Oncolytic immunovirotherapy as an agnostic vaccination against glioblastoma

High grade gliomas (HGG) are aggressive tumors characterized by high heterogeneity, immune modulated environment and resiliency to standard treatments. Despite the HGG frequency is about 5 people every 100000, the overall survival (OS) rate at one year is extremely low. Standard of care treatments are invasive and never resolving while alternative approaches like immunotherapy and oncolytic virotherapy are showing promising outcomes. In this thesis are discussed the results obtained by the employment of an innovative therapy based on the combination of immunotherapy and oncolytic virotherapy. A new oncolytic HSV, R-115, has been evaluated for its ability to increase survival and rescue immunocompetent mice previously injected with murine HGG. The effects of R-115 immunovirotherapy were tested in two murine models, based on completely or partially targetable cells, respectively. The analyses were also focused on the ability of R-115 treatment to induce an immune memory which prevented secondary transplanted tumors and prolonged survival when administered as cell therapy. Altogether, our investigations candidate R-115 as a valuable alternative to common treatments and encourage further investigation to evaluate its potential for clinical application

Dysregulation of microRNA biogenesis in cancer: the impact of mutant p53 on Drosha complex activity

A widespread decrease of mature microRNAs is often observed in human malignancies giving them potential to act as tumor suppressors. Thus, microRNAs may be potential targets for cancer therapy. The global miRNA deregulation is often the result of defects in the miRNA biogenesis pathway, such as genomic mutation or aberrant expression/localization of enzymes and cofactors responsible of miRNA maturation. Alterations in the miRNA biogenesis machinery impact on the establishment and development of cancer programs. Accumulation of pri-microRNAs and corresponding depletion of mature microRNAs occurs in human cancers compared to normal tissues, strongly indicating an impairment of crucial steps in microRNA biogenesis. In agreement, inhibition of microRNA biogenesis, by depletion of Dicer1 and Drosha, tends to enhance tumorigenesis in vivo. The p53 tumor suppressor gene, TP53, is mutated in half of human tumors resulting in an oncogene with Gain-Of-Function activities. In this review we discuss recent studies that have underlined a role of mutant p53 (mutp53) on the global regulation of miRNA biogenesis in cancer. In particular we describe how a new transcriptionally independent function of mutant p53 in miRNA maturation, through a mechanism by which this oncogene is able to interfere with the Drosha processing machinery, generally inhibits miRNA processing in cancer and consequently impacts on carcinogenesis

Pupillometry via smartphone for low-resource settings

The photopupillary reflex regulates the pupil reaction to changing light conditions. Being controlled by the autonomic nervous system, it is a proxy for brain trauma and for the conditions of patients in critical care. A prompt evaluation of brain traumas can save lives. With a simple penlight, skilled clinicians can do that, whereas less specialized ones have to resort to a digital pupilometer. However, many low-income countries lack both specialized clinicians and digital pupilometers. This paper presents the early results of our study aiming at designing, prototyping and validating an app for testing the photopupillary reflex via Android, following the European Medical Device Regulation and relevant standards. After a manual validation, the prototype underwent a technical validation against a commercial Infrared pupilometer. As a result, the proposed app performed as well as the manual measurements and better than the commercial solution, with lower errors, higher and significant correlations, and significantly better Bland-Altman plots for all the pupillometry-related measures. The design of this medical device was performed based on our expertise in low-resource settings. This kind of environments imposes more stringent design criteria due to contextual challenges, including the lack of specialized clinicians, funds, spare parts and consumables, poor maintenance, and harsh environmental conditions, which may hinder the safe operationalization of medical devices. This paper provides an overview of how these unique contextual characteristics are cascaded into the design of an app in order to contribute to the Sustainable Development Goal 3 of the World Health Organization: Good health and well-being

TLR3 Activation of Intratumoral CD103+ Dendritic Cells Modifies the Tumor Infiltrate Conferring Anti-tumor Immunity

