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Basal cell carcinoma (BCC) is the most common cancer in the white-skinned population accounting for about 15% of all neoplasms. Its incidence is increasing worldwide, at a rate of about 10% per year. BCC, although infrequently metastasizing, very often causes extensive tissue losses, due to the high propensity toward stromal infiltration, particularly in its dedifferentiated forms, with disfiguring and debilitating results. To date, there still is limited availability of therapeutic treatments alternative to surgery. We evaluated the immunohistochemical expression of the carbonic anhydrase IX (CAIX), one of the main markers of tissue hypoxia, in a set of 85 archived FFPE BCC tissues, including the main subtypes, with different clinical outcomes, to demonstrate a possible relationship between hypoxic phenotype and biological aggressiveness of these neoplasms. Our results showed that the expression level of the CAIX protein contributes to the stratification of BCC in the different risk classes for recurrence. We hypothesize for CAIX a potential therapeutic role as a target therapy in the treatment of more aggressive BCCs, thus providing an alternative to surgical and pharmacological therapy with Hedgehog inhibitors, a promising example of target therapy in BCCs.

Early events of basal cell carcinoma (BCC) tumorigenesis are triggered by inappropriate activation of SHH signaling, via the loss of Patched1 (Ptch1) or by activating mutations of Smoothened (Smo). TBX1 is a key regulator of pharyngeal development, mainly through expression in multipotent progenitor cells of the cardiopharyngeal lineage. This transcription factor is connected to several major signaling systems, such as FGF, WNT, and SHH, and it has been linked to cell proliferation and to the regulation of cell shape and cell dynamics. Here, we show that TBX1 was expressed in all of the 51 BCC samples that we have tested, while in healthy human skin it was only expressed in the hair follicle. Signal intensity and distribution was heterogeneous among tumor samples. Experiments performed on a cellular model of mouse BCC showed that Tbx1 is downstream to GLI2, a factor in the SHH signaling, and that, in turn, it regulates the expression of Dvl2, which encodes an adaptor protein that is necessary for the transduction of WNT signaling. Consistently, Tbx1 depletion in the cellular model significantly reduced cell migration. These results suggest that TBX1 is part of a core transcription network that promotes BCC tumorigenesis.

Purpose: To describe functional and histopathological findings after macular peeling using different dyes. Methods: Prospective, randomized, comparative, interventional, and immunohistochemical study. Forty-five eyes from 45 patients with idiopathic epiretinal membrane (ERM) underwent pars plana chromovitrectomy with ERM and inner limiting membrane (ILM) using trypan blue 0.15% + brilliant blue 0.05% + lutein 2% in group 1 (15 eyes), trypan blue 0.15% + brilliant blue 0.025% + polyethylene glycol 3350 4% in group 2 (15 eyes), and indocyanine green 0.05% in group 3 (15 eyes). We evaluated visual acuity (VA) and macular sensitivity (MS) preoperatively, 1, 3, and 6 months after surgery. The expression of glial fibrillary acidic protein (GFAP) and neurofilament protein (NF) was assessed immunohistochemically on the ILMs peeled as markers of glial and neuronal cells. Results: In group 1, both mean VA and MS were significantly better at 1 and 3 months after surgery (P < 0.05), whereas no significant difference was found after 6 months. GFAP and NF expression was significantly lower in group 1 (P < 0.05). Conclusions: The ERM/ILM peeling is thought to rip off the intraretinal tissue, based on the amounts of GFAP and NF in the specimens. The use of lutein dyes reduces iatrogenic stress to the retinal tissue and allows a faster functional recovery in the first 3 months after surgery, suggesting a less iatrogenic adhesion to the retinal tissue

