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A Novel Chromogenic In Situ Hybridization Assay for FGF23 mRNA in Phosphaturic Mesenchymal Tumors.

Carter JM, Caron BL, Dogan A, Folpe AL.

PMID: 25025444 | DOI: 10.1097/PAS.0000000000000290

American Journal of Surgical Pathology

2014 July 14

Abstract +

Oncofetal Long Noncoding RNA PVT1 promotes proliferation and stem cell-like property of hepatocellular carcinoma cells by stabilizing NOP2

Wang F, Yuan JH, Wang SB, Yang F, Yuan SX, Ye C, Yang N, Zhou WP, Li WL, Li W, Sun SH

PMID: - | DOI: 10.1002/hep.27239


2014 July 12

Abstract +

Many protein-coding oncofetal genes are highly expressed in murine and human fetal liver and silenced in adult liver. The protein products of these hepatic oncofetal genes have been used as clinical markers for the recurrence of hepatocellular carcinoma (HCC) and as therapeutic targets for HCC. Herein, we examined the expression profiles of long non-coding RNAs (lncRNAs) found in fetal and adult liver in mice. Many fetal hepatic lncRNAs were identified; one of these, lncRNA-mPvt1, is an oncofetal RNA that was found to promote cell proliferation, cell cycling and the expression of stem cell-like properties of murine cells. Interestingly, we found that human lncRNA-hPVT1 was up-regulated in HCC tissues and that patients with higher lncRNA-hPVT1 expression had poor clinical prognosis. The protumorigenic effects of lncRNA-hPVT1 on cell proliferation, cell cycling and stem cell-like properties of HCC cells were confirmed both in vitro and in vivo by gain-of-function and loss-of-function experiments. Moreover, mRNA expression profile data showed that lncRNA-hPVT1 up-regulated a series of cell cycle genes in SMMC-7721 cells. By RNA pulldown and mass spectrum experiments, we identified NOP2 as an RNA-binding protein that binds to lncRNA-hPVT1. We confirmed that lncRNA-hPVT1 up-regulated NOP2 by enhancing the stability of NOP2 proteins and that lncRNA-hPVT1 function depends on the presence of NOP2. Conclusion: Our study demonstrates that the expression of many lncRNAs is up-regulated in early liver development and that the fetal liver can be used to search for new diagnostic markers for HCC. LncRNA-hPVT1 promotes cell proliferation, cell cycling and the acquisition of stem cell-like properties in HCC cells by stabilizing NOP2 protein. Regulation of the lncRNA-hPVT1/NOP2 pathway may have beneficial effects in the treatment of HCC. (Hepatology2014;)

X-inactivation normalizes O-GlcNAc Transferase levels and generates an O-GlcNAc-depleted Barr body

Olivier-van Stichelen, S, J. A. Hanover

PMID: - | DOI: 10.3389/fgene.2014.00256

Frontiers in Genetics

2014 July 11

Abstract +

O-GlcNAc Transferase (OGT) catalyzes protein O-GlcNAcylation, an abundant and dynamic nuclear and cytosolic modification linked to epigenetic regulation of gene expression. The steady-state levels of O-GlcNAc are influenced by extracellular glucose concentrations suggesting that O-GlcNAcylation may serve as a metabolic sensor. Intriguingly, human OGT is located on the X-chromosome (Xq13) close to the X-inactivation center (XIC), suggesting that OGT levels may be controlled by dosage compensation. In human female cells, dosage compensation is accomplished by X-inactivation. Long noncoding RNAs and polycomb repression act together to produce an inactive X chromosome, or Barr body. Given that OGT has an established role in polycomb repression, it is uniquely poised to auto-regulate its own expression through X-inactivation. In this study, we examined OGT expression in male, female and triple-X female human fibroblasts, which differ in the number of inactive X chromosomes (Xi). We demonstrate that OGT is subjected to random X-inactivation in normal female and triple X cells to regulate OGT RNA levels. In addition, we used Chromosome isolation by RNA purification (ChIRP) and immunolocalization to examine O-GlcNAc levels in the Xi/Barr body. Despite the established role of O-GlcNAc in polycomb repression, OGT and target proteins bearing O-GlcNAc are largely depleted from the highly condensed Barr body. Thus, while O-GlcNAc is abundantly present elsewhere in the nucleus, its absence from the Barr body suggests that the transcriptional quiescence of the Xi does not require OGT or O-GlcNAc.

