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This review focuses on the immune checkpoint inhibitors (ICIs) in the context of breast cancer (BC) management. These innovative treatments, by targeting proteins expressed on both tumor and immune cells, aim to overcome tumor-induced immune suppression and reactivate the immune system. The potential of this approach is the subject of numerous clinical studies. Here, we explore the key studies and emerging therapies related to ICIs providing a detailed analysis of their specific and combined use in BC treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Checkpoint immunotherapy unleashes tumor control by T cells, but it is undermined in non-immunogenic tumors, e.g. with low MHC class I expression and low neoantigen burden, such as neuroblastoma (NB). Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an enzyme that trims peptides before loading on MHC class I molecules. Inhibition of ERAP1 results in the generation of new antigens able of inducing potent anti-tumor immune responses. Here, we identify a novel non-toxic combinatorial strategy based on genetic inhibition of ERAP1 and administration of the HDAC inhibitor (HDACi) entinostat that increase the immunogenicity of NB, making it responsive to PD-1 therapy., Methods: CRISPR/Cas9-mediated gene editing was used to knockout (KO) the ERAP1 gene in 9464D NB cells derived from spontaneous tumors of TH-MYCN transgenic mice. The expression of MHC class I and PD-L1 was evaluated by flow cytometry (FC). The immunopeptidome of these cells was studied by mass spectrometry. Cocultures of splenocytes derived from 9464D bearing mice and tumor cells allowed the assessment of the effect of ERAP1 inhibition on the secretion of inflammatory cytokines and activation and migration of immune cells towards ERAP1 KO cells by FC. Tumor cell killing was evaluated by Caspase 3/7 assay and flow cytometry analysis. The effect of ERAP1 inhibition on the immune content of tumors was analyzed by FC, immunohistochemistry and multiple immunofluorescence., Results: We found that inhibition of ERAP1 makes 9464D cells more susceptible to immune cell-mediated killing by increasing both the recall and activation of CD4 + and CD8 + T cells and NK cells. Treatment with entinostat induces the expression of MHC class I and PD-L1 molecules in 9464D both in vitro and in vivo. This results in pronounced changes in the immunopeptidome induced by ERAP1 inhibition, but also restrains the growth of ERAP1 KO tumors in vivo by remodelling the tumor-infiltrating T-cell compartment. Interestingly, the absence of ERAP1 in combination with entinostat and PD-1 blockade overcomes resistance to PD-1 immunotherapy and increases host survival., Conclusions: These findings demonstrate that ERAP1 inhibition combined with HDACi entinostat treatment and PD-1 blockade remodels the immune landscape of a non-immunogenic tumor such as NB, making it responsive to checkpoint immunotherapy., (© 2024. The Author(s).)

Natural Killer (NK) cells are integral to the innate immune system, renowned for their ability to target and eliminate cancer cells without the need for antigen presentation, sparing normal tissues. These cells are crucial in cancer immunosurveillance due to their diverse array of activating and inhibitory receptors that modulate their cytotoxic activity. However, the tumor microenvironment can suppress NK cell function through various mechanisms. Over recent decades, research has focused on overcoming these tumor escape mechanisms. Initially, efforts concentrated on enhancing T cell activity, leading to impressive results with immunotherapeutic approaches aimed at boosting T cell responses. Nevertheless, a substantial number of patients do not benefit from these treatments and continue to seek effective alternatives. In this context, NK cells present a promising avenue for developing new treatments, given their potent cytotoxic capabilities, safety profile, and activity against T cell-resistant tumors, such as those lacking HLA-I expression. Recent advancements in immunotherapy include strategies to restore and amplify NK cell activity through immune checkpoint inhibitors, cytokines, adoptive NK cell therapy, and CAR-NK cell technology. This review provides a comprehensive overview of NK cell receptors, the tumor escape mechanisms that hinder NK cell function, and the evolving field of NK cell-based cancer immunotherapy, highlighting ongoing efforts to develop more effective and targeted cancer treatment strategies., Competing Interests: Declaration of competing interest Federica Papaccio: private institutional research funding from Merck, travel support from Diatech Pharmacogenetics, ESMO Translational Research Fellowship sponsored by Amgen from 2018 to 2020. The other authors declare no conflicts of interest., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Natural killer (NK) cells and dendritic cells (DCs) are critical mediators of anti-cancer immune responses. In addition to their individual roles, NK cells and DCs are involved in intercellular crosstalk which is essential for the initiation and coordination of adaptive immunity against cancer. However, NK cell and DC activity is often compromised in the tumor microenvironment (TME). Recently, much attention has been paid to one of the major components of the TME, the cancer-associated fibroblasts (CAFs), which not only contribute to extracellular matrix (ECM) deposition and tumor progression but also suppress immune cell functions. It is now well established that CAFs support T cell exclusion from tumor nests and regulate their cytotoxic activity. In contrast, little is currently known about their interaction with NK cells, and DCs. In this review, we describe the interaction of CAFs with NK cells and DCs, by secreting and expressing various mediators in the TME of adult solid tumors. We also provide a detailed overview of ongoing clinical studies evaluating the targeting of stromal factors alone or in combination with immunotherapy based on immune checkpoint inhibitors. Finally, we discuss currently available strategies for the selective depletion of detrimental CAFs and for a better understanding of their interaction with NK cells and DCs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

