Giulia Masi

Loss of Growth Hormone Secretagogue Receptor 1a and Overexpression of Type 1b Receptor Transcripts in Human Adrenocortical Tumors

Objective and Methods: Quantitative analysis of mRNA expression of ghrelin and its receptors GHS-R1a and -R1b in a large series of normal and neoplastic human adrenocortical tissues. Evaluation of the effects of ghrelin on GHS-R expression and proliferation of human adrenocortical carcinoma (ACC) cell lines. Results: Ghrelin and GHS-R transcripts are expressed in normal adrenal cortex, with GHS-R1b mRNA levels being 5- to 10-fold higher than GHS-R1amRNA. A significant increase in ghrelin expression was observed in adrenocortical adenomas, but not in carcinomas. GHS-R1a was undetectable in about 60% of both benign and malignant tumor samples, except for cortisol-producing adenomas, which showed increased receptor expression. At variance, GHS-R1b was overexpressed in both benign and malignant adrenocortical tumors. In vitro studies in human ACC cell lines demonstrated that GHS-R1a is downregulated and GHS-R1bmRNA expression is upregulated by ghrelin, while inhibiting cell proliferation. Conclusion: Downregulation ofGHS-R1a in adrenal tumors and the presence of high levels of GHS-R1b transcripts in adrenocortical tissue suggest a role for these receptors in adrenal function and growth. In this regard, ghrelin inhibits cell proliferation and modulates GHS-R expression in ACC cells in vitro.

Whole genome sequencing and phylogenetic analysis of West Nile virus lineage 1 and lineage 2 from human cases of infection, Italy, August 2013.

A human outbreak of West Nile virus (WNV) infection caused by WNV lineage 2 is ongoing in northern Italy. Analysis of six WNV genome sequences obtained from clinical specimens demonstrated similarities with strains circulating in central Europe and Greece and the presence of unique amino acid changes that identify a new viral strain. In addition, WNV lineage 1 Livenza, responsible for a large outbreak in north-eastern Italy in 2012, was fully sequenced from a blood donor during this 2013 outbreak.

Clinical, genetic, and histopathologic investigation of CDC73-related familial hyperparathyroidism

CDC73 (HRPT2) germline mutations are responsible for more than half of cases of hyperparathyroidism-jaw tumor syndrome (HPT-JT) and for a subset of familial isolated HPT (FIHP). We performed a clinical, genetic, and histopathologic study in three unrelated Italian kindreds with HPT-JT and FIHP. We identified three germline inactivating mutations of the CDC73 gene in the probands and affected patients of the three kindreds, but also in some asymptomatic subjects. HPT-JT and FIHP patients had similar laboratory, clinical, and demographic features and shared primary HPT and other neoplasms, the most common of which was uterine polyposis. Genetic analysis of tumor samples demonstrated a second somatic CDC73 mutation only in a parathyroid adenoma and no cases with the loss of the wild-type allele or methylation of the CDC73 promoter, even though immunohistochemical analysis demonstrated the loss of nuclear parafibromin expression in all tumors, including a uterine polyp. In conclusion, our results indicate that FIHP and HPT-JT associated with CDC73 mutations do not have distinct clinical, genetic, and histopathologic features, but may represent variants of the same genetic disease. This study also confirms that uterine involvement represents a clinical manifestation of the syndrome.

Expression of aromatase and estrogen receptors in human adrenocortical tumors

We recently demonstrated that adrenocortical carcinoma cells express aromatase and estrogen receptors (ERs) and that 17β-estradiol enhances adrenocortical cell proliferation. To provide a clue to the role of estrogens in adrenal tumorigenesis, we investigated the expression profile of genes involved in sex steroid hormone production and activity in a large series of normal and neoplastic human adrenocortical tissues. Quantitative reverse transcriptase–polymerase chain reaction, Western blotting, and immunohistochemistry showed that ERα and ERβ, androgen receptor (AR), and aromatase were expressed in the adrenal cortex and in adrenocortical tumors. ERβ was the predominant ER subtype and was mainly expressed in the zona glomerulosa and fasciculata. Western blot analysis revealed the presence of a truncated form of AR in adrenocortical tissues. With respect to the normal adrenal cortex and adrenocortical adenomas, carcinomas were characterized by significantly lower ERβ levels, ERα upregulation, and aromatase overexpression. ER expression correlated with expression of nuclear hormone receptors, suggesting they could be involved in ER modulation. In agreement with our in vitro findings, the results of this study suggest that estrogens, locally produced by aromatase, could enhance adrenocortical cell proliferation though autocrine/paracrine mechanisms. This study opens new perspectives on the potential use of antiestrogens and aromatase inhibitors as therapeutic agents against ACC.

