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.

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.

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.

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.

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.

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.

The prognostic impact of cancer stem-like cell biomarker aldehyde dehydrogenase-1 (ALDH1) in ovarian cancer: A meta-analysis.

OBJECTIVE: To investigate the association of cancer stem cell biomarker aldehyde dehydrogenase-1 (ALDH1) with ovarian cancer patients' prognosis and clinico-pathological characteristics. METHODS: The electronic searches were performed in January 2018 through the databases PubMed, MEDLINE and Scopus by searching the terms: "ovarian cancer" AND "immunohistochemistry" AND ["aldehyde dehydrogenase-1" OR "ALDH1" OR "cancer stem cell"]. Studies evaluating the impact of ALDH1 expression on ovarian cancer survival and clinico-pathological variables were selected. RESULTS: 233 studies were retrieved. Thirteen studies including 1885 patients met all selection criteria. ALDH1-high expression was found to be significantly associated with poor 5-year OS (OR = 3.46; 95% CI: 1.61-7.42; P = 0.001, random effects model) and 5-year PFS (OR = 2.14; 95% CI: 1.11-4.13; P = 0.02, random effects model) in ovarian cancer patients. No correlation between ALDH1 expression and tumor histology (OR = 0.60; 95% CI: 0.36-1.02; P = 0.06, random effects model), FIGO Stage (OR = 0.65; 95% CI: 0.33-1.30; P = 0.22, random effects model), tumor grading (OR = 0.76; 95% CI: 0.40-1.45; P = 0.41, random effects model) lymph nodal status (OR = 2.05; 95% CI: 0.81-5.18; P = 0.13, random effects model) or patients' age at diagnosis (OR = 0.83; 95% CI: 0.54-1.29; P = 0.41, fixed effects model) was identified. CONCLUSIONS: Basing on the available evidence, this meta-analysis showed that high levels of ALDH1 expression correlate with worse OS and PFS in ovarian cancer patients.

Profiles of cancer stem cell subpopulations in cholangiocarcinomas.

Cholangiocarcinomas (CCAs) comprise a mucin-secreting form, intrahepatic or perihilar, and a mixed form located peripherally. We characterized cancer stem cells (CSCs) in CCA subtypes and evaluated their cancerogenic potential. CSC markers were investigated in 25 human CCAs in primary cultures and established cell lines. Tumorigenic potential was evaluated in vitro or in xenografted mice after s.c. or intrahepatic injection in normal and cirrhotic (carbon tetrachloride-induced) mice. CSCs comprised more than 30% of the tumor mass. Although the CSC profile was similar between mucin-intrahepatic and mucin-perihilar subtypes, CD13(+) CSCs characterized mixed-intrahepatic, whereas LGR5(+) characterized mucin-CCA subtypes. Many neoplastic cells expressed epithelial-mesenchymal transition markers and coexpressed mesenchymal and epithelial markers. In primary cultures, epithelial-mesenchymal transition markers, mesenchymal markers (vimentin, CD90), and CD13 largely predominated over epithelial markers (CD133, EpCAM, and LGR5). In vitro, CSCs expressing epithelial markers formed a higher number of spheroids than CD13(+) or CD90(+) CSCs. In s.c. tumor xenografts, tumors dominated by stromal markers were formed primarily by CD90(+) and CD13(+) cells. By contrast, in intrahepatic xenografts in cirrhotic livers, tumors were dominated by epithelial traits reproducing the original human CCAs. In conclusion, CSCs were rich in human CCAs, implicating CCAs as stem cell-based diseases. CSC subpopulations generate different types of cancers depending on the microenvironment. Remarkably, CSCs reproduce the original human CCAs when injected into cirrhotic livers.

Discovery of chemotherapy-associated ovarian cancer antigens by interrogating memory T cells.

According to the immunogenic cell death hypothesis, clinical chemotherapy treatments may result in CD8(+) and CD4(+) T-cell responses against tumor cells. To discover chemotherapy-associated antigens (CAAs), T cells derived from ovarian cancer (OC) patients (who had been treated with appropriate chemotherapy protocols) were interrogated with proteins isolated from primary OC cells. We screened for immunogenicity using two-dimensional electrophoresis gel-eluted OC proteins. Only the selected immunogenic antigens were molecularly characterized by mass-spectrometry-based analysis. Memory T cells that recognized antigens associated with apoptotic (but not live) OC cells were correlated with prolonged survival in response to chemotherapy, supporting the model of chemotherapy-induced apoptosis as an adjuvant of anti-tumor immunity. The strength of both memory CD4(+) and CD8(+) T cells producing either IFN-γ or IL-17 in response to apoptotic OC antigens was also significantly greater in Responders to chemotherapy than in nonresponders. Immunogenicity of some of these antigens was confirmed using recombinant proteins in an independent set of patients. The T-cell interrogation system represents a strategy of reverse tumor immunology that proposes to identify CAAs, which may then be validated as possible prognostic tumor biomarkers or cancer vaccines.

