Introduction: The quantification of circulating tumor DNA (ctDNA) on the liquid biopsy at the time of diagnosis allows to stratify the outcome of diffuse large B-cell lymphoma (DLBCL) patients but its integration with molecular clustering has not been evaluated so far. Methods: A multicenter cohort of newly diagnosed and homogeneously treated DLBCL provided with ctDNA and with genomic DNA from lymph node (LN) biopsy represented the training cohort. A validation cohort of newly diagnosed DLBCL patients provided with ctDNA was collected. The CAPP-Seq assay was used. Results: The training cohort included 77 newly diagnosed DLBCL patients treated with R-CHOP-based therapy. After a median follow-up of 33.9 months, the 40-month progression-free survival (PFS) and overall survival (OS) were 65.2% and 80.2%, respectively. Using the LymphGen tool on ctDNA, 9 patients were classified as MCD, 5 as ST2, 7 as EZB, 5 as BN2 and 1 as molecular composite (BN2/ST2). ST2 and BN2 patients in both ctDNA (p = 0.032) and in LN biopsy (p = 0.007) displayed an excellent PFS compared to other patients. By recursive partitioning, patients assigned to clusters ST2 or BN2 (N = 10) further split the outcome of patients with ctDNA levels ≥2.5Log10hGE (40-month PFS of 100% compared to 33.2% for patients classified as MCD or EZB or not classified, p = 0.009). Therefore, by combining ctDNA levels and molecular clusters identified on the liquid biopsy, patients with <2.5Log10hGE and/or assigned to cluster BN2 and ST2 presented a 40-month PFS and OS of 80.4% and 93.0%, compared to 33.2% and 54.8% for other patients, respectively (both p < 0.001) (Figure 1A). Interestingly, 80% of ST2/BN2 patients reached an early molecular response (EMR) and the 2 ST2/BN2 patients who did not achieve an EMR after 1 cycle of therapy are still in complete remission. To validate this finding, a validation cohort of 89 newly diagnosed DLBCL was collected. The PFS and OS of the training of the validation cohort were superimposable (p = 0.805 and p = 0.608, respectively). Also in the validation cohort, patients with <2.5Log10hGE and/or assigned to cluster BN2 and ST2 presented an excellent outcome with a 40-month PFS and OS of 73.2% and 79.7%, compared to 39.2% and 44.8% for other patients, respectively (both p = 0.001) (Figure 1B). By combining the training and the validation cohort, we performed a multivariate analysis on 166 patients. Both a ctDNA <2.5Log10hGE and the BN2/ST2 cluster maintained an independent association with an excellent outcome when adjusted for IPI and cell of origin (Figure 1C). Moreover, compared to ctDNA levels only, the addition of BN2/ST2 cluster improved the C statistics of the model (0.64 vs. 0.60 for PFS and 0.68 vs. 0.63 for OS, Figure 1D). Keywords: aggressive B-cell non-Hodgkin lymphoma, liquid biopsy The research was funded by: Molecular bases of disease dissemination in lymphoid malignancies to optimize curative therapeutic strategies, (5 x 1000 No. 21198), Associazione Italiana per la Ricerca sul Cancro Foundation Milan, Italy; Progetti di Rilevante Interesse Nazionale (PRIN; 2015ZMRFEA), Rome, Italy; the AGING Project—Department of Excellence—DIMET, Università del Piemonte Orientale, Novara, Italy; and Ricerca Finalizzata 2018 (project RF-2018-12365790), MoH, Rome, Italy; Swiss Cancer League, ID 3746, 4395 4660, and 4705, Bern, Switzerland; Research Advisory Board of the Ente Ospedaliero Cantonale, ABREOC 2019-22514, Bellinzona, Switzerland; European Research Council (ERC) Consolidator Grant CLLCLONE, ID: 772051; Swiss National Science Foundation, ID 320030_169670/1 and 310030_192439, Berne, Switzerland; Fondazione Fidinam, Lugano, Switzerland; Nelia & Amadeo Barletta Foundation, Lausanne, Switzerland; Fond’Action, Lausanne, Switzerland; The Leukemia & Lymphoma Society, Translational Research Program, ID 6594-20, New York. No conflicts of interests pertinent to the abstract.

