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Circulating tumor cells (CTCs) are one of the most important causes of tumor recurrence and distant metastases. Glioblastoma (GBM) has been considered restricted to the brain for many years. Nevertheless, in the past years, several pieces of evidence indicate that hematogenous dissemination is a reality, and this is also in the caseof GBM. Our aim was to optimize CTCs’ detection in GBM and define the genetic background of single CTCs compared to the primary GBM tumor and its recurrence to demonstrate that CTCs are indeed derived from the parental tumor. We collected blood samples from a recurrent IDH wt GBM patient. We genotyped the parental recurrent tumor tissue and the respective primary GBM tissue. CTCs were analyzed using the DEPArray system. CTCs Copy Number Alterations (CNAs) and sequencing analyses were performed to compare CTCs’ genetic background with the same patient’s primary and recurrent GBM tissues. We identified 210 common mutations in the primary and recurrent tumors. Among these, three somatic high-frequency mutations (in PRKCB, TBX1, and COG5 genes) were selected to investigate their presence in CTCs. Almost all sorted CTCs (9/13) had at least one of the mutations tested. The presence of TERT promoter mutations was also investigated and C228T variation was found in parental tumors and CTCs (C228T heterozygous and homozygous, respectively). We were able to isolate and genotype CTCs from a patient with GBM. We found common mutations but also exclusive molecular characteristics.

Conventional high-grade osteosarcoma (OS) is the most common primary cancer of bone and it typically affects the extremities of adolescents. OS has a complex karyotype, and molecular mechanisms related to carcinogenesis, progression and resistance to therapy are still largely unknown. For this reason, the current standard of care is associated with considerable adverse effects. In this study, our aim was to identify gene alterations in OS patients using whole exome sequencing (WES) to find new potential prognostic biomarkers and therapeutic targets. We performed WES on formalin-fixed paraffin-embedded (FFPE) biopsy materials collected from 19 patients affected by conventional high-grade OS. The clinical and genetic data were analyzed according to response to therapy, presence of metastasis and disease status. By comparing good and poor responders to neoadjuvant therapy, we detected a clear prevalence of mutations in the ARID1A, CREBBP, BRCA2 and RAD50 genes in poor responders that negatively influence the progression-free survival time. Moreover, higher tumor mutational burden values correlated with worse prognosis. The identification of mutations in ARID1A, CREBBP, BRCA2 and RAD50 may support the use of a more specific therapy for tumors harboring these alterations. In particular, BRCA2 and RAD50 are involved in homologous recombination repair, and could thus be used as specific therapy targets of inhibitors of the enzyme Poly ADP Ribose Polymerase (PARP). Finally, tumor mutational burden is found to be a potential prognostic marker for OS.

A protein corona-modulating peptide is integrated with non-persistent plasmonic nano-architectures containing a cisplatin prodrug to improve the efficacy of the nanomaterial-mediated chemotherapy in alternative in vivo models of oral carcinoma.

Background Glioblastoma growth impacts on the structure and physiology of peritumoral neuronal networks, altering the activity of pyramidal neurons which drives further tumor progression. It is therefore of paramount importance to identify glioma-induced changes in pyramidal neurons, since they represent a key therapeutic target. Methods We longitudinal monitored visual evoked potentials after the orthotopic implant of murine glioma cells into the mouse occipital cortex. With laser microdissection, we analyzed layer II-III pyramidal neurons molecular profile and with local field potentials recordings we evaluated the propensity to seizures in glioma-bearing animals with respect to control mice. Results We determine the time course of neuronal dysfunction of glioma-bearing mice and we identify a symptomatic stage, based on the decay of visual response. At that time point, we microdissect layer II-III pyramidal neurons and evaluate the expression of a panel of genes involved in synaptic transmission and neuronal excitability. Compared to the control group, peritumoral neurons show a decrease in the expression of the SNARE complex gene SNAP25 and the alpha1 subunit of the GABA-A receptor. No significant changes are detected in glutamatergic (ie, AMPA or NMDA receptor subunit) markers. Further reduction of GABA-A signaling by delivery of a benzodiazepine inverse agonist, DMCM (methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate) precipitates seizures in 2 mouse models of tumor-bearing mice. Conclusions These studies reveal novel molecular changes that occur in the principal cells of the tumor-adjacent zone. These modifications may be therapeutically targeted to ameliorate patients’ quality of life.

