ASPM Promotes the Progression of Anaplastic Thyroid Carcinomas by Regulating the Wnt/ß-Catenin Signaling Pathway.

Background: Abnormal spindle-like microcephaly-associated protein (ASPM) is closely correlated with several malignant tumors, whereas little is known about the role of ASPM in anaplastic thyroid cancer (ATC). Herein, we sought to investigate whether ASPM is involved in the pathogenesis of ATC and the underlying mechanisms. Methods: The data from two data sets (GSE76039 and GSE33630) were extracted and analyzed for the expression of ASPM, followed by a further validation in collected ATC patients using quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. The effect of ASPM on cell proliferation, migration, invasion, and cell cycle was explored in ATC cell lines by in vitro inhibition of ASPM, while ASPM-mediated tumorigenicity was investigated in a xenograft tumor model. The involvement of Wnt/ß-catenin signaling pathway was also investigated. Results: ASPM was overexpressed in ATC patients and cell lines. In vitro knockdown of ASPM inhibited the proliferation, migration, and invasion capabilities of ATC cells and induced cell cycle arrest. Wnt/ß-catenin signaling was suppressed in response to ASPM inhibition, while rescue of ß-catenin expression restored the impaired biological functions of ATC cells. In vivo transplantation of ASPM-knockdown cells inhibited the growth of tumors. Conclusions: Upregulation of ASPM promotes the malignant properties of ATC cells and contributes to tumorigenesis through the Wnt/ß-catenin signaling pathway.

Overexpression of centromere protein K (CENPK) gene in Differentiated Thyroid Carcinoma promote cell Proliferation and Migration.

Differentiated thyroid carcinoma (DTC) is one of the most common malignant tumors. Increasing evidence indicates that centromere protein K(CENPK) may play a key role in promoting carcinogenesis. The expression, biological functions, and clinical significance of CENPK in DTC are still unclear. The CENPK expression in the DTC specimen was confirmed using quantitative real-time PCR and Western blot. The expression of CENPK was silenced and promoted by lentivirus-mediated transfection with shRNA sequences or CENPK plasmid targeting CENPK in TPC1 and FTC-133 cells, respectively. Colony formation, Cell Counting Kit-8 (CCK-8), Transwell invasion, and scratch assays were performed to assess the malignant biological properties of FTC-133 and TPC1 cells. Tumorigenicity assay was performed using C57BL/6 mice to explore the influence of CENPK on the growth of TPC1. The present work suggested that the expression of CENPK remarkably increased in follicular thyroid cancer and papillary thyroid cancer  tissue samples at the mRNA level. Immunohistochemical staining also showed consistent results at the protein level. In addition, CENPK mRNA expression level showed great value in diagnosis of DTC. Knockdown of CENPK significantly inhibited the invasion and migration of TPC1 and FTC-133 cells. In contrast, CENPK overexpression promoted invasion and migration of TPC1 and FTC-133 cells. Knockdown and overexpression of CENPK showed consistent effect on DTC tumor growth and expression of Ki-67 invivo. Our results indicated that CENPK was evidently upregulated in DTC. Knocking down CENPK suppressed TPC1 cell proliferation, invasion and migration. Targeting the CENPK may be anovel therapeutic method for DTC.

Inhibiting miR-21 attenuates experimental hepatic fibrosis by suppressing both the ERK1 pathway in HSC and hepatocyte EMT.

MicroRNA-21 (miR-21) has emerged as a critical regulatory molecule and an important serum marker in hepatic fibrogenesis. The aim of the present study was to investigate the role of inhibiting miR-21 on hepatic fibrosis treatment. Serum miR-21 levels in 60 healthy individuals and 180 patients with different stages of liver cirrhosis were examined, miR-21 levels in normal or cirrhotic human liver tissues (n=10 each) were also detected. An adenoviral vector (Ad-TuD-21) carrying the sponging ToughDecoy (TuD)-RNA sequence against miR-21 was constructed to reduce miR-21 expression efficiently in vitro and in vivo Histological and immunohistological examinations were performed to evaluate the inhibitory effects and mechanism of Ad-TuD-21 delivery into carbon tetrachloride (CCl4) induced hepatic fibrosis rats by targeting extracellular signal-regulated kinase 1 (ERK1) signalling in hepatic stellate cells (HSC) and hepatocyte epithelial-mesenchymal transition (EMT). Our results revealed that enhanced miR-21 levels in cirrhotic patients were related to the severity and activity of liver cirrhosis. Ad-TuD-21 administered to liver fibrosis rats could remarkably suppress profibrotic gene expression, cause histological improvements in liver and attenuate hepatic fibrosis significantly. More importantly, after Ad-TuD-21 treatment, inhibition of both the ERK1 signalling pathway in HSC and hepatocyte EMT was confirmed, which paralleled the enhancement of miR-21 target genes-sprouty2 (SPRY2) and hepatocyte nuclear factor 4α (HNF4α)-expression in vivo These data demonstrated that miR-21 is a key regulator to promote hepatic fibrogenesis, and sponging miR-21 expression may present a novel potentially therapeutic option for hepatic fibrosis.

