Identification and Validation of a Diagnostic and Prognostic Multi-Gene Biomarker Panel for Pancreatic Ductal Adenocarcinoma.

Late diagnosis and systemic dissemination essentially contribute to the invariably poor prognosis of pancreatic ductal adenocarcinoma (PDAC). Therefore, the development of diagnostic biomarkers for PDAC are urgently needed to improve patient stratification and outcome in the clinic. By studying the transcriptomes of independent PDAC patient cohorts of tumor and non-tumor tissues, we identified 81 robustly regulated genes, through a novel, generally applicable meta-analysis. Using consensus clustering on co-expression values revealed four distinct clusters with genes originating from exocrine/endocrine pancreas, stromal and tumor cells. Three clusters were strongly associated with survival of PDAC patients based on TCGA database underlining the prognostic potential of the identified genes. With the added information of impact of survival and the robustness within the meta-analysis, we extracted a 17-gene subset for further validation. We show that it did not only discriminate PDAC from non-tumor tissue and stroma in fresh-frozen as well as formalin-fixed paraffin embedded samples, but also detected pancreatic precursor lesions and singled out pancreatitis samples. Moreover, the classifier discriminated PDAC from other cancers in the TCGA database. In addition, we experimentally validated the classifier in PDAC patients on transcript level using qPCR and exemplify the usage on protein level for three proteins (AHNAK2, LAMC2, TFF1) using immunohistochemistry and for two secreted proteins (TFF1, SERPINB5) using ELISA-based protein detection in blood-plasma. In conclusion, we present a novel robust diagnostic and prognostic gene signature for PDAC with future potential applicability in the clinic.

ZEB1-associated drug resistance in cancer cells is reversed by the class I HDAC inhibitor mocetinostat.

Therapy resistance is a major clinical problem in cancer medicine and crucial for disease relapse and progression. Therefore, the clinical need to overcome it, particularly for aggressive tumors such as pancreatic cancer, is very high. Aberrant activation of an epithelial-mesenchymal transition (EMT) and an associated cancer stem cell phenotype are considered a major cause of therapy resistance. Particularly, the EMT-activator ZEB1 was shown to confer stemness and resistance. We applied a systematic, stepwise strategy to interfere with ZEB1 function, aiming to overcome drug resistance. This led to the identification of both its target gene miR-203 as a major drug sensitizer and subsequently the class I HDAC inhibitor mocetinostat as epigenetic drug to interfere with ZEB1 function, restore miR-203 expression, repress stemness properties, and induce sensitivity against chemotherapy. Thereby, mocetinostat turned out to be more effective than other HDAC inhibitors, such as SAHA, indicating the relevance of the screening strategy. Our data encourage the application of mechanism-based combinations of selected epigenetic drugs with standard chemotherapy for the rational treatment of aggressive solid tumors, such as pancreatic cancer.

Radiotherapy for SMAD4-negative musculoskeletal lesions from pancreatic cancer: case report and review.

BACKGROUND: Pancreatic cancer (PC) predominantly metastasizes to liver, lung, and peritoneum. Metastatic disease correlates with SMAD4 status. Musculoskeletal metastases (MSM) are rare in pancreatic cancer. The role of radiation therapy (RT) in patients with musculoskeletal metastases is not clear. METHODS: We present a case of a woman with musculoskeletal metastases of PC evolving 4 years after Whipple's procedure and adjuvant therapy. She was treated with RT for 7 MSM. Radiation dose was 15-45 Gy, delivered in doses of 2.5-5 Gy per fraction. SMAD4 status was examined by immunohistochemistry. Furthermore we undertook a review of the literature to examine the value of RT in musculoskeletal metastasis of PC. RESULTS: In the presented patient we treated 7 MSM of SMAD4-mutant PC with RT. RT achieved local control in 4 of the 7 MSM. At the resection margin of one MSM recurrent tumor was observed after RT. The status of one MSM was unknown and one MSM showed local progression. Follow-up revealed progression of pain in 1 of the 7 MSM. Except of hyperpigmentation no side effects occurred. There was no dose-correlation effect on tumor control observed. A review of the literature showed that a musculoskeletotrophic phenotype of metastases is rare in PC. MSM of PC are rapidly increasing soft tissue masses causing pain and loss of anatomical function. RT as a treatment option for musculoskeletal metastasis is described in the current literature in only 2 cases. Radiotherapy aims to achieve local control, pain relief, and to maintain anatomical function. CONCLUSION: Radiotherapy is an effective and well-tolerated approach for multiple musculoskeletal metastases of PC.

A prospective clinical study evaluating the development of bowel wall edema during laparoscopic and open visceral surgery.

