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Background: Tumor molecular profile analysis by Next Generation Sequencing technology is currently widely applied in clinical practice and has enabled the detection of predictive biomarkers of response to targeted treatment. In parallel with targeted therapies, immunotherapies are also evolving, revolutionizing cancer therapy, with Programmed Death-ligand 1 (PD-L1), Microsatellite instability (MSI), and Tumor Mutational Burden (TMB) analysis being the biomarkers employed most commonly., Methods: In the present study, tumor molecular profile analysis was performed using a 161 gene NGS panel, containing the majority of clinically significant genes for cancer treatment selection. A variety of tumor types have been analyzed, including aggressive and hard to treat cancers such as pancreatic cancer. Besides, the clinical utility of immunotherapy biomarkers (TMB, MSI, PD-L1), was also studied., Results: Molecular profile analysis was conducted in 610 cancer patients, while in 393 of them a at least one biomarker for immunotherapy response was requested. An actionable alteration was detected in 77.87% of the patients. 54.75% of them received information related to on-label or off-label treatment (Tiers 1A.1, 1A.2, 2B, and 2C.1) and 21.31% received a variant that could be used for clinical trial inclusion. The addition to immunotherapy biomarker to targeted biomarkers' analysis in 191 cases increased the number of patients with an on-label treatment recommendation by 22.92%, while an option for on-label or off-label treatment was provided in 71.35% of the cases., Conclusions: Tumor molecular profile analysis using NGS is a first-tier method for a variety of tumor types and provides important information for decision making in the treatment of cancer patients. Importantly, simultaneous analysis for targeted therapy and immunotherapy biomarkers could lead to better tumor characterization and offer actionable information in the majority of patients. Furthermore, our data suggest that one in two patients may be eligible for on-label ICI treatment based on biomarker analysis. However, appropriate interpretation of results from such analysis is essential for implementation in clinical practice and accurate refinement of treatment strategy.

Background: High grade glioma molecular profiling is of particular interest in neurooncology. The role of telomerase reverse transcriptase (TERT) varies dependent upon other molecular parameters. We explored the role of TERT in 101 high-grade gliomas., Methods: A total of 101 patients (pts) with grade III-IV gliomas treated with standard of care and informative tumor genotypes were included in the present study. Of 55 genes targeted with the next-generation sequencing panel, mutations (muts) were found in 37; these were included in the analysis. TERT mut were tested with Sanger sequencing. MGMT promoter methylation status was determined by methylation specific PCR., Results: 270 mut were detected in 92/101 tumors (91.1%). TERT was the most frequently mutated gene (74.3%). IDH1/2 mut were mutually exclusive with mut in the neurofibromin 1 (NF1) gene. Mutated TERT was associated with wild-type (wt) IDH1/2 (p = 0.025). The 12-month overall survival (OS) rate was 74.3% (median OS: 22 months). Pts with TERT and NF1 wt had a median OS of 40.8 months, while among pts with NF1 wt/TERT mutant, the median OS was 18.5 months. NF1 and TERT mut univariately conferred shorter OS (HR = 3.19; p = 0.004 and HR = 2.28; p = 0.002). Upon multivariate analysis, mutated TERT showed marginal unfavorable prognostic significance for OS (p = 0.049), while NF1 lost its unfavorable significance (p = 0.151)., Conclusions: TERT is herein proven to confer poor prognosis in high grade gliomas, independent of IDH and MGMT. NF1 seems to also confer poor prognosis although our small numbers do not allow for firm conclusions., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: While recent genome-wide association studies have suggested novel low-grade glioma (LGG) stratification models based on a molecular classification, we explored the potential clinical utility of patient-derived cells. Specifically, we assayed glioma-associated stem cells (GASC) that are patient-derived and representative of the glioma microenvironment., Methods: By next-generation sequencing, we analyzed the transcriptional profile of GASC derived from patients who underwent anaplastic transformation either within 48 months (GASC-BAD) or ≥7 years (GASC-GOOD) after surgery. Gene set enrichment and pathway enrichment analyses were applied. The prognostic role of a nuclear factor-kappaB (NF-κB) signature derived from GASC-BAD was tested in 530 newly diagnosed diffuse LGG patients comprised within The Cancer Genome Atlas (TCGA) database. The prognostic value of the GASC upstream regulator p65 NF-κB was assessed, by univariate and multivariate Cox analyses, in a single center case study, including 146 grade II LGGs., Results: The key elements differentiating the transcriptome of GASC isolated from LGG with different prognoses were mostly related to hallmarks of cancer (eg, inflammatory/immune process, NF-κB activation). Consistently, the NF-κB signature extrapolated from the GASC study was prognostic in the dataset of TCGA. Finally, the nuclear expression of the NF-kB-p65 protein, assessed using an inexpensive immunohistochemical method, was an independent predictor of both overall survival and malignant progression-free survival in 146 grade II LGGs., Conclusion: This study demonstrates for the first time the independent prognostic role of NF-kB activation in LGG and outlines the role of patient-based stem cell models as a tool for precision medicine approaches.

