Procalcitonin--a Marker for Anastomotic Insufficiency after Pancreatoduodenectomy?

BACKGROUND: Anastomotic insufficiency after pancreatoduodenectomy (PD) represents a major complication in pancreatic surgery. Early detection and treatment of pancreatic fistulas (PF) are essential for the outcome of affected patients. Procalcitonin (PCT) is a biochemical marker which allows detection of bacterial infections. The aim of this study was to evaluate if PCT is suitable for early detection of PF after PD. METHODS: In this prospective study patients undergoing PD from 08/2010 to 09/2012 were included into three groups: (1) patients without complications (n = 19), (2) patients with postoperative infections (n = 14) and (3) PF (n = 7). Using a defined study protocol, clinical (e.g., vital signs, drain fluid, etc.) and laboratory parameters (full blood count, inflammatory markers) were assessed daily for the first ten postoperative days. RESULTS: 76 patients were assessed. 40 (52.6%) patients underwent PD and were included. CRP and PCT demonstrated an initial peak at the 1st to 3rd postoperative day with subsequent normalization. Patients with postoperative infections and PF showed a significant increase of PCT and CRP (p < 0.05) compared to patients without complications. Leucocyte counts demonstrated a variance in all three groups and clinical use for detection of complications was not evident. CONCLUSIONS: Patients with a postoperative complication revealed significantly increased levels of PCT and CRP without the expected normalization. PCT and/or CRP did not enable a distinction between patients with PF or postoperative infections. Thus, PCT does not seem to be suitable for detecting PF after PD and its use in the postoperative course after PD cannot be recommended.

Epigenetic regulation and role of metastasis suppressor genes in pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is distinguished by rapid dissemination. Thus, genetic and/or epigenetic deregulation of metastasis suppressor genes (MSG) is a likely event during early pancreatic carcinogenesis and a potential diagnostic marker for the disease. We investigated 9 known MSGs for their role in the dissemination of PDAC and examined their promoters for methylation and its use in PDAC detection. METHODS: MRNA expression of 9 MSGs was determined in 18 PDAC cell lines by quantitative RT-PCR and promoter methylation was analyzed by Methylation Specific PCR and validated by Bisulfite Sequencing PCR. These data were compared to the cell lines' in vivo metastatic and invasive potential that had been previously established. Statistical analysis was performed with SPSS 20 using 2-tailed Spearman's correlation with P < 0.05 being considered significant. RESULTS: Complete downregulation of MSG-mRNA expression in PDAC cell lines vs. normal pancreatic RNA occurred in only 1 of 9 investigated genes. 3 MSGs (CDH1, TIMP3 and KiSS-1) were significantly methylated. Methylation only correlated to loss of mRNA expression in CDH1 (P < 0.05). Bisulfite Sequencing PCR showed distinct methylation patterns, termed constant and variable methylation, which could distinguish methylation-regulated from non methylation-regulated genes. Higher MSG mRNA-expression did not correlate to less aggressive PDAC-phenotypes (P > 0.14). CONCLUSIONS: Genes with metastasis suppressing functions in other tumor entities did not show evidence of assuming the same role in PDAC. Inactivation of MSGs by promoter methylation was an infrequent event and unsuitable as a diagnostic marker of PDAC. A distinct methylation pattern was identified, that resulted in reduced mRNA expression in all cases. Thus, constant methylation patterns could predict regulatory significance of a promoter's methylation prior to expression analysis and hence present an additional tool during target gene selection.

Mimicry of human sepsis in a rat model--prospects and limitations.

