Immunological characterization of a long-lasting response in a patient with metastatic triple-negative breast cancer treated with PD-1 and LAG-3 blockade.

In patients with advanced triple-negative breast cancer (TNBC), translational research efforts are needed to improve the clinical efficacy of immunotherapy with checkpoint inhibitors. Here, we report on the immunological characterization of an exceptional, long-lasting, tumor complete response in a patient with metastatic TNBC treated with dual PD-1 and LAG-3 blockade within the phase I/II study CLAG525X2101C (NCT02460224) The pre-treatment tumor biopsy revealed the presence of a CD3+ and CD8+ cell infiltrate, with few PD1+ cells, rare CD4+ cells, and an absence of both NK cells and LAG3 expression. Conversely, tumor cells exhibited positive staining for the three primary LAG-3 ligands (HLA-DR, FGL-1, and galectin-3), while being negative for PD-L1. In peripheral blood, baseline expression of LAG-3 and PD-1 was observed in circulating immune cells. Following treatment initiation, there was a rapid increase in proliferating granzyme-B+ NK and T cells, including CD4+ T cells, alongside a reduction in myeloid-derived suppressor cells. The role of LAG-3 expression on circulating NK cells, as well as the expression of LAG-3 ligands on tumor cells and the early modulation of circulating cytotoxic CD4+ T cells warrant further investigation as exploitable predictive biomarkers for dual PD-1 and LAG-3 blockade.Trial registration: NCT02460224. Registered 02/06/2015.

Ecosystem degradation and the spread of Covid-19.

The linkages between the emergence of zoonotic diseases and ecosystem degradation have been widely acknowledged by the scientific community and policy makers. In this paper we investigate the relationship between human overexploitation of natural resources, represented by the Human Appropriation of Net Primary Production Index (HANPP) and the spread of Covid-19 cases during the first pandemic wave in 730 regions of 63 countries worldwide. Using a Bayesian estimation technique, we highlight the significant role of HANPP as a driver of Covid-19 diffusion, besides confirming the well-known impact of population size and the effects of other socio-economic variables. We believe that these findings could be relevant for policy makers in their effort towards a more sustainable intensive agriculture and responsible urbanisation.

Predictors of fractional flow reserve/instantaneous wave-free ratio discordance: impact of tailored diagnostic cut-offs on clinical outcomes of deferred lesions.

BACKGROUND: Patient-related and lesion-related factors may influence instantaneous wave-free ratio (iFR)/fractional flow reserve (FFR) concordance, potentially affecting the safety of revascularization deferral. METHODS: Consecutive patients with at least an intermediate coronary stenosis evaluated by both iFR and FFR were retrospectively enrolled. The agreement between iFR and FFR at their diagnostic cut-offs (FFR 0.80, iFR 0.89) was assessed. Predictors of discordance were assessed using multivariate analyses. Tailored iFR cut-offs according to predictors of discordance best matching an FFR of 0.80 were identified. The impact of reclassification according to tailored iFR cut-offs on major cardiovascular events (MACE: cardiovascular death, myocardial infarction or target-lesion revascularization) among deferred lesions was investigated. RESULTS: Two hundred and ninety-nine intermediate coronary stenosis [FFR 0.84 (0.78-0.89), iFR 0.91 (0.87-0.95), 202 left main/left anterior descending (LM/LAD) vessels, 67.6%] of 260 patients were studied. Discordance rate was 23.4% (n = 70, 10.7% iFR-negative discordant, 12.7% iFR-positive discordant). Predictors of discordance were LM/LAD disease, multivessel disease, non-ST-elevation myocardial infarction, smoking, reduced eGFR and hypertension. Lesion reclassification with tailored iFR cut-offs based on patient-level predictors carried no prognostic value among deferred lesions. Reclassification according to lesion location, which was entirely driven by LM/LAD lesions (iFR cut-offs: 0.93 for LM/LAD, 0.89 for non-LM/LAD), identified increased MACE among lesions deferred based on a negative FFR, between patients with a positive as compared with a negative iFR (19.4 vs. 6.1%, P = 0.044), whereas the same association was not observed with the conventional 0.89 iFR cut-off (15 vs. 8.6%, P = 0.303). CONCLUSION: Tailored vessel-based iFR cut-offs carry prognostic value among FFR-negative lesions, suggesting that a one-size-fit-all iFR cut-off might be clinically unsatisfactory.

