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MULTI-<i>OMIC</i> SEQUENCING REVEALS DISTINCTIVE GENE EXPRESSION AND DNA METHYLATION ALTERATIONS AS POTENTIAL PREDICTORS OF PRIMARY SCLEROSING CHOLANGITIS DEVELOPMENT IN TREATMENT-NAÏVE PAEDIATRIC ULCERATIVE COLITIS
Date
May 6, 2023
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Society: AASLD
Background The prevalence of chronic liver disease is increasing but it remains underdiagnosed, with frequent late presentation. Development in early adulthood is likely to associate with higher lifetime cirrhosis risk but early identification of asymptomatic patients can prove challenging. We have developed a novel case finding database1 that can rapidly search all blood test results in Somerset, UK (pop. 560,000) to identify young patients with persistently abnormal liver biochemistry. In this study we tested the hypotheses that a) prevalence of liver disease is increasing in young adults, b) these people are under investigated, c) our case finding database can identify them prior to the development of advanced fibrosis, allowing opportunity for treatment.
Methods Data within our case finding system was used to analyse results from patients born between 1/1/1980 – 31/12/1989 [blood results from 2005-2020 were available which covered age range 25-29: males 21,246, females 35,523]. The prevalence of abnormal ALTs and adequacy of subsequent investigation (with a non-invasive liver screen) was assessed. Where possible the system calculated a FIB-4 score.
Results For patients aged 25-29 the number of ALTs requested has increased over time. Comparing patients born in 1989 to those in 1980 there has been an 83% increase in tests, and a 63% increase in the number of abnormal results. The relationship was linear (for tests, R2=0.9749; p<10-6; for abnormal R2 = 0.8518; p=0.004). The number of patients with >2 abnormal results also increased (Fig 1; p=0.0005). However, there was no significant change in mean ALT (p=0.42) or proportion of tests which were abnormal (p=0.32) suggesting a true increase in disease prevalence.
Rates of follow up investigations for patients with persistently abnormal ALT (>40 for at least 90 days until last result) are shown in Table 1. For men and women respectively only 41% and 47% had sufficient viral serology and only 26% and 28% had an AST (allowing estimate of fibrosis). There were significantly more men than women (477 vs 176) because the laboratory definition of abnormal was used. Of those for whom calculation of FIB-4 was possible the result was low – mean 0.72 + 0.053 (men), 0.79 + 0.094 (women), excluding significant fibrosis in 94% and 87% respectively.
Conclusion The prevalence of liver disease in young adults in the UK is rising but is frequently under investigated. These patients mostly have early disease (as defined by FIB-4) and thus there is significant benefit of targeting them for treatment. We have demonstrated that our case finding database can identify these cohorts of patients in seconds, allowing subsequent follow up and treatment to reduce the future risk of cirrhosis.
1) Wesley E et al. A cumulative liver damage index (CLDI) identifies patients at risk of significant liver disease. Gut 2022:71:A4
Methods Data within our case finding system was used to analyse results from patients born between 1/1/1980 – 31/12/1989 [blood results from 2005-2020 were available which covered age range 25-29: males 21,246, females 35,523]. The prevalence of abnormal ALTs and adequacy of subsequent investigation (with a non-invasive liver screen) was assessed. Where possible the system calculated a FIB-4 score.
Results For patients aged 25-29 the number of ALTs requested has increased over time. Comparing patients born in 1989 to those in 1980 there has been an 83% increase in tests, and a 63% increase in the number of abnormal results. The relationship was linear (for tests, R2=0.9749; p<10-6; for abnormal R2 = 0.8518; p=0.004). The number of patients with >2 abnormal results also increased (Fig 1; p=0.0005). However, there was no significant change in mean ALT (p=0.42) or proportion of tests which were abnormal (p=0.32) suggesting a true increase in disease prevalence.
Rates of follow up investigations for patients with persistently abnormal ALT (>40 for at least 90 days until last result) are shown in Table 1. For men and women respectively only 41% and 47% had sufficient viral serology and only 26% and 28% had an AST (allowing estimate of fibrosis). There were significantly more men than women (477 vs 176) because the laboratory definition of abnormal was used. Of those for whom calculation of FIB-4 was possible the result was low – mean 0.72 + 0.053 (men), 0.79 + 0.094 (women), excluding significant fibrosis in 94% and 87% respectively.
Conclusion The prevalence of liver disease in young adults in the UK is rising but is frequently under investigated. These patients mostly have early disease (as defined by FIB-4) and thus there is significant benefit of targeting them for treatment. We have demonstrated that our case finding database can identify these cohorts of patients in seconds, allowing subsequent follow up and treatment to reduce the future risk of cirrhosis.
