Chemoresistance in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC), generally known as pancreatic cancer (PC), ranks the fourth leading cause of cancer-related deaths in the western world. While the incidence of pancreatic cancer is displaying a rising tendency every year, the mortality rate has not decreased significantly because of late diagnosis, early metastasis, and limited reaction to chemotherapy or radiotherapy. Adjuvant chemotherapy after surgical resection is typically the preferred option to treat early pancreatic cancer. Although 5-fluorouracil/leucovorin with irinotecan and oxaliplatin (FOLFIRINOX) and gemcitabine/nab-paclitaxel can profoundly improve the prognosis of advanced pancreatic cancer, the development of chemoresistance still leads to poor clinical outcomes. Chemoresistance is multifactorial as a result of the interaction among pancreatic cancer cells, cancer stem cells, and the tumor microenvironment. Nevertheless, more pancreatic cancer patients will benefit from precision treatment and targeted drugs. Therefore, we outline new perspectives for enhancing the efficacy of gemcitabine after reviewing the related factors of gemcitabine metabolism, mechanism of action, and chemoresistance

Interdisciplinary Surgical Approaches in Vaginal and Perineal Reconstruction of Advanced Rectal and Anal Female Cancer Patients

Relapsing or far advanced rectal and anal cancers remain difficult to treat and require interdisciplinary approaches. Due to modern standard protocols all patients receive irradiation and neoadjuvant chemotherapy—and in case of a relapse a second irradiation—rendering the surgical site prone to surgical site infections and oftentimes long lasting sinus and septic complications after exenteration in the pelvis. Despite an improved overall survival rate in these patients the downside of radical tumor surgery in the pelvis is a major loss of quality of life, especially in women when parts of the vagina need to be resected. Derived from our experince with over 300 patients receiving pelvic and perineal reconstruciton with a transpelvic vertical rectus abdominis myocutaneous (tpVRAM) flap we studied the impact of this surgical technique on the outcomes of female patients with or without vaginal reconstruction following pelvic exenteration. We found out that the tpVRAM flap is reliably perfused and helps to reduce long term wound healing desasters in the irradiated perineal/vaginal/gluteal region

Simultaneous gene silencing of Bcl-2, XIAP and Survivin re-sensitizes pancreatic cancer cells towards apoptosis

Abstract Background Pancreatic ductal adenocarcinoma shows a distinct apoptosis resistance, which contributes significantly to the aggressive nature of this tumor and constrains the effectiveness of new therapeutic strategies. Apoptosis resistance is determined by the net balance of the cells pro-and anti-apoptotic "control mechanisms". Numerous dysregulated anti-apoptotic genes have been identified in pancreatic cancer and seem to contribute to the high anti-apoptotic buffering capacity. We aimed to compare the benefit of simultaneous gene silencing (SGS) of several candidate genes with conventional gene silencing of single genes. Methods From literature search we identified the anti-apoptotic genes XIAP, Survivin and Bcl-2 as commonly upregulated in pancreatic cancer. We performed SGS and silencing of single candidate genes using siRNA molecules in two pancreatic cancer cell lines. Effectiveness of SGS was assessed by qRT-PCR and western blotting. Apoptosis induction was measured by flow cytometry and caspase activation. Results Simultaneous gene silencing reduced expression of the three target genes effectively. Compared to silencing of a single target or control, SGS of these genes resulted in a significant higher induction of apoptosis in pancreatic cancer cells. Conclusions In the present study we performed a subliminal silencing of different anti-apoptotic target genes simultaneously. Compared to silencing of single target genes, SGS had a significant higher impact on apoptosis induction in pancreatic cancer cells. Thereby, we give further evidence for the concept of an anti-apoptotic buffering capacity of pancreatic cancer cells.</p

Silenced ZNF154 Is Associated with Longer Survival in Resectable Pancreatic Cancer