Early colonizing Escherichia coli elicits remodeling of rat colonic epithelium shifting toward a new homeostatic state.

Tomas J, Reygner J, Mayeur C, Ducroc R, Bouet S, Bridonneau C, Cavin JB, Thomas M, Langella P, Cherbuy C.

PMID: 25012905 | DOI: doi:10.1038/ismej.2014.111.


2014 July 11

Abstract +

We investigated the effects of early colonizing bacteria on the colonic epithelium. We isolated dominant bacteria, Escherichia coli, Enterococcus faecalis, Lactobacillus intestinalis, Clostridium innocuum and a novel Fusobacterium spp., from the intestinal contents of conventional suckling rats and transferred them in different combinations into germfree (GF) adult rats. Animals were investigated after various times up to 21 days. Proliferative cell markers (Ki67, proliferating cell nuclear antigen, phospho-histone H3, cyclin A) were higher in rats monocolonized with E. coli than in GF at all time points, but not in rats monocolonized with E. faecalis. The mucin content of goblet cells declined shortly after E. coli administration whereas the mucus layer doubled in thickness. Fluorescence in situ hybridization analyses revealed that E. coli resides in this mucus layer. The epithelial mucin content progressively returned to baseline, following an increase in KLF4 and in the cell cycle arrest-related proteins p21CIP1 and p27KIP1. Markers of colonic differentiated cells involved in electrolyte (carbonic anhydrase II and slc26A3) and water (aquaglyceroporin3 (aqp3)) transport, and secretory responses to carbachol were modulated after E. coli inoculation suggesting that ion transport dynamics were also affected. The colonic responses to simplified microbiotas differed substantially according to whether or not E. coli was combined with the other four bacteria. Thus, proliferation markers increased substantially when E. coli was in the mix, but very much less when it was absent. This work demonstrates that a pioneer strain of E. coli elicits sequential epithelial remodeling affecting the structure, mucus layer and ionic movements and suggests this can result in a microbiota-compliant state.

Endogenous intrahepatic IFNs and association with IFN-free HCV treatment outcome.

Meissner EG, Wu D, Osinusi A, Bon D, Virtaneva K, Sturdevant D, Porcella S, Wang H, Herrmann E, McHutchison J, Suffredini AF, Polis M, Hewitt S, Prokunina-Olsson L, Masur H, Fauci AS, Kottilil S.

PMID: 24983321 | DOI: doi: 10.1172/JCI75938

J Clin Invest

2014 July 01

Abstract +

BACKGROUND. Hepatitis C virus (HCV) infects approximately 170 million people worldwide and may lead to cirrhosis and hepatocellular carcinoma in chronically infected individuals. Treatment is rapidly evolving from IFN-α-based therapies to IFN-α-free regimens that consist of directly acting antiviral agents (DAAs), which demonstrate improved efficacy and tolerability in clinical trials. Virologic relapse after DAA therapy is a common cause of treatment failure; however, it is not clear why relapse occurs or whether certain individuals are more prone to recurrent viremia. METHODS. We conducted a clinical trial using the DAA sofosbuvir plus ribavirin (SOF/RBV) and performed detailed mRNA expression analysis in liver and peripheral blood from patients who achieved either a sustained virologic response (SVR) or relapsed. RESULTS. On-treatment viral clearance was accompanied by rapid downregulation of IFN-stimulated genes (ISGs) in liver and blood, regardless of treatment outcome. Analysis of paired pretreatment and end of treatment (EOT) liver biopsies from SVR patients showed that viral clearance was accompanied by decreased expression of type II and III IFNs, but unexpectedly increased expression of the type I IFN IFNA2. mRNA expression of ISGs was higher in EOT liver biopsies of patients who achieved SVR than in patients who later relapsed. CONCLUSION. These results suggest that restoration of type I intrahepatic IFN signaling by EOT may facilitate HCV eradication and prevention of relapse upon withdrawal of SOF/RBV. TRIAL REGISTRATION. NCT01441180. FUNDING. Intramural Programs of the National Institute of Allergy and Infectious Diseases, National Institutes of Health Clinical Center, and National Cancer Institute; German Research Foundation.

