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© 2021 Link, Ilardi and Zannetti., Despite important advances in cancer treatments, metastasis remains the major cause of cancer mortality. In the complex tumor microenvironment, several malignant and non-malignant cell types, as well as components of the extracellular matrix (ECM), interact together to promote the metastatic process (1, 2). Stromal cells, which are already present in the tumor microenvironment (TME), are recruited from distant sites and “educated” by cancer cells through a dynamic cross-talk involving growth factors, cytokines, chemokines, miRNAs, and exosomes (3, 4). These primed cells, including cancer-associated fibroblasts, tumor-associated mesenchymal stem cells, tumor-associated macrophages, and other immune cells acquire a pro-metastatic function that supports tumor cells in each step of the metastatic cascade (4). Importantly, the bi-directional communication between tumor microenvironment and cancer cells induces the epithelial-mesenchymal-transition (EMT) that in turn elicits profound morphological and functional changes in tumor cells, triggering a mesenchymal-like phenotype with higher invasive potential (5). A crucial step in the establishment of metastases includes the formation of a pre-metastatic niche where recruited stromal cells contribute to creating a favorable microenvironment that permits cancer cell seeding (6). To successfully germ in a distant site, cancer cells need to find nutrients, an ECM that can support their attachment, and stromal cells that help them with paracrine signaling to survive and proliferate in the new environment (1). Responding to this complex scenario, this Research Topic aims to bring together more recent studies and discoveries that shed light on the molecular mechanisms and signaling pathways involved in the cross-talk between TME and cancer cells. The special issue on the “Tumor Microenvironment: Molecular Mechanisms and Signaling Pathways Involved in Metastatic Progression” includes 27 original articles, 11 review articles, and three mini-review articles, each of which advances knowledge in this field concerning different types of carcinomas., This work was supported by the Spanish Ministry of Science, Innovation, and Universities through Grant RTI2018-094629-B-I00 to WL. This work was supported by grants from from MIUR Progetti di Ricerca di Rilevante Interesse Nazionale (PRIN) Bando 2017-grant 2017MHJJ55, MIUR-PON “Ricerca e Innovazione” 2014-2020 (grant MOLIMONCOBRAIN LAB), from Regione Campania PO FESR 2014-2020 (grant eMORFORAD) to AZ.

Simple Summary NRP-1, a co-receptor of the EGFR, represented an interesting candidate to investigate in HNSCC, as Cetuximab, in combination with radio and chemotherapy, provided the first targeted therapy scheme approved by the FDA as a standard of care for patients with recurrent or metastatic HNSCC. High levels of NRP-1 expression significantly correlated with a shorter overall survival in both Oral Squamous Cell Carcinoma and Oropharyngeal Squamous Cell Carcinoma diagnosed patients, suggesting a prognostic role for this protein. In HNSCC cell lines in vitro experiments, NRP-1 sustained EGFR activation upon CDDP exposure, together with activation of downstream MAPK/AKT pathways. Furthermore, NRP-1 modulated the responsiveness to CDDP treatment. Abstract Head and neck squamous cell carcinoma (HNSCC) includes a group of aggressive malignancies characterized by the overexpression of the epidermal growth factor receptor (EGFR) in 90% of cases. Neuropilin-1 (NRP-1) acts as an EGFR co-receptor, enhancing, upon ligand stimulation, EGFR signaling in several cellular models. However, NRP-1 remains poorly characterized in HNSCC. By utilizing in vitro cellular models of HNSCC, we report that NRP-1 is involved in the regulation of EGFR signaling. In fact, NRP-1 can lead to cisplatin-induced EGFR phosphorylation, an escape mechanism activated by cancer cells upon cytotoxic stress. Furthermore, we evaluated Neuropilin-1 staining in tissue samples of an HNSCC case series (n = 218), unraveling a prognostic value for the Neuropilin-1 tissue expression. These data suggest a potential role for NRP-1 in HNSCC cancer progression, expanding the repertoire of signaling in which NRP-1 is involved and eliciting the need for further investigations on NRP-1 as a suitable target for HNSCC novel therapeutic approaches.

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.

