Liver Biopsy

Liver biopsy is recommended as the gold standard method to determine diagnosis, fibrosis staging, prognosis and therapeutic indications in patients with chronic liver disease. However, liver biopsy is an invasive procedure with a risk of complications which can be serious. This book provides the management of the complications in liver biopsy. Additionally, this book provides also the references for the new technology of liver biopsy including the non-invasive elastography, imaging methods and blood panels which could be the alternatives to liver biopsy. The non-invasive methods, especially the elastography, which is the new procedure in hot topics, which were frequently reported in these years. In this book, the professionals of elastography show the mechanism, availability and how to use this technology in a clinical field of elastography. The comprehension of elastography could be a great help for better dealing and for understanding of liver biopsy

Non-alcoholic fatty liver disease in type-2 diabetes mellitus: population analysis, metabolic profile and referral management pathway

Introduction: Non-alcoholic fatty liver disease is strongly associated with type-2 diabetes mellitus, with diabetic patients being at higher risk for adverse outcomes. The aim of this thesis was to explore in detail the clinical and metabolic phenotype of diabetics screened for NAFLD in primary care and to develop a referral management pathway for this population. Moreover, this thesis investigated the impact of alterations of the gut-liver axis on the severity of liver disease in such cohort. Methods: In this cross-sectional study, consecutive diabetic patients from primary care were screened for liver disease and NAFLD. Nuclear magnetic resonance and liquid chromatography-mass spectrometry were used to explore the metabolic profile of the patients against severity of liver disease. Stool meta-taxagenomics allowed for the analysis of the composition of the microbiome, while gut permeability was investigated using an in-vitro model and an ex-vivo measurement of faecal protease activity. Inflammatory cytokines profile was also analysed in serum as well as in faecal samples. Results: Clinically significant NAFLD was highly prevalent in the diabetic population in primary care. According to the results of this study, applying FIB-4 with a cut-off of 1.3 in this population would miss up to 38% of the patients with significant liver disease. The BIMAST score, which was derived based on simple clinical parameters, was validated both internally and externally, outperformed conventional screening methods and optimised risk-stratification in primary care. Among the metabolites, only lysine deficiency was associated with increased hepatic collagen content. Moreover, specific changes in gut microbiome were associated with more severe liver disease, while intestinal permeability tended to increase with liver disease severity. A combination of host and microbiota-related factors were associated with a leakier gut in this population. Conclusions: Current risk-stratification for NAFLD among diabetics in primary care can be improved. Exploring the gut-liver axis may offer diagnostic as well as therapeutical insights in this population.Open Acces