BACKGROUND: Sepsis and systemic inflammatory response syndrome (SIRS) continue to represent critical conditions with persistently high mortality and continue to need experimental and clinical research. We developed a rat model of gram-positive and gram-negative SIRS/sepsis with in vivo visualization of the pulmonary microcirculation to evaluate the optimal dosage and application path for SIRS/sepsis-inducing agents. METHODS: Male Sprague-Dawley rats (n = 8 per group) were assigned to control, lipopolysaccharide (LPS), alphatoxin, or living Staphylococcus aureus (strain 68/50) groups. SIRS/sepsis was induced by intraperitoneal injection of the differing agents. The onset of SIRS was determined through human sepsis parameters and fluorescence video microscopy-based measurement of platelet and leukocyte velocity within the pulmonary vascular system (injection of 5 × 10(6) calcein AM-labeled nonactivated platelets; leukocytes labeled in vivo by rhodamine). RESULTS: The optimal dosage to induce SIRS was 30 mg/250 g body weight for LPS (bolus injection) and 60 µg/250 g body weight for alphatoxin (2 h continuous perfusion). Sepsis was not achieved by injection of living S. aureus. The onset of SIRS was seen after 2-5 h for LPS and after 2-4 h for alphatoxin after intraperitoneal administration with a significantly increased heart rate, breathing rate, and body temperature (P < 0.05) and significantly decreased cell velocity (P < 0.05). CONCLUSION: Our study represents an effective approach for a gram-negative (LPS) and gram-positive (alphatoxin) SIRS model to mimic human sepsis. Human sepsis-based criteria were used to define SIRS in our rats to achieve an optimal analogy for the human system. In our model, higher dosages were needed for SIRS induction than have been previously reported. The resulting, considerable heterogeneity of current SIRS-inducing models suggests that additional studies in this field are required to define standard procedures.

Blended learning in surgery using the Inmedea Simulator.

BACKGROUND: Recently, medical education in surgery has experienced several modifications. We have implemented a blended learning module in our teaching curriculum to evaluate its effectiveness, applicability, and acceptance in surgical education. METHODS: In this prospective study, the traditional face-to-face learning of our teaching curriculum for fourth-year medical students (n = 116) was augmented by the Inmedea Simulator, a web-based E-learning system, with six virtual patient cases. Student results were documented by the system and learning success was determined by comparing patient cases with comparable diseases (second and sixth case). The acceptance among the students was evaluated with a questionnaire. RESULTS: After using the Inmedea Simulator, correct diagnoses were found significantly (P < 0.05) more often, while an incomplete diagnostic was seen significantly (P < 0.05) less often. Significant overall improvement (P < 0.05) was seen in sixth case (62.3 ± 5.6 %) vs. second case (53.9 ± 5.6 %). The questionnaire revealed that our students enjoyed the surgical seminar (score 2.1 ± 1.5) and preferred blended learning (score 2.5 ± 1.2) to conventional teaching. CONCLUSION: The blended learning approach using the Inmedea Simulator was highly appreciated by our medical students and resulted in a significant learning success. Blended learning appears to be a suitable tool to complement traditional teaching in surgery.

Pancreatoduodenectomy--current status of surgical and perioperative techniques in Germany.

BACKGROUND: Pancreatoduodenectomy in Germany is performed by a broad range of hospitals. A diversity of operative techniques is employed as no guidelines exist for intra- and perioperative management. We carried out a national survey to determine the de facto German standards for pancreatoduodenectomy, assess quality assurance measures, and identify relevant issues for further investigation. METHODS: A questionnaire evaluating major outcome variables, case load, preferred surgical procedures, and perioperative management during pancreatoduodenectomy was developed and sent to 211 German hospitals performing >12 pancreatoduodenectomies per year (requirement for certification as a pancreas center). Statistical analysis was carried out using the Fisher Exact, Mann-Whitney U, and Spearman tests. RESULTS: The final response rate was 86 % (182/211). The preferred technique and de facto German standard for pancreatoduodenectomy was pylorus-preserving pancreatoduodenectomy with pancreatojejunostomy carried out via duct-to-mucosa anastomosis with interrupted sutures using PDS 4.0. The minority of German pancreas centers were certified (18-48 %). The certification rate increased with higher capacity levels and case load (P < 0.05); however, significant correlations between the fistula rate and hospital case load, hospital capacity level, or hospital certification status were not seen. CONCLUSION: This study revealed a distinct variety of management strategies for pancreatic surgery and available evidence-based data was not necessarily translated into clinical practice. The limited certification rate represented a shortcoming of quality assurance. The data emphasize the need for further trials to answer the questions whether hospital certifications and omission of drains improve outcome after pancreatoduodenectomy and for the establishment of guidelines for pancreatoduodenectomy.

