Florian Primavesi

Comprehensive immunohistochemical analysis of histone deacetylases in pancreatic neuroendocrine tumors: HDAC5 as a predictor of poor clinical outcome ☆ ☆☆

Epigenetic factors contribute to carcinogenesis, tumor promotion, and chemoresistance. Histone deacetylases (HDACs) are epigenetic regulators that primarily cause chromatin compaction, leading to inaccessibility of promoter regions and eventually gene silencing. Many cancer entities feature overexpression of HDACs. Currently, the role of HDACs in pancreatic neuroendocrine tumors (pNETs) is unclear. We analyzed the expression patterns of all HDAC classes (classes I, IIA, IIB, III, and IV) in 5 human tissue microarrays representing 57 pNETs resected between 1997 and 2013 and corresponding control tissue. All pNET cases were characterized clinically and pathologically according to recent staging guidelines. The investigated cases included 32 (56.1%) female and 25 (43.9%) male pNET patients (total n=57, 47.4% immunohistochemically endocrine positive). Immunohistochemical profiling revealed a significant up-regulation of all HDAC classes in pNET versus control, with different levels of intensity and extensity ranging from 1.5- to >7-fold up-regulation. In addition, expression of several HDACs (HDAC1, HDAC2, HDAC5, HDAC11, and Sirt1) was significantly increased in G3 tumors. Correlation analysis showed a significant association between the protein expression of HDAC classes I, III, and IV and rate of the pHH3/Ki-67-associated mitotic and proliferation index. Furthermore, especially HDAC5 proved as a negative predictor of disease-free and overall survival in pNET patients. Overall, we demonstrate that specific members of all 4 HDAC classes are heterogeneously expressed in pNET. Moreover, expression of HDACs was associated with tumor grading, proliferation markers, and patient survival, therefore representing interesting new targets in pNET treatment.

Perioperative Von Willebrand Factor Dynamics are Associated with Liver Regeneration and Predict Outcome after Liver Resection

von Willebrand Factor (vWF) was found to mediate platelet influx during the early phase of liver regeneration in mice. Furthermore, increased vWF‐antigen (vWF‐Ag) levels were shown to be predictive for outcome of patients with chronic liver disease. Accordingly, we aimed to assess the relevance of perioperative vWF‐Ag dynamics in terms of liver regeneration and clinical outcome in patients undergoing liver resection (LR). Accordingly, we observed that vWF‐Ag and its activity—estimated by ristocetin cofactor measurement—increased immediately after induction of liver regeneration and was associated with platelet accumulation within the liver. However, a significant vWF‐Ag burst was only observed in patients with unaffected postoperative liver regeneration. E‐selectin, as an established marker for endothelial cell activation, was found to correlate with vWF‐Ag in the liver vein after induction of liver regeneration (R = 0.535, P = 0.022). Preoperative vWF‐Ag levels significantly predicted postoperative liver dysfunction (LD; N = 95; area under the curve, 0.725; P = 0.009). Furthermore, a cutoff of vWF‐Ag ≥182% was defined to identify patients with a higher risk for postoperative LD or morbidity. This was confirmed within an independent mulitcenter validation cohort (N = 133). Ultimately, multivariable analysis revealed that vWF‐Ag was an independent predictor of postoperative LD and morbidity. Conclusion: Within this study, we were able to provide evidence that an initial vWF burst is required to allow for adequate platelet accumulation and concomitant liver regeneration post‐LR and might be abolished as a consequence of intrahepatic endothelial cell dysfunction. We were further able to reveal and validate the potential of preoperative vWF‐antigen levels to predict poor postoperative outcome in patients undergoing LR. Despite the pathophysiological relevance of our findings, vWF‐Ag seems to be a valuable tool for preoperative risk assessment in patients undergoing LR. (Hepatology 2018;67:1516‐1530)

Relevance of MicroRNA200 Family and MicroRNA205 for Epithelial to Mesenchymal Transition and Clinical Outcome in Biliary Tract Cancer Patients