Background The muscle invasive form of urothelial bladder cancer (UBC) is a deadly disease. Currently, the therapeutic approach of UBC is mostly based on surgery and standard chemotherapy. Biomarkers to establish appropriate drugs usage are missing. Deficiency of the tumor suppressor CCDC6 determines PARP-inhibitor sensitivity. The CCDC6 levels are modulated by the deubiquitinase USP7. In this work we scored CCDC6 and USP7 expression levels in primary UBC and we evaluated the expression levels of CCDC6 in correlation with the effects of the PARP-inhibitors combined with the USP7 inhibitor, P5091, in vitro. Since PARP-inhibitors could be enhanced by conventional chemotherapy or DNA damage inducers, we tested the new agent RRx-001, able to induce DNA damage, to prove the benefit of combined treatments in bladder cancer cells. Methods The J82, T24, 5637 and KU-19-19 bladder cancer cells were exposed to USP7 inhibitor P5091 in presence of cycloheximide to analyse the CCDC6 stability. Upon the CCDC6 degradation induced by P5091, the cells sensitivity to PARP-inhibitor was evaluated by cell viability assays. The ability of the DNA damage inducer RRx-001 to modulate CCDC6 protein levels and H2AX phosphorylation was detected at immunoblot. The combination of USP7 inhibitor plus RRx-001 enhanced the PARP-inhibitor sensitivity, as evaluated by cell viability assays. The results of the scores and correlation of CCDC6 and USP7 expression levels obtained by UBC primary biopsies staining were used to cluster patients by a K-mean cluster analysis. Results P5091 determining CCDC6 degradation promoted bladder cancer cells sensitivity to PARP-inhibitor drugs. RRx-001, by inducing DNA damage, enhanced the effects of the combined treatment. The immunohistochemical staining of both CCDC6 and USP7 proteins allowed to cluster the high grade (G3) UBC patients, on the basis of CCDC6 expression levels. Conclusions In high grade UBC the identification of two clusters of patients based on CCDC6 and USP7 expession can possibly indicate the use of PARP-inhibitor drugs, in combination with USP7 inhibitor in addition to the DNA damage inducer RRx-001, that also acts as an immunomodulatory agent, offering novel therapeutic strategy for personalized medicine in bladder cancer patients. Electronic supplementary material The online version of this article (10.1186/s13046-019-1087-1) contains supplementary material, which is available to authorized users.

Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine, whose role in melanoma is controversial. Although high-dose TNF-α is approved for the treatment of patients with in transit-metastatic melanoma confined to the limb, diverse preclinical models of melanoma have shown that TNF-α can induce cell invasion. Biomarkers that can differentiate between the dual role of TNF-α are needed. TRAF2 is critical to TNF receptor-induced activation of nuclear factor-κB (NF-κB), allowing shifting from death to survival-signaling cascades. The large immunophilin FKBP51 acts as a scaffold and catalyst in the IκB kinase complex assembly and activation. Here, using microscopy and an electrophoretic mobility-shift assay, we provide further evidence in support of the essential role of FKBP51 in sustaining the TNF-α NF-κB signaling in melanoma. Through the cross-linking reaction with the chemical linker disuccinimidyl glutarate, we show that a direct interaction occurs between FKBP51 and TRAF2 in melanoma cells. Immunohistochemistry of tumor samples from 24 patients with cutaneous melanomas showed a correlation between the expressions of the two proteins. Given the association of FKBP51 and TRAF2 with TNF-α-induced NF-κB signaling and their correlation in tumor samples, we propose that the two proteins can be exploited as useful markers for the identification of those melanoma tumors that can benefit from TNF-α inhibition. Future studies will address this hypothesis.

Germ cell tumors (GCT) are the most common malignancies in males between 15 and 35 years of age. Despite the high cure rate, achieved through chemotherapy and/or surgery, the molecular basis of GCT etiology is still largely obscure. Here, we show a positive correlation between MAPK15 (ERK8; ERK7) expression and specific GCT subtypes, with the highest levels found in the aggressive embryonal carcinomas (EC). Indeed, in corresponding cellular models for EC, MAPK15 enhanced tumorigenicity in vivo and promoted cell proliferation in vitro, supporting a role for this kinase in human GCT. At molecular level, we demonstrated that endogenous MAPK15 is necessary to sustain cell cycle progression of EC cells, by limiting p53 activation and preventing the triggering of p53-dependent mechanisms resulting in cell cycle arrest. To understand MAPK15-dependent mechanisms impinging on p53 activation, we demonstrate that this kinase efficiently protects cells from DNA damage. Moreover, we show that the ability of MAPK15 to control the autophagic process is necessary for basal management of DNA damage and for tumor formation controlled by the kinase. In conclusion, our findings suggest that MAPK15 overexpression may contribute to the malignant transformation of germ cells by controlling a "stress support" autophagic pathway, able to prevent DNA damage and the consequent activation of the p53 tumor suppressor. Moreover, in light of these results, MAPK15-specific inhibitors might represent new tools to enhance the therapeutic index of cytotoxic therapy in GCT treatment, and to increase the sensitivity to DNA-damaging drugs in other chemotherapy-resistant human tumors.

Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by fibrosis, alteration in the microvasculature and immunologic abnormalities. It has been hypothesized that an abnormal redox state could regulate the persistent fibrotic phenotype in SSc patients. N-Formyl peptide receptors (FPRs) are chemotactic receptors overexpressed in fibroblasts derived from SSc patients. In this study, we demonstrated that stimulation of FPRs promotes the generation of reactive oxygen species (ROS) in skin fibroblasts. In fibroblast cells, ROS production was due to FPRs interaction with the urokinase receptor (uPAR) and to β1 integrin engagement. FPRs cross-talk with uPAR and integrins led to Rac1 and ERKs activation. FPRs stimulation increased gp91phox and p67phox expression as well as the direct interaction between GTP-Rac1 and p67phox, thus promoting assembly and activation of the NADPH oxidase complex. FPRs functions occur through interaction with a specific domain of uPAR (residues 88SRSRY92) that can be exposed on the cell membrane by protease-mediated receptor cleavage. Immunohistochemistry analysis with a specific anti-SRSRY antibody showed increased expression of uPAR in a cleaved form, which exposes the SRSRY sequence at its N-terminus (DIIDIII-uPAR88–92) in skin biopsies from SSc patients. As expected by the increased expression of both FPRs and DII-DIII-uPAR88-92, fibroblasts derived from SSc patients showed a significantly increase in ROS generation both at a basal level than after FPRs stimulation, as compared to fibroblasts from normal subjects. C37, a small molecule blocking the interaction between FPRs and uPAR, and selumetinib, a clinically approved MAPKK/ERK inhibitor, significantly inhibited FPRs-mediated ROS production in fibroblasts derived from SSc patients. Thus, FPRs, through the interaction with the uPA/uPAR system, can induce ROS generation in fibroblasts by activating the NADPH oxidase, playing a role in the alteration of the redox state observed in SSc.

Human Papilloma Virus (HPV) can play a causative role in the development of sinonasal tract malignancies. In fact, HPV may be the most significant causative agent implicated in sinonasal tumorigenesis and is implicated in as many as 21% of sinonasal carcinomas. To date, there are no definitive, reliable and cost-effective, diagnostic tests approved by the FDA for the unequivocal determination of HPV status in head and neck cancers. We followed an exhaustive algorithm to correctly test HPV infection, including a sequential approach with p16INK4a IHC, viral DNA genotyping and in situ hybridization for E6/E7 mRNA. Here, we report a case of sinonasal carcinoma with discordant results using HPV test assays. The tumor we describe showed an irregular immunoreactivity for p16INK4a, and it tested positive for HPV DNA; nevertheless, it was negative for HR-HPV mRNA. We discuss the possible meaning of this discrepancy. It would be advisable to test HPV transcriptional status of sinonasal carcinoma on a diagnostic routine basis, not only by p16INK4a IHC assay, but also by HPV DNA genotyping and HR-HPV mRNA assessment.

Introduction Epiretinal traction is not responsible only for epiretinal but also intraretinal changes. This study aims to describe structural and vascular intraretinal changes after macular peeling in idiopathic (iERM) vs diabetic ERM (dERM). Methods We conducted a prospective interventional study on forty-two eyes, 23 with iERMs and 19 with dERMs, undergoing ERM-ILM peeling. We performed SD-OCT preoperatively, 1 and 6 months postoperatively to assess central macular thickness (CMT), intraretinal cysts (IC) and/or continuous ectopic inner foveal layers (CEIFL), superficial and deep capillary free zone (CFZ) area on OCT-A. Glial fibrillary acidic protein (GFAP), as a Muller cells marker, was detected immunohistochemically on ILM specimens, to assess Muller cells iatrogenic damage. Results The CEIFLs were significantly more common in iERMs (12 (52.2%) in iERMs vs 2 (10.5%) in dERMs, p = 0.004), whereas ICs in dERMs (6 (26.1%) in iERMs vs 17 (89.5%) in dERMs, p

Granular cell tumor (GCT), a relatively rare neuroectodermal tumor occurring most often in the head and neck region, is not uncommonly associated with pseudoepitheliomatous hyperplasia of the overlying surface epithelium, which may be at times nonreadily distinguishable from well-differentiated squamous cell carcinoma (SCC). To the best of our knowledge, only a handful of coexisting SCC and GCT, mostly described in the esophagus, have been reported in (the current) literature so far. We herein report 2 new cases of coexisting GCT and SCC of the head and neck region, located, respectively, in larynx and tongue; comment on their clinical, imaging, and pathologic features; and discuss their management. In the present work, we also review the literature concerning this association to contribute to the head and neck pathologists’ and surgeons’ awareness regarding the possibility of this association for an adequate surgical excision and a better management of these patients.