Clinical Utilities of Transient Elastography

Chronic liver disease causes 1.75 million deaths globally and is within the top 10 leading causes of death in middle income countries. Chronic liver injury occurs via a process of inflammation and fibrosis formation. Patients often do not present to healthcare until advanced stages of disease and when there is already decompensated cirrhosis. Liver biopsy has been used to identify earlier stages of fibrosis, but it is poorly accepted by patients and has limitations. Transient Elastography (TE) using Fibroscan ® is a non-invasive tool for the diagnosis liver fibrosis. The clinical application of Fibroscan in non-alcoholic fatty disease (NAFLD), chronic hepatitis B (CHB), and methotrexate induced liver fibrosis were examined. Clinical Utility of Transient Elastography in non-alcoholic fatty liver disease: Chapters 2, 3 & 4 Non-alcoholic fatty liver disease affects 20-35% of the global population, but only a small subset develop the histological subtype of non-alcoholic steatohepatitis (NASH), which can lead to progressive liver disease by causing fibrosis and eventually cirrhosis. Fibroscan can potentially identify those patients who have fibrosis and are at increased risk of further progression. Patients with type 2 diabetes, who are at high risk of NASH, were assessed. A liver stiffness measurement (LSM) ≥9.8 kPa, used as a cut-off for advanced fibrosis (1), was found in 12% (10/77) of subjects. Higher LSM readings correlated with higher BMI and the use of insulin therapy. Patients on insulin had LSM ≥9.8 kPa with likelihood ratio (LR): 12.3, p=0.002 (Chapter 2). The study was limited by a small sample size, and a high failure rate as the medium (M) probe was only available. A systemic review evaluating all non-invasive methods for diagnosing NASH and NAFLD fibrosis was undertaken. This included a meta-analysis that focused on what was found to be the most widely studied markers of NASH and NAFLD fibrosis: cytokeratin-18 (CK-18) fragments and transient elastography respectively (Chapter 3). Not only was TE found to be the most extensively studied, it had also one of the highest diagnostic accuracies with pooled sensitivities and specificities to diagnose F≥2, 3 and 4 to be: 79% and 75%, 85% and 85%, and 92% and 92% respectively. We then proceeded to perform a much larger study in diabetic subjects using the latest generation of Fibroscan ® 502 touch model (Chapter 4). This included the extra-large (XL) probe for obese subjects and also featured the novel Controlled Attenuation Parameter (CAP), which assesses liver steatosis. A total of 1918 diabetes patients at Prince of Wales Hospital, Hong Kong were recruited. Each had a TE and CAP to assess liver stiffness and steatosis. Reliable scans were achieved in 98.2% of patients using the M or XL probes. The proportion of patients with increased CAP (suggestive of steatosis) and increased LSM (suggestive of advanced fibrosis) were 72.8% and 17.7% respectively. By multivariate analysis, female gender, higher body mass index, triglycerides, fasting plasma glucose and alanine aminotransferase, and non-insulin use were associated with increased CAP. Longer duration of diabetes, higher body mass index, alanine aminotransferase, spot urine albumin-creatinine ratio, and lower high-density lipoprotein-cholesterol were associated with increased LSM. The17.7% prevalence of advanced fibrosis suggests type 2 diabetic patients would benefit from routine screening for liver disease. Clinical Utility of Transient Elastography in chronic hepatitis B: Chapters 5 and 6 Transient elastography was initially applied for staging patients with chronic hepatitis C (CHC) with data rapidly growing on its utility for the assessment in patients with CHB infection. Our study contributes to this by further evaluating the diagnostic accuracy and usefulness of TE, and also comparing its performance against the FIB4 index, Aspartate Platelet Ratio Index (APRI), Aspartate Alanine Aminotransferase Ratio (AAR), Age Platelet Index (API), Fibrosis Index (FI) and Caffeine Breath Test (CBT) (Chapter 5). In 71 CHB patients, the diagnostic