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Pembrolizumab (an anti-PD1 antibody) alone or combined with chemotherapy represented the standard of care for advanced non-oncogene addicted non-small cell lung cancer (NSCLC) patients. These therapies induced early modifications of the immune response impacting the clinical outcome. Identifying early changes in the immune system was critical to directing the therapeutic choice and improving the clinical outcome. In this study, we aim to analyze the activating and inhibiting immune cells of NSCLC patients before and during therapy to identify patients who will benefit from immunotherapies. Forty-eight NSCLC patients were analyzed before (T0) and after the first cycle of immunotherapy (T1), evaluating several activating (CD137+and PD1+), proliferating (Ki67+) and immunosuppressing immune subsets (Tregs: total, active, resting, and non-suppressive; MDSCs: PMN(Lox1+)-MDSC and M-MDSCs) by cytofluorimetry. Concurrently, 14 soluble immune checkpoints were analyzed by Luminex assay. Immunotherapy significantly increased the levels of Ki67+(total and CD8+) T cells, PMN(Lox1+)-MDSCs, non-suppressive Tregs (nsTregs), and soluble PD1 from T0 to T1 in the entire NSCLC population, while decreased active Tregs. These changes were partially attributed to responding patients who showed an increase of Ki67+ and CD8+T cells and nsTregs at T1. CD137+(total, CD8+, and CD4+) T cells and soluble LAG3 were predictor factors at T0 and T1. A low ratio of Tregs/CD137+ T cells and high levels of Ki67+CD137+ T cells positively correlated with response to therapy at T0 and T1, respectively. Results highlighted that immunotherapy improved the immunological fitness of those patients who benefited from immunotherapy, changing the immunological balance towards immune activation.

Macrophages are integral part of the body's defense against pathogens and serve as vital regulators of inflammation. Adaptor molecules, featuring diverse domains, intricately orchestrate the recruitment and transmission of inflammatory responses through signaling cascades. Key domains involved in macrophage polarization include Toll-like receptors (TLRs), Src Homology2 (SH2) and other small domains, alongside receptor tyrosine kinases, crucial for pathway activation. This review aims to elucidate the enigmatic role of macrophage adaptor molecules in modulating macrophage activation, emphasizing their diverse roles and potential therapeutic and investigative avenues for further exploration.

Abstract Background Fibroblast growth factor receptor (FGFR) gene family alterations are found in several cancers, indicating their importance as potential therapeutic targets. The FGFR-tyrosine kinase inhibitor (TKI) pemigatinib has been introduced in the treatment of advanced cholangiocarcinoma and more recently for relapsed or refractory myeloid/lymphoid neoplasms with FGFR2 and FGFR1 rearrangements, respectively. Several clinical trials are currently investigating the possible combination of pemigatinib with immunotherapy. In this study, we analyzed the biological and molecular effects of pemigatinib on different cancer cell models (lung, bladder, and gastric), which are currently objective of clinical trial investigations. Methods NCI-H1581 lung, KATO III gastric and RT-112 bladder cancer cell lines were evaluated for FGFR expression by qRT-PCR and Western blot. Cell lines were treated with Pem and then characterized for cell proliferation, apoptosis, production of intracellular reactive oxygen species (ROS), and induction of senescence. The expression of microRNAs with tumor suppressor functions was analyzed by qRT-PCR, while modulation of the proteins coded by their target genes was evaluated by Western blot and mRNA. Descriptive statistics was used to analyze the various data and student’s t test to compare the analysis of two groups. Results Pemigatinib exposure triggered distinct signaling pathways and reduced the proliferative ability of all cancer cells, inducing G1 phase cell cycle arrest and strong intracellular stress resulting in ROS production, senescence and apoptosis. Pemigatinib treatment also caused the upregulation of microRNAs (miR-133b, miR-139, miR-186, miR-195) with tumor suppressor functions, along with the downregulation of validated protein targets with oncogenic roles (c-Myc, c-MET, CDK6, EGFR). Conclusions These results contribute to clarifying the biological effects and molecular mechanisms mediated by the anti-FGFR TKI pemigatinib in distinct tumor settings and support its exploitation for combined therapies.

