CD137+ and regulatory T cells as independent prognostic factors of survival in advanced non-oncogene addicted NSCLC patients treated with immunotherapy as first-line.

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.

Targeting FGFRs by pemigatinib induces G1 phase cell cycle arrest, cellular stress and upregulation of tumor suppressor microRNAs.

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.

A network approach to define the predictive role of immune profile on tumor response and toxicity of anti PD-1 single agent immunotherapy in patients with solid tumors.

Background: The 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. Methods: A 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). Results: A 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. Conclusions: A 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.

Proteomic characterization of extracellular vesicles released by third stage larvae of the zoonotic parasite Anisakis pegreffii (Nematoda: Anisakidae).

Introduction: Anisakis 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. Methods: Genetically 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 discussion: EVs 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.

Membrane and Soluble CD137 in Systemic Lupus Erythematosus: Role as Biomarkers for Disease Activity.

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.

Circulating CD137+ T Cell Levels Are Correlated with Response to Pembrolizumab Treatment in Advanced Head and Neck Cancer Patients.

Pembrolizumab, an anti-PD-1 antibody, has been approved as first-line treatment for recurrent or metastatic head and neck squamous cell carcinoma ((R/M) HNSCC). However, only a minority of patients benefit from immunotherapy, which highlights the need to identify novel biomarkers to optimize treatment strategies. CD137+ T cells have been identified as tumour-specific T cells correlated with immunotherapy responses in several solid tumours. In this study, we investigated the role of circulating CD137+ T cells in (R/M) HNSCC patients undergoing pembrolizumab treatment. PBMCs obtained from 40 (R/M) HNSCC patients with a PD-L1 combined positive score (CPS) ≥1 were analysed at baseline via cytofluorimetry for the expression of CD137, and it was found that the percentage of CD3+CD137+ cells is correlated with the clinical benefit rate (CBR), PFS, and OS. The results show that levels of circulating CD137+ T cells are significantly higher in responder patients than in non-responders (p = 0.03). Moreover, patients with CD3+CD137+ percentage ≥1.65% had prolonged OS (p = 0.02) and PFS (p = 0.02). Multivariate analysis, on a combination of biological and clinical parameters, showed that high levels of CD3+CD137+ cells (≥1.65%) and performance status (PS) = 0 are independent prognostic factors of PFS (CD137+ T cells, p = 0.007; PS, p = 0.002) and OS (CD137+ T cells, p = 0.006; PS, p = 0.001). Our results suggest that levels of circulating CD137+ T cells could serve as biomarkers for predicting the response of (R/M) HNSCC patients to pembrolizumab treatment, thus contributing to the success of anti-cancer treatment.

Immune-related toxicity and soluble profile in patients affected by solid tumors: a network approach.

BACKGROUND: Immune checkpoint inhibitors (ICIs) have particular, immune-related adverse events (irAEs), as a consequence of interfering with self-tolerance mechanisms. The incidence of irAEs varies depending on ICI class, administered dose and treatment schedule. The aim of this study was to define a baseline (T0) immune profile (IP) predictive of irAE development. METHODS: A prospective, multicenter study evaluating the immune profile (IP) of 79 patients with advanced cancer and treated with anti-programmed cell death protein 1 (anti-PD-1) drugs as a first- or second-line setting was performed. The results were then correlated with irAEs onset. The IP was studied by means of multiplex assay, evaluating circulating concentration of 12 cytokines, 5 chemokines, 13 soluble immune checkpoints and 3 adhesion molecules. Indoleamine 2, 3-dioxygenase (IDO) activity was measured through a modified liquid chromatography-tandem mass spectrometry using the high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS) method. A connectivity heatmap was obtained by calculating Spearman correlation coefficients. Two different networks of connectivity were constructed, based on the toxicity profile. RESULTS: Toxicity was predominantly of low/moderate grade. High-grade irAEs were relatively rare, while cumulative toxicity was high (35%). Positive and statistically significant correlations between the cumulative toxicity and IP10 and IL8, sLAG3, sPD-L2, sHVEM, sCD137, sCD27 and sICAM-1 serum concentration were found. Moreover, patients who experienced irAEs had a markedly different connectivity pattern, characterized by disruption of most of the paired connections between cytokines, chemokines and connections of sCD137, sCD27 and sCD28, while sPDL-2 pair-wise connectivity values seemed to be intensified. Network connectivity analysis identified a total of 187 statistically significant interactions in patients without toxicity and a total of 126 statistically significant interactions in patients with toxicity. Ninety-eight interactions were common to both networks, while 29 were specifically observed in patients who experienced toxicity. CONCLUSIONS: A particular, common pattern of immune dysregulation was defined in patients developing irAEs. This immune serological profile, if confirmed in a larger patient population, could lead to the design of a personalized therapeutic strategy in order to prevent, monitor and treat irAEs at an early stage.