Pancreatic cancer has a poor prognosis due to its aggressive nature and ability to metastasize at an early stage. Currently, its management is still a challenge because this neoplasm is resistant to conventional treatment approaches, among which is chemo-radiotherapy (CRT), due to the abundant stromal compartment involved in the mechanism of hypoxia. Hyperthermia, among other effects, counteracts hypoxia by promoting blood perfusion and thereby can enhance the therapeutic effect of radiotherapy (RT). Therefore, the establishment of integrated treatments would be a promising strategy for the management of pancreatic carcinoma. Here, the effects of joint radiotherapy/hyperthermia (RT/HT) on optimized chick embryo chorioallantoic membrane (CAM) pancreatic tumor models are investigated. This model enables a thorough assessment of the tumor-arresting effect of the combined approach as well as the quantitative evaluation of hypoxia and cell cycle-associated mechanisms by both gene expression analysis and histology. The analysis of the lower CAM allows to investigate the variation of the metastatic behaviors of the cancer cells associated with the treatments. Overall, this study provides a potentially effective combined strategy for the non-invasive management of pancreatic carcinoma.

MiRNAs represent a mechanism that regulates gene expression in many pathological conditions. Exosomes are known to be secreted from all types of cells, and the exosomes-released molecules are crucial messengers that can regulate cellular processes. We investigated the miRNAs content of exosomes released by cancer cells during the invasion . An invasion stimulus has been generated through scratches created on the confluent cells of cancer cell lines: glioblastoma, breast and prostate cancers. Several miRNAs were found to be significantly differentially abundant during the cell invasion , both in common among different cell lines and exclusive. Understanding the language codes among cells involved in invasion can lead to the development of therapies that can inhibit cellular communication, slowing or eventually stopping their activity.

Glioblastoma (GBM) is the most common form of malignant brain cancer and is considered the deadliest human cancer. Because of poor outcomes in this disease, there is an urgent need for progress in understanding the molecular mechanisms of GBM therapeutic resistance, as well as novel and innovative therapies for cancer prevention and treatment. The pentose phosphate pathway (PPP) is a metabolic pathway complementary to glycolysis, and several PPP enzymes have already been demonstrated as potential targets in cancer therapy. In this work, we aimed to evaluate the role of sedoheptulose kinase (SHPK), a key regulator of carbon flux that catalyzes the phosphorylation of sedoheptulose in the nonoxidative arm of the PPP. SHPK expression was investigated in patients with GBM using microarray data. SHPK was also overexpressed in GBM cells, and functional studies were conducted. SHPK expression in GBM shows a significant correlation with histology, prognosis, and survival. In particular, its increased expression is associated with a worse prognosis. Furthermore, its overexpression in GBM cells confirms an increase in cell proliferation. This work highlights for the first time the importance of SHPK in GBM for tumor progression and proposes this enzyme and the nonoxidative PPP as possible therapeutic targets.

Glioblastoma (GB) is a devastating primary brain malignancy. Recurrence of GB is inevitable despite the standard treatment of surgery, chemotherapy, and radiation, and the median survival is limited to around 15 months. Barriers to treatment include the complex interactions among the different cellular components inhabitant of the tumor microenvironment. These challenges are further compounded by extensive inter- and intra-tumor heterogeneity and by the dynamic plasticity of GB cells. The complex heterogeneous nature of GB cells is helped by the local inflammatory tumor microenvironment, which mostly induces tumor aggressiveness and drug resistance. More effective therapy development heavily depends on higher resolution molecular subtype signatures. All cellular components of a GB tumor are subjected to a continuous pressure inducing proliferation that might favor the accumulation of genetic mutations. Understanding the genetic background of several cellular component s belonging to a GB microenvironment could give insights into tumor behavior and progression. In the present study by using fluorescent multiple labeling and DEPArray cell separator, we recovered several single cells or groups of single cells from populations of different origins from IDH-WT GB samples. From each GB sample, we collected astrocytes (GFAP+), microglia (IBA+), stem cells (CD133+), and endothelial cells (CD105+) and performed Copy Number Aberration (CNA) analysis with a low sequencing depth. The same tumors were subjected to a bulk CNA analysis. The tumor partition in its single components allowed single-cell molecular subtyping which revealed new aspects of GB altered genetic background. Nowadays, single-cell approaches are leading to a new understanding of GB physiology and disease. Moreover, single-cell CNAs resource will permit new insights into genome heterogeneity, mutational processes, and clonal evolution in malignant tissues.