Uveal melanoma (UM) is the most frequent primary ocular malignancy of adults; it exhibits an almost invariably poor prognosis with onset of liver metastases within 10–15 years after the diagnosis. Serine and arginine-rich splicing factor 1 (SRSF1) is an RNA-binding protein with proto-oncogene functions, including stimulation of angiogenesis, cell migration and cell growth; regarding the complex regulation of tumor angiogenesis, it has been suggested that SRSF1 regulates the alternative splicing of vascular endothelial growth factor-α, promoting the formation of its pro-angiogenic isoform. The immunohistochemical expression of SRSF1 on a series of 85 primary UMs, including 39 metastasizing and 46 non-metastasizing cases, was investigated; to clarify the potential pathogenetic role of SRSF1 in this tumor and its effect on angiogenesis, we correlated our immunohistochemical findings with the clinico-pathological features, the prognostic data and blood vascular microvessel density (MVD) findings of the cases from our series. Cases with higher immunohistochemical expression of SRSF1 also had higher MVD, higher metastatic potential and shorter metastasis-free survival; conversely, cases with lower SRSF1 immunoexpression showed lower MVD, lower metastatic risk and longer metastasis-free survival times. Our results suggested that SRSF1 has a negative prognostic role and a pro-angiogenic function in UM.

Low-grade intraductal carcinoma is a rare neoplasia with an excellent prognosis, previously classified as low-grade cribriform cystadenocarcinoma and low-grade salivary duct carcinoma. The tumor mainly occurs in the parotid gland and presents a ductal phenotype and an intraductal/intracystic growth pattern. It resembles intraductal breast lesions such as atypical ductal hyperplasia, papillary and cribriform ductal carcinoma in situ. Despite its infrequency, discriminating low-grade intraductal carcinoma from other salivary gland tumors is crucial, especially because of its favorable prognosis. A 74-year-old woman with a history of neurofibromatosis underwent a superficial parotidectomy to remove a sharply demarcated multi-cystic mass, diagnosed as category 4 at FNAC. The histological examination revealed a demarcated but unencapsulated lesion composed of a bigger cyst surrounded by several smaller cysts, lined by a monolayer or bilayer epithelium alternated with a cribriform proliferation, characterized by “Roman-bridges”, with occasional micro-papillae. A myoepithelial component, with a basal disposition, was present, confirmed by intense staining for protein p63 and SMA. Immunohistochemical stains showed intense, strong uniform positivity for pan-cytokeratin, protein S100, and SOX10. The Ki67 proliferation index was low (< 10%). A diagnosis of Low-grade Intraductal Carcinoma (LGIC) of the parotid was made. We performed a literature search in PUBMED for “Intraductal carcinoma”, “Low-grade Intraductal Carcinoma”, “Cribriform Cystadenocarcinoma”, “Salivary Duct Carcinoma”, and “Low-Grade Salivary Duct Carcinoma”. We selected 17 papers published between 1983 and 2020; the most affected anatomical site was the parotid gland (77/90), followed by minor salivary glands (6/90), the intraparotid lymph nodes (3/90) and the submandibular gland (4/90). Their main histopathological features are reported in the paper. Here we present a case report and a review of scientific literature on this topic to provide some essential diagnostic tools to discriminate this rare entity.

Oral squamous cell carcinoma is the most common oral cancer. In this paper, we present a performance analysis of four different deep learning-based pixel-wise methods for lesion segmentation on oral carcinoma images. Two diverse image datasets, one for training and another one for testing, are used to generate and evaluate the models used for segmenting the images, thus allowing to assess the generalization capability of the considered deep network architectures. An important contribution of this work is the creation of the Oral Cancer Annotated (ORCA) dataset, containing ground-truth data derived from the well-known Cancer Genome Atlas (TCGA) dataset.

Uveal melanoma (UM) is the most common intraocular tumor in adults. Despite sharing the name and similar morphological features with cutaneous melanoma (CM), it is an entirely different neoplasia with a particular genetic background and clinical behavior. CDKN2A is a gene located at chromosome 9p21, encoding for P16INK4a and P14(ARF) proteins, whose role as a tumor suppressor has been clearly defined in many malignant tumors. CDKN2A frequently presents germline mutations in familial CM and epigenetic downregulation in a considerable percentage of sporadic CM. It has been hypothesized that CDKN2A alterations are early events in CM development, playing a central role in the malignant transformation of melanocytes. Alterations of the CDKN2A gene reduce the expression of P16INK4a in most CM subtypes. Immunohistochemical evaluation of P16INK4a is currently used, in association with Ki67 and HMB45, in pathology practice to discriminate between dysplastic nevi and melanoma. On the other hand, CKDN2A is rarely mutated in UM, and the immunohistochemical expression of P16INK4a has only been reported in small case series. We tested P16INK4a expression on paraffin-embedded tissue sections from 9 tissue microarrays (TMAs), built with 2 mm cores derived from 133 uveal melanoma FFPE blocks, collected from 1990 to 2018, and from selected paraffin-blocks of 3 UM liver metastases. The immunohistochemical expression of P16INK4a was assessed with a visual evaluation by light microscopy and then with a digital approach. Both approaches, with an acceptable concordance rate, revealed P16INK4a expression in a large proportion of UM cases and all liver metastases, opening new possibilities of using it in the differential diagnosis between cutaneous and uveal melanoma metastases in cases of unknown primary tumor or patients with two different primary melanomas.