An important determinant for oral squamous cell carcinoma (OSCC) onset and outcome is the composition of the tumor microenvironment (TME). Thus, the study of the interactions occurring among cancer cells, immune cells, and cancer-associated fibroblasts within the TME could facilitate the understanding of the mechanisms underlying OSCC development and progression, as well as of its sensitivity or resistance to the therapy. In this context, it must be highlighted that the characterization of TME proteins is enabled by proteomic methodologies, particularly mass spectrometry (MS). Aiming to identify TME protein markers employable for diagnosing and prognosticating OSCC, we have retrieved a total of 119 articles spanning 2001 to 2023, of which 17 have passed the selection process, satisfying all its criteria. We have found a total of 570 proteins detected by MS-based proteomics in the TME of OSCC; among them, 542 are identified by a single study, while 28 are cited by two or more studies. These 28 proteins participate in extracellular matrix remodeling and/or energy metabolism. Here, we propose them as markers that could be used to characterize the TME of OSCC for diagnostic/prognostic purposes. Noteworthy, most of the 28 individuated proteins share one feature: being modulated by the hypoxia that is present in the proliferating OSCC mass.

High levels of vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF)-2 and angiopoietin (ANG)-2 are found in tissues from oral squamous cell carcinoma (OSCC) and oral potentially malignant disorders (OPMDs). As might be expected, VEGF, FGF-2, and ANG-2 overexpression parallels the development of new blood and lymphatic vessels that nourish the growing OPMDs or OSCCs and provide the latter with metastatic routes. Notably, VEGF, FGF-2, and ANG-2 are also linked to the epithelial-to-mesenchymal transition (EMT), a trans-differentiation process that respectively promotes or exasperates the invasiveness of normal and neoplastic oral epithelial cells. Here, we have summarized published work regarding the impact that the interplay among VEGF, FGF-2, ANG-2, vessel generation, and EMT has on oral carcinogenesis. Results from the reviewed studies indicate that VEGF, FGF-2, and ANG-2 spark either protein kinase B (AKT) or mitogen-activated protein kinases (MAPK), two signaling pathways that can promote both EMT and new vessels' formation in OPMDs and OSCCs. Since EMT and vessel generation are key to the onset and progression of OSCC, as well as to its radio- and chemo-resistance, these data encourage including AKT or MAPK inhibitors and/or antiangiogenic drugs in the treatment of this malignancy., Competing Interests: The authors declare no conflicts of interest.

The polyphenols Curcumin (CUR) and Resveratrol (RES) are widely described for their antitumoral effects. However, their low bioavailability is a drawback for their use in therapy. The aim of this study was to explore whether CUR and RES, used at a bioavailable concentration, could modulate immune responses while retaining antitumor activity and to determine whether CUR and RES effects on the immune responses of peripheral blood mononuclear cells (PBMCs) and tumor growth inhibition could be improved by their combination. We demonstrate that the low-dose combination of CUR and RES reduced the survival of cancer cell lines but had no effect on the viability of PBMCs. Although following CUR + RES treatment T lymphocytes showed an enhanced activated state, RES counteracted the increased IFN-γ expression induced by CUR in T cells and the polyphenol combination increased IL-10 production by T regulatory cells. On the other hand, the combined treatment enhanced NK cell activity through the up- and downregulation of activating and inhibitory receptors and increased CD68 expression levels on monocytes/macrophages. Overall, our results indicate that the combination of CUR and RES at low doses differentially shapes immune cells while retaining antitumor activity, support the use of this polyphenol combinations in anticancer therapy and suggest its possible application as adjuvant for NK cell-based immunotherapies.

