Daniel Croagh

Identification of Candidate Murine Esophageal Stem Cells Using a Combination of Cell Kinetic Studies and Cell Surface Markers

The identification and characterization of esophageal stem cells are critical to our understanding of the biology of the esophageal epithelium in health and disease. However, the proliferative compartment within the mouse esophageal epithelium remains poorly characterized. Here, we report that the basal cells of the mouse esophagus can be separated into three phenotypically and functionally distinct subpopulations based on the expression of α6 integrin and transferrin receptor (CD71). Cells that express high levels of α6 integrin and low levels of CD71, termed α6briCD71dim, are a minor subpopulation of small and undifferentiated cells that are enriched for label‐retaining cells and thus represent a putative esophageal stem cell population. Conversely, cells expressing high levels of both α6 integrin and CD71 (α6briCD71bri), the majority of basal esophageal cells, are enriched for actively cycling cells and therefore represent a transit‐amplifying population. Kinetic analyses revealed that a third cell population, which is α6 integrin‐dim and CD71‐bright (α6dim), is destined to leave the basal layer and differentiate.

Sox9 drives columnar differentiation of esophageal squamous epithelium: a possible role in the pathogenesis of Barrett's esophagus

The molecular mechanism underlying the development of Barretts esophagus (BE), the precursor to esophageal adenocarcinoma, remains unknown. Our previous work implicated sonic hedgehog (Shh) signaling as a possible driver of BE and suggested that bone morphogenetic protein 4 (Bmp4) and Sox9 were downstream mediators. We have utilized a novel in vivo tissue reconstitution model to investigate the relative roles of Bmp4 and Sox9 in driving metaplasia. Epithelia reconstituted from squamous epithelial cells or empty vector-transduced cells had a stratified squamous phenotype, reminiscent of normal esophagus. Expression of Bmp4 in the stromal compartment activated signaling in the epithelium but did not alter the squamous phenotype. In contrast, expression of Sox9 in squamous epithelial cells induced formation of columnar-like epithelium with expression of the columnar differentiation marker cytokeratin 8 and the intestinal-specific glycoprotein A33. In patient tissue, A33 protein was expressed specifically in BE, but not in normal esophagus. Expression of Cdx2, another putative driver of BE, alone had no effect on reconstitution of a squamous epithelium. Furthermore, epithelium coexpressing Cdx2 and Sox9 had a phenotype similar to epithelium expressing Sox9 alone. Our results demonstrate that Sox9 is sufficient to drive columnar differentiation of squamous epithelium and expression of an intestinal differentiation marker, reminiscent of BE. These data suggest that Shh-mediated expression of Sox9 may be an important early event in the development of BE and that the potential for inhibitors of the hedgehog pathway to be used in the treatment of BE and/or esophageal adenocarcinoma could be tested in the near future.

Impact of an acute care surgery model on appendicectomy outcomes.

Monash Medical Centre introduced the acute surgical unit (ASU) in July 2011. The ASU is modelled on the concept of acute care surgery (ACS). This study reviews the impact of the ASU on the outcomes in an appendicectomy population.

Randomized clinical trial of intraoperative endoscopic retrograde cholangiopancreatography versus laparoscopic bile duct exploration in patients with choledocholithiasis

Various minimally invasive approaches exist for the management of choledocholithiasis at the time of laparoscopic cholecystectomy. The aim of this study was to compare endoscopic retrograde cholangiopancreatography (ERCP) with laparoscopic bile duct exploration (LBDE) and test the hypothesis that intraoperative ERCP is no different to LBDE in terms of rate of bile duct clearance or retained stones.

Reconstitution of stratified murine and human oesophageal epithelia in an in vivo transplant culture system.

Objective. The molecular and cellular events responsible for regulating development of the oesophageal epithelium are not well understood. At least in part, this is due to the lack of a suitable model system with which to study the process. Here, we report development of a manipulable in vivo transplant model for mouse or human oesophageal epithelium. Material and methods. Epithelial cells were isolated from mouse or human oesophagus and inoculated into de-epithelialized and devitalized rat tracheas. The rat trachea, containing cells, was placed subcutaneously under the dorsal skin of immunodeficient mice. Results. We show that a multilayered stratified squamous epithelium can be generated in 4–6 weeks from as few as 5×104 isolated oesophageal epithelial cells. The reconstituted epithelium recapitulates many of the structural and histological features of the normal oesophageal epithelium, including a basal layer of cuboidal-like cells, suprabasal layers of differentiating squamous cells and, in the case of murine cells, a superficial layer of cornified material. Conclusion. Our model can be used to generate a multilayered normal murine or human epithelium from a single cell suspension of oesophageal epithelial cells. The ability to genetically manipulate the cells prior to growth in the model is a powerful tool with which to study the molecular mechanisms involved in the development of normal oesophagus or in pathogenic processes such as Barretts metaplasia or tumorigenesis.