An important challenge in cancer immunotherapy is to expand the number of patients that benefit from immune checkpoint inhibitors (CI), a fact that has been related to the pre-existence of an efficient anti-tumor immune response. Different strategies are being proposed to promote tumor immunity and to be used in combined therapies with CI. Recently, we reported that intratumoral administration of naked poly A:U, a dsRNA mimetic empirically used in early clinical trials with some success, delays tumor growth and prolongs mice survival in several murine cancer models. Here, we show that CD103+ cDC1 and, to a much lesser extent CD11b+ cDC2, are the only populations expressing TLR3 at the tumor site, and consequently could be potential targets of poly A:U. Upon poly A:U administration these cells become activated and elicit profound changes in the composition of the tumor immune infiltrate, switching the immune suppressive tumor environment to anti-tumor immunity. The sole administration of naked poly A:U promotes striking changes within the lymphoid compartment, with all the anti-tumoral parameters being enhanced: a higher frequency of CD8+ Granzyme B+ T cells, (lower Treg/CD8+ ratio) and an important expansion of tumor-antigen specific CD8+ T cells. Also, PD1/PDL1 showed an increased expression indicating that neutralization of this axis could be exploited in combination with poly A:U. Our results shed new light to promote further assays in this dsRNA mimetic to the clinical field

Secondary Somatic Mutations in G-Protein-Related Pathways and Mutation Signatures in Uveal Melanoma

Background: Uveal melanoma (UM), a rare cancer of the eye, is characterized by initiating mutations in the genes G-protein subunit alpha Q (GNAQ), G-protein subunit alpha 11 (GNA11), cysteinyl leukotriene receptor 2 (CYSLTR2), and phospholipase C beta 4 (PLCB4) and by metastasis-promoting mutations in the genes splicing factor 3B1 (SF3B1), serine and arginine rich splicing factor 2 (SRSF2), and BRCA1-associated protein 1 (BAP1). Here, we tested the hypothesis that additional mutations, though occurring in only a few cases (\u201csecondary drivers\u201d), might influence tumor development. Methods: We analyzed all the 4125 mutations detected in exome sequencing datasets, comprising a total of 139 Ums, and tested the enrichment of secondary drivers in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that also contained the initiating mutations. We searched for additional mutations in the putative secondary driver gene protein tyrosine kinase 2 beta (PTK2B) and we developed new mutational signatures that explain the mutational pattern observed in UM. Results: Secondary drivers were significantly enriched in KEGG pathways that also contained GNAQ and GNA11, such as the calcium-signaling pathway. Many of the secondary drivers were known cancer driver genes and were strongly associated with metastasis and survival. We identified additional mutations in PTK2B. Sparse dictionary learning allowed for the identification of mutational signatures specific for UM. Conclusions: A considerable part of rare mutations that occur in addition to known driver mutations are likely to affect tumor development and progression

The cross-talk between the macro and micro-environment in precursor lesions of pancreatic cancer leads to new and promising circulating biomarkers

Pancreatic cancer (PC) is a clinically challenging tumor to combat due to its advanced stage at diagnosis as well as its resistance to currently available therapies. The absence of early symptoms and known detectable biomarkers renders this disease incredibly difficult to detect/manage. Recent advances in the understanding of PC biology have highlighted the importance of cancer-immune cell interactions, not only in the tumor micro-environment but also in distant systemic sites, like the bone marrow, spleen and circulating immune cells, the so-called macro-environment. The response of the macro-environment is emerging as a determining factor in tumor development by contributing to the formation of an increasingly immunogenic micro-environment promoting tumor homeostasis and progression. We will summarize the key events associated with the feedback loop between the t umor i mmune m icro- e nvironment (TIME) and the t umor i mmune ma croenvironment (TIMaE) in pancreatic precancerous lesions along with how it regulates disease development and progression. In addition, liquid biopsy biomarkers capable of diagnosing PC at an early stage of onset will also be discussed. A clearer understanding of the early crosstalk between micro-environment and macro-environment could contribute to identifying new molecular therapeutic targets and biomarkers, consequently improving early PC diagnosis and treatment