Treatment options for postoperatively infected abdominal wall wounds healing by secondary intention.

PURPOSE: We present our current clinical approach for the treatment of postoperatively infected wounds of the abdominal wall healing by secondary intention that may help in the design of a randomized controlled trial to develop a standardized wound treatment pathway. METHODS: Patients with postoperatively infected abdominal wounds treated with either Advanced Wound Care (AWC) dressings or vacuum-assisted closure (VAC) therapy were enrolled in the study. Follow-up was carried out prospectively for wound healing and incidence of incisional hernia at the earliest 3 years after surgery. RESULTS: Sixty-two patients were included and wounds were initially treated antiseptically for 5.19 ± 2.91 days. Prior to VAC therapy, AWC dressings were applied for 8.75 ± 2.93 days to reduce reinfection. Greater wound size (>12 × 6 × 6cm) and extensive secretion (>200 ml/day) argued for the VAC system. Overall incidence of incisional hernia was 20.4%, with 18.4% occurring in AWC-treated patients and 27.3% in VAC-treated patients. Based on these results, a wound treatment pathway was established in our department. CONCLUSION: The established wound treatment pathway has helped to increase both workflow efficacy and outcome in the treatment of abdominal wounds. Wound size, amount of secretion, and status of infection were the parameters we used for the determination of appropriate treatment. The observational data gathered during the initiation of our pathway lay the basis for future randomized controlled trials that will determine the most appropriate treatment options in the setting of a standardized wound treatment pathway.

Epigenetic markers for chemosensitivity and chemoresistance in pancreatic cancer--a review.

Adjuvant first-line gemcitabine monochemotherapy presents a standard treatment for patients with advanced pancreatic adenocarcinoma and improves overall survival in chemosensitive patients. Nonetheless, 6-month progression-free survival remains below 15%, despite interdisciplinary approaches. The success of gemcitabine treatment is disappointing and-in the absence of reliable tumor markers--challenging to quantify. Epigenetic alterations have been recently identified to take on important roles in cancer development and possibly cancer treatment. In this context, microRNAs are becoming increasingly acknowledged as useful biomarkers for classifying cancers and providing information on their chemo- and radiosensitivity. This review illustrates the potential of genetic and epigenetic markers in the prediction of chemosensitivity in pancreatic cancer patients and in the monitoring of their response rates to adjuvant therapy.

Role of tumor microenvironment on gene expression in pancreatic cancer tumor models.

OBJECTIVES: The microenvironment is known to be a relevant factor of influence on tumor growth and metastasis in pancreatic ductal adenocarcinoma (PDAC). To determine the influence of the microenvironment on changes in gene expression, we analyzed gene expression in different PDAC tissues. METHODS: Four human PDAC cell lines were introduced into a murine PDAC model with two insertion techniques: injection and implantation. Gene expression profiles of the cell lines growing in vitro and in vivo (ectopically and orthotopically) were established by microarray and validated by RT-PCR. RESULTS: Significant differences were found in the gene expression profiles of the in vitro versus in vivo tissues (P < 0.05), while no differences were found between the in vivo tissues. Analyzing the orthotopic tumors derived from the injection and implantation methods, similar gene expression patterns with 0%-18% significantly differentially expressed genes between tumors of the two different methods were observed (analysis of variance [ANOVA]; P < 0.0001). CONCLUSIONS: Gene expression from cell lines growing in vitro differed from the expression patterns of the same cells growing in vivo, while the localization of the growing tumor cells did not significantly alter gene expression. These data demonstrate that the implantation and injection techniques used in this study yield similar results and may be compared with each other.

Analyzing miRNAs in ductal adenocarcinomas of the pancreas.

