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Introduction Cannabinoids are a group of terpenophenolic compounds derived from the Cannabis sativa L. plant. There is a growing body of evidence from cell culture and animal studies in support of cannabinoids possessing anticancer properties. Method A database search of peer reviewed articles published in English as full texts between January 1970 and April 2021 in Google Scholar, MEDLINE, PubMed and Web of Science was undertaken. References of relevant literature were searched to identify additional studies to construct a narrative literature review of oncological effects of cannabinoids in pre-clinical and clinical studies in various cancer types. Results Phyto-, endogenous and synthetic cannabinoids demonstrated antitumour effects both in vitro and in vivo. However, these effects are dependent on cancer type, the concentration and preparation of the cannabinoid and the abundance of receptor targets. The mechanism of action of synthetic cannabinoids, (−)-trans-Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD) has mainly been described via the traditional cannabinoid receptors; CB1 and CB2, but reports have also indicated evidence of activity through GPR55, TRPM8 and other ion channels including TRPA1, TRPV1 and TRPV2. Conclusion Cannabinoids have shown to be efficacious both as a single agent and in combination with antineoplastic drugs. These effects have occurred through various receptors and ligands and modulation of signalling pathways involved in hallmarks of cancer pathology. There is a need for further studies to characterise its mode of action at the molecular level and to delineate efficacious dosage and route of administration in addition to synergistic regimes.

Metabolic syndrome (MetS) is a pathological condition characterized by abdominal obesity, insulin resistance, hypertension, and hyperlipidemia. Sirtuin 1 (SIRT1), a highly conserved histone deacetylase, is characterized as a key metabolic regulator and protector against aging-associated pathologies, including MetS. In this study, we investigate the therapeutic potential of activating SIRT1 using small activating RNAs (saRNA), thereby reducing inflammatory-like responses and re-establishing normal lipid metabolism. SIRT1 saRNA significantly increased SIRT1 messenger RNA (mRNA) and protein levels in both lipopolysaccharide-stimulated and nonstimulated macrophages. SIRT1 saRNA significantly decreased inflammatory-like responses, by reducing mRNA levels of key inflammatory cytokines, such as Tumor Necrosis Factor alpha, Interleukin 1 beta (IL-1β), Interleukin 6 (IL-6), and chemokines Monocyte Chemoattractant Protein-1 and keratinocyte chemoattractant. SIRT1 overexpression also significantly reduced phosphorylation of nuclear factor-κB and c-Jun N-terminal kinase, both key signaling molecules for the inflammatory pathway. To investigate the therapeutic effect of SIRT1 upregulation, we treated a high-fat diet model with SIRT1 saRNA conjugated to a transferrin receptor aptamer for delivery to the liver and cellular internalization. Animals in the SIRT1 saRNA treatment arm demonstrated significantly decreased weight gain with a significant reduction in white adipose tissue, triglycerides, fasting glucose levels, and intracellular lipid accumulation. These suggest treatment-induced changes to lipid and glucose metabolism in the animals. The results of this study demonstrate that targeted activation of SIRT1 by saRNAs is a potential strategy to reverse MetS.

This study aimed to identify drought-tolerant genotypes and to evaluate and compare the response of genotypes under normal conditions and humidity stress. The experiment was conducted in a Randomized Complete Block Design (RCBD) on 12 commercial single cross hybrids of maize (Zea mays L.) with three replications in two separate experiments under normal and stress conditions. GT biplot was used to compare genotypes under normal conditions and humidity stress. Based on the polygon diagrams’ graphical analysis, KSC206, KSC704, KSC705 and KSC706 genotypes were identified as desirable hybrids. The ranking diagram of genotypes based on ideal genotype also showed that the KSC704 genotype had high desirability in all evaluated traits in normal and stress conditions. TOL, MP, HARM, GMP, SSI and STI indices were used to identify drought-tolerant genotypes, and the genotypes were ranked based on this index. Based on this, KSC260, SC302 and KSC400 hybrids were selected as resistant hybrids. Based on the correlation analysis between drought-tolerance indices, a positive correlation was observed between MP, GMP, HARM and STI indices. Based on the analysis of the PCA on the indices, the first and second principal components were given the titles of grain yield tolerance component under humidity stress conditions and grain yield stability component under normal humidity conditions, respectively. KSC704 was superior to other hybrids in terms of grain yield under normal conditions and stress, and the KSC260 hybrid was identified as a tolerant hybrid in terms of all studied traits under drought stress.

Background There is an ongoing debate on the feasibility, safety, and oncological efficacy of the associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) technique. The aim of this study was to compare ALPPS, two-staged hepatectomy (TSH), and portal vein embolization (PVE)/ligation (PVL) using updated traditional meta-analysis and network meta-analysis (NMA). Data sources Electronic databases were used in a systematic literature search. Updated traditional meta-analysis and NMA were performed and compared. Mortality and major morbidity were selected as primary outcomes. Results Nineteen studies including 1200 patients were selected from the pool of 436 studies. Of these patients, 315 (31%) and 702 (69%) underwent ALPPS and portal vein occlusion (PVO), respectively. Ninety-day mortality based on updated traditional meta-analysis, subgroup analysis of the randomized controlled trials (RCTs), and both Bayesian and frequentist NMA did not demonstrate significant differences between the ALPPS cohort and the PVE, PVL, and TSH cohorts. Moreover, analysis of RCTs did not demonstrate significant differences of major morbidity between the ALPPS and PVO cohorts. The ALPPS cohort demonstrated significantly more favorable outcomes in hypertrophy parameters, time to operation, definitive hepatectomy, and R0 margins rates compared with the PVO cohort. In contrast, 1-year disease-free survival was significantly higher in the PVO cohort compared to the ALPPS cohort. Conclusions This study is the first to use updated traditional meta-analysis and both Bayesian and frequentist NMA and demonstrated no significant differences in 90-day mortality between the ALPPS and other hepatic hypertrophy approaches. Furthermore, two high quality RCTs including 147 patients demonstrated no significant differences in major morbidity between the ALPPS and PVO cohorts.

Advances in surgery, peri-operative care and systemic chemotherapy have not significantly improved the prognosis of pancreatic cancer for several decades. Early clinical trials of immunotherapy have yielded disappointing results proposing other means by which the tumour microenvironment serves to decrease the immune response. Additionally, the emergence of various subtypes of pancreatic cancer has emerged as a factor for treatment responses with immunogenic subtypes carrying a better prognosis. Herein we discuss the reasons for the poor response to checkpoint inhibitors and outline a rationale why combination treatments are likely to be most effective. We review the therapies which could provide optimal synergistic effects to immunotherapy including chemotherapy, agents targeting the stroma, co-stimulatory molecules, vaccinations and methods of immunogenic tumour priming including radiofrequency ablation. Finally, we discuss reasons why peri-operative and in particular neoadjuvant combination treatments are likely to be most effective and should be considered for early clinical trials.