Extensive stromal interaction is one reason for the dismal outcome of biliary tract cancer (BTC) patients. Epithelial to mesenchymal transition (EMT) is involved in tumor invasion and metastasis and is partly regulated by microRNAs (miRs). This study explores the expression of anti-EMT miR200 family (miR141, −200a/b/c, −429) and miR205 as well as the EMT-related proteins E-cadherin and vimentin in a panel of BTC cell lines and clinical specimens by quantitative real-time polymerase chain reaction, Western blot and immunohistochemistry, respectively. MicroRNA expression was correlated to (i) the expression patterns of E-cadherin and vimentin; (ii) clinicopathological characteristics; and (iii) survival data. MicroRNA-200 family and miR205 were expressed in all BTC cells and clinical specimens. E-cadherin and vimentin showed a mutually exclusive expression pattern in both, in vitro and in vivo. Expression of miR200 family members positively correlated with E-cadherin and negatively with vimentin expression in BTC cells and specimens. High expression of miR200 family members (but not miR205) and E-cadherin was associated with longer survival, while low miR200 family and high vimentin expression was a predictor of unfavorable survival. Overall, the current study demonstrates the relevance of the miR200 family in EMT of BTC tumors and suggests these miRs as predictors for positive outcome.

Thermographic real-time-monitoring of surgical radiofrequency and microwave ablation in a perfused porcine liver model

Radiofrequency ablation (RFA) and microwave ablation (MWA) are currently the dominant modalities to treat unresectable liver tumors. Monitoring the ablation process with b-mode-sonography is often hampered by artefacts. Furthermore, vessels may cause cooling in the adjacent tumor target (heat-sink-effect) with risk of local recurrence. The present study evaluated infrared-thermography to monitor surgical RFA/MWA and detect heat-sink-effects in real-time. RFA and MWA of perfused porcine livers was conducted at peripheral and central-vessel-adjacent locations, and monitored by real-time thermography. Ablation was measured and evaluated by gross pathology. The mean time for ablation was significantly longer in RFA compared with MWA (8 vs. 2 min). Although mean macroscopic ablation diameter was similar (RFA, 3.17 cm; MWA, 3.38 cm), RFA showed a significant heat-sink-effect compared with MWA. The surface temperature during central RFA near vessels was 1/3 lower compared with peripheral RFA (47.11±8.35°C vs. 68.72±12.70°C; P<0.001). There was no significant difference in MWA (50.52±8.35°C vs. 50.18±10.35°C; P=0.74). In conclusion, thermography is suitable to monitor the correct ablation with MWA and RFA. The results of the current study demonstrated a significant heat-sink-effect for RFA, but not MWA near vessels. MWA reaches consistent surface temperatures much faster than RFA. With further in vivo validation, thermography may be useful to ensure appropriate ablation particularly near vulnerable or vascular structures.

Hepatosteatosis from Lysosomal Acid Lipase Deficiency

A 21-year-old clinically well male (BMI 20.8 kg/m) with unremarkable family and medical history presented to the surgical outpatient clinic with a two-day history of left side inguinal pain and was diagnosed with uncomplicated hernia. Transabdominal preperitoneal patch repair was scheduled and performed several weeks later. During herniotomy, a Bmassive liver steatosis with completely orange surface^ was reported by the surgeon and liver biopsy specimens were obtained (Fig. 1). Pathological examination reported 50–60% microvesicular steatosis affecting hepatocytes and Kupffer cells with portal, periportal, and septal fibrosis (Fig. 2). Upon referral to the hepatologist, biochemical work-up revealed total cholesterol 265 mg/dL, LDL 211 mg/dL, HDL 39 mg/dL, triglycerides 164 mg/dL, ALT 56 IU/L, and a serum ferritin of 277 μg/L with otherwise normal laboratory reports. Infectious, autoimmune, or metabolic liver diseases such as Wilson’s disease were ruled out. The patient did not use and had never used any medication or illicit drugs and consumed moderate amounts of alcohol on weekends.