// Francesco Morra 1 , Francesco Merolla 2, 3 , Virginia Napolitano 1, 2 , Gennaro Ilardi 2 , Caterina Miro 1 , Simona Paladino 4 , Stefania Staibano 2 , Aniello Cerrato 1 , Angela Celetti 1 1 Institute for Experimental Endocrinology and Oncology, Research National Council, Naples, Italy 2 Department of Advanced Biomedical Sciences, University “Federico II”, Naples, Italy 3 Department of Medicine and Health Sciences “V. Tiberio”, University of Molise, Campobasso, Italy 4 Department of Molecular Medicine and Medical Biotechnology, University “Federico II”, Naples, Italy Correspondence to: Angela Celetti, email: celetti@unina.it Keywords: CCDC6, USP7, ARFL and V7, P5091, olaparib Received: December 08, 2016 Accepted: March 15, 2017 Published: March 22, 2017 ABSTRACT Purpose of the study: Reduced levels of the tumor suppressor protein CCDC6 sensitize cancer cells to the treatment with PARP-inhibitors. The turnover of CCDC6 protein is regulated by the de-ubiquitinase USP7, which also controls the androgen receptor (AR) stability. Here, we correlated the expression levels of CCDC6 and USP7 proteins in primary prostate cancers (PC). Moreover, we tested the efficacy of the USP7 inhibitors, in combination with PARP-inhibitors as a novel therapeutic option in advanced prostate cancer. Experimental techniques: PC cells were exposed to USP7 inhibitor, P5091, together with cycloheximide, to investigate the turnover of the USP7 substrates, AR and CCDC6. As outcome of the AR downregulation, transcription targets of AR and its variant V7 were examined by qPCR. As a result of CCDC6 degradation, the induction of PARP inhibitors sensitivity was evaluated by analyzing PC cells viability and foci formation. We scored and correlated CCDC6 and USP7 expression levels in a prostate cancer tissue microarray (TMA). Results: P5091 accelerated the degradation of AR and V7 isoform affecting PSA, UBE2C, CDC20 transcription and PC cells proliferation. Moreover, P5091 accelerated the degradation of CCDC6 sensitizing the cells to PARP-inhibitors, that acted sinergistically with genotoxic agents. The immunohistochemical analysis of both CCDC6 and USP7 proteins exhibited significant correlation for the intensity of staining ( p ≤ 0.05). Data interpretation: Thus, CCDC6 and USP7 represent predictive markers for the combined treatment of the USP7-inhibitors and PARP-inhibitors in advanced prostate cancer.

Objectives CCDC6 gene product is a tumor-suppressor pro-apoptotic protein, substrate of ATM, involved in DNA damage response and repair. Altered levels of CCDC6 expression are dependent on post-translational modifications, being the de-ubiquitinating enzyme USP7 responsible of the fine tuning of the CCDC6 stability. Thus, our aim was to investigate CCDC6 and USP7 expression levels in Lung-Neuroendocrine Tumors (L-NETs) to verify if they correlate and may be exploited as novel predictive therapeutic markers. Materials and methods Tumor tissues from 29 L-NET patients were investigated on tissue microarrays. CCDC6 levels were scored and correlated with immunoreactivity for USP7. Next generation sequencing (NGS) of a homogenous group of Large Cell Neuroendocrine Carcinoma (LCNEC) (N=8) was performed by Ion AmpliSeq NGS platform and the Ion AmpliSeq Cancer Hotspot Panel v2. The inhibition of USP7, using P5091, was assayed in vitro to accelerate CCDC6 turnover in order to sensitize the neuroendocrine cancer cells to PARP-inhibitors, alone or in association with cisplatinum. Results The immunostaining of 29 primary L-NETs showed that the intensity of CCDC6 staining correlated with the levels of USP7 expression (p≤0.05). The NGS analysis of 8 LCNEC revealed mutations in the hot spot regions of the p53 gene (in 6 out of 8). Moreover, gene polymorphisms were identified in the druggable STK11, MET and ALK genes. High intensity of p53 immunostaining was reported in the 6 tissues carrying the TP53 mutations. The inhibition of USP7 by P5091 accelerated the degradation of CCDC6 versus control in cycloheximide treated L-NET cells in vitro and sensitized the cells to PARP-inhibitors alone and in combination with cisplatinum. Conclusion Our data suggest that CCDC6 and USP7 have a predictive value for the clinical usage of USP7 inhibitors in combination with the PARP-inhibitors in L-NET in addition to standard therapy.