Soluble PD-L1 as a Prognostic Factor for Immunotherapy Treatment in Solid Tumors: Systematic Review and Meta-Analysis.

Blocking the Programmed Cell Death Protein 1 (PD-1)/programmed death ligand-1 (PD-L1) axis has demonstrated great efficacy in cancer immunotherapy treatment and remains the central modality of immune targeting. To support the rational and tailored use of these drugs, it is important to identify reliable biomarkers related to survival. The role of the soluble form of the PD-L1 (sPD-L1) as a prognostic biomarker related to survival in solid cancer patients treated with immunotherapy has not yet been consistently evaluated. A systematic literature search of original articles in PubMed, MEDLINE and Scopus was conducted. Studies reporting hazard ratios (HRs) with a 95% confidence interval (CI) or Kaplan−Meier curves or individual patient data for overall survival (OS) or progression-free survival (PFS) associated with baseline levels of sPD-L1 in cancer patients undergoing immunotherapy treatment were considered eligible. Twelve studies involving 1076 patients and different tumor types treated with immunotherapy were included in the analysis. High blood levels of sPD-L1 correlated with poorer OS and PFS in cancer patients treated with immunotherapy (HR = 1.49, 95%CI: 1.15, 1.93, p < 0.01, I2 = 77% for OS; HR = 1.59, 95%CI: 1.20, 2.12, p < 0.01, I2 = 82% for PFS). A subgroup analysis highlighted that high levels of sPD-L1 were associated with worse survival in patients affected by NSCLC (HR = 1.81 95%CI: 1.09−3.00, p = 0.02, I2 = 83% for OS; HR = 2.18, 95%CI: 1.27−3.76, p < 0.01, I2 = 88% for PFS). An HR > 1 indicated that patients with low levels of sPD-L1 have the highest rates of OS/PFS. In this meta-analysis, we clarified the role of sPD-L1 in different solid cancers treated exclusively with Immune checkpoint inhibitors (ICIs). sPD-L1 could represent a non-invasive biomarker that is easily dosable in the blood of patients. The pooled data from the selected studies showed that a high circulating concentration of sPD-L1 in cancer patients correlates with worse survival, suggesting that it may be a helpful prognostic biomarker for the selection of cancer patients before immunotherapy, thus improving the efficacy of ICIs and avoiding unnecessary treatment.

Glycan-Lectin Interactions as Novel Immunosuppression Drivers in Glioblastoma.