The Fas/Fas ligand apoptosis pathway underlies immunomodulatory properties of human biliary tree stem/progenitor cells.

BACKGROUND & AIMS: Human biliary tree stem/progenitor cells (hBTSCs) are multipotent epithelial stem cells, easily obtained from the biliary tree, with the potential for regenerative medicine in liver, biliary tree, and pancreas diseases. Recent reports indicate that human mesenchymal stem cells are able to modulate the T cell immune response. However, no information exists on the capabilities of hBTSCs to control the allogeneic response. The aims of this study were to evaluate FasL expression in hBTSCs, to study the in vitro interaction between hBTSCs and human lymphocytes, and the role of Fas/FasL modulation in inducing T cell apoptosis in hBTSCs/T cell co-cultures. METHODS: Fas and FasL expression were evaluated in situ and in vitro by immunofluorescence and western blotting. Co-cultures of hBTSCs with human leukocytes were used to analyze the influence of hBTSCs on lymphocytes activation and apoptosis. RESULTS: hBTSCs expressed HLA antigens and FasL in situ and in vitro. Western blot data demonstrated that hBTSCs constitutively expressed high levels of FasL that increased after co-culture with T cells. Confocal microscopy demonstrated that FasL expression was restricted to EpCAM(+)/LGR5(+) cells. FACS analysis of T cells co-cultured with hBTSCs indicated that hBTSCs were able to induce apoptosis in activated CD4(+) and CD8(+) T cell populations. Moreover, the Fas receptor appears to be more expressed in T cells co-cultured with hBTSCs than in resting T cells. CONCLUSIONS: Our data suggest that hBTSCs could modulate the T cell response through the production of FasL, which influences the lymphocyte Fas/FasL pathway by inducing "premature" apoptosis in CD4(+) and CD8(+) T cells.

Evidence for multipotent endodermal stem/progenitor cell populations in human gallbladder.

BACKGROUND & AIMS: Multipotent stem/progenitor cells are found in peribiliary glands throughout human biliary trees and are able to generate mature cells of hepato-biliary and pancreatic endocrine lineages. The presence of endodermal stem/progenitors in human gallbladder was explored. METHODS: Gallbladders were obtained from organ donors and laparoscopic surgery for symptomatic cholelithiasis. Tissues or isolated cells were characterized by immunohistochemistry and flow cytometry. EpCAM+ (Epithelial Cell Adhesion Molecule) cells were immunoselected by magnetic microbeads, plated onto plastic in self-replication conditions and subsequently transferred to distinct serum-free, hormonally defined media tailored for differentiation to specific adult fates. In vivo studies were conducted in an experimental model of liver cirrhosis. RESULTS: The gallbladder does not have peribiliary glands, but it has stem/progenitors organized instead in mucosal crypts. Most of these can be isolated by immune-selection for EpCAM. Approximately 10% of EpCAM+ cells in situ and of immunoselected EpCAM+ cells co-expressed multiple pluripotency genes and various stem cell markers; other EpCAM+ cells qualified as progenitors. Single EpCAM+ cells demonstrated clonogenic expansion ex vivo with maintenance of stemness in self-replication conditions. Freshly isolated or cultured EpCAM+ cells could be differentiated to multiple, distinct adult fates: cords of albumin-secreting hepatocytes, branching ducts of secretin receptor+ cholangiocytes, or glucose-responsive, insulin/glucagon-secreting neoislets. EpCAM+ cells transplanted in vivo in immune-compromised hosts gave rise to human albumin-producing hepatocytes and to human Cytokeratin7+ cholangiocytes occurring in higher numbers when transplanted in cirrhotic mice. CONCLUSIONS: Human gallbladders contain easily isolatable cells with phenotypic and biological properties of multipotent, endodermal stem cells.

Transplantation of human fetal biliary tree stem/progenitor cells into two patients with advanced liver cirrhosis.

