YAP/TAZ incorporation in the ß-catenin destruction complex orchestrates the Wnt response.

The Hippo transducers YAP/TAZ have been shown to play positive, as well as negative, roles in Wnt signaling, but the underlying mechanisms remain unclear. Here, we provide biochemical, functional, and genetic evidence that YAP and TAZ are integral components of the ß-catenin destruction complex that serves as cytoplasmic sink for YAP/TAZ. In Wnt-ON cells, YAP/TAZ are physically dislodged from the destruction complex, allowing their nuclear accumulation and activation of Wnt/YAP/TAZ-dependent biological effects. YAP/TAZ are required for intestinal crypt overgrowth induced by APC deficiency and for crypt regeneration ex vivo. In Wnt-OFF cells, YAP/TAZ are essential for ß-TrCP recruitment to the complex and ß-catenin inactivation. In Wnt-ON cells, release of YAP/TAZ from the complex is instrumental for Wnt/ß-catenin signaling. In line, the ß-catenin-dependent maintenance of ES cells in an undifferentiated state is sustained by loss of YAP/TAZ. This work reveals an unprecedented signaling framework relevant for organ size control, regeneration, and tumor suppression.

Snot what you think: Mucus or myxoid matrix with epithelioid cells and bubbly cytoplasm?

A case of progressive nasal obstruction in a 63 year old man is described. FNA cytology yielded dominant myxoid matrix with charateristic epithelioid cells, round nuclei with nucleoli, and eosinophilic, granular or vacuolated cytoplasm to allow a diagnosis tto be made.

Management of cytological material, pre-analytical procedures and bio-banking in effusion cytopathology.

Serous effusion fluid is one of the most commonly encountered specimens in routine cytopathology practice. It provides invaluable information about the patient and the clinical status; but to get the most of it, specimen handling and processing must be carried out properly. Cytomorphology is the basis of a successful analysis which should complemented by ancillary tests when needed. A wide spectrum of ancillary techniques - ranging from immunocytochemistry and flow cytometry to different assays of molecular pathology - can be applied to serous effusions. This article describes the acquisition and management of serous effusion fluids, methods for preservation and transportation, different techniques of cytopreparation, application of immunocytochemistry, flow cytometry, and fluorescence in-situ hybridization (FISH), as well as DNA extraction for polymerase chain reaction (PCR) and next generation sequencing (NGS). Principles of bio-banking of effusion samples are also discussed which is getting more important in correlation with the developments in personalized medicine.

Histological criteria for age determination of fatal venous thromboembolism.

In clinical and forensic practice, fatal thromboembolism is a major problem, particularly in a patient with no pre-existing risk factors. Out of a recent case, we discuss here the criteria for age determination of venous thrombi in a 46-year-old female with concomitant deep vein thrombosis in the common femoral left vein and in the right heart and fatal pulmonary embolism. At autopsy, histopathology and immunohistochemistry evidenced the different composition of thrombi in different sites and permitted to define the different timing. We discuss and review the histopathological criteria for age estimation of venous thrombi starting from the case and in relation to the acquired and inherited thrombophilic risk factors. The appropriateness of clinical management is also discussed.

Investigation of N-cadherin/ß-catenin expression in adrenocortical tumors.

