Data Science for Health Image Alignment: A User-Friendly Open-Source ImageJ/Fiji Plugin for Aligning Multimodality/Immunohistochemistry/Immunofluorescence 2D Microscopy Images.

Most of the time, the deep analysis of a biological sample requires the acquisition of images at different time points, using different modalities and/or different stainings. This information gives morphological, functional, and physiological insights, but the acquired images must be aligned to be able to proceed with the co-localisation analysis. Practically speaking, according to Aristotle's principle, "The whole is greater than the sum of its parts", multi-modal image registration is a challenging task that involves fusing complementary signals. In the past few years, several methods for image registration have been described in the literature, but unfortunately, there is not one method that works for all applications. In addition, there is currently no user-friendly solution for aligning images that does not require any computer skills. In this work, DS4H Image Alignment (DS4H-IA), an open-source ImageJ/Fiji plugin for aligning multimodality, immunohistochemistry (IHC), and/or immunofluorescence (IF) 2D microscopy images, designed with the goal of being extremely easy to use, is described. All of the available solutions for aligning 2D microscopy images have also been revised. The DS4H-IA source code; standalone applications for MAC, Linux, and Windows; video tutorials; manual documentation; and sample datasets are publicly available.

Immuno markers in newly diagnosed glioblastoma patients underwent Stupp protocol after neurosurgery: a retrospective series.

PURPOSE: The aims of our retrospective study investigated the role of immune system in glioblastoma (GBM), which is the most aggressive primary brain tumor in adults characterized by a poor prognosis. The recurrence rate remains high, probably due to "immune-desert" tumor microenvironment (TME) making GBM hidden from the anti-tumoral immune clearance. Considering this, we aimed to create a panel of prognostic markers from blood and tumor tissue correlating with overall survival (OS) and progression-free survival (PFS). METHODS: Firstly, we analyzed the inflammatory markers NLR and PLR as the ratio of the absolute neutrophil count and absolute platelet count by the absolute lymphocyte count respectively, collected at different time points in the peripheral blood of 95 patients. Furthermore, in 31 patients of the same cohort, we analyzed the formalin-fixed paraffin embedded samples to further compare the impact of circulating and inflammatory markers within the TME. RESULTS: Patients aged < 60 years and with methylated MGMT showed better OS. While, pre-chemotherapy Systemic Inflammatory Index (SII) < 480 was related to a better OS and PFS, we observed that only CD68+macrophage and CD66b+neutrophils expressed in vascular/perivascular area (V) showed a statistically significant prognostic role in median OS and PFS. CONCLUSIONS: Thus, we underscored a role of SII as predictive value of response to STUPP protocol. Regarding the TME-related markers, we suggested to take into consideration for future studies with new immunotherapy combinations, each component relating to expression of immune infiltrating subsets.

Follicular Lymphoma Microenvironment Traits Associated with Event-Free Survival.

The majority of patients with Follicular Lymphoma (FL) experience subsequent phases of remission and relapse, making the disease "virtually" incurable. To predict the outcome of FL patients at diagnosis, various clinical-based prognostic scores have been proposed; nonetheless, they continue to fail for a subset of patients. Gene expression profiling has highlighted the pivotal role of the tumor microenvironment (TME) in the FL prognosis; nevertheless, there is still a need to standardize the assessment of immune-infiltrating cells for the prognostic classification of patients with early or late progressing disease. We studied a retrospective cohort of 49 FL lymph node biopsies at the time of the initial diagnosis using pathologist-guided analysis on whole slide images, and we characterized the immune repertoire for both quantity and distribution (intrafollicular, IF and extrafollicular, EF) of cell subsets in relation to clinical outcome. We looked for the natural killer (CD56), T lymphocyte (CD8, CD4, PD1) and macrophage (CD68, CD163, MA4A4A)-associated markers. High CD163/CD8 EF ratios and high CD56/MS4A4A EF ratios, according to Kaplan-Meier estimates were linked with shorter EFS (event-free survival), with the former being the only one associated with POD24. In contrast to IF CD68+ cells, which represent a more homogeneous population, higher in non-progressing patients, EF CD68+ macrophages did not stratify according to survival. We also identify distinctive MS4A4A+CD163-macrophage populations with different prognostic weights. Enlarging the macrophage characterization and combining it with a lymphoid marker in the rituximab era, in our opinion, may enable prognostic stratification for low-/high-grade FL patients beyond POD24. These findings warrant validation across larger FL cohorts.

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.