Endoscopic ultrasound-guided fine-needle aspirate-derived preclinical pancreatic cancer models reveal panitumumab sensitivity in KRAS wild-type tumors

Pancreatic cancer (PC) is largely refractory to existing therapies used in unselected patient trials, thus emphasizing the pressing need for new approaches for patient selection in personalized medicine. KRAS mutations occur in 90% of PC patients and confer resistance to epidermal growth factor receptor (EGFR) inhibitors (e.g., panitumumab), suggesting that KRAS wild‐type PC patients may benefit from targeted panitumumab therapy. Here, we use tumor tissue procured by endoscopic ultrasound‐guided fine‐needle aspirate (EUS‐FNA) to compare the in vivo sensitivity in patient‐derived xenografts (PDXs) of KRAS wild‐type and mutant PC tumors to panitumumab, and to profile the molecular signature of these tumors in patients with metastatic or localized disease. Specifically, RNASeq of EUS‐FNA‐derived tumor RNA from localized (n = 20) and metastatic (n = 20) PC cases revealed a comparable transcriptome profile. Screening the KRAS mutation status of tumor genomic DNA obtained from EUS‐FNAs stratified PC patients into either KRAS wild‐type or mutant cohorts, and the engraftment of representative KRAS wild‐type and mutant EUS‐FNA tumor samples into NOD/SCID mice revealed that the growth of KRAS wild‐type, but not mutant, PDXs was selectively suppressed with panitumumab. Furthermore, in silico transcriptome interrogation of The Cancer Genome Atlas (TCGA)‐derived KRAS wild‐type (n = 38) and mutant (n = 132) PC tumors revealed 391 differentially expressed genes. Taken together, our study validates EUS‐FNA for the application of a novel translational pipeline comprising KRAS mutation screening and PDXs, applicable to all PC patients, to evaluate personalized anti‐EGFR therapy in patients with KRAS wild‐type tumors.

Management of choledocholithiasis in an emergency cohort undergoing laparoscopic cholecystectomy: a single‐centre experience

INTRODUCTION Minimally-invasive options for the management of choledocholithiasis in patients undergoing laparoscopic cholecystectomy include laparoscopic and endoscopic approaches. This study reviews the effectiveness of both approaches in an emergency setting. METHODS A retrospective chart review was performed for a cohort of patients who underwent laparoscopic cholecystectomy. Outcomes assessed were duct clearance, the number of procedures performed (NPP), length of stay (LOS) and complication rate. RESULTS A total of 182 patients who underwent emergency laparoscopic cholecystectomies received intervention for choledocholithiasis. The duct clearance rate was lower in the laparoscopic group, 63% versus 86% (P = 0.001). However, the median NPP was also lesser in the laparoscopic group, 1 (interquartile range (IQR) 1-2) versus 2 (IQR 2-2) (P < 0.001), as was the median LOS, 5 days (IQR 3-8) versus 7 days (IQR 6-10) (P = 0.009). Forty-eight laparoscopic endobiliary stents were attempted; stent deployment was successful in 37 patients. A larger proportion of patients with laparoscopic endobiliary stents had duct clearance by endoscopic retrograde cholangiopancreatography (ERCP) compared with those without, although this was not statistically significant (P = 0.208). CONCLUSION Laparoscopic clearance is not as effective as post-operative ERCP in an emergency cohort, but is associated with fewer procedures required and a shorter inpatient stay. Thus, laparoscopic clearance may still be an attractive option for surgeons especially where conditions are favourable during an emergency laparoscopic cholecystectomy.

Esophageal stem cells and genetics/epigenetics in esophageal cancer

The following, from the 12th OESO World Conference: Cancers of the Esophagus, includes commentaries on the relationship between stem cells, cancer, and the esophagus; the behavior of esophageal stem cells; and the role of genetics and epigenetics in approaches to translational research.

Interventional management of necrotizing pancreatitis: an Australian experience.

The interventional management of necrotizing pancreatitis has evolved from early open surgery to delayed endoscopic or percutaneous intervention. However, few studies have directly compared the three treatment modalities. We aim to compare the outcomes of patients who had endoscopic, percutaneous or surgical interventions for necrotizing pancreatitis at our institution.

Management of CBD stones in patients having laparoscopic cholecystectomy in a private setting in Australia.

Laparoscopic bile duct exploration at the time of laparoscopic cholecystectomy has been promoted as being equally successful as endoscopic bile duct clearance. Further, if successful it offers the possibility of reducing the number of interventions required and therefore reducing overall costs. However, there is little in the literature that describe current treatment patterns in the Australian environment.