Identification of a set of genes potentially responsible for resistance to ferroptosis in lung adenocarcinoma cancer stem cells.

Scientific literature supports the evidence that cancer stem cells (CSCs) retain inside low reactive oxygen species (ROS) levels and are, therefore, less susceptible to cell death, including ferroptosis, a type of cell death dependent on iron-driven lipid peroxidation. A collection of lung adenocarcinoma (LUAD) primary cell lines derived from malignant pleural effusions (MPEs) of patients was used to obtain 3D spheroids enriched for stem-like properties. We observed that the ferroptosis inducer RSL3 triggered lipid peroxidation and cell death in LUAD cells when grown in 2D conditions; however, when grown in 3D conditions, all cell lines underwent a phenotypic switch, exhibiting substantial resistance to RSL3 and, therefore, protection against ferroptotic cell death. Interestingly, this phenomenon was reversed by disrupting 3D cells and growing them back in adherence, supporting the idea of CSCs plasticity, which holds that cancer cells have the dynamic ability to transition between a CSC state and a non-CSC state. Molecular analyses showed that ferroptosis resistance in 3D spheroids correlated with an increased expression of antioxidant genes and high levels of proteins involved in iron storage and export, indicating protection against oxidative stress and low availability of iron for the initiation of ferroptosis. Moreover, transcriptomic analyses highlighted a novel subset of genes commonly modulated in 3D spheroids and potentially capable of driving ferroptosis protection in LUAD-CSCs, thus allowing to better understand the mechanisms of CSC-mediated drug resistance in tumors.

In-silico guided chemical exploration of KDM4A fragments hits.

BACKGROUND: Lysine demethylase enzymes (KDMs) are an emerging class of therapeutic targets, that catalyse the removal of methyl marks from histone lysine residues regulating chromatin structure and gene expression. KDM4A isoform plays an important role in the epigenetic dysregulation in various cancers and is linked to aggressive disease and poor clinical outcomes. Despite several efforts, the KDM4 family lacks successful specific molecular inhibitors. RESULTS: Herein, starting from a structure-based fragments virtual screening campaign we developed a synergic framework as a guide to rationally design efficient KDM4A inhibitors. Commercial libraries were used to create a fragments collection and perform a virtual screening campaign combining docking and pharmacophore approaches. The most promising compounds were tested in-vitro by a Homogeneous Time-Resolved Fluorescence-based assay developed for identifying selective substrate-competitive inhibitors by means of inhibition of H3K9me3 peptide demethylation. 2-(methylcarbamoyl)isonicotinic acid was identified as a preliminary active fragment, displaying inhibition of KDM4A enzymatic activity. Its chemical exploration was deeply investigated by computational and experimental approaches which allowed a rational fragment growing process. The in-silico studies guided the development of derivatives designed as expansion of the primary fragment hit and provided further knowledge on the structure-activity relationship. CONCLUSIONS: Our study describes useful insights into key ligand-KDM4A protein interaction and provides structural features for the development of successful selective KDM4A inhibitors.

Dynamics of humoral and cellular response to three doses of anti-SARS-CoV-2 BNT162b2 vaccine in patients with hematological malignancies and older subjects.