Non‑small cell lung cancer (NSCLC) is the most common type of lung cancer and a tumor with a broad spectrum of targeted therapies already available or in clinical trials. Thus, molecular characterization of the tumor using next generation sequencing (NGS) technology, has become a key tool for facilitating treatment decisions and the clinical management of NSCLC patients. The performance of a custom 23 gene multiplex amplification hot spot panel, based on Ion AmpliSeq™ technology, was evaluated for the analysis of tumor DNA extracted from formalin-fixed and paraffin-embedded (FFPE) tissues. Furthermore, the Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel was used for fusion RNA transcript analysis. The mutation spectrum of the tumors was determined in a cohort of 502 patients with NSCLC using the aforementioned targeted gene panels. The panel used for tumor DNA analysis in this study exhibited high rates (100%) of sensitivity, specificity and reproducibility at a mutation allelic frequency of 3%. At least one DNA mutation was detected in 374 patients (74.5%) and an RNA fusion was identified in 16 patients, (3.2%). In total, alterations in a cancer-driver gene were identified (including point mutations, gene rearrangements and MET amplifications) in 77.6% of the tumors tested. Among the NSCLC patients, 23% presented a mutation in a gene associated with approved or emerging targeted therapy. More specifically, 13.5% (68/502) presented a mutation in a gene with approved targeted therapy (EGFR, ALK, ROS1) and 9.4% (47/502) had an alteration in a gene related to emerging targeted therapies (ERBB2, BRAF, MET and RET). Furthermore, 51.6% of the patients had a mutation in a gene that could be related to an off label therapy or indicative for access to a clinical trial. Thus, the targeted NGS panel used in this study is a reliable approach for tumor molecular profiling and can be applied in personalized treatment decision making for NSCLC patients.

A major contributing factor to glioma development and progression is its ability to evade the immune system. Nano-meter sized vesicles, exosomes, secreted by glioma-stem cells (GSC) can act as mediators of intercellular communication to promote tumor immune escape. Here, we investigated the immunomodulatory properties of GCS-derived exosomes on different peripheral immune cell populations. Healthy donor peripheral blood mononuclear cells (PBMCs) stimulated with anti-CD3, anti-CD28 and IL-2, were treated with GSC-derived exosomes. Phenotypic characterization, cell proliferation, Th1/Th2 cytokine secretion and intracellular cytokine production were analysed by distinguishing among effector T cells, regulatory T cells and monocytes. In unfractionated PBMCs, GSC-derived exosomes inhibited T cell activation (CD25 and CD69 expression), proliferation and Th1 cytokine production, and did not affect cell viability or regulatory T-cell suppression ability. Furthermore, exosomes were able to enhance proliferation of purified CD4+ T cells. In PBMCs culture, glioma-derived exosomes directly promoted IL-10 and arginase-1 production and downregulation of HLA-DR by unstimulated CD14+ monocytic cells, that displayed an immunophenotype resembling that of monocytic myeloid-derived suppressor cells (Mo-MDSCs). Importantly, the removal of CD14+ monocytic cell fraction from PBMCs restored T-cell proliferation. The same results were observed with exosomes purified from plasma of glioblastoma patients. Our results indicate that glioma-derived exosomes suppress T-cell immune response by acting on monocyte maturation rather than on direct interaction with T cells. Selective targeting of Mo-MDSC to treat glioma should be considered with regard to how immune cells allow the acquirement of effector functions and therefore counteracting tumor progression., Competing Interests: The authors have declared that no competing interests exist.