Up-to-date, several molecular markers of prognosis have been studied in Oral Squamous Cell Carcinoma (OSCC), but none entered in the clinical setting. Therapy of OSCC tumors mainly relies on surgery, radiotherapy and partially on chemotherapy; there is an urgent need for biomarkers able to better stratify OSCC patients’ risk to address targeted therapeutic strategies. The role of immune response in the pathogenesis and biological behavior of OSCC has been investigated by several authors, and promising results have been obtained with immune checkpoint inhibitors. We already investigated the role of the immune modulator FK506-binding protein 51 (FKBP51), a FK506-binding immunophilin, in cutaneous melanoma biology, and its expression in several human solid tumors. In the present study, we aimed to assess the value of FKBP51 expression in OSCC tumor cells as a marker of outcome. We collected clinical data from 72 patients who underwent surgery for Squamous Cell Carcinoma (SCC) of the tongue, floor, lips and palate. FKBP51 expression was assessed by immunohistochemistry on paraffin-embedded tumor tissues. In addition, we evaluated the human papillomavirus (HPV) status of primary tumors by immunohistochemistry, viral subtyping and In Situ Hybridization (ISH) assay. We found that high FKBP51-expressing tumors characterized the OSCCs with the worst prognosis: the high immunohistochemical expression of FKBP51 associated with death occurring within five years from the diagnosis with a sensitivity of 88.46% and a specificity of 91.67%. The estimated positive predictive value of the test was 88.45% and negative predictive value 91.67%. We tested FKBP51 mRNA presence, by RT-PCR assay, in a selected series of OSCC tumors, and we found that mRNA correlated well to the protein expression and to the clinical outcome. Applying the Bayes formula, we estimated an 88% probability of dying within five years from the diagnosis of OSCC patients with a high FKBP51 immunohistochemical (IHC) test result (>51% of FKBP51 positive tumor cells). On the basis of our analysis, we propose tumor tissue expression of FKBP51 protein as a reliable prognostic marker for OSCC tumors.

Oral (OSCC) and oropharyngeal (OPSCC) squamous cell carcinomas show high morbidity and mortality rates. We aimed to investigate the role of the &ldquo, Chromatin Assembly Factor-1&rdquo, (CAF-1) p60 and p150 subunits, involved in DNA repair and replication, in OSCC and OPSCC progression and in response to Poly(ADP-ribose) polymerase (PARP)-inhibitors and exposure to ionizing radiation (IR). We immunostained tissue microarrays (TMAs), including 112 OSCC and 42 OPSCC, with anti-CAF-1/p60 and anti-CAF-1/p150 specific antibodies, correlating their expression with prognosis. Moreover, we assessed the sensitivity to PARP inhibitors and the double-strand breaks repair proficiency by cell viability and HR reporter assays, respectively, in HPV-positive and HPV-negative cell lines upon CAF-1/p60 and CAF-1/p150 depletion. The immunohistochemical analysis revealed a significant prognostic value of both tissue biomarkers combined expression in OSCC but not in OPSCC. In in vitro studies, the p60/150 CAF-1 subunits&rsquo, depletion impaired the proficiency of Homologous Recombination DNA damage repair, inducing sensitivity to the PARP-inhibitors, able to sensitize both the cell lines to IR. These results indicate that regardless of the prognostic meaning of p60/p150 tissue expression, the pharmacological depletion of CAF-1 complex&rsquo, s function, combined to PARP-inhibitors and/or IR treatment, could represent a valid therapeutic strategy for squamous cell carcinomas of head and neck region.

According to the American Academy of Aesthetic Plastic Surgeons, more than 11 million cosmetic surgical and nonsurgical procedures were performed by board-certified plastic surgeons, dermatologists and otolaryngologists in the United States, totaling more than 12 billion dollars. We performed a retrospective observational multi-centric study on patients treated with a non-animal origin cross-linked hyaluronic acid with different molecular weights for nasolabial folds, evaluating through a new imaging system, profilometric techniques with the confocal microscopy, the durability, the efficacy and the safety of this product. From 25 patients, 150 silicone casts were obtained: 75 casts of the right nasolabial fold and 75 casts of the left nasolabial fold. Roughness arithmetical average of the right fold at T2 decreased by 50% versus T0 and by 40% compared to T1; at T2, it decreased by the 45% versus T0 and by 35% compared to T1. No side effects were reported. Results proved that the analysis of the skin microreliefs through confocal microscopy is a new imaging system that allows to evaluate with precision and safety the results of aesthetic treatments such as fillers objectively.

