Impact of disease stage and aetiology on survival in hepatocellular carcinoma::implications for surveillance

Background: Variation in survival in hepatocellular carcinoma (HCC) has been attributed to different aetiologies or disease stages at presentation. While international guidelines recommend surveillance of high-risk groups to permit early diagnosis and curative treatment, the evidence that surveillance decreases disease-specific mortality is weak. Methods: We compared HCC survival figures from Japan (n=1174) and Hong Kong (n=1675) over similar time periods (Japan 2000–2013, Hong Kong, China 2003–2014). The former has an intensive national surveillance programme, while the latter has none. We also analysed changes in survival in Japan over a 50-year period including data from before and after institution of a national HCC surveillance programme. Results: In Japan, over 75% of cases are currently detected by surveillance, whereas in Hong Kong <20% of cases are detected presymptomatically. Median survival was 52 months in Japan and 17.8 months in Hong Kong; this survival advantage persisted after allowance for lead-time bias. Sixty-two per cent of Japanese patients had early disease at diagnosis and 63% received curative treatment. The comparable figures for Hong Kong were 31.7% and 44.1%, respectively. These differences could not be accounted for by disease aetiology, and patients in Hong Kong who were detected at an early stage had a similar survival to the analogous patients in Japan. Conclusions: The variation in survival is largely accounted for by stage at diagnosis, which in turn relates to the intensity of surveillance programmes and the consequent variation in curative therapeutic options

Identification of host cell proteins involved in Shigella flexneri pathogenesis.

Shigella flexneri is the etiological agent of bacillary dysentery (shigellosis). It is transmitted via the faecal-oral route and is a significant human pathogen due to the high morbidity among children <5 years in developing countries. The key pathogenic features of Shigella include cell death induction in myeloid immune cells and circumventing cell death in colonic epithelial cells, the site of bacterial infection. Shigella also interact with host proteins to initiate de novo actin synthesis to facilitate its intra- and intercellular spread to disseminate in the host. In this thesis, the role of three host proteins: myosin IIA, dynamin II, and dynamin-related protein 1 (Drp1) during Shigella cell-to-cell spreading was examined. The myosin IIA specific kinase, myosin like chain kinase (MLCK), was previously shown to be important for Shigella plaque formation. Myosin IIA and MLCK have also been implicated in septin caging of non-motile Shigella which are targeted for degradation. Chemical inhibition and siRNA knockdown of myosin IIA reduced Shigella plaque formation. Curiously HeLa cells infected with Shigella mutants defective in cell-to-cell spreading have significantly reduced myosin IIA levels when quantified by immunofluorescence microscopy. Dynamin II and Drp1 are members of the dynamin superfamily. Both proteins have self-assembly driven GTPase activation. Dynamin II is important for clathrin-mediated endocytosis and pinches the budding clathrin-coated vesicle, and Drp1 is essential for mitochondrial fission. It was hypothesized that Shigella protrusion formation into adjacent host cells resembles endocytic and exocytic processes, and components of these processes may facilitate Shigella dissemination. When dynamin II GTPase was inhibited with dynasore and dynamin II was knocked down with siRNA, Shigella cell-to-cell spreading was significantly reduced. The in vivo efficacy of dynasore was tested in a murine Sereny model. No significant reduction in inflammation was observed but mice were protected against weight loss during infection. Further experimentation suggested dynasore protected mice against cytotoxic effects from the three secretion system (TTSS) effectors expressed by Shigella during infection. Drp1 was investigated in this thesis as dynasore also inhibits the GTPase of this mitochondrial fission protein. Mitochondrial fission is important in maintaining mitochondrial dynamics and also in events downstream of intrinsic apoptosis and programmed necrosis pathways activation. Loss of mitochondrial function in Shigella-induced epithelial cell death has been reported previously. Hence the role of Drp1 in Shigella plaque formation and HeLa death was examined with the Drp1-specific inhibitor, Mdivi-1, and siRNA knockdown. HeLa cell death was significantly reduced; suggesting loss of mitochondrial function observed previously may now be attributed to Drp1 and subsequent Drp1-mediated mitochondrial fission. The impairment in Shigella cell-to-cell spreading in the absence of Drp1 suggests maintaining an intact mitochondrial network is essential for Shigella lateral spread since loss of Drp1 function would result in excessive mitochondrial fusion, leading to formation of netlike or perinuclear structures. The outcomes of this thesis highlight the importance of host proteins during different stages of Shigella infection. By improving our understanding on the host and bacteria interaction, future work on novel approaches to prevent Shigella dissemination can be developed.Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 201

Intraday analysis of dually-listed stock (Sime Darby Berhad)

