Analysis of EVs from patients with advanced pancreatic cancer identifies antigens and miRNAs with predictive value.

The identification of predictive factors for treatment of pancreatic cancer (PC) is an unmet clinical need. In the present work, we analyzed blood-derived extracellular vesicles (EVs) from patients with advanced PC in order to find a molecular signature predictive of response to therapy. We analyzed samples from 21 patients with advanced PC, all receiving first-line treatment with gemcitabine + nab-paclitaxel. Isolated EVs have been analyzed, and the results of laboratory have been matched with clinical data in order to investigate possible predictive factors. EV concentration and size were similar between responder and non-responder patients. Analysis of 37 EV surface epitopes showed a decreased expression of SSEA4 and CD81 in responder patients. We detected more than 450 expressed miRNAs in EVs. A comparative survey between responder and non-responder patients showed that at least 44 miRNAs were differently expressed. Some of these miRNAs have already been observed in relation to the survival and gemcitabine sensitivity of tumor cells. In conclusion, we showed the ability of our approach to identify EV-derived biomarkers with predictive value for therapy response in PC. Our findings are worthy of further investigation, including the analysis of samples from patients treated with different schedules and in different settings.

Effects of yoga practice on physiological distress, fatigue and QOL in patients affected by breast cancer undergoing adjuvant radiotherapy.

Background and purpose: In this study we want to evaluate the efficacy of yoga practice on dysfunctional stress, inflammation and QOL in breast cancer patients undergoing adjuvant radiotherapy. Patients and methods: Patients with stage 0 to III breast cancer were recruited before starting radiotherapy (XRT) and were randomly assigned to yoga group (YG) two times a week during XRT or control group (CG). Self-report measures of QOL, fatigue and sleep quality, and blood samples were collected at day 1 of treatment, day 15, end of treatment and 1, 3 and 6 months later. Cortisol blood level, IL6, IL10, IL1RA, TNFα and lymphocyte-to-monocyte ratio were analyzed as measures of dysfunctional stress and inflammation. Results: Patients started XRT and yoga classes in October 2019. Due to COVID-19 pandemic we closed the enrollment in March 2020. We analysed 24 patients, 12 YG and 12 CG. The analysis of blood cortisol levels revealed an interaction (p = 0.04) between yoga practice and time, in particular YG had lower cortisol levels at the end of XRT respect to CG (p-adj = 0.02). The analysis of IL-1RA revealed an interaction effect (p = 0.04) suggesting differences between groups at some time points that post-hoc tests were not able to detect. Conclusions: To our knowledge, this is the first study to evaluate the effects of yoga in a cancer population studying inflammation markers, cortisol trend and QOL during and until 6 months after XRT. This study suggests that yoga practice is able to reduce stress and inflammation levels over time. Besides including a larger number of patients to increase the power, future studies should consider other inflammatory or pro inflammatory factors and long-term yoga program to gain more evidence on yoga practice benefits.

T-Cell Receptor Repertoire Sequencing and Its Applications: Focus on Infectious Diseases and Cancer.

The immune system is a dynamic feature of each individual and a footprint of our unique internal and external exposures. Indeed, the type and level of exposure to physical and biological agents shape the development and behavior of this complex and diffuse system. Many pathological conditions depend on how our immune system responds or does not respond to a pathogen or a disease or on how the regulation of immunity is altered by the disease itself. T-cells are important players in adaptive immunity and, together with B-cells, define specificity and monitor the internal and external signals that our organism perceives through its specific receptors, TCRs and BCRs, respectively. Today, high-throughput sequencing (HTS) applied to the TCR repertoire has opened a window of opportunity to disclose T-cell repertoire development and behavior down to the clonal level. Although TCR repertoire sequencing is easily accessible today, it is important to deeply understand the available technologies for choosing the best fit for the specific experimental needs and questions. Here, we provide an updated overview of TCR repertoire sequencing strategies, providers and applications to infectious diseases and cancer to guide researchers' choice through the multitude of available options. The possibility of extending the TCR repertoire to HLA characterization will be of pivotal importance in the near future to understand how specific HLA genes shape T-cell responses in different pathological contexts and will add a level of comprehension that was unthinkable just a few years ago.

