Prime and pull of T cell responses against cancer-exogenous antigens is effective against CPI-resistant tumors.

Neoantigen (neoAg)-based cancer vaccines expand preexisting antitumor immunity and elicit novel cancer-specific T cells. However, at odds with prophylactic vaccines, therapeutic antitumor immunity must be induced when the tumor is present and has already established an immunosuppressive environment capable of rapidly impairing the function of anticancer neoAg T cells, thereby leading to lack of efficacy. To overcome tumor-induced immunosuppression, we first vaccinated mice bearing immune checkpoint inhibitor (CPI)-resistant tumors with an adenovirus vector encoding a set of potent cancer-exogenous CD8 and CD4 T cell epitopes (Ad-CAP1), and then "taught" cancer cells to express the same epitopes by using a tumor-retargeted herpesvirus vector (THV-CAP1). Potent CD8 effector T lymphocytes were elicited by Ad-CAP1, and subsequent THV-CAP1 delivery led to a significant delay in tumor growth and even cure.

Development of a Potency Assay for Nous-209, a Multivalent Neoantigens-Based Genetic Cancer Vaccine.

Quality control testing of vaccines, including potency assessment, is critical to ensure equivalence of clinical lots. We developed a potency assay to support the clinical advancement of Nous-209, a cancer vaccine based on heterologous prime/boost administration of two multivalent viral vector products: GAd-209 and MVA-209. These consist of a mix of four Adeno (Great Ape Adenovirus; GAd) and four Modified Vaccinia Ankara (MVA) vectors respectively, each containing a different transgene encoding a synthetic polypeptide composed of antigenic peptide fragments joined one after the other. The potency assay employs quantitative Reverse Transcription PCR (RT-Q-PCR) to quantitatively measure the transcripts from the four transgenes encoded by each product in in vitro infected cells, enabling simultaneous detection. Results showcase the assay's robustness and biological relevance, as it effectively detects potency loss in one component of the mixture comparably to in vivo immunogenicity testing. This report details the assay's setup and validation, offering valuable insights for the clinical development of similar genetic vaccines, particularly those encoding synthetic polypeptides.

Phase I trial of viral vector based personalized vaccination elicits robust neoantigen specific antitumor T cell responses.

PURPOSE: Personalized vaccines targeting multiple neoantigens (nAgs) are a promising strategy for eliciting a diversified antitumor T cell response to overcome tumor heterogeneity. NOUS-PEV is a vector based personalized vaccine, expressing 60 nAgs and consists of priming with a non-human Great Ape Adenoviral vector (GAd20) followed by boosts with Modified Vaccinia Ankara (MVA). Here, we report data of a phase Ib trial of NOUS-PEV in combination with pembrolizumab in treatment naïve metastatic melanoma patients (NCT04990479). EXPERIMENTAL DESIGN: The feasibility of this approach was demonstrated by producing, releasing and administering to six patients 11 out of 12 vaccines within 8 weeks from biopsy collection to GAd20 administration. RESULTS: The regimen was safe, with no treatment-related serious adverse events observed and mild vaccine-related reactions. Vaccine immunogenicity was demonstrated in all evaluable patients receiving the prime/boost regimen, with detection of robust neoantigen specific immune responses to multiple neoantigens comprising both CD4 and CD8 T cells. Expansion and diversification of vaccine-induced TCR clonotypes was observed in the post-treatment biopsies of patients with clinical response providing evidence of tumor infiltration by vaccine-induced neoantigen-specific T cell. CONCLUSIONS: These findings indicate the ability of NOUS-PEV to amplify and broaden the repertoire of tumor reactive T cells to empower a diverse, potent and durable antitumor immune response. Finally, a gene signature indicative for reduced presence of activated T cells together with very poor expression of the antigen processing machinery (APM) genes has been identified in pre-treatment biopsies as a potential biomarker of resistance to the treatment.

Tumor Burden Dictates the Neoantigen Features Required to Generate an Effective Cancer Vaccine.

