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Organ preservation is a concept proposed for patients with rectal cancer after a good clinical response to neoadjuvant chemotherapy, to potentially avoid morbidity and side-effects of rectal excision. The objective of this study was to compare local excision and total mesorectal excision in patients with a good response after chemoradiotherapy for lower rectal cancer. We did a prospective, randomised, open-label, multicentre, phase 3 trial at 15 tertiary centres in France that were experts in the treatment of rectal cancer. Patients aged 18 years and older with stage T2T3 lower rectal carcinoma, of maximum size 4 cm, who had a good clinical response to neoadjuvant chemoradiotherapy (residual tumour ≤2 cm) were centrally randomly assigned by the surgeon before surgery to either local excision or total mesorectal excision surgery. Randomisation, which was done via the internet, was not stratified and used permuted blocks of size eight. In the local excision group, a completion total mesorectal excision was required if tumour stage was ypT2–3. The primary endpoint was a composite outcome of death, recurrence, morbidity, and side-effects at 2 years after surgery, to show superiority of local excision over total mesorectal excision in the modified intention-to-treat (ITT) population (expected proportions of patients having at least one event were 25% vs 60% for superiority). This trial was registered with ClinicalTrials.gov, number NCT00427375. From March 1, 2007, to Sept 24, 2012, 186 patients received chemoradiotherapy and were enrolled in the study. 148 good clinical responders were randomly assigned to treatment, three were excluded (because they had metastatic disease, tumour >8 cm from anal verge, and withdrew consent), and 145 were analysed: 74 in the local excision group and 71 in the total mesorectal excision group. In the local excision group, 26 patients had a completion total mesorectal excision. At 2 years in the modified ITT population, one or more events from the composite primary outcome occurred in 41 (56%) of 73 patients in the local excision group and 33 (48%) of 69 in the total mesorectal excision group (odds ratio 1·33, 95% CI 0·62–2·86; p=0·43). In the modified ITT analysis, there was no difference between the groups in all components of the composite outcome, and superiority was not shown for local excision over total mesorectal excision. We failed to show superiority of local excision over total mesorectal excision, because many patients in the local excision group received a completion total mesorectal excision that probably increased morbidity and side-effects, and compromised the potential advantages of local excision. Better patient selection to avoid unnecessary completion total mesorectal excision could improve the strategy. National Cancer Institute of France, Sanofi, Roche Pharma.

The genome of Hepatitis B virus (HBV) persists in infected hepatocytes as a nuclear episome (cccDNA) that is responsible for the transcription of viral genes and viral rebound, following antiviral treatment arrest in chronically infected patients. There is currently no clinically approved therapeutic strategy able to efficiently target cccDNA (Lucifora J 2016). The development of alternative strategies aiming at permanently abrogating HBV RNA production requires a thorough understanding of cccDNA transcriptional and post-transcriptional regulation. In a previous study, we discovered that 1C8, a compound that inhibits the phosphorylation of some cellular RNA-binding proteins, could decrease the level of HBV RNAs. Here, we aimed at identifying kinases responsible for this effect. Among the kinases targeted by 1C8, we focused on DYRK1A, a dual-specificity kinase that controls the transcription of cellular genes by phosphorylating transcription factors, histones, chromatin regulators as well as RNA polymerase II. The results of a combination of genetic and chemical approaches using HBV-infected hepatocytes, indicated that DYRK1A positively regulates the production of HBV RNAs. In addition, we found that DYRK1A associates with cccDNA, and stimulates the production of HBV nascent RNAs. Finally, reporter gene assays showed that DYRK1A up-regulates the activity of the HBV enhancer 1/X promoter in a sequence-dependent manner. Altogether, these results indicate that DYRK1A is a proviral factor that may participate in the HBV life cycle by stimulating the production of HBx, a viral factor absolutely required to trigger the complete cccDNA transcriptional program.

FZD7 is one of the key players in the subset of WNT-TGFβ-activated hepatocellular carcinomas (HCC), but the consequences of its abnormal expression on hepatocarcinogenesis remain to be better understood. Herein, we aimed to investigate the role of the FZD7-mediated signaling in immature phenotype and aggressiveness of HCC. Firstly, 499 human HCCs were used for clinical and molecular comparisons regarding the expression of FZD7 and s temness-associated markers. We showed that FZD7 overexpression was associated with poor differentiation and, in combination with CD133 , predicted a poor outcome of patients with aggressive recurrence. Next, the impact of WNT3/FZD7 signaling on the differentiation of hepatic cells was assessed in HCC cell lines, as well in the non-transformed progenitor HepaRG cell line and in primary human hepatocytes, transduced with WNT3 and FZD7 -expressing lentiviruses. We demonstrated that the ectopic expression of WNT3 and FZD7 inhibited the differentiation behavior of HepaRG cells and human primary hepatocytes, amplified the pool of EpCAM (+) , CD90 (+) and CD133 (+) subsets of HCC cell lines, and increased their cancer stem cell features. Moreover, we found that WNT3/FZD7-mediated stemness properties of cancer cells were independent of the stemness-associated marker NANOG. In conclusion, we identified the FZD7 (+) /CD133 (+) signature as a potential prognosis marker and molecular therapeutic target, and we strengthened the hypothesis for the involvement of FZD7 in the enrichment of a cancer stem cell pool in HCC.