Confined 3D microenvironment regulates early differentiation in human pluripotent stem cells.

The therapeutic potential of human pluripotent stem (hPS) cells is threatened, among various problems, by the difficulty to homogenously direct cell differentiation into specific lineages. The transition from hPSC into committed differentiated cells is accompanied by secretome activity, remodeling of extracellular matrix and self‐organization into germ layers. In this work, we aimed to investigate how different three‐dimensional microenvironments regulate the early differentiation of the three germ layers in human embryonic stem (hES) cells derived embryoid bodies. In particular, a permeable, biocompatible, hydrogel microwell array was specifically designed for recreating a confined niche in which EB secreted molecules accumulate in accordance with hydrogel diffusional cut‐off. Fluorescence recovery after photobleaching technique was performed to accurately evaluate hydrogel permeability, mesh size and diffusional cutoff for soluble molecules. Three different culture conditions of EB culture were analyzed: suspension, confinement in microwells of width/depth ratio 1:1 and 1:2. Results show that EBs cultured in microwells are viable and have comparable average size after 8 days culture. Whole genome microarrays show that significative differential gene expression was observed between suspension and confined EBs culture. In particular, EBs culture in microwells promotes the expression of genes involved in pattern specification processes, brain development, ectoderm and endoderm differentiation. On the contrary, suspension EBs express instead genes involved in mesoderm specification and heart development. These results suggest that local accumulation of EBs secreted molecules drives differentiation patterns, as confirmed by immunofluorescence of germ layer markers, in hydrogel confined EB culture from both hES cells and human induced pluripotent stem (hiPS) cells. Our findings highlight an additional potential role of biomaterial in controlling hPSC differentiation through secreted factor niche specification. Biotechnol. Bioeng. 2012; 109: 3119–3132.

Investigation of BRAFand CTNNB1 activating mutations in adrenocortical tumors

Background: Activating mutations of the BRAF oncogene play a central role in the development of various cancer types, but their role in human adrenocortical tumors is unknown. At variance, activating mutations of another oncogene, CTNNB1, which encodes β-catenin, have been shown to be common events in both benign and malignant adrenocortical tumors. Aim: To investigate the prevalence of BRAF and CTNNB1 activating mutations in sporadic adrenocortical tumors. Materials and methods: Tissue samples from 15 adrenocortical carcinomas and 41 adrenocortical adenomas were investigated for the presence of BRAF and CTNNB1 activating mutations by PCR amplification and direct sequencing. Results: An advanced invasive non-functioning adrenocortical carcinoma carried a somatic heterozygous BRAF V600E mutation, while 4 functioning and 4 non-functioning adenomas and 3 functioning carcinomas carried different CTNNB1 activating mutations. Conclusions: Activating BRAF somatic mutations may be occasionally found in advanced adrenocortical carcinomas, while CTNNB1 activating mutations are early and common events in adrenal tumorigenesis.

Phylogenetic characterization of Central/Southern European lineage 2 West Nile virus: analysis of human outbreaks in Italy and Greece, 2013–2014

In recent years, West Nile virus (WNV) lineage 2 has been spreading and causing disease outbreaks in humans and animals in Europe. In order to characterize viral diversity, we performed full-length genome sequencing of WNV lineage 2 from human samples collected during outbreaks in Italy and Greece in 2013 and 2014. Phylogenetic analysis showed that these WNV lineage 2 genomes belonged to a monophyletic clade derived from a single introduction into Europe of the prototype Hungarian strain. Correlation of phylogenetic data with geospatial information showed geographical clustering of WNV genome sequences both in Italy and in Greece, indicating that the virus had evolved and diverged during its dispersal in Europe, leading to the emergence of novel genotypes, as it adapted to local ecological niches. These genotypes carried divergent conserved amino acid substitutions, which might have been relevant for viral adaptation, as suggested by selection pressure analysis and in silico and experimental modelling of sequence changes. In conclusion, the results of this study provide further information on WNV lineage 2 transmission dynamics in Europe, and emphasize the need for WNV surveillance activities to monitor viral evolution and diversity.