Radiotherapy and High-Dose Interleukin-2: Clinical and Immunological Results of a Proof of Principle Study in Metastatic Melanoma and Renal Cell Carcinoma.

High-dose interleukin-2 (HD IL-2) has curative potential in metastatic melanoma (MM) and renal cell carcinoma (RCC). Radiotherapy (RT) kills cancer cells and induces immunomodulatory effects. Prospective trials exploring clinical and immunological properties of combined RT/HD IL-2 are still needed. We designed a phase II, single-arm clinical trial for patients with MM and RCC. The treatment schedule consisted of 3 daily doses of 6-12 Gy of RT to 1-5 non-index metastatic fields, before IL-2 at the first and third treatment cycle. HD IL-2 was administered by continuous infusion for 72 hours and repeated every 3 weeks for up to 4 cycles, thereafter every 4 weeks for a maximum of 2 cycles. The primary endpoint was the immunological efficacy of the combined RT/HD IL-2 treatment (assessed by IFN-γ ELISPOT). Nineteen out of 22 patients were evaluable for immunological and clinical response. Partial response occurred in 3 (15.7%) patients and stable disease was observed in 7 (36.8%). The disease control rate was 52.6% after a median follow up of 39.2 months. According to Common Terminology Criteria for Adverse Events 4.0 (CTCAE 4.0), the majority of toxicities were grade 1-2. Immunological responses were frequent and detected in 16 (84.2%) patients. Increased levels of IL-8 and IL-10 in melanoma, circulating effector memory CD4+ and intratumoral CD8+ T cells in both tumor types were detected after therapy. Overall the treatment was well tolerated and immunologically active. Immunomonitoring and correlative data on tumor and peripheral blood cell subsets suggest that this combination treatment could be a promising strategy for patients progressing after standard treatments.

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.

Complementary vaccination protocol with dendritic cells pulsed with autologous tumour lysate in patients with resected stage III or IV melanoma: protocol for a phase II randomised trial (ACDC Adjuvant Trial).

INTRODUCTION: Surgery is one of the treatments of choice for patients with a single metastasis from melanoma but is rarely curative. Such patients could potentially benefit from consolidation immunotherapy. Vaccination with dendritic cells (DCs) loaded with tumour antigens elicits a tumour-specific immune response. In our experience, patients who developed delayed type hypersensitivity (DTH) after DC vaccination showed a median overall survival (OS) of 22.9 monthsvs4.8 months for DTH-negative cases. A phase II randomised trial showed an advantage OS of a DC vaccine over a tumour cell-based vaccine (2-year OS 72% vs31%, respectively). Given that there is no standard therapy after surgical resection of single metastases, we planned a study to compare vaccination with DCs pulsed with autologous tumour lysate versus follow-up. METHODS AND ANALYSIS: This is a randomised phase II trial in patients with resected stage III/IV melanoma. Assuming a median relapse-free survival (RFS) of 7.0 months for the standard group and 11.7 months for the experimental arm (HR 0.60), with a two-sided tailed alpha of 0.10, 60 patients per arm must be recruited. An interim futility analysis will be performed at 18 months. The DC vaccine, produced in accordance with Good Manufacturing Practice guidelines, consists of autologous DCs loaded with autologous tumour lysate and injected intradermally near lymph nodes. Vaccine doses will be administered every 4 weeks for six vaccinations and will be followed by 3 million unit /day of interleukin-2 for 5 days. Tumour restaging, blood sampling for immunological biomarkers and DTH testing will be performed every 12 weeks. ETHICS AND DISSEMINATION: The protocol, informed consent and accompanying material given to patients were submitted by the investigator to the Ethics Committee for review. The local Ethics Committee and the Italian Medicines Agency approved the protocol (EudraCT code no.2014-005123-27). Results will be published in a peer-reviewed international scientific journal. TRIAL REGISTRATION NUMBER: 2014-005123-27.

Dendritic cell vaccination for metastatic melanoma: a 14-year monoinstitutional experience.