Immunomodulatory Factors Control the Fate of Melanoma Tumor Initiating Cells.

Melanoma is a highly heterogeneous tumor for which recent evidence supports a model of dynamic stemness. Melanoma cells might temporally acquire tumor-initiating properties or switch from a status of tumor-initiating cells (TICs) to a more differentiated one depending on the tumor context. However, factors driving these functional changes are still unknown. We focused on the role of cyto/chemokines in shaping TICs isolated directly from tumor specimens of two melanoma patients, namely Me14346S and Me15888S. We analyzed the secretion profile of TICs and of their corresponding melanoma differentiated cells and we tested the ability of cyto/chemokines to influence TIC self-renewal and differentiation. We found that TICs, grown in vitro as melanospheres, had a complex secretory profile as compared to their differentiated counterparts. Some factors, such as CCL-2 and IL-8, also produced by adherent melanoma cells and melanocytes did not influence TIC properties. Conversely, IL-6, released by differentiated cells, reduced TIC self-renewal and induced TIC differentiation while IL-10, produced by Me15888S, strongly promoted TIC self-renewal through paracrine/autocrine actions. Complete neutralization of IL-10 activity by gene silencing and antibody-mediated blocking of the IL-10Rα was required to sensitize Me15888S to IL-6-induced differentiation. For the first time these results show that functional heterogeneity of melanoma could be directly influenced by inflammatory and suppressive soluble factors, with IL-6 favoring TIC differentiation, and IL-10 supporting TIC self-renewal. Thus, understanding the tumor microenvironment (TME) role in modulating melanoma TIC phenotype is fundamental to identifying novel therapeutic targets to achieve long-lasting regression of metastatic melanoma. Stem Cells 2016;34:2449-2460.

Modulation of the myeloid compartment of the immune system by angiogenic- and kinase inhibitor-targeted anti-cancer therapies.

Targeted therapies were rationally designed to inhibit molecular pathways in tumor cells critically involved in growth and survival; however, many drugs used in targeted therapies may affect the immune system. In addition, selected conventional chemotherapeutic agents have also been reported to be endowed with direct or indirect effects on immunity, for instance via immunogenic death of tumors. Thus, cancer therapies may have off-target effects, some of which are directed to the immune system. Here, we will review some of these effects in specific therapeutic approaches. We will examine the modulation of the immune contexture in human sarcoma and melanoma induced by anti-angiogenic therapies and by BRAF inhibitors, respectively. We will then discuss how the anti-tumor agent trabectedin is selectively cytotoxic to cells of the monocytic-macrophage lineage and how these immune-related effects can be part of the response to treatment.

Melan-A/MART-1 immunity in a EWS-ATF1 translocated clear cell sarcoma patient treated with sunitinib: a case report.

BACKGROUND: Clear cell sarcoma (CCS), initially named malignant melanoma of soft parts, is an aggressive soft tissue sarcoma (STS) that, due to MITF activation, shares with melanoma the expression of melanocyte differentiation antigens. CCS is poorly sensitive to chemotherapy. Multi-kinase inhibitors have been used as therapeutic agents. In the case we report here, treatment with sunitinib induced a long-lasting clinical response that was associated with an immune activation directed against Melan-A/MART-1 antigen. CASE PRESENTATION: A 28 years old female patient with an advanced molecularly confirmed CCS resistant to conventional chemotherapy was started in January 2012 on sunitinib, 37.5 mg/day, with evidence of radiologic and metabolic response at the primary and metastatic sites of disease. Pathologic response and loss of the Melan-A/MART-1 antigen were evidenced on residual tumor removed in April 2012. Immunological monitoring performed on patient's blood during pharmacological treatment revealed a systemic, Melan-A/MART-1 specific immunity and a low frequency of immunosuppressive cells. Sunitinib was restarted in May 2012, with a new response, and continued for 11 months although with repeatedly interruptions due to toxicity. Disease progression and new responses were documented at each treatment interruption and restart. Sunitinib was definitively interrupted in April 2013 for disease progression. CONCLUSION: The analysis of this case proves that antigens expressed by CCS, as for melanoma, can be immunogenic in vivo and that tumor-antigen specific T cells may exert anti-tumor activity in CCS patient. Thus, manipulation of the immune response may have therapeutic potential for this STS subtype and immunotherapy approaches, can be promising therapeutic options for these patients.

Effects of cyclophosphamide and IL-2 on regulatory CD4+ T cell frequency and function in melanoma patients vaccinated with HLA-class I peptides: impact on the antigen-specific T cell response.