Despite diagnostic and therapeutic improvements, glioblastoma (GB) remains one of the most threatening brain tumor in adults, underlining the urgent need of new therapeutic targets. Lectins are glycan-binding proteins that regulate several biological processes through the recognition of specific sugar motifs. Lectins and their ligands are found on immune cells, endothelial cells and, also, tumor cells, pointing out a strong correlation among immunity, tumor microenvironment and vascularization. In GB, altered glycans and lectins contribute to tumor progression and immune evasion, shaping the tumor-immune landscape promoting immunosuppressive cell subsets, such as myeloid-derived suppressor cells (MDSCs) and M2-macrophages, and affecting immunoeffector populations, such as CD8+ T cells and dendritic cells (DCs). Here, we discuss the latest knowledge on the immune cells, immune related lectin receptors (C-type lectins, Siglecs, galectins) and changes in glycosylation that are involved in immunosuppressive mechanisms in GB, highlighting their interest as possible novel therapeutical targets.

Immune effects of CDK4/6 inhibitors in patients with HR+/HER2- metastatic breast cancer: Relief from immunosuppression is associated with clinical response.

BACKGROUND: Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) are innovative small target molecules that, in combination with endocrine therapy, have recently been employed in the treatment of patients with HR+/HER2- metastatic breast cancer (mBC). In this prospective study, we investigate the impact of CDK4/6i on the immune profile of patients with HR+/HER2- mBC. METHODS: Immune cell subsets were analysed using flow cytometry of peripheral blood mononuclear cells (PBMCs) isolated from patients with HR+/HER2- mBC, both before and during treatment. Regulatory T cells (Tregs) were identified using the markers CD4, CD25, CTLA4, CD45RA, and intracellular FOXP3. Monocytic and polymorphonuclear myeloid-derived suppressor cells (M-MDSCs and PMN-MDSCs) and other immune populations were analysed using CD45, CD14, CD66b, CD11c, HLA-DR, CD3, CD8, CD28, CD137, PD1, CD45RA, CCR7, and Ki67. FINDINGS: The percentage of circulating Tregs and M/PMN-MDSCs was significantly downregulated from baseline during CDK4/6i-treatment (p<0.0001 and p<0.05, respectively). In particular, the effector Treg subset (CD4+CD25+FOXP3highCD45RA-) was strongly reduced (p<0.0001). The decrease in Treg levels was significantly greater in responder patients than in non-responder patients. Conversely, CDK4/6i treatment was associated with increased levels of CD4+ T cells and anti-tumour CD137+CD8+ T cells (p<0.05). INTERPRETATION: CDK4/6i treatment results in downregulation of Tregs, M-MDSCs, and PMN-MDSCs, thus weakening tumour immunosuppression. This decrease is associated with response to treatment, highlighting the importance of unleashing immunity in cancer treatment efficacy. These results suggest a novel mechanism of immunomodulation in mBC and provide valuable information for the future design of novel treatments combining CDK4/6i with immunotherapy in other cancer settings. FUNDING: Sapienza University of Rome.

Circulating CD137+ T Cells Correlate with Improved Response to Anti-PD1 Immunotherapy in Patients with Cancer.

PURPOSE: CD137 molecule is expressed by activated lymphocytes, and in patients with cancer identifies the tumor-reactive T cells. In solid tumors, high levels of circulating CD137+ T cells are associated with the clinical response and the disease-free status. Here, we examined the role of the CD137+ T cells in the improvement of patients' selection for immunotherapy treatment. EXPERIMENTAL DESIGN: Peripheral blood mononuclear cells derived from 109 patients with metastatic cancer (66 patients for the identification cohort and 43 for the validation cohort) were analyzed for the expression of CD3, CD4, CD8, CD137, and PD1 molecules before the beginning of anti-PD1 therapy. Twenty healthy donors were used as control. The soluble form of CD137 (sCD137) was also analyzed. The CD137+ T cell subsets and the sCD137 were correlated with the clinicopathologic characteristics. The distribution of CD137+ T cells was also examined in different tumor settings. RESULTS: The percentage of CD137+ T cells was higher in healthy donors and in those patients with a better clinical status (performance status = 0-1, n°metastasis≤2) and these high levels were ascribed to the CD8+CD137+ T cell population. The high frequency of CD137+ and CD8+CD137+ T cells resulted as a prognostic factor of overall survival (OS) and progression-free survival (PFS), respectively, and were confirmed in the validation cohort. High levels of CD3+CD137+PD1+ lymphocytes were associated with a low number of metastasis and longer survival. Instead, the high concentration of the immunosuppressive sCD137 in the serum is associated with a lower PFS and OS. In tumor bed, patients with a complete response showed a high percentage of CD137+ and CD8+ T cells. CONCLUSIONS: We propose the CD137+ T subset as an immune biomarker to define the wellness status of the immune system for successful anticancer immunotherapy.