1) Wesley E et al. A cumulative liver damage index (CLDI) identifies patients at risk of significant liver disease. Gut 2022:71:A4
Background: TAF, a novel prodrug of tenofovir, has demonstrated noninferior efficacy with superior bone and renal safety compared to tenofovir disoproxil fumarate and is approved for treatment in adults with CHB. TAF has shown superior efficacy and comparable safety to placebo (PBO) at Week 24 in a multicenter, randomized, double-blind (DB), placebo-controlled trial in children 6 years and older weighing ≥25 kg. Here we report efficacy and safety findings at Week 48.
Methods: Pediatric patients 12 to <18 y weighing ≥35 kg (Cohort 1) and 6 to <12 y weighing ≥25 kg (Cohort 2, Group 1), with HBV DNA ≥2 × 104 IU/mL, ALT ≥1.5 × ULN, and creatinine clearance (eGFR; Schwartz method) ≥80 mL/min were enrolled in this ongoing trial (NCT02932150) and randomized (2:1) to TAF 25 mg or PBO daily for 24 weeks (primary endpoint: % with HBV DNA <20 IU/mL), after which all patients received open-label (OL) TAF for up to Week 240 (ie, TAF vs PBO→TAF). At Week 48, efficacy was assessed based on the proportion with HBV DNA <20 IU/mL, serological, and biochemical responses. Safety assessments included serious adverse events (SAEs) and all adverse events (AEs), bone mineral density (BMD; spine and whole body [minus head]), and resistance surveillance.
Results: 88 patients were randomized and treated (Cohort 1: TAF 47, PBO 23; Cohort 2 group 1: TAF 12, PBO 6). Overall mean (range) age and mean (SD) weight were 14 (7-17) years and 50.9 (12.9) kg, respectively; 58% were male, 66% Asian, 68% had HBV DNA ≥8 log10 IU/mL and mean (SD) ALT 107 (118) U/L. 99% were HBeAg-positive, with genotype D (44%) being most common. Week 48 efficacy results were similar for TAF vs PBO→TAF by missing = failure analysis (Table). The proportion with HBV DNA <20 IU/mL in the TAF group increased from Week 24 to Week 48, 11/59 (19%) vs 22/59 (37%), respectively. A lower response rate with TAF treatment was observed in patients with genotype D; however, with longer-term treatment, an improved response was seen relative to Week 24 (17% vs 0%). Viral resistance was not detected through Week 48. Most AEs during the OL phase were mild-moderate; no patient had a Grade 3/4 AE or SAE related to study treatment, and none discontinued OL treatment due to an AE. At Week 48, the median change from baseline in eGFR mL/min/1.73m2 for TAF vs PBO→TAF was similar (Table), and no patient had eGFR <90 mL/min/1.73m2 through Week 48. BMD increased from baseline to Week 48, and mean % change was similar in both groups (Table).
Conclusion: Increasing rates of viral suppression were seen in pediatric CHB patients who completed 48 weeks of TAF treatment, while the safety profile remained favorable. Patients switching from PBO →TAF at Week 24 had similar safety and efficacy results relative to the TAF group during the DB phase.
Methods: Pediatric patients 12 to <18 y weighing ≥35 kg (Cohort 1) and 6 to <12 y weighing ≥25 kg (Cohort 2, Group 1), with HBV DNA ≥2 × 104 IU/mL, ALT ≥1.5 × ULN, and creatinine clearance (eGFR; Schwartz method) ≥80 mL/min were enrolled in this ongoing trial (NCT02932150) and randomized (2:1) to TAF 25 mg or PBO daily for 24 weeks (primary endpoint: % with HBV DNA <20 IU/mL), after which all patients received open-label (OL) TAF for up to Week 240 (ie, TAF vs PBO→TAF). At Week 48, efficacy was assessed based on the proportion with HBV DNA <20 IU/mL, serological, and biochemical responses. Safety assessments included serious adverse events (SAEs) and all adverse events (AEs), bone mineral density (BMD; spine and whole body [minus head]), and resistance surveillance.
Results: 88 patients were randomized and treated (Cohort 1: TAF 47, PBO 23; Cohort 2 group 1: TAF 12, PBO 6). Overall mean (range) age and mean (SD) weight were 14 (7-17) years and 50.9 (12.9) kg, respectively; 58% were male, 66% Asian, 68% had HBV DNA ≥8 log10 IU/mL and mean (SD) ALT 107 (118) U/L. 99% were HBeAg-positive, with genotype D (44%) being most common. Week 48 efficacy results were similar for TAF vs PBO→TAF by missing = failure analysis (Table). The proportion with HBV DNA <20 IU/mL in the TAF group increased from Week 24 to Week 48, 11/59 (19%) vs 22/59 (37%), respectively. A lower response rate with TAF treatment was observed in patients with genotype D; however, with longer-term treatment, an improved response was seen relative to Week 24 (17% vs 0%). Viral resistance was not detected through Week 48. Most AEs during the OL phase were mild-moderate; no patient had a Grade 3/4 AE or SAE related to study treatment, and none discontinued OL treatment due to an AE. At Week 48, the median change from baseline in eGFR mL/min/1.73m2 for TAF vs PBO→TAF was similar (Table), and no patient had eGFR <90 mL/min/1.73m2 through Week 48. BMD increased from baseline to Week 48, and mean % change was similar in both groups (Table).