We introduce a machine learning-based analysis to predict the immunohistochemical (IHC) labeling index for the cell proliferation marker Ki67/MIB1 on cancer tissues based on morphometrical features extracted from hematoxylin and eosin (H&amp, E)-stained formalin-fixed, paraffin-embedded (FFPE) tumor tissue samples. We provided a proof-of-concept prediction of the Ki67/MIB1 IHC positivity of cancer cells through the definition and quantitation of single nuclear features. In the first instance, we set our digital framework on Ki67/MIB1-stained OSCC (oral squamous cell carcinoma) tissue sample whole slide images, using QuPath as a working platform and its integrated algorithms, and we built a classifier in order to distinguish tumor and stroma classes and, within them, Ki67-positive and Ki67-negative cells, then, we sorted the morphometric features of tumor cells related to their Ki67 IHC status. Among the evaluated features, nuclear hematoxylin mean optical density (NHMOD) presented as the best one to distinguish Ki67/MIB1 positive from negative cells. We confirmed our findings in a single-cell level analysis of H&amp, E staining on Ki67-immunostained/H&amp, E-decolored tissue samples. Finally, we tested our digital framework on a case series of oral squamous cell carcinomas (OSCC), arranged in tissue microarrays, we selected two consecutive sections of each OSCC FFPE TMA (tissue microarray) block, respectively stained with H&amp, E and immuno-stained for Ki67/MIB1. We automatically detected tumor cells in H&amp, E slides and generated a &ldquo, false color map&rdquo, (FCM) based on NHMOD through the QuPath measurements map tool. FCM nearly coincided with the actual immunohistochemical result, allowing the prediction of Ki67/MIB1 positive cells in a direct visual fashion. Our proposed approach provides the pathologist with a fast method of identifying the proliferating compartment of the tumor through a quantitative assessment of the nuclear features on H&amp, E slides, readily appreciable by visual inspection. Although this technique needs to be fine-tuned and tested on larger series of tumors, the digital analysis approach appears to be a promising tool to quickly forecast the tumor&rsquo, s proliferation fraction directly on routinely H&amp, E-stained digital sections.

Epithelial tumor progression often involves epithelial-mesenchymal transition (EMT). We report that increased intracellular levels of thyroid hormone (TH) promote the EMT and malignant evolution of squamous cell carcinoma (SCC) cells. TH induces the EMT by transcriptionally up-regulating ZEB-1, mesenchymal genes and metalloproteases and suppresses E-cadherin expression. Accordingly, in human SCC, elevated D2 (the T3-producing enzyme) correlates with tumor grade and is associated with an increased risk of postsurgical relapse and shorter disease-free survival. These data provide the first in vivo demonstration that TH and its activating enzyme, D2, play an effective role not only in the EMT but also in the entire neoplastic cascade starting from tumor formation up to metastatic transformation, and supports the concept that TH is an EMT promoter. Our studies indicate that tumor progression relies on precise T3 availability, suggesting that pharmacological inactivation of D2 and TH signaling may suppress the metastatic proclivity of SCC.

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.

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

To date, a very small number of serum biomarkers have been identified for clinical use in squamous carcinomas of the head and neck region. Chromatin Assembly Factor-1 (CAF-1) heterotrimeric complex subunit CAF1/p60 expression levels have been reported to be of prognostic value in Oral Squamous Cell Carcinoma (OSCC), as well as in other human solid tumors. Here our aim was to detect and quantify CAF1/p60 in the peripheral blood of Head and Neck Squamous Cell Carcinoma (HNSCC) patients, and to investigate the possible associations between serum concentration of CAF-1/p60 and HNSCC tumors. A total of 63 HNSCC patients (51 OSCC, 8 OPSCC, 3 laryngeal SCC, and 1 rhinopharynx SCC) and 30 healthy controls were enrolled. The serum levels of CAF-1/p60 were measured by ELISA assay before and after surgery. Serum CAF-1/p60 concentration resulted significantly higher in cancer patients, compared with healthy controls, in pre-surgery samples (P < 0.05). Serum levels of CAF-1/p60 significantly decreased in serum samples taken after surgery (P < 0.05). Our results demonstrated that CAF-1/p60 may be detected in serum, suggesting a role for CAF-1/p60 as potential soluble biomarkers in HNSCC tumors.