performance of the LSM for Metavir fibrosis stage F≥1, 2, 3 and 4 were: Area under Receiver Operator Characteristic (AUROC) = 0.825, 0.792, 0.874 and 0.945 respectively. Patients with high ALT required higher LSM cut-offs. Dual cut-offs are needed to “rule in” and to “rule out” stage of fibrosis with a high level of certainty. Using normal vs high ALT specific cut-offs, F≥2 and F≥3 can be “ruled in” or “ruled out” with certainty in 49.3% and 57.7% of CHB patients respectively. TE was the superior non-invasive test when compared with FIB4-I, APRI, API, AAR and FI. Caffeine breath test compared well against TE in a small cohort, but is not as practical. Liver histology is limited by interobserver variability, with 44% of liver biopsies being classified a different stage on second evaluation, and the intraclass correlation coefficient showing moderate agreement (K =0.457). Although routinely compared, this highlights the limitations of assessing the accuracy of TE and other non-invasive tests against a reference standard that has such a degree of variation. The use of TE in the longitudinal monitoring of fibrosis is important in the follow up of patients with CHB (Chapter 6). Current literature was conflicting and seemed to suggest that decline in LSM was influenced more by the fall in ALT with decline in necroinflammatory activity, rather than fibrosis regression. We sought to evaluate the factors that affected LSM change and assess which clinical subgroups experienced an LSM decline. In 124 CHB patients who were followed for 31.2 months (SD 13.1), LSM decline was greatest in those who had active disease and were subsequently treated with antivirals. This is associated with ALT normalization, HBeAg seroconversion and viral suppression. In CHB patients with quiescent disease - ie did not require antiviral treatment, or who had persistently normal ALT irrespective of treatment - only a small or non-significant decline in LSM was observed. The change in LSM was strongly correlated with length of time and may suggest fibrosis regression. Further studies are required, as our findings are limited by a lack of correlation with liver biopsy, and the low baseline levels of liver stiffness in those with inactive CHB. Clinical Utility of Transient Elastography in methotrexate induced liver fibrosis: Chapter 7 Long term use of methotrexate has been associated with risk of liver fibrosis and the role of TE in this cohort was evaluated. The relationship between liver fibrosis and methotrexate dose, and other factors associated with moderate fibrosis (F2) using an LSM cut-off of ≥7.1 kPa were examined. In 39 patients with a mean intake dose of 5.3g of methotrexate, no correlation was found between the LSM and the cumulative dose or duration of treatment. Of the 7/39 cases of LSM≥7.1 kPa (17.9%), BMI≥30 was the only risk factor with a likelihood ratio (LR) of 4.442, p=0.029. One patient had cirrhosis (2.6%). This is much lower than rates reported from early studies [26% (2, 3)], and more in line with recent data [around 2% (4)], and lends support to the suggestion that early studies overestimated the risk of methotrexate induced fibrosis due to lack of controls for pre-existing liver disease (5). There was also no difference in the LSM of methotrexate subjects and matched population controls. Conclusion Our studies lend further support to the utility of LSM on identifying those at increased risk of liver fibrosis progression, which will continue to remain a significant clinical challenge in both individuals and as a public health burden. In particular we feel that major contributions have been made on the subject of screening for advanced fibrosis in a high-risk population of type II diabetic patients. Our longitudinal studies on the role of using TE in follow up and comparing its performance in CHB patients are also significant. Despite the small cohort of methotrexate users, this further supports the utility of TE in a wide range of liver diseases that manifest with progressive fibrosis. The next area of further development in the clinical use of TE is as a stand-alone marker that has prognostic significance

