The mutational landscape of human somatic and germline cells.

Over the course of an individual's lifetime, normal human cells accumulate mutations1. Here we compare the mutational landscape in 29 cell types from the soma and germline using multiple samples from the same individuals. Two ubiquitous mutational signatures, SBS1 and SBS5/40, accounted for the majority of acquired mutations in most cell types, but their absolute and relative contributions varied substantially. SBS18, which potentially reflects oxidative damage2, and several additional signatures attributed to exogenous and endogenous exposures contributed mutations to subsets of cell types. The rate of mutation was lowest in spermatogonia, the stem cells from which sperm are generated and from which most genetic variation in the human population is thought to originate. This was due to low rates of ubiquitous mutational processes and may be partially attributable to a low rate of cell division in basal spermatogonia. These results highlight similarities and differences in the maintenance of the germline and soma.

Parallel single-cell sequencing links transcriptional and epigenetic heterogeneity.

We report scM&T-seq, a method for parallel single-cell genome-wide methylome and transcriptome sequencing that allows for the discovery of associations between transcriptional and epigenetic variation. Profiling of 61 mouse embryonic stem cells confirmed known links between DNA methylation and transcription. Notably, the method revealed previously unrecognized associations between heterogeneously methylated distal regulatory elements and transcription of key pluripotency genes.

G&T-seq: parallel sequencing of single-cell genomes and transcriptomes.

The simultaneous sequencing of a single cell's genome and transcriptome offers a powerful means to dissect genetic variation and its effect on gene expression. Here we describe G&T-seq, a method for separating and sequencing genomic DNA and full-length mRNA from single cells. By applying G&T-seq to over 220 single cells from mice and humans, we discovered cellular properties that could not be inferred from DNA or RNA sequencing alone.

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.

International validation of the Chinese university prognostic index for staging of hepatocellular carcinoma: a joint United Kingdom and Hong Kong study.

The outcome of hepatocellular carcinoma (HCC) patients significantly differs between western and eastern population centers. Our group previously developed and validated the Chinese University Prognostic Index (CUPI) for the prognostication of HCC among the Asian HCC patient population. In the current study, we aimed to validate the CUPI using an international cohort of patients with HCC and to compare the CUPI to two widely used staging systems, the Barcelona Clinic Liver Cancer (BCLC) classification and the Cancer of the Liver Italian Program (CLIP). To accomplish this goal, two cohorts of patients were enrolled in the United Kingdom (UK; n = 567; 2006-2011) and Hong Kong (HK; n = 517; 2007-2012). The baseline clinical data were recorded. The performances of the CUPI, BCLC, and CLIP were compared in terms of a concordance index (C-index) and were evaluated in subgroups of patients according to treatment intent. The results revealed that the median follow-up durations of the UK and HK cohorts were 27.9 and 29.8 months, respectively. The median overall survival of the UK and HK cohorts were 22.9 and 8.6 months, respectively. The CUPI stratified the patients in both cohorts into three risk subgroups corresponding to distinct outcomes. The median overall survival of the CUPI low-, intermediate-, and high-risk subgroups were 3.15, 1.24, and 0.29 years, respectively, in the UK cohort and were 2.07, 0.32, and 0.10 years, respectively, in the HK cohort. For the patients who underwent curative treatment, the prognostic performance did not differ between the three staging systems, and all were suboptimal. For those who underwent palliative treatment, the CUPI displayed the highest C-index, indicating that this staging system was the most informative for both cohorts. In conclusion, the CUPI is applicable to both western and eastern HCC patient populations. The performances of the three staging systems differed according to treatment intent, and the CUPI was demonstrated to be optimal for those undergoing palliative treatment. A more precise staging system for early-stage disease patients is required.

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.

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.

Octreotide and the mTOR inhibitor RAD001 (everolimus) block proliferation and interact with the Akt-mTOR-p70S6K pathway in a neuro-endocrine tumour cell Line.