Despite the existence of a preventive vaccine, chronic infection with Hepatitis B virus (HBV) affects more than 250 million people and represents a major global cause of hepatocellular carcinoma (HCC) worldwide. Current clinical treatments, in most of cases, do not eliminate viral genome that persists as a DNA episome in the nucleus of hepatocytes and constitutes a stable template for the continuous expression of viral genes. Several studies suggest that, among viral factors, the HBV core protein (HBc), well-known for its structural role in the cytoplasm, could have critical regulatory functions in the nucleus of infected hepatocytes. To elucidate these functions, we performed a proteomic analysis of HBc-interacting host-factors in the nucleus of differentiated HepaRG, a surrogate model of human hepatocytes. The HBc interactome was found to consist primarily of RNA-binding proteins (RBPs), which are involved in various aspects of mRNA metabolism. Among them, we focused our studies on SRSF10, a RBP that was previously shown to regulate alternative splicing (AS) in a phosphorylation-dependent manner and to control stress and DNA damage responses, as well as viral replication. Functional studies combining SRSF10 knockdown and a pharmacological inhibitor of SRSF10 phosphorylation (1C8) showed that SRSF10 behaves as a restriction factor that regulates HBV RNAs levels and that its dephosphorylated form is likely responsible for the anti-viral effect. Surprisingly, neither SRSF10 knock-down nor 1C8 treatment modified the splicing of HBV RNAs but rather modulated the level of nascent HBV RNA. Altogether, our work suggests that in the nucleus of infected cells HBc interacts with multiple RBPs that regulate viral RNA metabolism. Our identification of SRSF10 as a new anti-HBV restriction factor offers new perspectives for the development of new host-targeted antiviral strategies.

Current therapies for chronic hepatitis B virus (HBV) infections are effective at decreasing the viral load in serum, but do not lead to viral eradication. Recent studies highlighted the therapeutic or “adjuvant” potential of immune-modulators. Our aim was to explore the direct anti-HBV effect of Toll-Like-Receptors (TLR) agonists in hepatocytes. HBV-infected primary human hepatocytes (PHH) or differentiated HepaRG cells (dHepaRG) were treated with various TLR agonists. Amongst all TLR ligands tested, Pam3CSK4 (TLR1/2-ligand) and poly(I:C)-(HMW) (TLR3/MDA5-ligand) were the best at reducing all HBV parameters. No or little viral rebound was observed after treatment arrest, implying a long-lasting effect on cccDNA. We also tested Riboxxol that features improved TLR3 specificity compared to poly(I:C)-(HMW). This agonist demonstrated a potent antiviral effect in HBV-infected PHH. Whereas, poly(I:C)-(HMW) and Pam3CSK4 mainly induced the expression of classical genes from the interferon or NF-κB pathway respectively, Riboxxol had a mixed phenotype. Moreover, TLR2 and TLR3 ligands can activate hepatocytes and immune cells, as demonstrated by antiviral cytokines produced by stimulated hepatocytes and peripheral blood mononuclear cells. In conclusion, our data highlight the potential of innate immunity activation in the direct control of HBV replication in hepatocytes, and support the development of TLR-based antiviral strategies.

Backround and aims Complex host-virus interactions account for adaptive and innate immunity dysfunctions and viral cccDNA mini-chromosome persistence, key features of HBV chronicity and challenges for HBV cure. The extent of HBV direct impact on liver transcriptome remains controversial. Transcriptional activation in eukaryotic cells is tightly linked with disruption of nucleosome organization at accessible genomic sites of remodeled chromatin. We sought to investigate the impact of HBV on chromatin accessibility and transcription. Methods We used ATAC-seq (Assay for Transposase Accessible Chromatin followed by high throughput sequencing) to detect early changes in chromatin accessibility coupled with RNA-seq in HBV-infected Primary Human Hepatocytes (PHHs). Results An increasing number of genomic sites change their nucleosome organization over time after HBV infection, with a prevalent, but not exclusive, reduction of chromatin accessibility at specific sites that is partially prevented by inhibiting HBV transcription and replication. ATAC-seq and RNA-seq integration showed that HBV infection impacts on liver fatty acids, bile acids, iron metabolism and liver cancer pathways. The upregulation of iron uptake genes leads to a significant increase of iron content in HBV-infected PHHs whereas iron chelation inhibits cccDNA transcription and viral replication. The chromatin accessibility and transcriptional changes imposed by HBV early after infection persist, as an epigenetic scar, in chronic HBV (CHB) patients and in HBV-related HCCs. These changes are to a large extent independent from viral replication levels and disease activity. Conclusions Altogether our results show that HBV infection impacts on host cell chromatin landscape and specific transcriptional programs including liver metabolism and liver cancer pathways. Re-wiring of iron metabolism boosts viral replication early after infection. The modulation of genes involved in cancer-related pathways may favor the development or the selection of a pro-neoplastic phenotype and persists in HBV-related HCCs.