Targeted delivery of oligonucleotides to liver hepatocytes using N-acetylgalactosamine (GalNAc) conjugates that bind to the asialoglycoprotein receptor has become a breakthrough approach in the therapeutic oligonucleotide field. This technology has led to the approval of givosiran for the treatment of acute hepatic porphyria, and there are another seven conjugates in registrational review or phase 3 trials and at least another 21 conjugates at earlier stages of clinical development. This review highlights some of the recent chemical and preclinical advances in this space, leading to a large number of clinical candidates against a diverse range of targets in liver hepatocytes. The review focuses on the use of this delivery system for small interfering RNAs (siRNAs) and antisense molecules that cause downregulation of target mRNA and protein. A number of other approaches such as anti-microRNAs and small activating RNAs are starting to exploit the technology, broadening the potential of this approach for therapeutic oligonucleotide intervention.

Hepatocellular carcinoma (HCC) is the most common primary liver tumor and has been considered a very immunogenic tumor. The treatment with radiofrequency ablation (RFA) has been established as the standard ablative therapy for early HCC, and is currently recognized as the main ablative tool for HCC tumors < 5 cm in size; however, progression and local recurrence remain the main disadvantages of this approach. To solve this clinical problem, recent efforts were concentrated on multimodal treatment, combining different strategies, including the combination of RFA and immunotherapy. This article reviewed the combination treatment of RFA with immunotherapy and found that this treatment strategy leads to an increased response of anti-tumor T cells, significantly reduces the risk of recurrence and improves survival rates compared to RFA alone. This review highlighted scientific evidence that supports the current recommendations for pre-clinical studies, and discuss the need for further research on this topic.

In this study, 12 maize hybrids were planted and evaluated to determine the effect of genotype and genotype-environment interaction (GEI) base GGE (genotype plus genotype-by-environment) using a Graphical biplot technique in four research stations (Arak, Birjand, Shiraz and Karaj) within two years using a Randomized Complete Blocks Design (RCBD). The combined analysis of variance showed that the effects of the environment, genotype and genotype-environment interaction (GEI) were significant in the one percent probability level. GGE biplot results indicated that the first and second principal components (PC1 and PC2) explained more than 83% of the grain performance variation. Simultaneous study of grain performance and hybrid stability using the biplot of average environment coordinates showed that the KSC705 genotype had the highest yield and stability. Polygon view divided the studied areas into two mega-environments (MEs) and identified the best genotypes in each mega-environment (ME). In the first mega-environment (ME1), the Karaj and Shiraz with KSC706 and KSC400 genotypes were detected, and were the best; and in the second mega-environment (ME2), Arak and Birjand with KSC704 and KSC707 genotypes performed better. The biplot graph for the correlation between the genotypes categorized the studied hybrids into four groups positively related to each other based on the angles between vectors. The KSC704 and KSC707 genotypes were desirable in the yield in Shiraz and Karaj and KSC706 were in Arak and Birjand. Additionally, Arak-Birjand, Karaj-Shiraz showed a positive and significant correlation. Birjand and Karaj had most genotype interaction with each other.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies; it preferentially metastasizes to the liver and is the main cause of death from this disease. In previous studies, small activating RNA against CCAAT/enhancer-binding protein-α (C/EBPα-saRNA) demonstrated efficacy of PDAC in a local subcutaneous tumor model. In this study, we focused on the efficacy of C/EBPα-saRNA in advanced stage PDAC. For targeted delivery, we selected a new anti-transferrin receptor aptamer (TR14), which demonstrated a high binding affinity to target proteins. The TR14 aptamer was internalized with clathrin-mediated endocytosis, distributed in early endosome, late endosome, and lysosome subcellularly. To investigate its anti-tumor effects to advanced PDAC, we conjugated C/EBPα-saRNA to TR14. Treatment of pancreatic cancer cells with the conjugates upregulated expression of C/EBPα and its downstream target p21, and inhibited cell proliferation. For in vivo assays, we established an advanced PDAC mouse model by engrafting luciferase reporter-PANC-1 cells directly into the livers of non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. After treatment of aptamer-C/EBPα conjugates, we observed significant reduction of tumor growth in this advanced PDAC mouse model. Combinational treatment of the conjugates with gemcitabine also demonstrated enhanced anti-tumor effects in advanced PDAC. This suggests that aptamer-C/EBPα conjugates could be used as an adjuvant, along with other conventional anti-cancer drugs in advanced PDAC. In conclusion, targeted delivery of C/EBPα-saRNAs by aptamers might have potential therapeutic effects in advanced PDAC. Keywords: C/EBPα-saRNA, targeted delivery, transferrin receptor aptamer, clathrin-mediated endocytosis, advanced PDAC, anti-tumor effects, adjuvant, truncation, albumin affinity tag

Purpose: To evaluate the mechanisms of how therapeutic upregulation of the transcription factor, CCAAT/enhancer-binding protein alpha (C/EBPα), prevents tumor progression in patients with advanced hepatocellular carcinoma (HCC) and in different mouse tumor models. Experimental Design: We conducted a phase I trial in 36 patients with HCC (NCT02716012) who received sorafenib as part of their standard care, and were given therapeutic C/EBPα small activating RNA (saRNA; MTL-CEBPA) as either neoadjuvant or adjuvant treatment. In the preclinical setting, the effects of MTL-CEBPA were assessed in several mouse models, including BNL-1ME liver cancer, Lewis lung carcinoma (LLC), and colon adenocarcinoma (MC38). Results: MTL-CEBPA treatment caused radiologic regression of tumors in 26.7% of HCC patients with an underlying viral etiology with 3 complete responders. MTL-CEBPA treatment in those patients caused a marked decrease in peripheral blood monocytic myeloid-derived suppressor cell (M-MDSC) numbers and an overall reduction in the numbers of protumoral M2 tumor-associated macrophages (TAM). Gene and protein analysis of patient leukocytes following treatment showed CEBPA activation affected regulation of factors involved in immune-suppressive activity. To corroborate this observation, treatment of all the mouse tumor models with MTL-CEBPA led to a reversal in the suppressive activity of M-MDSCs and TAMs, but not polymorphonuclear MDSCs (PMN-MDSC). The antitumor effects of MTL-CEBPA in these tumor models showed dependency on T cells. This was accentuated when MTL-CEBPA was combined with checkpoint inhibitors or with PMN-MDSC–targeted immunotherapy. Conclusions: This report demonstrates that therapeutic upregulation of the transcription factor C/EBPα causes inactivation of immune-suppressive myeloid cells with potent antitumor responses across different tumor models and in cancer patients. MTL-CEBPA is currently being investigated in combination with pembrolizumab in a phase I/Ib multicenter clinical study (NCT04105335).