Gastrointestinal malignancies are among the leading causes of cancer-related deaths worldwide. Like all human malignancies they are characterized by accumulation of mutations which lead to inactivation of tumor suppressor genes or activation of oncogenes. Advances in Molecular Biology techniques have allowed for more accurate analysis of tumors' genetic profiling using new breakthrough technologies such as next generation sequencing (NGS), leading to the development of targeted therapeutical approaches based upon biomarker-selection. During the last 10 years tremendous advances in the development of targeted therapies for patients with advanced cancer have been made, thus various targeted agents, associated with predictive biomarkers, have been developed or are in development for the treatment of patients with gastrointestinal cancer patients. This review summarizes the advances in the field of molecular biomarkers in tumors of the gastrointestinal tract, with focus on the available NGS platforms that enable comprehensive tumor molecular profile analysis., Competing Interests: Conflict-of-interest statement: No potential conflicts of interest. No financial support.

Background: Glioblastoma stem cells (GSC) have been extensively recognized as a plausible cause of glioblastoma resistance to therapy and recurrence resulting in high glioblastoma mortality. Abnormalities in the DNA repair pathways might be responsible for the inability of the currently used chemotherapeutics to eliminate the (GSC) subpopulation., Methods: In this work, we compared the expression of sixty DNA repair related genes between primary glioblastoma cell cultures and the glioblastoma enriched stem cell primary cultures. MTT test was used to analyze the effect of selected drugs and immunofluorescence to evaluate the load of DNA damage., Results: We found several differentially expressed genes and we identified topoisomerase IIβ (Top2β) as the gene with highest up-regulation in GSC. Also among the tested cell lines the expression of Top2β was the highest in NCH421k cells, a well-characterized glioblastoma cell line with all the stemness characteristics. On the other hand, Top2β expression markedly decreased upon the induction of differentiation by all trans-retinoic acid. Depletion of Top2β increased the sensitivity of NCH421k cells to replication stress inducing drugs, such as cisplatin, methyl-methanesulfonate, hydrogen peroxide, and temozolomide. Consistently, we found an increased load of DNA damage and increased Chk1 activation upon Top2β depletion in NCH421k cells., Conclusion: We suggest that Top2β may represent a new target for gene therapy in glioblastoma. In addition, the other genes that we found to be up-regulated in GSC versus glioblastoma primary cells should be further investigated as glioblastoma theranostics.

Introduction: Autologous fat grafting is commonly used to correct soft-tissue contour deformities. However, results are impaired by a variable and unpredictable resorption rate. Autologous adipose-derived stromal cells in combination with lipoinjection (cell-assisted lipotransfer) seem to favor a long-term persistence of fat grafts, thus fostering the development of devices to be used in the operating room at the point of care, to isolate the stromal vascular fraction (SVF) and produce SVF-enhanced fat grafts with safe and standardized protocols. Focusing on patients undergoing breast reconstruction by lipostructure, we analyzed a standard technique, a modification of the Coleman's procedure, and three different commercially available devices (Lipokit, Cytori, Fastem), in terms of 1) ability to enrich fat grafts in stem cells and 2) clinical outcome at 6 and 12 months., Methods: To evaluate the ability to enrich stem cells, we compared, for each patient (n=20), the standard lipoaspirate with the respective stem cell-enriched one, analyzing yield, immunophenotype and colony-forming capacity of the SVF cells as well as immunophenotype, clonogenicity and multipotency of the obtained adipose stem cells (ASCs). Regarding the clinical outcome, we compared, by ultrasonography imaging, changes at 6 and 12 months in the subcutaneous thickness of patients treated with stem-cell enriched (n=14) and standard lipoaspirates (n=16)., Results: Both methods relying on the enzymatic isolation of primitive cells led to significant increase in the frequency, in the fat grafts, of SVF cells as well as of clonogenic and multipotent ASCs, while the enrichment was less prominent for the device based on the mechanical isolation of the SVF. From a clinical point of view, patients treated with SVF-enhanced fat grafts demonstrated, at six months, a significant superior gain of thickness of both the central and superior-medial quadrants with respect to patients treated with standard lipotransfer. In the median-median quadrant the effect was still persistent at 12 months, confirming an advantage of lipotransfer technique in enriching improving long-term fat grafts., Conclusions: This comparative study, based on reproducible biological and clinical parameters and endpoints, showed an advantage of lipotransfer technique in enriching fat grafts in stem cells and in favoring, clinically, long-term fat grafts.

