GLA-modified RNA treatment lowers GB3 levels in iPSC-derived cardiomyocytes from Fabry-affected individuals.

Recent studies in non-human model systems have shown therapeutic potential of nucleoside-modified messenger RNA (modRNA) treatments for lysosomal storage diseases. Here, we assessed the efficacy of a modRNA treatment to restore the expression of the galactosidase alpha (GLA), which codes for α-Galactosidase A (α-GAL) enzyme, in a human cardiac model generated from induced pluripotent stem cells (iPSCs) derived from two individuals with Fabry disease. Consistent with the clinical phenotype, cardiomyocytes from iPSCs derived from Fabry-affected individuals showed accumulation of the glycosphingolipid Globotriaosylceramide (GB3), which is an α-galactosidase substrate. Furthermore, the Fabry cardiomyocytes displayed significant upregulation of lysosomal-associated proteins. Upon GLA modRNA treatment, a subset of lysosomal proteins were partially restored to wild-type levels, implying the rescue of the molecular phenotype associated with the Fabry genotype. Importantly, a significant reduction of GB3 levels was observed in GLA modRNA-treated cardiomyocytes, demonstrating that α-GAL enzymatic activity was restored. Together, our results validate the utility of iPSC-derived cardiomyocytes from affected individuals as a model to study disease processes in Fabry disease and the therapeutic potential of GLA modRNA treatment to reduce GB3 accumulation in the heart.

Messenger RNA rescues medium-chain acyl-CoA dehydrogenase deficiency in fibroblasts from patients and a murine model.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common inherited disorder of mitochondrial fatty acid ß-oxidation (FAO) in humans. Patients exhibit clinical episodes often associated with fasting. Symptoms include hypoketotic hypoglycemia and Reye-like episodes. With limited treatment options, we explored the use of human MCAD (hMCAD) mRNA in fibroblasts from patients with MCAD deficiency to provide functional MCAD protein and reverse the metabolic block. Transfection of hMCAD mRNA into MCAD- deficient patient cells resulted in an increased MCAD protein that localized to mitochondria, concomitant with increased enzyme activity in cell extracts. The therapeutic hMCAD mRNA-lipid nanoparticle (LNP) formulation was also tested in vivo in Acadm-/- mice. Administration of multiple intravenous doses of the hMCAD mRNA-LNP complex (LNP-MCAD) into Acadm-/- mice produced a significant level of MCAD protein with increased enzyme activity in liver, heart and skeletal muscle homogenates. Treated Acadm-/- mice were more resistant to cold stress and had decreased plasma levels of medium-chain acylcarnitines compared to untreated animals. Furthermore, hepatic steatosis in the liver from treated Acadm-/- mice was reduced compared to untreated ones. Results from this study support the potential therapeutic value of hMCAD mRNA-LNP complex treatment for MCAD deficiency.

Chemical conversion of human conventional PSCs to TSCs following transient naive gene activation.

In human embryos, naive pluripotent cells of the inner cell mass (ICM) generate epiblast, primitive endoderm and trophectoderm (TE) lineages, whence trophoblast cells derive. In vitro, naive pluripotent stem cells (PSCs) retain this potential and efficiently generate trophoblast stem cells (TSCs), while conventional PSCs form TSCs at low efficiency. Transient histone deacetylase and MEK inhibition combined with LIF stimulation is used to chemically reset conventional to naive PSCs. Here, we report that chemical resetting induces the expression of both naive and TSC markers and of placental imprinted genes. A modified chemical resetting protocol allows for the fast and efficient conversion of conventional PSCs into TSCs, entailing shutdown of pluripotency genes and full activation of the trophoblast master regulators, without induction of amnion markers. Chemical resetting generates a plastic intermediate state, characterised by co-expression of naive and TSC markers, after which cells steer towards one of the two fates in response to the signalling environment. The efficiency and rapidity of our system will be useful to study cell fate transitions and to generate models of placental disorders.