MGMT expression and promoter methylation status may depend on the site of surgical sample collection within glioblastoma: a possible pitfall in stratification of patients?

We recently described a three-layer concentric model of a glioblastoma (GBM) related to a specific distribution of molecular and phenotypic characteristics driven by the intratumoral hypoxic gradient in which the cancer stem cells niche is located in the hypoxic necrotic core of the tumour. The purpose of this study was to investigate the relationship between O(6)-methylguanine-DNA methyltransferase (MGMT) promoter methylation status and MGMT expression in GBM samples collected according to the three-layer concentric model. Multiple tissue samples were obtained, by means of image-guided surgery, from the three concentric layers of newly diagnosed GBM. Two samples from each layer were collected from 12 patients (total 72 samples). Immunohistochemical analysis was performed on formalin-fixed paraffin-embedded tissue samples. The methylation status of the MGMT promoter was determined by methylation-specific polymerase-chain-reaction analysis. In all tumours, MGMT protein expression decreased progressively from the inner to the outer layer, and methylation of the MGMT promoter was unrelated to tumour layer. In particular, the MGMT promoter was unmethylated in all layers in 41.7% of tumours, methylated in all layers in 25%, and variably methylated in the three layers in 33.3%. We recorded concordance between MGMT expression and MGMT promoter methylation status within the GBM in only 58.8% of the samples collected. Our data suggest that both MGMT expression and promoter methylation data may be variable throughout GBM and that they may, consequently, depend on the site of surgical sample collection, even in the same patient. However, whereas MGMT expression is pre-operatively predictable when sampling is performed according to the three-layer concentric model, MGMT promoter methylation is not. These results must be considered when sample collection is performed for assessment of MGMT data.

Genome sequencing of West Nile Virus from human cases in Greece, 2012.

A West Nile Virus (WNV) lineage 2 strain, named Nea Santa-Greece-2010, has been demonstrated to be responsible for the large outbreaks of neuroinvasive disease (WNND) that have been occurring in Greece since 2010, based on sequence similarities of viral isolates identified between 2010–2012. However, knowledge on the evolution of this strain is scarce because only partial WNV genome sequences are available from Greece. The aim of this study was to get the complete genome sequence of WNV from patients with infection. To this aim, plasma and urine samples collected during the 2012 Greek outbreak were retrospectively investigated. Full WNV genome sequence was obtained from a patient with WNND. The genome had 99.7% sequence identity to Nea Santa, higher than to other related WNV lineage 2 strains, and five amino acid changes apparently not relevant for viral pathogenicity or fitness. In addition, infection by WNV lineage 2 was confirmed in additional nine patients with WNND; in three of them the infection with WNV Nea Santa was demonstrated by sequencing. In conclusion, this study characterized for the first time a WNV full genome from a patient with WNND from Greece, demonstrated the persistence of the Nea Santa strain, and suggested that the virus might have locally evolved.

Characterization of a novel complex BRAF mutation in a follicular variant papillary thyroid carcinoma.

INTRODUCTION Activating mutations of the BRAF oncogene are frequently detected in papillary thyroid carcinoma (PTC) and have been associated with a worse prognosis. The amino acid substitution V600E accounts for 90% of all oncogenic BRAF mutations and is typically detected in classic PTCs, whereas other less frequent BRAF mutations seem to be associated with other PTC histotypes. CASE Screening for activating BRAF mutations in a series of 83 PTCs identified the most common V600E mutation in 39 cases (histologically, 38 classic PTCs and 1 sclerosing variant PTC) and a complex in-frame mutation involving amino acids V600-S605 in a stage III multicentric follicular variant PTC, occurring in a 50-year-old female patient, who was affected by hypothyroidism in autoimmune thyroiditis and had a family history of PTC and autoimmune thyroiditis. Since the identified BRAF mutation was novel in the literature, bioinformatic modeling was performed to predict its impact on BRAF activity. Although the mutation resulted in loss of a phosphorylation site in the activation loop of BRAF, it was predicted to increase BRAF kinase activity by mimicking an activating phosphorylation. CONCLUSIONS This study, which reports a new BRAF mutation, highlights the usefulness of bioinformatic modeling in the prediction of functional effects of new mutations and indicates that mutation-specific screening tests might miss some rare BRAF mutations. These facts should be taken into consideration in the molecular diagnosis of thyroid cancer and in the design of therapeutic protocols based on inhibitors of the BRAF pathway.