Establishment of patient-derived xenograft models and cell lines for malignancies of the upper gastrointestinal tract.

BACKGROUND: The upper gastrointestinal tract is home to some of most notorious cancers like esophagogastric and pancreatic cancer. Several factors contribute to the lethality of these tumors, but one that stands out for both tumor types is the strong inter- as well as intratumor heterogeneity. Unfortunately, genetic tumor models do not match this heterogeneity, and for esophageal cancer no adequate genetic models exist. To allow for an improved understanding of these diseases, tissue banks with sufficient amount of samples to cover the extent of diversity of human cancers are required. Additionally, xenograft models that faithfully mimic and span the breadth of human disease are essential to perform meaningful functional experiments. METHODS: We describe here the establishment of a tissue biobank, patient derived xenografts (PDXs) and cell line models of esophagogastric and pancreatic cancer patients. Biopsy material was grafted into immunocompromised mice and PDXs were used to establish primary cell cultures to perform functional studies. Expression of Hedgehog ligands in patient tumor and matching PDX was assessed by immunohistochemical staining, and quantitative real-time PCR as well as flow cytometry was used for cultured cells. Cocultures with Hedgehog reporter cells were performed to study paracrine signaling potency. Furthermore, SHH expression was modulated in primary cultures using lentiviral mediated knockdown. RESULTS: We have established a panel of 29 PDXs from esophagogastric and pancreatic cancers, and demonstrate that these PDXs mirror several of the (immuno)histological and biochemical characteristics of the original tumors. Derived cell lines can be genetically manipulated and used to further study tumor biology and signaling capacity. In addition, we demonstrate an active (paracrine) Hedgehog signaling mode by both tumor types, the magnitude of which has not been compared directly in previous studies. CONCLUSIONS: Our established PDXs and their matching primary cell lines retain important characteristics seen in the original tumors, and this should enable future studies to address the responses of these tumors to different treatment modalities, but also help in gaining mechanistic insight in how some tumors respond to certain regimens and others do not.

Expression of the antiapoptotic protein BAG3 is a feature of pancreatic adenocarcinoma and its overexpression is associated with poorer survival.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly cancers, being the fourth leading cause of cancer-related deaths. Long-term survival reaching 15% is achieved in less than 5% of patients who undergo surgery, and median survival is only 6 months in those with inoperable lesions. A deeper understanding of PDAC biologic characteristics as well as novel prognostic markers are therefore required to improve outcomes. Herein we report that BAG3, a protein with recognized anti-apoptotic activity, was expressed in 346 PDACs analyzed, but was not expressed in the surrounding nonneoplastic tissue. In a cohort of 66 patients who underwent radical resection (R0), survival was significantly shorter in patients with high BAG3 expression (median, 12 months) than in those with low BAG3 expression (median, 23 months) (P = 0.001). Furthermore, we report that BAG3 expression in PDAC-derived cell lines protects from apoptosis and confers resistance to gemcitabine, offering a partial explanation for the survival data. Our results indicate that BAG3 has a relevant role in PDAC biology, and suggest that BAG3 expression level might be a potential marker for prediction of patient outcome.

Unsupervised class discovery in pancreatic ductal adenocarcinoma reveals cell-intrinsic mesenchymal features and high concordance between existing classification systems.

Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis of all common cancers. However, divergent outcomes exist between patients, suggesting distinct underlying tumor biology. Here, we delineated this heterogeneity, compared interconnectivity between classification systems, and experimentally addressed the tumor biology that drives poor outcome. RNA-sequencing of 90 resected specimens and unsupervised classification revealed four subgroups associated with distinct outcomes. The worst-prognosis subtype was characterized by mesenchymal gene signatures. Comparative (network) analysis showed high interconnectivity with previously identified classification schemes and high robustness of the mesenchymal subtype. From species-specific transcript analysis of matching patient-derived xenografts we constructed dedicated classifiers for experimental models. Detailed assessments of tumor growth in subtyped experimental models revealed that a highly invasive growth pattern of mesenchymal subtype tumor cells is responsible for its poor outcome. Concluding, by developing a classification system tailored to experimental models, we have uncovered subtype-specific biology that should be further explored to improve treatment of a group of PDAC patients that currently has little therapeutic benefit from surgical treatment.

