Metastatic phenotype and immunosuppressive tumour microenvironment in pancreatic ductal adenocarcinoma: Key role of the urokinase plasminogen activator (PLAU).

Background: Previous studies have revealed the role of dysregulated urokinase plasminogen activator (encoded by PLAU) expression and activity in several pathways associated with cancer progression. However, systematic investigation into the association of PLAU expression with factors that modulate PDAC (pancreatic ductal adenocarcinoma) progression is lacking, such as those affecting stromal (pancreatic stellate cell, PSC)-cancer cell interactions, tumour immunity, PDAC subtypes and clinical outcomes from potential PLAU inhibition. Methods: This study used an integrated bioinformatics approach to identify prognostic markers correlated with PLAU expression using different transcriptomics, proteomics, and clinical data sets. We then determined the association of dysregulated PLAU and correlated signatures with oncogenic pathways, metastatic phenotypes, stroma, immunosuppressive tumour microenvironment (TME) and clinical outcome. Finally, using an in vivo orthotopic model of pancreatic cancer, we confirmed the predicted effect of inhibiting PLAU on tumour growth and metastasis. Results: Our analyses revealed that PLAU upregulation is not only associated with numerous other prognostic markers but also associated with the activation of various oncogenic signalling pathways, aggressive phenotypes relevant to PDAC growth and metastasis, such as proliferation, epithelial-mesenchymal transition (EMT), stemness, hypoxia, extracellular cell matrix (ECM) degradation, upregulation of stromal signatures, and immune suppression in the tumour microenvironment (TME). Moreover, the upregulation of PLAU was directly connected with signalling pathways known to mediate PSC-cancer cell interactions. Furthermore, PLAU upregulation was associated with the aggressive basal/squamous phenotype of PDAC and significantly reduced overall survival, indicating that this subset of patients may benefit from therapeutic interventions to inhibit PLAU activity. Our studies with a clinically relevant orthotopic pancreatic model showed that even short-term PLAU inhibition is sufficient to significantly halt tumour growth and, importantly, eliminate visible metastasis. Conclusion: Elevated PLAU correlates with increased aggressive phenotypes, stromal score, and immune suppression in PDAC. PLAU upregulation is also closely associated with the basal subtype type of PDAC; patients with this subtype are at high risk of mortality from the disease and may benefit from therapeutic targeting of PLAU.

HGF/c-Met Inhibition as Adjuvant Therapy Improves Outcomes in an Orthotopic Mouse Model of Pancreatic Cancer.

BACKGROUND: Inhibition of hepatocyte growth factor (HGF)/c-MET pathway, a major mediator of pancreatic stellate cell (PSC)-PC cell interactions, retards local and distant cancer progression. This study examines the use of this treatment in preventing PC progression after resection. We further investigate the postulated existence of circulating PSCs (cPSCs) as a mediator of metastatic PC. METHODS: Two orthotopic PC mouse models, produced by implantation of a mixture of luciferase-tagged human pancreatic cancer cells (AsPC-1), and human PSCs were used. Model 1 mice underwent distal pancreatectomy 3-weeks post-implantation (n = 62). One-week post-resection, mice were randomised to four treatments of 8 weeks: (i) IgG, (ii) gemcitabine (G), (iii) HGF/c-MET inhibition (HiCi) and (iv) HiCi + G. Tumour burden was assessed longitudinally by bioluminescence. Circulating tumour cells and cPSCs were enriched by filtration. Tumours of Model 2 mice progressed for 8 weeks prior to the collection of primary tumour, metastases and blood for single-cell RNA-sequencing (scRNA-seq). RESULTS: HiCi treatments: (1) reduced both the risk and rate of disease progression after resection; (2) demonstrated an anti-angiogenic effect on immunohistochemistry; (3) reduced cPSC counts. cPSCs were identified using immunocytochemistry (α-smooth muscle actin+, pan-cytokeratin-, CD45-), and by specific PSC markers. scRNA-seq confirmed the existence of cPSCs and identified potential genes associated with development into cPSCs. CONCLUSIONS: This study is the first to demonstrate the efficacy of adjuvant HGF/c-Met inhibition for PC and provides the first confirmation of the existence of circulating PSCs.

Day-only elective cholecystectomy: early experience and barriers to implementation in Australia.

