Comprehensive analysis of transcriptome profiles in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma is the second most deadly cancer with late presentation and limited treatment options, highlighting an urgent need to better understand HCC to facilitate the identification of early-stage biomarkers and uncover therapeutic targets for the development of novel therapies for HCC. METHODS: Deep transcriptome sequencing of tumor and paired non-tumor liver tissues was performed to comprehensively evaluate the profiles of both the host and HBV transcripts in HCC patients. Differential gene expression patterns and the dys-regulated genes associated with clinical outcomes were analyzed. Somatic mutations were identified from the sequencing data and the deleterious mutations were predicted. Lastly, human-HBV chimeric transcripts were identified, and their distribution, potential function and expression association were analyzed. RESULTS: Expression profiling identified the significantly upregulated TP73 as a nodal molecule modulating expression of apoptotic genes. Approximately 2.5% of dysregulated genes significantly correlated with HCC clinical characteristics. Of the 110 identified genes, those involved in post-translational modification, cell division and/or transcriptional regulation were upregulated, while those involved in redox reactions were downregulated in tumors of patients with poor prognosis. Mutation signature analysis identified that somatic mutations in HCC tumors were mainly non-synonymous, frequently affecting genes in the micro-environment and cancer pathways. Recurrent mutations occur mainly in ribosomal genes. The most frequently mutated genes were generally associated with a poorer clinical prognosis. Lastly, transcriptome sequencing suggest that HBV replication in the tumors of HCC patients is rare. HBV-human fusion transcripts are a common observation, with favored HBV and host insertion sites being the HBx C-terminus and gene introns (in tumors) and introns/intergenic-regions (in non-tumors), respectively. HBV-fused genes in tumors were mainly involved in RNA binding while those in non-tumors tissues varied widely. These observations suggest that while HBV may integrate randomly during chronic infection, selective expression of functional chimeric transcripts may occur during tumorigenesis. CONCLUSIONS: Transcriptome sequencing of HCC patients reveals key cancer molecules and clinically relevant pathways deregulated/mutated in HCC patients and suggests that while HBV may integrate randomly during chronic infection, selective expression of functional chimeric transcripts likely occur during the process of tumorigenesis.

Early experience with robot-assisted laparoscopic hepatobiliary and pancreatic surgery in Singapore: single-institution experience with 20 consecutive patients.

INTRODUCTION: Experience with robot-assisted laparoscopic (RAL) hepatobiliary and pancreatic (HPB) surgery remains limited worldwide. In this study, we report our early experience with RAL HPB surgery in Singapore. METHODS: A retrospective review of the first 20 consecutive patients who underwent RAL HPB surgery at a single institution over a 34-month period from February 2013 to November 2015 was conducted. The 20 cases were performed by three principal surgeons, of which 17 (85.0%) were performed by a single surgeon. RESULTS: The median age of patients was 56 (range 22-75) years and median tumour size was 4.0 (range 1.2-7.5) cm. The surgeries performed included left-sided pancreatectomies (n = 10), hepatectomies (n = 7), triple bypass with bile duct exploration for obstructing pancreatic head cancer with choledocholithiasis (n = 1), cholecystectomy for Mirizzi's syndrome (n = 1) and gastric resection for gastrointestinal stromal tumour (n = 1). The median operation time was 445 (range 80-825) minutes and median blood loss was 350 (range 0-1,200) mL. There was only 1 (5%) open conversion. There were 2 (10.0%) major morbidities (> Grade II on the Clavien-Dindo classification) and no 30-day/in-hospital mortalities. There was no reoperation for postoperative complications. The median postoperative stay was 5.5 (range 3-22) days. CONCLUSION: Our initial experience confirms the feasibility and safety of RAL HPB surgery.

Evolution of laparoscopic liver resection at Singapore General Hospital: a nine-year experience of 195 consecutive resections.

INTRODUCTION: We aimed to analyse the changing trends, safety and outcomes associated with the adoption of laparoscopic liver resection (LLR) at a single centre. METHODS: A retrospective review of patients who underwent LLR from 2006 to 2014 at our institution was performed. To explore the evolution of LLR, the study was divided into three equal consecutive time periods (Period 1: 2006-2008, Period 2: 2009-2011, and Period 3: 2012-2014). RESULTS: Among 195 patients who underwent LLR, 24 (12.3%) required open conversions, 68 (34.9%) had resection of tumours in the difficult posterosuperior segments and 12 (6.2%) underwent major (≥ 3 segments) hepatectomies. Median operation time was 210 (range 40-620) minutes and median postoperative stay was 4 (range 1-26) days. Major postoperative morbidity (> Grade II) occurred in 11 (5.6%) patients and 90-day/in-hospital mortality was 1 (0.5%). During the study, the number of LLRs performed showed an increasing trend (Period 1: n = 22; Period 2: n = 19; Period 3: n = 154). Other statistically significant trends were: (a) increase in malignant neoplasms resected; (b) increase in resections of difficult posterosuperior segments; (c) longer median operation time; and (d) decrease in open conversion rates. CONCLUSION: Over the study period, the number of LLRs increased rapidly. LLR was increasingly performed for malignant neoplasms and lesions located in the difficult posterosuperior segments, resulting in longer operation times. However, open conversion rates decreased, and there was no change in postoperative morbidity and other perioperative outcomes.