Molecular testing and cervical screening: will one test fit all?

INTRODUCTION: Cervical screening has undergone significant changes in recent years, with molecular human papillomavirus (HPV) testing for HPV 16 and 18 at the forefront of clinical practice. But is molecular testing more effective than morphologic testing for cervical screening? Does current information on HPV hold true across all populations? As a public health laboratory serving high-risk, underserved populations, these remain important considerations for our practice. MATERIALS AND METHODS: The subject population largely consisted of young women within 200% or less of the poverty line. Correlation of Papanicolaou and HPV results was performed via retrospective review, focusing on Papanicolaou cases with high-grade diagnoses and an associated HPV test using the cobas 4800 HPV test. Secondary HPV testing and typing was performed via PCR at an outside laboratory for 205 cases with sufficient residual material and negative for HPV 16/18 by cobas. RESULTS: Of 20,211 cytology tests reviewed from July 2013 to May 2015, 521 were diagnosed as high-grade; 387 had concurrent HPV tests. Of those with concurrent HPV tests, 58% (225 of 387) of the high-grade Papanicolaou cases were not HPV 16/18 positive; furthermore, no HPV was detected in 14% (55 of 387) of these cases. Secondary testing revealed the presence of 25 unique genotypes. CONCLUSIONS: With recent emphasis on molecular HPV testing, the results of this review are concerning. As we move forward with evolution of cervical screening practices, it will be important to explore these questions for the continued quality and integrity of women's health services.

Clinicopathological characteristics of chinese colorectal cancer patients under 30 years of age: implication in diagnosis and therapy.

OBJECTIVES: The incidence of colorectal cancer (CRC) is increasing in younger populations; the characteristics and prognosis of those younger patients are not fully understood. The aim of this retrospective study was to analyze the clinicopathological features of Chinese CRC patients under 30 years of age. METHODS: We reviewed the clinical and pathological features of 83 CRC patients (33 males and 50 females) aged 13-30 years (mean, 26.1 years) selected from consecutive 5,830 patients with primary CRC referred to Shanghai Changzheng Hospital between January 1995 and December 2013. RESULTS: The duration from the onset of symptoms to diagnosis ranged from 3 days to 24.0 months (average 4.6 months). The most common symptom at the time of diagnosis was bloody stool, occurring in 66.3% of the patients. 60.2% patients had tumors located in the rectum and 72.8% of them presented advanced diseases (TNM stage III or IV). More male patients presented as M1 stage than the female patients. Patients with CRC metastasis complained of more fatigue at the time of diagnosis than their counterparts without metastasis (31.0% vs. 5.8%, p = 0.002), but had less pronounced bowel habit change (38.0% vs. 65.4%, p = 0.017). Additionally, there were differences in histologic distributions of the tumors between patients with and without metastasis (p = 0.021). CONCLUSIONS: Compared with older CRC patients, younger CRC patients (<30 yr) have a higher frequency of mucinous adenocarcinomas, more aggressive diseases and poorer prognosis. Identification of clinicopathological characteristics in younger CRC patients would help diagnose and treat the disease in this unique group of CRC patients in the clinic.

MicroRNA-155 attenuates activation of hepatic stellate cell by simultaneously preventing EMT process and ERK1 signalling pathway.

BACKGROUND & AIMS: Epithelial-mesenchymal transition (EMT) process and extracellular signal-regulated kinase 1 (ERK1) signalling pathway play pivotal roles in hepatic stellate cell (HSC) activation, which is associated with the altered expression patterns of microRNAs (miRNAs). miR-155 is considered a typical multifunctional miRNA to regulate many biological processes. However, little attention has been given to the contributions of miR-155 to simultaneous regulation of EMT process and ERK1 pathway during HSC activation. METHODS: Differential expression of miR-155 was assessed in activated HSC, sera and liver tissues from cirrhotic patients. Whether miR-155 could directly interact with 3'-untranslated region (3'-UTR) of T cell factor 4 (TCF4) and angiotensin II receptor type 1 (AGTR1) respectively was detected by luciferase reporter assay. The effects of enhanced miR-155 on EMT process and ERK1 pathway, cell apoptosis in HSC activation were also evaluated. RESULTS: A significant decrease in miR-155 expression was observed in activated HSC, sera or liver tissues of cirrhotic patients. MiR-155 was found to simultaneously interact with 3'-UTR of TCF4 and AGTR1 mRNAs, which are known as important regulators associated with EMT and ERK1 pathway repectively. Inhibiting miR-155 expression could stimulate the EMT state and ERK1 pathway activity, thus contributing to HSC activation. Forced miR-155 expression markedly decreased the mesenchymal markers and phosphorylated ERK1 level, and enhanced E-cadherin expression, leading to the synchronous inhibitory effect on EMT and ERK1 pathway and inducing HSC apoptosis. CONCLUSIONS: Our results implicate that miR-155 plays an important role in regulating the pathological network involving EMT process and ERK1 pathway during HSC activation.

MiR-21 simultaneously regulates ERK1 signaling in HSC activation and hepatocyte EMT in hepatic fibrosis.