Background: Few data are available about the durability of the response, the induction of neutralizing antibodies, and the cellular response upon the third dose of the anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine in hemato-oncological patients. Objective: To investigate the antibody and cellular response to the BNT162b2 vaccine in patients with hematological malignancy. Methods: We measured SARS-CoV-2 anti-spike antibodies, anti-Omicron neutralizing antibodies, and T-cell responses 1 month after the third dose of vaccine in 93 fragile patients with hematological malignancy (FHM), 51 fragile not oncological subjects (FNO) aged 80-92, and 47 employees of the hospital (healthcare workers, (HW), aged 23-66 years. Blood samples were collected at day 0 (T0), 21 (T1), 35 (T2), 84 (T3), 168 (T4), 351 (T pre-3D), and 381 (T post-3D) after the first dose of vaccine. Serum IgG antibodies against S1/S2 antigens of SARS-CoV-2 spike protein were measured at every time point. Neutralizing antibodies were measured at T2, T3 (anti-Alpha), T4 (anti-Delta), and T post-3D (anti-Omicron). T cell response was assessed at T post-3D. Results: An increase in anti-S1/S2 antigen antibodies compared to T0 was observed in the three groups at T post-3D. After the third vaccine dose, the median antibody level of FHM subjects was higher than after the second dose and above the putative protection threshold, although lower than in the other groups. The neutralizing activity of antibodies against the Omicron variant of the virus was tested at T2 and T post-3D. 42.3% of FHM, 80,0% of FNO, and 90,0% of HW had anti-Omicron neutralizing antibodies at T post-3D. To get more insight into the breadth of antibody responses, we analyzed neutralizing capacity against BA.4/BA.5, BF.7, BQ.1, XBB.1.5 since also for the Omicron variants, different mutations have been reported especially for the spike protein. The memory T-cell response was lower in FHM than in FNO and HW cohorts. Data on breakthrough infections and deaths suggested that the positivity threshold of the test is protective after the third dose of the vaccine in all cohorts. Conclusion: FHM have a relevant response to the BNT162b2 vaccine, with increasing antibody levels after the third dose coupled with, although low, a T-cell response. FHM need repeated vaccine doses to attain a protective immunological response.

Identification of a miRNA-based non-invasive predictive biomarker of response to target therapy in BRAF-mutant melanoma.

Rationale: Metastatic melanoma is the most aggressive and dangerous form of skin cancer. The introduction of immunotherapy with Immune checkpoint Inhibitors (ICI) and of targeted therapy with BRAF and MEK inhibitors for BRAF mutated melanoma, has greatly improved the clinical outcome of these patients. Nevertheless, response to therapy remains highly variable and the development of drug resistance continues to be a daunting challenge. Within this context there is a need to develop diagnostic tools capable of predicting response or resistance to therapy in order to select the best therapeutic approach. Over the years, accumulating evidence brought to light the role of microRNAs (miRNAs) as disease biomarkers. Methods: In particular, the detection of miRNAs in whole blood or specific blood components such as serum or plasma, allows these molecules to be good candidates for diagnosis, prognosis and for monitoring response to anticancer therapy. In this paper, we evaluated circulating basal levels of 6 previously identified miRNAs in serum samples of 70 BRAF-mutant melanoma patients before starting targeted therapy. Results: Results show that the circulating levels of the oncosuppressor miR-579-3p and of the oncomiR miR-4488 are able to predict progression free survival (PFS) but not overall survival (OS). Most importantly, we observed that the best predictor of disease outcome is represented by the ratio of circulating miR-4488 vs. miR-579-3p (miRatio). Finally, the combination of the Lactate dehydrogenase (LDH) blood levels with the two circulating miRNAs alone or together did not produce any improvement in predicting PFS indicating that miR-579-3p and miR-4488 are independent predictors of PFS as compared to LDH. Conclusions: All together these data underscored the relevance of circulating miRNAs as suitable tools to predict therapy response in melanoma and maybe further developed as companion diagnostics in the clinic.

Peptidomimetics: An Overview of Recent Medicinal Chemistry Efforts toward the Discovery of Novel Small Molecule Inhibitors.

The use of peptides as therapeutics has often been associated with several drawbacks such as poor absorption, low stability to proteolytic digestion, and fast clearance. Peptidomimetics are developed by modifications of native peptides with the aim of obtaining molecules that are more suitable for clinical development and, for this reason, are widely used as tools in medicinal chemistry programs. The effort to disclose innovative peptidomimetic therapies is recurrent and constantly evolving as demonstrated by the new lead compounds in clinical trials. Synthetic strategies for the development of peptidomimetics have also been implemented with time. This perspective highlights some of the most recent efforts for the design and synthesis of peptidomimetic agents together with their biological evaluation toward a panel of targets.