The Glioblastoma (GB) field has been experiencing a therapeutic standstill since 2005, primarily due to ineffective preclinical approaches to test anti-cancer treatments. Therefore, better treatment screening approaches are required. We developed a NADH FLIM-based functional precision medicine approach, that within one week after surgery identified two groups of TMZ Responder and Non-Responder tumors. A 17 gene molecular signature able to classify with 100% precision the TMZ responder and non-responder samples was discovered and identified by Kaplan Meier analysis, a Low-Risk and a High-Risk survival group, interrogating the TCGA GB database (Hazard Ratio = 1.87 p=0.00098,n=148) and TCGA LGG database (Hazard Ratio = 7.66 p=1.197e−35, n=660). Same results were obtained in the CCGA datasets. The 17 gene signature power for independently predicting prognosis (Hazard Ratio = 1.9, p=0.002) was then confirmed by direct RNAseq analysis of a separate clinically characterized cohort of 235 GB patients. Then, we combined the methylation status of the MGMT promoter to analyze the survival status of patients. The survival analysis based on the 17 gene risk signature and MGMT promoter methylation status demonstrated remarkable stratification of the clinical courses into four subgroups. Patients with MGMT promoter unmethylation and 17 gene High-Risk score had the worst prognosis, while patients with MGMT promoter methylation and 17 gene Low-Risk score had the best prognosis. In the latter group, a significant 24-month increase in survival was observed with 10% of 100 months Long Survivors with a difference of 35 months compared to the other groups. Our data indicate a new statistically strong RNAseq-based prognostic survival and TMZ response tool for patients with malignant glioma. The accuracy of this functional precision medicine approach allowed the development of a new prognostic gene signature that can improve the clinical management of GB patients. The approach could be implemented on other cancers as well.

Recent evidence suggests that a loss of expression of caveolin in the stromal compartment (sCav-1) of human invasive breast carcinoma (IBC) may be a predictor of disease recurrence, metastasis and poor outcome. At present, there is little knowledge regarding the expression of sCav-1 at the metastatic sites. We therefore studied sCav-1 expression in IBCs and in their axillary lymph nodes to seek a correlation with cancer metastasis. 189 consecutive invasive IBCs (53 with axillary lymph node metastases and 136 without) were studied by immunohistochemistry, using a rabbit polyclonal anti-Cav-1 antibody. In IBCs sCav-1 was evaluated in fibroblasts scattered in the tumor stroma whereas in lymph nodes sCav-1 was assessed in fibroblast-like stromal cells. For the first time, we observed a statistically significant progressive loss of sCav-1 from normal/reactive axillary lymph nodes of tumors limited to the breast to metastatic axillary lymph nodes, through normal/reactive axillary lymph nodes of tumors with axillary metastatic spread. These data indicate that Cav-1 expressed by the stromal compartment of lymph nodes, somehow, may possibly contribute to metastatic spread in IBC.

// Sara Franceschi 1 , Francesca Lessi 1 , Paolo Aretini 1 , Valerio Ortenzi 2 , Cristian Scatena 2 , Michele Menicagli 1 , Marco La Ferla 1 , Prospero Civita 1 , Katia Zavaglia 3 , Claudia Scopelliti 1 , Alessandro Apollo 4 , Francesco Giovanni Carbone 2 , Riccardo Vannozzi 5 , Generoso Bevilacqua 6 , Francesco Pasqualetti 7 , Antonio Giuseppe Naccarato 2 and Chiara Maria Mazzanti 1 1 Fondazione Pisana per la Scienza, Onlus, Pisa, Italy 2 Department of Translational Research and New Technologies in Medicine, University Hospital of Pisa, Pisa, Italy 3 SOD Molecular Genetics, University Hospital of Pisa, Pisa, Italy 4 Tumor Cell Biology Unit, Istituto Tumori Toscana, Florence, Italy 5 Neurosurgery Department, University Hospital of Pisa, Pisa, Italy 6 Pathological Anatomy Section, San Rossore Clinic, Pisa, Italy 7 Radiotherapy Department, University Hospital of Pisa, Pisa, Italy Correspondence to: Chiara Maria Mazzanti, email: c.mazzanti@fpscience.it Keywords: glioblastoma; next generation sequencing (NGS); recurrence free survival (RFS); onco-drivers; tumor suppressors Received: November 17, 2017 Accepted: April 02, 2018 Published: May 08, 2018 ABSTRACT Glioblastoma is a devastating disease that despite all the information gathered so far, its optimal management remains elusive due to the absence of validated targets from clinical studies. A better clarification of the molecular mechanisms is needed. In this study, having access to IDH1 wild-type glioblastoma of patients with exceptionally long recurrence free survival (RFS), we decided to compare their mutational and gene expression profile to groups of IDH1 wild-type glioblastoma of patients with shorter RFS, by using NGS technology. The exome analysis revealed that Long-RFS tumors have a lower mutational rate compared to the other groups. A total of 158 genes were found differentially expressed among the groups, 112 of which distinguished the two RFS extreme groups. Overall, the exome data suggests that shorter RFS tumors could be, chronologically, in a more advanced state in the muli-step tumor process of sequential accumulation of mutations. New players in this kind of cancer emerge from the analysis, confirmed at the RNA/DNA level, identifying, therefore, possible oncodrivers or tumor suppressor genes.