BACKGROUND: Day-only laparoscopic cholecystectomy (DOLC) has been shown to be safe and feasible yet has not been widely implemented in Australia. This study explores the introduction of routine DOLC to Westmead Hospital, and highlights the barriers to its implementation. METHODS: Routine day-only cholecystectomy protocol was introduced at Westmead Hospital in 2014. A retrospective review of patients who underwent elective laparoscopic cholecystectomy during a 12-month period in 2014 was compared to a 12-month period in 2018, to examine the changes in practice after implementation of a unit protocol. Data were collected on patient demographics, admission category, outcomes and re-presentations. RESULTS: A total of 282 patients were included in the study, of these 169 were booked as day procedures, with 124 (73%) successfully discharged on the same day. There was a significant increase in the proportion of patients booked as day-only from 2014 to 2018 (48% versus 73%, P < 0.001). Day-only failure rates (unplanned overnight admissions), readmissions and complication rates were comparable between the two periods. The most common reason for unplanned overnight admissions were due to intraoperative findings (n = 28/45). CONCLUSION: Routine DOLC can be adopted in Australian hospitals without compromise to patient safety. Unplanned overnight admission is predominantly due to unexpected surgical pathology and can be reduced by protocols for the use of drains and planned outpatient endoscopic retrograde cholangiopancreatography. Unplanned outpatient review can be minimized by optimizing both intra- and post-operative pain management. Individual surgeon and anaesthetist preferences remain an obstacle to a standardized protocol in the Australian setting.

Circulating tumour cells in pancreatic cancer: A systematic review and meta-analysis of clinicopathological implications.

BACKGROUND: The detection and quantification of circulating tumour cells (CTCs) in pancreatic cancer (PC) has the potential to provide prognostic information. The aim of this review was to provide an overview of the literature surrounding CTCs in PC. METHODS: A systematic literature review on CTCs in PC between 2005-2020 was performed. Data based on peripheral vein samples were used to determine the positivity rate of CTCs, their prognostic significance and their relative numbers compared to portal vein (PV) samples. RESULTS: The overall CTC detection rate in forty-four articles was 65% (95%CI: 55-75%). Detection rate for CellSearch was 26% (95%CI: 14-38%), which was lower than for both filtration and microfluidic techniques. In nine studies with >50 patients, overall survival was worse with CTC positivity (HR 1.82; 95%CI: 1.61-2.05). Five of seven studies which described PV CTC collection provided patient-level data. PV CTC yield was 7.7-fold (95%CI 1.35-43.9) that of peripheral blood. CONCLUSIONS: CTCs were detected in the peripheral circulation of most patients with PC and may be related to prognosis and disease stage. PV blood contains more CTCs than peripheral blood sampling. This review points to the maturation of techniques of CTC enrichment, and its evidence base for eventual clinical deployment.

An Orthotopic Resectional Mouse Model of Pancreatic Cancer.

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer (PC). To address this deficiency, we describe a mouse model involving orthotopic implantation of PC followed by distal pancreatectomy and splenectomy. The model has been demonstrated to be safe and suitably flexible for the study of various therapeutic approaches in adjuvant and neo adjuvant settings. In this model, a pancreatic tumor is first generated by implanting a mixture of human pancreatic cancer cells (luciferase-tagged AsPC-1) and human cancer associated pancreatic stellate cells into the distal pancreas of Balb/c athymic nude mice. After three weeks, the cancer is resected by re-laparotomy, distal pancreatectomy and splenectomy. In this model, bioluminescence imaging can be used to follow the progress of cancer development and effects of resection/treatments. Following resection, adjuvant therapy can be given. Alternatively, neoadjuvant treatment can be given prior to resection. Representative data from 45 mice are presented. All mice underwent successful distal pancreatectomy/splenectomy with no issues of hemostasis. A macroscopic proximal pancreatic margin greater than 5 mm was achieved in 43 (96%) mice. The technical success rate of pancreatic resection was 100%, with 0% early mortality and morbidity. None of the animals died during the week after resection. In summary, we describe a robust and reproducible technique for a surgical resection model of pancreatic cancer in mice which mimics the clinical scenario. The model may be useful for the testing of both adjuvant and neoadjuvant treatments.