Neuroblastoma (NB) is a childhood tumor that originates in the peripheral sympathetic nervous system and is responsible for 15% of cancer-related deaths in the pediatric population. Despite intensive multimodal treatment, many patients with high-risk NB relapse and develop a therapy-resistant tumor. One of the phenomena related to therapeutic resistance is intratumor heterogeneity resulting from the adaptation of tumor cells in response to different selective environmental pressures. The transcriptional and epigenetic profiling of NB tissue has recently revealed the existence of two distinct cellular identities in the NB, termed adrenergic (ADRN) and mesenchymal (MES), which can spontaneously interconvert through epigenetic regulation. This phenomenon, known as tumor plasticity, has a major impact on cancer pathogenesis. The aim of this review is to describe the peculiarities of these two cell states, and how their plasticity affects the response to current therapeutic treatments, with special focus on the immunogenic potential of MES cells. Furthermore, we will discuss the opportunity to combine immunotherapy with chemotherapy to counteract NB phenotypic interconversion., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 D’Amico, Tempora, Gragera, Król, Melaiu, De Ioris, Locatelli and Fruci.)

Glioblastoma multiforme (GM) is a malignant tumor of the central nervous system considered to be highly aggressive and often carrying a terrible survival prognosis. An accurate prognosis is therefore pivotal for deciding a good treatment plan for patients. In this context, computational intelligence applied to data of electronic health records (EHRs) of patients diagnosed with this disease can be useful to predict the patients' survival time. In this study, we evaluated different machine learning models to predict survival time in patients suffering from glioblastoma and further investigated which features were the most predictive for survival time. We applied our computational methods to three different independent open datasets of EHRs of patients with glioblastoma: the Shieh dataset of 84 patients, the Berendsen dataset of 647 patients, and the Lammer dataset of 60 patients. Our survival time prediction techniques obtained concordance index (C-index) = 0.583 in the Shieh dataset, C-index = 0.776 in the Berendsen dataset, and C-index = 0.64 in the Lammer dataset, as best results in each dataset. Since the original studies regarding the three datasets analyzed here did not provide insights about the most predictive clinical features for survival time, we investigated the feature importance among these datasets. To this end, we then utilized Random Survival Forests, which is a decision tree-based algorithm able to model non-linear interaction between different features and might be able to better capture the highly complex clinical and genetic status of these patients. Our discoveries can impact clinical practice, aiding clinicians and patients alike to decide which therapy plan is best suited for their unique clinical status., Competing Interests: Conflict of interestThe authors declare no competing interests., (© The Author(s) 2023.)

DNAM-1 is a major NK cell activating receptor and, together with NKG2D and NCRs, by binding specific ligands, strongly contributes to mediating the killing of tumor or virus-infected cells. DNAM-1 specifically recognizes PVR and Nectin-2 ligands that are expressed on some virus-infected cells and on a broad spectrum of tumor cells of both hematological and solid malignancies. So far, while NK cells engineered for different antigen chimeric receptors (CARs) or chimeric NKG2D receptor have been extensively tested in preclinical and clinical studies, the use of DNAM-1 chimeric receptor-engineered NK cells has been proposed only in our recent proof-of-concept study and deserves further development. The aim of this perspective study is to describe the rationale for using this novel tool as a new anti-cancer immunotherapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Cifaldi, Melaiu, Giovannoni, Benvenuto, Focaccetti, Nardozi, Barillari and Bei.)

Immune checkpoint inhibitors (ICIs) have a modest clinical activity when administered as monotherapy against breast cancer (BC), the most common malignancy in women. Novel combinatorial strategies are currently being investigated to overcome resistance to ICIs and promote antitumor immune responses in a greater proportion of BC patients. Recent studies have shown that the BC abnormal vasculature is associated with immune suppression in patients, and hampers both drug delivery and immune effector cell trafficking to tumor nests. Thus, strategies directed at normalizing (i.e., at remodeling and stabilizing) the immature, abnormal tumor vessels are receiving much attention. In particular, the combination of ICIs with tumor vessel normalizing agents is thought to hold great promise for the treatment of BC patients. Indeed, a compelling body of evidence indicates that the addition of low doses of antiangiogenic drugs to ICIs substantially improves antitumor immunity. In this review, we outline the impact that the reciprocal interactions occurring between tumor angiogenesis and immune cells have on the immune evasion and clinical progression of BC. In addition, we overview preclinical and clinical studies that are presently evaluating the therapeutic effectiveness of combining ICIs with antiangiogenic drugs in BC patients.