Extracellular vesicles in anti-tumor immunity.

To what extent extracellular vesicles (EVs) can impact anti-tumor immune responses has only started to get unraveled. Their nanometer dimensions, their growing number of subtypes together with the difficulties in defining their origin hamper their investigation. The existence of tumor cell lines facilitated advance in cancer EV understanding, while capturing information about phenotypes and functions of immune cell EVs in this context is more complex. The advent of immunotherapy with immune checkpoint inhibitors has further deepened the need to dissect the impact of EVs during immune activation and response, not least to contribute unraveling and preventing the generation of resistance occurring in the majority of patients. Here we discuss the factors that influence anddrive the immune response in cancer patients in the context of cancer therapeutics and the roles or possible functions that EVs can have in this scenario. With immune cell-derived EVs as leitmotiv, we will journey from EV discovery and subtypes through physiological and pathological functions, from similarities with tumor EVs to measures to revert detrimental consequences on immune responses to cancer.

Molecular Determinants of Soft Tissue Sarcoma Immunity: Targets for Immune Intervention.

Soft tissue sarcomas (STSs) are a family of rare malignant tumors encompassing more than 80 histologies. Current therapies for metastatic STS, a condition that affects roughly half of patients, have limited efficacy, making innovative therapeutic strategies urgently needed. From a molecular point of view, STSs can be classified as translocation-related and those with a heavily rearranged genotype. Although only the latter display an increased mutational burden, molecular profiles suggestive of an "immune hot" tumor microenvironment are observed across STS histologies, and response to immunotherapy has been reported in both translocation-related and genetic complex STSs. These data reinforce the notion that immunity in STSs is multifaceted and influenced by both genetic and epigenetic determinants. Cumulative evidence indicates that a fine characterization of STSs at different levels is required to identify biomarkers predictive of immunotherapy response and to discover targetable pathways to switch on the immune sensitivity of "immune cold" tumors. In this review, we will summarize recent findings on the interplay between genetic landscape, molecular profiling and immunity in STSs. Immunological and molecular features will be discussed for their prognostic value in selected STS histologies. Finally, the local and systemic immunomodulatory effects of the targeted drugs imatinib and sunitinib will be discussed.

Integrated transcriptional-phenotypic analysis captures systemic immunomodulation following antiangiogenic therapy in renal cell carcinoma patients.

BACKGROUND: The combination of immune checkpoint blockade (ICB) with standard therapies is becoming a common approach for overcoming resistance to cancer immunotherapy in most human malignancies including metastatic renal cell carcinoma (mRCC). In this regard, insights into the immunomodulatory properties of antiangiogenic agents may help designing multidrug schedules based on specific immune synergisms. METHODS: We used orthogonal transcriptomic and phenotyping platforms combined with functional analytic pipelines to elucidate the immunomodulatory effect of the antiangiogenic agent pazopanib in mRCC patients. Nine patients were studied longitudinally over a period of 6 months. We also analyzed transcriptional data from The Cancer Genome Atlas (TCGA) RCC cohort (N = 571) to assess the prognostic implications of our findings. The effect of pazopanib was assessed in vitro on NK cells and T cells. Additionally, myeloid-derived suppressor (MDSC)-like cells were generated from CD14+ monocytes transfected with mimics of miRNAs associated with MDSC function in the presence or absence of pazopanib. RESULTS: Pazopanib administration caused a rapid and dramatic reshaping in terms of frequency and transcriptional activity of multiple blood immune cell subsets, with a downsizing of MDSC and regulatory T cells in favor of a strong enhancement in PD-1 expressing cytotoxic T and Natural Killer effectors. These changes were paired with an increase of the expression of transcripts reflecting activation of immune-effector functions. This immunomodulation was marked but transient, peaking at the third month of treatment. Moreover, the intratumoral expression level of a MDSC signature (MDSC INT) was strongly associated with poor prognosis in RCC patients. In vitro experiments indicate that the observed immunomodulation might be due to an inhibitory effect on MDSC-mediated suppression, rather than a direct effect on NK and T cells. CONCLUSIONS: The marked but transient nature of this immunomodulation, peaking at the third month of treatment, provides the rationale for the use of antiangiogenics as a preconditioning strategy to improve the efficacy of ICB.