Reliability of the primary triage process after the Volendam fire disaster.

In a major incident, correct triage is crucial to emergency treatment and transportation priority. The aim of this study was to evaluate the triage process pursued at the site of the fire disaster in Volendam, the Netherlands on January 1, 2001. On-site (OS) and Emergency Department (ED) data regarding total body surface area burned (TBSA) and inhalation injury (INH) were compared with the final (FIN) assessment of these two parameters after hospital admission. Finally, the effect of OS intubation and the time of arrival at a hospital were evaluated. There were 245 injured. Mean age was 17.3 years. Final median TBSA was 12%; 96 patients (39%) had inhalation injury. Agreement between TBSA-OS (n = 46) and TBSA-FIN was poor (Pearson's correlations coefficient [PCC] = 0.77; R(2) = 0.60). TBSA-ED (n = 78) was more accurate (PCC = 0.96; R(2) = 0.93). INH-OS (n = 79, sensitivity 100%, specificity 24%) and INH-ED (n = 198, sensitivity 99%, specificity 36%) were sensitive but not specific. Eight patients were intubated on-site. No differences in outcome were found between this group and the patients who were intubated in the hospital. There was no difference in time of arrival at a hospital (p = 0.55). TBSA was not estimated reliably in a non-clinical environment. The diagnosis of inhalation injury was adequate but resulted in over-triage on-site and at the ED. Triage did not lead to transport priorities for the severely wounded. In a major burn accident, a field triage protocol for rapid evaluation of burn injuries may be useful. Detailed assessment of injuries of burn casualties is practical only in a specialized clinical setting.

Dilemmas in the management of screen-detected lesions in patients at high risk for pancreatic cancer.

In 3-5 % of all cases of pancreatic ductal adenocarcinoma (PDAC), hereditary factors influence etiology. While surveillance of high-risk individuals may improve the prognosis, this study describes two very different outcomes in patients with screen-detected lesions. In 2000, a surveillance program of carriers of a CDKN2A/p16-Leiden-mutation consisting of annual MRI was initiated. Patients with a suspected pancreatic lesion undergo CT-scan and Endoscopic Ultrasound, and surgery is offered when a lesion is confirmed. In 2015, two patients with a screen-detected solid lesion were identified. In both patients, lesions were visible on MRI and CT scan, while the EUS was unremarkable. Surgical resection of the head of the pancreas resulted in nearly fatal complications in the first patient. This patient was shown to have a benign lesion. In contrast, timely identification of an early cancer in the second patient was accompanied by an uneventful postoperative course. These cases underline the risks inherent to a PDAC prevention program. All patients should be fully informed about the possible outcomes before joining a surveillance program.

The Volendam fire: lessons learned from disaster research.

INTRODUCTION: After the Volendam fire, a multidisciplinary, integral evaluation, called the Medical Evaluation of the Disaster in Volendam (MERV), was established. This article is a discussion of disaster research methodology. It describes the organizational framework of this project and the methodological problems. METHODS: A scientific steering group consisting of members from three hospitals prepared and guided the project. A research team wrote the final study protocol and performed the study. The project was funded by the Ministry of Health. The study protocol had a modular design in which each of the modules focused on one specific area or location. The main questions for each location were: (1) which treatment protocols were used; (2) what was the condition of the patient; and (3) was medical care provided according to existing protocols. After the fire, 241 victims were treated in hospitals; they all were included in the study. Most of the victims had burn injuries, and approximately one-third suffered from inhalation injury. All hospitals and ambulance services involved were visited in order to collect data, and interviewers obtained additional information. The government helped obtain permission for data-collection in three of the hospitals. Over 1,200 items of information about each patient and > 200,000 total items were collected. During data processing, the data were re-organized, categorized, and presented in a uniform and consistent style. A cross-sectional site analysis and a longitudinal patient analysis were conducted. This was facilitated by the use of several sub-databases. The modular approach made it possible to obtain a complete overview of the medical care provided. The project team was guided by a multidisciplinary steering group and the research was performed by a research team. This enabled the research team to focus on the scientific aspects. CONCLUSION: The evaluation of the Volendam fire indicates that a project approach with a modular design is effective for the analysis of complex incidents. The use of several sub-databases makes it easy to combine findings and conduct cross-sectional and longitudinal analyses. The government played an important role in the funding and support of the project. To limit and structure data collection and analysis, a pilot study based on several predefined main questions should be conducted. The questions then can be specified further based on the availability of data.