BACKGROUND: MicroRNAs (miRNAs) have gained attention as an epigenetic component involved in the development of pancreatic ductal adenocarcinoma (PDAC). Several methods for miRNA profiling are in common use, but the validity of these methods is not defined. The aim of this study was to define the optimal method for miRNA detection in PDAC. METHODS: miRNA expression was determined using different and partially redundant methods (miRNA microarray, TaqMan low density array (TLDA), single tube quantitative RT-PCR). The data from different methods were statistically evaluated and tested for intermethodic consistency and reliability of the results. Finally, the miRNA expression status and the cell lines' ability to metastasize were correlated. RESULTS: Comparing low and high metastatic cells, miRNA-microarrays identified fewer differentially expressed and only upregulated miRNAs (n=27; 27 up-regulated) compared with TLDAs (n=54; 19 up- and 35 down-regulated). Evaluating miRNAs that target tumor suppressor genes, expression of all single tube quantitative real-time reverse transcriptase PCR (qRT-PCR) validated miRNAs was detected to be significantly altered in TLDA analysis (100%). MiRNA microarrays detected only 25% of qRT-PCR validated miRNAs. Furthermore, results from TLDA analysis correlated well with data from qRT-PCR and presented ΔΔCt values from 3.5±1.86 (range 0.8-5.62) compared with 3.74±1.86 (range 0.78-5.95) in qRT-PCR. CONCLUSION: Notable differences comparing data obtained from different screening methods were found. While TLDA and qRT-PCR correlated well in quantity and quality of the measured miRNAs, several tumor suppressor gene targeting and down-regulated miRNAs were not detected by miRNA-microarrays. This heterogeneity shows that care must be exercised when comparing results from different methods in PDAC.

Economic Burden of Endoscopic Vacuum Therapy Compared to Alternative Therapy Methods in Patients with Anastomotic Leakage After Esophagectomy.

BACKGROUND: Endoscopic vacuum therapy (EVT) has become a promising option in the management of anastomotic leakage (AL) after esophagectomy. However, EVT is an effortful approach associated with multiple interventions. In this study, we conduct a comparative cost analysis for methods of management of AL. METHODS: All patients who experienced AL treated by EVT, stent, or reoperation following Ivor Lewis esophagectomy for esophageal cancer were included. Cases that were managed by more than one modality were excluded. For the remaining cases, in-patient treatment cost was collected for material, personnel, (par)enteral nutrition, intensive care, operating room, and imaging. RESULTS: 42 patients were treated as follows: EVT n = 25, stent n = 13, and reoperation n = 4. The mean duration of therapy as well as length of overall hospital stay was significantly shorter in the stent than the EVT group (30 vs. 44d, p = 0.046; 34 vs. 53d, p = 0.02). The total mean cost for stent was €33.685, and the total cost for EVT was €46.136, resulting in a delta increase of 37% for EVT vs. stent cost. 75% (€34.320, EVT), respectively, 80% (€26.900, stent) of total costs were caused by ICU stay. Mean pure costs for endoscopic management were relatively low and comparable between both groups (EVT: €1.900, stent: €1.100, p = 0.28). CONCLUSION: Management of AL represents an effortful approach that results in high overall costs. The expenses directly related to EVT and stent therapy were however comparatively low with more than 75% of costs being attributable to the ICU stay. Reduction of ICU care should be a central part of cost reduction strategies.

EP300--a miRNA-regulated metastasis suppressor gene in ductal adenocarcinomas of the pancreas.