BackgroundMTL-CEBPA is a novel immunotherapy targeting the myeloid cell lineage which has shown promising clinical activity as monotherapy and combination therapy with tyrosine kinase inhibitors in hepatocellular carcinoma (HCC). Immunosuppressive myeloid cells are associated with worse outcomes to checkpoint inhibitors. Pre-clinical data have shown that MTL-CEBPA potentiates the oncological effect of PD-1 inhibitors.MethodsThis phase 1A/B, first-in-human, open-label, multicenter study evaluates the safety, tolerability, PK, and efficacy of MTL-CEBPA in combination with a pembrolizumab in adult patients with advanced solid tumours across 3 dose cohorts (70mg/98mg/130mg/m2 MTL-CEBPA once weekly for 3 consecutive weeks with final week break per cycle, with 200mg pembrolizumab every 3 weeks). The primary endpoint is safety and ORR; key secondary endpoints include PK, CR rate & DCR. Key inclusion criteria: Patients with advanced solid tumours who have progressed on standard of care therapy or for whom no standard therapy is available, measurable disease, ECOG PS 3 months. A dose exploration will determine the maximum tolerated dose (MTD) or recommended phase 2 dose (RP2D).Results10 pts (3 men, 7 women; median age 50.5yrs), all with different tumor types (1 each of triple negative breast, methothelioma, squamous thymic, cholangiocarcinoma, eccrine, fibrolamellar hepatocellular, colorectal, pancreatic and 2 platinum resistant high-grade serous ovarian). 4 pts had ≥4 prior lines of treatment. All pts reported treatment-related AEs, 7 pts reported AEs considered related to MTL-CEBPA only and all were grade 1 or 2. The most common was nausea (n=3) followed by anaemia, headache, insomnia, neutropenia, pyrexia, transaminase increase and ventricular extrasystole (all n=1). Five pts reported AEs considered related to pembrolizumab only, 2AEs in 1 pt only were grade 3 (ALT and AST increases) There were no DLTs, SAEs or AEs leading to discontinuation or to death in the study. Tumor response was evaluated in 9 pts. 2 pts had a PR (epithelioid mesothelioma at 2 months with 83% tumour reduction, pt ongoing at 9 months & serous ovarian cancer at 2 months with 69% reduction in tumour, pt progressed at 6 months). Three pts had SD, 4 pts had PD as BOR, and 4 pts are continuing to receive treatment.ConclusionsMTL-CEBPA in combination with pembrolizumab demonstrated manageable toxicity at the dose levels tested and has shown antitumor activity. MTD was not reached and RP2D was determined at 130mg/m2 on day 1, 8 and 15 of a 28 day cycle. Enrolment into the dose expansion is ongoing.Trial RegistrationThis study was registered with ClinicalTrials.gov, number NCT04105335.Ethics ApprovalThe study was approved by the North East - Newcastle & North Tyneside 2 Research Ethics Committee, approval number 19/NE/0312.

RNA activation (RNAa) is a mechanism whereby RNA oligos complementary to genomic sequences around the promoter region of genes increase the transcription output of their target gene. Small activating RNA (saRNA) mediate RNAa through interaction with protein co-factors to facilitate RNA polymerase II activity and nucleosome remodeling. As saRNA are small, versatile and safe, they represent a new class of therapeutics that can rescue the downregulation of critical genes in disease settings. This review highlights our current understanding of saRNA biology and describes various examples of how saRNA are successfully used to treat various oncological, neurological and monogenic diseases. MTL-CEBPA, a first-in-class compound that reverses CEBPA downregulation in oncogenic processes using CEBPA-51 saRNA has entered clinical trial for the treatment of hepatocellular carcinoma (HCC). Preclinical models demonstrate that MTL-CEBPA reverses the immunosuppressive effects of myeloid cells and allows for the synergistic enhancement of other anticancer drugs. Encouraging results led to the initiation of a clinical trial combining MTL-CEBPA with a PD-1 inhibitor for treatment of solid tumors.

The transcription factor CEBPA is a master regulator of liver homeostasis, myeloid cell differentiation and is downregulated in several oncogenic diseases. MTL-CEBPA is a small activating RNA drug which upregulates gene expression of CEBPA for treatment of hepatocellular carcinoma (HCC). We investigate whether MTL-CEBPA has immune modulatory effects by combining MTL-CEBPA with an anti-PD-1 checkpoint inhibitor (CPI) and/or radiofrequency ablation (RFA) in two preclinical models. First, mice with two flanks of HCC tumors (BNL) were treated with combinations of RFA (right flank), anti-PD-1 or MTL-CEBPA. The reduction of the left flank tumors was most pronounced in the group treated with RFA+anti-PD1+MTL-CEBPA and 7/8 animals responded. This was the only group with a significant increase in CD8+ and CD49b+/CD45+ tumor infiltrating lymphocytes (TIL). Second, a combination of anti-PD-1+MTL-CEBPA was tested in a CT26 colon cancer model and this treatment significantly reduced tumor size, modulated the tumor immune microenvironment and increased TILs. These data suggest a clinical role for combination treatment with CPIs, RFA and MTL-CEBPA through synergistic priming of the immune tumor response, enabling RFA and CPIs to have a pronounced anti-tumor effect including activity in non-treated tumors in the case of RFA.