Oral cavity squamous cell cancer (OSCC) and Oropharynx squamous cell carcinoma (OPSCC) are the most frequent forms of Head and Neck Cancers (HNCs) [1]. About 300,000 new cases of oral cancers are being counted yearly worldwide, having registered an increase of incidence of 225% in U.S.A.

Objectives CCDC6 (coiled-coil domain containing 6) is a player of the HR response to DNA damage and has been predicted to interact with BAP1, another HR-DNA repair gene highly mutated in Malignant Pleural Mesothelioma (MPM), an aggressive cancer with poor prognosis. CCDC6 levels are modulated by the deubiquitinase USP7, and CCDC6 defects have been reported in several tumors determining PARP-inhibitors sensitivity. Our aim was to investigate the functional role of CCDC6 in MPM carcinogenesis and response to PARP-inhibitors. Materials and Methods The interaction between CCDC6 and BAP1 was confirmed in MPM cells, by co-immunoprecipitation. Upon USP7 inhibition, that induces CCDC6 degradation, the ability to repair the DSBs and the sensitivity to PARP inhibitors, was explored by HR reporter and by cells viability assays, respectively. A TMA including 34 MPM cores was immunostained for CCDC6, USP7 and BAP1 and the results correlated by statistical analysis. Results MPM cells depleted of CCDC6 showed defects in DSBs repair and sensitivity to PARP inhibitors. The silencing of CCDC6 when combined with the overexpression of BAP1-mutant (Δ221-238) enhanced the HR-DNA repair defects and the PARP inhibitors sensitivity. In the TMA of MPM primary samples, the staining of CCDC6 and of its de-ubiquitinase USP7 showed a significant correlation in the tested primary samples (p = 0.01). CCDC6 was barely detected in 30% of the tumors that also carried BAP1 defects. Conclusion The combination of CCDC6 and BAP1 staining may indicate therapeutic options for DDR targeting, acting in synergism with cisplatinum.

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.

Epithelial tumor progression often involves epithelial-mesenchymal transition (EMT). We report that increased intracellular levels of thyroid hormone (TH) promote the EMT and malignant evolution of squamous cell carcinoma (SCC) cells. TH induces the EMT by transcriptionally up-regulating ZEB-1, mesenchymal genes and metalloproteases and suppresses E-cadherin expression. Accordingly, in human SCC, elevated D2 (the T3-producing enzyme) correlates with tumor grade and is associated with an increased risk of postsurgical relapse and shorter disease-free survival. These data provide the first in vivo demonstration that TH and its activating enzyme, D2, play an effective role not only in the EMT but also in the entire neoplastic cascade starting from tumor formation up to metastatic transformation, and supports the concept that TH is an EMT promoter. Our studies indicate that tumor progression relies on precise T3 availability, suggesting that pharmacological inactivation of D2 and TH signaling may suppress the metastatic proclivity of SCC., SCOPUS: ar.j, info:eu-repo/semantics/published

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.

The aim of this study was to identify new disease-related circulating miRNAs with high diagnostic accuracy for breast cancer (BC) and to correlate their deregulation with the morpho-functional characteristics of the tumour, as assessed in vivo by positron emission tomography/magnetic resonance (PET/MR) imaging. A total of 77 untreated female BC patients underwent same-day PET/MR and blood collection, and 78 healthy donors were recruited as negative controls. The expression profile of 84 human miRNAs was screened by using miRNA PCR arrays and validated by real-time PCR. The validated miRNAs were correlated with the quantitative imaging parameters extracted from the primary BC samples. Circulating miR-125b-5p and miR-143-3p were upregulated in BC plasma and able to discriminate BC patients from healthy subjects (miR-125-5p area under the receiver operating characteristic ROC curve (AUC) = 0.85 and miR-143-3p AUC = 0.80). Circulating CA15-3, a soluble form of the transmembrane glycoprotein Mucin 1 (MUC-1) that is upregulated in epithelial cancer cells of different origins, was combined with miR-125b-5p and improved the diagnostic accuracy from 70% (CA15-3 alone) to 89% (CA15-3 plus miR-125b-5p). MiR-143-3p showed a strong and significant correlation with the stage of the disease, apparent diffusion coefficient (ADCmean), reverse efflux volume transfer constant (Kepmean) and maximum standardized uptake value (SUVmax), and it might represent a biomarker of tumour aggressiveness. Similarly, miR-125b-5p was correlated with stage and grade 2 but inversely correlated with the forward volume transfer constant (Ktransmean) and proliferation index (Ki67), suggesting a potential role as a biomarker of a relatively more favourable prognosis. In situ hybridization (ISH) experiments revealed that miR-143-3p was expressed in endothelial tumour cells, miR-125-5p in cancer-associated fibroblasts, and neither in epithelial tumour cells. Our results suggested that miR-125-5p and miR-143-3p are potential biomarkers for the risk stratification of BC, and Kaplan-Maier plots confirmed this hypothesis. In addition, the combined use of miR-125-b-5p and CA15-3 enhanced the diagnostic accuracy up to 89%. This is the first study that correlates circulating miRNAs with in vivo quantified tumour biology through PET/MR biomarkers. This integration elucidates the link between the plasmatic increase in these two potential circulating biomarkers and the biology of untreated BC. In conclusion, while miR-143-3b and miR-125b-5p provide valuable information for prognosis, a combination of miR-125b-5p with the tumour marker CA15-3 improves sensitivity for BC detection, which warrants consideration by further validation studies.