Genetic and epigenetic alterations during development of pancreatic ductal adenocarcinomas (PDACs) are well known. This study investigates genetic and epigenetic data together with tumor biology to find specific alterations responsible for metastasis formation. Using 16 human PDAC cell lines in a murine orthotopic PDAC model, local infiltration and metastatic spread were assessed by standardized dissemination scores. The cell lines were further classified into 3 hierarchical groups according to their metastatic potential. Their mRNA and microRNA (miRNA) expression was profiled via mRNA-microarray as well as Taqman Low Density Array, and validated by single quantitative RT-PCR and Western blotting. In the highly metastatic group, a significant induction of EP300 targeting miRNAs miR-194 (fold change: 26.88), miR-200b (fold change: 61.65), miR-200c (fold change: 19.44) and miR-429 (fold change: 21.67) (p < 0.05) was detected. Corresponding to this, decreased expression of EP300 mRNA (p < 0.0001) and protein (p < 0.05) were detected in the highly metastatic PDAC cell lines with liver metastases compared to the nonmetastatic or marginally metastatic cell lines, while no correlation with local tumor growth was found. In conclusion, epigenetic alterations with upregulated EP300 targeting miRNAs miR-194, miR-200b, miR-200c and miR-429 are related to reduced EP300 mRNA and protein in PDAC. These results demonstrate that miRNAs might be able to modulate the expression of metastasis-specific suppressor genes and metastatic behavior in PDAC, suggesting diagnostic and therapeutic opportunities for EP300 and its targeting miRNAs in PDAC.

Prognostic impact of underlying liver fibrosis and cirrhosis after curative resection of hepatocellular carcinoma.

BACKGROUND: In the case of hepatocellular carcinoma (HCC), underlying liver pathology may not only determine the feasibility of surgery but may also affect the postsurgical outcome. We report our experience after curative liver resection for HCC in patients with normal liver, liver fibrosis, and liver cirrhosis. METHODS: A total of 72 patients after liver resection with curative intention were analyzed. Histopathologic findings of tumor-unaffected liver tissue were used for retrospective classification: group A (normal liver); group B (liver fibrosis); group C (liver cirrhosis). The groups were compared for differences in short-term surgical results, total survival, and recurrence-free survival. RESULTS: The rate of major complications was 34.7% and did not significantly differ among groups. The overall perioperative mortality rate was 9.7%, with one patient dying in group A and three patients dying in each of the other two groups. Including perioperative mortality, the median overall survival for the whole group was 37.3 months (95% confidence interval 29.3-45.2 months). The respective 1-, 2-, and 5-year survival rates for group A (n = 21) were 86%, 71%, and 50% and for group C (n = 24) 62%, 50%, and 17%. The overall survival of group B (n = 27) was intermediate (log-rank, P = 0.032). The respective recurrence-free survival rates were 76%, 42%, and 20% for group A and 39%, 13%, and 4% for group C, with group B being intermediate (log-rank, P = 0.016). CONCLUSIONS: Our data demonstrate that liver resection in the presence of compensated liver cirrhosis is feasible but associated with a significantly impaired prognosis for overall and recurrence-free survival. The management of cirrhotic patients with compensated liver function and HCC therefore also requires the opportunity for transplantation.

MicroRNAs: novel diagnostic and therapeutic tools for pancreatic ductal adenocarcinoma?

Pancreatic ductal adenocarcinoma (PDAC) is known for its very poor overall prognosis, making tools for early diagnosis and new therapeutic modalities urgently needed. MicroRNAs (miRNAs), endogenous noncoding RNA molecules of approximately 22 nt, have gained attention as an epigenetic component involved in the development of many cancers, including PDAC. miRNA expression profiles of varying pancreatic tissues have identified a number of differentially expressed miRNAs and seem to be able to differentiate between three tissues of clinical importance: normal pancreas, chronic pancreatitis, and PDAC. This article gathers our current knowledge of differentially expressed miRNAs in pancreatic tissues with relevance to PDAC and presents potential diagnostic and therapeutic opportunities.

Involvement of CD40 targeting miR-224 and miR-486 on the progression of pancreatic ductal adenocarcinomas.