ß-catenin is a multifunctional protein; it is a key component of the Wnt signaling, and it plays a central role in cadherin-based adhesions. Cadherin loss promotes tumorigenesis by releasing membrane-bound ß-catenin, hence stimulating Wnt signaling. Cadherins seem to be involved in tumor development, but these findings are limited in adrenocortical tumors (ACTs). The objective of this study was to evaluate alterations in key components of cadherin/catenin adhesion system and of Wnt pathway. This study included eight normal adrenal samples (NA) and 95 ACT: 24 adrenocortical carcinomas (ACCs) and 71 adrenocortical adenomas (ACAs). ß-catenin mutations were evaluated by sequencing, and ß-catenin and cadherin (E-cadherin and N-cadherin) expression was analyzed by quantitative reverse transcription PCR (qRT-PCR) and by immunohistochemistry (IHC). We identified 18 genetic alterations in ß-catenin gene. qRT-PCR showed overexpression of ß-catenin in 50 % of ACC (12/24) and in 48 % of ACA (21/44). IHC data were in accordance with qRT-PCR results: 47 % of ACC (7/15) and 33 % of ACA (11/33) showed increased cytoplasmic or nuclear ß-catenin accumulation. N-cadherin downregulation has been found in 83 % of ACC (20/24) and in 59 % of ACA (26/44). Similar results were obtained by IHC: N-cadherin downregulation was observed in 100 % (15/15) of ACC and in 55 % (18/33) of ACA. ß-catenin overexpression together with the aberrant expression of N-cadherin may play important role in ACT tumorigenesis. The study of differentially expressed genes (such as N-cadherin and ß-catenin) may enhance our understanding of the biology of ACT and may contribute to the discovery of new diagnostic and prognostic tools.

Profiling of expression of human papillomavirus-related cancer miRNAs in penile squamous cell carcinomas.

Penile squamous cell carcinoma (PSCC) is a rare tumor associated with high-risk human papillomavirus (HR-HPV) infection in 30% to 60% of cases. Altered expression of miRNAs has been reported in HPV-related cervical and head and neck cancers, but such data have not been available for PSCC. We analyzed a series of 59 PSCCs and 8 condylomata for presence of HPV infection, for p16(INK4a), Ki-67, and p53 immunohistochemical expression, and for expression of a panel of cellular miRNAs (let-7c, miR-23b, miR-34a, miR-145, miR-146a, miR-196a, and miR-218) involved in HPV-related cancer. HR-HPV DNA (HPV16 in most cases) was detected in 17/59 (29%) PSCCs; all penile condylomata (8/8) were positive for low-risk HPV6 or HPV11. HR-HPV(+) PSCCs overexpressed p16(INK4a) in 88% cases and p53 in 35% of cases, whereas HR-HPV(-) PSCCs were positive for p16(INK4a) and p53 immunostaining in 9% and 44% of cases, respectively. Among the miRNAs investigated, expression of miR-218 was lower in PSCCs with HR-HPV infection and in p53(-) cancers. Hypermethylation of the promoter of the SLIT2 gene, which contains miR-218-1 in its intronic region, was frequently observed in PSCCs, mainly in those with low miR-218 expression. Epigenetic silencing of miR-218 is a common feature in HR-HPV(+) PSCCs and in HR-HPV(-) PSCCs without immunohistochemical detection of p53.

The prognostic role of the epithelial-mesenchymal transition markers E-cadherin and Slug in laryngeal squamous cell carcinoma.

AIMS: Laryngeal squamous cell carcinoma (LSCC) prognosis is definitely related to lymph node metastasis. Epithelial-mesenchymal transition (EMT) allows neoplastic cells to gain the plasticity and motility required for tumour progression and metastasis. The aim of this study was to investigate the role of EMT in the prognosis of LSCC. METHODS AND RESULTS: Immunohistochemical analysis of E-cadherin, N-cadherin, Snail, Slug, ZEB1, and ZEB2 was performed in 37 consecutive LSCC cases. Low E-cadherin levels and high Slug levels correlated with both disease recurrence (P = 0.02 and P =0.01, respectively) and shorter disease-free survival (DFS) (P = 0.04 and P = 0.02, respectively). Relative expression levels of CDH1, SNAI2, miR-1 and the miR-200 family were also evaluated. CDH1, miR-200a and miR-200c down-regulation and SNAI2 overexpression were significantly associated with disease recurrence (P = 0.03, P = 0.02, P = 0.04, and P = 0.04, respectively). CONCLUSIONS: EMT increases tumour recurrence risk and shortens DFS in LSCC. E-cadherin and Slug immunohistochemical analysis could be useful for identifying patients requiring more aggressive treatment after surgery.

Mitogen-Activated Protein Kinase Pathway: Genetic Analysis of 95 Adrenocortical Tumors.