Oral squamous cell carcinoma (OSCC) is a rapidly progressive cancer that often develops resistance against DNA damage inducers, such as radiotherapy and chemotherapy, which are still the standard of care regimens for this tumor. Thus, the identification of biomarkers capable of monitoring the clinical progression of OSCC and its responsiveness to therapy is strongly required. To meet this need, here we have employed Whole Genome Sequencing and RNA-seq data from a cohort of 316 patients retrieved from the TCGA Pan-Cancer Atlas to analyze the genomic and transcriptomic status of the DNA damage response (DDR) genes in OSCC. Then, we correlated the transcriptomic data with the clinical parameters of each patient. Finally, we relied on transcriptomic and drug sensitivity data from the CTRP v2 portal, performing Pearson's correlation analysis to identify putative vulnerabilities of OSCC cell lines correlated with DDR gene expression. Our results indicate that several DDR genes show a high frequency of genomic and transcriptomic alterations and that the expression of some of them correlates with OSCC grading and infection by the human papilloma virus. In addition, we have identified a signature of eight DDR genes (namely CCNB1 , CCNB2 , CDK2 , CDK4 , CHECK1 , E2F1 , FANCD2 , and PRKDC ) that could be predictive for OSCC response to the novel antitumor compounds sorafenib and tipifarnib-P1. Altogether, our data demonstrate that alterations in DDR genes could have an impact on the biology of OSCC. Moreover, here we propose a DDR gene signature whose expression could be predictive of OSCC responsiveness to therapy.

Background: Poor infiltration of functioning T cells renders tumors unresponsive to checkpoint-blocking immunotherapies. Here, we identified a combinatorial in situ immunomodulation strategy based on the administration of selected immunogenic drugs and immunotherapy to sensitize poorly T-cell-infiltrated neuroblastoma (NB) to the host antitumor immune response., Methods: 975A2 and 9464D NB cell lines derived from spontaneous tumors of TH-MYCN transgenic mice were employed to study drug combinations able of enhancing the antitumor immune response using in vivo and ex vivo approaches. Migration of immune cells towards drug-treated murine-derived organotypic tumor spheroids (MDOTS) were assessed by microfluidic devices. Activation status of immune cells co-cultured with drug-treated MDOTS was evaluated by flow cytometry analysis. The effect of drug treatment on the immune content of subcutaneous or orthotopic tumors was comprehensively analyzed by flow-cytometry, immunohistochemistry and multiplex immunofluorescence. The chemokine array assay was used to detect soluble factors released into the tumor microenvironment. Patient-derived organotypic tumor spheroids (PDOTS) were generated from human NB specimens. Migration and activation status of autologous immune cells to drug-treated PDOTS were performed., Results: We found that treatment with low-doses of mitoxantrone (MTX) recalled immune cells and promoted CD8 + T and NK cell activation in MDOTS when combined with TGFβ and PD-1 blockade. This combined immunotherapy strategy curbed NB growth resulting in the enrichment of a variety of both lymphoid and myeloid immune cells, especially intratumoral dendritic cells (DC) and IFNγ- and granzyme B-expressing CD8 + T cells and NK cells. A concomitant production of inflammatory chemokines involved in remodelling the tumor immune landscape was also detected. Interestingly, this treatment induced immune cell recruitment against PDOTS and activation of CD8 + T cells and NK cells., Conclusions: Combined treatment with low-dose of MTX and anti-TGFβ treatment with PD-1 blockade improves antitumor immunity by remodelling the tumor immune landscape and overcoming the immunosuppressive microenvironment of aggressive NB., (© 2022. The Author(s).)

Background/aim: The Axillary Reverse Mapping technique in breast cancer, was adopted in order to minimize the risk of upper limb lymphedema. Currently, there is only limited evidence available regarding its oncological safety. The aim of this study was to evaluate the presence of upper limb nodes in surgical specimens following axillary lymphadenectomy, and its relative predictive relevance., Patients and Methods: All patients undergoing axillary lymphadenectomy were enrolled in the current prospective observational study. Indocyanine green was injected into the ipsilateral arm, followed by the standard axillary surgical procedure. Subsequently, the surgical specimens were examined in order to identify any resected upper limb nodes., Results: Out of 22 patients, 5 (22.7%) exhibited fluorescent nodes in the surgical specimen. At univariate analysis, these patients presented statistically significant differences in terms of neoadjuvant treatment, estrogen receptor (ER), progesterone receptor (PR), Ki67 index and position of fluorescent lymph nodes (p=0.021, p=0.033, p=0.002, p=0.049 and p=0.001, respectively). At multivariate analysis, neoadjuvant chemotherapy and Ki67 index were associated with the risk of resecting fluorescent nodes during a standard lymphadenectomy (p=0.005 and p=0.018, respectively)., Conclusion: Axillary Reverse Mapping should be individually tailored for patients with advanced axillary breast cancer and those undergoing neoadjuvant treatment. Suspected metastases or upper limb nodes identified in unusual positions must always be resected., (Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Cluster of differentiation (CD)147, also termed extracellular matrix metalloprotease inducer or basigin, is a glycoprotein ubiquitously expressed throughout the human body, the oral cavity included. CD147 actively participates in physiological tissue development or growth and has important roles in reactive processes such as inflammation, immunity, and tissue repair. It is worth noting that deregulated expression and/or activity of CD147 is observed in chronic inflammatory or degenerative diseases, as well as in neoplasms. Among the latter, oral squamous cell carcinoma (OSCC) is characterized by an upregulation of CD147 in both the neoplastic and normal cells constituting the tumor mass. Most interestingly, the expression and/or activity of CD147 gradually increase as healthy oral mucosa becomes inflamed; hyperplastic/dysplastic lesions are then set on, and, eventually, OSCC develops. Based on these findings, here we summarize published studies which evaluate whether CD147 could be employed as a marker to monitor OSCC development and progression. Moreover, we describe CD147-promoted cellular and molecular events which are relevant to oral carcinogenesis, with the aim to provide useful information for assessing whether CD147 may be the target of novel therapeutic approaches directed against OSCC.