Deconvolution of malignant pleural effusions immune landscape unravels a novel macrophage signature associated with worse clinical outcome in lung adenocarcinoma patients.

BACKGROUND: Immune checkpoint inhibitors are still unable to provide clinical benefit to the large majority of non-small cell lung cancer (NSCLC) patients. A deeper characterization of the tumor immune microenvironment (TIME) is expected to shed light on the mechanisms of cancer immune evasion and resistance to immunotherapy. Here, we exploited malignant pleural effusions (MPEs) from lung adenocarcinoma (LUAD) patients as a model system to decipher TIME in metastatic NSCLC. METHODS: Mononuclear cells from MPEs (PEMC) and peripheral blood (PBMC), cell free pleural fluid and/or plasma were collected from a total of 24 LUAD patients and 12 healthy donors. Bulk-RNA sequencing was performed on total RNA extracted from PEMC and matched PBMC. The DEseq2 Bioconductor package was used to perform differential expression analysis and CIBERSORTx for the regression-based immune deconvolution of bulk gene expression data. Cytokinome analysis of cell-free pleural fluid and plasma samples was performed using a 48-Plex Assay panel. THP-1 monocytic cells were used to assess macrophage polarization. Survival analyses on NSCLC patients were performed using KM Plotter (LUAD, N=672; lung squamous cell carcinoma, N=271). RESULTS: Transcriptomic analysis of immune cells and cytokinome analysis of soluble factors in the pleural fluid depicted MPEs as a metastatic niche in which all the components required for an effective antitumor response are present, but conscripted in a wound-healing, proinflammatory and tumor-supportive mode. The bioinformatic deconvolution analysis revealed an immune landscape dominated by myeloid subsets with the prevalence of monocytes, protumoral macrophages and activated mast cells. Focusing on macrophages we identified an MPEs-distinctive signature associated with worse clinical outcome in LUAD patients. CONCLUSIONS: Our study reports for the first time a wide characterization of MPEs LUAD microenvironment, highlighting the importance of specific components of the myeloid compartment and opens new perspectives for the rational design of new therapies for metastatic NSCLC.

Multi-omic approach identifies a transcriptional network coupling innate immune response to proliferation in the blood of COVID-19 cancer patients.

Clinical outcomes of COVID-19 patients are worsened by the presence of co-morbidities, especially cancer leading to elevated mortality rates. SARS-CoV-2 infection is known to alter immune system homeostasis. Whether cancer patients developing COVID-19 present alterations of immune functions which might contribute to worse outcomes have so far been poorly investigated. We conducted a multi-omic analysis of immunological parameters in peripheral blood mononuclear cells (PBMCs) of COVID-19 patients with and without cancer. Healthy donors and SARS-CoV-2-negative cancer patients were also included as controls. At the infection peak, cytokine multiplex analysis of blood samples, cytometry by time of flight (CyTOF) cell population analyses, and Nanostring gene expression using Pancancer array on PBMCs were performed. We found that eight pro-inflammatory factors (IL-6, IL-8, IL-13, IL-1ra, MIP-1a, IP-10) out of 27 analyzed serum cytokines were modulated in COVID-19 patients irrespective of cancer status. Diverse subpopulations of T lymphocytes such as CD8+T, CD4+T central memory, Mucosal-associated invariant T (MAIT), natural killer (NK), and γδ T cells were reduced, while B plasmablasts were expanded in COVID-19 cancer patients. Our findings illustrate a repertoire of aberrant alterations of gene expression in circulating immune cells of COVID-19 cancer patients. A 19-gene expression signature of PBMCs is able to discriminate COVID-19 patients with and without solid cancers. Gene set enrichment analysis highlights an increased gene expression linked to Interferon α, γ, α/ß response and signaling which paired with aberrant cell cycle regulation in cancer patients. Ten out of the 19 genes, validated in a real-world consecutive cohort, were specific of COVID-19 cancer patients independently from different cancer types and stages of the diseases, and useful to stratify patients in a COVID-19 disease severity-manner. We also unveil a transcriptional network involving gene regulators of both inflammation response and proliferation in PBMCs of COVID-19 cancer patients.