BACKGROUND: To examine bowel wall edema development in laparoscopic and open major visceral surgery. METHODS: In a prospective study, 47 consecutively operated patients with gastric and pancreatic resections were included. Twenty-seven patients were operated in a conventional open procedure (open group) and 20 in a laparoscopic fashion (lap group). In all procedures, a small jejunal segment was resected during standard preparation, of which we measured the dry-wet ratio. Furthermore, HE staining was performed for measuring of bowel wall thickness and edema assessment. RESULTS: Mean value (±std) of dry-wet ratio was significantly lower in the open than in the lap group (0.169 ± 0.017 versus 0.179 ± 0.015; p = 0.03) with the same amount of fluid administration in both groups and a longer infusion interval during laparoscopic surgery. Subgroup analyses (only pancreatic resections) still showed similar results. Histologic examination depicted a significantly larger bowel wall thickness in the open group. CONCLUSIONS: Laparoscopic surgery does not seem to lead to the bowel wall edema observed to occur in open surgery regardless of the degree of intravenous fluid administration, thus supporting its use even in major visceral surgery.

Hybrid laparoscopic versus open pylorus-preserving pancreatoduodenectomy: retrospective matched case comparison in 80 patients.

PURPOSE: We compared the outcome of hybrid laparoscopic pylorus-preserving pancreatoduodenectomy (lapPPPD) and open PPPD (oPPPD) in a retrospective case-matched study. METHODS: Patients operated from 2010 to 2013 by lapPPPD were matched 1:1 for age, sex, histopathology, American Society of Anesthesiologists category and body mass index to oPPPD patients operated from 1996 to 2013. RESULTS: Patients eligible for lapPPPD are a risk group due to a high rate of soft pancreata. Complication rate and mortality were comparable to oPPPD. There was a significantly reduced transfusion requirement and a trend towards shorter operation time, less delayed gastric emptying, and reduced hospital stay. The main reason for conversion was portal venous tumor adhesion. Patient selection changed and operation time and hospital stay decreased with the surgeons' experience. CONCLUSION: In selected patients, a hybrid laparoscopic technique of pancreatoduodenectomy is feasible with complication rates comparable to the open procedure. There seem to be advantages in terms of transfusion requirement, operation time, and hospital stay.

Impact of routinely employed procedures for tissue processing on the proteomic analysis of formalin-fixed paraffin-embedded tissue.

PURPOSE: FFPE (formalin fixed, paraffin embedded) tissue cohorts represent an enduring archive of clinical specimens. Proteomic analysis of FFPE tissues is gaining interest for the in-depth analysis of aberrant proteome composition. Procedures for FFPE tissue processing are standardized but there is diversity regarding the different processing systems. This work focuses on three different processing methods commonly used in large European pathology institutes. EXPERIMENTAL DESIGN: Formalin fixed tissue specimens of different tumors were serially sliced and processed with three different processing systems (xylene, ethanol/vacuum or microwave based). After paraffin embedding, they were subjected to MS-based proteomic analysis to investigate the impact of tissue processing techniques on the quality of proteomic analysis. Results were compared with proteomic analysis of corresponding cryopreserved tissue specimens. RESULTS: All processing techniques achieved very good proteome coverage similar to the cryopreserved counterpart. Gene ontology profiles, relative protein abundances, and peptide modifications such as methionine oxidation or proteolytic truncation were highly similar for all techniques as well as for the cryopreserved samples. CONCLUSIONS AND CLINICAL RELEVANCE: The results show that different processing procedures do not impede proteomic analysis as a robust and powerful approach for the identification of protein determinants and markers of disease processes and highlights the general robustness of FFPE-tissue based proteomics.

Impact of intraoperative temperature and humidity on healing of intestinal anastomoses.

PURPOSE: Clinical data indicate that laparoscopic surgery has a beneficial effect on intestinal wound healing and is associated with a lower incidence of anastomotic leakage. This observation is based on weak evidence, and little is known about the impact of intraoperative parameters during laparoscopic surgery, e.g., temperature and humidity. METHODS: A small-bowel anastomosis was formed in rats inside an incubator, in an environment of stable humidity and temperature. Three groups of ten Wistar rats were operated: a control group (G1) in an open surgical environment and two groups (G2 and G3) in the incubator at a humidity of 60 % and a temperature of 30 and 37 °C (G2 and G3, respectively). After 4 days, bursting pressure and hydroxyproline concentration of the anastomosis were analyzed. The tissue was histologically examined. Serum levels of C-reactive-protein (CRP) were measured. RESULTS: No significant changes were seen in the evaluation of anastomotic stability. Bursting pressure was very similar among the groups. Hydroxyproline concentration in G3 (36.3 µg/g) was lower by trend (p = 0.072) than in G1 (51.7 µg/g) and G2 (46.4 µg/g). The histological evaluation showed similar results regarding necrosis, inflammatory cells, edema, and epithelization for all groups. G3 (2.56) showed a distinctly worse score for submucosal bridging (p = 0.061) than G1 (1.68). A highly significant increase (p = 0.008) in CRP was detected in G3 (598.96 ng/ml) compared to G1 (439.49 ng/ml) and G2 (460 ng/ml). CONCLUSION: A combination of high temperature and humidity during surgery induces an increased systemic inflammatory response and seems to be attenuating the early regeneration process in the anastomotic tissue.