Background Mobilization of endothelial progenitor cells (EPCs) into circulation from bone marrow in patients with acute myocardial infarction has strong scientific evidence; less is known about EPC mobilization in patients with stable coronary artery disease (CAD). The aim of this study was to investigate the association of stable ischemic heart disease with EPC levels in tissue and blood. Methods Fifty-five consecutive patients admitted to a single treatment center for valve or coronary artery bypass grafting (CABG) surgeries were included in the study. Blood samples were collected in the morning before surgery and analyzed by flow-cytometry to determine peripheral EPC levels (EPC/ml). Tissue EPC (CD34+VEGFR2+) levels were assessed on a right atrial appendage segment. Results Mean age was 76±5years, 48% were men, and 53% had CAD The number of CD34+ VEGFR2+ cells in the tissue of patients with CAD was significantly higher ( p Conclusion Patients with stable CAD had higher EPC density values (EPC/mm2) and were more likely to have lower EPC blood levels when compare with normal controls.

Simple Summary Mouse mammary tumour virus-like (MMTV-like) is suspected to be involved in human breast cancer and feline mammary carcinomas (FMCs). We previously reported the identification of MMTV-like sequences and viral protein in six of 78 FMCs collected in Tuscany, Italy. To corroborate this finding, FMCs samples collected from a different geographic area were investigated. MMTV-like sequences and p14 protein were identified in three of 24 FMCs collected at the University of Bologna, one tubular carcinoma, one tubulopapillary carcinoma and one ductal carcinoma. All the examined FMCs from Pisa and Bologna were submitted to immunohistochemistry for molecular phenotype characterization. Of the nine positive FMCs, six were basal-like and three luminal-like. This study highlights the presence of MMTV-like sequences and protein in FMCs of different geographic areas. The characterization of molecular phenotype could contribute to understand the possible role of MMTV-like virus in FMC biological behaviour. Abstract In the last few years MMTV-like nucleotide sequences were detected in some feline and canine mammary tumours. Due to the confirmed role of cats in the epidemiology of the MMTV-like virus, the aim of this study was to investigate the main pathological features of positive feline mammary carcinomas (FMCs). Twenty-four FMCs were collected at the University of Bologna, submitted to laser microdissection and analysed by nested fluorescence-PCR using primer sets specific for MMTV env sequence. For immunohistochemistry, an antibody against MMTV protein 14 (p14) was used. MMTV-like sequences were detected in three out of 24 FMCs (12.5%), one tubular carcinoma, one tubulopapillary carcinoma and one ductal carcinoma. All PCR-positive tumours were also positive for p14. Multiple nucleotide alignment has shown similarity to MMTV ranging from 98% to 100%. All the 102 examined FMCs were submitted to immunohistochemistry for molecular phenotyping. Of the nine MMTV-like positive FMCs, six were basal-like and three luminal-like. Our results demonstrate MMTV-like sequences and protein in FMCs of different geographic areas. Molecular phenotyping could contribute to understand the possible role of MMTV-like virus in FMC tumor biology.

Laser capture microdissection (LCM) coupled with RNA-seq is a powerful tool to identify genes that are differentially expressed in specific histological tumor subtypes. To better understand the role of single tumor cell populations in the complex heterogeneity of glioblastoma, we paired microdissection and NGS technology to study intra-tumoral differences into specific histological regions and cells of human GBM FFPE tumors. We here isolated astrocytes, neurons and endothelial cells in 6 different histological contexts: tumor core astrocytes, pseudopalisading astrocytes, perineuronal astrocytes in satellitosis, neurons with satellitosis, tumor blood vessels, and normal blood vessels. A customized protocol was developed for RNA amplification, library construction, and whole transcriptome analysis of each single portion. We first validated our protocol comparing the obtained RNA expression pattern with the gene expression levels of RNA-seq raw data experiments from the BioProject NCBI database, using Spearman's correlation coefficients calculation. We found a good concordance for pseudopalisading and tumor core astrocytes compartments (0.5 Spearman correlation) and a high concordance for perineuronal astrocytes, neurons, normal, and tumor endothelial cells compartments (0.7 Spearman correlation). Then, Principal Component Analysis and differential expression analysis were employed to find differences between tumor compartments and control tissue and between same cell types into distinct tumor contexts. Data consistent with the literature emerged, in which multiple therapeutic targets significant for glioblastoma (such as Integrins, Extracellular Matrix, transmembrane transport, and metabolic processes) play a fundamental role in the disease progression. Moreover, specific cellular processes have been associated with certain cellular subtypes within the tumor. Our results are promising and suggest a compelling method for studying glioblastoma heterogeneity in FFPE samples and its application in both prospective and retrospective studies.