Human Conchal Cartilage and Temporal Fascia: An Evidence-based Roadmap from Rhinoplasty to an In Vivo Study and Beyond.

Cimpean AM, Crăiniceanu Z, Mihailovici D, Bratu T, Raica M.

PMID: 24982216 | DOI:

In Vivo.

2014 July 01

Abstract +

Conchal cartilage or cartilage/ temporal fascia composite grafting (DC-F) used for rhinoplasty is applied by plastic surgeons for reconstructive purposes. Previous studies on experimental models such as mice or rabbits have elucidated on the late events following grafting, with tissue specimens being harvested two months after implantation. Early microscopic and molecular events following DC-F grafting are completely unknown. We designed a chick embryo chorioallantoic membrane model for human grafts study, regarding the dynamic observation of graft survival and its mutual interrelation with the chick embryo chorioallantoic membrane microenvironment. The DC-F graft preserved its cartilage component in a normal state compared to cartilage graft-only because of protective factors provided by temporal fascia. Its strong adherence to the cartilage, lack of angiogenic factors and high content of collagen IV-derived fragments with anti-angiogenic effects make the temporal fascia a good protective tissue to prevent implanted cartilage degeneration. The cartilage graft produced high inflammation, stromal fibrosis and activated angiogenic cascade through VEGF-mediated pathways followed by cartilage degeneration. Also, high content of podoplanin from conchal cartilage chondrocytes exerted a major role in inflammation accompanying cartilage graft. The presently employed experimental model allowed us to characterize the early histological and molecular events triggered by temporal fascia, cartilage or composite graft DC-F implanted on chick embryo chorioallantoic membrane. Our microscopic and molecular observations may help explain some post-surgical complications generated after using cartilage alone as biomaterial for nasal augmentation, supporting the use of DC-F composite graft, with the aim to reduce unwanted post-surgical events.

PROX1 Expression in Gastric Cancer: From Hypothesis to Evidence.

Taban O, Cimpean AM, Raica M, Olariu S.

PMID: 24982352 | DOI:

Anticancer Res.

2014 July 01

Abstract +

Background: PROX1 is involved in cancer development and progression as both a tumor suppressor and oncogene. Immunohistochemical (IHC) PROX1 nuclear expression is a widely accepted pattern. Scattered data reported PROX1 IHC cytoplasmic expression in different tumors, including gastric cancer but it is not clear if this holds true. Materials and Methods: Evaluation of the cytoplasmic expression of PROX1 in normal gastric mucosa and gastric cancer was performed by IHC followed by RNAscope, an in situ hybridization-based method for detecting PROX1 mRNA amplification on paraffin-embedded samples and to evaluate its clinical impact. Results: Twenty five out of 48 cases of gastric cancer showed PROX1 nuclear and cytoplasmic immunohistochemical expression. Twelve out of these 20 cases positive for PROX1 on IHC (54.5%) had PROX1 mRNA gene amplification. The overlapping of PROX1 cytoplasmic expression assessed by immunohistochemistry and cytoplasmic RNAscope amplification was statistically significant (p=0.031). PROX1 mRNA gene amplification correlated with tumor grade (p=0.05) and regional lymph node metastasis as well (p=0.033). No significant correlation was obtained between PROX1 and histopathology, tumor size or distal metastasis. Conclusion: A significant correlation was found between IHC and RNAscope PROX1 expression in the cytoplasm of normal and gastric cancer cells. This strongly supports its validation as a true expression on immunohistochemistry. A strong correlation between PROX1 mRNA amplification and regional lymph node metastasis supports its implications in cancer spreading and metastasis and sustains its utility, not only as a lymphatic marker, but also as a potential tumor marker in various tumor types, including gastric cancer.