A comprehensive investigation of histotype-specific microRNA and their variants in Stage I epithelial ovarian cancers.

isomiRs, the sequence-variants of microRNA, are known to be tissue and cell type specific but their physiological role is largely unknown. In our study, we explored for the first time the expression of isomiRs across different Stage I epithelial ovarian cancer (EOC) histological subtypes, in order to shed new light on their biological role in tumor growth and progression. In a multicentric retrospective cohort of tumor biopsies (n = 215) we sequenced small RNAs finding 971 expressed miRNAs, 64% of which are isomiRs. Among them, 42 isomiRs showed a clear histotype specific pattern, confirming our previously identified miRNA markers (miR192/194 and miR30a-3p/5p for mucinous and clear cell subtypes, respectively) and uncovering new biomarkers for all the five subtypes. Using integrative models, we found that the 38% of these miRNA expression alterations is the result of copy number variations while the 17% of differential transcriptional activities. Our work represents the first attempt to characterize isomiRs expression in Stage I EOC within and across subtypes and to contextualize their alterations in the framework of the large genomic heterogeneity of this tumor.

Synthetic mRNA rescues very long-chain acyl-CoA dehydrogenase deficiency in patient fibroblasts and a murine model.

Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is an inborn error of long chain fatty acid ß-oxidation (FAO) with limited treatment options. Patients present with heterogeneous clinical phenotypes affecting predominantly heart, liver, and skeletal muscle. While VLCAD deficiency is a systemic disease, restoration of liver FAO has the potential to improve symptoms more broadly due to increased total body ATP production and reduced accumulation of potentially toxic metabolites. We explored the use of synthetic human VLCAD (hVLCAD) mRNA and lipid nanoparticle encapsulated hVLCAD mRNA (LNP-VLCAD) to generate functional VLCAD enzyme in patient fibroblasts derived from VLCAD deficient patients, mouse embryonic fibroblasts, hepatocytes isolated from VLCAD knockout (Acadvl-/-) mice, and Acadvl-/- mice to reverse the metabolic effects of the deficiency. Transfection of all cell types with hVLCAD mRNA resulted in high level expression of protein that localized to mitochondria with increased enzyme activity. Intravenous administration of LNP-VLCAD to Acadvl-/- mice produced a significant amount of VLCAD protein in liver, which declined over a week. Treated Acadvl-/- mice showed reduced hepatic steatosis, were more resistant to cold stress, and accumulated less toxic metabolites in blood than untreated animals. Results from this study support the potential for hVLCAD mRNA for treatment of VLCAD deficiency.

Amnio acid substitution at position 298 of human glucose-6 phosphatase-α significantly impacts its stability in mammalian cells.

Glucose-6-phosphatase-α (G6Pase-α) catalyzes the hydrolysis of glucose-6-phosphate to glucose and functions as a key regulator in maintaining blood glucose homeostasis. Deficiency in G6Pase-α causes glycogen storage disease 1a (GSD1a), an inherited disorder characterized by life-threatening hypoglycemia and other long-term complications. We have developed a potential mRNA-based therapy for GSD1a and demonstrated that a human G6Pase-α (hG6Pase-α) variant harboring a single serine (S) to cysteine (C) substitution at the amino acid site 298 (S298C) had > twofold increase in protein expression, resulting in improved in vivo efficacy. Here, we sought to investigate the mechanisms contributing to the increased expression of the S298C variant. Mutagenesis of hG6Pase-α identified distinct protein variants at the 298 amino acid position with substantial reduction in protein expression in cultured cells. Kinetic analysis of expression and subcellular localization in mammalian cells, combined with cell-free in vitro translation assays, revealed that altered protein expression stemmed from differences in cellular protein stability rather than biosynthetic rates. Site-specific mutagenesis studies targeting other cysteines of the hG6Pase-α S298C variant suggest the observed improvements in stability are not due to additional disulfide bond formation. The glycosylation at Asparagine (N)-96 is critical in maintaining enzymatic activity and mutations at position 298 mainly affected glycosylated forms of hG6Pase-α. Finally, proteasome inhibition by lactacystin improved expression levels of unstable hG6Pase-α variants. Taken together, these data uncover a critical role for a single amino acid substitution impacting the stability of G6Pase-α and provide insights into the molecular genetics of GSD1a and protein engineering for therapeutic development.

The incidence of movement disorder increases with age and contrasts with subtle and limited neuroimaging abnormalities in argininosuccinic aciduria.