Tumor neoantigens (nAg) represent a promising target for cancer immunotherapy. The identification of nAgs that can generate T-cell responses and have therapeutic activity has been challenging. Here, we sought to unravel the features of nAgs required to induce tumor rejection. We selected clinically validated Great Ape-derived adenoviral vectors (GAd) as a nAg delivery system for differing numbers and combinations of nAgs. We assessed their immunogenicity and efficacy in murine models of low to high disease burden, comparing multi-epitope versus mono-epitope vaccines. We demonstrated that the breadth of immune response is critical for vaccine efficacy and having multiple immunogenic nAgs encoded in a single vaccine improves efficacy. The contribution of each single neoantigen was examined, leading to the identification of 2 nAgs able to induce CD8+ T cell-mediated tumor rejection. They were both active as individual nAgs in a setting of prophylactic vaccination, although to different extents. However, the efficacy of these single nAgs was lost in a setting of therapeutic vaccination in tumor-bearing mice. The presence of CD4+ T-cell help restored the efficacy for only the most expressed of the two nAgs, demonstrating a key role for CD4+ T cells in sustaining CD8+ T-cell responses and the necessity of an efficient recognition of the targeted epitopes on cancer cells by CD8+ T cells for an effective antitumor response. This study provides insight into understanding the determinants of nAgs relevant for effective treatment and highlights features that could contribute to more effective antitumor vaccines. See related Spotlight by Slingluff Jr, p. 382.

Personalized Cancer Vaccines Go Viral: Viral Vectors in the Era of Personalized Immunotherapy of Cancer.

The aim of personalized cancer vaccines is to elicit potent and tumor-specific immune responses against neoantigens specific to each patient and to establish durable immunity, while minimizing the adverse events. Over recent years, there has been a renewed interest in personalized cancer vaccines, primarily due to the advancement of innovative technologies for the identification of neoantigens and novel vaccine delivery platforms. Here, we review the emerging field of personalized cancer vaccination, with a focus on the use of viral vectors as a vaccine platform. The recent advancements in viral vector technology have led to the development of efficient production processes, positioning personalized viral vaccines as one of the preferred technologies. Many clinical trials have shown the feasibility, safety, immunogenicity and, more recently, preliminary evidence of the anti-tumor activity of personalized vaccination, fostering active research in the field, including further clinical trials for different tumor types and in different clinical settings.

The common H232 STING allele shows impaired activities in DNA sensing, susceptibility to viral infection, and in monocyte cell function, while the HAQ variant possesses wild-type properties.

Different innate immune pathways converge to Stimulator of interferon genes (STING) and trigger type I interferon responses after recognition of abnormal nucleic acids in the cells. This non-redundant function renders STING a major player in immunosurveillance, and an emerging target for cancer and infectious diseases therapeutics. Beyond somatic mutations that often occur in cancer, the human gene encoding STING protein, TMEM173 (STING1), holds great genetic heterogeneity; R232, HAQ (R71H-G230A-R293Q) and H232 are the most common alleles. Although some of these alleles are likely to be hypomorphic, their function is still debated, due to the available functional assessments, which have been performed in biased biological systems. Here, by using genetic background-matched models, we report on the functional evaluation of R232, HAQ and H232 variants on STING function, and on how these genotypes affect the susceptibility to clinically relevant viruses, thus supporting a potential contributing cause to differences in inter-individual responses to infections. Our findings also demonstrate a novel toll-like receptor-independent role of STING in modulating monocytic cell function and differentiation into macrophages. We further supported the interplay of STING1 variants and human biology by demonstrating how monocytes bearing the H232 allele were impaired in M1/M2 differentiation, interferon response and antigen presentation. Finally, we assessed the response to PD-1 inhibitor in a small cohort of melanoma patients stratified according to STING genotype. Given the contribution of the STING protein in sensing DNA viruses, bacterial pathogens and misplaced cancer DNA, these data may support the development of novel therapeutic options for infectious diseases and cancer.

Adenovirus Encoded Adjuvant (AdEnA) anti-CTLA-4, a novel strategy to improve Adenovirus based vaccines against infectious diseases and cancer.