Abstract Object In this study, we evaluated health, social inequalities and risk to gender violence of women living in a disadvantaged degraded suburb of Rome Metropolitan City, during COVID-19 pandemic. Methods The study included 779 women referring to primary care services of Medicina Solidale Institute for gynecological/breast examinations (209), medical and support aid for the children (383) and COVID-19 test execution (187). Results The data show that most women (68%) were unemployed or had an irregular job. The request of support varied depending on the ethnicity: while healthcare support was requested mostly by African female community, the COVID-19 test, mandatory for public transportation and work, was a need of the east-european community. Both these communities referred to Medical Solidale primary care service for the healthcare and food/clothing support for their children. It is interesting to note that the requests from the Italian women community was elevated in terms of personal healthcare, support for the children and COVID-19 test execution. The access to the national health system (NHS) resulted a complex administrative procedure despite the original social-ethnic communities. The vast majority of women lacked awareness of their crucial role for supporting the family entity, while inadequacy was commonly reported. Conclusions This study confirms a critical condition for women living in disadvantaged neighborhoods, whose vulnerability is further worsened by the limited access to primary care assistance with serious consequences for health and quality of life. Prevention and treatment, especially for the most vulnerable subjects, should be a priority for the public health system.

BackgroundThe immune profile of each patient could be considered as a portrait of the fitness of his/her own immune system. The predictive role of the immune profile in immune-related toxicities (irAEs) development and tumour response to treatment was investigated.MethodsA prospective, multicenter study evaluating, through a multiplex assay, the soluble immune profile at the baseline of 53 patients with advanced cancer, treated with immunotherapy as single agent was performed. Four connectivity heat maps and networks were obtained by calculating the Spearman correlation coefficients for each group: responder patients who developed cumulative toxicity (R-T), responders who did not develop cumulative toxicity (R-NT), non-responders who developed cumulative toxicity (NR-T), non-responders who did not develop cumulative toxicity (NR-NT).ResultsA statistically significant up-regulation of IL-17A, sCTLA4, sCD80, I-CAM-1, sP-Selectin and sEselectin in NR-T was detected. A clear loss of connectivity of most of the soluble immune checkpoints and cytokines characterized the immune profile of patients with toxicity, while an inversion of the correlation for ICAM-1 and sP-selectin was observed in NR-T. Four connectivity networks were built for each group. The highest number of connections characterized the NR-T.ConclusionsA connectivity network of immune dysregulation was defined for each subgroup of patients, regardless of tumor type. In patients with the worst prognosis (NR-T) the peculiar connectivity model could facilitate their early and timely identification, as well as the design of a personalized treatment approach to improve outcomes or prevent irAEs.

Pembrolizumab (an anti-PD1 antibody) alone or combined with chemotherapy represented the standard of care for advanced non-oncogene addicted non-small cell lung cancer (NSCLC) patients. These therapies induced early modifications of the immune response impacting the clinical outcome. Identifying early changes in the immune system was critical to directing the therapeutic choice and improving the clinical outcome. In this study, we aim to analyze the activating and inhibiting immune cells of NSCLC patients before and during therapy to identify patients who will benefit from immunotherapies. Forty-eight NSCLC patients were analyzed before (T0) and after the first cycle of immunotherapy (T1), evaluating several activating (CD137+and PD1+), proliferating (Ki67+) and immunosuppressing immune subsets (Tregs: total, active, resting, and non-suppressive; MDSCs: PMN(Lox1+)-MDSC and M-MDSCs) by cytofluorimetry. Concurrently, 14 soluble immune checkpoints were analyzed by Luminex assay. Immunotherapy significantly increased the levels of Ki67+(total and CD8+) T cells, PMN(Lox1+)-MDSCs, non-suppressive Tregs (nsTregs), and soluble PD1 from T0 to T1 in the entire NSCLC population, while decreased active Tregs. These changes were partially attributed to responding patients who showed an increase of Ki67+ and CD8+T cells and nsTregs at T1. CD137+(total, CD8+, and CD4+) T cells and soluble LAG3 were predictor factors at T0 and T1. A low ratio of Tregs/CD137+ T cells and high levels of Ki67+CD137+ T cells positively correlated with response to therapy at T0 and T1, respectively. Results highlighted that immunotherapy improved the immunological fitness of those patients who benefited from immunotherapy, changing the immunological balance towards immune activation. [ABSTRACT FROM AUTHOR]