Prognostic significance of p62/SQSTM1 subcellular localization and LC3B in oral squamous cell carcinoma

Liu JL, Chen FF, Lung J, Lo CH, Lee FH, Lu YC, Hung CH.

PMID: 24983366 | DOI: doi: 10.1038/bjc.2014.355.

Br J Cancer.

2014 July 01

Abstract +

Background:Autophagy is a programmed cell survival mechanism that has a key role in both physiologic and pathologic conditions. The relationship between autophagy and cancer is complex because autophagy can act as either a tumour suppressor or as a tumour promoter. The role of autophagy in oral squamous cell carcinoma (OSCC) is controversial. Several studies have claimed that either a high or low expression of autophagy-related proteins was associated with poor prognosis of OSCCs. The aims of the study were to compare autophagy in OSCCs, verrucous hyperplasias, and normal oral mucosas, and to inspect the prognostic role of autophagy in OSCCs.Methods:We used the autophagosome marker, LC3B, and autophagy flux marker, p62/SQSTM1 (p62), by using immunohistochemistry, and examined p62 mRNA by RNA in situ hybridization, to evaluate autophagy in 195 OSCCs, 47 verrucous hyperplasias, and 37 normal oral mucosas. The prognostic roles of LC3B and p62 protein expressions in OSCCs were investigated.Results:We discovered that the normal oral mucosa exhibited limited LC3B punctae and weak cytoplasmic p62 staining, whereas the OSCCs exhibited a marked increase in LC3B punctae and cytoplasmic p62 expression. The expression pattern of LC3B and cytoplasmic p62 of the verrucous hyperplasias were between normal oral mucosas and OSCCs. The normal oral mucosas, verrucous hyperplasias, and OSCCs presented no differences in nuclear p62 expression and the p62 mRNA level. p62 mRNA expression was elevated in a minority of cases. High p62 mRNA expression was associated with high p62 protein expression in the cytoplasm. Increased LC3B punctae, high cytoplasmic p62, and low nuclear p62 expressions in OSCCs were associated with aggressive clinicopathologic features and unfavourable prognosis. In addition, low nuclear p62 expression was an independent prognostic factor for overall and disease-specific survival rates. Furthermore, we disclosed that high cytoplasmic p62 expression accompanied with either a low or high LC3B expression, which indicated autophagy impairment under basal or activated autophagic activity, was associated with aggressive behaviour in advanced OSCCs.Conclusions:We suggested that autophagy was altered during cancer initiation and progression. Autophagy impairment contributed to cancer progression in advanced OSCCs.British Journal of Cancer advance online publication, 1 July 2014; doi:10.1038/bjc.2014.355

In Situ Quantitative Measurement of HER2mRNA Predicts Benefit from Trastuzumab-Containing Chemotherapy in a Cohort of Metastatic Breast Cancer Patients

Vassilakopoulou M, Togun T, Dafni U, Cheng H1, Bordeaux J, Neumeister VM, Bobos M, Pentheroudakis G, Skarlos DV, Pectasides D, Kotoula V, Fountzilas G, Rimm DL, Psyrri A.

PMID: 24968015 | DOI: 10.1371/journal.pone.0099131. eCollection 2014.

PLoS One.