The frequency and function of regulatory T cells (Tregs) were studied in stage II-III melanoma patients who were enrolled in a phase II randomized trial of vaccination with HLA-A*0201-modified tumor peptides versus observation. The vaccinated patients received low-dose cyclophosphamide (CTX) and low-dose interleukin-2 (IL-2). Tregs were analyzed in the lymph nodes (LNs) of stage III patients who were undergoing complete lymph node dissection and in peripheral blood mononuclear cells (PBMCs) collected before vaccination and at different time points during the vaccination period. The LNs of the vaccinated patients, which were surgically removed after two rounds of vaccination and one dose of CTX, displayed a low frequency of Tregs and a less immunosuppressive environment compared with those of the untreated patients. The accurate time-course analysis of the PBMCs of patients enrolled in the vaccination arm indicated a limited and transient modulation in the frequencies of Tregs in PBMCs collected after low-dose CTX administration and a strong Treg boost in those PBMCs collected after low-dose IL-2 administration. However, a fraction of the IL-2-boosted Tregs was functionally modulated to a Th-1-like phenotype in the vaccinated patients. Moreover, low-dose IL-2 promoted the concomitant expansion of conventional activated CD4(+) T cells. Despite the amplification of Tregs, IL-2 administration maintained or further increased the number of antigen-specific CD8(+) T cells that were induced by vaccination as demonstrated by the ex vivo human leukocyte antigen-multimer staining and IFN-γ ELISpot assays. Our study suggests that the use of CTX as a Treg modulator should be revised in terms of the administration schedule and of patients who may benefit from this drug treatment. Despite the Treg expansion that was observed in this study, low-dose IL-2 is not detrimental to the functional activities of vaccine-primed CD8(+) T cell effectors when used in the inflammatory environment of vaccination.

Structured myeloid cells and anti-angiogenic therapy in alveolar soft part sarcoma.

Alveolar soft part sarcoma (ASPS) is a rare soft tissue sarcoma and the clinical management of patients with unresectable, metastatic disease is still challenging. ASPS expresses an array of potentially therapeutically targetable, angiogenesis-related molecules and, importantly, it has a distinctive angiogenic phenotype marked by a peculiar tumor-associated vasculature. Several studies, conducted in transgenic mouse models and in a large variety of human tumors of different histotype, clearly proved the substantial contribution of tumor-infiltrating myeloid cells, such as myeloid derived suppressor cells, monocytes and macrophages, in the formation and maintenance of abnormal blood vessels in tumors. By immunohistochemistry we thus explored the presence and the distribution of cells expressing myeloid markers in the inflammatory infiltrate of surgical treated metastatic ASPS. Indeed, we found that myeloid cells expressing CD14 and CD163 markers constitute the prominent cells in the inflammatory infiltrate of ASPS. These macrophage-like cells form a network surrounding the endothelial cells, or, interspersed in the tumor nest, they keep deep contact with tumor cells. In this commentary, we discussed our findings in relation to the recently published paper by Kummar and colleagues reporting the clinical and molecular results of a phase II clinical trial in patients with unresectable, metastatic ASPS treated with the anti-angiogenic drug cediranib, targeting the VEGFR-1,-2,-3 tyrosine kinases.

Circulating hsa-miR-5096 predicts 18F-FDG PET/CT positivity and modulates somatostatin receptor 2 expression: a novel miR-based assay for pancreatic neuroendocrine tumors.