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) gene. About 2000 mutations have been described so far. We setup an ex vivo model of human nasal epithelial cells (HNECs) to study CF patients testing the effect of novel mutations and molecular therapies. We performed sampling (by brushing), followed by culture and analysis of HNECs using a series of molecular techniques. We performed 50 brushings from CF patients and controls. Using cultured cells, we: i) demonstrated the widely heterogeneous CFTR expression in patients and in controls; ii) defined the splicing effect of a CFTR mutation; iii) assessed the CFTR gating activity in patients bearing different mutations; iv) demonstrated that butyrate significantly enhances CFTR expression. Based on our data, we can conclude: 1) HNEC brushing is performed without anaesthesia and is well tolerated in all CF patients (children and adults); 2) HNECs can be preserved for up to 48 hours before culture allowings multicentre studies; 3) HNECs culture can be considered a suitable model to study the molecular effects of new CFTR gene mutations and/or uncertain meaning specific mutations of carriers; 4) an ex vivo model of HNECs may be used to evaluate, before human use, the effect of new drugs on patients' cells bearing specific CFTR mutations; 5) the methodology is adequate for a quantitative measurement, by fluorescence, of the CFTR gating activity of the HNECs from patients with different genotypes identifying: a) CF patients bearing two severe mutations with an activity10% (compared to controls - 100%); b) CF patients bearing at least a mild mutation with an activity of 10-20%; c) CF carriers (heterozygous subjects) with an activity between 40-70%.La fibrosi cistica (FC) è una malattia autosomica recessiva causata da mutazioni nel gene CFTR (Cystic Fibrosis Transmembrane conductance Regulator). Finora sono state descritte circa 2000 mutazioni, ma per la maggior parte di esse è difficile definirne l’effetto senza complesse procedure in vitro. Abbiamo effettuato il campionamento (mediante brushing), la cultura e l’analisi di cellule epiteliali nasali umane (HNEC) utilizzando una serie di tecniche che possono aiutare a testare l’effetto delle mutazioni CFTR. Abbiamo eseguito 50 brushing da pazienti FC e controlli, e in 45 casi si è ottenuta una coltura positiva. Utilizzando cellule in coltura: i) abbiamo dimostrato l’espressione ampiamente eterogenea del CFTR nei pazienti e nei controlli; ii) abbiamo definito l’effetto di splicing di una mutazione sul gene CFTR; iii) abbiamo valutato l’attività di gating di CFTR in pazienti portatori di differenti mutazioni; iv) abbiamo dimostrato che il butirrato migliora in modo significativo l’espressione di CFTR. I dati provenienti dal nostro studio sperimentale dimostrano che l’uso del modello ex-vivo di cellule epiteliali nasali è un importante e valido strumento di ricerca e di diagnosi nella studio della FC e può anche essere mirato alla sperimentazione ed alla verifica di nuovi farmaci. In definitiva, in base ai nostri dati è possibile esprimere le seguenti conclusioni: 1) il prelievo delle cellule epiteliali nasali mediante brushing è applicabile senza alcuna anestesia ed è ben tollerato da tutti i pazienti affetti da FC (bambini e adulti), è scarsamente invasivo e facilmente ripetibile, è anche in grado di ottenere una sufficiente quantità di HNECs rappresentative, ben conservate, idonee allo studio della funzionalità di CFTR; 2) la conservazione delle cellule prelevate è possibile fino a 48 ore prima che si provveda all’allestimento della coltura e ciò permette di avviare studi multicentrici con prelievi in ogni sede e quindi di ottenere una ampia numerosità campionaria; 3) la coltura di cellule epiteliali nasali può essere considerata un modello adatto a studiare l’effetto molecolare di nuove mutazioni del gene CFTR e/o mutazioni specifiche di pazienti “carriers” dal significato incerto; 4) il modello ex-vivo delle HNECs consente inoltre di valutare, prima dell’impiego nell’uomo, l’effetto di farmaci (potenziatori e/o correttori) sulle cellule di pazienti portatori di mutazioni specifiche di CFTR; tali farmaci possono modulare l’espressione genica del canale CFTR aprendo così nuove frontiere terapeutiche e migliori prospettive di vita per pazienti affetti da una patologia cronica come la Fibrosi Cistica; 5) la metodologia da noi istituita risulta essere idonea alla misura quantitativa, mediante fluorescenza, dell’attività di gating del canale CFTR presente nelle membrane delle cellule epiteliali nasali prelevate da pazienti portatori di differenti genotipi; in tal modo è possibile individuare: a) pazienti FC portatori di 2 mutazioni gravi con un’attività10% (in rapporto ai controlli -100%), b) soggetti FC portatori contemporaneamente di una mutazione grave e di una lieve con un’attività tra 10-30%, c) i cosiddetti portatori “carriers”- eterozigoti - con un’attività tra 40-70%. In conclusione la possibilità di misurare l’attività del canale CFTR in HNECs fornisce un importante contributo alla diagnosi di FC, mediante individuazione di un “cut-off diagnostico”, ed anche alla previsione della gravità fenotipica della malattia; quindi quanto rilevabile dalla misura del suddetto canale permette di prospettare per il futuro la possibilità di valutare meglio i pazienti per i quali il test del sudore ha dato risultati ambigui (borderline o negativi). La metodica da noi sperimentata consente anche di monitorare i pazienti durante il trattamento farmacologico, valutando in tal modo i reali effetti delle nuove terapie.