Conclusion: Increasing rates of viral suppression were seen in pediatric CHB patients who completed 48 weeks of TAF treatment, while the safety profile remained favorable. Patients switching from PBO →TAF at Week 24 had similar safety and efficacy results relative to the TAF group during the DB phase.
Background: Primary sclerosing cholangitis (PSC) is a progressive choleostatic disease and up to 80% of patients also have ulcerative colitis (PSC-UC). This presents a clinical challenge owing to difficulty in diagnosis and increased risk for developing cancer. While several multifactorial processes including inflammation and microbial dysbiosis have been associated with PSC-UC pathogenesis, the precise molecular factors that regulate the phenotype of this disease subtype remain unknown.
Methods: Here, we applied methyl-capture sequencing and mRNA sequencing to colonic mucosal biopsies to identify transcriptomic and epigenetic alterations differentiating UC from PSC-UC. Samples were taken from 3 groups of treatment-naïve children at diagnosis who participated in the DOCHAS study (GEN-193/11) - UC (n=10), PSC-UC (n=10) and healthy controls(n=10).
Results: Differential gene expression between UC and PSC-UC identified disease-associated genes that were either up- or down-regulated in UC or PSC relative to controls. Furthermore, the expression of these genes was intricately regulated by master transcriptional regulators (pro-caspases, IL7RA) and transcription factors (AR, p53, JUND, CEBPA). Importantly, gene expression comparison between UC vs PSC-UC revealed 4 up-regulated genes in PSC-UC (RAB31, TENM3, KLHL17 and COL7A1). Notably, RAB31 and TENM3 are also significantly up-regulated in gastrointestinal (GI) cancers. We also identified 4 down-regulated genes in PSC-UC (H3C15, SLC37A2, SLC14A2 and IL20RA).
Differential methylation analysis between healthy control biopsies vs PSC-UC and UC demonstrated >1000 differentially methylated regions (DMRs, 5Kb), with the majority of these sites displaying hypermethylation. Interestingly, PSC-UC vs UC analysis identified 53 regions and 59 DMRs that are hyper- and hypomethylated respectively in PSC-UC, with a large proportion of these DMRs located in gene promoters and putative enhancer regions and notably impacted gene expression of differentially expressed genes identified from the mRNAseq analysis.
Conclusion: Taken together, our study for the first time identifies distinct gene expression and DNA methylation alterations that differentiate UC from PSC-UC at diagnosis in treatment-naïve paediatric patients, some of which are associated with GI cancers. Their potential utility as predictive biomarkers of PSC development in UC or dysplasia risk in PSC-UC warrants further validation through larger patient cohorts.
Methods: Here, we applied methyl-capture sequencing and mRNA sequencing to colonic mucosal biopsies to identify transcriptomic and epigenetic alterations differentiating UC from PSC-UC. Samples were taken from 3 groups of treatment-naïve children at diagnosis who participated in the DOCHAS study (GEN-193/11) - UC (n=10), PSC-UC (n=10) and healthy controls(n=10).
Results: Differential gene expression between UC and PSC-UC identified disease-associated genes that were either up- or down-regulated in UC or PSC relative to controls. Furthermore, the expression of these genes was intricately regulated by master transcriptional regulators (pro-caspases, IL7RA) and transcription factors (AR, p53, JUND, CEBPA). Importantly, gene expression comparison between UC vs PSC-UC revealed 4 up-regulated genes in PSC-UC (RAB31, TENM3, KLHL17 and COL7A1). Notably, RAB31 and TENM3 are also significantly up-regulated in gastrointestinal (GI) cancers. We also identified 4 down-regulated genes in PSC-UC (H3C15, SLC37A2, SLC14A2 and IL20RA).
Differential methylation analysis between healthy control biopsies vs PSC-UC and UC demonstrated >1000 differentially methylated regions (DMRs, 5Kb), with the majority of these sites displaying hypermethylation. Interestingly, PSC-UC vs UC analysis identified 53 regions and 59 DMRs that are hyper- and hypomethylated respectively in PSC-UC, with a large proportion of these DMRs located in gene promoters and putative enhancer regions and notably impacted gene expression of differentially expressed genes identified from the mRNAseq analysis.
Conclusion: Taken together, our study for the first time identifies distinct gene expression and DNA methylation alterations that differentiate UC from PSC-UC at diagnosis in treatment-naïve paediatric patients, some of which are associated with GI cancers. Their potential utility as predictive biomarkers of PSC development in UC or dysplasia risk in PSC-UC warrants further validation through larger patient cohorts.
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