Antibody Persistence 6 Months Post-Vaccination with BNT162b2 among Health Care Workers.

BACKGROUND: We present immunogenicity data 6 months after the first dose of BNT162b2 in correlation with age, gender, BMI, comorbidities and previous SARS-CoV-2 infection. METHODS: An immunogenicity evaluation was carried out among health care workers (HCW) vaccinated at the Istituti Fisioterapici Ospitalieri (IFO). All HCW were asked to be vaccine by the national vaccine campaign at the beginning of 2021. Serum samples were collected on day 1 just prior to the first dose of the vaccine and on day 21 just prior to the second vaccination dose. Thereafter sera samples were collected 28, 49, 84 and 168 days after the first dose of BNT162b2. Quantitative measurement of IgG antibodies against S1/S2 antigens of SARS-CoV-2 was performed with a commercial chemiluminescent immunoassay. RESULTS: Two hundred seventy-four HWCs were analyzed, 175 women (63.9%) and 99 men (36.1%). The maximum antibody geometric mean concentration (AbGMC) was reached at T2 (299.89 AU/mL; 95% CI: 263.53-339.52) with a significant increase compared to baseline (p < 0.0001). Thereafter, a progressive decrease was observed. At T5, a median decrease of 59.6% in COVID-19 negative, and of 67.8% in COVID-19 positive individuals were identified with respect to the highest antibody response. At T1, age and previous COVID-19 were associated with differences in antibody response, while at T2 and T3 differences in immune response were associated with age, gender and previous COVID-19. At T4 and T5, only COVID-19 positive participants demonstrated a greater antibody response, whereas no other variables seemed to influence antibody levels. CONCLUSIONS: Overall our study clearly shows antibody persistence at 6 months, albeit with a certain decline. Thus, the use of this vaccine in addressing the COVID-19 pandemic is supported by our results that in turn open debate about the need for further boosts.

Early Onset of SARS-COV-2 Antibodies after First Dose of BNT162b2: Correlation with Age, Gender and BMI.

BACKGROUND: The first goal of the study was to analyse the antibody titre 21 days after the first dose of the BNT162b2 vaccine in a group of 252 healthcare workers (HCW). The second goal was to analyse how the antibody titre changes in correlation with age, gender and body mass index (BMI). METHODS: Participants had a nasopharyngeal swab for SARS-CoV-2 and were assessed for the presence of SARS-CoV-2 antibodies at baseline and 21 days after the BNT162b2 priming dose. RESULTS: First dose of BNT162b2 activated immune responses in 98% of the participants. Five HWC had no increase in antibody titre 21 days after the first dose. Antibody titre was greater in young (<38 years) vs. older participants (<38 vs. 47-56 p = 0.002; <38 vs. >56 p = 0.001). Higher antibody levels were detected in underweight vs. pre-obesity group (p = 0.026) and in normal-weight vs. pre-obesity group (p = 0.007). This association was confirmed after adjusting for age (p = 0.0001) and gender (p = 0.00001). CONCLUSIONS: Our study demonstrates that a single dose of BNT162b2 activates the immune response, and being young and normal-weight correlate positively with this response. Larger specifically designed clinical trials are needed to validate these results.

Basic principles of biobanking: from biological samples to precision medicine for patients.

The term "biobanking" is often misapplied to any collection of human biological materials (biospecimens) regardless of requirements related to ethical and legal issues or the standardization of different processes involved in tissue collection. A proper definition of biobanks is large collections of biospecimens linked to relevant personal and health information (health records, family history, lifestyle, genetic information) that are held predominantly for use in health and medical research. In addition, the International Organization for Standardization, in illustrating the requirements for biobanking (ISO 20387:2018), stresses the concept of biobanks being legal entities driving the process of acquisition and storage together with some or all of the activities related to collection, preparation, preservation, testing, analysing and distributing defined biological material as well as related information and data. In this review article, we aim to discuss the basic principles of biobanking, spanning from definitions to classification systems, standardization processes and documents, sustainability and ethical and legal requirements. We also deal with emerging specimens that are currently being generated and shaping the so-called next-generation biobanking, and we provide pragmatic examples of cancer-associated biobanking by discussing the process behind the construction of a biobank and the infrastructures supporting the implementation of biobanking in scientific research.