Argininosuccinate lyase (ASL) is integral to the urea cycle detoxifying neurotoxic ammonia and the nitric oxide (NO) biosynthesis cycle. Inherited ASL deficiency causes argininosuccinic aciduria (ASA), a rare disease with hyperammonemia and NO deficiency. Patients present with developmental delay, epilepsy and movement disorder, associated with NO-mediated downregulation of central catecholamine biosynthesis. A neurodegenerative phenotype has been proposed in ASA. To better characterise this neurodegenerative phenotype in ASA, we conducted a retrospective study in six paediatric and adult metabolic centres in the UK in 2022. We identified 60 patients and specifically looked for neurodegeneration-related symptoms: movement disorder such as ataxia, tremor and dystonia, hypotonia/fatigue and abnormal behaviour. We analysed neuroimaging with diffusion tensor imaging (DTI) magnetic resonance imaging (MRI) in an individual with ASA with movement disorders. We assessed conventional and DTI MRI alongside single photon emission computer tomography (SPECT) with dopamine analogue radionuclide 123 I-ioflupane, in Asl-deficient mice treated by hASL mRNA with normalised ureagenesis. Movement disorders in ASA appear in the second and third decades of life, becoming more prevalent with ageing and independent from the age of onset of hyperammonemia. Neuroimaging can show abnormal DTI features affecting both grey and white matter, preferentially basal ganglia. ASA mouse model with normalised ureagenesis did not recapitulate these DTI findings and showed normal 123 I-ioflupane SPECT and cerebral dopamine metabolomics. Altogether these findings support the pathophysiology of a late-onset movement disorder with cell-autonomous functional central catecholamine dysregulation but without or limited neurodegeneration of dopaminergic neurons, making these symptoms amenable to targeted therapy.

Standardized right artery first approach during laparoscopic pancreaticoduodenectomy for periampullary neoplasms: technical aspects and perioperative outcomes.

BACKGROUND: The most debated aspects of laparoscopic pancreaticoduodenectomy (LPD) concern the dissection of the pancreas from the surrounding vessels and the achievement of adequate resection margins, especially in patients with pancreatic cancer. METHODS: Data of consecutive patients undergoing LPD with right artery first approach from September 2020 to September 2021 for periampullary neoplasms (pancreatic, ampullary, duodenal, distal common biliary duct) were prospectively collected and retrospectively analyzed. The overall cohort was divided into two groups: patients affected by pancreatic carcinoma (PC) and patients affected by other periampullary neoplasms (OP). Surgical and postoperative outcomes between PC and OP were compared. RESULTS: Thirty-one patients (15 PC and 16 OP) were selected. No difference was found between PC and OP in terms of baseline characteristics. Median resection time and overall surgical time of the entire cohort were 275 min and 530 min, respectively, without difference between the groups (p = 0.599 and 0.052, respectively). Blood loss was similar between the groups, being 350 ml in PC and 325 ml in OP (p = 0.762). One patient (3.2%) was converted to laparotomy. No difference was found between the groups in terms of pathological outcomes. Median number of retrieved lymph nodes was 17. The majority of the patients (83.9%) received an R0 resection (73.3% and 93.7% in PC and OP, respectively; p = 0.172). Postoperative surgical outcomes did not differ between the groups, excepting for overall complication rate that was higher in the OP group (26.7% vs 68.7% in PC and OP, respectively; p = 0.032). CONCLUSION: Standardized right artery first approach during LPD was feasible and did not show worse surgical and postoperative outcomes in patients with pancreatic cancer as compared to those affected by other periampullary neoplasms, except for a higher rate of minor complications.

Multidimensional evaluation of the learning curve for totally laparoscopic pancreaticoduodenectomy: a risk-adjusted cumulative summation analysis.

INTRODUCTION: Laparoscopic pancreaticoduodenectomy (LPD) is a challenging procedure. We investigated the learning curve (LC) for LPD with a multidimensional analysis. METHODS: Data of patients undergoing LPD between 2017 and 2021, operated by a single surgeon, were considered. A multidimensional assessment of the LC was performed through Cumulative Sum (CUSUM) and Risk-Adjusted (RA)-CUSUM analysis. RESULTS: 113 patients were selected. Rates of conversion, overall postoperative complication, severe complication and mortality were 4%, 53%, 29% and 4%, respectively. RA-CUSUM analysis showed a LC with three phases: competency (procedures 1-51), proficiency (procedures 52-94), and mastery (after procedure 94). Operative time was lower in both phase two (588.17 vs 541.13 min, p = 0.001) and three (534.72 vs 541.13 min, p = 0.004) with respect to phase one. Severe complication rate was lower in mastery as compared to competency phase (42% vs 6%, p = 0.005). During mastery phase a greater number of lymph nodes was harvested in comparison to proficiency phase. CONCLUSIONS: According to our LC analysis, 52 procedures were required to achieve technical competency in LPD. Mastery, which corresponded to a reduction in operative time and surgical failures, was acquired after 94 procedures.