Abstract Objectives Anisakis pegreffii is a zoonotic parasite requiring marine organisms to complete its life-history. Human infection (anisakiasis) occurs when the third stage larvae (L3) are accidentally ingested with raw or undercooked infected fish or squids. A new de novo transcriptome of A. pegreffii was here generated aiming to provide a robust bulk of data to be used for a comprehensive "ready-to-use" resource for detecting functional studies on genes and gene products of A. pegreffii involved in the molecular mechanisms of parasite-host interaction. Data description A RNA-seq library of A. pegreffii L3 was here newly generated by using Illumina TruSeq platform. It was combined with other five RNA-seq datasets previously gathered from L3 of the same species stored in SRA of NCBI. The final dataset was analyzed by launching three assembler programs and two validation tools. The use of a robust pipeline produced a high-confidence protein-coding transcriptome of A. pegreffii. These data represent a more robust and complete transcriptome of this species with respect to the actually existing resources. This is of importance for understanding the involved adaptive and immunomodulatory genes implicated in the “cross talk” between the parasite and its hosts, including the accidental one (humans).

IntroductionAnisakis pegreffii is a sibling species within the A. simplex (s.l.) complex requiring marine homeothermic (mainly cetaceans) and heterothermic (crustaceans, fish, and cephalopods) organisms to complete its life cycle. It is also a zoonotic species, able to accidentally infect humans (anisakiasis). To investigate the molecular signals involved in this host-parasite interaction and pathogenesis, the proteomic composition of the extracellular vesicles (EVs) released by the third-stage larvae (L3) of A. pegreffii, was characterized.MethodsGenetically identified L3 of A. pegreffii were maintained for 24 h at 37°C and EVs were isolated by serial centrifugation and ultracentrifugation of culture media. Proteomic analysis was performed by Shotgun Analysis.Results and discussionEVs showed spherical shaped structure (size 65-295 nm). Proteomic results were blasted against the A. pegreffii specific transcriptomic database, and 153 unique proteins were identified. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis predicted several proteins belonging to distinct metabolic pathways. The similarity search employing selected parasitic nematodes database revealed that proteins associated with A. pegreffii EVs might be involved in parasite survival and adaptation, as well as in pathogenic processes. Further, a possible link between the A. pegreffii EVs proteins versus those of human and cetaceans’ hosts, were predicted by using HPIDB database. The results, herein described, expand knowledge concerning the proteins possibly implied in the host-parasite interactions between this parasite and its natural and accidental hosts.

Objective. The role of T cells in the pathogenesis of systemic lupus erythematosus (SLE) has recently gained attention. Costimulatory molecules are membrane proteins strictly associated with T-cell receptor (TCR), acting by activating or inhibiting T cells and antigen-presenting cells (APC) through direct and reverse signaling, thus becoming responsible for the development of effector T cells or regulatory T cells. The primary objective of the present case–control study was to evaluate the cell membrane expression of CD137 on T cells and the serum concentration of CD137 (sCD137) in a SLE cohort. Materials. We enrolled SLE patients and sex/age-matched healthy subjects (HS). Disease activity was assessed by SLEDAI-2K. By application of flow cytometry, we evaluated the expression of CD137 on CD4+ and CD8+ lymphocytes. ELISA test was performed to evaluate serum levels of sCD137. Results. Twenty-one SLE patients (M/F 1/20; median age 48 years (IQR 17); median disease duration 144 months (IQR 204)) were evaluated. SLE patients showed %CD3+CD137+ cells significantly higher compared to HS (median 5.32 (IQR 6.11) versus 3.3 (IQR 1.8), p=0.001). In SLE patients, %CD4+CD137+ cells positively correlated with SLEDAI-2K (p=0.0082, r = 0.58, CI (0.15–0.82); indeed, %CD4+CD137+ cells were significantly lower in SLE patients with a remission status compared to those not reaching this condition (median 1.07 (IQR 0.91) versus 1.58 (IQR 2.42), p=0.013). Accordingly, sCD137 levels were significantly lower in remission status (31.30 pg/mL (IQR 102.2 versus median 122.8 pg/mL (IQR 536); p=0.03) and correlated with %CD4+CD137+ cells (p=0.012, r = 0.60, CI (0.15–0.84)). Conclusion. Our results suggest a possible involvement of CD137-CD137L axis in SLE pathogenesis, as demonstrated by higher expression of CD137 on CD4+ cells in SLE compared with HS. Furthermore, the positive correlation between SLEDAI-2K and membrane CD137 expression on CD4+ cells, as well as soluble CD137, indicates a possible use as biomarkers for disease activity.