Co-infection with the hepatitis B virus and hepatitis delta virus (HDV) leads to the most aggressive form of viral hepatitis. Using in vitro infection models, we confirmed that IL-1β, a crucial innate immune molecule for pathogen control, was very potent against HBV from different genotypes. Additionally, we demonstrated for the first time a strong and rapid antiviral effect induced by very low doses of IL-1β against HDV. In parallel, using co-culture assays, we demonstrated that monocytes exposed to HBV, and in particular to HBsAg, during differentiation into pro-inflammatory macrophages secreted less IL-1β. Altogether, our data emphasize the importance of developing combined antiviral strategies that would, for instance, reduce the secretion of HBsAg and stimulate the immune system to produce endogenous IL-1β efficient against both HBV and HDV.

Surgery is the only curative treatment in patients with colorectal liver metastases (CLM), but only 10-20% of patients are eligible. High Intensity Focused Ultrasound (HIFU) technology is of proven value in several indications, notably prostate cancer. Its intra-operative use in patients with CLM has not previously been studied. Preclinical work suggested the safety and feasibility of a new HIFU device capable of ablating volumes of up to 2cm x 2cm in a few seconds. We conducted a prospective, single-centre phase I-IIa trial. HIFU was delivered immediately before scheduled hepatectomy. To demonstrate the safety and efficacy of rapidly ablating liver parenchyma, ablations were performed on healthy tissue within the areas scheduled for resection. In total, 30 ablations were carried out in 15 patients. These ablations were all generated within 40 seconds and on average measured 27.5mm x 21.0mm. The phase I study (n = 6) showed that use of the HIFU device was feasible and safe and did not damage neighbouring tissue. The phase IIa study (n = 9) showed both that the area of ablation could be precisely targeted on a previously implanted metallic mark (used to represent a major anatomical structure) and that ablations could be undertaken deliberately to avoid such a mark. Ablations were achieved with a precision of 1-2 mm. HIFU was feasible, safe and effective in ablating areas of liver scheduled for resection. The next stage is a phase IIb study which will attempt ablation of small metastases with a 5 mm margin, again prior to planned resection. ClinicalTrials.govNCT01489787.

The management of patients with colorectal cancer (CRC) and potentially resectable liver metastases (LM) requires quick assessment of mutational status and of response to pre-operative systemic therapy. In a prospective phase II trial (NCT01442935), we investigated the clinical validity of circulating tumor cell (CTC) and circulating tumor DNA (ctDNA) detection. CRC patients with potentially resectable LM were treated with first-line triplet or doublet chemotherapy combined with targeted therapy. CTC (Cellsearch®) and Kirsten RAt Sarcoma (KRAS) ctDNA (droplet digital polymerase chain reaction (PCR)) levels were assessed at inclusion, after 4 weeks of therapy and before LM surgery. 153 patients were enrolled. The proportion of patients with high CTC counts (≥3 CTC/7.5mL) decreased during therapy: 19% (25/132) at baseline, 3% (3/108) at week 4 and 0/57 before surgery. ctDNA detection sensitivity at baseline was 91% (N=42/46) and also decreased during treatment. Interestingly, persistently detectable KRAS ctDNA (p = 0.01) at 4 weeks was associated with a lower R0/R1 LM resection rate. Among patients who had a R0/R1 LM resection, those with detectable ctDNA levels before liver surgery had a shorter overall survival (p < 0.001). In CRC patients with limited metastatic spread, ctDNA could be used as liquid biopsy tool. Therefore, ctDNA detection could help to select patients eligible for LM resection.

Background The portal vascularization of segment IV (S4) of the liver has not been well described. Knowledge of the portal supply to S4 is of great interest for liver surgery and for interventional radiological procedures. This study aimed to analyse the distribution of portal vein branches supplying S4. Methods We retrospectively analysed data from patients operated on for liver tumours between 2007 and 2016. Patients with involvement of S4 branches or the left portal vein, previous liver surgery or poor quality imaging were excluded. Branches originating from the right portal vein and/or from the transverse part of the left portal vein (TPLPV) and/or from the umbilical part of the portal vein (UPLPV) were identified. Results In 102 patients who underwent a right hepatectomy, S4 was vascularized by 2–8 branches of the left portal vein, with 84.3 % of patients having 3–6 branches. Only eleven patients (10.8 %) had portal branches originating from the TPLPV, with no impact on the number of branches coming from the UPLPV. Three patients (2.9 %) had one branch from the right portal vein. In patients with only two or three branches supplying S4, the branches had a larger diameter and typically arose from a short common trunk which divided further within its first centimetres. Conclusions Portal vascularization of S4 varies widely (2–8 branches) between patients and originates predominantly from the junction between the left portal vein and the round ligament. There is no anatomical rationale to divide S4 into S4a and S4b.