BACKGROUND: Efforts to identify cell sources and approaches for cell therapy of liver diseases are ongoing, taking into consideration the limits recognized for adult liver tissue and for other forms of stem cells. In the present study, we described the first procedure of via hepatic artery transplantation of human fetal biliary tree stem cells in patients with advanced cirrhosis. METHODS: The cells were immune-sorted from human fetal biliary tree by protocols in accordance with current good manufacturing practice (cGMP) and extensively characterized. Two patients with advanced liver cirrhosis (Child-Pugh C) have been submitted to the procedure and observed through a 12 months follow-up. RESULTS: The resulting procedure was found absolutely safe. Immuno-suppressants were not required, and the patients did not display any adverse effects correlated with cell transplantation or suggestive of immunological complications. From a clinical point of view, both patients showed biochemical and clinical improvement during the 6 month follow-up and the second patient maintained a stable improvement for 12 months. CONCLUSION: This report represents proof of the concept that the human fetal biliary tree stem cells are a suitable and large source for cell therapy of liver cirrhosis. The isolation procedure can be carried out under cGMP conditions and, finally, the infusion procedure is easy and safe for the patients. This represents the basis for forthcoming controlled clinical trials.

Targeting of macrophage galactose-type C-type lectin (MGL) induces DC signaling and activation.

Dendritic cells (DCs) sense the microenvironment through several types of receptors recognizing pathogen-associated molecular patterns. In particular, C-type lectins, expressed by distinct subsets of DCs, recognize and internalize specific carbohydrate antigen in a Ca(2+) -dependent manner. Targeting of these receptors is becoming an efficient strategy of delivering antigens in DC-based anticancer immunotherapy. Here we investigated the role of the macrophage galactose type C-lectin receptor (MGL), expressed by immature DCs (iDCs), as a molecular target for α-N-acetylgalactosamine (GalNAc or Tn)-carrying tumor-associated antigens to improve DC performance. MGL expressed by ex vivo-generated iDCs from healthy donors was engaged by a 60-mer MUC1(9Tn) -glycopeptide as a Tn-carrying tumor-associated antigen, and an anti-MGL antibody, as a specific MGL binder. We demonstrated that MGL engagement induced homotrimers and homodimers, triggering the phosphorylation of extracellular signal-regulated kinase 1,2 (ERK1,2) and nuclear factor-κB activation. Analysis of DC phenotype and function demonstrated that MGL engagement improved DC performance as antigen-presenting cells, promoting the upregulation of maturation markers, a decrease in phagocytosis, an enhancement of motility, and most importantly an increase in antigen-specific CD8(+) T-cell activation. These results demonstrate that the targeting of MGL receptor on human DCs has an adjuvant effect and that this strategy can be used to design novel anticancer vaccines.

A standardized laboratory and surgical method for in vitro culture isolation and expansion of primary human Tenon's fibroblasts.

Good manufacturing practices guidelines require safer and standardized cell substrates especially for those cell therapy products to treat ocular diseases where fibroblasts are used as feeder layers. However, if these are unavailable for stem cells culturing, murine fibroblasts are regularly used, raising critical issues as accidentally transplanting xenogenous graft and adversely affecting stem cell clinical trials. Moreover, human fibroblasts play a significant role in testing novel ophthalmologic drugs. Accordingly, we developed a standardized laboratory and surgical approach to isolate normal and undamaged Tenon's fibroblasts to implement the setting up of banks for both stem cells-based ocular cell therapy and in vitro drug testing. A 2-3 cm(2) undamaged Tenon's biopsy was surgically obtained from 28 patients without mutually correlated ocular diseases. Nineteen dermal biopsies were used as control. Fibroblasts were isolated with or without collagenase, cultured in autologous, fetal bovine or AB serum, tested for viability by trypan blue, vimentin expression and standardized until passage 6. Successful Tenon's fibroblasts isolation was age dependent (P = 0.001) but not sex, pathology or surgery related. A significant rate of successful cultures were obtained when biopsies were not digested by collagenase (P = 0.013). Moreover, cultures in autologous or fetal bovine serum had comparable proliferative properties (P = 0.77; P = 0.82). Through our surgical and laboratory standardized procedure, we elucidated for the first time key points of this human primary culture system, the role of the autologous serum, comparing Tenon's and dermal fibroblasts. Our protocol may be clinically useful to reduce the risk above mentioned and may be potentially more effective for ophthalmological clinical purposes.

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.

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.

Thinking twice before abandoning first-line chemotherapy in ovarian cancer: report of two cases and literature review. Passing from tri-weekly to weekly regimens.

We present two cases of advanced ovarian cancer treated with neoadjuvant chemotherapy with standard tri-weekly carboplatin and paclitaxel. Therapy was converted to weekly regimens because of disease progression, resulting in disease response. Weekly regimens could overcome drug resistance and this strategy should be attempted before abandoning first-line chemotherapy in favor of palliation.

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.