Systemic mRNA Therapy for the Treatment of Fabry Disease: Preclinical Studies in Wild-Type Mice, Fabry Mouse Model, and Wild-Type Non-human Primates.

Fabry disease is an X-linked lysosomal storage disease caused by loss of alpha galactosidase A (α-Gal A) activity and is characterized by progressive accumulation of globotriaosylceramide and its analogs in all cells and tissues. Although enzyme replacement therapy (ERT) is considered standard of care, the long-term effects of ERT on renal and cardiac manifestations remain uncertain and thus novel therapies are desirable. We herein report preclinical studies evaluating systemic messenger RNA (mRNA) encoding human α-Gal A in wild-type (WT) mice, α-Gal A-deficient mice, and WT non-human primates (NHPs). The pharmacokinetics and distribution of h-α-Gal A mRNA encoded protein in WT mice demonstrated prolonged half-lives of α-Gal A in tissues and plasma. Single intravenous administration of h-α-Gal A mRNA to Gla-deficient mice showed dose-dependent protein activity and substrate reduction. Moreover, long duration (up to 6 weeks) of substrate reductions in tissues and plasma were observed after a single injection. Furthermore, repeat i.v. administration of h-α-Gal A mRNA showed a sustained pharmacodynamic response and efficacy in Fabry mice model. Lastly, multiple administrations to non-human primates confirmed safety and translatability. Taken together, these studies across species demonstrate preclinical proof-of-concept of systemic mRNA therapy for the treatment of Fabry disease and this approach may be useful for other lysosomal storage disorders.

Novel mRNA therapy restores GALT protein and enzyme activity in a zebrafish model of classic galactosemia.

Messenger RNA (mRNA) has emerged as a novel therapeutic approach for inborn errors of metabolism. Classic galactosemia (CG) is an inborn error of galactose metabolism caused by a severe deficiency of galactose-1-phosphate:uridylyltransferase (GALT) activity leading to neonatal illness and chronic impairments affecting the brain and female gonads. In this proof of concept study, we used our zebrafish model for CG to evaluate the potential of human GALT mRNA (hGALT mRNA) packaged in two different lipid nanoparticles to restore GALT expression and activity at early stages of development. Both one cell-stage and intravenous single-dose injections resulted in hGALT protein expression and enzyme activity in the CG zebrafish (galt knockout) at 5 days post fertilization (dpf). Moreover, the levels of galactose-1-phosphate (Gal-1-P) and galactonate, metabolites that accumulate because of the deficiency, showed a decreasing trend. LNP-packaged mRNA was effectively translated and processed in the CG zebrafish without signs of toxicity. This study shows that mRNA therapy restores GALT protein and enzyme activity in the CG zebrafish model, and that the zebrafish is a suitable system to test this approach. Further studies are warranted to assess whether repeated injections safely mitigate the chronic impairments of this disease.

Laparoscopic gastrectomy for stage II and III advanced gastric cancer: long­term follow­up data from a Western multicenter retrospective study.

INTRODUCTION: There has been an increasing interest for the laparoscopic treatment of early gastric cancer, especially among Eastern surgeons. However, the oncological effectiveness of Laparoscopic Gastrectomy (LG) for Advanced Gastric Cancer (AGC) remains a subject of debate, especially in Western countries where limited reports have been published. The aim of this paper is to retrospectively analyze short- and long-term results of LG for AGC in a real-life Western practice. MATERIALS AND METHODS: All consecutive cases of LG with D2 lymphadenectomy for AGC performed from January 2005 to December 2019 at seven different surgical departments were analyzed retrospectively. The primary outcome was diseases-free survival (DFS). Secondary outcomes were overall survival (OS), number of retrieved lymph nodes, postoperative morbidity and conversion rate. RESULTS: A total of 366 patients with stage II and III AGC underwent either total or subtotal LG. The mean number of harvested lymph nodes was 25 ± 14. The mean hospital stay was 13 ± 10 days and overall postoperative morbidity rate 27.32%, with severe complications (grade ≥ III) accounting for 9.29%. The median follow-up was 36 ± 16 months during which 90 deaths occurred, all due to disease progression. The DFS and OS probability was equal to 0.85 (95% CI 0.81-0.89) and 0.94 (95% CI 0.92-0.97) at 1 year, 0.62 (95% CI 0.55-0.69) and 0.63 (95% CI 0.56-0.71) at 5 years, respectively. CONCLUSION: Our study has led us to conclude that LG for AGC is feasible and safe in the general practice of Western institutions when performed by trained surgeons.