Novel markers of liver fibrosis

With chronic liver disease rising, the need to stage of liver disease and fibrosis accurately is paramount as it helps guide therapy and informs prognosis. Liver biopsy is a flawed gold standard, associated with morbidity and mortality. Application of simple non-invasive tests to assess fibrosis could provide a safe way of identifying patients in greatest need of intervention and of monitoring response to therapy. I have shown in this thesis that transient elastography is an excellent tool for ruling out significant fibrosis in patients with chronic liver disease. It is easy to learn and successful scanning correlates well with histological liver fibrosis. I have also shown that Use of APRI with a cut off of >1.5-2 and Fib-4 >3.25 can provide prognostic value for overall and liver-related mortality in patients with viral hepatitis. Finally I have assessed a range of potential new biomarkers showing that combining measuring serum levels of the chemokine CXCL10 and the endothelial adhesion receptor VAP-1 can increase the correlation strength with fibrosis stage. Using morphometric analysis of liver fibrosis I show that the same markers can be linked to quantitatively measured fibrosis, removing subjective bias and reducing inter and intra-operator variance in histological assessment

Primary sclerosing cholangitis: Surrogate markers of natural history, disease severity, and prognosis

Sammendrag Bakgrunn: Primær skleroserende kolangitt (PSC) er en immunrelatert leversykdom av ukjent årsak som karakteriseres av kolestase, inflammasjon og strikturer i galletreet og vanligvis utvikler seg til generell leverfibrose, cirrhose og endestadium leversykdom. Til tross for at tilstanden er sjelden, har den i flere tiår vært den vanligste årsaken til levertransplantasjoner i Norge. Sykdomsforløpet er høyst varierende og uforutsigbart. Mangelen på etablerte biomarkører til stratifisering av risiko og sykdomsaktivitet er det største hinderet for å utvikle effektiv behandling. Følgelig er nye biomarkører sterkt etterspurt for å bedre pasientseleksjon og effektmåling i kliniske studier. Mål: Målsetningen for studien var å karakterisere prognostiske biomarkører ved PSC og identifisere potensielle nye biomarkører. Vi har derfor vurdert variasjon over tid innen og mellom personer med PSC for dagens to mest lovende prediktive markører, «enhanced liver fibrosis test» (ELF) og leverstivhetsmålinger (LSM) (Artikkel I). I tillegg ønsket vi å undersøke om et panel med flere biomarkører ga bedret prediktiv verdi ved PSC sammenlignet med nåværende kliniske risikoscorer og enkeltmarkører (Artikkel II). Til slutt har vi studert markører for mitokondriefunksjon ved PSC (Artikkel III). Metoder: I Artikkel I ble det brukt en longitudinell blandet modell for å analysere ELF og LSM ved skjærebølge-elastografi i repeterte målinger fra et prospektivt pasientpanel med 113 pasienter fra Bergen og Oslo. I Artikkel II brukte vi elastisk nettverk og multivariat regresjon for å identifisere et prognostisk multimarkørpanel for PSC, basert på tverrsnittsdata fra et retrospektivt panel med 138 personer med PSC fra NoPSC biobank. I Artikkel III utførte vi omfattende analyser av lipidomsetning og anvendte romlig regresjonsanalyse. Her gjorde vi tverrsnittsanalyser av markører på mitokondriefunksjon i plasma fra 191 pasienter og 100 friske kontroller, samt levervev fra personer med PSC og ikke-kolestatiske leversykdommer som kontrollgruppe fra NoPSC biobank. Resultater: I Artikkel I fant vi en signifikant økning av ELF og LSM over tid, men subgruppeanalyse viste at økningen kun forekom i gruppen med høy ALP. Fem år fra baseline hadde ca. 30 til 40% av pasientene reduksjon i LSM og ELF. En undergruppe på 10% av pasientene viste samvariasjon med reduksjon i ELF, LSM og ALP, og understrekker behovet for bedre forståelse og definisjon av hva som utgjør klinisk signifikant reduksjon. Effekter mellom pasienter forklarte 78% av variasjonen i ELF og 56% av variasjonen i LSM, og foreslår at ELF muligens har bedre evne for risikostratifisering sammenlignet med LSM. I Artikkel II illustrerte vi hvordan prognostiske biomarkører i PSC dannet tre grupper med tett korrelerte variabler. Vi demonstrerte at et panel bestående av biomarkører fra ulike deler av patogenesen i PSC hadde den den beste prediktive evnen, det vil si fibrose (ELF), inflammasjon (kynurenin-tryptofan ratio; KT-ratio) og en mikrobiell metabolitt (pyridoxal 5’-fosfat; PLP). I Artikkel III viste vi at det er uttalte forskjeller i fettsyreprofilen i plasma ved PSC sammenlignet med friske kontroller, inkludert økning av enumettede fettsyrer (MUFA), reduksjon av langkjedete mettede fettsyrer (SFA), total n-3 og n-6 flerumettede fettsyrer (PUFA). Funnene våre indikerte at mitokondriell dysfunksjon er fremtredende ved PSC og mer uttalt med økende kolestase og sykdomsstadium. Konklusjon: Våre funn understreker behovet for å forstå variasjonen i biomarkører ved PSC og å etablere klare definisjoner av hva som er klinisk signifikante endringer. Videre har vi vist at det er mulig å bedre prediksjonen ved å kombinere biomarkører fra ulike sykdomsprosesser ved PSC. Dette fordrer videre studier i større og uavhengige pasientpaneler. Til slutt har vi vist endringer i lipidomsetning og mitokondriefunksjon, som gir et behov for videre studier for utforsking av biomarkører og mulige behandlingsmål.  