Pharmacological Inhibition of Class IIA HDACs by LMK-235 in Pancreatic Neuroendocrine Tumor Cells

Histone deacetylases (HDACs) play a key role in epigenetic mechanisms in health and disease and their dysfunction is implied in several cancer entities. Analysis of expression patterns in pancreatic neuroendocrine tumors (pNETs) indicated HDAC5 to be a potential target for future therapies. As a first step towards a possible treatment, the aim of this study was to evaluate the in vitro cellular and molecular effects of HDAC5 inhibition in pNET cells. Two pNET cell lines, BON-1 and QGP-1, were incubated with different concentrations of the selective class IIA HDAC inhibitor, LMK-235. Effects on cell viability were determined using the resazurin-assay, the caspase-assay, and Annexin-V staining. Western Blot and immunofluorescence microscopy were performed to assess the effects on HDAC5 functionality. LMK-235 lowered overall cell viability by inducing apoptosis in a dose- and time-dependent manner. Furthermore, acetylation of histone-H3 increased with higher LMK-235 concentrations, indicating functional inhibition of HDAC4/5. Immunocytochemical analysis showed that proliferative activity (phosphohistone H3 and Ki-67) decreased at highest concentrations of LMK-235 while chromogranin and somatostatin receptor 2 (SSTR2) expression increased in a dose-dependent manner. HDAC5 expression was found to be largely unaffected by LMK-235. These findings indicate LMK-235 to be a potential therapeutic approach for the development of an effective and selective pNET treatment.

HDAC-Linked “Proliferative” miRNA Expression Pattern in Pancreatic Neuroendocrine Tumors

Epigenetic factors are essentially involved in carcinogenesis, tumor promotion, and chemoresistance. Two epigenetic key players are miRNAs and histone deacetylases (HDACs). As previously shown by own theoretical databank analysis, the crosstalk between miRNAs and HDACs is relevant in different human chronic diseases and cancerogenic pathways. We aimed to investigate a potential connection between the expression of a well-defined subset of “proliferation-associated” miRNAs and the expression of HDACs as well as clinical parameters in pancreatic neuroendocrine tumors (pNETs). Materials and Methods: Expression levels of miRNA132-3p, miRNA145-5p, miRNA183-5p, miRNA34a-5p, and miRNA449a in 57 pNETs resected between 1997 and 2015 were measured and linked to the immunohistochemical expression pattern of members of the four HDAC classes on human tissue microarrays. All pNET cases were clinically and pathologically characterized according to published guidelines. Correlation analysis revealed a significant association between expression of specific miRNAs and two members of the HDAC family (HDAC3 and HDAC4). Additionally, a linkage between miRNA expression and clinico-pathological parameters like grading, TNM-staging, and hormone activity was found. Moreover, overall and disease-free survival is statistically correlated with the expression of the investigated miRNAs. Overall, we demonstrated that specific miRNAs could be linked to HDAC expression in pNETs. Especially miRNA449a (associated with HDAC3/4) seems to play an important role in pNET proliferation and could be a potential prognostic factor for poor survival. These first data could help, to improve our knowledge of the complex interactions of the epigenetic drivers in pNETs for further therapeutic approaches.

Exploring the surgical landscape of pancreatic neuroendocrine neoplasia in Austria: Results from the ASSO pNEN study group

INTRODUCTION Pancreatic neuroendocrine neoplasia (pNEN) show increasing incidence and management is complex due to biological heterogeneity. Most publications report isolated high-volume single-centre data. This Austrian multi-centre study on surgical management of pNENs provides a comprehensive real-life picture of quality indicators, recurrence-patterns, survival factors and systemic treatments. METHODS Retrospective, national cohort-study from 7 medium-/high-volume centres in Austria, coordinated under the auspices of the Austrian Society of Surgical Oncology (ASSO). RESULTS Two-hundred patients underwent resection for pNEN, 177 had non-functioning tumours and 31 showed stage 4 disease. Participating centres were responsible for 2/3 of pNEN resections in Austria within the last years. The mean rate of completeness of variables was 98.6%. Ninety-days mortality was 3.5%, overall rate of complications was 42.5%. Morbidity did not influence long-term survival. The 5-year overall-survival (OS) was 81.3%, 10-year-OS 52.5% and 5-year recurrence-free-survival (RFS) 69.8%. Recurrence was most common in the liver (68.1%). Four out of five patients with recurrence underwent further treatment, most commonly with medical therapy or chemotherapy. Multivariable analysis revealed grading (HR:2.7) and metastasis (HR:2.5) as significant factors for relapse. Tumours-size ≥2 cm (HR:5.9), age ≥60 years (HR:3.1), metastasis (HR:2.3) and grading (HR:2.0) were associated with OS. Tumours <2 cm showed 93.9% 10-year-OS, but 33% had G2/G3 grading, 12.5% positive lymph-nodes and 4.7% metastasis at diagnosis, each associated with significant worse survival. CONCLUSION Resection of pNENs in Austria is performed with internationally comparable safety. Analysed factors allow for risk-stratification in clinical treatment and future prospective trials. A watch-and-wait strategy purely based on tumour-size cannot be recommended.