Immunogenic Cell Death and Immunomodulatory Effects of Cabozantinib.

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 ß-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.

The Role of Soluble LAG3 and Soluble Immune Checkpoints Profile in Advanced Head and Neck Cancer: A Pilot Study.

Unresectable recurrent and/or metastatic head and neck squamous cell carcinoma (R/M HNSCC) has a very poor prognosis. Soluble immune checkpoints (sICs) are circulating proteins that result from the alternative splicing of membrane proteins and can modulate the immune response to cancer cells. The aim of our pilot study was to determine the possible role of a comprehensive evaluation of sICs in the classification of prognosis and response to treatment in patients with advanced disease. We evaluated several sICs (CD137, CTLA-4, PD-1, PD-L1, PD-L2, TIM3, LAG3, GITR, HVEM, BTLA, IDO, CD80, CD27, and CD28) from peripheral blood at baseline and investigated the association with clinical characteristics and outcomes. A high baseline soluble LAG3 (sLAG3 > 377 pg/mL) resulted in an association with poor PFS and OS (p = 0.047 and p = 0.003, respectively). Moreover, sLAG3 emerged as an independent prognostic factor using an MVA (p = 0.005). The evaluation of sICs, in particular sLAG3, may be relevant for identifying patients with worse prognoses, or resistance to treatments, and may lead to the development of novel targeted strategies.

Metformin exerts anti-cancerogenic effects and reverses epithelial-to-mesenchymal transition trait in primary human intrahepatic cholangiocarcinoma cells.

Intrahepatic cholangiocarcinoma (iCCA) is a highly aggressive cancer with marked resistance to chemotherapeutics without therapies. The tumour microenvironment of iCCA is enriched of Cancer-Stem-Cells expressing Epithelial-to-Mesenchymal Transition (EMT) traits, being these features associated with aggressiveness and drug resistance. Treatment with the anti-diabetic drug Metformin, has been recently associated with reduced incidence of iCCA. We aimed to evaluate the anti-cancerogenic effects of Metformin in vitro and in vivo on primary cultures of human iCCA. Our results showed that Metformin inhibited cell proliferation and induced dose- and time-dependent apoptosis of iCCA. The migration and invasion of iCCA cells in an extracellular bio-matrix was also significantly reduced upon treatments. Metformin increased the AMPK and FOXO3 and induced phosphorylation of activating FOXO3 in iCCA cells. After 12 days of treatment, a marked decrease of mesenchymal and EMT genes and an increase of epithelial genes were observed. After 2 months of treatment, in order to simulate chronic administration, Cytokeratin-19 positive cells constituted the majority of cell cultures paralleled by decreased Vimentin protein expression. Subcutaneous injection of iCCA cells previously treated with Metformin, in Balb/c-nude mice failed to induce tumour development. In conclusion, Metformin reverts the mesenchymal and EMT traits in iCCA by activating AMPK-FOXO3 related pathways suggesting it might have therapeutic implications.

Soluble Immune Checkpoints, Gut Metabolites and Performance Status as Parameters of Response to Nivolumab Treatment in NSCLC Patients.