The microRNA let-7b-5p Is Negatively Associated with Inflammation and Disease Severity in Multiple Sclerosis.

The identification of microRNAs in biological fluids for diagnosis and prognosis is receiving great attention in the field of multiple sclerosis (MS) research but it is still in its infancy. In the present study, we observed in a large sample of MS patients that let-7b-5p levels in the cerebrospinal fluid (CSF) were highly correlated with a number of microRNAs implicated in MS, as well as with a variety of inflammation-related protein factors, showing specific expression patterns coherent with let-7b-5p-mediated regulation. Additionally, we found that the CSF let-7b-5p levels were significantly reduced in patients with the progressive MS compared to patients with relapsing-remitting MS and were negatively correlated with characteristic hallmark processes of the two phases of the disease. Indeed, in the non-progressive phase, let-7b-5p inversely associated with both central and peripheral inflammation; whereas, in progressive MS, the CSF levels of let-7b-5p negatively correlated with clinical disability at disease onset and after a follow-up period. Overall, our results uncovered, by the means of a multidisciplinary approach and multiple statistical analyses, a new possible pleiotropic action of let-7b-5p in MS, with potential utility as a biomarker of MS course.

Back to simplicity: a four-marker blood cell score to quantify prognostically relevant myeloid cells in melanoma patients.

BACKGROUND: Myeloid-derived suppressor cells (MDSC), a cornerstone of cancer-related immunosuppression, influence response to therapy and disease outcomes in melanoma patients. Nevertheless, their quantification is far from being integrated into routine clinical practice mostly because of the complex and still evolving phenotypic signatures applied to define the cell subsets. Here, we used a multistep downsizing process to verify whether a core of few markers could be sufficient to capture the prognostic potential of myeloid cells in peripheral blood mononuclear cells (PBMC) of metastatic melanoma patients. METHODS: In baseline frozen PBMC from a total of 143 stage IIIc to IV melanoma patients, we first assessed the relevant or redundant expression of myeloid and MDSC-related markers by flow cytometry (screening set, n=23 patients). Subsequently, we applied the identified panel to the development set samples (n=59 patients undergoing first/second-line therapy) to obtain prognostic variables associated with overall survival (OS) and progression-free survival (PFS) by machine learning adaptive index modeling. Finally, the identified score was confirmed in a validation set (n=61) and compared with standard clinical prognostic factors to assess its additive value in patient prognostication. RESULTS: This selection process led to the identification of what we defined myeloid index score (MIS), which is composed by four cell subsets (CD14+, CD14+HLA-DRneg, CD14+PD-L1+ and CD15+ cells), whose frequencies above cut-offs stratified melanoma patients according to progressively worse prognosis. Patients with a MIS=0, showing no over-threshold value of MIS subsets, had the best clinical outcome, with a median survival of >33.6 months, while in patients with MIS 1→3, OS deteriorated from 10.9 to 6.8 and 6.0 months as the MIS increased (p<0.0001, c-index=0.745). MIS clustered patients into risk groups also according to PFS (p<0.0001). The inverse correlation between MIS and survival was confirmed in the validation set, was independent of the type of therapy and was not interfered by clinical prognostic factors. MIS HR was remarkably superior to that of lactate dehydrogenase, tumor burden and neutrophil-to-lymphocyte ratio. CONCLUSION: The MIS >0 identifies melanoma patients with a more aggressive disease, thus acting as a simple blood biomarker that can help tailoring therapeutic choices in real-life oncology.

microRNAs Shape Myeloid Cell-Mediated Resistance to Cancer Immunotherapy.