Simple Summary: This study investigates the molecular characterization of never-smoking patients (NS-pts) with high tumor mutation burden (H-TMB) in advanced non-small cell lung cancer (aNSCLC) and their response to immune checkpoint inhibitors (IO). Clinical data from 142 aNSCLC patients with PD-L1 ≥ 50% treated with pembrolizumab were analyzed. Next-generation sequencing identified mutations across 11 main pathways, revealing that NS-pts with H-TMB were enriched in β-catenin/Wnt and DDR pathway mutations. Validation using data from the POPLAR and OAK trials confirmed DDR pathway mutations were significantly associated with improved outcomes in NS/H-TMB patients. Patient similarity network analysis further showed that NS-pts with DDR mutations had better overall survival. The findings suggest that DDR mutations contribute to H-TMB in NS-pts and may identify a subgroup of patients who could benefit from IO therapy, offering potential for improved prognosis and targeted treatment strategies. Purpose: Single-agent immune checkpoint inhibitor (IO) therapy is the standard for non-oncogene-addicted advanced non-small cell lung cancer (aNSCLC) with PD-L1 tumor proportion score ≥ 50%. Smoking-induced harm generates high tumor mutation burden (H-TMB) in smoking patients (S-pts), while never-smoking patients (NS-pts) typically have low TMB (L-TMB) and are unresponsive to IO. However, the molecular characterization of NS-pts with H-TMB remains unclear. Experimental design: Clinical data of 142 aNSCLC patients with PD-L1 ≥ 50% treated with first line pembrolizumab were retrospectively collected. Next-generation sequencing was performed using the FoundationOne®CDx assay to correlate genomic alterations with clinical characteristics and response outcomes. Detected mutations were classified into eleven main pathways and enrichment analysis identified patient subgroups based on mutated pathways. Additionally, a patient similarity network was constructed to analyze molecular characterization. Results were validated using data from 853 aNSCLC patients in POPLAR and OAK trials. Results: Among the patients, S-pts had higher TMB than NS-pts. Interestingly, 11 (8%) NS-pts exhibited H-TMB and were enriched in β-catenin/Wnt and DDR pathway mutations. DDR pathway mutations were confirmed to be enriched in NS-pts with H-TMB using data from POPLAR and OAK trials. In the real-world cohort, the NS/H-TMB subgroup with DDR pathway mutations demonstrated improved IO outcome. Patient similarity network analysis confirmed the clustering of NS/H-TMB patients with DDR mutations and their association with improved overall survival in both the real-world cohort and the trials. Conclusions: The DDR signature has a potential role as an additional generator of H-TMB in NS-pts. This subgroup of IO-responsive NS-pts may have better prognosis. Our findings suggest that DDR-based mutational profiling may help identify NS-pts who could benefit from IO therapy. [ABSTRACT FROM AUTHOR]

Cabozantinib (XL-184) is a multitarget tyrosine kinase inhibitor (TKI) targeting receptor tyrosine kinases (RTKs) involved in oncogenesis and angiogenesis. It is currently the standard therapy for medullary thyroid cancer (MTC), metastatic renal cell carcinoma (mRCC), and hepatocellular carcinoma (HCC). Combination of Cabozantinib with immunotherapy is now a standard treatment in metastatic renal cancer, and its efficacy is being tested in ongoing clinical trial in prostate cancer patients. Here, we report that Cabozantinib may exert an immunostimulatory role by inducing immunogenic stress of prostate cancer cells and directly modulating dendritic cells (DCs). Cabozantinib treatment arrested the cell cycle and triggered immunogenic cell death (ICD) in prostate cancer cells in vitro. Cabozantinib had a direct effect on DCs by the down-modulation of β-catenin and change in migratory and costimulatory phenotype of the DCs. These results may suggest possible immunomodulatory effects induced by Cabozantinib that could be exploited to optimize patient-tailored immunotherapeutic treatments.