2014 June 24

Abstract +

BACKGROUND: We sought to determine the predictive value of in situ mRNA measurement compared to traditional methods on a cohort of trastuzumab-treated metastatic breast cancer patients.

METHODS: A tissue microarray composed of 149, classified as HER2-positive, metastatic breast cancers treated with various trastuzumab-containing chemotherapy regimens was constructed. HER2 intracellular domain(ICD), HER2 extracellular domain(ECD) and HER2 mRNA were assessed using AQUA. For HER2 protein evaluation, CB11 was used to measure ICD and SP3 to measure ECD of the HER2 receptor. In addition, HER2 mRNA status was assessed using RNAscope assay ERRB2 probe. Kaplan - Meier estimates were used for depicting time-to-event endpoints. Multivariate Cox regression models with backward elimination were used to assess the performance of markers as predictors of TTP and OS, after adjusting for important covariates.

RESULTS: HER2 mRNA was correlated with ICD HER2, as measured by CB11 HER2, with ECD HER2 as measured by SP3 (Pearson's Correlation Coefficient, r = 0.66 and 0.51 respectively) and with FISH HER2 (Spearman's Correlation Coefficient, r = 0.75). All markers, HER2 mRNA, ICD HER2 and ECD HER2, along with FISH HER2, were found prognostic for OS (Log-rank p = 0.007, 0.005, 0.009 and 0.043 respectively), and except for FISH HER2, they were also prognostic for TTP Log-rank p = 0.036, 0.068 and 0.066 respectively) in this trastuzumab- treated cohort. Multivariate analysis showed that in the presence of pre-specified set of prognostic factors, among all biomarkers only ECD HER2, as measured by SP3, is strong prognostic factor for both TTP (HR = 0.54, 95% CI: 0.31-0.93, p = 0.027) and OS (HR = 0.39, 95%CI: 0.22-0.70, p = 0.002).

CONCLUSIONS: The expression of HER2 ICD and ECD as well as HER2 mRNA levels was significantly associated with TTP and OS in this trastuzumab-treated metastatic cohort. In situ assessment of HER2 mRNA has the potential to identify breast cancer patients who derive benefit from Trastuzumab treatment.

Functional ex vivo assay to select Homologous Recombination deficient breast tumors for PARP inhibitor treatment

Naipal KA, Verkaik NS, Ameziane N, van Deurzen CH, Ter Brugge P, Meijers M, Sieuwerts AM, Martens J, O'Connor MJ, Vrieling H, Hoeijmakers JH, Jonkers J, Kanaar R, de Winter J, Vreeswijk M, Jager A, van Gent DC.

PMID: 24963051 | DOI:

Clin Cancer Res.

2014 June 24

Abstract +

Purpose: Poly(ADP-Ribose) Polymerase (PARP) inhibitors are promising targeted treatment options for hereditary breast tumors with a Homologous Recombination (HR) deficiency caused by BRCA1 or BRCA2 mutations. However, the functional consequence of BRCA gene mutations is not always known and tumors can be HR deficient for other reasons than BRCA gene mutations. Therefore, we aimed to develop a functional test to determine HR activity in tumor samples to facilitate selection of patients eligible for PARP inhibitor treatment. Experimental design: We obtained 54 fresh primary breast tumor samples from patients undergoing surgery. We determined their HR capacity by studying the formation of ionizing radiation induced foci (IRIF) of the HR protein RAD51 after ex vivo irradiation of these organotypic breast tumor samples. Tumors showing impaired RAD51 IRIF formation were subjected to genetic and epigenetic analysis. Results: Five out of 45 primary breast tumors with sufficient numbers of proliferating tumor cells were RAD51 IRIF formation deficient (11%, 95%CI: 5%-24%). This HR defect was significantly associated with Triple Negative Breast Cancer (OR:57, 95%CI: 3.9-825, p=0.003). Two out of five HR deficient tumors were not caused by mutations in the BRCA genes, but by BRCA1 promoter hypermethylation. Conclusion: The functional RAD51 IRIF assay faithfully identifies HR deficient tumors and has clear advantages over gene sequencing. It is a relatively easy assay that can be performed on biopsy material, making it a powerful tool to select patients with an HR-deficient cancer for PARP inhibitor treatment in the clinic.