Immunotherapy with immune checkpoint inhibitors can achieve long-term tumor control in subsets of patients. However, its effect can be blunted by myeloid-induced resistance mechanisms. Myeloid cells are highly plastic and physiologically devoted to wound healing and to immune homeostasis maintenance. In cancer, their physiological activities can be modulated, leading to an expansion of pro-inflammatory and immunosuppressive cells, the myeloid-derived suppressor cells (MDSCs), with detrimental consequences. The involvement of MDSCs in tumor development and progression has been widely investigated and MDSC-induced immunosuppression is acknowledged as a mechanism hindering effective immune checkpoint blockade. Small non-coding RNA molecules, the microRNAs (miRs), contribute to myeloid cell regulation at different levels, comprising metabolism and function, as well as their skewing to a MDSC phenotype. miR expression can be indirectly induced by cancer-derived factors or through direct miR import via extracellular vesicles. Due to their structural stability and their presence in body fluids miRs represent promising predictive biomarkers of resistance, as we recently found by investigating plasma samples of melanoma patients undergoing immune checkpoint blockade. Dissection of the miR-driven involved mechanisms would pave the way for the identification of new druggable targets. Here, we discuss the role of these miRs in shaping myeloid resistance to immunotherapy with a special focus on immunosuppression and immune escape.

Impact of extracorporeal shockwave myocardial revascularization on the ischemic burden of refractory angina patients: a single photon emission computed tomography study.

BACKGROUND: Extracorporeal shockwave myocardial revascularization (ESMR) is a non-invasive treatment designed to improve symptoms in refractory angina (RA) patients. Enhanced perfusion through local vasodilation and neo-capillarization is postulated to be the mechanism of the observed clinical benefit. However, the impact of ESMR on the ischemic burden of RA patients has not been adequately assessed. METHODS: One-hundred twenty-one consecutive RA patients suitable for ESMR were treated. Twenty-nine RA patients not suitable for treatment were clinically followed-up as a control group for clinical endpoints. ESMR-treated patients underwent baseline and 6-month single photon emission computed tomography (SPECT) to evaluate the changes in ischemic burden. The operator was blinded to the pre/post-treatment status of the SPECT exam. The primary endpoint was the difference in summed stress score (SSS) and summed difference score (SDS) between follow-up and baseline SPECTs. Secondary endpoints included the changes in Canadian Cardiovascular Society (CCS) angina class and nitroglycerin use between 6-month follow-up and baseline. Clinical endpoints were further compared between ESMR-treated patients and the control group. RESULTS: Following ESMR, a significant reduction in the ischemic burden was observed (follow-up SSS: 14.2±10 vs. baseline SSS: 21.2±9.42, P<0.0001; follow-up SDS: 4.6±5.9 vs. baseline SDS 10.2±7.9, P<0.0001) including less patients with moderate to severe ischemia (19% vs. 46% P<0.0001). CCS class and nitroglycerin use were significantly reduced (CCS: 1.5±0.6 vs. 2.7±0.6, P<0.0001; patients needing nitroglycerin: 24% vs. 64%, P<0.0001). When compared to the control group, CCS class reduction, nitroglycerin use and hospitalizations were significantly lower for ESMR treated vs. non-treated RA patients at 6-month follow-up. CONCLUSIONS: In this single-center cohort of RA patients undergoing ESMR treatment and serial myocardial perfusion imaging, ESMR was associated with a significant reduction in the ischemic burden. These findings provide a physiological rationale and mechanism for the observed clinical benefit.

Immune microenvironment profiling of gastrointestinal stromal tumors (GIST) shows gene expression patterns associated to immune checkpoint inhibitors response.

Few studies were conducted investigating the immunological profiles in gastrointestinal stromal tumors (GIST). Adaptive and innate immune cells are present in the tumor microenvironment, indicating GIST as inflamed tumors. In addition, murine models suggested a potential interaction between immune components and imatinib. In this retrospective study, the GIST immunological profile was investigated through in silico analysis and immunohistochemistry (IHC), exploring the basis for immunotherapy approaches. Gene expression profiles (GEP) from 31 KIT/PDGFRA-mutant GIST were analyzed to evaluate the tumor microenvironment and immunotherapy predictive signatures such as the expanded IFN-γ-induced immune signature (EIIS) and the T-cell-inflamed signature (TIS). GEP and IHC supported the presence of immune infiltrate in GIST, with dominance of CD4+ and CD8+ T cells and M2 macrophages showing a remarkable similarity with melanoma microenvironment. The EIIS genes were expressed in most of GIST samples and positively correlated with PD-L1 abundance (p < .0001). Co-expression was also found between PD-L1 and CD8A (p < .0001) or CD8B (p = .0003). Moreover, the median TIS score for GIST was between the 65th and 70th percentile of the Cancer Genome Atlas dataset, in the same range of tumors responding to anti-PD-1/PD-L1. Analysis of the Gene Expression Omnibus database GIST samples pre- and post-treatment confirmed that imatinib downregulates PD-L1 and IRF1 expression through the inhibition of KIT and PDGFRA, thus contributing to counteract the suppressed adaptive immune response against GIST. The presence of a rich immune infiltrate in GIST along with the presence of TIS and EIIS suggests that GIST may benefit from immunotherapy along with tyrosine kinase inhibitors.

