; physiology ; Rectal Neoplasms ; genetics ; metabolism ; pathology ; Spindle Apparatus ; metabolism

OBJECTIVETo study the expression of centromere protein A (CENP-A) and its significance in hepatocellular carcinoma (HCC) and adjacent non-neoplastic liver tissue.

METHODSThe expression levels of CENP-A mRNA in 20 samples of HCC and adjacent non-neoplastic liver tissue were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and real-time quantitative polymerase chain reaction (qRT-PCR). Immunohistochemical study for CENP-A and p53 proteins was also performed on tissue microarrays containing 80 samples of HCC and adjacent liver tissue.

RESULTSThe expression level of CENP-A mRNA in HCC (0.64 +/- 0.18) was higher than that in adjacent non-neoplastic liver tissue (0.09 +/- 0.09) (t = 12.78, P < 0.01). Of the 80 samples of HCC, 57 cases (71.25%) and 60 cases (75%) expressed CENP-A and p53 proteins respectively. The positivity rates of CENP-A and p53 proteins in non-neoplastic liver tissue were 43.75% (35/80) and 16.25% (13/80) respectively. There was a statistically significant difference in CENP-A and p53 protein expression between HCC and non-neoplastic liver tissue (P < 0.01). The coincident rate between CENP-A and p53 expression was 88.75% (71/80). Expression of CENP-A protein showed a positive correlation with that of p53 protein (r = 0.57, P < 0.01).

CONCLUSIONThe over-expression of CENP-A occurs at transcriptional level and may be related to malignant proliferation of HCC via possible interaction with p53 gene.

Autoantigens ; biosynthesis ; genetics ; Carcinoma, Hepatocellular ; genetics ; metabolism ; pathology ; Centromere Protein A ; Chromosomal Proteins, Non-Histone ; biosynthesis ; genetics ; Gene Expression Regulation, Neoplastic ; Humans ; Immunohistochemistry ; Liver ; metabolism ; pathology ; Liver Neoplasms ; genetics ; metabolism ; pathology ; RNA, Messenger ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Suppressor Protein p53 ; metabolism

OBJECTIVETo study the influence of siRNA inhibition of CENP-A expression on the biological behavior of HepG2 cells.

METHODSThree pairs of 21 bp reverse repeated motifs of CENP-A target sequence with 9 spacer were synthesized and inserted into vector pSilencer 2.1-U6 neo to generate siRNA eukaryotic expression plasmids. After stable transfection into HepG2 cells, cell growth, apoptosis, cell cycles and plate clone forming efficiency were investigated. Expressions of CENP-A mRNA was monitored by the reverse transcriptase polymerase chain reaction (RT-PCR). The protein expression of CENP-A, bcl-2, Bax, p53, p21waf1 and mdm2 were detected by Western-blotting.

RESULTSTwo eukaryotic expression plasmids with significant siRNA specific inhibition to the CENP-A gene were created. Compared with control cells, HepG2 cells transfected with the constructs showed G1 phase delay (P < 0.01) and cell number decrease in the S phase (P < 0.001), along with an increased apoptotic rate (P = 0.003), significant increase of Bax expression and decreased bcl-2 expression (P< or =0.001). The protein expressions of p21waf1 was higher and mdm2 was lower than those of the control groups. However, the wild type p53 protein expression was not effected by CENP-A siRNA.

CONCLUSIONSAn altered expression of CENP-A may be related to the proliferation of hepatocellular carcinoma through cell cycle regulation involving an altered bcl-2/Bax expression, that may be p53 independent.

Autoantigens ; drug effects ; genetics ; Carcinoma, Hepatocellular ; pathology ; Cell Line, Tumor ; Centromere Protein A ; Chromosomal Proteins, Non-Histone ; drug effects ; genetics ; Gene Expression Regulation, Neoplastic ; drug effects ; Hep G2 Cells ; Humans ; RNA Interference ; drug effects ; RNA, Small Interfering ; drug effects ; pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Cells, Cultured