Despite the significant clinical advances with the use of immune checkpoint inhibitors (ICIs) in a wide range of cancer patients, response rates to the therapy are variable and do not always result in long-term tumor regression. The development of ICI-resistant disease is one of the pressing issue in clinical oncology, and the identification of new targets and combination therapies is a crucial point to improve response rates and duration. Antigen processing and presentation (APP) pathway is a key element for an efficient response to ICI therapy. Indeed, malignancies that do not express tumor antigens are typically poor infiltrated by T cells and unresponsive to ICIs. Therefore, improving tumor immunogenicity potentially increases the success rate of ICI therapy. In this review, we provide an overview of the key elements of the APP machinery that can be exploited to enhance tumor immunogenicity and increase the efficacy of ICI-based immunotherapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 D’Amico, Tempora, Melaiu, Lucarini, Cifaldi, Locatelli and Fruci.)

Immune checkpoint inhibitors (ICIs) have shown unprecedented benefits in various adult cancers, and this success has prompted the exploration of ICI therapy even in childhood malignances. Although the use of ICIs as individual agents has achieved disappointing response rates, combinational therapies are likely to promise better results. However, only a subset of patients experienced prolonged clinical effects, thus suggesting the need to identify robust bio-markers that predict individual clinical response or resistance to ICI therapy as the main challenge. In this review, we focus on how the use of ICIs in adult cancers can be translated into pediatric malignances. We discuss the physiological mechanism of action of each IC, including PD-1, PD-L1 and CTLA-4 and the new emerging ones, LAG-3, TIM-3, TIGIT, B7-H3, BTLA and IDO-1, and evaluate their prognostic value in both adult and childhood tumors. Furthermore, we offer an overview of preclinical models and clinical trials currently under investigation to improve the effectiveness of cancer immunotherapies in these patients. Finally, we outline the main predictive factors that influence the efficacy of ICIs, in order to lay the basis for the development of a pan-cancer immunogenomic model, able to direct young patients towards more specific immunotherapy., (Copyright © 2020. Published by Elsevier Ltd.)

The endoplasmic reticulum aminopeptidase ERAP1 regulates innate and adaptive immune responses by trimming peptides for presentation by major histocompatibility complex (MHC) class I molecules. Previously, we have shown that genetic or pharmacological inhibition of ERAP1 on murine and human tumor cell lines perturbs the engagement of NK cell inhibitory receptors Ly49C/I and Killer-cell Immunoglobulin-like receptors (KIRs), respectively, by their specific ligands (MHC class I molecules), thus leading to NK cell killing. However, the effect of ERAP1 inhibition in tumor cells was highly variable, suggesting that its efficacy may depend on several factors, including MHC class I typing. To identify MHC class I alleles and KIRs that are more sensitive to ERAP1 depletion, we stably silenced ERAP1 expression in human HLA class I-negative B lymphoblastoid cell line 721.221 (referred to as 221) transfected with a panel of KIR ligands (i.e. HLA-B*51:01, -Cw3, -Cw4 and -Cw7), or HLA-A2 which does not bind any KIR, and tested their ability to induce NK cell degranulation and cytotoxicity. No change in HLA class I surface expression was detected in all 221 transfectant cells after ERAP1 depletion. In contrast, CD107a expression levels were significantly increased on NK cells stimulated with 221-B*51:01 cells lacking ERAP1, particularly in the KIR3DL1-positive NK cell subset. Consistently, genetic or pharmacological inhibition of ERAP1 impaired the recognition of HLA-B*51:01 by the YTS NK cell overexpressing KIR3DL1*001, suggesting that ERAP1 inhibition renders HLA-B*51:01 molecules less eligible for binding to KIR3DL1. Overall, these results identify HLA-B*51:01/KIR3DL1 as one of the most susceptible combinations for ERAP1 inhibition, suggesting that individuals carrying HLA-B*51:01-like antigens may be candidates for immunotherapy based on pharmacological inhibition of ERAP1., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 D’Amico, D’Alicandro, Compagnone, Tempora, Guida, Romania, Lucarini, Melaiu, Falco, Algeri, Pende, Cifaldi and Fruci.)