BACKGROUND/AIM: The mode of action of the somatostatin analog octreotide on neuro-endocrine tumour proliferation is largely unknown. Overexpression of the proto-oncogene Akt/PKB (protein kinase B) has been demonstrated in certain neuro-endocrine tumours: Akt activates downstream proteins including mTOR and p70S6K, which play an important role in cell proliferation. RAD001 (everolimus) is a novel agent that is being trialled in the treatment of neuro-endocrine tumours, and is known to interact with mTOR. We explored the mechanism of action of octreotide, RAD001, and their combination on cell proliferation and kinase activation in a neuro-endocrine tumour cell line (rat insulinoma cell line, INS1). METHODS: Proliferation assays were used to determine the effects of octreotide, RAD001, and their combination on cell proliferation. Western blotting was used to characterize the expression of phosphorylated Akt, phosphorylated TSC2, phosphorylated mTOR, and phosphorylated 70S6K. RESULTS: Treatment with octreotide and RAD001 inhibited proliferation and attenuated phosphorylation of all downstream targets of Akt: TSC2, mTOR, and p70S6K. CONCLUSIONS: In this cell model, octreotide and RAD001 appear to act through a similar pathway and inhibit the Akt-mTOR-p70S6 kinase pathway downstream of Akt. There may be some overlapping effects of the two inhibitors on the mTOR pathway, although it is likely that other additional effects may differentiate the two agents.

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.

Assessment of liver function in patients with hepatocellular carcinoma: a new evidence-based approach-the ALBI grade.

PURPOSE: Most patients with hepatocellular carcinoma (HCC) have associated chronic liver disease, the severity of which is currently assessed by the Child-Pugh (C-P) grade. In this international collaboration, we identify objective measures of liver function/dysfunction that independently influence survival in patients with HCC and then combine these into a model that could be compared with the conventional C-P grade. PATIENTS AND METHODS: We developed a simple model to assess liver function, based on 1,313 patients with HCC of all stages from Japan, that involved only serum bilirubin and albumin levels. We then tested the model using similar cohorts from other geographical regions (n = 5,097) and other clinical situations (patients undergoing resection [n = 525] or sorafenib treatment for advanced HCC [n = 1,132]). The specificity of the model for liver (dys)function was tested in patients with chronic liver disease but without HCC (n = 501). RESULTS: The model, the Albumin-Bilirubin (ALBI) grade, performed at least as well as the C-P grade in all geographic regions. The majority of patients with HCC had C-P grade A disease at presentation, and within this C-P grade, ALBI revealed two classes with clearly different prognoses. Its utility in patients with chronic liver disease alone supported the contention that the ALBI grade was indeed an index of liver (dys)function. CONCLUSION: The ALBI grade offers a simple, evidence-based, objective, and discriminatory method of assessing liver function in HCC that has been extensively tested in an international setting. This new model eliminates the need for subjective variables such as ascites and encephalopathy, a requirement in the conventional C-P grade.

The detection of hepatocellular carcinoma using a prospectively developed and validated model based on serological biomarkers.

BACKGROUND: Hepatocellular carcinoma is a common complication of chronic liver disease (CLD), and is conventionally diagnosed by radiological means. We aimed to build a statistical model that could determine the risk of hepatocellular carcinoma in individual patients with CLD using objective measures, particularly serological tumor markers. METHODS: A total of 670 patients with either CLD alone or hepatocellular carcinoma were recruited from a single UK center into a case-control study. Sera were collected prospectively and specifically for this study. A logistic regression analysis was used to determine independent factors associated with hepatocellular carcinoma and a model built and assessed in terms of sensitivity, specificity, and proportion of correct diagnoses. RESULTS: The final model involving gender, age, AFP-L3, α fetoprotein (AFP), and des-carboxy-prothrombin ("GALAD") was developed in a "discovery" data set and validated in independent data sets both from the same institution and from an external institution. When optimized for sensitivity and specificity, the model gave values of more than 0.88 irrespective of the disease stage. CONCLUSIONS: The presence of hepatocellular carcinoma can be detected in patients with CLD on the basis of a model involving objective clinical and serological factors. It is now necessary to test the model's performance in a prospective manner and in a routine clinical practice setting, to determine if it may replace or, more likely, enhance current radiological approaches. IMPACT: Our data provide evidence that an entirely objective serum biomarker-based model may facilitate the detection and diagnosis of hepatocellular carcinoma and form the basis for a prospective study comparing this approach with the standard radiological approaches.

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.

Single-centre comparative study of laparoscopic versus open right hepatectomy.

BACKGROUND: Expansion 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. METHODS: Patients undergoing open right hepatectomy (ORH) were matched with patients undergoing laparoscopic right hepatectomy (LRH) and compared for perioperative outcomes. RESULTS: Seventy 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 < 0.0001). Intensive care unit (median, 2 days for LRH vs. 4 days for ORH; p < 0.0001) and postoperative length of stay (5 days for LRH vs. 9 days for ORH; p < 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. CONCLUSIONS: LRH can be a safe, effective, and oncologically efficient alternative to open resection in selected cases. Extensive experience in hepatic and laparoscopic surgery is required.