The inheritance of mutated suppressor genes, such as BRCA1 and BRCA2, is acknowledged as an etiological factor in hereditary breast carcinoma (HBC). Two different molecular mechanisms are possible; the Knudson’s “two hits” or the gene haploinsufficiency. Etiology of sporadic breast carcinoma (SBC) is not known, although data support the possible role of the betaretrovirus Mouse Mammary Tumor Virus (MMTV). This study analyzes the presence of MMTV exogenous sequences in two representative groups of HBC and SBC, excluding any contamination by murine and retroviral material and endogenous betaretroviruses. The 30.3% of 56 SBC contained MMTV sequences, against the 4.2% of 47 HBC (p < 0.001). Cases positive for viral sequences showed the presence of p14, signal peptide of the MMTV envelope precursor. This result was expected based on the fact that HBCs, having a specific genetic etiology, do not need the action of a carcinogenetic viral agent. Moreover, the striking results obtained by comparing two groups of vastly different tumors represent an additional element of quality control: the distinction between HBC and SBC is so well-defined that results cannot be ascribed to mere coincidence. This paper strengthens the hypothesis for a viral etiology for human sporadic breast carcinoma.

Background Tumors develop by progression through a series of stages. Every cell of the tumor microenvironment is constantly changing in the flow of the cancer progression. It has become clear in recent years that stroma is essential for tumor maintenance and growth. Here, we aimed to give a chronological order of gene expression changes given in the dynamical framework of microinvasive breast cancer microenvironment. Methods RNA-seq was performed on seven microinvasive breast cancers. For each of them we microdissected seven different portions of the tumor, four related to the breast epithelium and three to the stroma. Breast epithelium was chronologically subdivided in normal breast epithelium (NBE), carcinoma in situ (CIS), emerging invasive fingers (EIF) and invasive breast cancer (IBC). For each of the breast epithelium subdivisions we collected the adjacent stroma (S): S-NBE, S-EIF and S-IBC. Results The overall differentially expressed genes (DEGs) in all the compartments were analysed and evaluated to understand the pathways involved in tumor progression. Then we analysed the DEGs of the epithelial and stromal portions in comparison with the normal portions. We observed that the stromal cells are necessary for the development and the maintenance of the tumor, especially in tumor progression. Moreover the most important genes involved in the main metabolic pathways were analysed and the communications within the different cell compartments were highlighted. Conclusions As a future perspective, a deeply study of the identified key genes, particularly in the stromal cells, will be crucial to develop an anticancer therapy that is undergoing a conversion from a cancer cell-centric strategy to a stroma-centric strategy, more genomically stable. Electronic supplementary material The online version of this article (10.1186/s12967-019-1936-x) contains supplementary material, which is available to authorized users.

Purpose The purpose of this study is to determine whether mouse mammary tumor virus (MMTV)-associated human breast cancer has the same or similar histology to MMTV-associated mouse mammary tumors. Such associations may indicate a role for MMTV in human breast cancer. Methods Immunohistochemical techniques (using antibodies directed against the signal peptide p14 of the envelope precursor protein of MMTV) and polymerase chain reaction (PCR) analyses were used to identify MMTV proteins and MMTV-like envelope gene sequences in a series of breast cancers from Australian women. The histological characteristics of these human breast cancer specimens were compared with MMTV positive mouse mammary tumors. The same methods were used to study benign breast tissues which 1–11 years later developed into breast cancer. Results MMTV p14 proteins were identified in 27 (54%) of 50 human breast cancers. MMTV env gene sequences were identified by PCR in 12 (27%) of 45 human breast cancers. There was a significant correlation between the presence of MMTV (identified by p14 immunohistochemistry) in human breast cancers and histological characteristics similar to MMTV positive mouse mammary tumors (p = 0.001). There was a non-significant correlation between the presence of MMTV env gene sequences (identified by PCR) in human breast cancers and histological characteristics similar to MMTV positive mouse mammary tumors (p = 0.290). MMTV p14 proteins were identified in 7 (54%) of 13 benign breast specimens that later developed into human breast cancers. MMTV by PCR was identified in two benign specimens one of whom later developed MMTV positive breast cancer. Discussion These observations offer evidence that MMTV may be associated with characteristic human breast cancer histology. p14-based immunohistochemistry appears to be a more reliable technique than PCR for the identification of MMTV in human breast cancer. Identification of MMTV-associated p14 proteins in benign breast tissues confirms prior PCR-based studies that MMTV infection occurs before the development of MMTV positive breast cancer. Conclusion Many MMTV positive human breast cancers have similar histology to MMTV positive mouse mammary tumors. MMTV infection identified in benign breast tissues precedes development of MMTV positive human breast cancer. When considered in the context of prior studies, these observations indicate a likely role for MMTV in human breast cancer.