BackgroundMost patients with pancreatic ductal adenocarcinoma (PDAC) are metastatic at presentation with dismal prognosis warranting improved systemic therapy options. Longitudinal sampling for assessment of treatment response poses a challenge for validating novel therapies. In this feasibility study, we evaluate the role of endoscopic ultrasound (EUS)-guided serial fine-needle aspiration biopsies (FNABs) to study the molecular changes associated with radiofrequency ablation (RFA).MethodsTwo stage III inoperable gemcitabine-treated PDAC patients were recruited in the treatment arm of ARDEO (ethically approved Phase-II prospective randomized clinical study). Post examination, targeted RFA was delivered thrice, and sequential FNABs of tumour were taken before and after treatment and analysed using a custom NanoString panel (144 genes) consisting of cancer and cancer-associated fibroblast (CAFs) subtypes and immune changes. CAF culture was established from one FNAB and characterised by immunofluorescence and immunoblotting.ResultsSerial EUS-RFA treatments were well tolerated with no complications. Both patients had stable disease immediately after EUS-RFA, however, with different survival outcomes. Two-course RFA led to upregulation of CD1E gene (involved in antigen presentation) in both, patient 1 and 2 (4.5 and 3.9-fold) compared to baseline. Patient 1 showed increased T cell genes (CD4 – 8.7-fold, CD8 – 35.7-fold), cytolytic function (6.4-fold) and inflammatory response (8-fold). Greater than 2-fold upregulation of immune checkpoint genes was observed post 2nd RFA in patient 1, patient 2 or both. Further, two-course RFA led to increased PDGFRa (4.5-fold) and CAF subtypes B and C genes in patient 1 and subtypes A, B and D genes in patient 2. Patient2-derived CAFs post 1st RFA showed expression of PDGFRα, POSTN and MYH11 proteins. Finally, RFA led to downregulation of classical PDA subtype in both patients. ConclusionsFor the first time, this feasibility study validates longitudinal sampling by EUS-FNABs as an appropriate research tool to study tumour microenvironmental changes associated with local PDAC immunomodulatory treatments like RFA.

Excessive or inappropriate inflammatory responses can cause serious and even fatal diseases. The CCAAT/enhancer-binding protein alpha (CEBPA) gene encodes C/EBPα, a transcription factor that plays a fundamental role in controlling maturation of the myeloid lineage and is also expressed during the late phase of inflammatory responses when signs of inflammation are decreasing. MTL-CEBPA, a small activating RNA targeting for upregulation of C/EBPα, is currently being evaluated in a phase 1b trial for treatment of hepatocellular carcinoma. After dosing, subjects had reduced levels of pro-inflammatory cytokines, and we therefore hypothesized that MTL-CEBPA has anti-inflammatory potential. The current study was conducted to determine the effects of C/EBPα saRNA - CEBPA-51 - on inflammation in vitro and in vivo after endotoxin challenge. CEBPA-51 led to increased expression of the C/EBPα gene and inhibition of pro-inflammatory cytokines in THP-1 monocytes previously stimulated by E. coli-derived lipopolysaccharide (LPS). Treatment with MTL-CEBPA in an LPS-challenged humanized mouse model upregulated C/EBPα mRNA, increased neutrophils, and attenuated production of several key pro-inflammatory cytokines, including TNF-α, IL-6, IL-1β, and IFN-γ. In addition, a Luminex analysis of mouse serum revealed that MTL-CEBPA reduced pro-inflammatory cytokines and increased the anti-inflammatory cytokine IL-10. Collectively, the data support further investigation of MTL-CEBPA in acute and chronic inflammatory diseases where this mechanism has pathogenic importance., MTL-CEBPA is a small activating RNA that targets the upregulation of transcription factor C/EBPα, currently being evaluated in a phase 1b trial for treatment of hepatocellular carcinoma. Zhou et al. demonstrate that MTL-CEBPA increases the expression of C/EBPα and displays anti-inflammatory effects in a tissue culture system and a humanized mouse model.

RNA activation (RNAa) allows specific gene upregulation mediated by a small activating RNA (saRNA). Harnessing this process would help in developing novel therapeutics for undruggable diseases. Since its discovery in mid 2000s, improvements of saRNA design, synthetic chemistry and understanding of the biology have matured the way to apply RNAa. Indeed, MiNA therapeutics Ltd has conducted the first RNAa clinical trial for advanced hepatocellular carcinoma patients with promising outcomes. However, to fully realize the RNAa potential better saRNA delivery strategies are needed to target other diseases. Currently, saRNA can be delivered in vivo by lipid nanoparticles, dendrimers, lipid and polymer hybrids and aptamers. Further developing these delivery technologies and novel application of RNAa will prove to be invaluable for new treatment development.

Human glioblastoma (GBM) is the most aggressive malignancy of the CNS, with less than 5% survival. Despite great efforts to find effective therapeutics, current options remain very limited. To develop a targeted cancer therapeutic, we selected RNA aptamers against platelet-derived growth factor receptor α (PDGFRα), which is a receptor tyrosine kinase. One RNA aptamer (PDR3) with high affinity (0.25 nM) showed PDGFRα specificity and was internalized in U251-MG cells. Following treatment with the PDR3 aptamer, expression of the transcription factor STAT3 (signal transducer and activator of transcription 3) was inhibited, whereas the expression of the histone demethylase JMJD3 and the tumor suppressor p53 were upregulated. PDR3 also upregulated serine phosphorylation of p53, which subsequently mediated apoptosis through the death receptors: tumor necrosis factor (TNF)-related apoptosis-inducing ligand receptors 1/2 (TRAIL-R1/R2), Fas-associated via death domain (FADD), and Fas. PDR3 significantly decreased cell viability in a dose-dependent manner. Furthermore, translocation of PDR3 into the nucleus induced hypomethylation at the promoters of cyclin D2. To assess the feasibility of targeted delivery, we conjugated PDR3 aptamer with STAT3-siRNA for a chimera. The PDR3-siSTAT3 chimera successfully inhibited the expression of target genes and showed significant inhibition of cell viability. In summary, our results show that well-tailored RNA aptamers targeting the PDGFRα-STAT3 axis have the potential to act as anti-cancer therapeutics in GBM. Keywords: PDGFRα aptamer, STAT3, p53, apoptosis, GBM

Hepatocellular carcinoma (HCC) is generally accompanied by high mortality and low cure rate. CCAAT enhancer-binding proteins (CEBPs) are transcriptional regulators that play a key role in maintaining liver function. Altered expression of C/EBPα and C/EBPβ occurs in many tumours including HCC. saRNAs are small double-stranded RNAs that enhance target gene expression at the transcriptional level. In this report, we activate CEPBA with saRNAs and suppress CEBPB with siRNAs in cells that represent three different degrees of HCC. We performed functional assays to investigate the effects of enhancing C/EBPα and its downstream targets, p21 and albumin across these lines. We also used Mass-spectrometry (MS) subsequent to a ChIP pull-down assay to characterise the components of the protein complex involved in regulating saRNA function. Putative saRNA interacting protein candidates that were identified by MS were knocked-down with siRNAs to investigate its impact on saRNA activity. We confirmed CEBPA-saRNA decreased proliferation and migration in the differentiated lines (HepG3/Hep3B). The undifferentiated line (PLCPRF5) showed saRNA-induced increase in CEBPA but with no loss in proliferation. This effect was reversed when CEBPB was suppressed with CEBPB-siRNA. When interrogating saRNA mode of action; three saRNA interacting proteins, CTR9, HnRNPA2/B1 and DDX5 were identified by MS. Targeted knock-down of these two proteins (by siRNA) abrogated saRNA activity. This study provides insight into how different HCC lines are affected by CEBPA-saRNAs and that endogenous abundance of CEBPB and saRNA accessory proteins may dictate efficacy of CEBPA-saRNA when used in a therapeutic context.