Patients with non-small cell lung cancer (NSCLC) have been shown to benefit from the introduction of anti-PD1 treatment. However, not all patients experience tumor regression and durable response. The identification of a string of markers that are direct or indirect indicators of the immune system fitness is needed to choose optimal therapeutic schedules in the management of NSCLC patients. We analyzed 34 immuno-related molecules (14 soluble immune checkpoints, 17 cytokines/chemokines, 3 adhesion molecules) released in the serum of 22 NSCLC patients under Nivolumab treatment and the gut metabolomic profile at baseline. These parameters were correlated with performance status (PS) and/or response to treatment. Nivolumab affected the release of soluble immune checkpoints (sICs). Patients with a better clinical outcome and with an optimal PS (PS = 0) showed a decreased level of PD1 and maintained low levels of several sICs at first clinical evaluation. Low levels of PDL1, PDL2, Tim3, CD137 and BTLA4 were also correlated with a long response to treatment. Moreover, responding patients showed a high proportion of eubiosis-associated gut metabolites. In this exploratory study, we propose a combination of immunological and clinical parameters (sICs, PS and gut metabolites) for the identification of patients more suitable for Nivolumab treatment. This string of parameters validated in a network analysis on a larger cohort of patients could help oncologists to improve their decision-making in an NSCLC setting.

Investigating Patterns of Immune Interaction in Ovarian Cancer: Probing the O-glycoproteome by the Macrophage Galactose-Like C-type Lectin (MGL).

Glycosylation, the posttranslational linking of sugar molecules to proteins, is notoriously altered during tumor transformation. More specifically in carcinomas, GalNAc-type O-glycosylation, is characterized by biosynthetically immature truncated glycans present on the cancer cell surface, which profoundly impact anti-tumor immune recognition. The tumor-associated glycan pattern may thus be regarded as a biomarker of immune modulation. In epithelial ovarian cancer (EOC) there is a particular lack of specific biomarkers and molecular targets to aid early diagnosis and develop novel therapeutic interventions. The aim of this study was to investigate the ovarian cancer O-glycoproteome and identify tumor-associated glycoproteins relevant in tumor-dendritic cell (DC) interactions, mediated by macrophage galactose-like C type lectin (MGL), which recognizes the tumor-associated Tn O-glycan. Lectin weak affinity chromatography (LWAC) was employed to probe the O-glycopeptidome by MGL and Vicia villosa agglutinin (VVA) lectin using glycoengineered ovarian cancer cell lines and ovarian cancer tissues as input material. Biochemical and bioinformatics analysis gave information on the glycan arrangement recognized by MGL in tumor cells. The potential MGL binders identified were located, as expected, at the cell membrane, but also within the intracellular compartment and the matrisome, suggesting that MGL in vivo may play a complex role in sensing microenvironmental cues. The tumor glycoproteins binders for MGL may become relevant to characterize the interaction between the immune system and tumor progression and contribute to the design of glycan targeting-based strategies for EOC immunotherapeutic interventions.

Exploratory Pilot Study of Circulating Biomarkers in Metastatic Renal Cell Carcinoma.

With the introduction of immune checkpoint inhibitors (ICIs) and next-generation vascular endothelial growth factor receptor-tyrosine kinase inhibitors (VEGFR-TKIs), the survival of patients with advanced renal cell carcinoma (RCC) has improved remarkably. However, not all patients have benefited from treatments, and to date, there are still no validated biomarkers that can be included in the therapeutic algorithm. Thus, the identification of predictive biomarkers is necessary to increase the number of responsive patients and to understand the underlying immunity. The clinical outcome of RCC patients is, in fact, associated with immune response. In this exploratory pilot study, we assessed the immune effect of TKI therapy in order to evaluate the immune status of metastatic renal cell carcinoma (mRCC) patients so that we could define a combination of immunological biomarkers relevant to improving patient outcomes. We profiled the circulating levels in 20 mRCC patients of exhausted/activated/regulatory T cell subsets through flow cytometry and of 14 immune checkpoint-related proteins and 20 inflammation cytokines/chemokines using multiplex Luminex assay, both at baseline and during TKI therapy. We identified the CD3+CD8+CD137+ and CD3+CD137+PD1+ T cell populations, as well as seven soluble immune molecules (i.e., IFNγ, sPDL2, sHVEM, sPD1, sGITR, sPDL1, and sCTLA4) associated with the clinical responses of mRCC patients, either modulated by TKI therapy or not. These results suggest an immunological profile of mRCC patients, which will help to improve clinical decision-making for RCC patients in terms of the best combination of strategies, as well as the optimal timing and therapeutic sequence.