Mitogen-activated protein kinase (MAPK) pathway is often deregulated in adrenocortical tumors (ACT) but with no concrete data confirming alteration rate. The objective of this study was to evaluate genetic alterations in key components of MAPK pathway. We found one BRAF mutation (p.V600E) and four HRAS silent mutations. No alteration was found in NRAS, KRAS, EGFR genes. The patient carrying BRAF mutation was further characterized by investigating his biomolecular and clinico-pathological findings. Therefore, even if MAPK signaling is activated in ACT, our results suggest that genetic alterations do not seem to represent a frequent mechanism of ACT tumorigenesis.

Changes in microRNA expression during disease progression in patients with chronic viral hepatitis.

BACKGROUND & AIMS: MicroRNAs (miRNAs) have been involved in hepatocarcinogenesis, but little is known on their role in the progression of chronic viral hepatitis. Aim of this study was to identify miRNA signatures associated with stages of disease progression in patients with chronic viral hepatitis. METHODS: MiRNA expression profile was investigated in liver biopsies from patients with chronic viral hepatitis and correlated with clinical, virological and histopathological features. Relevant miRNAs were further investigated. RESULTS: Most of the significant changes in miRNA expression were associated with liver fibrosis stages and included the significant up-regulation of a group of miRNAs that were demonstrated to target the master regulators of epithelial-mesenchymal transition ZEB1 and ZEB2 and involved in the preservation of epithelial cell differentiation, but also in cell proliferation and fibrogenesis. In agreement with miRNA data, immunostaining of liver biopsies showed that expression of the epithelial marker E-cadherin was maintained in severe fibrosis/cirrhosis while expression of ZEBs and other markers of epithelial-mesenchymal transition were low or absent. Severe liver fibrosis was also significantly associated with the down-regulation of miRNAs with antiproliferative and tumour suppressor activity. Similar changes in miRNA and target gene expression were demonstrated along with disease progression in a mouse model of carbon tetrachloride (CCl4)-induced liver fibrosis, suggesting that they might represent a general response to liver injury. CONCLUSION: Chronic viral hepatitis progression is associated with the activation of miRNA pathways that promote cell proliferation and fibrogenesis, but preserve the differentiated hepatocyte phenotype.

Complete response to weekly carboplatin-docetaxel chemotherapy in a 91-year-old woman with anaplastic thyroid cancer.

Anaplastic thyroid cancer (ATC) is an unusual tumor with the worst prognosis among thyroid malignancies. Treatment of the patients diagnosed with ATC is not standardized and the feasible options include surgery, radiotherapy and chemotherapy. ATC cannot be regarded as a very chemo-sensitive tumor. Herein, we reported a case of a 91-year-old woman with complete response after induction chemotherapy (weekly carboplatin and docetaxel) that underwent subsequent radiotherapy.

Guidelines for the cytopathologic diagnosis of epithelioid and mixed-type malignant mesothelioma. Complementary statement from the International Mesothelioma Interest Group, also endorsed by the International Academy of Cytology and the Papanicolaou Society of Cytopathology.

OBJECTIVE: To provide practical guidelines for the cytopathologic diagnosis of malignant mesothelioma. DATA SOURCES: Cytopathologists with an interest in the field involved in the International Mesothelioma Interest Group (IMIG) and the International Academy of Cytology (IAC) contributed to this update. Reference material includes peer-reviewed publications and textbooks. RATIONALE: This article is the result of discussions during and after the IMIG 2012 conference in Boston, followed by thorough discussions during the 2013 IAC meeting in Paris. Additional contributions have been obtained from cytopathologists and scientists who could not attend these meetings, with final discussions and input during the IMIG 2014 conference in Cape Town.

Inflammation and pancreatic cancer: molecular and functional interactions between S100A8, S100A9, NT-S100A8 and TGFß1.