Targeting the HGF/c-MET pathway in advanced pancreatic cancer: a key element of treatment that limits primary tumour growth and eliminates metastasis.

BACKGROUND: Stromal-tumour interactions facilitate pancreatic cancer (PC) progression. The hepatocyte growth factor (HGF)/c-MET pathway is upregulated in PC and mediates the interaction between cancer cells and stromal pancreatic stellate cells (PSCs). This study assessed the effect of HGF/c-MET inhibition plus gemcitabine (G) on the progression of advanced PC. METHODS: Orthotopic PC was produced by implantation of luciferase-tagged human cancer cells + human PSCs into mouse pancreas. Tumours were allowed to develop without treatment for 4 weeks. Mice were then treated for 6 weeks with one of the following: IgG, G, HGF inhibitor (Hi), c-MET inhibitor (Ci), Hi + Ci, Hi + G, Ci + G, or Hi + Ci + G. RESULTS: Bioluminescence imaging showed similar tumour sizes in all mice at the initiation of treatments. Triple therapy (Hi + Ci + G): (1) completely eliminated metastasis; (2) significantly reduced tumour size as assessed by bioluminescence and at necropsy; (3) significantly reduced proliferating cancer cell density and stem cell marker DCLK1 expression in tumours. In vitro 3D culture studies supported our in vivo findings. CONCLUSION: Even at an advanced disease stage, a two-pronged approach, targeting (a) HGF/c-MET with relevant inhibitors and (b) cancer cells with chemotherapy, completely eliminated metastasis and significantly decreased tumour growth, suggesting that this is a promising treatment approach for PC.

Pancreatic stellate cells: what's new?

PURPOSE OF REVIEW: Pancreatic stellate cells (PSCs) play an integral role in the pathogenesis of pancreatitis and pancreatic cancer. With the developing knowledge of this important cell type, we are at the cusp of developing effective therapies for the above diseases based upon targeting the PSC and modulating its function. RECENT FINDINGS: The major themes of the recent PSC literature include: PSC interactions with the extracellular matrix and other stromal components; intracellular calcium physiology as drivers of mechanical interactions and necrosis; the relationship between proinflammatory, protumoural, angiogenic, and metabolic pathways in pancreatic necrosis, fibrosis, and carcinogenesis; and targeting of the stroma for antitumoural and antifibrotic effects. SUMMARY: Traditionally, there have been few treatment options for pancreatitis and pancreatic cancer. The elucidation of the wide-ranging functions of PSCs provide an opportunity for treatments based on stromal reprogramming.

Circulating pancreatic stellate (stromal) cells in pancreatic cancer-a fertile area for novel research.

Pancreatic stellate cells (PSCs) are known to play an important role in facilitating pancreatic cancer progression-both in terms of local tumour growth as well as the establishment of metastases. We have previously demonstrated that PSCs from the primary cancer seed to distant metastatic sites. We therefore hypothesise that PSCs circulate along with pancreatic cancer cells (circulating tumour cells-CTCs) to help create a growth permissive microenvironment at distant metastatic sites. This review aims to explore the concept of circulating PSCs in pancreatic cancer and suggests future directions for research in this area.

Validity of the Barcelona Clinic Liver Cancer and Hong Kong Liver Cancer staging systems for hepatocellular carcinoma in Singapore.

BACKGROUND: Staging is vital in guiding therapeutic approach in patients diagnosed with hepatocellular carcinoma (HCC). Our study's goal is to compare paradigms in the Barcelona Clinic Liver Cancer (BCLC) and Hong Kong Liver Cancer (HKLC) systems, and evaluate the use of both in a local context, comparing their prognostic ability and therapeutic efficacy in the management of HCC. METHODS: Seven hundred and sixty-six patients diagnosed between 2010 and 2015 were identified and staged according to BCLC and HKLC. Both system's performances were compared using Akaike information criterion (AIC), bootstrap concordance-index (c-index), and through Kaplan-Meier survival curves of patients who came under HKLC stages 1, 2, and 3 and the individual BCLC stages. Independent prognostic factors of survival were identified using univariate and multivariate analyses. RESULTS: According to AIC and c-index, HKLC (AIC = 5,711, c-index = 0.74) has equivalent prognosticating value as BCLC (AIC = 5,764, c-index = 0.72). Through Kaplan-Meier curves, we determined that more aggressive treatments resulted in better outcomes. Particularly for patients under BCLC stage C, patients who followed the HKLC system's recommended treatments performed markedly better. CONCLUSIONS: In our patient population, the HKLC system is comparable to the BCLC system in prognosticating patients, but is suggested to have better performance in guiding treatment.