Prognostic significance of KIT exon 11 deletion mutation in intermediate-risk gastrointestinal stromal tumor.

AIM: Benefit of adjuvant imatinib therapy following curative resection in patients with intermediate-risk gastrointestinal stromal tumor (GIST) is unclear. GIST-specific exon mutations, in particular exon 11 deletions, have been shown to be prognostic. We hypothesize that specific KIT mutations may improve risk stratification in patients with intermediate-risk GIST, identifying a subgroup of patients who may benefit from adjuvant therapy. METHODS: In total, 142 GIST patients with complete clinicopathologic and mutational data from two sites were included. Risk classification was based on the modified National Institute of Health (NIH) criteria. RESULTS: In this cohort, 74% (n = 105) of patients harbored a KIT mutation; 61% (n = 86) were found in exon 11 of which nearly 70% were KIT exon 11 deletions (n = 60). A total of 18% (n = 25) of cases were classified as having intermediate-risk disease. Univariate analysis confirmed tumor size, mitotic index, nongastric origin, presence of tumor rupture and modified NIH criteria were adversely prognostic for relapse-free survival (RFS). Among KIT/PDGFRA mutants, KIT exon 11 deletions had a significantly worse prognosis (hazard ratio 2.31; 95% confidence interval, 1.30-4.10; P = 0.003). Multivariate analysis confirmed KIT exon 11 deletion (P = 0.003) and clinical risk classification (P < 0.001) as independent adverse prognostic factors for RFS. Intermediate-risk patients harboring KIT exon 11 deletions had RFS outcomes similar to high-risk patients. CONCLUSION: The presence of KIT exon 11 deletion mutation in patients with intermediate-risk GIST is associated with an inferior clinical outcome with RFS similar to high-risk patients.

First experience with robotic spleen-saving, vessel-preserving distal pancreatectomy in Singapore: a report of three consecutive cases.

INTRODUCTION: The use of laparoscopic distal pancreatectomy (LDP) has increased worldwide due to the reported advantages associated with this minimally invasive procedure. However, widespread adoption is hindered by its technical complexity. Robotic distal pancreatectomy (RDP) was introduced to overcome this limitation, but worldwide experience with RDP is still lacking. There is presently evidence that RDP is associated with decreased conversion rate and increased splenic preservation as compared to LDP. METHODS: We conducted a prospective study on our initial experience with robotic spleen-saving, vessel-preserving distal pancreatectomy (SSVP-DP) between July 2013 and April 2014. RESULTS: Three consecutive patients underwent attempted robotic SSVP-DP. The indications were a 2.1-cm indeterminate cystic neoplasm, 4.5-cm solid pseudopapillary neoplasm and 1.2-cm pancreatic neuroendocrine tumour. For all three patients, the procedure was completed without conversion, and the spleen, with its main vessels, was successfully conserved. The median total operation time, blood loss and postoperative stay were 350 (range 300-540) minutes, 200 (range 50-300) mL and 7 (range 6-14) days, respectively. Two patients had minor Clavien-Dindo Grade I complications (one Grade A pancreatic fistula and one postoperative ileus). One patient had a Clavien-Dindo Grade IIIa complication (Grade B pancreatic fistula requiring percutaneous drainage). All patients were well at the time of reporting after at least six months of follow-up. CONCLUSION: Our preliminary experience with robotic SSVP-DP confirmed the feasibility of the procedure.

High-depth sequencing of over 750 genes supports linear progression of primary tumors and metastases in most patients with liver-limited metastatic colorectal cancer.

BACKGROUND: Colorectal cancer with metastases limited to the liver (liver-limited mCRC) is a distinct clinical subset characterized by possible cure with surgery. We performed high-depth sequencing of over 750 cancer-associated genes and copy number profiling in matched primary, metastasis and normal tissues to characterize genomic progression in 18 patients with liver-limited mCRC. RESULTS: High depth Illumina sequencing and use of three different variant callers enable comprehensive and accurate identification of somatic variants down to 2.5% variant allele frequency. We identify a median of 11 somatic single nucleotide variants (SNVs) per tumor. Across patients, a median of 79.3% of somatic SNVs present in the primary are present in the metastasis and 81.7% of all alterations present in the metastasis are present in the primary. Private alterations are found at lower allele frequencies; a different mutational signature characterized shared and private variants, suggesting distinct mutational processes. Using B-allele frequencies of heterozygous germline SNPs and copy number profiling, we find that broad regions of allelic imbalance and focal copy number changes, respectively, are generally shared between the primary tumor and metastasis. CONCLUSIONS: Our analyses point to high genomic concordance of primary tumor and metastasis, with a thick common trunk and smaller genomic branches in general support of the linear progression model in most patients with liver-limited mCRC. More extensive studies are warranted to further characterize genomic progression in this important clinical population.