Diabetes mellitus (DM) is associated with pancreatic cancer (PC) as insulin is produced in the pancreatic beta cells. Whereas large epidemiological studies are available investigating the risk and prevalence of DM patients developing PC, only little is known about the impact of DM on the outcome of PC patients. PC patients treated at the Medical University of Vienna between 1997 and 2020 with available information about their diabetic status at initial diagnosis of PC were identified from the PC database of our institution. We investigated the association of DM, antidiabetic drugs as well as random blood glucose and HbA1c levels with overall survival (OS, interval from initial diagnosis to death or last date of contact) in the overall patient cohort and subgroups of early and metastatic PC patients. Among 667 patients included into this analysis, 221 (33.1%) presented with DM at PC diagnosis, 53 (8.0%) developed DM later during the course of disease and 393 (58.9%) never developed DM. Presence of DM at initial diagnosis was significantly associated with a shorter OS in the overall patient population (13 vs. 17 months, hazard ratio (HR): 1.23, 95% confidence interval (CI 9.3 - 12.8; 6.6 - 11.3), p=0.024) as well as in patients with resectable disease (20 vs. 29 months, HR: 1.44, CI (13.4 - 31.1; 22.2 - 35.1), p=0.034). but showed no association in metastatic patients (8 vs. 11 months, HR: 1.21, CI (6.6 - 11.3; 9.3 - 12.8), p=0.200). Median OS was comparable between DM PC patients receiving metformin, insulin or other antidiabetic drugs (13 vs 15 vs 8 months, p=0.221, log-rank test). A random blood glucose at diagnosis of >300mg/dl compared to <200mg/dl (HR 2.29, 95%CI 0.06-3.53; p=0.005) as well as an HbA1c of >8% compared to <6.5% (HR 1.88, 95%CI 1.03-3.44; p=0.040) was associated with a significantly shorter OS. Within this large cohort of PC patients, one third presented with DM at initial diagnosis. We observed a negative prognostic impact of DM especially in the subgroup of early PC patients. Different antidiabetic drugs thereby did not influence prognosis substantially. Only highly elevated levels of blood glucose and HbA1c seem to impact prognosis, whereas moderately elevated levels did not.

BACKGROUND:Statins are cholesterol-lowering drugs prescribed for the prevention and treatment of cardiovascular disease. Moreover, statins may possess anticancer properties and interact with receptor activator of nuclear factor κB ligand expression. We aimed at evaluating a hypothetical synergistic effect of statins with denosumab in early-stage breast cancer (BC) patients from the Austrian Breast and Colorectal Cancer Study Group (ABCSG) trial 18.PATIENTS AND METHODS:ABCSG-18 (NCT00556374) is a prospective, randomized, double-blind, phase III study; postmenopausal patients with hormone receptor-positive BC receiving a nonsteroidal aromatase inhibitor were randomly assigned to denosumab or placebo. In this post hoc analysis, we investigated the effects of concomitant statin therapy on recurrence risk (RR) of BC, fracture risk and bone mineral density (BMD).RESULTS:In the study population (n = 3420), statin therapy (n = 824) was associated with worse disease-free survival (DFS) [hazard ratio (HR) 1.35, 95% confidence interval (CI) 1.04-1.75; P = 0.023]. While no significant effect of lipophilic statins (n = 710) on RR was observed (HR 1.30, 95% CI 0.99-1.72; P = 0.062), patients on hydrophilic statins (n = 87) had worse DFS compared with patients not receiving any statins (HR 2.00, 95% CI 1.09-3.66; P = 0.026). This finding was mainly driven by the effect of hydrophilic statins on DFS in the denosumab arm (HR 2.63, 95% CI 1.21-5.68; P = 0.014). However, this effect subsided after correction for confounders in the sensitivity analysis. No association between statin use and fracture risk or osteoporosis was observed.CONCLUSION:According to this analysis, hydrophilic statins showed a detrimental effect on DFS in the main model, which was attenuated after correction for confounders. Our data need to be interpreted with caution due to their retrospective nature and the low number of patients receiving hydrophilic statins.