Abstract Background: Primary sclerosing cholangitis (PSC) is an immune-associated liver disease of unknown aetiology characterized by cholestasis, inflammation, and stricturing of the biliary tree, which typically progresses to general liver fibrosis, cirrhosis, and end-stage liver disease. Despite its status as a rare disease, it has been the leading cause of liver transplantation in Norway for decades. The disease course is highly variable and notoriously unpredictable. The lack of established biomarkers to stratify risk and assess disease activity is a major hurdle to developing effective therapy. Hence, new biomarkers are highly warranted to improve patient selection and effect assessment in clinical trials. Aims: The objective of the present study was to further characterize biomarkers of prognosis in PSC and explore novel potential biomarkers. Thus, we aimed to evaluate the within- and between-patient variability over time in PSC for the two currently most promising predictive markers, the enhanced liver fibrosis test (ELF) and liver stiffness measurement (LSM) (Paper I). Moreover, we aimed to identify a panel of multiple biomarkers with improved predictive abilities in people with PSC compared to current clinical risk scores or single biomarkers (Paper II). Lastly, we aimed to study markers of mitochondrial function in PSC (Paper III). Methods: In Paper I, we applied a longitudinal mixed model to analyse ELF and LSM by point shear wave elastography in repeated measurements from a prospective patient panel of 113 patients from Bergen and Oslo. In Paper II, we used elastic net- and multivariate regression to identify a prognostic multimarker panel for PSC based on cross-sectional, retrospective data from a panel of 138 PSC patients from the NoPSC biobank. In Paper III, we performed comprehensive lipidomic analyses and applied spatial regression. Here we explored markers of mitochondrial function cross-sectionally in plasma from 191 patients and 100 healthy controls and in liver tissue from people with PSC and non-cholestatic liver disease controls from the NoPSC biobank. Main findings: In Paper I, we found a significant increase in ELF and LSM over time, which was restricted to the high-ALP group (>1.5 x upper limit of normal) in subgroup analysis. Five years from baseline, about 30 to 40% of patients had reduced LSM and ELF values. A subgroup of 10% of patients showed a concomitant decrease in ELF, LSM, and ALP, underscoring the need to understand and define a clinically significant reduction. Between-patient effects explained 78% of ELF variation and 56% of LSM variation, suggesting that ELF may have superior reliability for risk stratification compared to LSM. In Paper II, we illustrated how prognostic biomarkers proposed in PSC seemed to form three groups of tightly intercorrelated variables. We demonstrated the best predictive ability in a panel consisting of biomarkers reflecting different aspects of PSC pathogenesis, i.e., fibrosis (ELF), inflammation (kynurenine-tryptophan ratio; KT-ratio), and a microbiota metabolite (pyridoxal 5’-phosphate; PLP). In Paper III, we demonstrated extensive differences in fatty acid profile in plasma from PSC patients compared to healthy controls, including increased mono-unsaturated fatty acids (MUFA), decreased long-chain saturated fatty acids (SFA), total n-3 and n-6 polyunsaturated fatty acids (PUFA). Moreover, our findings clearly indicated mitochondrial dysfunction as a prominent feature in PSC, which was more pronounced with increasing cholestasis and disease stage. Conclusion: Our findings underscore the need to understand the variation in biomarkers in PSC and establish a definition of clinically significant change. Furthermore, we have demonstrated the potential to improve the predictive abilities in PSC by combining biomarkers reflecting several pathogenic processes, warranting further studies in large and independent patient panels. Finally, we demonstrated lipidomic changes and mitochondrial dysfunction, which need further exploration to identify biomarkers or putative therapeutic targets.Doktorgradsavhandlin