Is routine CT scan after pancreaticoduodenectomy a useful tool in the early detection of complications? A single center retrospective analysis.

PURPOSE: The clinical impact of routine CT imaging after pancreaticoduodenectomy (PD) has not been properly investigated. The aim of this study was to investigate the role of routine CT scan after PD for the detection of postoperative complications. METHODS: Prospectively collected data of consecutive patients undergoing PD and receiving routine postoperative CT imaging were retrospectively analyzed. The primary endpoint was accuracy of CT imaging in identifying major complications. The secondary endpoint was identification of preoperative and intraoperative factors associated with severe complications. A subgroup analysis of CT scan accuracy in identifying severe complications in patients stratified by fistula risk score (FRS) and presence of early clinical alterations was also performed. RESULTS: A total of 145 patients were included. Routine CT scan had low specificity (Sp = 0.36) and high sensitivity (Sn = 0.98) for predicting major complications, with an accuracy of 0.57. At multivariate logistic regression analysis, only fistula moderate-high FRS (p = 0.029) was independently associated with severe complications. In patients with negligible-low FRS, CT scan showed a Sp of 0.63 and a Sn of 1.0 with an accuracy of 0.69. In patients with moderate-high FRS, CT scan had a Sp of 0.19, a Sn of 0.97 and an accuracy of 0.5. In the 20 (14%) patients with negligible-low FRS and no clinical alterations, no deaths or readmissions occurred regardless of CT findings, while one severe complication occurred in the positive CT scan group. In all other groups, no deaths or readmissions occurred in case of negative CT, with only one severe complication in the moderate-high FRS group with clinical alterations. In case of positive CT, the rate of severe complications was 47% in case of negligible-low FRS and clinical alterations, 40% in case of moderate-high FRS with no clinical alterations, and 45% in case of moderate-high FRS and clinical alterations. CONCLUSIONS: Routine postoperative CT scan after PD should not be performed in patients with negligible-low FRS and no clinical alterations. In all other patients, a negative CT scan appears to be highly accurate in identifying patients who will have an uneventful course and who could benefit from early discharge.

Lipid nanoparticle-targeted mRNA therapy as a treatment for the inherited metabolic liver disorder arginase deficiency.

Arginase deficiency is caused by biallelic mutations in arginase 1 (ARG1), the final step of the urea cycle, and results biochemically in hyperargininemia and the presence of guanidino compounds, while it is clinically notable for developmental delays, spastic diplegia, psychomotor function loss, and (uncommonly) death. There is currently no completely effective medical treatment available. While preclinical strategies have been demonstrated, disadvantages with viral-based episomal-expressing gene therapy vectors include the risk of insertional mutagenesis and limited efficacy due to hepatocellular division. Recent advances in messenger RNA (mRNA) codon optimization, synthesis, and encapsulation within biodegradable liver-targeted lipid nanoparticles (LNPs) have potentially enabled a new generation of safer, albeit temporary, treatments to restore liver metabolic function in patients with urea cycle disorders, including ARG1 deficiency. In this study, we applied such technologies to successfully treat an ARG1-deficient murine model. Mice were administered LNPs encapsulating human codon-optimized ARG1 mRNA every 3 d. Mice demonstrated 100% survival with no signs of hyperammonemia or weight loss to beyond 11 wk, compared with controls that perished by day 22. Plasma ammonia, arginine, and glutamine demonstrated good control without elevation of guanidinoacetic acid, a guanidino compound. Evidence of urea cycle activity restoration was demonstrated by the ability to fully metabolize an ammonium challenge and by achieving near-normal ureagenesis; liver arginase activity achieved 54% of wild type. Biochemical and microscopic data showed no evidence of hepatotoxicity. These results suggest that delivery of ARG1 mRNA by liver-targeted nanoparticles may be a viable gene-based therapeutic for the treatment of arginase deficiency.

Novel biodegradable internal stent as a mitigation strategy in high-risk pancreaticojejunostomy: technical notes and preliminary results.