Pancreatic cancer has become the third leading cause of cancer-related death in the Western world despite advances in therapy of other cancerous lesions. Late diagnosis due to a lack of symptoms during early disease allows metastatic spread of the tumor. Most patients are considered incurable because of metastasized disease. On a cellular level, pancreatic cancer proves to be rather resistant to chemotherapy. Hence, early detection and new therapeutic targets might improve outcomes. The detection of DNA promoter hypermethylation has been described as a method to identify putative genes of interest in cancer entities. These genes might serve as either biomarkers or might lead to a better understanding of the molecular mechanisms involved. We checked tumor specimens from 80 patients who had undergone pancreatic resection for promoter hypermethylation of the zinc finger protein ZNF154. Then, we further characterized the effects of ZNF154 on cell viability and gene expression by in vitro experiments. We found a significant association between ZNF154 hypermethylation and better survival in patients with resectable pancreatic cancer. Moreover, we suspect that the cell growth suppressor SLFN5 might be linked to a silenced ZNF154 in pancreatic cancer

CRISPR/Cas9-Mediated Knock-Out of KrasG12D Mutated Pancreatic Cancer Cell Lines

In 90% of pancreatic ductal adenocarcinoma cases, genetic alteration of the proto-oncogene Kras has occurred, leading to uncontrolled proliferation of cancerous cells. Targeting Kras has proven to be difficult and the battle against pancreatic cancer is ongoing. A promising approach to combat cancer was the discovery of the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system, which can be used to genetically modify cells. To assess the potential of a CRISPR/CRISPR-associated protein 9 (Cas9) method to eliminate Kras mutations in cells, we aimed to knock-out the c.35G>A (p.G12D) Kras mutation. Therefore, three cell lines with a heterozygous Kras mutation (the human cell lines SUIT-2 and Panc-1 and the cell line TB32047 from a KPC mouse model) were used. After transfection, puromycin selection and single-cell cloning, proteins from two negative controls and five to seven clones were isolated to verify the knock-out and to analyze changes in key signal transduction proteins. Western blots showed a specific knock-out in the KrasG12D protein, but wildtype Kras was expressed by all of the cells. Signal transduction analysis (for Erk, Akt, Stat3, AMPKα, and c-myc) revealed expression levels similar to the wildtype. The results described herein indicate that knocking-out the KrasG12D mutation by CRISPR/Cas9 is possible. Additionally, under regular growth conditions, the knock-out clones resembled wildtype cells

Apparent diffusion coefficient measurements of the pancreas, pancreas carcinoma, and mass-forming focal pancreatitis

Background Mass-forming focal pancreatitis (FP) may mimic pancreatic cancer (PC) on magnetic resonance (MR) imaging, and the preoperative differential diagnosis is often difficult. Recently, the usefulness of diffusion-weighted imaging (DWI) in the diagnosis of pancreatic cancer has been reported in several studies. Purpose To investigate if apparent diffusion coefficient (ADC) measurements based on diffusion-weighted echo-planar imaging (DW-EPI) may distinguish between normal pancreas parenchyma, mass-forming focal pancreatitis, and pancreas carcinoma. Material and Methods MRI was performed on 64 patients: 24 with pancreas carcinoma (PC), 20 with mass-forming focal pancreatitis (FP), three patients with other focal pancreatic disease as well as 17 controls without any known pancreatic disease. Diffusion-weighted sequence with ADC maps and T2-weighted sequence for anatomical information was performed. Apparent diffusion coefficient (ADC) maps were automatically created and analyzed using a dedicated user interface. In the group with pancreas disease the abnormal parenchyma was detected by using T1- and T2-weighted images and the region of interest (ROI) was transferred exactly to the ADC map and the coefficients were registered. In the control group the ROI was set to the head of the pancreas followed by a similar registration of the ADCs. Results ADC values for mass-forming FP and PC differed significantly from ADC values for normal pancreas parenchyma (P = 0.001/P = 0.002). Mean ADC values for mass-forming FP were 0.69 ± 0.18 × 10−3 mm2/s. ADC values for PC were 0.78 ± 0.11 × 10−3 mm2/s, compared to ADC values of 0.17 ± 0.06 × 10−3 mm2/s in the control group. However there was no significant difference in ADCs between PC and mass-forming FP (P = 0.15). Conclusion ADC measurements clearly differentiated between normal pancreatic tissue and abnormal pancreas parenchyma (PC and mass-forming FP). However there is an overlap in values of PC and mass-forming FP, with the consequent problem of their correct identification