Management of multiple burn casualties from the Volendam disaster in the emergency departments of general hospitals.

OBJECTIVE: To establish the level of medical care provided in the emergency department of general hospitals to the victims of the Volendam café fire on 1 January 2001. METHODS: A retrospective review was done based on a standardized chart, for all victims seen at the emergency departments of 19 hospitals. Diagnostic findings and logistic aspects were inventoried. Treatment described in the Emergency Management of Severe Burns protocol was used as a gold standard against which the care provided to the victims was assessed. RESULTS: Data from 233 patients were included in the analysis. The documentation rate was low. Suspected inhalation injury and burns were the most frequently documented diagnoses. Most patients with suspected inhalation injury, for whom treatment records were available, received oxygen therapy (81%). Intubation was performed in 43% of patients with suspected inhalation injury and 14% of the remaining patients required intubation after admission to the intensive care unit. Most patients with circulatory problems (83%) and/or more than 15% of total body surface area burned (97%), for whom treatment records were available, received intravenous lines. Pain treatment seemed to have had low priority. Two patients (3%) were re-admitted after having been released earlier from the emergency department. CONCLUSION: Treatment and triage of the burn casualties after the Volendam café fire was adequate. The documentation rate was low. Not all steps in diagnosis and treatment may be of equal importance in disaster circumstances.

Analysis of the pre-incident education and subsequent performance of emergency medical responders to the Volendam café fire.

OBJECTIVE: At this moment, in the Netherlands, rescue workers are not given any specific standardized training in disaster response or disaster management. After the café fire in Volendam, the Netherlands, on New Year's Eve 2000, around 200 rescue workers were deployed on-site. The aim of this study is to investigate the rescue workers' experiences with regard to their level of preparation for the emergency response. METHODS: In 2002, 30 members of the medical and paramedical personnel were requested to participate in a structured interview, focused on education, task perception, triage and registration. RESULTS: Twenty-seven participated. Twenty-two rescue workers received previous training in emergency medicine. During the alarm phase, 11 rescue workers had a clear perception of their tasks. Twenty-four were involved in triage and injury assessment. Three rescue workers used a protocol for triage and 15 for injury assessment. Twenty-five rescue workers gave on-scene treatment and 15 used a protocol. Eight registered their findings. CONCLUSIONS: Preparation for the emergency response lacked standardized procedures. The use of triage protocols was extremely poor, as was documentation of actions. Slightly more than half of the personnel followed treatment protocols. It is advisable that all rescue workers become familiar with the basic uniform principles and protocols regarding disaster management. A dedicated and standardized national disaster management course is needed for all rescue workers.

What is the evidence for rest, ice, compression, and elevation therapy in the treatment of ankle sprains in adults?

CONTEXT: Ankle sprains are common problems in acute medical care. The variation in treatment observed for the acutely injured lateral ankle ligament complex in the first week after the injury suggests a lack of evidence-based management strategies for this problem. OBJECTIVE: To analyze the effectiveness of applying rest, ice, compression, and elevation (RICE) therapy begun within 72 hours after trauma for patients in the initial period after ankle sprain. STUDY SELECTION: Eligible studies were published original randomized or quasi-randomized controlled trials concerning at least 1 of the 4 subtreatments of RICE therapy in the treatment of acute ankle sprains in adults. DATA SOURCES: MEDLINE, Cochrane Clinical Trial Register, CINAHL, and EMBASE. The lists of references of retrieved publications also were checked manually. DATA EXTRACTION: We extracted relevant data on treatment outcome (pain, swelling, ankle mobility or range of motion, return to sports, return to work, complications, and patient satisfaction) and assessed the quality of included studies. If feasible, the results of comparable studies were pooled using fixed- or random-effects models. DATA SYNTHESIS: After deduction of the overlaps among the different databases, evaluation of the abstracts, and contact with some authors, 24 potentially eligible trials remained. The full texts of these articles were retrieved and thoroughly assessed as described. This resulted in the inclusion of 11 trials involving 868 patients. The main reason for exclusion was that the authors did not describe a well-defined control group without the intervention of interest. CONCLUSIONS: Insufficient evidence is available from randomized controlled trials to determine the relative effectiveness of RICE therapy for acute ankle sprains in adults. Treatment decisions must be made on an individual basis, carefully weighing the relative benefits and risks of each option, and must be based on expert opinions and national guidelines.