We introduce here a novel machine learning (ML) framework to address the issue of the quantitative assessment of the immune content in neuroblastoma (NB) specimens. First, the EUNet, a U-Net with an EfficientNet encoder, is trained to detect lymphocytes on tissue digital slides stained with the CD3 T-cell marker. The training set consists of 3782 images extracted from an original collection of 54 whole slide images (WSIs), manually annotated for a total of 73,751 lymphocytes. Resampling strategies, data augmentation, and transfer learning approaches are adopted to warrant reproducibility and to reduce the risk of overfitting and selection bias. Topological data analysis (TDA) is then used to define activation maps from different layers of the neural network at different stages of the training process, described by persistence diagrams (PD) and Betti curves. TDA is further integrated with the uniform manifold approximation and projection (UMAP) dimensionality reduction and the hierarchical density-based spatial clustering of applications with noise (HDBSCAN) algorithm for clustering, by the deep features, the relevant subgroups and structures, across different levels of the neural network. Finally, the recent TwoNN approach is leveraged to study the variation of the intrinsic dimensionality of the U-Net model. As the main task, the proposed pipeline is employed to evaluate the density of lymphocytes over the whole tissue area of the WSIs. The model achieves good results with mean absolute error 3.1 on test set, showing significant agreement between densities estimated by our EUNet model and by trained pathologists, thus indicating the potentialities of a promising new strategy in the quantification of the immune content in NB specimens. Moreover, the UMAP algorithm unveiled interesting patterns compatible with pathological characteristics, also highlighting novel insights into the dynamics of the intrinsic dataset dimensionality at different stages of the training process. All the experiments were run on the Microsoft Azure cloud platform.

Objectives Malignant pleural mesothelioma (MPM) is an occupational disease mainly due to asbestos exposure. Effective therapies for MPM are lacking, making this tumour type a fatal disease. Materials and Methods In order to meet this need and in view of a future "drug repositioning" approach, here we screened five MPM (Mero-14, Mero-25, IST-Mes2, NCI-H28 and MSTO-211H) and one SV40-immortalized mesothelial cell line (MeT-5A) as a non-malignant model, with a library of 1170 FDA-approved drugs. Results Among several potential compounds, we found that fludarabine (F-araA) and, to a lesser extent, risedronic acid (RIS) were cytotoxic in MPM cells, in comparison to the non-malignant Met-5A cells. In particular, F-araA reduced the proliferation and the colony formation ability of the MPM malignant cells, in comparison to the non-malignant control cells, as demonstrated by proliferation and colony formation assays, in addition to measurement of the phospho-ERK/total-ERK ratio. We have shown that the response to F-araA was not dependent upon the expression of DCK and NT5E enzymes, nor upon their functional polymorphisms (rs11544786 and rs2295890, respectively). Conclusion This drug repositioning screening approach has identified that F-araA could be therapeutically active against MPM cells, in addition to other tumour types, by inhibiting STAT1 expression and nucleic acids synthesis. Further experiments are required to fully investigate this.

We evaluated the association between germline genetic variants located within the 3'-untranlsated region (polymorphic 3'UTR, ie, p3UTR) of candidate genes involved in multiple myeloma (MM). We performed a case-control study within the International Multiple Myeloma rESEarch (IMMEnSE) consortium, consisting of 3056 MM patients and 1960 controls recruited from eight countries. We selected p3UTR of six genes known to act in different pathways relevant in MM pathogenesis, namely KRAS (rs12587 and rs7973623), VEGFA (rs10434), SPP1 (rs1126772), IRF4 (rs12211228) and IL10 (rs3024496). We found that IL10-rs3024496 was associated with increased risk of developing MM and with a worse overall survival of MM patients. The variant allele was assayed in a vector expressing eGFP chimerized with the IL10 3'-UTR and it was found functionally active following transfection in human myeloma cells. In this experiment, the A-allele caused a lower expression of the reporter gene and this was also in agreement with the in vivo expression of mRNA measured in whole blood as reported in the GTEx portal. Overall, these data are suggestive of an effect of the IL10-rs3024496 SNP on the regulation of IL10 mRNA expression and it could have clinical implications for better characterization of MM patients in terms of prognosis., (© 2020 Union for International Cancer Control.)