Immunohistochemical Characterization of Immune Infiltrate in Tumor Microenvironment of Glioblastoma.

BACKGROUND: Glioblastoma (GBM) is the most common primary malignant brain cancer in adults, with very limited therapeutic options. It is characterized by a severe immunosuppressive milieu mostly triggered by suppressive CD163+ tumor-associated macrophages (TAMs). The efficacy of immune checkpoint inhibitor interventions aimed at rescuing anti-tumor immunity has not been proved to date. Thus, it is critically important to investigate the immunomodulatory mechanisms acting within the GBM microenvironment for the better design of immunotherapeutic strategies. METHODS: The immunohistochemical analysis of a panel of immune biomarkers (CD3, FoxP3, CD163, IDO, PDL-1, PD-1 and TIGIT) was performed in paired samples of the tumor core (TC) and peritumoral area (PTA) of nine GBM patients. RESULTS: CD163+ cells were the most common cell type in both the PTA and TC. IDO and PDL-1 were expressed in most of the TC samples, frequently accompanied by TIGIT expression; on the contrary, they were almost absent in the PTA. CD3+ cells were present in both the TC and PTA, to a lesser extent than CD163+ cells; they often were accompanied by PD-1 expression, especially in the TC. FoxP3 was scarcely present. CONCLUSION: Distinct inhibitory mechanisms can act simultaneously in both the TC and PTA to contribute to the strong immunosuppression observed within the GBM microenvironment. Nevertheless, the PTA shows strongly reduced immunosuppression when compared to the TC, thus representing a potential target for immunotherapies. Moreover, our results support the working hypothesis that immunosuppression and T-cell exhaustion can be simultaneously targeted to rescue anti-tumor immunity in GBM patients.

Bevacizumab-Based Chemotherapy Triggers Immunological Effects in Responding Multi-Treated Recurrent Ovarian Cancer Patients by Favoring the Recruitment of Effector T Cell Subsets.

Increasing evidence strongly suggests that bevacizumab compound impacts the immunological signature of cancer patients and normalizes tumor vasculature. This study aims to investigate the correlation between the clinical response to bevacizumab-based chemotherapy and the improvement of immune fitness of multi-treated ovarian cancer patients. Peripheral blood mononuclear cells (PBMCs) of 20 consecutive recurrent ovarian cancer patients retrospectively selected to have received bevacizumab or non-bevacizumab-based chemotherapy (Bev group and Ctrl group, respectively) were analyzed. CD4, CD8, and regulatory T cell (Treg) subsets were monitored at the beginning (T0) and after three and six cycles of treatment, together with IL10 production. A lower activated and resting Treg subset was found in the Bev group compared with the Ctrl group until the third therapy cycle, suggesting a reduced immunosuppressive signature. Indeed, clinically responding patients in the Bev group showed a high percentage of non-suppressive Treg and a significant lower IL10 production compared with non-responding patients in the Bev group after three cycles. Furthermore, clinically responding patients showed a discrete population of effector T cell at T0 independent of the therapeutic regimen. This subset was maintained throughout the therapy in only the Bev group. This study evidences that bevacizumab could affect the clinical response of cancer patients, reducing the percentage of Treg and sustaining the circulation of the effector T cells. Results also provide a first rationale regarding the positive immunologic synergism of combining bevacizumab with immunotherapy in multi-treated ovarian cancer patients.