DNA damage response has been clearly described as an anti-cancer barrier in early human tumorigenesis. Moreover, interestingly, testicular germ cell tumors (TGCTs) have been reported to lack the DNA Damage Response (DDR) pathway activation. CCDC6 is a pro-apoptotic phosphoprotein substrate of the kinase ataxia telangectasia mutated (ATM) able to sustain DNA damage checkpoint in response to genotoxic stress and is commonly rearranged in malignancies upon fusion with different partners. In our study we sought to determine whether CCDC6 could have a role in the patho-genesis of testicular germ cell tumors. To achieve this aim, analysis for CCDC6 expression has been evaluated on serial sections of the mouse testis by immunohistochemistry and on separate populations of murine testicular cells by western blot. Next, the resistance to DNA damage-induced apoptosis and the production of reactive oxygen species has been investigated in GC1 cells, derived from immortalized type B murine germ cells, following CCDC6 silencing. Finally, the CCDC6 expression in normal human testicular cells, in Intratubular Germ Cell Neoplasia Unclassified (IGCNU), in a large series of male germ cell tumours and in the unique human seminoma TCam2 cell line has been evaluated by immunohistochemistry and by Western Blot analyses. The analysis of the CCDC6 expression revealed its presence in Sertoli cells and in spermatogonial cells. CCDC6 loss was the most consistent feature among the primary tumours and TCam2 cells. Interestingly, following treatment with low doses of H2O2, the silencing of CCDC6 in GC1 cells caused a decrease in the oxidized form of cytochrome c and low detection of Bad, PARP-1 and Caspase 3 proteins. Moreover, in the silenced cells, upon oxidative damage, the cell viability was protected, the γH2AX activation was impaired and the Reactive Oxygen Species (ROS) release was decreased. Therefore, our results suggest that the loss of CCDC6 could aid the spermatogonial cells to be part of a pro-survival pathway that helps to evade the toxic effects of endogenous oxidants and contributes to testicular neoplastic growth.

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.

Oral and oropharyngeal cancers represent the two most common malignancies of the head and neck region. The major risk factors for these cancers include alcohol consumption, tobacco use (via smoking or chewing) and high-risk human papillomavirus infection. The transition from normal epithelium to premalignant tissue and finally carcinoma is in part caused by a summation of genetic and epigenetic modifications. Epigenetic refers to modifications in the way the genome is expressed in cells. The most common examples of epigenetic control of gene expression are DNA methylation, histone modification and regulation by small non-coding RNAs. The aim of the current paper was to review the recent studies on the main epigenetic changes that have been suggested to serve a role in the carcinogenesis process and progression of oral and oropharyngeal cancers. Furthermore, it is discussed how the epigenetic changes may be used as potential predictive biomarkers and how recent findings in the field may impact the personalized cancer therapy approach for these 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.

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.

Basal-cell carcinoma (BCC) is the most common non-melanoma skin cancer (NMSC) in white individuals. The main risk factor of BCC is intense exposure to ultraviolet radiation. The gold standard of diagnosis of BCC is histopathology. Treatment options for BCC consist of surgery, vismodegib, radiotherapy and topical or ablative treatments. Surgical excision is usually the preferred treatment for nodular and aggressive BCC subtypes. Here in, we report the case of a 51 year-old Caucasian woman successfully treated with ingenol mebutate (IM) 0.05% gel on a large-sized (ø3 cm) superficial BCC (sBCC) localized on the right shoulder. IM appears to be an effective, simple, safe and comfortable treatment even for large-sized sBCC with good cosmetic results.