Preoperative von willebrand factor – antigen predicts clinical outcome after liver resection: a prospective, international, multicenter trial

Backgroundand Aims:vWF-antigenhasbeenshowntobeincreased in patients with portal hypertension and to predict mortality in patients with chronic liver disease. This study aimed to assess the clinical utility of preoperative vWF-antigen levels to predict poor postoperative outcome in patients undergoing liver resection in a routine clinical setting. Methods: 95patientsundergoingliverresectionservedasprospective exploration cohort and results were validated in an independent cohort of 133 patients for 4 different institutions. VWF-Ag was evaluated perioperativelyand postoperative outcomewas recorded. Results: Preoperative vWF-antigen levels significantly predicted postoperative liver dysfunction (LD, area under the curve [AUC]: 0.725, P=0.009). Furthermore, a cut-off of vWF-antigen ≥182% was defined to identify patients with a higher incidence of postoperative LDormorbidity(LD:≥182%:33.3%,<182%:5.9%,P<0.001;morbidity: ≥182%: 74.1%, <182%: 44.1%, P=0.008). We confirmed our results within a prospective validation cohort (LD: ≥182%: 20.0%, <182%: 5.2%, P=0.008; morbidity: ≥182%: 56.4%, <182%: 35.1%, P=0.015). Analyzing the entire cohort, we found that patients exceeding the cut-off suffered from a significantly increased incidence of postoperative LD, morbidity, prolonged hospitalization, intensive care unit stayand mortality. Conclusions: Within this study we were able to reveal and subsequently validate the potential of preoperative vWF-antigen levels to predict poor postoperative outcome in patients undergoing liver resection. As vWF-antigen is easily determined and available in most standard laboratories, it seems to be avaluable clinical marker to allow for preoperative risk-stratification of patients undergoing liver resection.

Fatal long-term consequence of an allegedly safe and promising procedure: case report of gallbladder cancer 22 years after extracorporeal shockwave lithotripsy for gallstones

SummaryBackgroundExtracorporeal shockwave lithotripsy (ESWL) was first introduced in 1985 as a noninvasive, promising treatment option for gallbladder stones and extensively used especially in central European countries the following years. Due to high stone recurrence rates with secondarily indicated cholecystectomy and the introduction of laparoscopic surgery, ESWL gradually disappeared and is nowadays only rarely performed. In new techniques, long-term issues are sometimes not recognized initially.MethodsWe present a case of gallbladder cancer 22 years after ESWL in a 46-year-old female patient with fatal outcome. A short review on the history and technique of ESWL is presented and a possible relation to gallbladder cancer is discussed.ResultsAlthough gallbladder cancer after other gallbladder-preserving treatments for cholecystolithiasis‒such as cholecystostomy‒is a well-described issue, the present case is the only published report after ESWL in the English literature.In our patient, gallbladder cancer is probably not a direct cause of ESWL shock waves. Most likely chronic inflammation due to recurrent stones caused cancer. The fatal course of this case could have been prevented if regular follow-up and cholecystectomy would have been performed earlier.ConclusionsThousands of patients were treated with ESWL for gallbladder stones in the 1980s. Stone recurrence rates are high, but late complications such as malignancy are rare. On the other hand, since many patients were treated at a young age, regular long-term follow-up should be discussed in this cohort. We aim to draw attention to this topic and possibly prevent further cases.