[Categorization of occult tumour cells in lymph nodes in patients with colon cancer not reliable enough]. / Indeling occulte tumorcellen in de lymfklieren bij patiënten met darmkanker onvoldoende betrouwbaar.

OBJECTIVE: To assess interobserver agreement between pathologists in judging photographs of lymph node preparations of occult tumour cells of patients with colon cancer. DESIGN: Descriptive and comparative study of interobserver variability. METHODS: All lymph nodes of 82 pN0 patients with colon cancer were analysed using three monoclonal antibodies against epithelial cells. Digital pictures of the 37 lesions detected were placed on a secured website. Forty pathologists selected at random were asked to examine the pictures and to categorize the lesions into 'micro metastases', 'isolated tumour cells' or something else. The degree of agreement was calculated by the Kendall W coefficient (with a range of 0.0-1.0). RESULTS: Thirty-five pathologists (88%) categorized the 37 lesions. Five lesions (14%) were categorized unanimously as micro metastases or isolated tumour cells. In 26 pictures (70%) the degree of agreement was poor to moderate. When the analysis was performed only on those diagnoses of which the pathologists were confident about their judgment, the percentage of lesions with good agreement rose to 49%. Differences in agreement were principally associated with multifocal lesions, clusters of tumour cells < 0.2 mm with proliferation characteristics in the parenchyma of the lymph node and lymphangio invasion. CONCLUSION: The differentiation between micro metastases and isolated tumour cells in lymph nodes of patients with colon cancer was not uniform. If this classification has clinical relevance to colon cancer then better definitions are needed.

[Jaundice and a pancreatic tumour caused by auto-immune pancreatitis]. / Auto-immune pancreatitis als oorzaak van geelzucht en een tumor in het pancreas.

Three male patients aged between 50 and 70 years were referred with jaundice and weight loss. Imaging showed a pancreatic mass and changes in the calibre of the choledochal or pancreatic duct, suggestive of malignancy. Two patients were operated on. One patient was considered to have an unresectable carcinoma but showed remarkable clinical improvement after steroids were given for his poor condition. In the other patient a resection was performed. Histology showed IgG4-positive plasma cell infiltration without signs of malignancy. Eventually these patients were diagnosed with auto-immune pancreatitis (AIP). In the third patient AIP was considered beforehand and this patient was treated with steroids. He responded quickly both clinically and radiologically. CT imaging showed complete remission of the mass. AIP is a benign inflammatory process which can mimic pancreatic carcinoma. In doubtful cases, a short trial of steroids might be considered.

Interhospital Transportation of Mass Burn Casualties.

AIM OF STUDY: To establish the impact of the transportation on the condition and outcome of the victims of the Volendam fire incident. METHODS: Medical and logistic parameters from all victims in the Intensive Care Unit (ICU) were retrospectively collected. Physiologic parameters in the first 24 h and outcome parameters were compared between the transported and the non-transported patients. RESULTS: The first 24 h, 105 patients were admitted to an ICU: 47 of them were relocated during that same day. The pH value was significantly lower in the transported group (p = 0.016). Systolic blood pressure, bicarbonate, carbon dioxide, temperature, APACHE II score and fluctuation during the first day, as well as condition during the second day did not differ significantly. The origin of the acidosis seemed to be mainly metabolic. The number of hospitalization days was larger in the transported group with severe burn injury (≥25% total body surface area burnt), comparing to the non-transported group (p = 0.015). Ventilation days and mortality did not differ significantly. CONCLUSIONS: The transported patients had a lower pH the first day after transportation, but condition during the second day as well as ventilation day and mortality did not differ between the transported and the non-transported group. Therefore, transportation during the unstable phase, the first day post-burn, seemed not to have had a negative impact on patient outcome.