Introduction: Virus vectored genetic vaccines (Vvgv) represent a promising approach for eliciting immune protection against infectious diseases and cancer. However, at variance with classical vaccines to date, no adjuvant has been combined with clinically approved genetic vaccines, possibly due to the detrimental effect of the adjuvant-induced innate response on the expression driven by the genetic vaccine vector. We reasoned that a potential novel approach to develop adjuvants for genetic vaccines would be to "synchronize" in time and space the activity of the adjuvant with that of the vaccine. Methods: To this aim, we generated an Adenovirus vector encoding a murine anti-CTLA-4 monoclonal antibody (Ad-9D9) as a genetic adjuvant for Adenovirus based vaccines. Results: The co-delivery of Ad-9D9 with an Adeno-based COVID-19 vaccine encoding the Spike protein resulted in stronger cellular and humoral immune responses. In contrast, only a modest adjuvant effect was achieved when combining the vaccine with the same anti-CTLA-4 in its proteinaceous form. Importantly, the administration of the adjuvant vector at different sites of the vaccine vector abrogates the immunostimulatory effect. We showed that the adjuvant activity of Ad-α-CTLA-4 is independent from the vaccine antigen as it improved the immune response and efficacy of an Adenovirus based polyepitope vaccine encoding tumor neoantigens. Discussion: Our study demonstrated that the combination of Adenovirus Encoded Adjuvant (AdEnA) with an Adeno-encoded antigen vaccine enhances immune responses to viral and tumor antigens, representing a potent approach to develop more effective genetic vaccines.

Vector Aided Microenvironment programming (VAMP): reprogramming the TME with MVA virus expressing IL-12 for effective antitumor activity.

BACKGROUND: Tumor microenvironment (TME) represents a critical hurdle in cancer immunotherapy, given its ability to suppress antitumor immunity. Several efforts are made to overcome this hostile TME with the development of new therapeutic strategies modifying TME to boost antitumor immunity. Among these, cytokine-based approaches have been pursued for their known immunomodulatory effects on different cell populations within the TME. IL-12 is a potent pro-inflammatory cytokine that demonstrates striking immune activation and tumor control but causes severe adverse effects when systemically administered. Thus, local administration is considered a potential strategy to achieve high cytokine concentrations at the tumor site while sparing systemic adverse effects. METHODS: Modified Vaccinia Ankara (MVA) vector is a potent inducer of pro-inflammatory response. Here, we cloned IL-12 into the genome of MVA for intratumoral immunotherapy, combining the immunomodulatory properties of both the vector and the cargo. The antitumor activity of MVA-IL-12 and its effect on TME reprogramming were investigated in preclinical tumor models. RNA sequencing (RNA-Seq) analysis was performed to assess changes in the TME in treated and distal tumors and the effect on the intratumoral T-cell receptor repertoire. RESULTS: Intratumoral injection of MVA-IL-12 resulted in strong antitumor activity with the complete remission of established tumors in multiple murine models, including those resistant to checkpoint inhibitors. The therapeutic activity of MVA-IL-12 was associated with very low levels of circulating cytokine. Effective TME reprogramming was demonstrated on treatment, with the reduction of immunosuppressive M2 macrophages while increasing pro-inflammatory M1, and recruitment of dendritic cells. TME switch from immunosuppressive into immunostimulatory environment allowed for CD8 T cells priming and expansion leading to tumor attack. CONCLUSIONS: Intratumoral administration of MVA-IL-12 turns immunologically 'cold' tumors 'hot' and overcomes resistance to programmed cell death protein-1 blockade.

Systems Biology Approaches for the Improvement of Oncolytic Virus-Based Immunotherapies.

Oncolytic virus (OV)-based immunotherapy is mainly dependent on establishing an efficient cell-mediated antitumor immunity. OV-mediated antitumor immunity elicits a renewed antitumor reactivity, stimulating a T-cell response against tumor-associated antigens (TAAs) and recruiting natural killer cells within the tumor microenvironment (TME). Despite the fact that OVs are unspecific cancer vaccine platforms, to further enhance antitumor immunity, it is crucial to identify the potentially immunogenic T-cell restricted TAAs, the main key orchestrators in evoking a specific and durable cytotoxic T-cell response. Today, innovative approaches derived from systems biology are exploited to improve target discovery in several types of cancer and to identify the MHC-I and II restricted peptide repertoire recognized by T-cells. Using specific computation pipelines, it is possible to select the best tumor peptide candidates that can be efficiently vectorized and delivered by numerous OV-based platforms, in order to reinforce anticancer immune responses. Beyond the identification of TAAs, system biology can also support the engineering of OVs with improved oncotropism to reduce toxicity and maintain a sufficient portion of the wild-type virus virulence. Finally, these technologies can also pave the way towards a more rational design of armed OVs where a transgene of interest can be delivered to TME to develop an intratumoral gene therapy to enhance specific immune stimuli.