Although immunomodulating antibodies are highly effective in metastatic melanoma, their toxicity, related to the activation of T lymphocytes, can be severe. Anticancer vaccines promote a fairly specific response and are very well tolerated, but their effectiveness has yet to be demonstrated. We have been treating patients with advanced melanoma with an autologous dendritic cell vaccine since 2001; to better characterize the safety and efficacy of our product, we designed a retrospective study on all of our patients treated with the vaccine to date. We retrospectively reviewed both case report forms of patients included in clinical trials and medical records of those treated within a compassionate use program. Response was assessed according to the Response Evaluation Criteria In Solid Tumors criteria and toxicity has been graded according to CTCAE 4.0. Although the response rate has been rather low, the median overall survival of 11.4 months and the 1-year survival rate of 46.9% are encouraging, especially considering the fact that data were obtained in a heavily pretreated population and only about one quarter of the patients had received ipilimumab and/or BRAF inhibitors. Multivariate analysis confirmed that the development of an immune response was significantly correlated with a better prognosis (hazard ratio 0.54; P=0.019). The adverse events observed were generally mild and self-limiting. Our analysis confirms the excellent tolerability of our vaccine, making it a potential candidate for combination therapies. As efficacy seems largely restricted to immunoresponsive patients, future strategies should aim to increase the number of these patients.

The Comprehensive Cancer Care Network of Romagna: the opportunities generated by the OECI accreditation program.

Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS is a public-private partnership among 3 public sector bodies and 6 private nonprofit organizations and represents the hub of the Oncology Network of Romagna, which provides a wide range of services for the population ranging from primary prevention to palliative care. In 2012, IRST took part in the ministerial research project of the Organisation of European Cancer Institutes (OECI) accreditation program for Comprehensive Cancer Centers. The self-assessment period lasted 6 months and was coordinated by a multidisciplinary project team headed by a project leader. Each project team member coordinated a subgroup whose task was to analyze specific standards from qualitative and quantitative questionnaires. During the self-assessment period for the areas in which IRST did not meet OECI requirements, the project team outlined several improvement plans. At the end of the self-assessment period, the OECI Accreditation & Designation Board approved the documentation presented by IRST and a peer review visit was scheduled. The OECI report suggested establishing a more specific and stronger centralized management and leadership of all the oncologic activities carried out in other centers. In accordance with these suggestions, IRST and the Local Health Authority of Romagna laid the foundations for a new management model for the Oncology Network of Romagna: the Comprehensive Cancer Care Network (CCCN). The CCCN is a territory-oriented model (population approach) based on a principle of cooperation and collaboration among the network nodes.

Vaccination with autologous dendritic cells loaded with autologous tumor lysate or homogenate combined with immunomodulating radiotherapy and/or preleukapheresis IFN-α in patients with metastatic melanoma: a randomised "proof-of-principle" phase II study.

BACKGROUND: Vaccination with dendritic cells (DC) loaded with tumor antigens elicits tumor-specific immune responses capable of killing cancer cells without inducing meaningful side-effects. Patients with advanced melanoma enrolled onto our phase II clinical studies have been treated with autologous DC loaded with autologous tumor lysate/homogenate matured with a cytokine cocktail, showing a clinical benefit (PR + SD) in 55.5% of evaluable cases to date. The beneficial effects of the vaccine were mainly restricted to patients who developed vaccine-specific immune response after treatment. However, immunological responses were only induced in about two-thirds of patients, and treatments aimed at improving immunological responsiveness to the vaccine are needed. METHODS/DESIGN: This is a phase II, "proof-of-principle", randomized, open-label trial of vaccination with autologous DC loaded with tumor lysate or homogenate in metastatic melanoma patients combined with immunomodulating RT and/or preleukapheresis IFN-α. All patients will receive four bi-weekly doses of the vaccine during the induction phase and monthly doses thereafter for up to a maximum of 14 vaccinations or until confirmed progression. Patients will be randomized to receive:(1.) three daily doses of 8 Gy up to 12 Gy radiotherapy delivered to one non-index metastatic field between vaccine doses 1 and 2 and, optionally, between doses 7 and 8, using IMRT-IMAT techniques;(2.) daily 3 MU subcutaneous IFN-α for 7 days before leukapheresis;(3.) both 1 and 2;(4.) neither 1 nor 2.At least six patients eligible for treatment will be enrolled per arm. Daily 3 MU IL-2 will be administered subcutaneously for 5 days starting from the second day after each vaccine dose. Serial DTH testing and blood sampling to evaluate treatment-induced immune response will be performed. Objective response will be evaluated according to immune-related response criteria (irRC). DISCUSSION: Based upon the emerging role of radiotherapy as an immunologic modifier, we designed a randomized phase II trial adding radiotherapy and/or preleukapheresis IFN-α to our DC vaccine in metastatic melanoma patients. Our aim was to find the best combination of complementary interventions to enhance anti-tumor response induced by DC vaccination, which could ultimately lead to better survival and milder toxicity.