BACKGROUND: In order to gain further insight on the crosstalk between pancreatic cancer (PDAC) and stromal cells, we investigated interactions occurring between TGFß1 and the inflammatory proteins S100A8, S100A9 and NT-S100A8, a PDAC-associated S100A8 derived peptide, in cell signaling, intracellular calcium (Cai2+) and epithelial to mesenchymal transition (EMT). NF-κB, Akt and mTOR pathways, Cai2+ and EMT were studied in well (Capan1 and BxPC3) and poorly differentiated (Panc1 and MiaPaCa2) cell lines. RESULTS: NT-S100A8, one of the low molecular weight N-terminal peptides from S100A8 to be released by PDAC-derived proteases, shared many effects on NF-κB, Akt and mTOR signaling with S100A8, but mainly with TGFß1. The chief effects of S100A8, S100A9 and NT-S100A8 were to inhibit NF-κB and stimulate mTOR; the molecules inhibited Akt in Smad4-expressing, while stimulated Akt in Smad4 negative cells. By restoring Smad4 expression in BxPC3 and silencing it in MiaPaCa2, S100A8 and NT-S100A8 were shown to inhibit NF-κB and Akt in the presence of an intact TGFß1 canonical signaling pathway. TGFß1 counteracted S100A8, S100A9 and NT-S100A8 effects in Smad4 expressing, not in Smad4 negative cells, while it synergized with NT-S100A8 in altering Cai2+ and stimulating PDAC cell growth. The effects of TGFß1 on both EMT (increased Twist and decreased N-Cadherin expression) and Cai2+ were antagonized by S100A9, which formed heterodimers with TGFß1 (MALDI-TOF/MS and co-immuno-precipitation). CONCLUSIONS: The effects of S100A8 and S100A9 on PDAC cell signaling appear to be cell-type and context dependent. NT-S100A8 mimics the effects of TGFß1 on cell signaling, and the formation of complexes between TGFß1 with S100A9 appears to be the molecular mechanism underlying the reciprocal antagonism of these molecules on cell signaling, Cai2+ and EMT.

Role of SDHAF2 and SDHD in von Hippel-Lindau associated pheochromocytomas.

BACKGROUND: Pheochromocytomas (PCCs) develop from the adrenal medulla and are often part of a hereditary syndrome such as von Hippel-Lindau (VHL) syndrome. In VHL, only about 30 % of patients with a VHL missense mutation develop PCCs. Thus, additional genetic events leading to formation of such tumors in patients with VHL syndrome are sought. SDHAF2 (previously termed SDH5) and SDHD are both located on chromosome 11q and are required for the function of mitochondrial complex II. While SDHAF2 has been shown to be mutated in patients with paragangliomas (PGLs), SDHD mutations have been found both in patients with PCCs and in patients with PGLs. MATERIALS AND METHODS: Because loss of 11q is a common event in VHL-associated PCCs, we aimed to investigate whether SDHAF2 and SDHD are targets. In the present study, 41 VHL-associated PCCs were screened for mutations and loss of heterozygosity (LOH) in SDHAF2 or SDHD. Promoter methylation, as well as mRNA expression of SDHAF2 and SDHD, was studied. In addition, immunohistochemistry (IHC) of SDHB, known to be a universal marker for loss of any part the SDH complex, was conducted. RESULTS AND CONCLUSIONS: LOH was found in more than 50 % of the VHL-associated PCCs, and was correlated with a significant decrease (p < 0.05) in both SDHAF2 and SDHD mRNA expression, which may be suggestive of a pathogenic role. However, while SDHB protein expression as determined by IHC in a small cohort of tumors was lower in PCCs than in the surrounding adrenal cortex, there was no obvious correlation with LOH or the level of SDHAF2/SDHD mRNA expression. In addition, the lack of mutations and promoter methylation in the investigated samples indicates that other events on chromosome 11 might be involved in the development of PCCs in association with VHL syndrome.

Predicting late recurrence in surgically treated patients with Cushing's disease.