Adenoviral-based vaccine promotes neoantigen-specific CD8+ T cell stemness and tumor rejection.

Upon chronic antigen exposure, CD8+ T cells become exhausted, acquiring a dysfunctional state correlated with the inability to control infection or tumor progression. In contrast, stem-like CD8+ T progenitors maintain the ability to promote and sustain effective immunity. Adenovirus (Ad)-vectored vaccines encoding tumor neoantigens have been shown to eradicate large tumors when combined with anti-programmed cell death protein 1 (αPD-1) in murine models; however, the mechanisms and translational potential have not yet been elucidated. Here, we show that gorilla Ad vaccine targeting tumor neoepitopes enhances responses to αPD-1 therapy by improving immunogenicity and antitumor efficacy. Single-cell RNA sequencing demonstrated that the combination of Ad vaccine and αPD-1 increased the number of murine polyfunctional neoantigen-specific CD8+ T cells over αPD-1 monotherapy, with an accumulation of Tcf1+ stem-like progenitors in draining lymph nodes and effector CD8+ T cells in tumors. Combined T cell receptor (TCR) sequencing analysis highlighted a broader spectrum of neoantigen-specific CD8+ T cells upon vaccination compared to αPD-1 monotherapy. The translational relevance of these data is supported by results obtained in the first 12 patients with metastatic deficient mismatch repair (dMMR) tumors vaccinated with an Ad vaccine encoding shared neoantigens. Expansion and diversification of TCRs were observed in post-treatment biopsies of patients with clinical response, as well as an increase in tumor-infiltrating T cells with an effector memory signature. These findings indicate a promising mechanism to overcome resistance to PD-1 blockade by promoting immunogenicity and broadening the spectrum and magnitude of neoantigen-specific T cells infiltrating tumors.

Maximizing cancer therapy via complementary mechanisms of immune activation: PD-1 blockade, neoantigen vaccination, and Tregs depletion.

BACKGROUND: A number of different immune pathways are involved in the effective killing of cancer cells, collectively named as the 'Cancer Immunity Cycle'. Anti-PD-1 checkpoint blockade (CPB) therapy is active on one of these pathways and reinvigorates anticancer T cell immunity, leading to long-term responses in a limited fraction of patients with cancer. We have previously shown that neoantigens-based adenovirus vectored vaccine in combination with anti-PD-1 further expands pre-existing anticancer immunity and elicits novel neoantigen-specific T cells thereby increasing efficacy to 50% of tumor clearance in mice. Here we added a third component to the CPB plus vaccine combination, which is able to modify the suppressive tumor microenvironment by reducing the number of tumor-infiltrating regulatory T cells (Tregs), as strategy for improving the therapeutic efficacy and overcoming resistance. METHODS: The antitumor efficacy of anti-PD-1, neoantigen vaccine and Treg modulating agents, either Bempegaldesleukin (BEMPEG: NKTR-214) or an anti-CTLA-4 mAb with Treg-depleting activity, was investigated in murine tumor models. We evaluated tumor growth in treated animals, neoantigen-specific T cells in tumors, tumor-infiltrating lymphocytes (TILs) and intratumoral Tregs. RESULTS: The addition of BEMPEG or anti-CTLA-4 to the combination of vaccine and anti-PD-1 led to complete eradication of large tumors in nearby 100% of treated animals, in association with expansion and activation of cancer neoantigen-specific T cells and reduction of tumor-infiltrating Tregs. CONCLUSION: These data support the notion that the integrated regulation of three steps of the cancer immunity cycle, including expansion of neoantigen-specific T cells, reversal of the exhausted T cell phenotype together with the reduction of intratumoral Tregs may represent a novel rationally designed drug combination approach to achieve higher cure rates.

VENUS, a Novel Selection Approach to Improve the Accuracy of Neoantigens' Prediction.