Malignant pleural mesothelioma (MPM) is a fatal tumor lacking effective therapies. The characterization of overexpressed genes could constitute a strategy for identifying drivers of tumor progression as targets for novel therapies. Thus, we performed an integrated gene-expression analysis on RNAseq data of 85 MPM patients from TCGA dataset and reference samples from the GEO. The gene list was further refined by using published studies, a functional enrichment analysis, and the correlation between expression and patients' overall survival. Three molecular signatures defined by 15 genes were detected. Seven genes were involved in cell adhesion and extracellular matrix organization, with the others in control of the mitotic cell division or apoptosis inhibition. Using Western blot analyses, we found that ADAMTS1, PODXL, CIT, KIF23, MAD2L1, TNNT1, and TRAF2 were overexpressed in a limited number of cell lines. On the other hand, interestingly, CTHRC1, E-selectin, SPARC, UHRF1, PRSS23, BAG2, and MDK were abundantly expressed in over 50% of the six MPM cell lines analyzed. Thus, these proteins are candidates as drivers for sustaining the tumorigenic process. More studies with small-molecule inhibitors or silencing RNAs are fully justified and need to be undertaken to better evaluate the cancer-driving role of the targets herewith identified.

Patients with coronavirus disease 2019 (COVID-19) have a wide variety of clinical outcomes ranging from asymptomatic to severe respiratory syndrome that can progress to life-threatening lung lesions. The identification of prognostic factors can help to improve the risk stratification of patients by promptly defining for each the most effective therapy to resolve the disease. The etiological agent causing COVID-19 is a new coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that enters cells via the ACE2 receptor. SARS-CoV-2 infection causes a reduction in ACE2 levels, leading to an imbalance in the renin-angiotensin system (RAS), and consequently, in blood pressure and systemic vascular resistance. ERAP1 and ERAP2 are two RAS regulators and key components of MHC class I antigen processing. Their polymorphisms have been associated with autoimmune and inflammatory conditions, hypertension, and cancer. Based on their involvement in the RAS, we believe that the dysfunctional status of ERAP1 and ERAP2 enzymes may exacerbate the effect of SARS-CoV-2 infection, aggravating the symptomatology and clinical outcome of the disease. In this review, we discuss this hypothesis.

In this study, we explored whether Nutlin-3a, a well-known, nontoxic small-molecule compound antagonizing the inhibitory interaction of MDM2 with the tumor suppressor p53, may restore ligands for natural killer (NK) cell-activating receptors (NK-AR) on neuroblastoma cells to enhance the NK cell-mediated killing. Neuroblastoma cell lines were treated with Nutlin-3a, and the expression of ligands for NKG2D and DNAM-1 NK-ARs and the neuroblastoma susceptibility to NK cells were evaluated. Adoptive transfer of human NK cells in a xenograft neuroblastoma-bearing NSG murine model was assessed. Two data sets of neuroblastoma patients were explored to correlate p53 expression with ligand expression. Luciferase assays and chromatin immunoprecipitation analysis of p53 functional binding on PVR promoter were performed. Primary neuroblastoma cells were also treated with Nutlin-3a, and neuroblastoma spheroids obtained from one high-risk patient were assayed for NK-cell cytotoxicity. We provide evidence showing that the Nutlin-3a-dependent rescue of p53 function in neuroblastoma cells resulted in (i) increased surface expression of ligands for NK-ARs, thus rendering neuroblastoma cell lines significantly more susceptible to NK cell-mediated killing; (ii) shrinkage of human neuroblastoma tumor masses that correlated with overall survival upon adoptive transfer of NK cells in neuroblastoma-bearing mice; (iii) and increased expression of ligands in primary neuroblastoma cells and boosting of NK cell-mediated disaggregation of neuroblastoma spheroids. We also found that p53 was a direct transcription factor regulating the expression of PVR ligand recognized by DNAM-1. Our findings demonstrated an immunomodulatory role of Nutlin-3a, which might be prospectively used for a novel NK cell-based immunotherapy for neuroblastoma., (©2020 American Association for Cancer Research.)

Tumor-infiltrating CD8 + T cells have been shown to play a crucial role in controlling tumor progression. However, the recruitment and activation of these immune cells at the tumor site are strictly dependent on several factors, including the presence of dendritic cells (DCs), the main orchestrators of the antitumor immune responses. Among the various DC subsets, the role of cDC1s has been demonstrated in several preclinical experimental mouse models. In addition, the high density of tumor-infiltrating cDC1s has been associated with improved survival in many cancer patients. The ability of cDC1s to modulate antitumor activity depends on their interaction with other immune populations, such as NK cells. This evidence has led to the development of new strategies aimed at increasing the abundance and activity of cDC1s in tumors, thus providing attractive new avenues to enhance antitumor immunity for both established and novel anticancer immunotherapies. In this review, we provide an overview of the various subsets of DCs, focusing in particular on the role of cDC1s, their ability to interact with other intratumoral immune cells, and their prognostic significance on solid tumors. Finally, we outline key therapeutic strategies that promote the immunogenic functions of DCs in cancer immunotherapy., Competing Interests: The authors declare no conflict of interest.