OBJECTIVE: Cushing's disease (CD) has an uncertain prognosis because patients achieving remission after transsphenoidal pituitary neurosurgery (TSS) may relapse. We aimed to identify factors predicting relapse, focusing on desmopressin (DDAVP) and corticotropin-releasing hormone (CRH) tests after surgery. MATERIALS AND METHODS: Fifty-seven patients with CD (mean age 36 years) after TSS experienced remission (24 cases), late relapse (LR) (15 cases), or persistent disease (18 cases). RESULTS: The median time to relapse was 40 months. ACTH levels increased after both DDAVP and CRH stimulation, with a significantly higher response in the late recurrence group, showing this to be an indicator of increased risk of relapse. In the logistic regression model, a rise in ACTH >9 pg/ml after DDAVP and >36·7 pg/ml after CRH showed a sensitivity of 93% and 73%, respectively, a specificity of 82% and 76% in LR group. The area under the curve was 0·91 for DDAVP, 0·80 for CRH and 0·95 for DDAVP+CRH test, i.e. the combined tests performed better than each test alone, but not to a statistically significant degree. A response to both tests resulted in a positive predictive value (PPV) of 100%, while no response to either test in a negative predictive value (NPV) of 100%. CONCLUSIONS: ACTH hyper-responsiveness to DDAVP stimulation proved a valuable indicator of relapsing patients with high sensitivity and specificity; in selected cases when a clear high increment of ACTH level is not evident, the CRH test might be used as additional tool to confirm the risk of future relapses.

The miR-17-92 microRNA cluster: a novel diagnostic tool in large B-cell malignancies.

Diffuse large B-cell lymphoma (DLBCL) can present as de novo or can arise through the transformation of many indolent lymphomas, including follicular lymphoma (FL). The morphological differentiation between germinal center-DLBCL (GC-DLBCL) and high-grade (grade 3) FL could be challenging; the accurate sub-classification of large B-cell lymphomas is mandatory in order to select the most appropriate among the new-targeted therapies. Recent expression profiling studies reported microRNAs (miRNAs) (and miR-17-92 cluster, in particular) as useful tools in differentiating DLBCL and FL. However, these preliminary results are based on cell line-derived data or did not consider grade 3 FL cases. To investigate this point, 36 cases of GC-DLBCL and 18 cases of grade 3 non-transforming FL were considered. All diagnoses were based on the World Health Organization criteria and were confirmed by clinical, histological, and immunohistochemical data. Six members of the miR-17-92 cluster (ie, miR-18b, miR-19b, miR-20a, miR-92, miR-93, and miR-106a) and two control miRNAs (ie, miR-150 and miR-210) were quantified by quantitative reverse transcription-polymerase chain reaction. All the considered miR-17-92 cluster miRNAs were significantly overexpressed in GC-DLBCL, being miR-20a and miR-106a the most dysregulated (P<0.001). Receiver operating characteristics (ROCs) analysis was used to find the optimal cut-off in distinguishing the two histotypes. The ROC estimated thresholds for miR-18b, miR-19b, miR-20a, miR-92, and miR-106a displayed a sensitivity level higher than 0.80 in achieving the GC-DLBCL diagnosis. The classification tree built on the six thresholds allowed the correct identification of 35/36 GC-DLBCL (97.2%). Profiling the miR-17-92 cluster is a promising investigative method for differentiating GC-DLBCL from high-grade FL. Subject to the validation of these findings in further larger studies; miR-17-92 cluster could represent a reliable, standardizable diagnostic tool for the sub-classification of large B-cell lymphoid neoplasm.

Epithelial-mesenchymal transition in malignant mesothelioma.

Epithelial-mesenchymal transition is a physiopathological process by which epithelial cells acquire mesenchymal shape and properties. Malignant mesothelioma is histologically characterized by the concomitant presence of epithelioid and sarcomatoid features, the latter being associated to worse prognosis, thus suggesting a role of epithelial-mesenchymal transition in this dual phenotype. We studied 109 malignant mesotheliomas (58 epithelioid, 26 sarcomatoid, and 25 biphasic) by immunohistochemistry and qRT-PCR analysis, and demonstrated a substantial switch from epithelial markers (E-cadherin, ß-catenin, and cytokeratins 5/6) to mesenchymal markers (N-cadherin, vimentin, α-smooth muscle actin, Snail, Slug, Twist, ZEB1, ZEB2, S100A4, MMP2, and MMP9) through epithelioid to biphasic and sarcomatoid histotypes. In agreement with these findings, the ectopic expression of miR-205 (a repressor of ZEB1 and ZEB2 expression) in MeT-5A (mesothelial cell line), H2452 (an epithelioid malignant mesothelioma cell line) and MSTO-211H (a biphasic malignant mesothelioma cell line) not only induced a significant reduction of ZEB1 and ZEB2 and a consequent up-regulation of E-cadherin gene expression, but also inhibited migration and invasion. Moreover, miR-205 was significantly down-regulated in biphasic and sarcomatoid histotypes (qRT-PCR and in situ hybridization analyses). Collectively, our findings indicate that epithelial-mesenchymal transition has a significant part in the morphological features of malignant mesothelioma. In particular, miR-205 down-regulation correlated significantly with both a mesenchymal phenotype and a more aggressive behavior.