Chronic hepatitis B virus (HBV) infection is a global health problem that presents as a spectrum of liver disease, reflecting an interplay between the virus and the host immune system. HBV genomes exist as episomal covalently closed circular DNA (cccDNA) or chromosomal integrants. The relative contribution of these genomes to the viral transcriptome in chronic hepatitis B (CHB) is not well-understood. We developed a qPCR method to estimate the abundance of HBV cccDNA- and integrant-derived viral transcripts and applied this to a cohort of patients diagnosed with CHB in the HBe antigen negative phase of disease. We noted a variable pattern of HBV transcripts from both DNA templates, with preS1/S2 mRNAs predominating and a significant association between increasing age and the expression of integrant-derived mRNAs, but not with inflammatory status. In contrast, cccDNA-derived transcripts were associated with markers of liver inflammation. Analysis of the inflammatory hepatic transcriptome identified 24 genes significantly associated with cccDNA transcriptional activity. Our study uncovers an immune gene signature that associates with HBV cccDNA transcription and increases our understanding of viral persistence.

Background and Aims Liver fibrosis holds a relevant prognostic meaning in primary biliary cholangitis (PBC). Noninvasive fibrosis evaluation using vibration‐controlled transient elastography (VCTE) is routinely performed. However, there is limited evidence on its accuracy at diagnosis in PBC. We aimed to estimate the diagnostic accuracy of VCTE in assessing advanced fibrosis (AF) at disease presentation in PBC. Approach and Results We collected data from 167 consecutive treatment‐naïve PBC patients who underwent liver biopsy (LB) at diagnosis at six Italian centers. VCTE examinations were completed within 12 weeks of LB. Biopsies were scored by two blinded expert pathologists, according to the Ludwig system. Diagnostic accuracy was estimated using the area under the receiver operating characteristic curves (AUROCs) for AF (Ludwig stage ≥III). Effects of biochemical and clinical parameters on liver stiffness measurement (LSM) were appraised. The derivation cohort consisted of 126 patients with valid LSM and LB; VCTE identified patients with AF with an AUROC of 0.89. LSM cutoffs ≤6.5 and >11.0 kPa enabled to exclude and confirm, respectively, AF (negative predictive value [NPV] = 0.94; positive predictive value [PPV] = 0.89; error rate = 5.6%). These values were externally validated in an independent cohort of 91 PBC patients (NPV = 0.93; PPV = 0.89; error rate = 8.6%). Multivariable analysis found that the only parameter affecting LSM was fibrosis stage. No association was found with BMI and liver biochemistry. Conclusions In a multicenter study of treatment‐naïve PBC patients, we identified two cutoffs (LSM ≤6.5 and >11.0 kPa) able to discriminate at diagnosis the absence or presence, respectively, of AF in PBC patients, with external validation. In patients with LSM between these two cutoffs, VCTE is not reliable and liver biopsy should be evaluated for accurate disease staging. BMI and liver biochemistry did not affect LSMs.

Hepatocellular carcinoma (HCC) incidence is increasing worldwide and prognostic biomarkers are urgently needed to guide treatment and reduce mortality. Circulating cell‐free DNA of tumour origin (ctDNA) is a novel, minimally invasive means of determining genetic alterations in cancer. We determined the utility of ctDNA as a prognostic biomarker of survival in HCC.

Among clinicians, the users of medical calculators have expanded in recent years to an unprecedented number. The usefulness of some of these calculators is sometimes debatable, and experienced professionals may at times be right in avoiding their use; however, many may simply be unaware of the very existence of medical calculators applicable to their field of interest. The authors felt that this latter scenario might possibly apply to hepatocellular carcinoma (HCC). Hence, the authors concisely reviewed 10 free online medical calculators proposed in the last 8 years, categorizing them on the basis of the purpose for which they were developed (risk of harboring or developing HCC, N=4; prognostication in established HCC, N=6). In addition, the authors tried to establish the success each calculator has had so far in the medical community, by 2 criteria: having been included in the more popular app of medical calculators and being highly cited in the scientific literature.

Angioleiomyoma is a benign smooth muscle and vessel tumor; laryngeal localization is extremely rare with only 24 cases described in the literature; moreover, it should be considered in the differential diagnosis of laryngeal mass. Endoscopic complete surgical excision with dissection along capsule is now considered the gold-standard treatment for small and well-circumscribed laryngeal angioleiomyoma. We present a case of laryngeal angioleiomyoma successfully treated with carbon dioxide laser technology which resulted in a bleeding reduction and adequate hemostasis with less tissue damage and good functional outcome.