The Abdominal Reapproximation Anchor Device.

INTRODUCTION: Achieving primary fascial closure after damage control laparostomy can be challenging. A number of devices are in use, with none having yet emerged as best practice. In July 2013, at Westmead Hospital, we started using the abdominal reapproximation anchor (ABRA; Canica Design, Almonte, Ontario, Canada) device. We report on our experience. METHODS: A retrospective review of medical records for patients who had open abdomens managed with the ABRA device between July to December 2013 was done. Data extracted included age, sex, body mass index (BMI), reason for the open abdomen, Acute Physiology and Chronic Health Evaluation II (APACHE II) score, number of laparostomies prior to ABRA placement, duration of placement, device complications, length of hospital and intensive care unit (ICU) stay, and outcomes. RESULTS: Four cases of open abdomens managed using the ABRA device were identified, with 3 a consequence of intra-abdominal sepsis and 1 a consequence of penetrating trauma. Mean BMI was 33.5 kg/m2, APACHE II score was 14.5, duration with open abdomen prior to ABRA placement was 11.75 days, duration with ABRA in situ was 9 days, duration of hospital stay was 64.25 days, and ICU stay was 37.75 days. Three patients (75%) achieved fascial closure, and 1 achieved skin closure. No incidences of enterocutaneous fistulae occurred. CONCLUSION: The ABRA is a unique emerging alternative to aid in achieving fascial closure in patients managed with open abdomens. Our case series demonstrates that it can be used effectively in selected patients. Studies are needed to compare its efficacy with more traditional methods.

Index cholecystectomy in grade II and III acute calculous cholecystitis is feasible and safe.

BACKGROUND: According to the Tokyo Guidelines, recommendation on management of moderate and severe cholecystitis are cholecystostomy in severe cases and either cholecystostomy or emergency cholecystectomy in moderate cases depending on surgical experience. The rationale for this is that percutaneous cholecystostomy is a short procedure while laparoscopic cholecystectomy may be associated with a larger physiological insult. The aim of this study was to determine the safety and efficacy of cholecystectomy in moderate and severe acute calculous cholecystitis (ACC) at our institution. METHODS: A retrospective review of patients presenting to Westmead Hospital with ACC between 2011 and 2012 was performed. Patients were classified according to the Tokyo Guidelines and only grade II and grade III patients were included. Clinical and complication details were recorded from the clinical notes. RESULTS: Of the 84 patients, 60 had grade II and 24 had grade III ACC. The mean age was 52 years and 59% were female. In both groups, index cholecystectomy was performed in 88% of patients. None of the grade II ACC patients and three (12%) of grade III ACC underwent cholecystostomy. Length of stay (5 versus 12, P < 0.001) and conversion rate (2% versus 27%, P = 0.006) was higher in the grade III group. There were no deaths in patient who underwent surgery in either group. Severe complications were not significantly different (2% versus 9%, P = 0.219). CONCLUSION: Index cholecystectomy is feasible with low morbidity and no mortality even in severe ACC. Emergency cholecystectomy in the setting of severe cholecystitis appear to be safe and technically feasible option.

Immunohistochemical validation of overexpressed genes identified by global expression microarrays in adrenocortical carcinoma reveals potential predictive and prognostic biomarkers.