BACKGROUND: Genetic and epigenetic alterations during development of pancreatic ductal adenocarcinomas (PDAC) are well known. Genetic and epigenetic data were correlated with tumor biology to find specific alterations responsible for invasion and metastasis in pancreatic ductal adenocarcinomas. METHODS: A total of 16 human PDAC cell lines were used in murine orthotopic PDAC models. By means of standardized dissemination scores, local invasion and metastatic spread were assessed. mRNA and microRNA expression were studied by microarray and TaqMan low-density array. Quantitative real-time-polymerase chain reaction and flow cytometry were used for expression validation. RESULTS: CD40 was detected as a relevant target gene for differentially expressed miRNAs observed in highly invasive and metastatic PDAC only. A significant overexpression (P < .05) of CD40-related miRNAs miR-224 and miR-486 was detected in highly invasive and metastatic PDAC, whereas CD40 mRNA expression was not significantly altered. Instead, CD40 protein expression at cell surfaces of these highly invasive and metastatic PDAC was significantly reduced (P < .01). CONCLUSIONS: Epigenetic alterations with upregulated CD40-targeting miR-224 and miR-486 are related to downregulated CD40 protein expression at cell surfaces in highly invasive and metastatic PDAC. Thus, miRNA-regulated CD40 expression seems to play an important role in progression of PDAC. These data suggest a diagnostic and therapeutic potential for CD40 and/or its targeting miRNAs in PDAC.

SERPINB5 Promoter Hypomethylation Differentiates Pancreatic Ductal Adenocarcinoma From Pancreatitis.

OBJECTIVES: The diagnosis of pancreatic ductal adenocarcinoma (PDAC) is challenging in the setting of pancreatitis. We investigated SERPINB5 for its impact on PDAC tumor biology and its use as a diagnostic marker for PDAC in the setting of pancreatitis. METHODS: Patient samples from PDAC primary tumors, PDAC lymph node metastases, and pancreatitis were investigated for SERPINB5 promoter methylation by methylation-specific polymerase chain reaction (PCR). Six PDAC cell lines were investigated in vitro and in vivo using an orthotopic mouse model to generate primary tumors and metastases. SERPINB5 mRNA expression, protein expression, and promoter methylation were determined by quantitative reverse transcriptase-PCR, methylation-specific PCR, and Western Blot. RESULTS: In patient samples, detection of an unmethylated SERPINB5 promoter differentiated pancreatitis from PDAC with a sensitivity of 57% and a specificity of 95% (P < 0.001). SERPINB5 was not deregulated in primary tumors versus metastases, but primary tumors without SERPINB5 protein expression had significantly reduced viability (P = 0.02). CONCLUSIONS: SERPINB5 seems to assume an oncogenic role in PDAC. In clinical samples, detection of unmethylated SERPINB5 was a specific marker for PDAC even in the context of pancreatitis and may provide the basis for a liquid biopsy option to detect PDAC.

MicroRNA-100 and microRNA-21 as markers of survival and chemotherapy response in pancreatic ductal adenocarcinoma UICC stage II.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) remains a highly chemoresistant tumor entity for which no reliable molecular targets exist to predict or influence the success of chemotherapy. Recently, we identified a panel of microRNAs associated with induced gemcitabine chemoresistance in human PDAC cell lines. This clinical study evaluates these microRNAs and associated molecular markers as prognostic markers of outcome in 98 PDAC patients Union Internationale Contre le Cancer (UICC) stage II undergoing curative surgery with adjuvant gemcitabine chemotherapy. The primary end points of this study are recurrence-free survival and overall survival. RESULTS: Poor response to chemotherapy was significantly correlated to overexpression of microRNA-21 (p = 0.029), microRNA-99a (p = 0.037), microRNA-100 (p = 0.028), and microRNA-210 (p = 0.021) in tissue samples of PDAC patients UICC stage II. Upregulation of these microRNAs was associated with a significantly shorter overall survival and recurrence-free survival (p < 0.05). Overexpression of phosphatase and tensin homolog (PTEN) (p = 0.039) and low expression of multidrug resistance (MDR)-1 (p = 0.043) and breast cancer resistance protein (BCRP)-1 (p = 0.038) were significantly correlated to improved response to adjuvant chemotherapy. Adjuvant gemcitabine treatment (p < 0.0001) and low tumor grading (p = 0.047) were correlated to better outcome. MicroRNA-100, microRNA-21, and its targets PTEN and MDR-1 were independent factors of survival in multivariate analysis. CONCLUSIONS: Multivariate survival analyses identified microRNA-21 and microRNA-100 as unfavorable prognostic factors in resected and adjuvant treated PDAC UICC stage II patients.