Ultraconserved regions (UCRs) have been shown to originate non-coding RNA transcripts (T-UCRs) that have different expression profiles and play functional roles in the pathophysiology of multiple cancers. The relevance of these functions to the pathogenesis of bladder cancer (BlCa) is speculative. To elucidate this relevance, we first used genome-wide profiling to evaluate the expression of T-UCRs in BlCa tissues. Analysis of two datasets comprising normal bladder tissues and BlCa specimens with a custom T-UCR microarray identified ultraconserved RNA (uc.) 8+ as the most upregulated T-UCR in BlCa tissues, although its expression was lower than in pericancerous bladder tissues. These results were confirmed on BlCa tissues by real-time PCR and by in situ hybridization. Although uc.8+ is located within intron 1 of CASZ1, a zinc-finger transcription factor, the transcribed non-coding RNA encoding uc.8+ is expressed independently of CASZ1. In vitro experiments evaluating the effects of uc.8+ silencing, showed significantly decreased capacities for cancer cell invasion, migration, and proliferation. From this, we proposed and validated a model of interaction in which uc.8+ shuttles from the nucleus to the cytoplasm of BlCa cells, interacts with microRNA (miR)-596, and cooperates in the promotion and development of BlCa. Using computational analysis, we investigated the miR-binding domain accessibility, as determined by base-pairing interactions within the uc.8+ predicted secondary structure, RNA binding affinity, and RNA species abundance in bladder tissues and showed that uc.8+ is a natural decoy for miR-596. Thus uc.8+ upregulation results in increased expression of MMP9, increasing the invasive potential of BlCa cells. These interactions between evolutionarily conserved regions of DNA suggest that natural selection has preserved this potentially regulatory layer that uses RNA to modulate miR levels, opening up the possibility for development of useful markers for early diagnosis and prognosis as well as for development of new RNA-based cancer therapies.

Genome integrity maintenance is crucial for cell survival and for counteracting cancer onset and progression. Mammary cells invest great amount of energy in DNA repair, in order to avoid errors accumulation in DNA sequence. Nucleotide Excision Repair (NER) removes a broad spectrum of DNA damages, mainly bulky DNA lesions. Tissues of Head and Neck region are heavily exposed to bulky lesions inducing carcinogens, this making NER process of great interest in the field. Here we review the recent literature about NER in HNC and we also discuss the role played by NER in HNSCC in the chromatin context; to this aim we particularly focus on the role played by histones chaperon CAF-1, essential in restoring the chromatin structure following DNA replication and DNA damage repair, including NER. A better understanding of basic mechanisms underlying the DNA damage response, particularly involving NER, especially in the chromatin context, will provide us with new promising way to bypass the repair block, possibly becoming an unexpected mode of "transversal" control also of the proliferative deregulation, classically observed in HNSCC.

Hereditary spherocytosis (HS) and Chronic myelocytic leukemia (CML) are both life threatening hemotologic diseases. They are rarely seen to occur simultaneously in one individual patient. Here we demonstrate a case of HS associated with CML in this study. The patient is a young female, diagnosed with HS in 2005, and was given partial embolization of the splenic artery. She got significant remission after the procedure. In 2008, she was found abnormal in blood routine test, after bone marrow routine, chromosome and fusion gene tests, she was diagnosed with CML (chronic phase). She did not receive regular treatment until 3 months prior, and is currently being treated with Dasatimib. She achieved hematological remission, but had no significant improvement in chromosome and fusion gene figures. Due to her severe condition of hemolysis, a splenectomy or an allogeneic hematopoietic stem cell transplantation is considered.