Gastro-entero-pancreatic neuroendocrine tumors (GEP-NETs) are rare diseases encompassing pancreatic (PanNETs) and ileal NETs (SINETs), characterized by heterogeneous somatostatin receptors (SSTRs) expression. Treatments for inoperable GEP-NETs are limited, and SSTR-targeted Peptide Receptor Radionuclide Therapy (PRRT) achieves variable responses. Prognostic biomarkers for the management of GEP-NET patients are required. 18F-FDG uptake is a prognostic indicator of aggressiveness in GEP-NETs. This study aims to identify circulating and measurable prognostic miRNAs associated with 18F-FDG-PET/CT status, higher risk and lower response to PRRT. Methods: Whole miRNOme NGS profiling was conducted on plasma samples obtained from well-differentiated advanced, metastatic, inoperable G1, G2 and G3 GEP-NET patients enrolled in the non-randomized LUX (NCT02736500) and LUNET (NCT02489604) clinical trials prior to PRRT (screening set, n= 24). Differential expression analysis was performed between 18F-FDG positive (n=12) and negative (n=12) patients. Validation was conducted by Real Time quantitative PCR in two distinct well-differentiated GEP-NET validation cohorts, considering the primary site of origin (PanNETs n=38 and SINETs n=30). The Cox regression was applied to assess independent clinical parameters and imaging for progression-free survival (PFS) in PanNETs. In situ RNA hybridization combined with immunohistochemistry was performed to simultaneously detect miR and protein expression in the same tissue specimens. This novel semi-automated miR-protein protocol was applied in PanNET FFPE specimens (n=9). In vitro functional experiments were performed in PanNET models. Results: While no miRNAs emerged to be deregulated in SINETs, hsa-miR-5096, hsa-let-7i-3p and hsa-miR-4311 were found to correlate with 18F-FDG-PET/CT in PanNETs (p-value:<0.005). Statistical analysis has shown that, hsa-miR-5096 can predict 6-month PFS (p-value:<0.001) and 12-month Overall Survival upon PRRT treatment (p-value:<0.05), as well as identify 18F-FDG-PET/CT positive PanNETs with worse prognosis after PRRT (p-value:<0.005). In addition, hsa-miR-5096 inversely correlated with both SSTR2 expression in PanNET tissue and with the 68Gallium-DOTATOC captation values (p-value:<0.05), and accordingly it was able to decrease SSTR2 when ectopically expressed in PanNET cells (p-value:<0.01). Conclusions: hsa-miR-5096 well performs as a biomarker for 18F-FDG-PET/CT and as independent predictor of PFS. Moreover, exosome-mediated delivery of hsa-miR-5096 may promote SSTR2 heterogeneity and thus resistance to PRRT.

Immunotherapy in soft tissue sarcoma: current evidence and future perspectives in a variegated family of different tumor.

INTRODUCTION: In the last few years, steps forward in the knowledge of the biology of soft tissue sarcomas (STS) have led to the development of new therapeutic strategies, including immunotherapy. AREAS COVERED: This review outlines the recent findings on immunological features and provides a synopsis of the results of clinical trials with different immunotherapy approaches in STS, discussing criticisms, and how the efficacy of immunotherapy could be improved. EXPERT OPINION: The heterogeneity of STS has limited generalized approaches of immunotherapy in the disease. Clinical decisions should encompass a comprehensive characterization of the tumor microenvironment (TME), marked by intra-histotype diversity. Profiling of immune cells, checkpoint molecules, and antigen target/HLA expression is deemed to reshape the classical histotype classification for a selection of the most appropriate immune-based treatment. In a synergistic view, tumor-directed treatments, designed on the genetic and epigenetic histotype make-up, should be monitored for their immunomodulant effect and applied to ensure or amplify immunotherapy response. In light of the dynamic nature of the TME, this immunomonitoring should be conducted at baseline and during treatment, for improved therapeutic decisions and rational sequence of treatment combination, pursuing an immunological marker approach by histotype guidance.

Stability Program in Dendritic Cell Vaccines: A "Real-World" Experience in the Immuno-Gene Therapy Factory of Romagna Cancer Center.

Advanced therapy medical products (ATMPs) are rapidly growing as innovative medicines for the treatment of several diseases. Hence, the role of quality analytical tests to ensure consistent product safety and quality has become highly relevant. Several clinical trials involving dendritic cell (DC)-based vaccines for cancer treatment are ongoing at our institute. The DC-based vaccine is prepared via CD14+ monocyte differentiation. A fresh dose of 10 million DCs is administered to the patient, while the remaining DCs are aliquoted, frozen, and stored in nitrogen vapor for subsequent treatment doses. To evaluate the maintenance of quality parameters and to establish a shelf life of frozen vaccine aliquots, a stability program was developed. Several parameters of the DC final product at 0, 6, 12, 18, and 24 months were evaluated. Our results reveal that after 24 months of storage in nitrogen vapor, the cell viability is in a range between 82% and 99%, the expression of maturation markers remains inside the criteria for batch release, the sterility tests are compliant, and the cell costimulatory capacity unchanged. Thus, the data collected demonstrate that freezing and thawing do not perturb the DC vaccine product maintaining over time its functional and quality characteristics.

CAR T cells targeting options in the fight against multiple myeloma.

