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

Liver Elastography in Primary Sclerosing Cholangitis Patients Using Three Different Scanner Systems

The aim of the study described here was to characterize three different liver elastography methods in primary sclerosing cholangitis (PSC) patients, for the first time exploring 2-D shear wave elastography (2-D-SWE) in PSC patients and its putative advantages over point shear wave elastography (pSWE). Sixty-six adult PSC patients (51 males, 77%) underwent liver elastography: Transient elastography (TE), pSWE and 2-D-SWE were applied head-to-head after B-mode ultrasonography and blood tests. Liver stiffness measurements (LSMs) by pSWE yielded lower values than those by TE; 2-D-SWE had less steep slope but was overall not significantly different from TE. Correlation between LSMs by pSWE and TE was excellent (intraclass correlation coefficient = 0.92); correlation for 2-D-SWE with either pSWE or TE was moderate but improved with exclusion of overweight individuals. LSMs correlated with the Enhanced Liver Fibrosis test (ELF) across all scanner systems. Our study indicates that LSM by different systems is feasible in PSC patients and that 2-D-SWE tends to underestimate stiffness compared with TE.publishedVersio

Aqp 9 and Brain Tumour Stem Cells

Several studies have implicated the aquaporins (aqp) 1, 4, and 9 in the pathogenesis of malignant brain tumours, suggesting that they contribute to motility, invasiveness, and oedema formation and facilitate metabolism in tumour cells under hypoxic conditions. We have studied the expression of aqp1, 4, and 9 in biopsies from glioblastomas, isolated tumour stem cells grown in a tumoursphere assay and analyzed the progenitor and differentiated cells from these cultures. We have compared these to the situation in normal rat brain, its stem cells, and differentiated cells derived thereof. In short, qPCR in tumour tissue showed presence of aqp1, 4, and 9. In the tumour progenitor population, aqp9 was markedly more highly expressed, whilst in tumour-derived differentiated cells, aqp4 was downregulated. However, immunostaining did not reveal increased protein expression of aqp9 in the tumourspheres containing progenitor cells; in contrast, its expression (both mRNA and protein) was high in differentiated cultures. We, therefore, propose that aquaporin 9 may have a central role in the tumorigenesis of glioblastoma

Fluctuating biomarkers in primary sclerosing cholangitis: A longitudinal comparison of alkaline phosphatase, liver stiffness, and ELF

Background & Aims Primary sclerosing cholangitis (PSC) is a progressive liver disease characterised by fluctuating liver biochemistries and highly variable disease progression. The Enhanced Liver Fibrosis (ELF®) test and liver stiffness measurements (LSMs) reflect fibrosis and predict clinical outcomes in PSC; however, longitudinal assessments are missing. We aimed to characterise the systematic change in ELF and LSM over time in a prospective cohort of patients with PSC, along with their longitudinal relationship to alkaline phosphatase (ALP) and bilirubin. Methods We included 113 non-transplant PSC patients (86 males [76.1%]; mean age 43.3 ± 15.7 years) with annual study visits between 2013 and 2019 at 2 Norwegian centres. ELF test, LSM, clinical data, liver biochemistries, and revised Mayo risk score were measured. We used linear mixed-effects models to estimate change over time, intraclass correlations (ICCs), and their relationship with ALP and bilirubin. Results At baseline, the median (range) ELF test was 9.3 (7.5–12.9) and median LSM 1.26 m/s (0.66–3.04 m/s). ELF and LSM increased over time (0.09 point/year, 95% CI [0.03, 0.15], p = 0.005, vs. 0.12 point/year, 95% CI [0.03, 0.21], p = 0.009). Between-patient effects explained 78% of ELF variation (ICC 0.78) and 56% of LSM variation (ICC 0.56). ALP also increased and showed the highest ICC (0.86). Conclusions ELF and LSM increased over a 5-year period. Longitudinal analyses demonstrated differences regarding within- and between-patient effects, suggesting that the ELF test may have superior reliability for risk stratification compared with LSM in PSC.publishedVersio

Liver Elastography in Primary Sclerosing Cholangitis Patients Using Three Different Scanner Systems

The aim of the study described here was to characterize three different liver elastography methods in primary sclerosing cholangitis (PSC) patients, for the first time exploring 2-D shear wave elastography (2-D-SWE) in PSC patients and its putative advantages over point shear wave elastography (pSWE). Sixty-six adult PSC patients (51 males, 77%) underwent liver elastography: Transient elastography (TE), pSWE and 2-D-SWE were applied head-to-head after B-mode ultrasonography and blood tests. Liver stiffness measurements (LSMs) by pSWE yielded lower values than those by TE; 2-D-SWE had less steep slope but was overall not significantly different from TE. Correlation between LSMs by pSWE and TE was excellent (intraclass correlation coefficient = 0.92); correlation for 2-D-SWE with either pSWE or TE was moderate but improved with exclusion of overweight individuals. LSMs correlated with the Enhanced Liver Fibrosis test (ELF) across all scanner systems. Our study indicates that LSM by different systems is feasible in PSC patients and that 2-D-SWE tends to underestimate stiffness compared with TE

Fluctuating biomarkers in primary sclerosing cholangitis: A longitudinal comparison of alkaline phosphatase, liver stiffness, and ELF

Background & Aims Primary sclerosing cholangitis (PSC) is a progressive liver disease characterised by fluctuating liver biochemistries and highly variable disease progression. The Enhanced Liver Fibrosis (ELF®) test and liver stiffness measurements (LSMs) reflect fibrosis and predict clinical outcomes in PSC; however, longitudinal assessments are missing. We aimed to characterise the systematic change in ELF and LSM over time in a prospective cohort of patients with PSC, along with their longitudinal relationship to alkaline phosphatase (ALP) and bilirubin. Methods We included 113 non-transplant PSC patients (86 males [76.1%]; mean age 43.3 ± 15.7 years) with annual study visits between 2013 and 2019 at 2 Norwegian centres. ELF test, LSM, clinical data, liver biochemistries, and revised Mayo risk score were measured. We used linear mixed-effects models to estimate change over time, intraclass correlations (ICCs), and their relationship with ALP and bilirubin. Results At baseline, the median (range) ELF test was 9.3 (7.5–12.9) and median LSM 1.26 m/s (0.66–3.04 m/s). ELF and LSM increased over time (0.09 point/year, 95% CI [0.03, 0.15], p = 0.005, vs. 0.12 point/year, 95% CI [0.03, 0.21], p = 0.009). Between-patient effects explained 78% of ELF variation (ICC 0.78) and 56% of LSM variation (ICC 0.56). ALP also increased and showed the highest ICC (0.86). Conclusions ELF and LSM increased over a 5-year period. Longitudinal analyses demonstrated differences regarding within- and between-patient effects, suggesting that the ELF test may have superior reliability for risk stratification compared with LSM in PSC