BACKGROUND: Adrenocortical carcinoma (ACC) is a rare malignancy with a poor prognosis. The aim of this study was to identify novel protein signatures that would predict clinical outcomes in a large cohort of patients with ACC based on data from previous gene expression microarray studies. MATERIALS AND METHODS: A tissue microarray was generated from the paraffin tissue blocks of 61 patients with clinical outcomes data. Selected protein biomarkers based on previous gene expression microarray profiling studies were selected, and immunohistochemistry staining was performed. Staining patterns were correlated with clinical outcomes, and a multivariate analysis was undertaken to identify potential biomarkers of prognosis. RESULTS: Median overall survival was 45 months, with a 5-year overall survival rate of 44%. Median disease-free survival was 58 months, with a 5-year disease-free survival rate of 44%. The proliferation marker Ki-67 and DNA topoisomerase TOP2A were associated with significantly poorer overall and disease-free survival. The results also showed strong correlation between the transcriptional repressor EZH2 and TOP2A expression, suggesting a novel role for EZH2 as an additional marker of prognosis. In contrast, increased expression of the BARD1 protein, with its ubiquitin ligase function, was associated with significantly improved overall and disease-free survival, which has yet to be documented for ACC. CONCLUSION: We present novel biomarkers that assist in determining prognosis for patients with ACC. Ki-67, TOP2A, and EZH2 were all significantly associated with poorer outcomes, whereas BARD1 was associated with improved overall survival. It is hoped that these biomarkers may help tailor additional therapy and be potential targets for directed therapy.

Chemical burns in children: Aetiology and prevention.

BACKGROUND: Chemical burns account for a small proportion of total burns in children, but may require specific first aid and different modes of prevention. METHODS: A retrospective study between 2006 and 2012 of children ≤16 years treated with chemical burns at a specialist paediatric burn centre. Data were extracted from a prospectively maintained database. RESULTS: 56 episodes of chemical burns occurred during the study period. The majority (54%) occurred in boys. There were 39 (72%) patients <10 years and 17 (39%) ≥10 years. Median total body surface area burnt was 1% with nine (16%) patients requiring skin grafting. Only 24 (45%) had adequate first aid. The majority (n=46, 82%) of chemical burns occurred in the domestic setting, especially in the <10 years age group (P=0.052). Non-intentional exposure of chemicals by an unattended child accounted for half of all (n=22, 49%) chemical burns <10 years of age. Eight (47%) burns in patients ≥10 years resulted from self-harm. The most common aetiological agents were household cleaners and aerosols in the younger and older age groups respectively. CONCLUSION: Chemical burns remain infrequent but potentially preventable. These burns mainly occur in the domestic setting due to non-intentional exposure of household chemicals in children <10, and due to deliberate self-harm in children ≥10. The use of child-resistant packaging, similar to that used for medications, and improved parental practices could help decrease the incidence of burns in children <10.

Analysis of actual healthcare costs of early versus interval cholecystectomy in acute cholecystitis.

BACKGROUND: Healthcare cost modeling have favored early (ELC) over interval laparoscopic cholecystectomy (ILC) for acute cholecystitis (AC). However, actual costs of treatment have never been studied. The aim of the present study was to compare actual hospital costs involved in ELC and ILC in patients with AC. METHODS: Retrospective study of patients who underwent laparoscopic cholecystectomy for AC was conducted. Demographic, clinical, operative data and costs were extracted and analyzed. RESULTS: Between 2011 and 2013, 201 had laparoscopic surgery for AC at Tan Tock Seng Hospital, Singapore. One hundred and thirty-four (67%) patients underwent ELC (≤7 days of presentation, within index admission). Median total length of stay (LOS) was 4.6 and 6.8 days for ELC and ILC groups, respectively (P = 0.006). Patients who had ELC also had significantly lesser total number of admissions (P < 0.001). The median (IQR) total inpatient costs were €4.4 × 10(3) (3.6-5.6) and €5.5 × 10(3) (4.0-7.5) for ELC and ILC patients, respectively (P < 0.007). Costs associated with investigations were significantly higher in the ILC group (P = 0.039), of which serological costs made most difference (P < 0.005). The ward costs were also significantly higher in the ILC group. CONCLUSION: The cost differences reflect the significantly increased total LOS, and repeat presentations associated with ILC. Therefore, ELC should be the preferred management strategy for AC.

Improving Outcomes in Adrenocortical Cancer: An Australian Perspective.