59 p.In this report, we analyse the intraday trading patterns on Sime Darby Berhad a Malaysian conglomerate dually listed on Kuala Lumpur Stock Exchange (KLSE) and Stock Exchange of Singapore-Central Limit Order Book (SES-CLOB), and investigate the influence of lagging returns and volume of one listing on another. In this report, we analyse the intraday trading patterns on Sime Darby Berhad a Malaysian conglomerate dually listed on Kuala Lumpur Stock Exchange (KLSE) and Stock Exchange of Singapore-Central Limit Order Book (SES-CLOB), and investigate the influence of lagging returns and volume of one listing on another.BUSINES

Separation and parallel sequencing of the genomes and transcriptomes of single cells using G&T-seq

Parallel sequencing of a single cell's genome and transcriptome provides a powerful tool for dissecting genetic variation and its relationship with gene expression. Here we present a detailed protocol for G&T-seq, a method for separation and parallel sequencing of genomic DNA and full-length polyA(+) mRNA from single cells. We provide step-by-step instructions for the isolation and lysis of single cells; the physical separation of polyA(+) mRNA from genomic DNA using a modified oligo-dT bead capture and the respective whole-transcriptome and whole-genome amplifications; and library preparation and sequence analyses of these amplification products. The method allows the detection of thousands of transcripts in parallel with the genetic variants captured by the DNA-seq data from the same single cell. G&T-seq differs from other currently available methods for parallel DNA and RNA sequencing from single cells, as it involves physical separation of the DNA and RNA and does not require bespoke microfluidics platforms. The process can be implemented manually or through automation. When performed manually, paired genome and transcriptome sequencing libraries from eight single cells can be produced in ∼3 d by researchers experienced in molecular laboratory work. For users with experience in the programming and operation of liquid-handling robots, paired DNA and RNA libraries from 96 single cells can be produced in the same time frame. Sequence analysis and integration of single-cell G&T-seq DNA and RNA data requires a high level of bioinformatics expertise and familiarity with a wide range of informatics tools.status: publishe

Laparoscopic right hepatectomy: a challenging, but feasible, safe and efficient procedure

BACKGROUND: Few centers are undertaking major laparoscopic liver resections, because of the well-recognized technical difficulties and lack of training opportunities.METHODS: The authors describe their technique for laparoscopic right hepatectomy, highlighting relevant details for accomplishing a safe and efficient procedure. Patients were chronologically divided into 2 groups to evaluate the impact of increasing experience on the surgical outcomes.RESULTS: Group I included 17 patients and group II 18 patients. The conversion rate to open or hybrid techniques significantly decreased from 36% in group I to 6% in group II (P = .03). The hospital stay decreased from a median of 6 days in group I to a median of 4 days in group II (P = .05). Complications occurred in 4 patients (11%), of whom 3 were in group I. The mortality was zero.CONCLUSIONS: Laparoscopic right hepatectomy is a safe and efficient procedure when performed at specialized centers with extensive experience in hepatic surgery. Long-term training is necessary to acquire adequate expertise.<br/

Single-centre comparative study of laparoscopic versus open right hepatectomy

BackgroundExpansion of laparoscopic major hepatectomy is still limited mainly due to the well-recognised technical difficulties compared to open surgery, and doubts regarding the oncological efficiency when major resections are required.MethodsPatients undergoing open right hepatectomy (ORH) were matched with patients undergoing laparoscopic right hepatectomy (LRH) and compared for perioperative outcomes.ResultsSeventy patients were included: 36 patients underwent LRH and 34 ORH. Operative time was significantly longer for LRH (median, 300 min vs. 180 min for ORH; p?&lt;?0.0001). Intensive care unit (median, 2 days for LRH vs. 4 days for ORH; p?&lt;?0.0001) and postoperative length of stay (5 days for LRH vs. 9 days for ORH; p?&lt;?0.0001) were significantly shorter for LRH. Four laparoscopic cases were converted to open surgery. No significant difference in postoperative complications and mortality was observed between LRH and ORH. Among patients with colorectal carcinoma liver metastases, R0 resection was obtained in 20/21 (95%) cases after LRH, and in 20/25 (80%) after ORH (p?=?0.198). Mid-term overall survival did not significantly differ between the laparoscopic and the open group.ConclusionsLRH can be a safe, effective, and oncologically efficient alternative to open resection in selected cases. Extensive experience in hepatic and laparoscopic surgery is required

Surgical management of benign and indeterminate hepatic lesions in the era of laparoscopic liver surgery