cGAS-cGAMP-STING is essential for sensing foreign DNA from pathogens or self-DNA from dying cancer cells. Activation of this pathway is critical for the innate immune response to cancer and is necessary for the full efficacy of various cancer treatments including checkpoint and PARP inhibitors, radiotherapy, and CAR T-cells. Intense efforts have focused on triggering this pathway with cGAMP analogs, which are small-molecule activators of STING. However, recent reports show that many cancer cells downregulate STING by promoter methylation. Furthermore, T-cells activated by cGAMP undergo apoptosis, which limits the efficacy of T-cells based tumor eradication. Indeed, emerging evidence suggests that the critical cells in which STING activation achieves maximal anti-cancer efficacy is myeloid cells in the tumor microenvironment (TME). saRNAs are small double-stranded complementary RNA oligos that increase target mRNA expression, they directly modulate traditional "undruggable" targets such as STING. Using single cell RNA sequencing data, we demonstrated that STING expression is decreased in myeloid cells within the TME of cancer patients. Therefore, our hypothesis was that saRNAs targeted against STING would restore its expression to levels that are necessary for pathway activation either alone or in combination with other drugs, including cGAMP analogs. Using our proprietary algorithm, we designed saRNA sequences that are complementary to positions +2000 to -2000 nucleotides around the transcriptional start site of STING. The lead sequence (named saSTING) was further optimized by our discovery pipeline. Upregulation of STING mRNA was detected up to 168 hours post-transfection of saSTING in multiple cell lines, with an increase in the range of 4 to 11 fold. The upregulation of STING expression was further validated at the protein level. Strand and sequence specificity of saSTING was demonstrated by mutational analysis. Concerning the mechanism of transcriptional activation via saSTING, we show that saSTING regulates STING expression by increasing both nascent pre-mRNA and mature mRNA. In essence, saSTING causes transcriptional activation across the whole STING locus, which is recapitulated in an upregulation of spliced and polyadenylated (mature) STING mRNA. Development candidate MTL-STING encapsulates saSTING in NOV340 liposomes for in vivo administration. NOV340 liposomes have previously been clinically validated to efficiently deliver saRNA to myeloid cells, which is the critical cellular subset to benefit from STING activation from an anti-tumor perspective. MTL-STING is pre-clinical development as a combination treatment administered intra-tumorally and is expected to enter Phase 1 in 2023. Citation Format: Choon Ping Tan, Konstantina Skourti Stathaki, Brid M Ryan, Valentí Gomez, Laura Sinigaglia, Siv Anita Hegre, Robert Habib, John J Rossi, Nagy A Habib. MTL-STING restores endogenous STING expression for improving efficacy of cancer therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 382.

4093 Background: Early-stage hepatocellular carcinoma (HCC) can be treated with liver resection (LR), but up to 70% of patients experience relapse within two years after surgery. Despite their established use in advanced disease, immune checkpoint inhibitors (ICPI) are still under investigation in the peri-operatory setting. Methods: PRIME‐HCC is a phase Ib study investigating safety and bioactivity of the nivolumab (3 mg/kg, day 1 and day 22) plus ipilimumab (1mg/kg, day 1 only) combination (Nivo+Ipi) prior to LR in early-stage HCC. The primary safety analysis assessed treatment-related adverse events (trAE) and delays to surgery. Secondary endpoint included objective response rate (ORR) by RECIST v1.1 and pathologic response rate on resection specimens. Results: At data censoring on the 27th of January 2022, 17 patients were enrolled, of whom 82% (n = 14) were male, with a median age of 64 years (range 47-76). Performance status was 0 in 88% of patients (n = 15) according to the Eastern Cooperative Oncology Group scale. Liver cirrhosis was found in 65% (n = 11) of the patients, mostly secondary to viral hepatitis (41%, n = 7). All patients were Child-Pugh A, with 53% (n = 9) classified as albumin-bilirubin (ALBI) grade 2, and the rest grade 1. Median tumour diameter was 3.4 cm (interquartile range [IQR] 2.4-4.0), and the median number of liver nodules was 1 (range 1-3). Any-grade trAEs were reported by 73% of the patients receiving at least one dose of treatment (n = 11, tot n = 15). Four patients (27%) reported grade 2 trAEs including hypothyroidism (n = 2), diarrhoea (n = 1), and fatigue (n = 1), and one (7%) grade 3 ALT/AST elevation. After a median follow-up of 6.3 months (IQR 1.9-23.0), no deaths had occurred. One patient had experienced relapse 20.8 months after treatment commencement, and he achieved partial response to subsequent treatment with atezolizumab plus bevacizumab. Median time to LR from screening was 2.5 months (IQR 2.3-3.2). Only one patient had a surgery delay due to liver function worsening (ICPI-unrelated) and experienced disease progression 12.4 months post-screening. One patient was found to have cholangiocarcinoma (CCA) on LR specimen and was excluded from efficacy analyses. Of the 13 patients with an available radiological assessment, ORR was 23%, with two partial responses and one complete response. Disease control rate was 92%, with one patient with mixed HCC/CCA histology showing primary progression. Of the nine pathologically evaluable patients, seven (78%) achieved a pathological response, including two (22%) complete responses. Conclusions: Nivo+Ipi can be safely administered in the neoadjuvant setting for HCC and does not delay LR. The combination demonstrates promising evidence of anti-tumour efficacy in terms of radiological and pathological response. Clinical trial information: NCT03682276.

