Comprehensive tumor molecular profile analysis in clinical practice.

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.

An NF-κB signature predicts low-grade glioma prognosis: a precision medicine approach based on patient-derived stem cells.

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.

Systemic T Cells Immunosuppression of Glioma Stem Cell-Derived Exosomes Is Mediated by Monocytic Myeloid-Derived Suppressor Cells.

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.

Topoisomerase IIß mediates the resistance of glioblastoma stem cells to replication stress-inducing drugs.

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 IIß (Top2ß) as the gene with highest up-regulation in GSC. Also among the tested cell lines the expression of Top2ß was the highest in NCH421k cells, a well-characterized glioblastoma cell line with all the stemness characteristics. On the other hand, Top2ß expression markedly decreased upon the induction of differentiation by all trans-retinoic acid. Depletion of Top2ß 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 Top2ß depletion in NCH421k cells. CONCLUSION: We suggest that Top2ß 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.

Adipose tissue derived stem cells: in vitro and in vivo analysis of a standard and three commercially available cell-assisted lipotransfer techniques.

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.

TRIM28 and ß-actin identified via nanobody-based reverse proteomics approach as possible human glioblastoma biomarkers.

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 ß-actin, that were up-regulated in the GBM stem-like cells compared to the controls.

Glioma-associated stem cells: a novel class of tumor-supporting cells able to predict prognosis of human low-grade gliomas.

BACKGROUND: Translational medicine aims at transferring advances in basic science research into new approaches for diagnosis and treatment of diseases. Low-grade gliomas (LGG) have a heterogeneous clinical behavior that can be only partially predicted employing current state-of-the-art markers, hindering the decision-making process. To deepen our comprehension on tumor heterogeneity, we dissected the mechanism of interaction between tumor cells and relevant components of the neoplastic environment, isolating, from LGG and high-grade gliomas (HGG), proliferating stem cell lines from both the glioma stroma and, where possible, the neoplasm. METHODS AND FINDINGS: We isolated glioma-associated stem cells (GASC) from LGG (n=40) and HGG (n=73). GASC showed stem cell features, anchorage-independent growth, and supported the malignant properties of both A172 cells and human glioma-stem cells, mainly through the release of exosomes. Finally, starting from GASC obtained from HGG (n=13) and LGG (n=12) we defined a score, based on the expression of 9 GASC surface markers, whose prognostic value was assayed on 40 subsequent LGG-patients. At the multivariate Cox analysis, the GASC-based score was the only independent predictor of overall survival and malignant progression free-survival. CONCLUSIONS: The microenvironment of both LGG and HGG hosts non-tumorigenic multipotent stem cells that can increase in vitro the biological aggressiveness of glioma-initiating cells through the release of exosomes. The clinical importance of this finding is supported by the strong prognostic value associated with the characteristics of GASC. This patient-based approach can provide a groundbreaking method to predict prognosis and to exploit novel strategies that target the tumor stroma.

Role of tumor associated fibroblasts in human liver regeneration, cirrhosis, and cancer.

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.