Low-dose temozolomide before dendritic-cell vaccination reduces (specifically) CD4+CD25++Foxp3+ regulatory T-cells in advanced melanoma patients.

BACKGROUND: In cancer immunotherapy, dendritic cells (DCs) play a fundamental role in the dialog between innate and adaptive immune response, but several immunosuppressive mechanisms remain to be overcome. For example, a high number of CD4+CD25++Foxp3+ regulatory T-cells (Foxp3+Tregs) have been observed in the peripheral blood and tumor microenvironment of cancer patients. On the basis of this, we conducted a study on DC-based vaccination in advanced melanoma, adding low-dose temozolomide to obtain lymphodepletion. METHODS: Twenty-one patients were entered onto our vaccination protocol using autologous DCs pulsed with autologous tumor lysate and keyhole limpet hemocyanin. Patients received low-dose temozolomide before vaccination and 5 days of low-dose interleukin-2 (IL-2) after vaccination. Circulating Foxp3+Tregs were evaluated before and after temozolomide, and after IL-2. RESULTS: Among the 17 evaluable patients we observed 1 partial response (PR), 6 stable disease (SD) and 10 progressive disease (PD). The disease control rate (PR+SD = DCR) was 41% and median overall survival was 10 months. Temozolomide reduced circulating Foxp3+Treg cells in all patients. A statistically significant reduction of 60% was observed in Foxp3+Tregs after the first cycle, whereas the absolute lymphocyte count decreased by only 14%. Conversely, IL-2 increased Foxp3+Treg cell count by 75.4%. Of note the effect of this cytokine, albeit not statistically significant, on the DCR subgroup led to a further 33.8% reduction in Foxp3+Treg cells. CONCLUSIONS: Our results suggest that the combined immunological therapy, at least as far as the DCR subgroup is concerned, effectively reduced the number of Foxp3+Treg cells, which exerted a blunting effect on the growth-stimulating effect of IL-2. However, this regimen, with its current modality, would not seem to be capable of improving clinical outcome.

Accreditation for excellence of cancer research institutes: recommendations from the Italian Network of Comprehensive Cancer Centers.

A panel of experts from Italian Comprehensive Cancer Centers defines the recommendations for external quality control programs aimed to accreditation to excellence of these institutes. After definition of the process as a systematic, periodic evaluation performed by an external agency to verify whether a health organization possesses certain prerequisites regarding structural, organizational and operational conditions that are thought to affect health care quality, the panel reviews models internationally available and makes final recommendations on aspects considered of main interest. This position paper has been produced within a special project of the Ministry of Health of the Italian Government aimed to accredit, according to OECI model, 11 Italian cancer centers in the period 2012-2014. The Project represents the effort undertaken by this network of Comprehensive Cancer Centers to find a common denominator for the experience of all Institutes in external quality control programs. Fourteen shared "statements" are put forth, designed to offer some indications on the main aspects of this subject, based on literature evidence or expert opinions. They deal with the need for "accountability" and involvement of the entire organization, the effectiveness of self-evaluation, the temporal continuity and the educational value of the experience, the use of indicators and measurement tools, additionally for intra- and inter-organization comparison, the system of evaluation models used, the provision for specific requisites for oncology, and the opportunity for mutual exchange of evaluation experiences.

Dendritic cell-based vaccine in advanced melanoma: update of clinical outcome.