Neoantigens are tumor-specific antigens able to induce T-cell responses, generated by mutations in protein-coding regions of expressed genes. Previous studies demonstrated that only a limited subset of mutations generates neoantigens in microsatellite stable tumors. We developed a method, called VENUS (Vaccine-Encoded Neoantigens Unrestricted Selection), to prioritize mutated peptides with high potential to be neoantigens. Our method assigns to each mutation a weighted score that combines the mutation allelic frequency, the abundance of the transcript coding for the mutation, and the likelihood to bind the patient's class-I major histocompatibility complex alleles. By ranking mutated peptides encoded by mutations detected in nine cancer patients, VENUS was able to select in the top 60 ranked peptides, the 95% of neoantigens experimentally validated including both CD8 and CD4 T cell specificities. VENUS was evaluated in a murine model in the context of vaccination with an adeno vector encoding the top ranked mutations prioritized in the MC38 cell line. Efficacy studies demonstrated anti tumoral activity of the vaccine when used in combination with checkpoint inhibitors. The results obtained highlight the importance of a combined scoring system taking into account multiple features of each tumor mutation to improve the accuracy of neoantigen prediction.

Integrity of the Antiviral STING-mediated DNA Sensing in Tumor Cells Is Required to Sustain the Immunotherapeutic Efficacy of Herpes Simplex Oncolytic Virus.

The dichotomic contribution of cancer cell lysis and tumor immunogenicity is considered essential for effective oncovirotherapy, suggesting that the innate antiviral immune response is a hurdle for efficacy of oncolytic viruses. However, emerging evidence is resizing this view. By sensing cytosolic DNA, the cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) axis can both counteract viral spread and contribute to the elicitation of adaptive immunity via type I interferon responses. In this paper, we analyzed the tumor-resident function of Sting-mediated DNA sensing in a combined approach of oncovirotherapy and PD-1 immune checkpoint blockade, in an immunocompetent murine model. While supporting increased lytic potential by oncolytic HER2-retargeted HSV-1 in vitro and in vivo, Sting-knockout tumors showed molecular signatures of an immunosuppressive tumor microenvironment. These signatures were correspondingly associated with ineffectiveness of the combination therapy in a model of established tumors. Results suggest that the impairment in antiviral response of Sting-knockout tumors, while favoring viral replication, is not able to elicit an adequate immunotherapeutic effect, due to lack of immunogenic cell death and the inability of Sting-knockout cancer cells to promote anti-tumor adaptive immune responses. Accordingly, we propose that antiviral, tumor-resident Sting provides fundamental contributions to immunotherapeutic efficacy of oncolytic viruses.

A Genetic Vaccine Encoding Shared Cancer Neoantigens to Treat Tumors with Microsatellite Instability.

Tumors with microsatellite instability (MSI) are caused by a defective DNA mismatch repair system that leads to the accumulation of mutations within microsatellite regions. Indels in microsatellites of coding genes can result in the synthesis of frameshift peptides (FSP). FSPs are tumor-specific neoantigens shared across patients with MSI. In this study, we developed a neoantigen-based vaccine for the treatment of MSI tumors. Genetic sequences from 320 MSI tumor biopsies and matched healthy tissues in The Cancer Genome Atlas database were analyzed to select shared FSPs. Two hundred nine FSPs were selected and cloned into nonhuman Great Ape Adenoviral and Modified Vaccinia Ankara vectors to generate a viral-vectored vaccine, referred to as Nous-209. Sequencing tumor biopsies of 20 independent patients with MSI colorectal cancer revealed that a median number of 31 FSPs out of the 209 encoded by the vaccine was detected both in DNA and mRNA extracted from each tumor biopsy. A relevant number of peptides encoded by the vaccine were predicted to bind patient HLA haplotypes. Vaccine immunogenicity was demonstrated in mice with potent and broad induction of FSP-specific CD8 and CD4 T-cell responses. Moreover, a vaccine-encoded FSP was processed in vitro by human antigen-presenting cells and was subsequently able to activate human CD8 T cells. Nous-209 is an "off-the-shelf" cancer vaccine encoding many neoantigens shared across sporadic and hereditary MSI tumors. These results indicate that Nous-209 can induce the optimal breadth of immune responses that might achieve clinical benefit to treat and prevent MSI tumors. SIGNIFICANCE: These findings demonstrate the feasibility of an "off-the-shelf" vaccine for treatment and prevention of tumors harboring frameshift mutations and neoantigenic peptides as a result of microsatellite instability.