Volatile aldehydes are enriched in esophageal adenocarcinoma (EAC) patients’ breath and could improve early diagnosis, however the mechanisms of their production are unknown. Here, we show that weak aldehyde detoxification characterizes EAC, which is sufficient to cause endogenous aldehyde accumulation in vitro. Two aldehyde groups are significantly enriched in EAC biopsies and adjacent tissue: (i) short-chain alkanals, and (ii) medium-chain alkanals, including decanal. The short-chain alkanals form DNA-adducts, which demonstrates genotoxicity and confirms inadequate detoxification. Metformin, a putative aldehyde scavenger, reduces this toxicity. Tissue and breath concentrations of the medium-chain alkanal decanal are correlated, and increased decanal is linked to reduced ALDH3A2 expression, TP53 deletion, and adverse clinical features. Thus, we present a model for increased exhaled aldehydes based on endogenous accumulation from reduced detoxification, which also causes therapeutically actionable genotoxicity. These results support EAC early diagnosis trials using exhaled aldehyde analysis., Volatile aldehydes can be enriched in esophageal adenocarcinoma patients’ breath. Here, the authors reveal corresponding metabolic changes in EAC tumours, which may be caused by impaired detoxification of endogenous metabolites.

Background After liver resection (LR), patients with hepatocellular cancer (HCC) are at high risk of recurrence. There are no approved anti-cancer therapies known to affect such risk, highlighting the acute need for novel systemic therapies to control the probability of disease relapse. Immunotherapy is expanding as a novel treatment option for HCC. Emerging data from cohort 4 of the CA209–040 study, which investigated the safety and preliminary efficacy of nivolumab/ipilimumab co-administration in advanced HCC, suggest that the combination can be delivered safely with an acceptable proportion of reversible grade 3–4 toxicities (27.1%) and a low discontinuation rate (2%) in patients with HCC. Here, we describe the design and rationale of PRIME-HCC, a two-part, multi-centre, phase Ib study to assess safety and bioactivity of the nivolumab/ipilimumab combination prior to LR in early-stage HCC. Methods The study involves an initial safety run-in phase (Part 1) to allow for preliminary safety characterisation within the first 6 patients enrolled and a subsequent expansion (Part 2). Ipilimumab will be administered once only on Day 1. Nivolumab will be administered on Day 1 and Day 22 (± 3 days) for a total of two 21-day cycles (i.e. 6 weeks of treatment). The primary objective of the study is to determine the safety and tolerability of the nivolumab/ipilimumab combination prior to LR. The secondary objective is to preliminarily characterize the efficacy of the combination prior to LR, including objective response rate (ORR) and pathologic response rates. Additional exploratory objectives include preliminary evidence of long-term disease control and to identify predictive correlates of response to the nivolumab/ipilimumab combination in HCC. Discussion The results of this study will help define the positioning of neoadjuvant nivolumab/ipilimumab combination in the perioperative management of HCC, with potential to improve survival outcomes in this patient population. Trial registration EudraCT Number: 2018–000987-27 Clinical trial registry & ID: ClinicalTrials.gov: NCT03682276.

A majority of hepatocellular carcinoma (HCC) develops in the setting of persistent chronic inflammation as immunological mechanisms have been shown to play a vital role in the initiation, growth and progression of tumours. The index review has been intended to highlight ongoing immunological changes in the hepatic parenchyma responsible for the genesis and progression of HCC. The in-situ vaccine effect of radiofrequency (RF) is through generation tumour-associated antigens (TAAs), following necrosis and apoptosis of tumour cells, which not only re-activates the antitumour immune response but can also act in synergism with checkpoint inhibitors to generate a superlative effect with intent to treat primary cancer and distant metastasis. An improved understanding of oncogenic responses of immune cells and their integration into signaling pathways of the tumour microenvironment will help in modulating the antitumour immune response. Finally, we analyzed contemporary literature and summarised the recent advances made in the field of targeted immunotherapy involving checkpoint inhibitors along with RF application with the intent to reinstate antitumour immunity and outline future directives in very early and early stages of HCC.

International audience; Non-alcoholic fatty liver disease (NAFLD) culminates in insulin resistance and metabolic syndrome. Because there are no approved pharmacological treatment agents for non-alcoholic steatohepatitis (NASH) and NAFLD, different signaling pathways are under investigation for drug development with the focus on metabolic pathways. Hepatocyte nuclear factor 4-alpha (HNF4A) is at the center of a complex transcriptional network where its disruption is directly linked to glucose and lipid metabolism. Resetting HNF4A expression in NAFLD is therefore crucial for re-establishing normal liver function. Here, small activating RNA (saRNA) specific for upregulating HNF4A was injected into rats fed a high-fat diet for 16 weeks. Intravenous delivery was carried out using 5-(G 5)-triethanolamine-core pol-yamidoamine (PAMAM) dendrimers. We observed a significant reduction in liver triglyceride, increased high-density lipopro-tein/low-density lipoprotein (HDL/LDL) ratio, and decreased white adipose tissue/body weight ratio, all parameters to suggest that HNF4A-saRNA treatment induced a favorable metabolic profile. Proteomic analysis showed significant regulation of genes involved in sphingolipid metabolism, fatty acid b-oxidation , ketogenesis, detoxification of reactive oxygen species, and lipid transport. We demonstrate that HNF4A activation by oligonucleotide therapy may represent a novel single agent for the treatment of NAFLD and insulin resistance.

Purpose: Transcription factor C/EBP-α (CCAAT/enhancer-binding protein alpha) acts as a master regulator of hepatic and myeloid functions and multiple oncogenic processes. MTL-CEBPA is a first-in-class small activating RNA oligonucleotide drug that upregulates C/EBP-α. Patients and Methods: We conducted a phase I, open-label, dose-escalation trial of MTL-CEBPA in adults with advanced hepatocellular carcinoma (HCC) with cirrhosis, or resulting from nonalcoholic steatohepatitis or with liver metastases. Patients received intravenous MTL-CEBPA once a week for 3 weeks followed by a rest period of 1 week per treatment cycle in the dose-escalation phase (3+3 design). Results: Thirty-eight participants have been treated across six dose levels (28–160 mg/m2) and three dosing schedules. Thirty-four patients were evaluable for safety endpoints at 28 days. MTL-CEBPA treatment–related adverse events were not associated with dose, and no maximum dose was reached across the three schedules evaluated. Grade 3 treatment-related adverse events occurred in nine (24%) patients. In 24 patients with HCC evaluable for efficacy, an objective tumor response was achieved in one patient [4%; partial response (PR) for over 2 years] and stable disease (SD) in 12 (50%). After discontinuation of MTL-CEBPA, seven patients were treated with tyrosine kinase inhibitors (TKIs); three patients had a complete response with one further PR and two with SD. Conclusions: MTL-CEBPA is the first saRNA in clinical trials and demonstrates an acceptable safety profile and potential synergistic efficacy with TKIs in HCC. These encouraging phase I data validate targeting of C/EBP-α and have prompted MTL-CEBPA + sorafenib combination studies in HCC.