L-citrulline prevents alveolar and vascular derangement in a rat model of moderate hyperoxia-induced lung injury.

BACKGROUND: Moderate normobaric hyperoxia causes alveolar and vascular lung derangement in the newborn rat. Endogenous nitric oxide (NO), which promotes lung growth, is produced from the metabolism of L-arginine to L-citrulline in endothelial cells. We investigated whether administering L-citrulline by raising the serum levels of L-arginine and enhancing NO endogenous synthesis attenuates moderate hyperoxia-induced lung injury. METHODS: Newborn rats were exposed to FiO(2) = 0.6 or room air for 14 days to induce lung derangement and then were administered L-citrulline or a vehicle (sham). Lung histopathology was studied with morphometric features. Lung tissues and bronchoalveolar lavage fluid (BALF) were collected for analysis. Lung vascular endothelial growth factor (VEGF), nitric oxide synthase (eNOS), and matrix metalloproteinase 2 (MMP2) gene and protein expressions were assessed. RESULTS: Serum L-arginine rose in the L-citr + hyperoxia group (p = 0.05), as well as the Von Willebrand factor stained vessels count (p = 0.0008). Lung VEGF immune staining, localized on endothelial cells, was weaker in the sections under hyperoxia than the L-citr + hyperoxia and room air groups. This pattern was comparable with the VEGF gene and protein expression profiles. Mean alveolar size increased in the untreated hyperoxia and sham-treated groups compared with the groups reared in room air or treated with L-citrulline under exposure to hyperoxia (p = 0.0001). Lung VEGF and eNOS increased in the L-citrulline-treated rats, though this treatment did not change MMP2 gene expression but regulated the MMP2 active protein, which rose in BALF (p = 0.003). CONCLUSIONS: We conclude that administering L: -citrulline proved effective in improving alveolar and vascular growth in a model of oxygen-induced pulmonary damage, suggesting better lung growth and matrix regulation than in untreated groups.

Necroptosis-driving genes RIPK1, RIPK3 and MLKL-p are associated with intratumoral CD3+ and CD8+ T cell density and predict prognosis in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a highly lethal cancer and the second leading cause of cancer-related deaths worldwide. As demonstrated in other solid neoplasms and HCC, infiltrating CD8+ T cells seem to be related to a better prognosis, but the mechanisms affecting the immune landscape in HCC are still mostly unknown. Necroptosis is a programmed, caspase-independent cell death that, unlike apoptosis, evokes immune response by releasing damage-associated molecular factors. However, in HCC, the relationship between the necroptotic machinery and the tumor-infiltrating lymphocytes has not been fully investigated so far. METHODS: We investigated the association between the main necroptosis-related genes, that is, RIPK1, RIPK3, MLKL-p, and CD3+/CD8+ tumor-infiltrating T cell by RNA-seq data analysis in 371 patients with primary HCC from The Cancer Genome Atlas and then by immunohistochemistry in two independent cohorts of HCC patients from Italy (82) and Japan (86). RESULTS: Our findings highlighted the immunogenetic role of necroptosis and its potential prognostic role in HCC: RIPK1, RIPK3 and MLKL-p were found significantly associated with intratumoral CD3+ and CD8+ T cells. In addition, multivariate survival analysis showed that the expression of RIPK1, RIPK3 and MLKL-p was associated with better overall survival in the two independent cohorts. CONCLUSIONS: Our results confirmed the immunogenetic properties of necroptosis (NCP) in human HCC, showing that tumor-infiltrating lymphocytes (TILs) and, specifically, CD8+ T cells accumulate in tumors with higher expression of the necroptosis-related genes. These results suggest the importance of further studies to better assess the specific composition, as well as the functional features of the immune environment associated with a necroptotic signature in order to explore new possible diagnostic and immunotherapeutic scenarios.