Involvement of miR-190b in Xbp1 mRNA Splicing upon Tocotrienol Treatment.

We previously demonstrated that apoptosis induced by tocotrienols (γ and δT3) in HeLa cells is preceded by Ca2+ release from the endoplasmic reticulum. This event is eventually followed by the induction of specific calcium-dependent signals, leading to the expression and activation of the gene encoding for the IRE1α protein and, in turn, to the alternative splicing of the pro-apoptotic protein sXbp1 and other molecules involved in the unfolded protein response, the core pathway coping with EndoR stress. Here, we showed that treatment with T3s induces the expression of a specific set of miRNAs in HeLa cells. Data interrogation based on the intersection of this set of miRNAs with a set of genes previously differentially expressed after γT3 treatment provided a few miRNA candidates to be the effectors of EndoR-stress-induced apoptosis. To identify the best candidate to act as the effector of the Xbp1-mediated apoptotic response to γT3, we performed in silico analysis based on the evaluation of the highest ∆ in Gibbs energy of different mRNA-miRNA-Argonaute (AGO) protein complexes. The involvement of the best candidate identified in silico, miR-190b, in Xbp1 splicing was confirmed in vitro using T3-treated cells pre-incubated with the specific miRNA inhibitor, providing a preliminary indication of its role as an effector of EndoR-stress-induced apoptosis.

Adenoviral vaccine targeting multiple neoantigens as strategy to eradicate large tumors combined with checkpoint blockade.

Neoantigens (nAgs) are promising tumor antigens for cancer vaccination with the potential of inducing robust and selective T cell responses. Genetic vaccines based on Adenoviruses derived from non-human Great Apes (GAd) elicit strong and effective T cell-mediated immunity in humans. Here, we investigate for the first time the potency and efficacy of a novel GAd encoding multiple neoantigens. Prophylactic or early therapeutic vaccination with GAd efficiently control tumor growth in mice. In contrast, combination of the vaccine with checkpoint inhibitors is required to eradicate large tumors. Gene expression profile of tumors in regression shows abundance of activated tumor infiltrating T cells with a more diversified TCR repertoire in animals treated with GAd and anti-PD1 compared to anti-PD1. Data suggest that effectiveness of vaccination in the presence of high tumor burden correlates with the breadth of nAgs-specific T cells and requires concomitant reversal of tumor suppression by checkpoint blockade.

Longitudinal transcriptomic and genetic landscape of radiotherapy response in canine melanoma.

Canine malignant melanoma (MM) is a highly aggressive tumour with a low survival rate and represents an ideal spontaneous model for the human counterpart. Considerable progress has been recently obtained, but the therapeutic success for canine melanoma is still challenging. Little is known about the mechanisms beyond pathogenesis and melanoma development, and the molecular response to radiotherapy has never been explored before. A faster and deeper understanding of cancer mutational processes and developing mechanisms are now possible through next generation sequencing technologies. In this study, we matched whole exome and transcriptome sequencing in four dogs affected by MM at diagnosis and at disease progression to identify possible genetic mechanisms associated with therapy failure. According to previous studies, a genetic similarity between canine MM and its human counterpart was observed. Several somatic mutations were functionally related to MAPK, PI3K/AKT and p53 signalling pathways, but located in genes other than BRAF, RAS and KIT. At disease progression, several mutations were related to therapy effects. Natural killer cell-mediated cytotoxicity and several immune-system-related pathways resulted activated opening a new scenario on the microenvironment in this tumour. In conclusion, this study suggests a potential role of the immune system associated to radiotherapy in canine melanoma, but a larger sample size associated with functional studies are needed.

Proteomic Analysis of Zn Depletion/Repletion in the Hormone-Secreting Thyroid Follicular Cell Line FRTL-5.