Lower response to BNT162b2 vaccine in patients with myelofibrosis compared to polycythemia vera and essential thrombocythemia.

In a population of 42 Philadelphia negative myeloproliferative neoplasm patients, all on systemic active treatment, the likelihood of responding to anti-SARS-CoV-2 BNT162b2 vaccine at 2 weeks after the second dose was significantly lower in the ten patients with myelofibrosis compared to the 32 with essential thrombocythemia (n = 17) and polycythemia vera (n = 15) grouped together, both in terms of neutralizing anti-SARS-CoV-2 IgG titers and seroprotection rates (32.47 AU/mL vs 217.97 AU/mL, p = 0.003 and 60% vs 93.8%, p = 0.021, respectively). Ruxolitinib, which was the ongoing treatment in five patients with myelofibrosis and three with polycythemia vera, may be implicated in reducing vaccine immunogenicity (p = 0.076), though large prospective study is needed to address this issue.

Initial observations on age, gender, BMI and hypertension in antibody responses to SARS-CoV-2 BNT162b2 vaccine.

BACKGROUND: Literature data suggests that age, gender and body mass index (BMI) could be associated with difference in immune responses to vaccines. The first goal of the study was to analyze the antibody titre seven days after the second dose of BNT162b2 vaccine in a group of 248 healthcare workers (HCWs). The second goal was to analyze how antibody titre changes in correlation with age, gender, BMI and hypertension. METHODS: An immunogenicity evaluation was carried out among HCWs vaccinated at the Istituti Fisioterapici Ospitalieri (IFO), Rome, Italy. All HCWs were asked to be vaccinated by the Italian national vaccine campaign at the beginning of 2021. 260 vaccinated HCWs were enrolled in the study. All eligible participants were assigned to receive the priming dose in two weeks' time and the booster dose exactly 21 days thereafter. Blood and nasopharyngeal swabs were collected at baseline and 7 days after second dose of vaccine. Quantitative measurements of IgG antibodies against S1/S2 antigens of SARS-CoV-2 were performed with a commercial chemiluminescent immunoassay. Presence of SARS-Cov-2 in nasopharyngeal swab was determined by commercial RT-PCR testing. FINDINGS: 248 HWCs were analyzed, 158 women (63.7%) and 90 men (36.3%). After the second dose of BNT162b2 vaccine, 99.5% of participants developed a humoral immune response. The geometric mean concentration of antibodies among the vaccinated subjects after booster dose (285.9 AU/mL 95% CI: 249.5-327.7) was higher than that of human convalescent sera (39.4 AU/mL, 95% CI: 33.1-46.9), with p<0.0001. Multivariate linear regression analysis of AU/mL by age, gender and BMI multivariate was performed by the inclusion of covariates. This analysis demonstrated that age (p<0.0001) and gender (p = 0.038) are statistically associated with differences in antibody response after vaccination, whereas BMI and hypertension have no statistically significant association (p = 0.078 and p = 0.52 respectively). INTERPRETATION: 99.5% of HCW developed a humoral immune response and female and young participants seem to have an increased capacity to mount humoral immune responses. BMI and hypertension seem not associated with difference in immune response to the vaccine. FUNDING: None.

Fifth-week immunogenicity and safety of anti-SARS-CoV-2 BNT162b2 vaccine in patients with multiple myeloma and myeloproliferative malignancies on active treatment: preliminary data from a single institution.