BACKGROUND:The risk of life-threatening complications, such as visceral disseminated varicella zoster virus (VZV) infection, is greater in immunosuppressed individuals, such as systemic lupus erythematosus (SLE) patients.CASE PRESENTATION:Here, a case is reported of a Caucasian woman diagnosed with lupus nephritis and anti-phospholipid syndrome, who was subjected to mycophenolate mofetil and high-dose steroid remission-induction therapy. Two months later she developed abdominal pain followed by a fatal rapid multi-organ failure. As no typical skin rashes were evident, death was initially attributed to catastrophic anti-phospholipid syndrome. However, autopsy and virological examinations on archival material revealed a disseminated VZV infection.CONCLUSIONS:Overall, this case highlights the importance of having a high clinical suspicion of fatal VZV infections in heavily immunosuppressed SLE patients even when typical signs and symptoms are lacking.

Serous effusions complicating the course of lymphomas occur commonly in the pleural space but seldom in the peritoneum, where they most often present as chylous ascites with diagnostic cytology. Almost invariably, in these rare cases, the serum to ascites albumin gradient is low. We describe a 28-year-old woman with anasarca, ascites and a serum to ascites albumin gradient of 1.1 g/dl, consistent with portal hypertension. No tumour cells were detected in the ascitic fluid. However, a CT scan of the chest and abdomen disclosed liver and spleen enlargement and multiple enlarged retroperitoneal lymph nodes, suspicious for a lymphoproliferative disorder. Bone marrow aspiration and biopsy were not diagnostic, so a decision was made to proceed with a splenectomy despite the onset of low-grade disseminated intravascular coagulation. Surgery was uneventful. Diffuse large B cell lymphoma was diagnosed. A liver biopsy taken at the time of surgery demonstrated that the liver parenchyma was massively infiltrated by reactive T lymphocytes surrounding rare large CD20+ tumour cells. This infiltrate had likely led to increased portal pressure attended by ascites formation, which resolved completely after chemotherapy. The case emphasizes the rewards of pursuing a diagnosis supported by a high prior probability even in the presence of apparently discordant laboratory findings, as well as the importance of performing a diagnostic splenectomy in case of splenomegaly with unexplained focal lesions. LEARNING POINTS Lymphomas may present with serous effusion, which is usually chylous and with positive cytology when represented by ascites accumulation; non-chylous effusions can be due to altered lymphatic drainage, extrinsic compression of the portal vein by enlarged lymph nodes as well as massive infiltration of the liver by lymphoma. If the cause of splenomegaly is unclear, diagnostic splenectomy remains a viable option. The diagnosis of lymphoma should always be pursued, even if it requires apparently unwise surgery, since this type of cancer can be treated effectively only if thoroughly characterized pathologically and molecularly.

Background: Hepatocellular carcinoma (HCC) is increasing globally. Prognostic biomarkers are urgently needed to guide treatment and reduce mortality. Tumour-derived circulating cell-free DNA (ctDNA) is a novel, minimally invasive means of determining genetic alterations in cancer. We evaluate the accuracy of ctDNA as a biomarker in HCC. Methods: Plasma cell-free DNA, matched germline DNA and HCC tissue DNA were isolated from patients with HCC (n = 51) and liver cirrhosis (n = 10). Targeted, multiplex polymerase chain reaction ultra-deep sequencing was performed using a liver cancer-specific primer panel for genes ARID1A, ARID2, AXIN1, ATM, CTNNB1, HNF1A and TP53. Concordance of mutations in plasma ctDNA and HCC tissue DNA was determined, and associations with clinical outcomes were analysed. Results: Plasma cell-free DNA was detected in all samples. Lower plasma cell-free DNA levels were seen in Barcelona Clinic Liver Cancer (BCLC A compared with BCLC stage B/C/D (median concentration 122.89 ng/mL versus 168.21 ng/mL, p = 0.041). 29 mutations in the eight genes (21 unique mutations) were detected in 18/51 patients (35%), median 1.5 mutations per patient (interquartile range 1-2). Mutations were most frequently detected in ARID1A (11.7%), followed by CTNNB1 (7.8%) and TP53 (7.8%). In patients with matched tissue DNA, all mutations detected in plasma ctDNA detected were confirmed in HCC DNA; however, 71% of patients had mutations identified in HCC tissue DNA that were not detected in matched ctDNA. Conclusion: ctDNA is quantifiable across all HCC stages and allows detection of mutations in key driver genes of hepatic carcinogenesis. This study demonstrates high specificity but low sensitivity of plasma ctDNA for detecting mutations in matched HCC tissue. Crown Copyright (C) 2019 Published by Elsevier Ltd. All rights reserved.