GATA4 is essential for bone mineralization via ERα and TGFβ/BMP pathways

Güemes M, Garcia AJ, Rigueur D, Runke S, Wang W, Zhao G, Mayorga VH, Atti E, Tetradis S, Péault B, Lyons K, Miranda-Carboni GA, Krum SA.

PMID: 24932701 | DOI: doi: 10.1002/jbmr.2296.

J Bone Miner Res. 2014 Jun 16.

2014 June 16

Abstract +

Osteoporosis is a disease characterized by low bone mass, leading to an increased risk of fragility fractures. GATA4 is a zinc-finger transcription factor that is important in several tissues, such as the heart and intestines, and has recently been shown to be a pioneer factor for estrogen receptor alpha (ERα) in osteoblast-like cells. Herein, we demonstrate that GATA4 is necessary for estrogen-mediated transcription and estrogen-independent mineralization in vitro. In vivo deletion of GATA4, driven by Cre-recombinase in osteoblasts, results in perinatal lethality, decreased trabecular bone properties and abnormal bone development. Microarray analysis revealed GATA4 suppression of TGFβ signaling, necessary for osteoblast progenitor maintenance and concomitant activation of BMP signaling, necessary for mineralization. Indeed, pSMAD1/5/8 signaling, downstream of BMP signaling, is decreased in the trabecular region of conditional knockout femurs, and pSMAD2/3, downstream of TGFβ signaling, is increased in the same region. Together these experiments demonstrate the necessity of GATA4 in osteoblasts. Understanding the role of GATA4 to regulate the tissue specificity of estrogen-mediated osteoblast gene regulation and estrogen-independent bone differentiation may help to develop therapies for post-menopausal osteoporosis. © 2014 American Society for Bone and Mineral Research

Hippo pathway activity influences liver cell fate.

Yimlamai D, Christodoulou C, Galli GG, Yanger K, Pepe-Mooney B, Gurung B, Shrestha K, Cahan P, Stanger BZ, Camargo FD.

PMID: 24906150 | DOI: doi: 10.1016/j.cell.2014.03.060.


2014 June 05

Abstract +

The Hippo-signaling pathway is an important regulator of cellular proliferation and organ size. However, little is known about the role of this cascade in the control of cell fate. Employing a combination of lineage tracing, clonal analysis, and organoid culture approaches, we demonstrate that Hippo pathway activity is essential for the maintenance of the differentiated hepatocyte state. Remarkably, acute inactivation of Hippo pathway signaling in vivo is sufficient to dedifferentiate, at very high efficiencies, adult hepatocytes into cells bearing progenitor characteristics. These hepatocyte-derived progenitor cells demonstrate self-renewal and engraftment capacity at the single-cell level. We also identify the NOTCH-signaling pathway as a functional important effector downstream of the Hippo transducer YAP. Our findings uncover a potent role for Hippo/YAP signaling in controlling liver cell fate and reveal an unprecedented level of phenotypic plasticity in mature hepatocytes, which has implications for the understanding and manipulation of liver regeneration.

SIRT6 Is Required for Normal Retinal Function.

Silberman DM, Ross K, Sande PH, Kubota S, Ramaswamy S, Apte RS, Mostoslavsky R.

PMID: 24896097 | DOI: doi: 10.1371/journal.pone.0098831

PLoS One.