Systematic ileal transposition in Zucker rats shows advantage for long segment distal transposition.

INTRODUCTION: The possibility of achieving diabetes remission through bariatric surgery has dramatically changed treatment options for this disease. Ileal transposition (IT), specifically designed to provoke diabetes remission, has so far shown great success in rodent studies. However, it remains uncertain which combination of ileal length and origin produces best results. METHODS: Forty male Zucker rats underwent transposition of 25% distal, 50% distal, and 50% proximal ileum or sham surgery. Glucose control, insulin, and glucagon-like peptide (GLP)-1 serum levels were analyzed after 1, 3, and 6 months. Body weight was recorded weekly. RESULTS: In relation to sham-operated animals, the 50% distal IT presented with improved glucose tolerance after 1, 3, and 6 months (2-way analysis of variance [ANOVA]: P < .05, < .0001, and < .0001, respectively). The 25% distal and 50% proximal IT only showed improved glucose control after 3 months, suggesting a fading effect in long-term observation (2-way ANOVA: P < .0001 for both). Glucose-stimulated GLP-1 levels were steadily elevated only in the 2 distal IT groups (Mann-Whitney sham versus 50% distal, P < .01, < .01, and < .05; sham versus 25% distal, P < .01, = .001, < .05 for 1, 3, and 6 months, respectively). IT had no impact on serum insulin levels. CONCLUSION: The current study restates the findings of improved glucose tolerance and GLP-1 stimulation after IT, but is the first to demonstrate a fading glycemic effect in long-term observation. Systematic comparison of length and ileal origin revealed that long and distal transposition delivers best results.

Long segment ileal transposition leads to early amelioration of glucose control in the diabetic obese Zucker rat.

INTRODUCTION: Fast delivery of food to the terminal ileum is thought to be pathophysiologically responsible for type 2 diabetes remission after obesity surgery. Imitating this effect, ileal transposition (IT) is designed as initiating diabetes remission for non-obese patients. AIM: To date, it is not clear which length of the transposed segment achieves the best glucose lowering results. As previous rodent data mostly rely on a 10 cm IT, the current study evaluated a long segment IT (20 cm) in the diabetic obese Zucker rat. MATERIAL AND METHODS: Twenty male diabetic obese Zucker rats (Crl:ZUC-Lepr(fa)) were randomly assigned to undergo either a long segment (20 cm; ∼ 50% of ileum) IT or sham surgery. Glucose control was determined by an oral glucose tolerance test (OGTT) on day -7, 0, 14 and 20. Analysis of the incretin hormones glucagon-like peptide 1 (GLP-1), peptide YY (PYY) and insulin was included in the first and third OGTT. RESULTS: Ileal transposition animals showed an early improvement of glucose control after 14 days (area under the curve: IT vs. baseline 314.7 ±229.0 mmol/l × min vs. 564.6 ±268.5 mmol/l × min; p < 0.05). Compared to sham animals, glucose-stimulated GLP-1 and PYY levels were raised (5.75 ±3.73 pmol/l vs. 18.52 ±14.22 pmol/l, p < 0.05; 129.7 ±64.62 pmol/l vs. 164.0 ±62.26 pmol/l, p < 0.05). Body weight gain from postoperative day 5 was greater for sham animals (50.22 ±20.93 γ vs. 16.4 ±25.93 g; p < 0.01). CONCLUSIONS: Long segment IT shows a rapid rise in GLP-1 and PYY levels, thus leading to early amelioration of glucose control.

Total laparoscopic partial pancreatoduodenectomy and reconstruction via laparoscopic pancreatogastrostomy.

INTRODUCTION: Minimally invasive surgery has conquered almost all niches of abdominal surgery. Even though some surgeons have shown equal lymph node ratio and oncologic radicality for laparoscopic surgery of pancreatic cancer, oncologic surgeons still take reasonably conservative views of the use of minimally invasive techniques for the treatment of pancreatic cancer, especially if located in the head of the pancreas. Laparoscopic abdominal approaches on the other hand have a potential advantage of better visualization, decreased postoperative pain, decreased use of analgetics, and shorter hospital stay. We demonstrate in this technical surgical report the first description of a total laparoscopic pancreatoduodenectomy and reconstruction via laparoscopic pancreatogastrostomy in a 74-year-old female patient with a periampullary tumor. DISCUSSION: After pylorus-preserving pancreatoduodenectomy by superior mesenteric artery, first approach including standard lymphadenectomy, the reconstruction involved total laparoscopic end-to-side running-suture hepaticojejunostomy, double-layer running-suture antecolic pylorojejunostomy to the first jejunal loop, and pancreatogastrostomy via posterior gastrotomy secured by two anchoring and purse-string sutures.