Malignant gliomas are among the rarest brain tumours, and they have the worst prognosis. Grade IV astrocytoma, known as glioblastoma multiforme (GBM), is a highly lethal disease where the standard therapies of surgery, followed by radiation and chemotherapy, cannot significantly prolong the life expectancy of the patients. Tumour recurrence shows more aggressive form compared to the primary tumour, and results in patient survival from 12 to 15 months only. Although still controversial, the cancer stem cell hypothesis postulates that cancer stem cells are responsible for early relapse of the disease after surgical intervention due to their high resistance to therapy. Alternative strategies for GBM therapy are thus urgently needed. Nanobodies are single-domain antigen-binding fragments of heavy-chain antibodies, and together with classical antibodies, they are part of the camelid immune system. Nanobodies are small and stable, and they share a high degree of sequence identity to the human heavy chain variable domain, and these characteristics offer them advantages over classical antibodies or antibody fragments. We first immunised an alpaca with a human GBM stem-like cell line prepared from primary GBM cultures. Next, a nanobody library was constructed in a phage-display vector. Using nanobody phage-display technology, we selected specific GBM stem-like cell binders through a number of affinity selections, using whole cell protein extracts and membrane protein-enriched extracts from eight different GBM patients, and membrane protein-enriched extracts from two established GBM stem-like cell lines (NCH644 and NCH421K cells). After the enrichment, periplasmic extract ELISA was used to screen for specific clones. These nanobody clones were recloned into the pHEN6 vector, expressed in Escherichia coli WK6, and purified using immobilised metal affinity chromatography and size-exclusion chromatography. Specific nanobody:antigen pairs were obtained and mass spectrometry analysis revealed two proteins, TRIM28 and β-actin, that were up-regulated in the GBM stem-like cells compared to the controls.

Active cell migration and invasion is a peculiar feature of glioma that makes this tumor able to rapidly infiltrate into the surrounding brain tissue. In our recent work, we identified a novel class of glioma-associated-stem cells (defined as GASC for high-grade glioma--HG--and Gasc for low-grade glioma--LG) that, although not tumorigenic, act supporting the biological aggressiveness of glioma-initiating stem cells (defined as GSC for HG and Gsc for LG) favoring also their motility. Migrating cancer cells undergo considerable molecular and cellular changes by remodeling their cytoskeleton and cell interactions with surrounding environment. To get a better understanding about the role of the glioma-associated-stem cells in tumor progression, cell deformability and interactions between glioma-initiating stem cells and glioma-associated-stem cells were investigated. Adhesion of HG/LG-cancer cells on HG/LG-glioma-associated stem cells was studied by time-lapse microscopy, while cell deformability and cell-cell adhesion strengths were quantified by indentation measurements by atomic force microscopy and single cell force spectroscopy. Our results demonstrate that for both HG and LG glioma, cancer-initiating-stem cells are softer than glioma-associated-stem cells, in agreement with their neoplastic features. The adhesion strength of GSC on GASC appears to be significantly lower than that observed for Gsc on Gasc. Whereas, GSC spread and firmly adhere on Gasc with an adhesion strength increased as compared to that obtained on GASC. These findings highlight that the grade of glioma-associated-stem cells plays an important role in modulating cancer cell adhesion, which could affect glioma cell migration, invasion and thus cancer aggressiveness. Moreover this work provides evidence about the importance of investigating cell adhesion and elasticity for new developments in disease diagnostics and therapeutics.

Tumor associated fibroblasts (TAFs) are considered a microenvironmental element critical for tumor growth and progression. Experimental studies suggest that their origin could be from mesenchymal stem cells (MSCs) derived from the bone marrow. However, the role played by TAFs in cirrhosis, hepatocellular carcinoma development, and progression is largely unknown, and in vitro human models are missing. This paper for the first time demonstrates that (1) human neoplastic livers possess a population of multipotent adult stem cells (MASCs) with properties of TAFs; (2) a population of MASC-derived TAFs is already present in cirrhotic, not yet neoplastic, livers; (3) MASCs isolated from nonneoplastic and noncirrhotic liver scan acquire a TAF phenotype when grown in a medium conditioned by tumor cell lines, supporting the notion that TAF could originate from resident primitive cells (MASCs), possibly through a paracrine mechanism.