Inter- and intraobserver agreement in whole-slide digital ThinPrep samples of low-grade squamous lesions of the cervix uteri with known high-risk HPV status: A multicentric international study.

BACKGROUND: High-risk human papilloma virus (HR HPV) testing and liquid-based cytology are used for primary cervical screening. Digital cytology, based on whole-slide scanned samples, is a promising technique for teaching and diagnostic purposes. The aim of our study was to evaluate the interobserver and intraobserver variation in low-grade squamous lesions, HR HPV status bias, and the use of whole-slide scanned digital cervical cytology slides. METHODS: Fifteen expert cytopathologists evaluated 71 digitalized ThinPrep slides (31 atypical squamous cells of undetermined significance [ASC-US], 21 negative for intraepithelial lesion or malignancy, and 19 low-grade squamous intraepithelial lesion cases). HR HPV data were accessible only in the second round. RESULTS: In interobserver analysis, Kendall's coefficient of concordance was 0.52 in the first round and 0.58 in the second round. Fleiss' kappa values were 0.29 in the first round and 0.31 in the second round. In the ASC-US category, Fleiss kappa increased from 0.19 to 0.22 in the second round and the increase was even higher expressed by Kendall's coefficient: from 0.42 to 0.52. In intraobserver analysis, personal scores were higher in the second round. CONCLUSIONS: The interobserver and intraobserver variability in low-grade squamous lesions was within fair agreement values in the present study, in line with previous works. The comparison of two rounds showed that expert cytopathologists are generally unbiased by the knowledge of HR HPV data, but that being informed of the HR HPV status leads to a better agreement. Stain quality and back discomfort were highlighted as factors affecting digital cytopathology use.

Antitumor activity of gold(III)-dithiocarbamato derivatives on prostate cancer cells and xenografts.

Among the nonplatinum antitumor drugs, gold(III)-dithiocarbamato derivatives have recently attracted considerable attention due to their strong in vitro and in vivo antiproliferative activity and reduced renal toxicity. Some of them, namely [AuCl(2) (DMDT)] (compound 1) and [AuBr(2) (ESDT)] (compound 2), have shown to be highly active against the androgen-resistant prostate cancer cell lines PC3 and DU145, both inhibiting cell proliferation in a dose-dependent way, and are more active than the reference drug cisplatin (cis-[PtCl(2) (NH(3) )(2) ]). In particular, [AuCl(2) (DMDT)] was proved cytotoxic against cisplatin-resistant R-PC3 cells, with activity levels comparable to those induced on the parent cisplatin-sensitive PC3 cells, ruling out the occurrence of cross-resistance phenomena. Moreover, it causes early cell damage, slightly affecting the cell cycle, thus suggesting a different mechanism of action from clinically established platinum-based drugs. In fact, the investigated gold(III) complex alters mitochondrial functions, promoting mitochondrial membrane permeabilization and Cyt-c release, stimulating ROS generation, and strongly inhibiting the activity of the selenoenzyme TrxR, which is overexpressed in prostate cancer and associated with the onset of drug resistance. In addition, it induces apoptosis, caspase activation, Bcl-2 downregulation and Bax upregulation, reduces the expression of the phosphorylated form of the EGFR, and it inhibits PC3 cell migration. Finally, the treatment of PC3 prostate tumor-bearing nude mice with [AuCl(2) (DMDT)] significantly inhibited tumor growth in vivo, causing minimal systemic toxicity. Altogether, our results confirm that these gold(III)-dithiocarbamato derivatives have potential for the treatment of prostate cancer.