INTRODUCTION: Multiple myeloma (MM) is a hematological malignancy in which patients present with bone marrow infiltration of clonal terminally-differentiated plasma cells. Monoclonal protein in the serum and/or urine is frequently detected. Over the past decade, important progress has been made in the comprehension of disease biology and treatment personalization. Much work has been put into the development of chimeric antigen receptor (CAR) gene-modified T-cell therapy thought to be a promising therapeutic option for pluritreated patients with refractory MM. EVIDENCE ACQUISITION: We performed an analysis of clinical trials registered at the international repository clinicaltrials.gov using "CAR" OR "CAR T" AND "multiple myeloma" as search terms to understand what were the antigens targeted by CAR T strategies and what was the trade-off of their exploitation. The search retrieved a list of 103 trials that was manually filtered to eliminate follow-up and observational or not-pertinent trials. EVIDENCE SYNTHESIS: Most studies employed anti-BCMA targeting either alone (62/94; 66%), or in combination with a second target (12/94; 13%). The second target most studied was SLAMF7 (CD319) explored by 4/94 (4%) clinical trials. Other antigens investigated and described here include: CD44v6, CD38, CD138, MUC1, CD56, CD19, Igk light chain, Lewis Y, CD229 and GPRC5D. CONCLUSIONS: Targeting an appropriate antigen(s) is the key to both safety and efficacy of CAR T approaches in MM as there is dearth of tumor-specific antigens. Most antigens tested are merely enriched on MM cells. Working with tumor-enriched antigens requires careful assessment of the balance between harm (toxicity) and benefit (disease eradication) to the patient. This review provides an up-to-date overview of the avenues that are being explored.

Effects of a Diet Based on Foods from Symbiotic Agriculture on the Gut Microbiota of Subjects at Risk for Metabolic Syndrome.

Diet is a major driver of gut microbiota variation and plays a role in metabolic disorders, including metabolic syndrome (MS). Mycorrhized foods from symbiotic agriculture (SA) exhibit improved nutritional properties, but potential benefits have never been investigated in humans. We conducted a pilot interventional study on 60 adults with ≥ 1 risk factors for MS, of whom 33 consumed SA-derived fresh foods and 27 received probiotics over 30 days, with a 15-day follow-up. Stool, urine and blood were collected over time to explore changes in gut microbiota, metabolome, and biochemical, inflammatory and immunologic parameters; previous dietary habits were investigated through a validated food-frequency questionnaire. The baseline microbiota showed alterations typical of metabolic disorders, mainly an increase in Coriobacteriaceae and a decrease in health-associated taxa, which were partly reversed after the SA-based diet. Improvements were observed in metabolome, MS presence (two out of six subjects no longer had MS) or components. Changes were more pronounced with less healthy baseline diets. Probiotics had a marginal, not entirely favorable, effect, although one out of three subjects no longer suffered from MS. These findings suggest that improved dietary patterns can modulate the host microbiota and metabolome, counteracting the risk of developing MS.

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.

Intradermal DNA electroporation induces survivin-specific CTLs, suppresses angiogenesis and confers protection against mouse melanoma.

Survivin is an intracellular tumor-associated antigen that is broadly expressed in a large variety of tumors and also in tumor associated endothelial cells but mostly absent in differentiated tissues. Naked DNA vaccines targeting survivin have been shown to induce T cell as well as humoral immune responses in mice. However, the lack of epitope-specific CD8+ T cell detection and modest tumor protection observed highlight the need for further improvements to develop effective survivin DNA vaccination approaches. Here, the efficacy of a human survivin DNA vaccine delivered by intradermal electroporation (EP) was tested. The CD8+ T cell epitope surv(20-28) restricted to H-2 Db was identified based on in-silico epitope prediction algorithms and binding to MHC class I molecules. Intradermal DNA EP of mice with a human survivin encoding plasmid generated CD8+ cytotoxic T lymphocyte (CTL) responses cross-reactive with the mouse epitope surv(20-28), as determined by intracellular IFN-gamma staining, suggesting that self-tolerance has been broken. Survivin-specific CTLs displayed an activated effector phenotype as determined by CD44 and CD107 up-regulation. Vaccinated mice displayed specific cytotoxic activity against B16 and peptide-pulsed RMA-S cells in vitro as well as against surv(20-28) peptide-pulsed target cells in vivo. Importantly, intradermal EP with a survivin DNA vaccine suppressed angiogenesis in vivo and elicited protection against highly aggressive syngeneic B16 melanoma tumor challenge. We conclude that intradermal EP is an attractive method for delivering a survivin DNA vaccine that should be explored also in clinical studies.