SummaryBackground Mycosis fungoides (MF) is the major subtype of cutaneous T-cell lymphomas (CTCL). It usually has a prolonged indolent clinical course with a minority of cases acquiring a more aggressive biological profile and resistance to conventional therapies, partially attributed to the persistent activation of Nuclear Factor-kappa B (NF-κB) pathway. In the last decade, several papers suggested an important role for the FK506-binding protein 51 (FKBP51), an immunophilin initially cloned in lymphocytes, in the control of NF-κB pathway in different types of human malignancies. Objectives We aimed to investigate the possible value of FKBP51 expression as a new reliable marker of outcome in MF patients. Methods We assessed by immunohistochemistry (IHC) FKBP51 expression in 44 patients with MF, representative of different stages of the disease. Immunohistochemical results were subsequently confirmed at mRNA level with quantitative PCR (qPCR) in a subset of enrolled patients. In addition, IHC and qPCR served to study the expression of some NF-κB target genes, including the tumour necrosis factor receptor-associated factor 2 (TRAF2). Results Our results show that FKBP51 was expressed in all evaluated cases, with the highest level of expression characterizing MFs with the worst prognosis. Moreover, a significant correlation subsisted between FKBP51 and TRAF2 IHC expression scores. Conclusions we hypothesize a role for FKBP51 as a prognostic marker for MF and suggest an involvement of this immunophilin in deregulated NF-κB pathway of this CTCL. This article is protected by copyright. All rights reserved.

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

[This corrects the article DOI: 10.1371/journal.pone.0197065.].

Non-small cell lung cancer (NSCLC) is the main cause of cancer-related death worldwide and new therapeutic strategies are urgently needed. In this study, we have characterized a panel of NSC lung cancer cell lines for the expression of coiled-coil-domain containing 6 (CCDC6), a tumor suppressor gene involved in apoptosis and DNA damage response. We show that low CCDC6 protein levels are associated with a weak response to DNA damage and a low number of Rad51 positive foci. Moreover, CCDC6 deficient lung cancer cells show defects in DNA repair via homologous recombination. In accordance with its role in the DNA damage response, CCDC6 attenuation confers resistance to cisplatinum, the current treatment of choice for NSCLC, but sensitizes the cells to olaparib, a small molecule inhibitor of the repair enzymes PARP1/2. Remarkably, the combination of the two drugs is more effective than each agent individually, as demonstrated by a combination index

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.

Oral cavity squamous cell cancer (OSCC) and Oropharynx squamous cell carcinoma (OPSCC) are the most frequent forms of Head and Neck Cancers (HNCs) [1]. About 300,000 new cases of oral cancers are being counted yearly worldwide, having registered an increase of incidence of 225% in U.S.A. in the last 20 years, with about 50% of related deaths [2,3]. The relevant advances in treatments of OSCC during the last decades, allowed updated surgery techniques, robotic surgery, intensive induction chemotherapy and hyperfractionated radiotherapy to be currently applied to the advanced cases. Unfortunately, most of these therapies often carry severe acute and chronic side effects, heavily impacting on patients’ quality of life.

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.

// 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.

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.

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.

Background: Malignant tumours of minor salivary glands are uncommon, representing only 2-4% of all head and neck cancers. In the larynx, minor salivary gland tumours rarely occur and constitute less than 1% of laryngeal neoplasm. Most of the minor salivary gland tumours arise in the subglottis; however, they can also occur in the supraglottis, in the false vocal cords, aryepiglottic folds and caudal portion of the epiglottis. The most common type of malignant minor salivary gland tumour is adenoid cystic carcinoma. Methods: We present a unusual case of adenoid cystic carcinoma of glottic-subglottic region in a 61-year-old woman. Follow-up endoscopy and laryngeal magnetic resonance imaging (MRI) at three years after treatment showed no recurrence of the tumour. Results: The diagnosis of glottic-subglottic adenoid cystic carcinoma should be considered in patients who are characterized by dyspnea, cough and stridor, but do not respond to pharmacologic approach. Conclusions: Adenoid cystic carcinoma is usually a very slow growing cancer, invested by an apparently normal laryngeal mucosa, so that it can show no clear symptoms for a long time. For these reasons the increasing number of diagnostic mistakes or late diagnosis that may be fatal in some cases

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.