Background Glioblastoma (GBM) is the most frequent and malignant primary brain tumor in adults and despite the progress in surgical procedures and therapy options, the overall survival remains very poor. Glutamate and α-KG are fundamental elements necessary to support the growth and proliferation of GBM cells. Glutamate oxidative deamination, catalyzed by GLUD2, is the predominant pathway for the production of α-KG. Methods GLUD2 emerged from the RNA-seq analysis of 13 GBM patients, performed in our laboratory and a microarray analysis of 77 high-grade gliomas available on the Geo database. Thereafter, we investigated GLUD2 relevance in cancer cell behavior by GLUD2 overexpression and silencing in two different human GBM cell lines. Finally, we overexpressed GLUD2 in-vivo by using zebrafish embryos and monitored the developing central nervous system. Findings GLUD2 expression was found associated to the histopathological classification, prognosis and survival of GBM patients. Moreover, through in-vitro functional studies, we showed that differences in GLUD2 expression level affected cell proliferation, migration, invasion, colony formation abilities, cell cycle phases, mitochondrial function and ROS production. In support of these findings, we also demonstrated, with in-vivo studies, that GLUD2 overexpression affects glial cell proliferation without affecting neuronal development in zebrafish embryos. Interpretation We concluded that GLUD2 overexpression inhibited GBM cell growth suggesting a novel potential drug target for control of GBM progression. The possibility to enhance GLUD2 activity in GBM could result in a blocked/reduced proliferation of GBM cells without affecting the survival of the surrounding neurons.

Papillary thyroid carcinoma is the most frequent histologic type of thyroid tumor. Few studies investigated the role of c-KIT expression in thyroid tumors, suggesting a role for this receptor and its ligand in differentiation and growth control of thyroid epithelium and a receptor loss following malignant transformation. We investigated and correlated c-KIT expression levels and two known markers of thyrocytes differentiation, PAX8 and TTF-1, in malignant and benign cytological thyroid samples. Moreover, we performed functional studies on human papillary thyroid carcinoma cell line to associated c-KIT expression to thyrocytes differentiation and tumor proliferation. c-KIT and PAX8 expression resulted higher in benign samples compared to the malignant ones, and the expression levels of these two genes were significantly correlated to each other. We also observed that c-KIT overexpression led to an increase of PAX8 expression level together with a decrease of proliferation. Furthermore, c-KIT overexpressing cells showed a regression of typical morphological features of malignancy. Taken together these results suggest that c-KIT could be involved in the differentiation of thyroid cells and in tumor progression.

The neuromuscular junction has retained through evolution the capacity to regenerate after damage, but little is known on the inter‐cellular signals involved in its functional recovery from trauma, autoimmune attacks, or neurotoxins. We report here that CXCL12α, also abbreviated as stromal‐derived factor‐1 (SDF‐1), is produced specifically by perisynaptic Schwann cells following motor axon terminal degeneration induced by α‐latrotoxin. CXCL12α acts via binding to the neuronal CXCR4 receptor. A CXCL12α‐neutralizing antibody or a specific CXCR4 inhibitor strongly delays recovery from motor neuron degeneration in vivo . Recombinant CXCL12α in vivo accelerates neurotransmission rescue upon damage and very effectively stimulates the axon growth of spinal cord motor neurons in vitro . These findings indicate that the CXCL12α‐CXCR4 axis plays an important role in the regeneration of the neuromuscular junction after motor axon injury. The present results have important implications in the effort to find therapeutics and protocols to improve recovery of function after different forms of motor axon terminal damage.

Mouse mammary tumour virus-like (MMTV-like) is suspected to be involved in human breast cancer and it has been hypothesized that companion animals might have a role in viral transmission. The aim of our study was to investigate the presence of MMTV-like nucleotide sequences and viral protein in a larger number of feline (FMCs) and canine mammary carcinomas (CMCs) by nested PCR and immunohistochemistry. Results showed that the presence of MMTV-like env sequence in FMCs was 7% (6/86), while all the CMCs and canine dysplastic lesions scored negative. All PCR-positive FMCs scored positive for the MMTV p14 signal peptide of the envelope precursor protein of the virus. In contrast, all PCR-negative FMCs and canine mammary lesions were also negative for immunohistochemistry analysis. Canine and feline normal mammary gland tissues scored negative for both PCR and MMTV-p14 protein. Multiple nucleotide alignment of MMTV-like env gene sequences isolated from cat showed 97% and 99% similarity with HMTV and MMTV, respectively, while the others two presented some polimorphisms. Particularly the sequences of one of these two tumors showed a polymorphism (c.7575 A> G), that causes a previously unreported amino acid substitution (Thr > Ala). In conclusion, the results of our study showed the presence of MMTV-like sequences and viral protein in some FMCs. Further studies are needed to understand whether this virus does play a role in the development of FMCs, if MMTV-like is an exogenous virus as these data suggest and, in such a case, how and from whom this virus was acquired.