2014 June 04

Abstract +

The retina is one of the major energy consuming tissues within the body. In this context, synaptic transmission between light-excited rod and cone photoreceptors and downstream ON-bipolar neurons is a highly demanding energy consuming process. Sirtuin 6 (SIRT6), a NAD-dependent deacylase, plays a key role in regulating glucose metabolism. In this study, we demonstrate that SIRT6 is highly expressed in the retina, controlling levels of histone H3K9 and H3K56 acetylation. Notably, despite apparent normal histology, SIRT6 deficiency caused major retinal transmission defects concomitant to changes in expression of glycolytic genes and glutamate receptors, as well as elevated levels of apoptosis in inner retina cells. Our results identify SIRT6 as a critical modulator of retinal function, likely through its effects on chromatin.

Detection of human papillomavirus (HPV) in clinical samples: Evolving methods and strategies for the accurate determination of HPV status of head and neck carcinomas

Westra WH

PMID: 1 | DOI: DOI: 10.1016/j.oraloncology.2014.05.004

Oral Oncology, 2014

2014 June 02

Abstract +

Much recent attention has highlighted a subset of head and neck squamous cell carcinomas (HNSCCs) related to human papillomavirus (HPV) that has an epidemiologic, demographic, molecular and clinical profile which is distinct from non-HPV-related HNSCC. The clinical significance of detecting HPV in a HNSCC has resulted in a growing expectation for HPV testing of HNSCCs. Although the growing demand for routine testing is understandable and appropriate, it has impelled an undisciplined approach that has been largely unsystematic. The current state of the art has now arrived at a point where a better understanding of HPV-related tumorigenesis and a growing experience with HPV testing can now move wide scale, indiscriminant and non-standardized testing towards a more directed, clinically relevant and standardized approach. This review will address the current state of HPV detection; and will focus on why HPV testing is important, when HPV testing is appropriate, and how to test for the presence of HPV in various clinical samples. As no single test has been universally accepted as a best method, this review will consider the strengths and weaknesses of some of the more commonly used assays, and will emphasize some emerging techniques that may improve the efficiency of HPV testing of clinical samples including cytologic specimens.

HER2 Status in Colorectal Cancer: Its Clinical Significance and the Relationship between HER2 Gene Amplification and Expression.

Seo AN, Kwak Y, Kim DW, Kang SB, Choe G, Kim WH, Lee HS.

PMID: 24879338 | DOI: doi: 10.1371/journal.pone.0098528

PLoS One

2014 May 30

Abstract +

This study aimed at determining the incidence and clinical implications of HER2 status in primary colorectal cancer (CRC). HER2 status was investigated in two retrospective cohorts of 365 consecutive CRC patients (cohort 1) and 174 advanced CRC patients with synchronous or metachronous distant metastasis (cohort 2). HER2 status was determined by performing dual-color silver in-situ hybridization (SISH), mRNA in-situ hybridization (ISH), and immunohistochemistry (IHC). The incidence of HER2 protein overexpression (IHC 2+/3+) was approximately 6% (22 of 365 in cohort 1; 10 of 174 in cohort 2). HER2 gene amplification was observed in 5.8% of the patients from cohort 1 and 6.3% of the patients from cohort 2.HER2 gene amplification was more frequently observed in CRCs located in the rectum than in the right and left colon (P = 0.013 in cohort 1; P = 0.009 in cohort 2). HER2 status, determined by IHC, ISH, and dual-color SISH, was not significantly associated with aggressive CRC behaviour or patients' prognosis in both the cohorts. Of the combined cohort with a total of 539 cases, the concordance rate was 95.5% between dual-color SISH and IHC detection methods. On excluding equivocally immunostained cases (IHC 2+), the concordance rate was 97.7%. HER2 mRNA overtranscription, detected by ISH, significantly correlated with protein overexpression and gene amplification (P<0.001). HER2 gene amplification was identified in a minority of CRC patients with high concordance rates between dual-color SISH and IHC detection methods. Although HER2 status did not predict patients' prognosis, our findings may serve as a basis for future studies on patient selection for HER2 targeted therapy.

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