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.

Coexistence of metastasis from squamous cell carcinoma and tuberculosis within lymph nodes is rare. We report a case of 86 years old woman with a mass in the left laterocervical region. The patient had undergone excision of a poorly differentiated squamous cell carcinoma from the mucosa of the left cheek, a few months before. Histological examination of a mass of few fused lymph nodes, isolated from left laterocervical lymphadenectomy, showed metastatic squamous cell carcinoma with concomitant granulomatous inflammation. A diagnosis of tuberculosis associated with malignancy was posed. The suspect was confirmed by a positive anamnestic finding of a previous tuberculosis infection. The granulomatous reaction may be associated with many types of tumor, and can be found in the draining lymph nodes. The possibility that this reaction is also due to a tuberculosis infection should be kept in mind for elderly oncology patient.

Despite the progressive increase of early diagnosis, a subset of prostate cancers show a metastasizing and lethal course, not always predictable upon the traditional prognostic parameters. The object of this study was to investigate the role of the survival co-chaperone protein BAG3 as a new prognostic marker for prostate cancer. BAG3 was detected by immunohistochemistry in 55 specimens of surgically removed prostate carcinomas and in 15 surgical specimens of non-neoplastic prostate tissues. Results were compared with clinic-pathological data and outcome of patients and statistically evaluated. BAG3 resulted expressed in all the cases: Non-neoplastic prostate tissue showed a cytoplasmatic staining with apical reinforcement, a finding which appears consistent with the reported connection of the protein with the membrane focal cell-adhesion complexes. In prostate carcinomas, BAG3 showed a progressive decrease of the expression level from well- to low-differentiated carcinoma, coupled with the loss of polarisation of the signal in metastasizing cases. These results indicate that BAG3 intra-cytoplasmic delocalisation is a specific feature of cancer versus non-neoplastic prostate and a candidate new marker for prediction of prostate cancer invasiveness and behaviour.

Melanoma of the anal cavity is an uncommon malignant tumor with an aggressive clinical behavior. The presence of nonmelanocytic cell or tissue components, designated as divergent differentiation, is an unusual but well-documented phenomenon in melanoma. We experienced a rare case of amelanotic melanoma with neuroendocrine differentiation of the anal canal, occurring in a 68-year old woman. This tumor was characterized by a clear-cut radial growth phase and an invasive component composed of a diffuse small cells population positive for neuroendocrine markers with a focal but convincing co-expression of S100 protein. To the best of our knowledge, this represents the first case of neuroendocrine differentiation in a primary melanoma of the anal cavity. Although anal melanoma with neuroendocrine differentiation is exceptional, clinical practitioners should be aware of its possibility at this site.

// Francesco Morra 1,2,* , Chiara Luise 1,* , Francesco Merolla 1,3 , Ina Poser 4 , Roberta Visconti 1 , Gennaro Ilardi 3 , Simona Paladino 2 , Hiroyuki Inuzuka 5 , Gianluca Guggino 6 , Roberto Monaco 7 , David Colecchia 8 , Guglielmo Monaco 6 , Aniello Cerrato 1 , Mario Chiariello 8 , Krista Denning 9 , Pier Paolo Claudio 10 , Stefania Staibano 3 and Angela Celetti 1 1 Istituto per l’Endocrinologia e l’Oncologia Sperimentale “Gaetano Salvatore”, CNR, Napoli, Italy 2 Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Universita Federico II, Napoli, Italy 3 Dipartimento di Scienze Biomediche Avanzate, Universita Federico II, Napoli, Italy 4 Max Plank Institute, MPI-CBG Dresden, Germany 5 Harvard Medical School, Beth Israel Deaconess Medical Center, MA, USA 6 UOC Chirurgia Toracica, Azienda Ospedaliera di Rilievo Nazionale “A.Cardarelli”, Napoli, Italy 7 UOC Anatomia Patologica, Azienda Ospedaliera di Rilievo Nazionale “A.Cardarelli”, Napoli, Italy 8 Istituto Toscano Tumori, Core Research Laboratory, Siena, Italy 9 Department of Pathology, Joan C. Edwards Cancer Center, Huntington, WV, USA 10 Department of Biochemistry and Microbiology & Dept. of Surgery, Marshall University, Joan C. Edwards Cancer Center, Huntington, WV, USA * These authors have contributed equally to this work Correspondence to: Angela Celetti, email: // Keywords : CCDC6, FBXW7, USP7, mitotic kinases, cisplatinum Received : January 20, 2015 Accepted : March 03, 2015 Published : March 30, 2015 Abstract CCDC6 gene product is a pro-apoptotic protein substrate of ATM, whose loss or inactivation enhances tumour progression. In primary tumours, the impaired function of CCDC6 protein has been ascribed to CCDC6 rearrangements and to somatic mutations in several neoplasia. Recently, low levels of CCDC6 protein, in NSCLC, have been correlated with tumor prognosis. However, the mechanisms responsible for the variable levels of CCDC6 in primary tumors have not been described yet. We show that CCDC6 turnover is regulated in a cell cycle dependent manner. CCDC6 undergoes a cyclic variation in the phosphorylated status and in protein levels that peak at G2 and decrease in mitosis. The reduced stability of CCDC6 in the M phase is dependent on mitotic kinases and on degron motifs that are present in CCDC6 and direct the recruitment of CCDC6 to the FBXW7 E3 Ubl. The de-ubiquitinase enzyme USP7 appears responsible of the fine tuning of the CCDC6 stability, affecting cells behaviour and drug response. Thus, we propose that the amount of CCDC6 protein in primary tumors, as reported in lung, may depend on the impairment of the CCDC6 turnover due to altered protein-protein interaction and post-translational modifications and may be critical in optimizing personalized therapy.