TLRs are main actors of the innate immune response against HPV. There are very few studies on the role of TLRs mediated HPV clearance in Head and Neck oncology. Our aim was to evaluate whether TLR4 expression identifies HPV infection and/or HR-HPV integration status in oral and oropharyngeal cancers. By immunohistochemistry we assessed TLR4 levels in OSCC/OPSCC. To detect viral integration or episomic status In situ hybridization for HPV-DNA and Pyro-sequencing techniques have been performed. The relationship between TLR4 expression with HPV infection status has been investigated. ISH HPV positive samples have reported lower levels of TLR4 intensity than negative samples (p = .002). There was no statistical correlation between TLR4 intensity and PCR HPV results (p more than 0.0.5). Point-biserial correlation coefficient revealed significant association between TLR4 expression and HR-HPV integration status (p = .0001) and between TLR4 expression index and HR-HPV infection (p = .001). These data have shown that TLR4 down-regulation is strongly associated to both HPV-16 infection and its integration into the host DNA.

// Paolo D’Arrigo 1, * , Michele Russo 1, * , Anna Rea 1 , Martina Tufano 1 , Elia Guadagno 2 , Maria Laura Del Basso De Caro 2 , Roberto Pacelli 2 , Felix Hausch 3 , Stefania Staibano 2 , Gennaro Ilardi 2 , Silvia Parisi 1 , Maria Fiammetta Romano 1 and Simona Romano 1 1 Department of Molecular Medicine and Medical Biotechnologies, Federico II University, Naples, Italy 2 Department of Advanced Biomedical Sciences, Federico II University, Naples, Italy 3 Technical University Darmstadt Institute of Organic Chemistry and Biochemistry, Darmstadt, Germany * These authors have contributed equally to this work Correspondence to: Maria Fiammetta Romano, email: mariafiammetta.romano@unina.it , romano@dbbm.unina.it Keywords: PD-L1, glioblastoma multiforme, FKBP51, ionizing radiation, SAFit Received: March 10, 2017 Accepted: June 11, 2017 Published: July 17, 2017 ABSTRACT Background: FKBP51 is a co-chaperone with isomerase activity, abundantly expressed in glioma. We previously identified a spliced isoform (FKBP51s) and highlighted a role for this protein in the upregulation of Programmed Death Ligand 1 (PD-L1) expression in melanoma. Because gliomas can express PD-L1 causing a defective host anti-tumoral immunity, we investigated whether FKBP51s was expressed in glioma and played a role in PD-L1 regulation in this tumour. Methods: We used D54 and U251 glioblastoma cell lines that constitutively expressed PD-L1. FKBP51s was measured by immunoblot, flow cytometry and microscopy. In patient tumours, IHC and qPCR were used to measure protein and mRNA levels respectively. FKBP51s depletion was achieved by siRNAs, and its enzymatic function was inhibited using selective inhibitors (SAFit). We investigated protein maturation using N-glycosidase and cell fractionation approaches. Results: FKBP51s was expressed at high levels in glioma cells. Glycosylated-PD-L1 was increased and reduced by FKBP51s overexpression or silencing, respectively. Naive PD-L1 was found in the endoplasmic reticulum (ER) of glioma cells complexed with FKBP51s, whereas the glycosylated form was measured in the Golgi apparatus. SAFit reduced PD-L1 levels (constitutively expressed and ionizing radiation-induced). SAFit reduced cell death of PBMC co-cultured with glioma. Conclusions: Here we addressed the mechanism of post-translational regulation of PD-L1 protein in glioma. FKBP51s upregulated PD-L1 expression on the plasma membrane by catalysing the protein folding required for subsequent glycosylation. Inhibition of FKBP51s isomerase activity by SAFit decreased PD-L1 levels. These findings suggest that FKBP51s is a potential target of immunomodulatory strategies for glioblastoma treatment.

Melanoma is the most aggressive skin cancer; there is no cure in advanced stages. Identifying molecular participants in melanoma progression may provide useful diagnostic and therapeutic tools. FK506 binding protein 51 (FKBP51), an immunophilin with a relevant role in developmental stages, is highly expressed in melanoma and correlates with aggressiveness and therapy resistance. We hypothesized a role for FKBP51 in melanoma invasive behaviour. FKBP51 promoted activation of epithelial-to-mesenchymal transition (EMT) genes and improved melanoma cell migration and invasion. In addition, FKBP51 induced some melanoma stem cell (MCSC) genes. Purified MCSCs expressed high EMT genes levels, suggesting that genetic programs of EMT and MCSCs overlap. Immunohistochemistry of samples from patients showed intense FKBP51 nuclear signal and cytoplasmic positivity for the stem cell marker nestin in extravasating melanoma cells and metastatic brains. In addition, FKBP51 targeting by small interfering RNA (siRNA) prevented the massive metastatic substitution of liver and lung in a mouse model of experimental metastasis. The present study provides evidence that the genetic programs of cancer stemness and invasiveness overlap in melanoma, and that FKBP51 plays a pivotal role in sustaining such a program.