Pazopanib for treatment of advanced extraskeletal myxoid chondrosarcoma: a multicentre, single-arm, phase 2 trial.

BACKGROUND: Extraskeletal myxoid chondrosarcoma is a rare sarcoma with low sensitivity to cytotoxic chemotherapy. Retrospective evidence suggests that antiangiogenic drugs could be a treatment option. We aimed to investigate the activity of pazopanib, an antiangiogenic drug, in patients with advanced extraskeletal myxoid chondrosarcoma. METHODS: In this single-arm, open-label phase 2 trial, three parallel independent cohorts of different histotypes of advanced sarcomas were recruited (extraskeletal myxoid chondrosarcoma, typical solitary fibrous tumour, and malignant-dedifferentiated solitary fibrous tumour). In each cohort, patients received pazopanib. In this Article, we report the results of the cohort of patients with advanced extraskeletal myxoid chondrosarcoma. Separate reporting of the three cohorts was prespecified in the study protocol. In this cohort, adult patients (aged ≥18 years) with a diagnosis of NR4A3-translocated, metastatic, or unresectable extraskeletal myxoid chondrosarcoma, who had Response Evaluation Criteria in Solid Tumors (RECIST) progression in the previous 6 months, and had an Eastern Cooperative Oncology Group performance status of 0-2, were enrolled at 11 study sites of the Spanish, Italian, and French sarcoma groups. Patients received oral pazopanib (800 mg/day) continuously, until disease progression, unacceptable toxicity, death, non-compliance, patient refusal, or investigator's decision. The primary endpoint was the proportion of patients achieving an objective response according to RECIST 1·1 in the modified intention-to-treat population (patients who provided consent and had a central molecularly confirmed diagnosis of extraskeletal myxoid chondrosarcoma). The safety analysis included all patients who received at least one dose of pazopanib. This study is registered with ClinicalTrials.gov, number NCT02066285. FINDINGS: Between June 24, 2014, and Jan 17, 2017, 26 patients entered the study and started pazopanib. Of these, 23 met the eligibility criteria for the modified intention-to-treat analysis. Median follow-up was 27 months (IQR 18-30). 22 patients (one patient died before the primary analysis) were evaluable for the primary endpoint: four (18% [95% CI 1-36]) had a RECIST objective response. No deaths or grade 4 adverse events occurred. The most frequent grade 3 adverse events were hypertension (nine [35%] of 26 patients), increased concentration of alanine aminotransferase (six [23%]), and increased aspartate aminotransferase (five [19%]). INTERPRETATION: Pazopanib had clinically meaningful antitumour activity in patients with progressive and advanced extraskeletal myxoid chondrosarcoma, and could be considered a suitable option after failure to respond to first-line anthracycline-based chemotherapy in these patients. FUNDING: Spanish Group for Research on Sarcomas, Italian Sarcoma Group, French Sarcoma Group, GlaxoSmithKline, and Novartis.

Hepatocellular Carcinoma Is Associated With Gut Microbiota Profile and Inflammation in Nonalcoholic Fatty Liver Disease.