BACKGROUND: Oligonucleotide drug development has revolutionised the drug discovery field allowing the notoriously "undruggable" genome to potentially become "druggable". Within this field, 'small' or 'short' activating RNAs (saRNA) are a more recently discovered category of short double stranded RNA with clinical potential. SaRNAs promote endogenous transcription from target loci, a phenomenon widely observed in mammals known as RNA activation (RNAa). The ability to target a particular gene is dependent on the sequence of the saRNA. Hence, the potential clinical application of saRNA is to increase target gene expression in a sequence specific manner. SaRNA based oligonucleotide therapeutics present great promise in expanding the "druggable" genome with particular areas of interest including transcription factor activation and haploinsufficency. Review and Conclusion: In this mini-review, we describe the pre-clinical development of the first saRNA drug to enter the clinic. This saRNA, referred to as MTL-CEBPA, induces transcription of the transcription factor CCAAT/enhancer-binding protein alpha (CEBPα), a tumour suppressor and critical regulator of hepatocyte function. MTL-CEBPA is presently in Phase I clinical trials for hepatocellular carcinoma (HCC). The clinical development of MTL-CEBPA will demonstrate "proof of concept", showing that saRNAs can provide the basis for drugs which enhance targeted gene expression and consequently improve disease outcome in patients.

Background Gemcitabine is used to treat a wide range of tumours but its efficacy is limited by cancer cell resistance mechanisms. NUC-1031, a phosphoramidate modification of gemcitabine, is the first anti-cancer ProTide to enter the clinic and is designed to overcome these key resistance mechanisms. Methods Sixty-eight patients with advanced solid tumours who had relapsed after treatment with standard therapy were recruited to a dose escalation study to determine the recommended Phase II dose (RP2D) and assess safety of NUC-1031. Pharmacokinetics and anti-tumour activity was also assessed. Results Sixty-eight patients received treatment, 50% of whom had prior exposure to gemcitabine. NUC-1031 was well-tolerated with the most common Grade 3/4 adverse events of neutropaenia, lymphopaenia and fatigue occurring in 13 patients each (19%). In 49 response-evaluable patients, 5 (10%) achieved a partial response and 33 (67%) had stable disease, resulting in a 78% disease control rate. Cmax levels of the active intracellular metabolite, dFdCTP, were 217-times greater than those reported for equimolar doses of gemcitabine, with minimal toxic metabolite accumulation. The RP2D was determined as 825 mg/m2 on Days 1, 8 and 15 of a 28-day cycle. Conclusion NUC-1031 was well-tolerated and demonstrated clinically significant anti-tumour activity, even in patients with prior gemcitabine exposure and in cancers not traditionally perceived as gemcitabine-responsive. Trial registration: clinicaltrials.gov identifier: NCT01621854

Background: Most patients with pancreatic ductal adenocarcinoma (PDAC) are metastatic at presentation with dismal prognosis warranting improved systemic therapy options. Longitudinal sampling for assessment of treatment response poses a challenge for validating novel therapies. In this proof-of-principle study, we evaluate the role of endoscopic ultrasound (EUS)-guided serial fine-needle aspiration biopsies (FNABs) to study the mechanism of action of radiofrequency ablation (RFA). Methods: Patients with stage III inoperable PDAC with prior exposure to gemcitabine were selected into ARDEO (ethically approved Phase-II prospective clinical study of EUS-RFA). Post examination, targeted RF was delivered thrice and sequential FNABs of tumor were taken before and after treatment. Transcriptomic profiling of 6 FNABs from 2 patients was performed using a custom NanoString gene panel (144 genes) consisting of cancer and cancer-associated fibroblast (CAFs) subtypes along with immune changes. CAF culture was established from one FNAB and characterised by immunofluorescence and immunoblotting. Results: RFA treatments were well tolerated without any complications and both patients had stable disease immediately after EUS-RFA. Two-course RFA led to upregulation of CD1E gene (participates in antigen presentation) in both, patient 1 and 2 (4.5 and 3.9-fold) compared to baseline. Patient 1 showed increased expression of T cell genes (CD4 – 8.7-fold, CD8 – 35.7-fold), cytolytic function (6.4-fold) and inflammatory response (8-fold) post-RFA. Greater than 2-fold upregulation of CD274 (PDL1), IDO1, PDCD1 and TNFRSF18 (GITR) was observed post 2nd RFA in both the patients. Further, two-course RFA led to increased PDGFRa (4.5 and 9-fold) in both patients along with enrichment of pCAF subtypes B and C in patient 1 and subtypes A, B and D in patient 2. Immunofluorescence staining revealed expression of PDGFRa, aSMA, VIM, POSTN and MYH11 on patient2-derived CAFs post 1st RFA; validated by immunoblotting. Finally, RFA led to downregulation of classical PDA subtype in both patients. Conclusions: This feasibility study validates longitudinal sampling by EUS-FNABs as an appropriate research tool to study tumor microenvironmental changes associated with local pancreatic immunomodulatory techniques like RFA. Citation Format: Krisha Desai, Patrick Varun Lawrence, Christopher Wadsworth, Nagina Mangal, Nagy Habib, Anguraj Sadanandam, Mikael Sodergren. Characterization of longitudinally collected fine needle aspiration biopsies of pancreatic ductal adenocarcinoma upon endoscopic ultrasound guided radiofrequency ablation [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-101.

Purpose To study the safety, efficacy, and long-term outcomes of percutaneous pancreatic duct drainage (PPDD) for treating pancreatic duct (PD) obstruction. Materials and methods This prospective observational cohort study included 73 patients with PD obstruction between December 2010 and June 2020. Patients underwent PPDD under ultrasound and fluoroscopy guidance, computed tomography (CT) and fluoroscopy guidance, or CT guidance only. They were categorized into two groups: nonmalignant (26 patients with PD obstruction due to acute and chronic pancreatitis or postoperative stricture) and malignant (47 patients with pancreatic head and ampullary tumors). Results Overall technical success rate was 98.6% (72/73). No major complications were encountered; however, severe weakness, lack of appetite, and tachycardia were observed in 4.1% (3/73) of patients managed with intravenous resuscitation. Multivariate analysis demonstrated that diagnosis type (pancreatic head tumor: P = .049 and odds ratio = 1.95 [1.11-2.25] and chronic pancreatitis: P = .048 and odds ratio = 6.25 [1.74-22.22]) were associated with mortality. The median survival time was 16.3 months. Moreover, 15.1% (11/73) of the patients were alive 4 years after the PPDD procedure, and the mean overall survival time of nonmalignant and malignant patients was 35.1 and 21.4 months, respectively. Conclusions Image-guided PPDD appears to be feasible and safe, and provides a valuable therapeutic option for managing patients with PD obstruction.