BACKGROUND: Adrenocortical carcinoma (ACC) is a rare malignancy that carries a poor prognosis. There has yet to be a large Australian series that documents the characteristics of ACC and there are a paucity of data on management and the long-term outcomes. We sought to provide a unique insight into the management of ACC in Australia as well as to identify factors associated with prognosis and survival. METHODS: A multivariate analysis of a cohort of patients identified with ACC between 1998 and 2013 was undertaken. Recurrence-free survival (RFS) and overall survival (OS) were assessed as the main outcome measures and correlated with multiple clinical variables in order to identify prognostic markers. RESULTS: Of the 104 patients identified, a total of 98 patients with complete clinical and outcome data were included in the study. Median OS was 56 months, with the 5-year survival being 48 % (95 % confidence interval 36-59). On multivariate analysis, age ≥50 years, metastases at presentation, and evidence of extra-adrenal invasion were found to be statistically associated with reduced OS. RFS was analyzed in patients without metastases. On multivariate analysis, extra-adrenal invasion and no preoperative endocrine investigations were found to be statistically significant poor prognostic factors, with a non-significant trend for higher individual surgeon volume to be associated with improved resection margins and RFS. CONCLUSIONS: We present clinical outcomes and prognostic factors for patients with ACC in a landmark Australian series. We suggest that management in a specialized tertiary endocrine and/or surgical oncology unit is more likely to lead to improved outcomes.

Mutations in KCNJ5 determines presentation and likelihood of cure in primary hyperaldosteronism.

INTRODUCTION: Primary hyperaldosteronism (PA) is a common cause of secondary hypertension. Two recurrent mutations (G151R and L168R) in the potassium channel gene KCNJ5 have been identified that affect the Kir3.4 potassium channel found in the cells of the zona glomerulosa of the adrenal gland. The aim of this study was to determine the prevalence of KCNJ5 mutations in an Australian cohort of patients and to correlate these findings with clinical outcome data, in order to describe the clinical impact on patients who harbour this mutation. METHODS: Direct Sanger sequencing for KCNJ5 on DNA from adrenal tumour tissue of 83 patients with PA in a cohort study was undertaken and mutation status correlated with clinical outcome data. RESULTS: Seventy-one of 83 patients (86%) had adrenocortical adenomas and 12 patients (14%) had bilateral adrenal hyperplasia. A total of 34 (41%) patients were found to have heterozygous somatic mutations in KCNJ5, G151R and L168R. No germ line mutations were identified. Patients with mutations were predominately female (68% versus 49%) and significantly younger at presentation (48 versus 55 years). When correlated with clinical data, our results demonstrated that patients with KCNJ5 mutations were more likely to be cured following surgery without the requirement for ongoing medications. CONCLUSIONS: Our findings in a large Australian cohort show that patients with mutations in KCNJ5 present earlier with the signs and symptoms of PA benefit from surgical intervention. Moreover, our results highlight the importance of a thorough workup and management plan for younger patients who present with hypertension.

Systematic review and meta-analysis of laparoscopic versus open distal gastrectomy.

BACKGROUND: Laparoscopic distal gastrectomy has been increasingly utilized in the treatment of gastric adenocarcinoma. This study aims to compare the morbidity/mortality and postoperative outcomes of laparoscopic-assisted versus open distal gastrectomy since 2000. METHODS: A comprehensive search of MEDLINE and EMBASE was conducted including studies published between 2000 and present. RESULTS: Seventeen studies with a total of 7,109 distal gastrectomies (3,496 lap vs 3,613 open) were included. Across all studies, postoperative morbidity rates for laparoscopic gastrectomy were lower than that of open [median (range) 10 (0-36) % vs 17 (0-43) %]. Meta-analysis of postoperative morbidity rates in prospective studies only yielded pooled odds ratio of 0.52 (95 % CI 0.33-0.81) (P = 0.004). In-hospital mortality rates were comparable between the two (range: laparoscopic 0-3.3 vs open 0-6.7 %). The long-term oncological outcomes of resection were difficult to analyze given variable reporting but appeared similar between the two. Meta-analysis of prospective studies showed that laparoscopic-assisted distal gastrectomy was associated with significantly shorter hospital length of stay [standard mean difference (SMD) = -0.78 (95 % CI = -1.0 to -0.56)], comparable intraoperative bleeding [SMD = 0.64 (95 % CI = -1.3-0.0430) P = 0.066] and longer operative time compared to open gastrectomy [1.9 (95 % CI 0.05-3.8) P = 0.045, with P < 0.001]. CONCLUSION: This study supports the use of laparoscopic-assisted distal gastrectomy for treatment of gastric adenocarcinoma with evidence of comparable, if not better, short-term postoperative parameters when compared to open distal gastrectomy. The long-term oncological outcomes appear similar but may require more evaluation.