Metabolic dysfunction-associated steatotic liver disease:A wide-angled perspective on a multifaceted problem

In dit proefschrift is metabole dysfunctie-geassocieerde steatotische leverziekte (MASLD) onderzocht, opgedeeld in drie delen:Deel 1 omvat een MRI-onderzoek naar vetstapeling in de lever en pancreas, en een genoombrede associatiestudie in de Amsterdamse multi-etnische populatie, waarbij een relatie tussen het gen MRC1 en niet-invasieve leverfibrosetesten wordt gevonden. Dit gen vertoont variaties tussen etnische groepen, wat wijst op een rol van MRC1 in de bestaande verschillen in MASLD tussen bevolkingsgroepen van verschillende afkomst.In deel 2 is gericht op nieuwe niet-invasieve levertesten van fibrosevorming in mensen met MASLD. Ten eerste een systematische review van de marker Pro-C3 voor het detecteren van fibrose, en ten tweede een onderzoek naar een nieuw niet-invasief biomarkerpanel, van ontdekking in muisstudies tot bevestiging in humane cohorten.Deel 3 van het proefschrift beschrijft de potentie van het darmmicrobioom om MASLD te beïnvloeden. Een fecestransplantatiestudie toont aan dat het manipuleren van het darmmicrobioom leidt tot veranderingen in circulerende metabolieten en lever-DNA-methylatie. Daarnaast beschrijft een muisonderzoek het effect van de boterzuurproducerende bacterie A. soehngenii op de ernst van MASLD, waarbij toediening ervan de suikerhuishouding verbeterde zonder verbetering van de leverhistologie.Gezamenlijk leveren de studies die in dit proefschrift beschreven zijn waardevolle inzichten in de complexiteit van MASLD en bieden ze verschillende potentiële mogelijkheden om de zorg voor mensen met MASLD te verbeteren door middel van genetische, metabole en microbioom-gerichte benaderingen

FUNCTIONAL LIVER TESTS IN LIVER DISEASE

The liver is subject to several insults, whether from ingested chemicals, pathogens or genetic diseases. Many of these are transient and full function returns. However, chronic injury may lead to scarring, the volume and effect of which may be viewed along a spectrum inclusive of fibrosis, cirrhosis and eventually death. Several complications may arise from this process, including decompensation and hepatocellular carcinoma. Recognising this damage in a timely, objectively reproducible and, most importantly, safe manner is clinically critical and great strides in this regard have recently been made. Several non-invasive technologies have been produced, the most ubiquitous being transient elastography (TE), for detecting fibrosis. To measure the actual function of the remaining liver, indocyanine green excretion allows for direct and minimally-invasive testing. This thesis focusses on the use of non-invasive testing to identify significant liver disease in a variety of different disorders. I have studied clinically relevant changes in liver fibrosis and function and related these to conventional liver function tests in an attempt to develop clinically useful markers of liver function that can be used by clinicians to risk stratify patients at risk of liver disease. Hepatitis C treatment has recently been revolutionised with the introduction of direct-acting antivirals, not only allowing a greater level of treatment success but also widening those patients we could treat to include those with cirrhosis. However, the benefits of therapy to those with advanced fibrosis are unclear and it is not established which patients are likely to undergo functional recovery following viral clearance nor is it clear which patients are at greatest risk of liver cancer. We studied liver fibrosis and indocyanine green excretion in a cohort of patients with advanced fibrosis and here I show that patients (n=43) with mild functional impairment (ICGR15<20.9%) are likely to recover but others are unlikely to undergo significant functional recovery. I studied the massive HCV Research UK database to identify risk factors for post-treatment development of HCC and I showed that virological treatment failure and pre-existing liver lesions predispose to future development of liver cancer. Recent medical advances have improved the prognosis for patients with cystic fibrosis and sickle cell disease. This improved life expectancy has unveiled new long-term complications of these disorders, including liver disease. To investigate the prevalence of liver disease in adults with these genetic disorders I conducted two clinical audits using non-invasive testing and here I show that the use of a TE in a sickle cell disease clinic adds value and identifies people with liver fibrosis. However, in an adult cystic fibrosis clinic the use of TE was of limited value as some patients with evident fibrosis were not detected by this technique and other diagnostic criteria need to be applied. In summary this work has identified prognostic factors for patients with hepatitis C and cirrhosis who have undergone effective antiviral therapy and demonstrated the value of non-invasive testing in haematological conditions whilst identifying weaknesses in the use of TE in those with cystic fibrosis