The gut-liver axis plays a pivotal role in the pathogenesis of nonalcoholic fatty liver disease (NAFLD), which is the third leading cause of hepatocellular carcinoma (HCC) worldwide. However, the link between gut microbiota and hepatocarcinogenesis remains to be clarified. The aim of this study was to explore what features of the gut microbiota are associated with HCC in patients with cirrhosis and NAFLD. A consecutive series of patients with NAFLD-related cirrhosis and HCC (group 1, 21 patients), NAFLD-related cirrhosis without HCC (group 2, 20 patients), and healthy controls (group 3, 20 patients) was studied for gut microbiota profile, intestinal permeability, inflammatory status, and circulating mononuclear cells. We finally constructed a model depicting the most relevant correlations among these features, possibly involved in hepatocarcinogenesis. Patients with HCC showed increased levels of fecal calprotectin, while intestinal permeability was similar to patients with cirrhosis but without HCC. Plasma levels of interleukin 8 (IL8), IL13, chemokine (C-C motif) ligand (CCL) 3, CCL4, and CCL5 were higher in the HCC group and associated with an activated status of circulating monocytes. The fecal microbiota of the whole group of patients with cirrhosis showed higher abundance of Enterobacteriaceae and Streptococcus and a reduction in Akkermansia. Bacteroides and Ruminococcaceae were increased in the HCC group, while Bifidobacterium was reduced. Akkermansia and Bifidobacterium were inversely correlated with calprotectin concentration, which in turn was associated with humoral and cellular inflammatory markers. A similar behavior was also observed for Bacteroides. Conclusion: Our results suggest that in patients with cirrhosis and NAFLD the gut microbiota profile and systemic inflammation are significantly correlated and can concur in the process of hepatocarcinogenesis.

Immunosuppressive circuits in tumor microenvironment and their influence on cancer treatment efficacy.

It has been for long conceived that hallmarks of cancer were intrinsic genetic features driving tumor development, proliferation, and progression, and that targeting such cell-autonomous pathways could be sufficient to achieve therapeutic cancer control. Clinical ex vivo data demonstrated that treatment efficacy often relied on the contribution of host immune responses, hence introducing the concept of tumor microenvironment (TME), namely the existence, along with tumor cells, of non-tumor components that could significantly influence tumor growth and survival. Among the complex network of TME-driving forces, immunity plays a key role and the balance between antitumor and protumor immune responses is a major driver in contrasting or promoting cancer spreading. TME is usually a very immunosuppressed milieu because of a vast array of local alterations contrasting antitumor adaptive immunity, where metabolic changes contribute to cancer dissemination by impairing T cell infiltration and favoring the accrual and activation of regulatory cells. Subcellular structures known as extracellular vesicles then help spreading immunosuppression at systemic levels by distributing genetic and protein tumor repertoire in distant tissues. A major improvement in the knowledge of TME is now pointing the attention back to tumor cells; indeed, recent findings are showing how oncogenic pathways and specific mutations in tumor cells can actually dictate the nature and the function of immune infiltrate. As our information on the reciprocal interactions regulating TME increases, finding a strategy to interfere with TME crosstalk becomes more complex and challenging. Nevertheless, TME interactions represent a promising field for the discovery of novel biomarkers and therapeutic targets for improving treatment efficacy in cancer.

Circulating mir-320a promotes immunosuppressive macrophages M2 phenotype associated with lung cancer risk.

miRNAs play a central role in the complex signaling network of cancer cells with the tumor microenvironment. Little is known on the origin of circulating miRNAs and their relationship with the tumor microenvironment in lung cancer. Here, we focused on the cellular source and relative contribution of different cell types to circulating miRNAs composing our risk classifier of lung cancer using in vitro/in vivo models and clinical samples. A cell-type specific expression pattern and topography of several miRNAs such as mir-145 in fibroblasts, mir-126 in endothelial cells, mir-133a in skeletal muscle cells was observed in normal and lung cancer tissues. Granulocytes and platelets are the major contributors of miRNAs release in blood. miRNAs modulation observed in plasma of lung cancer subjects was consistent with de-regulation of the same miRNAs observed during immunosuppressive conversion of immune cells. In particular, activated neutrophils showed a miRNA profile mirroring that observed in plasma of lung cancer subjects. Interestingly mir-320a secreted by neutrophils of high-risk heavy-smokers promoted an M2-like protumorigenic phenotype through downregulation of STAT4 when shuttled into macrophages. These findings suggest a multifactorial and nonepithelial cell-autonomous origin of circulating miRNAs associated with risk of lung cancer and that circulating miRNAs may act in paracrine signaling with causative role in lung carcinogenesis and immunosuppression.

Complex Immune Contextures Characterise Malignant Peritoneal Mesothelioma: Loss of Adaptive Immunological Signature in the More Aggressive Histological Types.