Clinically relevant postoperative pancreatic fistula (CR-POPF) is the most feared complication after pancreaticoduodenectomy (PD), as it can lead to extremely poor outcomes. We herein report the preliminary results of an anastomotic technique based on the use of a novel internal biodegradable stent (IBS) to mitigate POPF sequelae. Between October 2020 and May 2021, all patients undergoing PD with high-risk pancreatic anastomosis received a pancreato-jejunal (PJ) anastomosis with an Archimedes™ IBS placement. Fifteen patients comprised our study cohort. In 11 cases, a 2-mm Archimedes™ stent was used, and in the remaining four patients, a 2.6-mm stent was used. Overall postoperative complications occurred in eight patients, with four cases being severe. Two patients developed CR-POPF, with one of them dying. In our small preliminary series, PJ anastomosis with an Archimedes™ IBS showed encouraging results in terms of CR-POPF incidence. Further studies are needed to confirm these findings.

Zebrafish Mutant Lines Reveal the Interplay between nr3c1 and nr3c2 in the GC-Dependent Regulation of Gene Transcription.

Glucocorticoids mainly exert their biological functions through their cognate receptor, encoded by the nr3c1 gene. Here, we analysed the glucocorticoids mechanism of action taking advantage of the availability of different zebrafish mutant lines for their receptor. The differences in gene expression patterns between the zebrafish gr knock-out and the grs357 mutant line, in which a point mutation prevents binding of the receptor to the hormone-responsive elements, reveal an intricate network of GC-dependent transcription. Particularly, we show that Stat3 transcriptional activity mainly relies on glucocorticoid receptor GR tethering activity: several Stat3 target genes are induced upon glucocorticoid GC exposure both in wild type and in grs357/s357 larvae, but not in gr knock-out zebrafish. To understand the interplay between GC, their receptor, and the mineralocorticoid receptor, which is evolutionarily and structurally related to the GR, we generated an mr knock-out line and observed that several GC-target genes also need a functional mineralocorticoid receptor MR to be correctly transcribed. All in all, zebrafish mutants and transgenic models allow in vivo analysis of GR transcriptional activities and interactions with other transcription factors such as MR and Stat3 in an in-depth and rapid way.

Synthetic human ABCB4 mRNA therapy rescues severe liver disease phenotype in a BALB/c.Abcb4-/- mouse model of PFIC3.

BACKGROUND & AIMS: Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a rare lethal autosomal recessive liver disorder caused by loss-of-function variations of the ABCB4 gene, encoding a phosphatidylcholine transporter (ABCB4/MDR3). Currently, no effective treatment exists for PFIC3 outside of liver transplantation. METHODS: We have produced and screened chemically and genetically modified mRNA variants encoding human ABCB4 (hABCB4 mRNA) encapsulated in lipid nanoparticles (LNPs). We examined their pharmacological effects in a cell-based model and in a new in vivo mouse model resembling human PFIC3 as a result of homozygous disruption of the Abcb4 gene in fibrosis-susceptible BALB/c.Abcb4-/- mice. RESULTS: We show that treatment with liver-targeted hABCB4 mRNA resulted in de novo expression of functional hABCB4 protein and restored phospholipid transport in cultured cells and in PFIC3 mouse livers. Importantly, repeated injections of the hABCB4 mRNA effectively rescued the severe disease phenotype in young Abcb4-/- mice, with rapid and dramatic normalisation of all clinically relevant parameters such as inflammation, ductular reaction, and liver fibrosis. Synthetic mRNA therapy also promoted favourable hepatocyte-driven liver regeneration to restore normal homeostasis, including liver weight, body weight, liver enzymes, and portal vein blood pressure. CONCLUSIONS: Our data provide strong preclinical proof-of-concept for hABCB4 mRNA therapy as a potential treatment option for patients with PFIC3. LAY SUMMARY: This report describes the development of an innovative mRNA therapy as a potential treatment for PFIC3, a devastating rare paediatric liver disease with no treatment options except liver transplantation. We show that administration of our mRNA construct completely rescues severe liver disease in a genetic model of PFIC3 in mice.

Magnetic resonance (MR) for mural nodule detection studying Intraductal papillary mucinous neoplasms (IPMN) of pancreas: Imaging-pathologic correlation.