Background: Immune checkpoint inhibitors (ICIs), administered alone or combined with chemotherapy, are the standard of care in advanced non-oncogene addicted Non-Small Cell Lung Cancer (NSCLC). Despite these treatments' success, most long-term survival benefit is restricted to approximately 20% of patients, highlighting the need to identify novel biomarkers to optimize treatment strategies. In several solid tumors, immune soluble factors, the activatory CD137+ Tcells, and the immunosuppressive cell subsets Tregs and MDSCs (PMN(Lox1+)-MDSC and M-MDSCs) correlated with responses to ICIs and clinical outcomes thus becoming appealing predictive and prognostic factors. This study investigated the role of distinct CD137+ Tcell subsets, Tregs, MDSCs, and immune-soluble factors in NSCLC patients as possible biomarkers. Methods: The levels of T cells, MDSCs and soluble factors were evaluated in 89 metastatic NSCLC patients who underwent ICIs as first- or second-line treatment. T cell analysis was performed by cytoflurimetry evaluating Tregs and different CD137+ Tcell subsets also combined with CD3+, CD8+, PD1+, and Ki67+ markers. Circulating cytokines and immune checkpoints were also evaluated by Luminex analysis. All these parameters were correlated with several clinical factors (age, sex, smoking status, PS and TPS), response to therapy, PFS , and OS. The analyses were conducted in the overall population and in patients treated with ICIs as first-line (naïve patients). Results: In both groups of patients, high levels of circulating CD137+ and CD137+PD1+ T cells (total, CD4 and CD8) and the soluble factor LAG3 positively correlated with response to therapy. In naïve patients, PMN(Lox1+)-MDSCs negatively correlated with clinical response, and a high percentage of Tregs was associated with favorable survival. Moreover, the balance between Treg/CD137+ Tcells or PMN(Lox1+)-MDSC/CD137+ Tcells was higher in non-responding patients and was associated with poor survival. CD137+ Tcells and Tregs resulted as two positive independent prognostic factors. Conclusion: High levels of CD137+, CD137+PD1+ Tcells and sLAG3 could predict the response to ICIs in NSCLC patients independently by previous therapy. Combining the evaluation of CD137+ Tcells and Tregs also as Treg/CD137+ T cells ratio it is possible to identify naive patients with longer survival. [ABSTRACT FROM AUTHOR]

Background. Personalised medicine in oncology needs standardised immunological assays. Flow cytometry (FCM) methods represent an essential tool for immunomonitoring, and their harmonisation is crucial to obtain comparable data in multicentre clinical trials. The objective of this study was to design a harmonisation workflow able to address the most effective issues contributing to intra- and interoperator variabilities in a multicentre project. Methods. The Italian National Institute of Health (Istituto Superiore di Sanità, ISS) managed a multiparametric flow cytometric panel harmonisation among thirteen operators belonging to five clinical and research centres of Lazio region (Italy). The panel was based on a backbone mixture of dried antibodies (anti-CD3, anti-CD4, anti-CD8, anti-CD45RA, and anti-CCR7) to detect naïve/memory T cells, recognised as potential prognostic/predictive immunological biomarkers in cancer immunotherapies. The coordinating centre distributed frozen peripheral blood mononuclear cells (PBMCs) and fresh whole blood (WB) samples from healthy donors, reagents, and Standard Operating Procedures (SOPs) to participants who performed experiments by their own equipment, in order to mimic a real-life scenario. Operators returned raw and locally analysed data to ISS for central analysis and statistical elaboration. Results. Harmonised and reproducible results were obtained by sharing experimental set-up and procedures along with centralising data analysis, leading to a reduction of cross-centre variability for naïve/memory subset frequencies particularly in the whole blood setting. Conclusion. Our experimental and analytical working process proved to be suitable for the harmonisation of FCM assays in a multicentre setting, where high-quality data are required to evaluate potential immunological markers, which may contribute to select better therapeutic options.