Tumor associated macrophages (TAM) and myeloid-derived suppressor cells (MDSC) are widely implicated in the suppression of immune responses in cancer and are associated with negative clinical outcomes. We report that in mouse tumor models, therapeutic up-regulation of the transcriptional factor C/EBPα with small activating RNA (MTL-CEBPA) blocks the suppressive activity of monocytic (M) MDSC and TAM without affecting polymorphonuclear (PMN) MDSC. MTL-CEBPA treatment demonstrated antitumor activity that was dependent on T cells. Combination of MTL-CEBPA and anti-PD1 antibody or with PMN-MDSC targeted therapy resulted in marked antitumor effect. A phase I trial was conducted in 36 patients with advanced hepatocellular carcinoma (HCC). Marked decrease in M-MDSC and the expression of genes and proteins involved in immune suppressive activity of these cells were observed. Combined treatment with MTL-CEBPA and sorafenib resulted in a 27% objective radiological response and three complete responses in patients with virally associated HCC. Tissue biopsies from these patients demonstrated decrease in M2 polarized macrophages within the tumors. Thus, therapeutic up-regulation of C/EBPα inhibited suppressive myeloid cells that resulted in potent antitumor effect in mice and with encouraging clinical response in cancer patients. We are currently recruiting to a multi-centre study of MTL-CEBPA in combination with a PD-1 inhibitor (pembrolizumab) in adult patients with advanced solid tumors (TIMEPOINT - ClinicalTrials.gov Identifier: NCT04105335). Citation Format: Ayumi Hashimoto, Debashis Sarker, Vikash Reebye, Sheba Jarvis, Mikael H. Sodergren, Andrew Kossenkov, Nina Raulf, Jenni Vasara, Pinelopi Andrikakou, Tim Meyer, Kai-Wen Huang, Ruth Plummer, Cheng Ean Chee, Duncan Spalding, Madhava Pai, David J. Pinato, Rohini Sharma, Bristi Basu, Daniel Palmer, Yuk-Ting Ma, Robert Habib, Anna Martirosyan, Naouel Elasri, Adeline Reynaud, John Rossi, Mark Cobbold, Nagy Habib, Dmitry Gabrilovich. Up-regulation of C/EBPα inhibits suppressive activity of myeloid cells and potentiates antitumor response in mice and cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1730.

The high mortality rates of cancer patients receiving standard treatments emphasize the crucial need for alternative treatments. One promising strategy is to use organic nanoparticles (NPs) for gene delivery. Although multifunctional NPs theoretically have many desirable properties as gene vectors, there are several practical barriers to successful gene delivery. In this review we discuss the properties of NPs and overview in vitro and preclinical studies of organic NPs over the past 5 years. The results of recent clinical trials suggest that the NP field holds significant promise. The annual market value for successful gene delivery could exceed US$30 billion, and this will encourage researchers to study the application of NPs to therapeutic gene delivery.

Background: Colorectal cancer (CRC) is the third most common cancer worldwide and the second leading cause of cancer-related deaths in developed countries. The liver is the most prevalent site of metastasis from CRC. Currently, the gold-standard treatment for colorectal liver metastases (CLMs) is surgical resection. However, depending on the pattern of the disease, a significant number of patients may require different approaches alone or in combination with surgery, including thermal ablation (radiofrequency (RFA) or microwave (MWA) ablation) or transarterial liver-directed therapies, although the latter is not yet part of the standard treatment for CRC liver metastases. Methods and Results: We present the case of a 63-yearold man with bilobar CLM who was treated with transarterial embolization (TAE) and RFA followed by chemotherapy. A post-RFA study of immune parameters revealed the downregulation of CD39 expression in the circulating CD4+ T cell population and a reduction of the serum levels of cytokines IL-10, TGF-β, IFN-gamma and IL-17, which positively correlated with the diminished serum level of gamma-glutamyl transferase (GGT) and the subdued inflammatory markers: the neutrophil/lymphocyte ratio (NLR) and platelet/lymphocyte ratio (PLR). Later, the patient underwent chemotherapy. Liver failure developed within two years and nine months following tumour ablation, leading to the death of the patient. Conclusions: However, the denial of adjuvant chemotherapy by the patient gave us the opportunity to assess the immunomodulatory changes following RFA in the absence of any other therapeutic modalities.

Introduction: Hepatocellular carcinoma (HCC) often develops on a background of chronic inflammation and a complex immunosuppressive network with increased regulatory T cells, impaired CD8+ T cells and the secretion of immunosuppressive cytokines. Previous clinical studies have reported a superior disease-free survival (DFS) following a radiofrequency-based ablation or resection in HCC tumours compared to conventional liver resection techniques. The aim of this study was to investigate whether there is any correlation with the use of a radiofrequency-assisted liver resection and clinical outcome. Material and Methods: Patients&rsquo, peripheral blood was collected prior and 7 days following surgery from patients undergoing a liver resection for HCC. There were 5 liver resections performed using CUSA and 6 liver resections with the RF-based device, HabibTM 4X. The primary endpoint of the study was to assess the immunological parameters of circulating immune cell populations as well as serum cytokines. The Student&rsquo, s t-test, chi-square or Fisher&rsquo, s Exact test were applied for statistical comparisons, as appropriate. Results: Patients undergoing an RF-assisted liver resection with HabibTM 4X had a significant decrease in the inhibitory Treg cells (p = 0.002) and a significant increase in CD8+ T lymphocytes (p = 0.050) and CD4+CD45RO+/CD4+ memory T cells (p = 0.002) compared to those patients undergoing a liver resection with CUSA. It was also noted that the RF-assisted liver resection group had a significant decrease in circulating TGF-&szlig, (p = 0.000), IL10 (p = 0.000) and a significant increase in IFN-gamma (p = 0. 027) and IL-17 compared to the CUSA group. Conclusion: A liver resection with RF-based device HabibTM 4X was associated with positive immunomodulatory changes in circulating immune cells and circulating cytokines which could explain the significant improvement in DFS.