Dendritic cells (DCs) are unique specialized antigen-presenting cells capable of priming naive T cells and inducing antigen-specific cytotoxic T lymphocytes. This study presents an update of clinical results from a DC-based phase I-II clinical vaccine trial in stage IV melanoma. From 2003 to 2010, 27 patients with metastatic melanoma were treated with mature DCs pulsed with autologous tumor lysate and keyhole limpet hemocyanin and with subcutaneous low-dose interleukin-2. Delayed-type hypersensitivity (DTH) tests for in-vivo immunomonitoring were performed at baseline and every four vaccinations thereafter. Two complete, two mixed and six partial responses, and five stable diseases were observed (overall response, 37.0%; clinical benefit, 55.5%). All 15 responders showed DTH positivity. A median overall survival of 22.9 months [95% confidence interval (CI): 13.4-61.3] for DTH-positive patients (19) and 4.8 months (95% CI: 3.9-11.9) for DTH-negative patients (8; log rank=7.26; P=0.007) was observed. The overall median overall survival was 16 months (95% CI: 9-33). Our results would seem to highlight a relationship between positive-DTH test and an improved survival.

Unexpected high response rate to traditional therapy after dendritic cell-based vaccine in advanced melanoma: update of clinical outcome and subgroup analysis.

We reviewed the clinical results of a dendritic cell-based phase II clinical vaccine trial in stage IV melanoma and analyzed a patient subgroup treated with standard therapies after stopping vaccination. From 2003 to 2009, 24 metastatic melanoma patients were treated with mature dendritic cells pulsed with autologous tumor lysate and keyhole limpet hemocyanin and low-dose interleukin-2. Overall response (OR) to vaccination was 37.5% with a clinical benefit of 54.1%. All 14 responders showed delayed type hypersensitivity positivity. Median overall survival (OS) was 15 months (95% CI, 8-33). Eleven patients underwent other treatments (3 surgery, 2 biotherapy, 2 radiotherapy, 2 chemotherapy, and 4 biochemotherapy) after stopping vaccination. Of these, 2 patients had a complete response and 5 a partial response, with an OR of 63.6%. Median OS was 34 months (range 16-61). Our results suggest that therapeutic DC vaccination could favor clinical response in patients after more than one line of therapy.

FRET microscopy autologous tumor lysate processing in mature dendritic cell vaccine therapy.

BACKGROUND: Antigen processing by dendritic cells (DC) exposed to specific stimuli has been well characterized in biological studies. Nonetheless, the question of whether autologous whole tumor lysates (as used in clinical trials) are similarly processed by these cells has not yet been resolved. METHODS: In this study, we examined the transfer of peptides from whole tumor lysates to major histocompatibility complex class II molecules (MHC II) in mature dendritic cells (mDC) from a patient with advanced melanoma. Tumor antigenic peptides-MHC II proximity was revealed by Förster Resonance Energy Transfer (FRET) measurements, which effectively extends the application of fluorescence microscopy to the molecular level (<100A). Tumor lysates were labelled with Alexa-488, as the donor, and mDC MHC II HLA-DR molecules were labelled with Alexa-546-conjugated IgG, as the acceptor. RESULTS: We detected significant energy transfer between donor and acceptor-labelled antibodies against HLA-DR at the membrane surface of mDC. FRET data indicated that fluorescent peptide-loaded MHC II molecules start to accumulate on mDC membranes at 16 hr from the maturation stimulus, steeply increasing at 22 hr with sustained higher FRET detected up to 46 hr. CONCLUSIONS: The results obtained imply that the patient mDC correctly processed the tumor specific antigens and their display on the mDC surface may be effective for several days. These observations support the rationale for immunogenic efficacy of autologous tumor lysates.

[Os(bpy)2(CO)(enIA)][OTf]2: a novel sulfhydryl-specific metal-ligand complex.

The synthesis and physical-chemical characterization of the metal-ligand complex [Os(bpy)2(CO)(enIA)][OTf]2 (where enIA = ethylenediamine iodoacetamide) with a sulfhydryl-specific functional group is described. The UV and visible absorption and luminescence emission, including lifetime and steady-state anisotropy, are reported for the free probe and the probe covalently linked to two test proteins. The spectroscopic properties of the probe are unaffected by chemical modification and subsequent covalent linkage to the proteins. The luminescence lifetime in aqueous buffer is approximately 200 ns and the limiting anisotropy is greater than 0.125, suggesting a potentially useful probe for biophysical investigations.