PURPOSE: Magnetic Resonance (MR) is recommended to diagnose Intraductal Papillary Mucinous Neoplasms (IPMN) and in the follow-up of borderline lesions. The purpose of this work is to evaluate the diagnostic accuracy of dynamic MR with Diffusion Weighted Imaging (DWI) in the identification of mural nodules of pancreatic IPMN by using pathological analysis as gold standard. MATERIALS AND METHODS: Ninety-one preoperative MR with histopathological diagnosis of IPMN were reviewed by two radiologists. Presence, number and size of mural nodule, signal intensity of the nodule on T1-weighted imaging (T1-WI) after contrast medium administration and on DWI. Inter-observer agreement was evaluated. RESULTS: Significant correlation (p < 0.0001) were found for presence of nodules > 5 mm on MR and pathological specimen, size and number of mural nodules evaluated on pathological review and degree of dysplasia, size and number of mural nodules evaluated on MR and tumoral dysplasia, presence of nodule > 5 mm with enhancement after contrast medium administration and hyperintensity on DWI and degree of dysplasia. Interobserver agreement was moderate for the presence of mural nodule (K = 0.56), for the presence of high signal intensity on DWI (K = 0.57) and enhancement of mural nodule (K = 0.58). Apparent Diffusion Coefficient (ADC) map histogram analysis showed a correlation between Entropy of the entire cystic lesion and the degree of dysplasia (p < 0.034). CONCLUSIONS: MR with dynamic and DWI sequences was an accurate method for the identification of ≥ 5 mm solid nodules of the IPMNs and correlate with the lesion malignancy. Entropy, calculated from the histogram analysis of the IPMN ADC map, correlated with the lesion dysplasia.

Prognostic value of pathological tumor regression grade in locally advanced gastric cancer: New perspectives from a single-center experience.

BACKGROUND AND OBJECTIVE: Perioperative chemotherapy (PC) with radical surgery represents the gold standard of treatment for resectable advanced gastric cancer (GC). The prognostic value of pathological tumor regression grade (TRG) induced by neoadjuvant chemotherapy (NACT) is not clearly established. This study aimed to investigate the correlation between TRG and survival in GC. METHODS: Patients affected by advanced GC undergoing PC and radical surgery were considered. TRG was assessed for each patient according to Becker's grading system. The correlation between TRG and survival was investigated. RESULTS: One-hundred patients were selected; 25 showed a good response (GR) (TRG 1a/1b), while 75 had a poor response (PR) (TRG 2/3) to NACT. GR patients showed better disease-free survival (DFS) (52 vs. 19 months, p < .001) and disease-specific survival (DSS) (57 vs. 25 months, p < .0001) when compared to PR patients. On univariate analysis, TRG, lymph node ratio (LNR), tumor size, grading, and post-neoadjuvant therapy TNM stage were significantly correlated with survival. On multivariate analysis, TRG, LNR and tumor size were independent prognostic factors for DFS and DSS. CONCLUSIONS: TRG, LNR, and tumor size are independent prognostic factors for DFS and DSS in patients with advanced GC undergoing NACT.

Impact of neoadjuvant therapy followed by laparoscopic radical gastrectomy with D2 lymph node dissection in Western population: A multi-institutional propensity score-matched study.

BACKGROUND AND OBJECTIVES: In the setting of a minimally invasive approach, we aimed to compare short and long-term postoperative outcomes of patients treated with neoadjuvant therapy (NAT) + surgery or upfront surgery in Western population. METHODS: All consecutive patients from six Italian and one Serbian center with locally advanced gastric cancer who had undergone laparoscopic gastrectomy with D2 lymph node dissection were selected between 2005 and 2019. After propensity score-matching, postoperative morbidity and oncologic outcomes were investigated. RESULTS: After matching, 97 patients were allocated in each cohort with a mean age of 69.4 and 70.5 years. The two groups showed no difference in operative details except for a higher conversion rate in the NAT group (p = 0.038). The overall postoperative complications rate significantly differed between NAT + surgery (38.1%) and US (21.6%) group (p = 0.019). NAT was found to be related to a higher risk of postoperative morbidity in patients older than 60 years old (p = 0.013) but not in patients younger (p = 0.620). Conversely, no difference in overall survival (p = 0.41) and disease-free-survival (p = 0.34) was found between groups. CONCLUSIONS: NAT appears to be related to a higher postoperative complication rate and equivalent oncological outcomes when compared with surgery alone. However, poor short-term outcomes are more evident in patients over 60 years old receiving NAT.