BACKGROUND: Safety and immunogenicity of BNT162b2 mRNA vaccine are unknown in hematological patients; both were evaluated prospectively in 42 patients with multiple myeloma (MM) and 50 with myeloproliferative malignancies (MPM) (20 chronic myeloid leukemias and 30 myeloproliferative neoplasms), all of them on active anti-cancer treatment, in comparison with 36 elderly controls not suffering from cancer. Subjects serologically and/or molecularly (by nasal/throat swab) positives at basal for SARS-CoV-2 were excluded. Primary endpoint was to compare titers of neutralizing anti-SARS-CoV-2 IgG and seroprotection rates among the cohorts at 3 and 5 weeks from first dose. METHODS: Titration was done using LIAISON® SARS-CoV-2 S1/S2 IgG test, a quantitative chemiluminescent immunoassay approved by FDA on the basis of robust evidences of concordance (94.4%) between the test at cutoff of 15 AU/mL and the Plaque Reduction Neutralization Test 90% at 1:40 ratio. Cutoff of 15 AU/mL was assumed to discriminate responders to vaccination with a protective titer. Cohorts were compared using Fisher' exact test and the Mann-Whitney test as appropriated. Geometric mean concentrations (GMCs), geometric mean ratios and response rates after 1st and 2nd dose were compared in each cohort by Wilcoxon and McNemar tests, respectively. RESULTS: At 5 weeks, GMC of IgG in elderly controls was 353.3 AU/mL versus 106.7 in MM (p = 0.003) and 172.9 in MPM patients (p = 0.049). Seroprotection rate at cutoff of 15 AU/mL was 100% in controls compared to 78.6% in MM (p = 0.003) and 88% in MPM patients (p = 0.038). In terms of logarithm of IgG titer, in a generalized multivariate linear model, no gender effect was observed (p = 0.913), while there was a significant trend toward lower titers by increasing age (p < 0.001) and in disease cohorts with respect to controls (MM: p < 0.001 and MPM: p < 0.001). An ongoing treatment without daratumumab was associated with higher likelihood of response in MM patients (p = 0.003). No swabs resulted positive on each time point. No safety concerns were observed. CONCLUSIONS: BNT162b2 has demonstrated to be immunogenic at different extent among the cohorts. Response was 88% and robust in MPM patients. MM patients responded significantly less, particularly those on anti-CD38-based treatment. These latter patients should be advised to maintain masks and social distancing regardless of vaccination status, and their cohabiting family members need to be vaccinated in order to reduce the risk of contagion from the family. Additional boosters and titer monitoring could be considered. Trial registration Study was formally approved by the IRCCS Central Ethical Committee of Regione Lazio in January 2021 (Prot. N-1463/21).

A 17-Gene Expression Signature for Early Identification of Poor Prognosis in Clear Cell Renal Cell Carcinoma.

The Identification of reliable Biomarkers able to predict the outcome after nephrectomy of patients with clear cell renal cell carcinoma (ccRCC) is an unmet need. The gene expression analysis in tumor tissues represents a promising tool for better stratification of ccRCC subtypes and patients' evaluation. METHODS: In our study we retrospectively analyzed using Next-Generation expression analysis (NanoString), the expression of a gene panel in tumor tissue from 46 consecutive patients treated with nephrectomy for non-metastatic ccRCC at two Italian Oncological Centres. Significant differences in expression levels of selected genes was sought. Additionally, we performed a univariate and a multivariate analysis on overall survival according to Cox regression model. RESULTS: A 17-gene expression signature of patients with a recurrence-free survival (RFS) < 1 year (unfavorable genomic signature (UGS)) and of patients with a RFS > 5 years (favorable genomic signature (FGS)) was identified and resulted in being significantly correlated with overall survival of the patients included in this analysis (HR 51.37, p < 0.0001). CONCLUSIONS: The identified Genomic Signatures may serve as potential biomarkers for prognosis prediction of non-metastatic RCC and could drive both follow-up and treatment personalization in RCC management.

Design, Synthesis, and Biological Evaluation of a Series of Oxazolone Carboxamides as a Novel Class of Acid Ceramidase Inhibitors.