Dynamics of humoral and cellular response to three doses of anti-SARS-CoV-2 BNT162b2 vaccine in patients with hematological malignancies and older subjects

Background: Few data are available about the durability of the response, the induction of neutralizing antibodies, and the cellular response upon the third dose of the anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine in hemato-oncological patients. Objective: To investigate the antibody and cellular response to the BNT162b2 vaccine in patients with hematological malignancy. Methods: We measured SARS-CoV-2 anti-spike antibodies, anti-Omicron neutralizing antibodies, and T-cell responses 1 month after the third dose of vaccine in 93 fragile patients with hematological malignancy (FHM), 51 fragile not oncological subjects (FNO) aged 80-92, and 47 employees of the hospital (healthcare workers, (HW), aged 23-66 years. Blood samples were collected at day 0 (T0), 21 (T1), 35 (T2), 84 (T3), 168 (T4), 351 (T pre-3D), and 381 (T post-3D) after the first dose of vaccine. Serum IgG antibodies against S1/S2 antigens of SARS-CoV-2 spike protein were measured at every time point. Neutralizing antibodies were measured at T2, T3 (anti-Alpha), T4 (anti-Delta), and T post-3D (anti-Omicron). T cell response was assessed at T post-3D. Results: An increase in anti-S1/S2 antigen antibodies compared to T0 was observed in the three groups at T post-3D. After the third vaccine dose, the median antibody level of FHM subjects was higher than after the second dose and above the putative protection threshold, although lower than in the other groups. The neutralizing activity of antibodies against the Omicron variant of the virus was tested at T2 and T post-3D. 42.3% of FHM, 80,0% of FNO, and 90,0% of HW had anti-Omicron neutralizing antibodies at T post-3D. To get more insight into the breadth of antibody responses, we analyzed neutralizing capacity against BA.4/BA.5, BF.7, BQ.1, XBB.1.5 since also for the Omicron variants, different mutations have been reported especially for the spike protein. The memory T-cell response was lower in FHM than in FNO and HW cohorts. Data on breakthrough infections and deaths suggested that the positivity threshold of the test is protective after the third dose of the vaccine in all cohorts. Conclusion: FHM have a relevant response to the BNT162b2 vaccine, with increasing antibody levels after the third dose coupled with, although low, a T-cell response. FHM need repeated vaccine doses to attain a protective immunological response

Neutralizing antibodies to Omicron after the fourth SARS-CoV-2 mRNA vaccine dose in immunocompromised patients highlight the need of additional boosters

IntroductionImmunocompromised patients have been shown to have an impaired immune response to COVID-19 vaccines.MethodsHere we compared the B-cell, T-cell and neutralizing antibody response to WT and Omicron BA.2 SARS-CoV-2 virus after the fourth dose of mRNA COVID-19 vaccines in patients with hematological malignancies (HM, n=71), solid tumors (ST, n=39) and immune-rheumatological (IR, n=25) diseases. The humoral and T-cell responses to SARS-CoV-2 vaccination were analyzed by quantifying the anti-RBD antibodies, their neutralization activity and the IFN-γ released after spike specific stimulation.ResultsWe show that the T-cell response is similarly boosted by the fourth dose across the different subgroups, while the antibody response is improved only in patients not receiving B-cell targeted therapies, independent on the pathology. However, 9% of patients with anti-RBD antibodies did not have neutralizing antibodies to either virus variants, while an additional 5.7% did not have neutralizing antibodies to Omicron BA.2, making these patients particularly vulnerable to SARS-CoV-2 infection. The increment of neutralizing antibodies was very similar towards Omicron BA.2 and WT virus after the third or fourth dose of vaccine, suggesting that there is no preferential skewing towards either virus variant with the booster dose. The only limited step is the amount of antibodies that are elicited after vaccination, thus increasing the probability of developing neutralizing antibodies to both variants of virus.DiscussionThese data support the recommendation of additional booster doses in frail patients to enhance the development of a B-cell response directed against Omicron and/or to enhance the T-cell response in patients treated with anti-CD20