Dendritic Cell Vaccination in Metastatic Melanoma Turns "Non-T Cell Inflamed" Into "T-Cell Inflamed" Tumors.

Dendritic cell (DC)-based vaccination effectively induces anti-tumor immunity, although in the majority of cases this does not translate into a durable clinical response. However, DC vaccination is characterized by a robust safety profile, making this treatment a potential candidate for effective combination cancer immunotherapy. To explore this possibility, understanding changes occurring in the tumor microenvironment (TME) upon DC vaccination is required. In this line, quantitative and qualitative changes in tumor-infiltrating T lymphocytes (TILs) induced by vaccination with autologous tumor lysate/homogenate loaded DCs were investigated in a series of 16 patients with metastatic melanoma. Immunohistochemistry for CD4, CD8, Foxp3, Granzyme B (GZMB), PDL1, and HLA class I was performed in tumor biopsies collected before and after DC vaccination. The density of each marker was quantified by automated digital pathology analysis on whole slide images. Co-expression of markers defining functional phenotypes, i.e., Foxp3+ regulatory CD4+ T cells (Treg) and GZMB+ cytotoxic CD8+ T cells, was assessed with sequential immunohistochemistry. A significant increase of CD8+ TILs was found in post-vaccine biopsies of patients who were not previously treated with immune-modulating cytokines or Ipilimumab. Interestingly, along with a maintained tumoral HLA class I expression, after DC vaccination we observed a significant increase of PDL1+ tumor cells, which significantly correlated with intratumoral CD8+ T cell density. This observation might explain the lack of a significant concurrent cytotoxic reactivation of CD8+ T cell, as measured by the numbers of GZMB+ T cells. Altogether these findings indicate that DC vaccination exerts an important role in sustaining or de novo inducing a T cell inflamed TME. However, the strength of the intratumoral T cell activation detected in post-DC therapy lesions is lessened by an occurring phenomenon of adaptive immune resistance, yet the concomitant PDL1 up-regulation. Overall, this study sheds light on DC immunotherapy-induced TME changes, lending the rationale for the design of smarter immune-combination therapies.

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.

Adaptive Immunity in Fibrosarcomatous Dermatofibrosarcoma Protuberans and Response to Imatinib Treatment.

Dermatofibrosarcoma protuberans (DFSP), although rare, is the most frequent skin sarcoma. Here, we focus on DFSP carrying the fibrosarcomatous transformation (FS-DFSP). FS-DFSP responds to imatinib (IM); however, tumor relapse often occurs. In a series of 21 pre- and post-treatment FS-DFSP samples, the present study explored the events that occur at the tumor site during IM therapy. Gene expression profile and immunohistochemistry analyses documented the occurrence of IM-induced senescence phenotype in the tumor cells and showed the accumulation of activated CD3+ T cells and CD163+CD14+ myeloid cells expressing the CD209 marker in post-therapy lesions. In post-IM specimens, the pathological response and tumor apoptosis were tightly associated with T-cell infiltration, thus suggesting the presence of an ongoing anti-tumor response, which was further confirmed by in vitro functional assays with CD3+ T cells isolated from an IM-responding FS-DFSP lesion. The integration of targeted therapies with immune therapies is currently under investigation to achieve longer tumor control. Our data outline the in situ immunological effects of IM and classify IM-treated FS-DFSP as potentially sensitive to immunotherapy, thus providing the rationale for further investigations of combination treatment for this soft-tissue sarcoma.

Targeting Immune Regulatory Networks to Counteract Immune Suppression in Cancer.

The onset of cancer is unavoidably accompanied by suppression of antitumor immunity. This occurs through mechanisms ranging from the progressive accumulation of regulatory immune cells associated with chronic immune stimulation and inflammation, to the expression of immunosuppressive molecules. Some of them are being successfully exploited as therapeutic targets, with impressive clinical results achieved in patients, as in the case of immune checkpoint inhibitors. To limit immune attack, tumor cells exploit specific pathways to render the tumor microenvironment hostile for antitumor effector cells. Local acidification might, in fact, anergize activated T cells and facilitate the accumulation of immune suppressive cells. Moreover, the release of extracellular vesicles by tumor cells can condition distant immune sites contributing to the onset of systemic immune suppression. Understanding which mechanisms may be prevalent in specific cancers or disease stages, and identifying possible strategies to counterbalance would majorly contribute to improving clinical efficacy of cancer immunotherapy. Here, we intend to highlight these mechanisms, how they could be targeted and the tools that might be available in the near future to achieve this goal.