BACKGROUND: MicroRNA-21 (miR-21) plays an important role in the pathogenesis and progression of liver fibrosis. Here, we determined the serum and hepatic content of miR-21 in patients with liver cirrhosis and rats with dimethylnitrosamine-induced hepatic cirrhosis and examined the effects of miR-21 on SPRY2 and HNF4α in modulating ERK1 signaling in hepatic stellate cells (HSCs) and epithelial-mesenchymal transition (EMT) of hepatocytes. METHODS: Quantitative RT-PCR was used to determine miR-21 and the expression of SPRY2, HNF4α and other genes. Immunoblotting assay was carried out to examine the expression of relevant proteins. Luciferase reporter assay was performed to assess the effects of miR-21 on its predicted target genes SPRY2 and HNF4α. Primary HSCs and hepatocytes were treated with miR-21 mimics/inhibitors or appropriate adenoviral vectors to examine the relation between miR-21 and SPRY2 or HNF4α. RESULTS: The serum and hepatic content of miR-21 was significantly higher in cirrhotic patients and rats. SPRY2 and HNF4α mRNA levels were markedly lower in the cirrhotic liver. MiR-21 overexpression was associated with enhanced ERK1 signaling and EMT in liver fibrosis. Luciferase assay revealed suppressed SPRY2 and HNF4α expression by miR-21. Ectopic miR-21 stimulated ERK1 signaling in HSCs and induced hepatocyte EMT by targeting SPRY2 or HNF4α. Downregulating miR-21 suppressed ERK1 signaling, inhibited HSC activation, and blocked EMT in TGFß1-treated hepatocytes. CONCLUSIONS: MiR-21 modulates ERK1 signaling and EMT in liver fibrosis by regulating SPRY2 and HNF4α expression. MiR-21 may serve as a potentially biomarker as well as intervention target for hepatic cirrhosis.

Virtual microscopy: an educator's tool for the enhancement of cytotechnology students' locator skills.

Virtual microscopy (VM) is being utilized as an educational tool in many areas of pathology. The aim of this study is to analyze the locator and diagnostic skills of cytotechnology students by using the Aperio T3 ScanScope, and examine VM's viability as an educational tool in cytotechnology. Ten validated cytology slides were digitized and reviewed by three senior cytotechnologist instructors. Each technologist made annotations indicating diagnostic areas on the virtual slide. A subset of the slides was used for locator skill evaluation. Cytotechnology students examined a pristine copy of the virtual slide and made annotations for comparison to those made by experienced instructors. Annotations of the subset were then scored based on the degree of correlation between students and cytotechnologists. A cytopathologist performed a final review of the students' marks; points were then added or subtracted based on this interpretation. Students were graded based on their correlation to senior cytotechnologists. A statistical analysis using modified interrater calculations ranked the students as to locator ability, producing illuminating results. This study shows that VM has promise as a cytotechnology educational tool by allowing the instructor to evaluate students' locator and diagnostic abilities. We have attempted to implement a simple scoring system for evaluation of locator skills where students are compared versus expert cytotechnologists. We anticipate further technological improvements as the products mature.

Virtual microscopy for cytology proficiency testing: are we there yet?

BACKGROUND: The objective of this study was to investigate the potential of virtual microscopy (VM) as an avenue for the delivery of mandatory cytology proficiency tests). METHODS: Three senior cytotechnologists and 2 board-certified cytopathologists participated in 3 virtual proficiency tests. Each set consisted of 10 ThinPrep slides that were digitized by an Aperio T3 ScanScope. The cytologic diagnoses covered the range of interpretive guidelines provided by the Centers for Medicare and Medicaid Services (CMS). Each cytotechnologist followed the requirement of a primary screener with the cytopathologists utilizing the secondary screener option. RESULTS: Analysis of the diagnostic interpretation of the first proficiency test showed correct classification of 100% of normal and abnormal cells for primary and secondary screeners. The second proficiency test analysis revealed a 93.3% correct classification (100% using CMS guidelines) among the primary screeners. The secondary screeners gave a 100% correct classification. The final proficiency test had primary screeners and secondary screeners with 100% correct classification. CONCLUSIONS: The current results confirmed the feasibility of VM for proficiency tests with 2 main problems noted. First, primary screeners had difficulties meeting the mandatory time allocation; however, with increased familiarity with the software, the screening times decreased. Second, the 3-dimensional nature of certain lesions made them difficult to interpret even on monolayered, liquid-based preparations. Creation of a more user-friendly software interface and better methods to capture depth of focus should make this a valid measure of cervicovaginal cytopathologic interpretive competence.

Clear-cell endocervical adenocarcinoma in a 19-year-old woman.

The incidence of adenocarcinoma of the cervix is increasing within the US, but this diagnostic category is not typically associated with teenaged patients. A report on a case of a 19-year-old woman, with no history of diethylbestrol exposure in uteri, diagnosed with clear-cell endocervical adenocarcinoma is made. Malignant glandular cells were detected on both conventional and Thin Prep gynecologic tests with histologic confirmation. Human papilloma virus (HPV) high-risk type results were negative in this particular case. The patient's unremarkable medical and sexual history prompted us to review the diagnostic criteria and pathogenesis of the disease.