Choroid plexus carcinomas in four dogs (three male, one female) aged small middle 2.5 to 10 years, were examined by light microscopy and immunohistochemistry. The dogs showed progressive neurological signs including ataxia, seizures, vestibular disease and cranial nerve deficits, lasting for several months in some cases. Primary tumours were localized in the lateral (one case), third (one case), and fourth (two cases) ventricles. Hydrocephalus was evident at post-mortem examination in one case. In two cases the neoplastic cells closely resembled the structure of normal choroid plexus, with a distinct papillary pattern, composed of well-differentiated columnar epithelium. In the other two cases, cellular pleomorphism, nuclear atypia, increased mitotic activity and necrosis were observed. In all cases, dissemination of neoplastic cell clusters was detected within the subarachnoid space or the ventricular cavity. Immunohistochemical examination showed a multifocal labelling pattern for pankeratin and cytokeratin AE1 and diffuse vimentin positivity in poorly differentiated tumours. Well-differentiated choroid plexus carcinomas showed multifocal immunoreactivity for cytokeratin AE3, multifocal to diffuse immunoreactivity for vimentin and occasional positivity for carcinoembryonic antigen. Epithelial membrane antigen, Ber EP4 and S-100 were negative in all cases. Glial fibrillary acidic protein labelling occurred only in a single, poorly differentiated tumour. Occasional reactions for proliferating cell nuclear antigen and MIB-1 were seen in two cases. It was concluded that at least two morphological and possibly phenotypic subtypes (well-differentiated and anaplastic) of choroid plexus carcinoma of the dog could be identified.

Endothelialprogenitorcells(EPCs)aremobilizedfrombonemarrowinto peripheral blood, contributing to the revascularization of ischemicareas, to endothelial repair and to the physiological maintenance ofvascularization. EPC mobilization and homing have been primarilylinked to ischemia and inflammation presence [1]. EPCs are directlycorrelated with endothelial function and inversely correlated withcardiovascular risk factors and atherosclerosis progression [2].EPClevelshavealsobeencorrelatedtoprognosisevaluationincardiovascu-lar disease [3].Regarding EPC correlation with coronary artery disease (CAD)presence and severity, an inverse relationship with CAD severity, inde-pendent of traditional risk factors, was demonstrated by EPC colonycounting [4],whileahighnumberofEPCsassociatedwithCADandcorre-latedwithstenosisseveritywereshownby flowcytometry [5].Recentlyanew protocol, adapted from the standardized ISHAGE protocol for hema-topoieticstemcells,hasbeendevelopedforEPClevelevaluationtoenablecomparison of clinical and laboratory data [6].While thepresence of circulatingEPCshasbeenwidely evaluated indifferent diseases, few studies tried to evaluate the presence of EPCs inhuman vital myocardium.TheaimofourstudywastoinvestigateEPClevelsbothinperipheralbloodand inmyocardium in thesame patients atthesame time, evalu-atingthecorrelationwith CADpresence. In bothsampleswequantifiedCD34

Von Hipple–Lindau gene (VHL) inactivation represents the most frequent abnormality in clear cell renal cell carcinoma (ccRCC). Hypoxia-inducible factor-1α (HIF-1α) expression is regulated by O2 level. In normal O2 conditions, VHL binds HIF-1α and allows HIF-1α proteasomal degradation. A single-nucleotide polymorphism (SNP) has been found located in the oxygen-dependent degradation domain at codon 582 (C1772T, rs11549465, Pro582Ser). In hypoxia, VHL/HIF-1α interaction is abolished and HIF-1α activates target genes in the nucleus. This study analyzes the impact of genetic alterations and protein expression of VHL and the C1772T SNP of HIF-1α gene (HIF-1α) on prognosis in early-stage ccRCC (pT1a, pT1b, and pT2). Mutational analysis of the entire VHL sequence and the genotyping of HIF-1α C1772T SNP were performed together with VHL promoter methylation analysis and loss of heterozygosis (LOH) analysis at (3p25) locus. Data obtained were correlated with VHL and HIF-1α protein expression and with tumor-specific survival (TSS). VHL mutations, methylation status, and LOH were detected in 51, 11, and 12 % of cases, respectively. Our results support the association between biallelic alterations and/or VHL silencing with a worse TSS. Moreover, we found a significant association between the HIF-1α C1772C genotype and a worse TSS. The same association was found when testing the presence of HIF-1α protein in the nucleus. Our results highlight the role of VHL/HIF-1α pathway in RCC and support the molecular heterogeneity of early-stage ccRCC. More important, we show the involvement of HIF-1α C1772T SNP in ccRCC progression.