Tumor-infiltrating lymphocytes play an essential role in improving clinical outcome of neuroblastoma (NB) patients, but their relationship with other tumor-infiltrating immune cells in the T cell-inflamed tumors remains poorly investigated. Here we show that dendritic cells (DCs) and natural killer (NK) cells are positively correlated with T-cell infiltration in human NB, both at transcriptional and protein levels, and associate with a favorable prognosis. Multiplex imaging displays DC/NK/T cell conjugates in the tumor microenvironment of low-risk NB. Remarkably, this connection is further strengthened by the identification of gene signatures related to DCs and NK cells able to predict survival of NB patients and strongly correlate with the expression of PD-1 and PD-L1. In summary, our findings unveil a key prognostic role of DCs and NK cells and indicate their related gene signatures as promising tools for the identification of clinical biomarkers to better define risk stratification and survival of NB patients.

Background: Sporadic medullary thyroid carcinoma (sMTC) is an uncommon neoplasia arising from the calcitonin-producing parafollicular cells of the thyroid. Previous studies evaluated whether single nucleotide polymorphisms (SNPs) within RET (a pivotal proto-oncogene for this disease) are associated with the risk for developing sMTC, but the results are inconclusive. Methods: In this work, we evaluated the association of RET -SNPs c.74-126G>T (rs2565206), p.Gly691Ser (rs1799939, G>A), p.Leu769 = (rs1800861, G>T), p.Ser836 = (rs1800862, C>T), and p.Ser904 = (rs1800863, C>G) (listed in the order of their chromosomal location) with sMTC. This is one of the largest case - control association studies carried out on sMTC, including 585 sMTC cases (negative for germline mutations within RET ), 1529 patients affected by sporadic nonmedullary thyroid carcinoma (sNMTC), and 989 healthy controls, from central and southern Italy and collected in the period 2000-2017. Results: sNMTC patients showed similar genotype and allele frequencies compared with healthy controls. On the other hand, among sMTC patients, the T-allele of p.Leu769 = was less frequent (OR = 0.70 [CI 0.58-0.84], p  = 1.9 × 10 -4 ) and rare homozygotes TT showed an OR = 0.32 ([CI 0.17-0.60], p  = 2.3 × 10 -4 ). Moreover, a statistically significant excess of the haplotype 2 (characterized by the alleles T-G-G-C-C of the listed SNPs) was observed ( p  = 3.9 × 10 -3 ). The SNPs were not associated with the expression of RET mRNA, that is, they did not exert an effect in cis as quantitative trait locus (cis-eQTL). However, a strong eQTL association was found for p.Leu769 = and the neighboring gene CSGALNACT2 ( p  = 9.3 × 10 -50 ; effect-size = -0.65), whose function in cancer is unknown. Conclusions: This study shows that specific RET haplotypes, in particular haplotype 2 and the T-allele of p.Leu769 = , are associated with a reduced risk of sMTC in Italians.

In our study, we investigated the role of CD39 on tumor-infiltrating CD8 + T lymphocytes (CD8 + TILs) in colorectal, head and neck and pancreatic cancers. Partially confirming recent observations correlating the CD39 expression with T-cell exhaustion, we demonstrated a divergent functional activity in CD39 + CD8 + TILs. On the one hand, CD39 + CD8 + TILs (as compared to their CD39 - counterparts) produced significantly lower IFN-γ and IL-2 amounts, expressed higher PD-1, and inversely correlated with perforin and granzyme B expression. On the other, they displayed a significantly higher proliferative capacity ex vivo that was inversely correlated with the PD-1 expression. Therefore, CD39 + CD8 + TILs, including those co-expressing the CD103 (a marker of T resident memory [TRM] cells), were defined as partially dysfunctional T cells that correlate with tumor patients with initial progression stages. Interestingly, our results identified for the first time a single nucleotide polymorphism (SNP rs10748643 A>G), as a genetic factor associated with CD39 expression in CD8 + TILs. Finally, we demonstrated that compounds inhibiting CD39-related ATPases improved CD39 + CD8 + T-cell effector function ex vivo, and that CD39 + CD8 + TILs displayed effective suppression function in vitro. Overall these data suggest that the SNP analysis may represent a suitable predictor of CD39 + CD8 + T-cell expression in cancer patients, and propose the modulation of CD39 as a new strategy to restore partially exhausted CD8 + TILs., (© 2020 UICC.)