FKBP51 (gene FKBP5) is an immunophilin capable of immunosuppression expressed in melanoma and lymphocytes. We found increased levels of a spliced FKBP5 variant in the PBMCs of 124 patients with melanoma. This variant encodes for an unknown isoform (FKBP51s). We hypothesized that FKBP51s resulted from tumour interaction with immune cells, through PDL-1/PD-1. To address this issue, we performed melanoma/PBMC cocultures. Furthermore, the immunohistochemistry of 76 melanoma specimens served to investigate whether FKBP51s stained tumour infiltrating lymphocytes. Our results showed that PBMCs expressed FKBP51s when cocultured with melanoma. Tumour PDL-1 knockdown or anti-PD-1 reduced FKBP51s expression in cocultured PBMCs. IHC showed a strong FKBP51s signal in tumour infiltrating lymphocytes, and lymphocytes of the invasion front of the tumour, along with melanoma PDL-1 expression. When overexpressed in melanoma, FKBP51s facilitated PDL-1 expression on the cell surface. In conclusion, our study shows that FKBP51s marks the PBMCs of patients with melanoma and is exploited by the tumour to immunomodulate through PDL-1.

Background Breast neoplasms include different histopathological entities, varying from benign tumors to highly aggressive cancers. Despite the key role of imaging, traditional histology is still required for a definitive diagnosis. Confocal Laser Endomicroscopy (CLE) is a new technique, which enables to obtain histopathological images in vivo, currently used in the diagnosis of gastrointestinal diseases. This is a single-center pilot feasibility study; the main aim is to describe the basic morphological patterns of Confocal Laser Endomicroscopy in normal breast tissue besides benign and malignant lesions. Methods Thirteen female patients (mean age 52.7, range from 22 to 86) who underwent surgical resection for a palpable breast nodule were enrolled. CLE was performed soon after resection with the Cellvizio® Endomicroscopy System (Mauna Kea Technologies, Paris, France), by using a Coloflex UHD-type probe; intravenous fluorescein was used as contrast-enhancing agent. The surgical specimen was cut along the main axis; dynamic images were obtained and recorded using a hand-held probe directly applied both to the internal part of the lesion and to several areas of surrounding normal tissue. Each specimen was then sent for definitive histologic examination. Results Histopathology revealed a benign lesion in six patients (46%), while a breast cancer was diagnosed in seven women (54%). Confocal laser endomicroscopy showed some peculiar morphological patterns. Normal breast tissue was characterized by a honeycomb appearance with regular, dark, round or hexagonal glandular lobules on a bright stroma background; tubular structures, representing ducts or blood vessels, were also visible in some frames. Benign lesions were characterized by a well-demarcated “slit-like” structure or by lobular structures in abundant bright stroma. Finally, breast cancer was characterized by a complete architectural subversion: ductal carcinoma was characterized by ill-defined structures, with dark borders and irregular ductal shape, formingribbons, tubules or nests; mucinous carcinoma showed smaller cells organized in clusters, floating in an amorphous extracellular matrix. Conclusions This is the first pilot study to investigate the potential role of confocal laser imaging as a diagnostic tool in breast diseases. Further studies are required to validate these results and establish the clinical impact of this technique.