We studied the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptors (GM-CSF.R) in 20 human brain gliomas with different tumor gradings and demonstrated constitutive high levels of both mRNA gene expression and protein production exclusively in the highest-grade tumors (WHO, III-IV grade). Five astrocytic cell lines were isolated in vitro from glioma cells, which had selectively adhered to plates pre-coated with rhGM-CSF. These cells were tumorigenic when xenografted to athymic mice, and produced GM-CSF constitutively in culture. Two lines, particularly lines AS1 and PG1, each from a patient with glioblastoma multiforme, constitutively over-expressed both GM-CSF and GM-CSF.R genes and secreted into their culture media biologically active GM-CSF. Different clones of the AS1 line, isolated after subsequent passages in vitro and then transplanted to athymic mice, demonstrated higher tumorigenic capacity with increasing passages in vivo. Cell proliferation was stimulated by rhGM-CSF in late-stage malignant clones, whereas apoptosis occurred at high frequency in the presence of blocking anti-GM-CSF antibodies. In contrast, rhGM-CSF did not induce any apparent effect in early-stage clones expressing neither GM-CSF nor GM-CSF.R. The addition of rhGM-CSF or rhIL-1β, to cultures induced the overproduction of both GM-CSF and its receptors and increased gene activation for several functional proteins (e.g. NGF, VEGF, VEGF.R1, G-CSF, MHC-II), indicating that these cells may undergo dynamic changes in response to environmental stimuli. These findings thus revealed: (1) that the co-expression of both autocrine GM-CSF and GM-CSF.R correlates with the advanced tumor stage; (2) that an important contribution of GM-CSF in malignant glioma cells is the prevention of apoptosis. These results imply that GM-CSF has an effective role in the evolution and pathogenesis of gliomas.

Ductal pancreatic carcinoma (DPC) is a deadly disease with an incidence of 9 cases in 100,000 people per year and a mortality rate close to 100%. Allelic losses in the long arm of chromosome 9 are commonly encountered in many human malignancies but no data are yet available about DPC. We screened 40 laser-microdissected DPC samples and 6 pre-invasive lesions for 9 microsatellite mapping markers of region 9q21.3 through 9q34.2. A small overlapping region of deletion, spanning 8 million base pairs, was identified between D9S127 and D9S105. Two genes, RSG3 and KLF4, mapped to 9q31.1 through 9q32, were further investigated. A highly significant association was found between KLF4 gene expression levels and genomic status. Similarly, absence of immunohistochemical expression of KLF4 protein was found in 86.8% cases of DPC (33/38). Overexpression of KLF4 in a human pancreatic carcinoma cell line induced a significant decrease in the proliferation associated with up-regulation of p21 and the down-regulation of cyclin D1. In conclusion, we identified a novel oncosuppressor region located at the 9q 31.1-3 locus that is lost in DPC at high frequency. Loss of KLF4 expression is closely related to the genomic loss, and its restoration inhibits cancer cell proliferation, suggesting a key suppressor role in pancreatic tumorigenesis.

Context: Serum pituitary antibodies (Pit Abs) and tumor-infiltrating lymphocytes (TILs) have been described in pituitary adenomas, but their clinical significance remains unknown. Objective: The objective of the study was to assess Pit Abs and TILs prevalence in pituitary adenomas and their influence on clinical outcome. Design: This was a prevalence case-control study. Patients and Setting: Two hundred ninety-one pituitary adenoma cases (110 non-secreting, 30 ACTH-69 GH-71 prolactin- and 13 TSH-secreting adenoma; 177 operated and 114 untreated), 409 healthy controls, and 14 autoimmune hypophysitis were enrolled in a tertiary referral center. Intervention: Pit Abs were measured using immunofluorescence in all cases and controls (n = 714). The presence of TILs was evaluated using CD45 staining in a subset of adenomas surgically treated (n = 72). Main Outcome Measure: Clinical response of pituitary adenoma after surgery was evaluated. Results: Pit Abs prevalence was higher in adenomas (5.1%) than healthy subjects (0.7%, P < 0.0001) and lower than in autoimmune hypophysitis patients (57%, P < 0.0001). Similarly, TILs prevalence was higher in adenomas than normal pituitary (P = 0.01) and lower than in autoimmune hypophysitis (P < 0.0001). No correlation between Pit Abs and TILs was found (P = 0.78). A poor clinical outcome was more common in adenoma patients with TILs (11 of 18, 61%) than in those without (17 of 54, 31%, P = 0.026). Multivariate regression analysis identified the presence of TILs as independent prognostic factor for persistence/recurrence of pituitary adenoma. Conclusions: TILs and Pit Abs are present in a significant number of pituitary adenoma patients. Cell-mediated immunity appears to be predictive of a less favorable clinical outcome.