Deep sequencing of the hepatitis B virus in hepatocellular carcinoma patients reveals enriched integration events, structural alterations and sequence variations.

Chronic hepatitis B virus (HBV) infection is epidemiologically associated with hepatocellular carcinoma (HCC), but its role in HCC remains poorly understood due to technological limitations. In this study, we systematically characterize HBV in HCC patients. HBV sequences were enriched from 48 HCC patients using an oligo-bead-based strategy, pooled together and sequenced using the FLX-Genome-Sequencer. In the tumors, preferential integration of HBV into promoters of genes (P < 0.001) and significant enrichment of integration into chromosome 10 (P < 0.01) were observed. Integration into chromosome 10 was significantly associated with poorly differentiated tumors (P < 0.05). Notably, in the tumors, recurrent integration into the promoter of the human telomerase reverse transcriptase (TERT) gene was found to correlate with increased TERT expression. The preferred region within the HBV genome involved in integration and viral structural alteration is at the 3'-end of hepatitis B virus X protein (HBx), where viral replication/transcription initiates. Upon integration, the 3'-end of the HBx is often deleted. HBx-human chimeric transcripts, the most common type of chimeric transcripts, can be expressed as chimeric proteins. Sequence variation resulting in non-conservative amino acid substitutions are commonly observed in HBV genome. This study highlights HBV as highly mutable in HCC patients with preferential regions within the host and virus genome for HBV integration/structural alterations.

Adult choledochal cysts: an audit of surgical management.

BACKGROUND: Choledochal cysts are rare congenital cystic dilatations of the biliary tree. Surgical management has evolved with regards to timing and surgical procedure of choice. We conducted a retrospective review of clinical presentation and surgical management of adult choledochal cysts. METHODS: Thirty-two patients with choledochal cysts who underwent surgery between April 1991 and January 2005 were reviewed. There were 27 Todani Type I, 2 Type II, 2 Type IVA and 1 Type V cysts. Eighty-four per cent of patients underwent complete cystectomy and hepaticojejunostomy. Seven patients had revision surgery comprising completion cystectomy and hepaticojejunostomy. RESULTS: There were no perioperative mortalities. Perioperative morbidity rate was 44% and the commonest complication perioperatively was wound infection (19%). Malignancy was noted in one histological specimen. This patient was disease free for 1 year postoperatively and was subsequently lost to follow up. No further malignancy was found on median follow up of 3.9 years (range, 1-14 years) for the other 31 patients. CONCLUSION: Adult choledochal cysts are rare and are often non-specific in their clinical presentation. In managing patients with choledochal cysts, it is important to first treat complications such as sepsis and pancreatitis before imaging of the biliary tree with endoscopic retrograde cholangiopancreatography or magnetic resonance cholangiopancreatography to evaluate the full extent and type of choledochal cyst. Surgical management should be planned single-stage surgery comprising complete cyst resection, cholecystectomy and Roux-en-Y hepaticojejunostomy and should be carried out by hepatobiliary specialists. Excellent perioperative morbidity and mortality results are possible with this strategy. Malignancy is rare and was only noted in 3% but close follow up is warranted.

Factors affecting early mortality in spontaneous rupture of hepatocellular carcinoma.

BACKGROUND: Spontaneous rupture of hepatocellular carcinoma (HCC) is a catastrophic surgical emergency with high mortality rates. The aim of this study is to determine the factors associated with the prognosis and to assess the outcome of different management strategies. METHODS: A retrospective study of 34 consecutive patients with spontaneous rupture of HCC was conducted from January 1996 to January 2004. Clinical, biochemical and operative factors influencing 30-day mortality were analysed. RESULTS: In our study, 30-day mortality rate was 32% (n = 11). Presence of cirrhosis, Child's C status, shock on admission, higher blood transfusion requirement, raised alpha-fetoprotein, raised alkaline phosphatase, raised aspartate transaminase, and raised indocyanine green at 15 min were all associated with increased risk of 30-day mortality on univariate analysis (P < 0.05). On multivariate analysis, only shock on admission (P = 0.001) and higher blood transfusion requirement (P = 0.01) were significant independent factors affecting early mortality. Surgical intervention was associated with a better 30-day survival as compared with medical therapy or transarterial embolization. The median survival time of patients undergoing curative resection was significantly longer than that of patients who had surgery for haemostasis only (420 vs 205 days). The overall median survival was 161 days. CONCLUSIONS: Spontaneous rupture of HCC is a potentially salvageable complication of HCC. Poor prognosis is associated with poor liver reserve, advanced disease and severity of haemorrhage. Shock and blood transfusion requirement are the only independent factors affecting early mortality.