Isolation of Human Islets from Partially Pancreatectomized Patients

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 diabetic islets from organ donors2. Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area

The impact of surgical experience and frequency of practice on perioperative outcomes in pancreatic surgery

Objective We aimed to determine the impact of surgical experience and frequency of practice on perioperative morbidity and mortality in pancreatic surgery. Methods 1281 patients that underwent pancreatic resections from 1993 to 2013 were retrospectively analyzed using logistic regression models. All cases were stratified according to the surgeon’s level of experience, which was based on the number of previously performed pancreatic resections and the extent of received supervision (novice: n  90 / none). Additional stratification was based on the frequency of practice (sporadic: 3 resections > 6 weeks, frequent: 3 resections ≤6 weeks). Results The novice and experienced categories were related to a decreased risk of postoperative pancreatic fistulas (odds ratio [OR] 0.46, 95% confidence interval [CI] 0.26–0.82 and 0.54, 95% CI 0.36–0.82) and in-hospital mortality (OR 0.45, 95% CI 0.17–1.16 and 0.42, 95% CI 0.21–0.83) compared to the intermediate category. Frequent practice was associated with a significantly lower risk of delayed gastric emptying (OR 0.56, 95% CI 0.38–0.83), postpancreatectomy hemorrhage (OR 0.64, 95% CI 0.42–0.98) and in-hospital mortality (OR 0.45, 95% CI 0.24–0.87). Conclusions Our results emphasize the importance of supervision within a pancreatic surgery training program. In addition, our data underline the need of a sufficient patient caseload to ensure frequent practice

Metabolic biomarker signature to differentiate pancreatic ductal adenocarcinoma from chronic pancreatitis

Objective Current non-invasive diagnostic tests can distinguish between pancreatic cancer (pancreatic ductal adenocarcinoma (PDAC)) and chronic pancreatitis (CP) in only about two thirds of patients. We have searched for blood-derived metabolite biomarkers for this diagnostic purpose. Design For a case-control study in three tertiary referral centres, 914 subjects were prospectively recruited with PDAC (n=271), CP (n=282), liver cirrhosis (n=100) or healthy as well as non-pancreatic disease controls (n=261) in three consecutive studies. Metabolomic profiles of plasma and serum samples were generated from 477 metabolites identified by gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry. Results A biomarker signature (nine metabolites and additionally CA19-9) was identified for the differential diagnosis between PDAC and CP. The biomarker signature distinguished PDAC from CP in the training set with an area under the curve (AUC) of 0.96 (95% CI 0.93-0.98). The biomarker signature cut-off of 0.384 at 85% fixed specificity showed a sensitivity of 94.9% (95% CI 87.0%-97.0%). In the test set, an AUC of 0.94 (95% CI 0.91-0.97) and, using the same cut-off, a sensitivity of 89.9% (95% CI 81.0%-95.5%) and a specificity of 91.3% (95% CI 82.8%-96.4%) were achieved, successfully validating the biomarker signature. Conclusions In patients with CP with an increased risk for pancreatic cancer (cumulative incidence 1.95%), the performance of this biomarker signature results in a negative predictive value of 99.9% (95% CI 99.7%-99.9%) (training set) and 99.8% (95% CI 99.6%-99.9%) (test set). In one third of our patients, the clinical use of this biomarker signature would have improved diagnosis and treatment stratification in comparison to CA19-9