Acid ceramidase (AC) is a cysteine hydrolase that plays a crucial role in the metabolism of lysosomal ceramides, important members of the sphingolipid family, a diversified class of bioactive molecules that mediate many biological processes ranging from cell structural integrity, signaling, and cell proliferation to cell death. In the effort to expand the structural diversity of the existing collection of AC inhibitors, a novel class of substituted oxazol-2-one-3-carboxamides were designed and synthesized. Herein, we present the chemical optimization of our initial hits, 2-oxo-4-phenyl-N-(4-phenylbutyl)oxazole-3-carboxamide 8a and 2-oxo-5-phenyl-N-(4-phenylbutyl)oxazole-3-carboxamide 12a, which resulted in the identification of 5-[4-fluoro-2-(1-methyl-4-piperidyl)phenyl]-2-oxo-N-pentyl-oxazole-3-carboxamide 32b as a potent AC inhibitor with optimal physicochemical and metabolic properties, showing target engagement in human neuroblastoma SH-SY5Y cells and a desirable pharmacokinetic profile in mice, following intravenous and oral administration. 32b enriches the arsenal of promising lead compounds that may therefore act as useful pharmacological tools for investigating the potential therapeutic effects of AC inhibition in relevant sphingolipid-mediated disorders.

Thymic Epithelial Tumors as a Model of Networking: Development of a Synergistic Strategy for Clinical and Translational Research Purposes.

Among the group of thymic epithelial tumors (TET), thymomas often show either uncertain or explicit malignant biological behavior, local invasiveness, and intrathoracic relapse and are often difficult to manage. From the initial stages, thymic carcinomas tend to show aggressive behavior and extrathoracic spread. Moreover, the interplay of epithelial cells and thymocytes in thymomas causes complex immune derangement and related systemic autoimmune diseases. Due to their rare occurrence and to the limited funding opportunities available for rare tumors, it is challenging to make advances in clinical and translational research in TET. The authors of this paper are all members of a multidisciplinary clinical and research thoracic tumor team. Strong input was given to the team by long-standing expertise in TET in the Pathology Department. In addition, thanks to the collaboration between research units at our Institute as well as to national collaborations, over the last 10 years we were able to perform several tissue-based research studies. The most recent studies focused on microRNA and on functional studies on the thymic carcinoma cell line 1889c. The recent implementation of our biobank now provides us with a new tool for networking collaborative research activities. Moreover, the participation in a worldwide community such as ITMIG (International Thymic Malignancy Interest Group) has allowed us to significantly contribute toward fundamental projects/research both in tissue-based studies (The Cancer Genome Atlas) and in clinical studies (TNM staging of TET). Our achievements derive from constant commitment and long-standing experience in diagnosis and research in TET. New perspectives opened up due to the establishment of national [the Italian Collaborative Group for ThYmic MalignanciEs (TYME)] and European reference networks such as EURACAN, for an empowered joint clinical action in adult solid rare tumors. The challenge we face still lies in the advancement of clinical and basic science in thymic epithelial malignancies.

Lead Optimization of Benzoxazolone Carboxamides as Orally Bioavailable and CNS Penetrant Acid Ceramidase Inhibitors.

Sphingolipids (SphLs) are a diverse class of molecules that are regulated by a complex network of enzymatic pathways. A disturbance in these pathways leads to lipid accumulation and initiation of several SphL-related disorders. Acid ceramidase is one of the key enzymes that regulate the metabolism of ceramides and glycosphingolipids, which are important members of the SphL family. Herein, we describe the lead optimization studies of benzoxazolone carboxamides resulting in piperidine 22m, where we demonstrated target engagement in two animal models of neuropathic lysosomal storage diseases (LSDs), Gaucher's and Krabbe's diseases. After daily intraperitoneal administration at 90 mg kg-1, 22m significantly reduced the brain levels of the toxic lipids glucosylsphingosine (GluSph) in 4L;C* mice and galactosylsphingosine (GalSph) in Twitcher mice. We believe that 22m is a lead molecule that can be further developed for the correction of severe neurological LSDs where GluSph or GalSph play a significant role in disease pathogenesis.