ABSTRACT
Familial amyloidotic polyneuropathy (FAP) is caused by a mutation in the transthyretin (TTR) gene. In addition, deposition of wild-type TTR can cause senile systemic amyloidosis (SSA). To date, we have produced several transgenic mouse models for FAP and SSA by introducing TTR genes with different promoters or mutations. However, mouse TTR can associate with human TTR to produce hybrid tetramers in transgenic mice. Thus, these transgenic mice cannot be used to test the efficacy of a new therapy. In this study, we attempted to construct an optimized mouse model to verify a new therapy. The TTR gene consists of 4 exons and 3 introns. We prepared two gRNAs, one for the exon 1 and the other for exon 4, and a single donor vector carrying the whole TTR gene in which mouse exons were replaced with human exons. Using these vectors, we produced a TTR exon-humanized mouse with human exons and mouse introns using genome editing technology. These TTR exon-humanized mice showed normal TTR expression patterns in terms of serum TTR level and spatial specificity. These TTR exon-humanized mice will be useful for devising new treatment methods for FAP, including gene therapy.
Subject(s)
Polyneuropathies/etiology , Prealbumin/genetics , Animals , Disease Models, Animal , Exons , Gene Expression Regulation , Humans , Mice, Inbred C57BL , Mice, Inbred ICR , Mice, Transgenic , Polyneuropathies/therapy , Prealbumin/analysis , RNA, Guide, Kinetoplastida/geneticsABSTRACT
BACKGROUND: HER2 testing for samples from recurrent or metastatic disease is recommended by the 2013 update of the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines and cytological analysis can be applied to several types of metastatic lesions. However, the practical method to assess the HER2 testing of breast cancer cytology specimens has yet to be resolved. Therefore, we conducted the bright-field HER2 dual in situ hybridization (DISH) assay on cell blocks (CBs) prepared from breast cancer cell samples as a validation study before clinical use. METHODS: CBs were prepared from tumor cell samples collected from 54 surgically excised breast tumors. The cells were fixed in 10 % buffered formalin for 16-28 h, and embedded in paraffin. The INFORM HER2/neu Dual ISH DNA Probe Cocktail was used for the DISH assay on the Ventana BenchMark ULTRA (Roche Diagnostics). RESULTS: Successful results were obtained in 51 of 54 CB specimens, and the results from the CB specimens were in agreement with those from the histological sections in 48 of the 51 cases (concordance rate, 94 %; kappa, 0.846). The intraclass correlation coefficient (ICC) between the CB and histological specimens in the continuous HER2/CEP17 signal count ratio was 0.89 (95 % CI 0.81-0.93), and the Pearson's CC was 0.91 (95 % CI 0.85-0.94). CONCLUSION: The HER2 DISH assay, utilizing 10 % buffered formalin-fixed CB, would be a reliable and ideal method to assess the HER2 gene status of breast cancer cytological specimens.
Subject(s)
Biomarkers, Tumor/analysis , Breast Neoplasms/pathology , In Situ Hybridization/methods , Receptor, ErbB-2/analysis , Breast Neoplasms/metabolism , Breast Neoplasms/surgery , Female , Humans , Specimen Handling/methodsABSTRACT
BACKGROUND: Human epidermal growth factor receptor 2 (HER2) testing of samples from recurrent or metastatic breast cancer is recommended by the 2013 update of the American Society of Clinical Oncology/College of American Pathologists guidelines. Although cytological analysis can be applied to several types of metastatic lesions, the practical method for HER2 testing of cytological specimens is yet to be resolved. We conducted immunohistochemical (IHC) staining for HER2 in breast cancer cell blocks (CBs) and compared the results with those from the corresponding histological specimens. In cases of discrepancy between the two types of specimen, the bright-field HER2 dual in situ hybridization (DISH) assay was performed. METHODS: CBs were prepared from 54 surgically excised breast cancers. The cells were fixed in 10% buffered formalin and embedded in paraffin. A Ventana BenchMark ULTRA (Roche Diagnostics) with anti-HER-2/neu (4B5) rabbit monoclonal primary antibody and INFORM HER2/neu Dual ISH DNA Probe Cocktail was used for the assays. RESULTS: Successful results were obtained in 52 of 54 CBs. Forty cases showed agreement between CBs and the histological specimens. No discrepancy was observed between the two types of specimens in cases where HER2 expression was positive. IHC results of CB in 12 discrepant cases were HER2 intermediate or negative. The DISH results of 11 of these cases were negative. CONCLUSION: IHC staining of HER2 for breast cancer CBs can be used in the same way as that used for histological specimens, although the number of equivocal cases in CBs is greater than that in histological specimens. Diagn. Cytopathol. 2016;44:274-279. © 2016 The Authors Diagnostic Cytopathology Published by Wiley Periodicals, Inc.
Subject(s)
Biomarkers, Tumor/genetics , Breast Neoplasms/diagnosis , Immunohistochemistry/standards , Receptor, ErbB-2/genetics , Antibodies, Monoclonal/chemistry , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Breast Neoplasms/surgery , Female , Gene Expression , Humans , In Situ Hybridization, Fluorescence , Microtomy , Neoplasm Metastasis , Practice Guidelines as Topic , Sensitivity and Specificity , Tissue Embedding , Tissue FixationABSTRACT
BACKGROUND: While HER2 gene detection in cytological specimens using fluorescence in situ hybridization (FISH) has been reported, the appropriate criteria for such specimens remain controversial. METHODS: Fine needle aspiration (FNA) samples collected from surgically resected breast cancer specimens were rinsed in a cytopreservative solution containing fixative. Then, slides of the FNA samples were prepared by liquid-based cytology (LBC) (ThinPrep system, Hologic) according to the manufacturer's instructions, and a PathVision HER2 DNA probe kit (Abbott) was used for FISH staining. The results were evaluated using an automated MetaCyte imaging system (MetaSystems, Altlussheim, Germany). HER2 gene amplification was scored using the HER2/chromosome enumeration probe 17 (CEP17) signal count ratio as follows: amplified, >2.2; equivocal, 1.8-2.2; and unamplified, <1.8. The cytology results were compared with the histology results from concordant cases. RESULTS: Successful results were obtained in 98 of 100 cases, and results from the FNA specimens were in agreement with those from the histological sections in 97 of these 98 cases (accuracy rate, 99 %; kappa, 0.962). CONCLUSIONS: FISH-based assessment of the HER2 gene status is consistent between histological sections and cytological specimens of breast cancer.