Background: We recently derived and validated a model including alkaline phosphatase (ALP), bilirubin, aminotransferase, age at diagnosis, and waiting time before receiving ursodeoxycholic acid (UDCA), i.e. the UDCA-response score (URS). This score predicts future response to UDCA prior to initiation of therapy in patients with primary biliary cholangitis (PBC) and enables pre-treatment selection of high-risk patients for second-line therapy earlier in the disease course, hence delivering effective care.

Radiofrequency ablation (RFA) achieves tumor necrosis by cell protein denaturation, induced by tissue heating above 45°C to obtain irreversible cellular injury. Energy is supplied by a generator connected to an active electrode tip inserted into the tumor. RFA was developed in the early nineties for percutaneous or intraoperative treatment of hepatocellular carcinoma (HCC). It is recommended by the EASL–EORTC Clinical Practice Guidelines for early-stage disease (not eligible for surgery).[1] Today, RFA is widely used in the treatment of HCC and has been given attention as a potential treatment for many tumors (e.g., kidney, lung, and bone). Recently, new devices have been used to perform RFA through linear echoendoscopes, for targeting focal pancreatic lesions (neuroendocrine tumors, adenocarcinomas, and pancreatic cysts).[234] Moreover, the left lobe of the liver can be well visualized using endoscopic ultrasound (EUS), making it amenable to EUS-guided interventional procedures, including ablations.[5] A 76-year-old HBV/HCV-cirrhotic female presenting with portal hypertension and ascites had been diagnosed with HCC on radiological imaging (Barcelona Clinic Liver Cancer Stage A: Child–Pugh B8/Performance status: 1), but she was deemed unfit for surgery. She was, therefore, referred for EUS-guided sampling of hilar lymph nodes, in view of further management of a suspicious HCC in the fourth segment of her liver [Figure 1a–c]. Sampling of the hepatic nodule and the lymph nodes resulted in a diagnosis of well-differentiated HCC and reactive adenopathies [Figure 2].Figure 1: Abdominal computed tomography shows a 30-mm nodule with diffuse hyperenhancement in the arterial phase, and slow wash out in the portal and late phases (a-c). After 1 month, an abdominal computed tomography scan shows a large ablated area in the arterial, portal, and late phases (d-f)Figure 2: High-power images show pseudoglandular aggregates of atypical epithelial cells (a) (H and E, ×400). Gomori's staining (b) highlights the presence of neoplastic pseudoglandular structures (×400). CD34 immunostaining demonstrates sinusoidal capillarization (c), a typical change that characterizes the endothelial cells of hepatocellular carcinoma (×200)Considering the easy approach provided by EUS and obstacles to the percutaneous route (large umbilical hernia), the nodule was ablated with EUS guidance (GIF-160 Olympus, Tokyo, Japan) using a 20-mm active tip water-cooled electrode needle (EUSRA™ RF Electrode-VIVA RF Generator, STARmed, Seoul Korea). The procedure required three passes at five positions, using a multi-pass technique that allowed a larger ablation. Video 1 shows the insertion of the needle inside the HCC and the development of air bubbles near the electrode tip (first pass). Later, a panoramic view shows a large hyperechoic halo occupying the nodule. This marked the conclusion of the procedure, after a total of 30 min of energy application (starting power 30 W). An ultrasound imaging contrast (Sonovue®, Bracco, Italy) was administered during the procedure for marking the ablated area (nonenhancing tissue) and targeting further insertions. No adverse event was observed, and the patient was discharged on the 3rd postoperative day. One month later, computed tomography scan showed a large ablated area [Figure 1d–f]. This case highlights the possibilities for an EUS-guided transluminal approach to perform RFA of extrapancreatic neoplasms, such as a liver lesion, when percutaneous or surgical treatments are technically challenging or prohibitive. Declaration of patient consent The authors certify that they have obtained all appropriate patient consent forms. In the form the patient has given her consent for her images and other clinical information to be reported in the journal. The patient understands that her name and initial will not be published and due efforts will be made to conceal her identity, but anonymity cannot be guaranteed. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.