Background/Aims: The expansion of the laparoscopic approach for the management of benign liver lesions has raised concerns regarding the risk of widening surgical indications and compromising safety. Large single-centre series focusing on laparoscopic management of benign liver lesions are sporadic. Methods: We reviewed a prospectively collected database of patients undergoing pure laparoscopic liver resection (LLR) for benign liver lesions. All cases were individually discussed at a multidisciplinary team meeting. Results: Forty-six patients underwent 50 LLRs for benign disease. Indications for surgery were: symptomatic lesions, preoperative diagnosis of adenoma or cystadenoma, and lesions with an indeterminate diagnosis. The preoperative diagnosis was uncertain in 11 cases. Of these, histological diagnosis was hepatocellular carcinoma in one (9%) and benign lesion in 10 patients (91%). Thirteen patients (28%) required major hepatectomy. Three patients (7%) developed postoperative complications. Mortality was nil. The median postoperative hospital stay following major and minor hepatectomy was 4 and 3 days, respectively. Conclusion: The laparoscopic approach represents a safe option for the management of benign and indeterminate liver lesions, even when major hepatectomy is required. LLR should be only performed in specialized centres to ensure safety and strict adherence to orthodox surgical indication

Circulating tumour DNA (ctDNA) in metastatic melanoma, a systematic review and meta-analysis.

INTRODUCTION Circulating tumour DNA (ctDNA) is an emerging biomarker in melanoma. We performed a systematic review and meta-analysis to explore its clinical utility as a prognostic, pharmacodynamic (PD) and predictive biomarker. METHODS A systematic search was conducted from Jan 2015 to April 2021, of the electronic databases PubMed, Cochrane Library and Ovid MEDLINE to identify studies. Studies were restricted to those published in English within the last 5 years, evaluating ctDNA in humans in ≥10 patients. Survival data were extracted for meta-analysis using pooled treatment effect (TE), i.e. log hazard ratios (HRs) and corresponding standard error of TE for progression-free survival or overall survival differences in patients who were ctDNA positive or negative. PRISMA statement guidelines were followed. RESULTS A meta-analysis of 19 studies grouped according to methodology of ctDNA detection, revealed a combined estimate for HR of progression-free survival (13 studies using droplet digital Polymerase Chain Reaction (ddPCR) methodology (N = 1002) of 2.10 (95% CI: 1.71-2.59) revealing a poorer prognosis when ctDNA was detected. This result was confirmed in the smaller analysis of (non-ddPCR, N = 347) five studies: HR = 2.45 (95% CI: 1.29-4.63). Similar findings were found in the overall survival analysis of nine studies (ddPCR methodology, N = 841) where the combined HR was 2.78 (95% CI: 2.21-3.49) and of the five studies (non-ddPCR methodology, N = 326) where the combined HR was 2.58 (95% CI: 1.74-3.84). Serial ctDNA levels on treatment showed a pharmacodynamic role reflecting response or resistance earlier than radiological assessment. CONCLUSIONS Circulating tumour DNA is a predictive, prognostic and PD biomarker in melanoma. Technical standardisation of assays is required before clinical adoption

The Impact of Diabetes and Glucose-Lowering Therapies on Hepatocellular Carcinoma Incidence and Overall Survival.

PURPOSE The incidence of hepatocellular carcinoma (HCC) in the United Kingdom has increased 60% in the past 10 years. The epidemics of obesity and type 2 diabetes are contributing factors. In this article, we examine the impact of diabetes and glucose-lowering treatments on HCC incidence and overall survival (OS). METHODS Data from 1064 patients diagnosed with chronic liver disease (CLD) (n = 340) or HCC (n = 724) were collected from 2007 to 2012. Patients with HCC were followed up prospectively. Univariate and multivariate logistic regression determined HCC risk factors. Kaplan-Meier curves were used to examine survival and Cox proportional hazards analysis estimated hazard ratios (HRs) for death according to use of glucose-lowering therapies. FINDINGS Diabetes prevalence was 39.6% and 10.6% within the HCC and CLD cohorts, respectively. The odds ratio for having HCC in patients with diabetes was 5.55 (P < 0.001). Univariate analysis found an increased association of HCC with age, sex, cirrhosis, hemochromatosis, alcohol abuse, diabetes, and Child's Pugh score. In multivariate analysis age, sex, cirrhosis, Child's Pugh score, diabetes status, and insulin use retained significance. Diabetes status did not significantly affect OS in HCC; however, in people with diabetes and HCC, metformin treatment was associated with improved OS (mean survival, 31 vs 24 months; P =0.016; HR for death = 0.75; P = 0.032). IMPLICATIONS Diabetes is significantly associated with HCC in the United Kingdom. Metformin treatment is associated with improved OS after HCC diagnosis. Treatment of diabetes should be appropriately reviewed in high-risk populations, with specific consideration of the potential hepatoprotective effects of metformin in HCC