Malignant peritoneal mesothelioma (MpM), arising in the setting of local inflammation, is a rare aggressive tumour with a poor prognosis and limited therapeutic options. The three major MpM histological variants, epithelioid (E-MpMs), biphasic, and sarcomatoid MpMs (S-MpMs), are characterised by an increased aggressiveness and enhanced levels of EZH2 expression. To investigate the MpM immune contexture along the spectrum of MpM histotypes, an extended in situ analysis was performed on a series of 14 cases. Tumour-infiltrating immune cells and their functionality were assessed by immunohistochemistry, immunofluorescence, qRT-PCR, and flow cytometry analysis. MpMs are featured by a complex immune landscape modulated along the spectrum of MpM variants. Tumour-infiltrating T cells and evidence for pre-existing antitumour immunity are mainly confined to E-MpMs. However, Th1-related immunological features are progressively impaired in the more aggressive forms of E-MpMs and completely lost in S-MpM. Concomitantly, E-MpMs show also signs of active immune suppression, such as the occurrence of Tregs and Bregs and the expression of the immune checkpoint inhibitory molecules PD1 and PDL1. This study enriches the rising rationale for immunotherapy in MpM and points to the E-MpMs as the most immune-sensitive MpM histotypes, but it also suggests that synergistic interventions aimed at modifying the tumour microenvironment (TME) should be considered to make immunotherapy beneficial for these patients.

Immune landscape and in vivo immunogenicity of NY-ESO-1 tumor antigen in advanced neuroblastoma patients.

BACKGROUND: Indirect evidence suggesting the immunosensitivity/immunogenicity of neuroblastoma is accumulating. The aims of this study were to investigate the immune landscape of neuroblastoma and to evaluate the in vivo immunogenicity of the NY-ESO-1 tumor antigen in advanced neuroblastoma patients. METHODS: The immune infiltrating cells of the NY-ESO-1+ tumors from three HLA*A201 patients with metastatic neuroblastoma who relapsed after conventional treatments were evaluated by immunohistochemistry. The patients were vaccinated with the HLA-A*0201-restricted peptide NY-ESO-1157-165(V). The peptide was emulsified in Montanide ISA51 and given subcutaneously in a phase I pilot study. The immunogenicity of NY-ESO-1 antigen was evaluated by monitoring mononuclear cells in patient peripheral blood, pre- and post-vaccine, by short-term in vitro sensitization, HLA-multimer staining and IFN-γ ELISpot analysis. RESULTS: Both CD3 T cells and CD163 myeloid cells were present in pre-vaccine tumors and PD-1 and PD-L1 expression was mainly found in the immune infiltrate. Despite the advanced stage of the disease, the vaccination induced systemic NY-ESO-1 specific CD8 T cells releasing IFN-γ in response to activation with the NY-ESO-1 peptide and an HLA-A2 positive neuroblastoma cell line. CONCLUSIONS: Our results indicate that vaccination with a tumor-associated peptide is able to boost NY-ESO-1-specific, functionally active T cells in advanced neuroblastoma patients with lymphocyte infiltration in their pre-vaccine tumors. TRIAL REGISTRATION: EudraCT #2006-002859-33.

Modulation of Pulmonary Microbiota by Antibiotic or Probiotic Aerosol Therapy: A Strategy to Promote Immunosurveillance against Lung Metastases.

Pulmonary immunological tolerance to inhaled particulates might create a permissive milieu for lung metastasis. Lung microbiota contribute to pulmonary tolerance; here, we explored whether its manipulation via antibiotic or probiotic aerosolization favors immune response against melanoma metastasis. In lungs of vancomycin/neomycin-aerosolized mice, a decrease in bacterial load was associated with reduced regulatory T cells and enhanced T cell and NK cell activation that paralleled a significant reduction of melanoma B16 lung metastases. Reduction of metastases also occurred in lungs transplanted with bacterial isolates from antibiotic-treated lungs. Aerosolized Lactobacillus rhamnosus strongly promoted immunity against B16 lung metastases as well. Furthermore, probiotics or antibiotics improved chemotherapy activity against advanced B16 metastases. Thus, we identify a role for lung microbiota in metastasis and show that its targeting via aerosolization is a therapy that can prevent metastases and enhance responses to chemotherapy.