Introduction: The scarcity of donor livers has triggered efforts to expand the existing donor pool by using marginal donor livers, such as steatotic livers. However, steatotic donor livers are at a high risk of primary graft non-function, early allograft dysfunction, and graft loss due to their enhanced susceptibility to ischemia–reperfusion (I/R) injury. Necroptosis is a novel form of cell death and has been implicated in I/R injury. Receptor-interacting protein kinase 3 (RIPK3) is thought to be instrumental in the execution of necroptosis. However, necroptosis and RIPK3 have not been fully examined in steatotic liver undergoing I/R injury. In this study, we developed an in vitro hepatic steatosis model undergoing I/R injury to further study mechanisms of cell death. Methods: AML-12 cells were cultured in media containing increasing concentrations (0.25, 0.5, 1.0, 2.0) mM of free fatty acid (FFA) for 24 hours to induce hepatic steatosis. Further, FFA-treated cells were subjected to oxygen-glucose deprivation (OGD) conditions by culturing in glucose-free media under hypoxic conditions (1% O2, 5% CO2, and 94% N2) for 12 hours to mimic ischemia. Oil Red O staining was performed to assess the hepatocellular steatosis. Protein expression was detected by western blot analysis, and quantitative polymerase chain reaction was used for mRNA expression quantification. Cell viability was determined by CellTiter-Blue Cell Viability Assay (Promega). Results: A dose-dependent increase in fat accumulation was observed after 24 hours of FFA treatment. There was no significant decrease in cell viability after FFA exposure (P = 0.17). A concentration of 2 mM FFA was considered to be optimal as the cells maintained viability and FFA deposition even after 48 hours of FFA exposure. The hypoxia-sensitive genes, solute carrier family 2, facilitated glucose transporter member 1 (Slca1), and vascular endothelial growth factor (Vegf) were increased (2.6and 2.7-fold, respectively) after OGD. Treatment with FFA + OGD reduced cell viability after 12 hours of OGD (P < 0.01). RIPK3 protein was significantly upregulated in OGD-treated cells compared with control FFA-treated cells (2.4-fold; P = 0.01). Lack of cleaved-CASPASE3 expression indicated apoptosis was not an active pathway in our model. Nuclear factor NF-κB, which plays a role in regulating the DNA damage-repairing system, decreased in OGD-treated cells (4.6-fold). Similarly, phosphoglycerate mutase family 5 (Pgam5), a gene that protects the cells from necroptosis, was downregulated in OGD-treated cells (1.5-fold). Conclusion: Our findings suggest that necroptosis may contribute to I/R injury in our in vitro model. Future studies will investigate the use of RIPK3 inhibition during the re-oxygenation stage as a therapeutic agent to reduce the consequences of I/R injury. In conclusion, these findings suggest our model may be used to study the cell death pathways active during steatosis and hepatic I/R injury. H-Ferritin and iron activate inflammatory pathways in adipocytes LA JASKOWSKI,* KR BRIDLE,* LJ BRITTON,* A JAYACHANDRAN,* GA RAMM, DHG CRAWFORD* *Gallipoli Medical Research Institute, School of Clinical Medicine, University of Queensland, and QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia

Bariatrische Chirurgie geht mit einer Reihe von metabolischen Veränderungen einher. So wurden zum Beispiel ein erhöhtes Risiko für hyperinsulinämische Hypoglykämien als Folge eines gestörten Kohlenhydratresorptions-/Insulinwirkungsverhältnisses nach Roux-en-Y Magenbypass beschrieben. Mögliche Auswirkungen auf die Schwangerschaft wurden bislang allerdings nicht untersucht.