Zinc deficiency predisposes to a wide spectrum of chronic diseases. The human Zn proteome was predicted to represent about 10% of the total human proteome, reflecting the broad array of metabolic functions in which this micronutrient is known to participate. In the thyroid, Zn was reported to regulate cellular homeostasis, with a yet elusive mechanism. The Fischer Rat Thyroid Cell Line FRTL-5 cell model, derived from a Fischer rat thyroid and displaying a follicular cell phenotype, was used to investigate a possible causal relationship between intracellular Zn levels and thyroid function. A proteomic approach was applied to compare proteins expressed in Zn deficiency, obtained by treating cells with the Zn-specific chelator N,N,N',N'-tetrakis (2-pyridylmethyl) ethylene-diamine (TPEN), with Zn repleted cells. Quantitative proteomic analysis of whole cell protein extracts was performed using stable isotope dimethyl labelling coupled to nano-ultra performance liquid chromatography-mass spectrometry (UPLC-MS). TPEN treatment led to almost undetectable intracellular Zn, while decreasing thyroglobulin secretion. Subsequent addition of ZnSO4 fully reversed these phenotypes. Comparative proteomic analysis of Zn depleted/repleted cells identified 108 proteins modulated by either treatment. Biological process enrichment analysis identified functions involved in calcium release and the regulation of translation as the most strongly regulated processes in Zn depleted cells.

Immune Profiling of Premalignant Lesions in Patients With Lynch Syndrome.

Importance: Colorectal carcinomas in patients with Lynch syndrome (LS) arise in a background of mismatch repair (MMR) deficiency, display a unique immune profile with upregulation of immune checkpoints, and response to immunotherapy. However, there is still a gap in understanding the pathogenesis of MMR-deficient colorectal premalignant lesions, which is essential for the development of novel preventive strategies for LS. Objective: To characterize the immune profile of premalignant lesions from a cohort of patients with LS. Design, Setting, and Participants: Whole-genome transcriptomic analysis using next-generation sequencing was performed in colorectal polyps and carcinomas of patients with LS. As comparator and model of MMR-proficient colorectal carcinogenesis, we used samples from patients with familial adenomatous polyposis (FAP). In addition, a total of 47 colorectal carcinomas (6 hypermutants and 41 nonhypermutants) were obtained from The Cancer Genome Atlas (TCGA) for comparisons. Samples were obtained from the University of Texas MD Anderson Cancer Center and "Regina Elena" National Cancer Institute, Rome, Italy. All diagnoses were confirmed by genetic testing. Polyps were collected at the time of endoscopic surveillance and tumors were collected at the time of surgical resection. The data were analyzed from October 2016 to November 2017. Main Outcomes and Measures: Assessment of the immune profile, mutational signature, mutational and neoantigen rate, and pathway enrichment analysis of neoantigens in LS premalignant lesions and their comparison with FAP premalignant lesions, LS carcinoma, and sporadic colorectal cancers from TCGA. Results: The analysis was performed in a total of 28 polyps (26 tubular adenomas and 2 hyperplastic polyps) and 3 early-stage LS colorectal tumors from 24 patients (15 [62%] female; mean [SD] age, 48.12 [15.38] years) diagnosed with FAP (n = 10) and LS (n = 14). Overall, LS polyps presented with low mutational and neoantigen rates but displayed a striking immune activation profile characterized by CD4 T cells, proinflammatory (tumor necrosis factor, interleukin 12) and checkpoint molecules (LAG3 [lymphocyte activation gene 3] and PD-L1 [programmed cell death 1 ligand 1]). This immune profile was independent of mutational rate, neoantigen formation, and MMR status. In addition, we identified a small subset of LS polyps with high mutational and neoantigen rates that were comparable to hypermutant tumors and displayed additional checkpoint (CTLA4 [cytotoxic T-lymphocyte-associated protein 4]) and neoantigens involved in DNA damage response (ATM and BRCA1 signaling). Conclusions and Relevance: These findings challenge the canonical model, based on the observations made in carcinomas, that emphasizes a dependency of immune activation on the acquisition of high levels of mutations and neoantigens, thus opening the door to the implementation of immune checkpoint inhibitors and vaccines for cancer prevention in LS.

A simple microsatellite-based method for hazelnut oil DNA analysis.

Molecular food traceability requires continuous updates to identify more robust, efficient and affordable methodologies to guarantee food quality and safety and especially consumers' health. Available commercial kits are often unsatisfactory and require modifications to successfully detect single components on complex and transformed food matrices. Here we report a simple method for molecular traceability of cold-pressed hazelnut oil based on microsatellite DNA markers. Different genomic extraction methodologies were tested and a total genome pre-amplification step was applied on PCR-negative samples. PCR-capillary electrophoresis using nine microsatellites demonstrates the accuracy of the fingerprint analysis even for filtered oil.